MOLECULAR AND RADIATION STUDIES ON IMPROVING THE AJMALICINE PRODUCTION IN Catharanthus roseus.
By ISLAM MOHAMED SALAMA EL-SAYED B. Sc. Agric. (Agric. Botany - Genetics), Fac. of Agric. Al-Azhar University 2001 M. Sc. (Agric Botany. - Genetics), Fac. of Agric. Al-Azhar University 2006
THESIS Submitted in partial fulfillment of the Requirements for the Degree
Of DOCTOR OF PHILOSOPHY
In AGRICULTURE SCIENCES (Agric. Botany - Genetics)
Department of Agric. Botany Faculty of Agriculture, Cairo Al-Azhar University
1434 A. H. 2013 A. D. TITLE: MOLECULAR AND RADIATION STUDIES ON IMPROVING THE AJMALICINE PRODUCTION IN Catharanthus roseus. NAME: ISLAM MOHAMED SALAMA EL-SAYED
THESIS Submitted in partial fulfillment of the Requirements for the Degree
Of DOCTOR OF PHILOSOPHY
In AGRICULTURE SCIENCES (Agric. Botany - Genetics)
Department of Agric. Botany Faculty of Agriculture, Cairo Al-Azhar University
1434 A. H. 2013 A. D.
Supervision committee: Prof. Dr. ABD EL-HADI IBRAHIM HASSN SAYED. Prof. of Genetics, Department of Agricultural Botany, Faculty of Agriculture, Al-Azhar University. Prof. Dr. MOHAMED ALI ABD EL-RAHMAN. Prof. of Genetics, Department of Agricultural Botany, Faculty of Agriculture, Al-Azhar University. APPROVAL SHEET
NAME: ISLAM MOHAMED SALAMA EL-SAYED TITLE: MOLECULAR AND RADIATION STUDIES ON IMPROVING THE AJMALICINE PRODUCTION IN Catharanthus roseus.
THESIS Submitted in partial fulfillment of the Requirements for the Degree
Of DOCTOR OF PHILOSOPHY
In AGRICULTURE SCIENCES (Agric. Botany - Genetics)
Department of Agric. Botany Faculty of Agriculture, Cairo Al-Azhar University 1434 A. H. 2013 A. D.
Approved by: Prof. Dr. Gomaah Ali Bahgat El – Fadly ………………... Prof. of Genetics, Faculty of Agriculture, Kafrelsheikh University. Prof. Dr. Shafik Ibrahim EL - Morsy El – Bosty ………………... Prof. of Genetics, Faculty of Agriculture, Al-Azhar University. Prof. Dr. Abd El-Hadi Ibrahim Hassn Sayed. ………………... Prof. of Genetics, Faculty of Agriculture, Al-Azhar University. Prof. Dr. Mohamed Ali Abd El-Rahman ………………... Prof. of Genetics, Faculty of Agriculture, Al-Azhar University.
Date: 23 / 1 / 2013 A.D. CONTENTS page LIST OF TABLES ii LIST OF FIGURES iii І. INTRODUCTION 1 ІІ. REVIEW OF LITTERATURE 7 II.1. Effect of Radiation on Indole alkalids biosynthesis. 7 II.2. Radiation doses effect. 14 II.3. Isomerism of Ajmalicine. 15 II.4. Effect of radiation on Isozymes banding patterns. 20 II.5. Effect of radiation on Proteins banding patterns. 30 II.6. Random amplified polymorphic DNA (RAPD). 31 ІІІ. MATERIALS AND METHODS 40 III.1. Materials 40 III.2. Methods 40 III.2.1. Gamma radiation treatment. 41 III.2.2. Indole alkalids determination. 41 III.2.3. Isozymes banding patterns analysis. 43 III.2.4. Proteins banding patterns analysis. 48 III.2.5. Randomly amplified polymorphic DNA (RABD). 54 ІV. RESULTS AND DISCUSSION 63 VI.1. Radiation treatment. 63 VI.5.Effect of radiation on Indole alkaloids biosynthesis. 64 a- First variety LM 64 b- Second variety CP3 72 VI.5.1. compare of Ajmalicine production in LM & CP3 variety in Catharanthus rouses. 78 VI.3. Effect of radiation on Isozymes banding patterns. 85 VI.3.1.Tryptophandecarpoxylase enzyme (TDC). 85 VI.3.2.Strrictosidinesynthase enzyme (STR). 90 VI.4.Effect of radiation on Protein banding patterns. 93 VI.2. DNA finger print analysis. 98 VI.2.1. Random amplified polymorphic DNA (RAPD) 98 . a- First variety LM 98 VI.2.1.2. RAPD markers of the 11 radiation treatments with 5 RAPD primers. 107 VI.2.1.3. Genetic similarity and cluster analysis based on RAPDs markers. 110 b- Second variety CP3 113 VI.2.1.4. RAPD markers of the 16 Krad radiation treatments with 10 RAPD primers. 126 VI.2.1.5. Genetic similarity and cluster analysis based on RAPDs markers. 128 VI.2.2. Similarity and unsimilarity between (LM) & (CP3) Catharanthus roseus Varieties in Genomic under radiation stress. 130 V. SUMMARY 133 VI. REFERENCES 141 ARABIC SUMMARY
i LIST OF TABELS Page Table 1: Isomerism of Ajmalicine 17 Table 2: stock solution for isozymes analysis. 43 Table 3: List of primer names and their nucleotide sequences used in variety LM (RAPD). 54 Table 4: List of primer names and their nucleotide sequences used in variety CP3 (RAPD). 55 Table 5: Effect of the radiation treatments on the plants survivor. 63 Table 6: Effect of Gamma radiation treatment on biosynthesis of Indole alkaloids Catharanthus roseus variety LM concentration by (µg). 72 Table 7: Effect of gamma radiation on the Ajmalicine content in C. roseus variety CP3. 77 Table 8: Molecular weight and Intensity of Tryptophan decarpoxylase (TDC) and Strictosidine synthase (SSS) Enzymes in Catharanthus roseus variety LM which was treated by Gamma radiation. 93 Table 9: SDS – Page protein analysis of the variety LM which is treated by Gamma radiation. 98 Table 10: RAPD profiles of the Catharanthus roseas variety LM which were treated by Gamma radiation amplified with primer OP-B01. 99 Table 11: RAPD profiles of the Catharanthus roseas variety LM which were treated by Gamma radiation amplified with primer OP-B07 103 Table 12: RAPD profiles of the Catharanthus roseas variety LM which were treated by Gamma radiation amplified with primer OP-B11 105 Table 13: RAPD profiles of the Catharanthus roseas variety LM which were treated by Gamma radiation amplified with primer OP-B12. 106 Table 14: RAPD profiles of the Catharanthus roseas variety LM which were treated by Gamma radiation amplified with primer OP-F06. 107 Table 15: RAPD markers of the 12 radiation treatment with 5 RAPD primers. 110 Table 16: Similarity indices among the 12 radiation treatment Taxa Based on RAPD-PCR using 5 primers. 112 Table 17: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-B09. 114 Table 18: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-C10. 115 Table 19: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-C13. 116 Table 20: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-C15. 117 Table 21: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-G17. 118 Table 22: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-L12. 119 Table 23: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-L13. 119 Table 24: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-L16. 120 Table 25: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-L20. 120 Table 26: RAPD profiles of the Catharanthus roseas variety CP3 which were treated by Gamma radiation amplified with primer OP-Z03. 122 Table 27: RAPD markers of the 16Krad with 10 RAPD primers. 127 Table 28: Similarity indices among the 8 deferent time Taxa Based on RAPD- PCR using 10 primers. 129 ii LIST OF FIGURES Figures Page Figure 1: The biosynthetic pathway of some phenolic compounds a small- dashed line means multi-steps reactions. 11 Figure 2: Summary of effects reported for various plant hormones and signal compounds in Catharanthus roseus cell cultures. 12 Figure 3: Proposed model for UV-B mediated signal transduction pathway leading to activation of the TIA pathway. 15 Figure 4: type of organic components isomerism. 16 Figure 5: Construction of the binary plant expression vector pBDH5. 23 Figure 6: Structure of the Str1 gene from C. roseas. 27 Figure 7: Overview of transcription factors that can interact with the STR and TDC promoters. 28 Figure 8: Model for elicitor signal transduction leading to STR expression. 30 Figure 9: Molecular architecture of the activation tagging vectors pVICE n4HPT and pSKI015. 34 Figure 10: Recently developed activation tagging vectors. 34 Figure 11: Linkage map of Catharanthus roseas in the F2 population of the cross ‘Pink Delhi’ × gsr8. 39 Figure 12: Indole alkaloids determination by HPLC in Catharanthus roseas variety LM which is treated by gamma radiation 67 Figure 13: Effect of time development on the Ajmalicine production in C. ruses variety CP3 at 16 Krad. 78 Figure 14: HPLC analysis of time development on the Ajmalicine production in C. ruses. under radiation does rate 16 Krad. 79 Figure 15: Tryptophan decarpoxylase diagram for the Catharanthus roseus Which are treatments by Gamma radiation. 87 Figure 16: Strictosidine synthase diagram for the Catharanthus roseus Which are treatments by Gamma radiation. 91 Figure 17: Protein diagram for the Catharanthus roseus which were treated by Gamma radiation. 96 Figure 18: Figure (12): RAPD profiles of the Catharanthus roseas which are treated by using gamma radiation amplified with 5 primers. 101 Figure 19: Dendrogram for the genetic distances relationships among the12 Radiation treatments taxa based on similarity indices data of RAPD analysis. 112 Figure 20: RAPD profiles of the 8 times treatment which are Gamma Irradiated at 16 Krad. amplified with 10 primers. 122 Figure 21: Dendrogram for the genetic distances relationships among the8 deferent time taxa based on similarity indices data of RAPD analysis. 130
iii ______abstract
ABSTRACT
NAME: Islam Mohamed Salama EL-Sayed TITLE: ‘‘Molecular and radiation studies on improving the Ajmalicine production in Catharanthus roseus” Elicitations are considered to be an important strategy towards improve in vitro production of secondary metabolites. In seedling cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cultures to low dose of Gamma irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (TDC) and strictosidine synthase (STR). In the present study, the signaling pathway mediating Gamma irradiation -induced catharanthine accumulation in C. roseus seedling cultures were investigated. Catharanthus roseus seedling cultures were exposed to different low dose of Gamma irradiation in order to induce alkaloid metabolism. The exposure to Gamma irradiation elicitors resulted in the transcriptional activation of tryptophan decarboxylase and in the accumulation of the monoterpenoid indole alkaloids ajmalicine and catharanthine, ______abstract but not of vindoline. The inability of the seedling cultures to produce vindoline was related to a lack of expression of the tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes. Acknowledgement DEEP THANKS TO ALLAH The author wishes to express his sincere gratitude and appreciation to Prof. Dr. ABD EL-HADI IBRAHIM HASSN SAYED Professor of Genetics, Department of Agricultural Botany, Faculty of Agriculture, AL-Azhar University. For his supervision, encouragement, valuable advises and his unlimited helps in writing thesis. Special thanks to Prof. Dr. MOHAMED ALI ABD EL-RAHMAN Professor of Genetics, Department of Agricultural Botany, Faculty of Agriculture, AL-Azhar University. For his supervision and help. The other wishes to acknowledge all members of Genetics, Department of Agricultural Botany, Faculty of Agriculture, AL-Azhar University. And all members of Department of Natural Product Research, National Center for Radiation Research and Technology, Atomic Energy Authority. The author wishes to extend his deep thanks and appreciation to Genetics, Department of Natural Product Research, National Center for Radiation Research and Technology, Atomic Energy Authority. for helping and providing facilities during the experimental work. ______Introduction
I- Introduction
Catharanthus roseas is a medicinal plant that produces clinically useful drugs, such as ajmalicine and vinblastine.
Tryptophan decarboxylase (TDC) and strictosidine synthase
(STR) are two enzymes that act early in the biosynthetic pathway leading to terpenoid indole alkaloids. Knowledge of the regulation of these biosynthetic genes will be helpful for metabolic engineering of terpenoid indole alkaloid productivity. In suspension-cultured cells, the genes encoding these enzymes are induced by fungal elicitors, such as
Pythium aphanidermatum culture filtrate or yeast extract. The mRNA levels of TDC and STR in response to elicitor treatment of the suspension cultured cells can be visualized by Northern blotting using radioactively labeled cDNA probes. This system is used in our laboratory as a bioassay to help identify the elicitor in fractionated yeast extract. Here we report the successful use of digoxigenin-labeled probes in this system. Frank et al., (1996).
1 ______Introduction
Although the production of most of the current medicines is based on chemical synthesis, more than 25% of the current prescribed drugs contain at least one active ingredient of plant origin. Examples of important plant- derived pharmaceuticals include the antitumoral taxol and vinblastine, the antimalarial drug quinine and artemisinin, the analgesical morphine and codeine. In addition, it has been estimated that more than 80% of the world’s population in developing countries depends primarily on herbal medicine for basic healthcare needs. There is also a revival of traditional medicine in developed countries and an increase in the use of herbal remedies. The world market of herbal medicines’, including herbal and raw material, has been estimated to have an annual growth rate between 5-15%.
Total global herbal drug market is estimated as US $ 62 billion and it is expected to grow to US $ 5 trillion by the year 2050. At same time, there is a growing concern on loss of genetic diversity since about 75% of the 50,000 different
2 ______Introduction medicinal plant species in use are collected from the wild.
Moreover, to rely solely on wild spontaneous plants
Antonella et al., (2007).
Catharanthus roseas plant is still the only source for the powerful antitumor drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, e.g., ajmalicine and serpentine are also of economical importance.
These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially.
Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. More than 130 C. roseas alkaloids have been identified, they are sharing many biosynthetic steps. The early stages of alkaloid biosynthesis in C. roseas involve the formation of secologanin derived from the terpenoid biosynthesis and its condensation with tryptamine to produce the central intermediate strictosidine,
3 ______Introduction the common precursor for the monoterpenoid indole alkaloids. Over twenty enzymes steps are involved in the biosynthesis of terpenoid indole alkaloids (TIAs) in C. roseas. Whereas, reported these enzymes take place in at least three subcellular compartments, the cytosol, the plastids, and the vacuol. Furthermore, the full characterization of C. roseas's alkaloid pathway is not yet achieved. A significant amount of researchs has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, the available knowledge of the regulation of this pathway is still sparse.
The conversion of L-tryptophan into tryptamine is catalysed by the enzyme tryptophan decarboxylase (TDC). This enzyme is regarded as a putative site for regulatory control of alkaloid biosynthesis and operates at the interface between primary and secondary metabolism. The stereospecific condensation of tryptamine and secolaganin is catalyzes by strictosidine synthase enzyme (STR, EC 4.3.3.2) to form the
4 ______Introduction key indole alkaloid 3 alpha (S)-strictosidine. The STR gene of C. roseas has been cloned and its nucleotide sequence was furthermore, determined. Hussein et al., (2008) Reported that cells of C. roseas (L.) Don. were genetically engineered to over-express the enzymes strictosidine synthase and
Tryptophan decarboxylase. Cultures transgenic for STR consistently showed ten fold higher STR. Two such lines accumulated over 200 mg / L of the glucoalkaloid -1 strictosidine and / or strictosidine-derived terpenoid indole alkaloids (TIAs), including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity.
In this study tryptophan decarboxylase and strictosidine synthase genes will be manipulated in order to determine the genes behavior in C. roseas which are treated by gamma radiation. The effect of gene within the obtained, in terpenoid
Indole alkaloid production will be investigated. The selection
5 ______Introduction of the best dose rate will be chosen depending on the resistance of radiation treatments.
6 ______Review of Literature
II. Review of Literature II.1. Effect of radiation on Indole alkaloids biosynthesis and other components: Sharabash (1970). reported that no signification effect on chlorophyll concentration in tissue on onion seedling occurred
when exposed to 50 or 5000 rad. of Gamma irradiation.
Sharabash et al., (1972). found that the dose of 10 Krad. of Gamma irradiation induced a marked increase in the chlorophyll contents in wheat seedlings. Tikhonoy et al., (1980). found that irradiation of Datura
innoxia. seeds with Gamma irradiation at 0.5 – 1.0 Krad. reduced N content. Schmauder et al., (1985). Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L- tryptophan exerts a stimulatorv effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzvmes was investigated in non-supplemented and Tryptophan supplemented Cinchona cell cultures. A remarkable increase of trvptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under nonregulated
7 ______Review of Literature
(normal) pH conditions. Abbreviations: TDC = tryptophan decarboxylase, tyr = L-tyrosine, phe = L-phenylalanine, DAHP = 3-deoxy-D-arabino-heptulosonic acid-7-phosphate, trp = L- tryptophan, E-4-P = erythrose-4-phosphate, PEP = phospheenolpyruvate, NDH = malate dehydrogenase, G-6-PDH = glucose-6-phosphate dehydrogenase, 6-PG-DH = 6- phosphogluconate dehydrogenase, Ch-mutase = chorismate mutase, AS-synthase = anthranilate svnthase, n.d. = not determined Georgiveva (1987). reported that increasing the Gamma irradiation doses caused increases in the content of quiones in pollen tubes of Lilum regali. and Beta vulgares. Lucumi et al., (2001). A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors were converted into indole alkaloids such as strictosidine, vallesamine, O- acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of sub culturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways. Felipe et al., (2002). Catharanthus roseus cell cultures were exposed to different conditions in order to induce alkaloid metabolism. The exposure to jasmonate and fungal elicitors resulted in the transcriptional activation of Tryptophan
8 ______Review of Literature
decarboxylase and in the accumulation of the monoterpenoid indole alkaloids ajmalicine and catharanthine, but not of vindoline. The inability of the cell cultures to produce vindoline was related to a lack of expression of the desacetoxyvindoline 4- hydroxylase (D4h) gene. Southern blot analysis revealed that D4h gene was not lost in the cell cultures. Felipe and Victor (2003). The Scientific Research Center of Yucatan (CICY, for its Spanish acronym) was founded in November 1979 as part of an effort to decentralize scientific activities from Mexico City. Several of the research programs carried out at CICY makes use of plant tissue culture techniques for their development. For this article, we have reviewed results obtained in research projects oriented towards basic plant biology questions, as well as towards the micropropagation of economically important species, and the production of secondary metabolites. Young et al., (2003). Production of camptothecin (CPT) from callus cultures of Camptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and
9 ______Review of Literature
CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light, 90:10. Carolyn et al., (2004). The optimum growth stage for enhancing ajmalicine production in Catharanthus roseus cultures with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10 or 100 µM on day 6 gave a maximum ajmalicine production of 10.2 mg l−1, a 300% increase over that of non-elicited cultures. Natali and Robert (2007). Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole alkaloid biosynthesis for chorismate, an important intermediate in plant metabolism. The biosynthesis C6C1 compounds are induced by biotic elicitors.
10 ______Review of Literature
Fig. (1). The biosynthetic pathway of some phenolic compounds. A small-dashed line means multi-steps reactions.
11 ______Review of Literature
Fig. (2). Summary of effects reported for various plant hormones and signal compounds in Catharanthus roseus cell cultures. A continued-line means one-step reaction. A small-dashed line means multi-step reactions. A bigdashed line with + or – indicates activation or inhibition of gene(s) expression, enzyme activity or end product level. A big-dashed line with both + and-means a concentration-dependent activation or inhibition. A strong activation or-inhibition is indicated by ++ or – –
Antonio et al., (2008). For nutritional purposes, a survey of the vitamin B6 levels from a variety of commercial presentations of table olives was carried out, taking into account the three main processing types (Spanish-style, directly brined and ripe olives). The analysis was performed by HPLC, following the official
12 ______Review of Literature
French method for vitamin B6 determination in foodstuffs. In- house validation data for two commercial table olives showed that themethod precision was good (coefficient of variation <6%) and recovery was quantitative (104% on average). There was a wide range of values for vitamin B6 in table olives (0–69.3 μg/100 g edible portion). The highest mean content was found in directly brined olives (33.9 μg/ 100 g edible portion) followed by Spanish- style (14.4 μg/ 100 g) and ripe olives (4.3 μg/100 g). On average, samples of the Gordal and Carrasqueña cultivars showed the highest vitamin B6 content in the case of Spanish-style olives, but in directly brined olives as well as in ripe olives the effect of cultivar was not statistically significant (p<0.05). Hussein et al., (2008). Suspension, calli and leaves of Egyptian Catharanthus roseas (L.) were genetically engineered to over-express the two enzymes Tryptophandecarboxylase and Strrictosidine syntheses, which catalyze key steps in the biosynthesis of terpenoid indole alkaloids, using Agrobacterium- mediated transformation with the two corresponding genes. The percentages of total alkaloids, vinblastine and vincristine were recorded as relative to C. roseas intact plant. The highest values of total alkaloids (14.47%), vinblastine (13.62 %) and vincristine (11.5%) of transgenic leaf cell cultures were recorded with (CS7). However, (C4) transformed leaf calli cultures gave 10.48, 8.3 and 6.19 (%) for total alkaloid, vinblastine and vincristine, respectively. On other hand, the maximum values of total alkaloids (16.47 %), vinblastine (18.09 %) and vincristine (14.16
13 ______Review of Literature
%) were recorded with (L3) transgenic leaf in vitro derived germinated seeds) as compared with other selected cell lines. II.2. Radiation doses effect Shilpa and Jayabaskaran (2007). Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitorsignaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding Tryptophan decarboxylase (Tdc) and strictosidine synthase (STR). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated.
14 ______Review of Literature
Proposed model for UV-B mediated signal transduction pathway leading to activate Fig. (3). Proposed model for UV-B mediated signal transduction pathway leading to activation of the TIA pathway.
II.3. Isomerism of Ajmalicine
Isomers are molecules that have the same molecular formula, but have a different arrangement of the atoms in space. That excludes any different arrangements which are simply due to the molecule rotating as a whole, or rotating about particular bonds. In structural isomerism, the atoms are arranged in a completely different order. This is easier to see with specific examples. What follows looks at some of the ways that structural isomers can arise. The names of the various forms of structural isomerism probably don't matter all that much, but you must be
15 ______Review of Literature aware of the different possibilities when you come to draw isomers. www.pubchemsubstance.com
Fig. (4) type of organic components isomerism
16 ______Review of Literature
Table No. (1) Isomerism of Ajmalicine ] tricture mol - / g D [ 2 3 O 2 ) N 6 24 H 21 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 3 1 0 0 1 Isomerism ( Isomerism Stricture - D TetrahydroalstoninePrestwick 3 StereoCenter Count StereoCenter Bonded Unit Count Unit Bonded - AA - 3 Bond Donor Bond Acceptor Bond - - Molecular Weight Molecular Formula Molecular XLogP H H Count Bond Rotatable Exact Mass Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Formal Charge Complexity Count Atom Isotope Atom Defined Count StereoCenter Atom Undefined Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently ] mol / g [ 3 O tricture 2 - N D 24 2 ) H 5 21 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 4 0 0 0 1 Ajmalicine - epi - Isomerism ( Isomerism 19 Stricture - D or Bonded Unit Count Unit Bonded 3 - AA - ned Atom StereoCenter Count StereoCenter Atom ned 3 Bond Don Bond Acceptor Bond - - Molecular Weight Molecular Formula Molecular XLogP H H Count Bond Rotatable Mass Exact Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Charge Formal Complexity Count Atom Isotope Count StereoCenter Atom Defined Undefi Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently ] mol / 3 g O [ 2 Stricture ClN - 25 D H 2 21 388.8878 C 2 5 2 388.15537 388.15537 54.6 27 0 606 0 0 4 0 0 2 ) 4 r Count r Isomerism ( Isomerism Ajmalicine HCL Ajmalicine tor Bonded Unit Count Unit Bonded - D not available not D 3 Bond Donor Bond Accep Bond - - Molecular Weight Molecular Formula Molecular H H Count Bond Rotatable Mass Exact Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Charge Formal Complexity Count Atom Isotope Count StereoCenter Atom Defined StereoCente Atom Undefined Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently
17 ______Review of Literature Table No. (1) Continue ] tricture mol - / g D [ 2 3 O 2 ) N 6 ( 24 H 21 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 3 1 0 0 1 Isomerism Stricture - D TetrahydroalstoninePrestwick 3 StereoCenter Count StereoCenter Bonded Unit Count Unit Bonded - AA - 3 Bond Donor Bond Acceptor Bond - - Molecular Weight Molecular Formula Molecular XLogP H H Count Bond Rotatable Exact Mass Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Formal Charge Complexity Count Atom Isotope Atom Defined Count StereoCenter Atom Undefined Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently ] mol / g [ 3 O tricture 2 - N D 24 2 ) H 5 21 ( 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 4 0 0 0 1 Ajmalicine - epi - Isomerism 19 Stricture - D or Bonded Unit Count Unit Bonded 3 - AA - ned Atom StereoCenter Count StereoCenter Atom ned 3 Bond Don Bond Acceptor Bond - - Molecular Weight Molecular Formula Molecular XLogP H H Count Bond Rotatable Mass Exact Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Charge Formal Complexity Count Atom Isotope Count StereoCenter Atom Defined Undefi Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently ] mol / 3 g O [ 2 Stricture ClN - 25 D H 2 21 388.8878 C 2 5 2 388.15537 388.15537 54.6 27 0 606 0 0 4 0 0 2 ) 4 ( r Count r Isomerism Ajmalicine HCL Ajmalicine tor Bonded Unit Count Unit Bonded - D not available not D 3 Bond Donor Bond Accep Bond - - Molecular Weight Molecular Formula Molecular H H Count Bond Rotatable Mass Exact Mass MonoIsotopic Area Surface Polar Topological Count Atom Heavy Charge Formal Complexity Count Atom Isotope Count StereoCenter Atom Defined StereoCente Atom Undefined Count StereoCenter Bond Defined Count StereoCenter Bond Undefined Covalently
18 ______Review of Literature Table No. (1) Continue tricture - D tetrahydro 2 - ) ] 9 ( mol / g 3,4,5,6 [ 3 , O 2 N 24 H 21 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 4 0 0 0 1 Isomerism Stricture - D Ajmalicine Alstonine Ajmalicine Polar Surface Area Bonded Unit Count 3 - AA - 3 Bond Donor Bond Acceptor - - Molecular Weight Molecular Formula XLogP H H Rotatable Bond Count Exact Mass MonoIsotopic Mass Topological Heavy Atom Count Formal Charge Complexity Isotope Atom Count Defined Atom StereoCenter Count Undefined Atom StereoCenter Count Defined Bond StereoCenter Count Undefined Bond StereoCenter Count Covalently Stricture - D 2 ) 8 ( Isomerism Ajmalicine methyl acetal methyl Ajmalicine Properties not available not Properties D not available not D 3 Stricture - D ) 2 ] 7 ( mol / g [ 3 O 2 N 24 H 21 352.42686 C 2.7 1 5 2 352.178693 352.178693 54.6 26 0 606 0 4 0 0 0 1 Isomerism Tetrahydroalstonine not available not StereoCenter Count D Bonded Unit Count 3 - AA - 3 Bond Donor Bond Acceptor - - Molecular Weight Molecular Formula XLogP H H Rotatable Bond Count Exact Mass MonoIsotopic Mass Topological Polar Surface Area Heavy Atom Count Formal Charge Complexity Isotope Atom Count Defined Atom Undefined Atom StereoCenter Count Defined Bond StereoCenter Count Undefined Bond StereoCenter Count Covalently
19 ______Review of Literature
II.4. Effect of radiation on Isozymes banding patterns. Flaig and Schmid (1966). reported that differences in the content on different compounds or enzyme activities have been observed in irradiated seeds, seedling and plant organs. Thus the content of sucrose and other sugars increased with increasing doses of irradiation. Changes in the activities of different enzymes, such as acid phosphatase, peroxidase, polyphenralase has been observed, sometimes an increase in activity could be observed using low doses. For example, respiration is often increased and content of chlorophyll can be increased after irradiation. Toth et al., (1983). Said that there were differences in isoperoxidase patterns within and between from seeds of Digitalis lanata treated with Gamma rays and another untreated plants.Georgieva (1987). reported that increasing the Gamma irradiation doses enhanced peroxidase activity Lilium regali. and Beta vulgaris. Mollenschott and Berlin (1984). The purification of Tryptophandecarboxylase from Catharanthus roseas (TDC, E.C.: 4.1.1.27), to apparent homogeneity, the enzyme represents a soluble protein with a molecular weight of 115 000 + 3 000, consisting of 2 identical subunits of 54 000 + 1 000. The pI was estimated to be 5.9 and the K m for L-tryptophan was found to be 7.5 × 10 -5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog
20 ______Review of Literature
kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed. Peter and Frank (1991) suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In-vitro assays for the specific activity of tryptophan decarboxylase (TDC) and Tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension- cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible
21 ______Review of Literature for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension- cultured cells only after the addition of exogenous secologanin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on Indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to Indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of Indole alkaloid biosynthetic enzymes in our system beyond, and including, Strrictosidinesynthase. The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Oscar et al., (1995). Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a TDC cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein Level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production was
22 ______Review of Literature observed compared to empty-vector-transformed tumor calluses. In turnout calluses containing the TDC antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.
Fig. (5). Construction of the binary plant expression vector pBDH5 and generation of transcriptional fusions between an artificially completed tdc cDNA sequence and the CaMV 35S promoter and terminator in pBDH5. Construct pTDCs leads to overexpression of tdc mRNA, while pTDCa leads to expression of antisense tdc RNA. Abbreviations, M.C.S., multiple cloning site; LB, RB, left and right T-DNA border repeats; P35S and Pnos refer to the CaMV 35S and nopaline synthase promoter sequences; T35S and Trios refer to the CaMV 35S and nopaline synthase terminator sequences; NPTII, neomycin phosphotransferase gene; Km, bacterial kanamycin resistance selection marker; Cb, bacterial carbenicillin resistance selection marker.
23 ______Review of Literature
Serap et al., (1998). The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4- dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the
24 ______Review of Literature
central intermediate strictosidine, and desacetoxyvindoline 4- hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. TDC and STR1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. Benoit et al., (1999) in marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the Vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce Vindoline by cell culture technology have failed. Giancarlo et al., (1999). Strictosidine synthase (STR) is a key enzyme in the biosynthesis of terpenoid indole alkaloids. This class of secondary metabolites harbours several pharmaceutically important compounds used, among other applications, in cancer treatment. Terpenoid indole alkaloid biosynthesis and expression
25 ______Review of Literature of biosynthetic genes including Str1 is induced by fungal elicitors. To identify elicitor-responsive regulatory promoter elements and trans-acting factors, the single-copy Str1 gene was isolated from the subtropical plant species Catharanthus roseus (Madagascar periwinkle). Str1 upstream sequences conferred elicitor- responsive expression to the _-glucuronidase (gusA) reporter gene in transgenic tobacco plants. Main enhancer sequences within the Str1 promoter region studied were shown to be located between _339 and _145. This region and two other regions of the promoter bound the tobacco nuclear protein factor GT-1. A G-box located around position _105 bound nuclear and cloned G-box-binding factors (GBFs). A mutation that knocked out GBF binding had no measurable effect on expression, which indicates that the G-box is not essential for the elicitor responsiveness of the Str1 promoter. No obvious homologies with promoter elements identified in other elicitor-responsive genes were observed, suggesting that the Str1 gene may depend on novel regulatory mechanisms.
Fig. (6). Structure of the Str1 gene from C. roseas. a. Restriction map of the insert of the genomic clone pGCR18. Restriction sites are indicated for XhoI (X), BglII (B), HindIII (H), EcoRI (E) and BamHI (Ba). The outer XhoI sites are derived from the lambda vector. The region indicated with a hatched bar was sequenced, and a detailed map is shown in b. b. Detailed map of the
26 ______Review of Literature
Str1 gene and flanking sequences. The transcribed region is indicated with black and white boxes. The black portions represent coding sequences. The 50- and 30-untranslated regions as well as two introns are shown in white. The promoter region shown in Figure 2 is represented by a hatched bar. Restriction sites are indicated as in a. The XhoI site is derived from the lambda vector.
Leslie et al., (2000). Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (STR) is mediated via the plant stress hormone jasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the STR promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the STR promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor- responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.
27 ______Review of Literature
FIG. (7). Overview of transcription factors that can interact with the STR and TDC promoters. Perception of YE leads to an increase in JA levels, which is necessary for the activation of the ORCA transcription factors. Although the cellular location of the YE receptor is unknown, it is tentatively placed in the plasma membrane. The ORCA transcription factors can activate gene expression via interaction with the TDC promoter and the RV fragment of the STR promoter. Although the ORCA binding site in the TDC promoter has not been precisely mapped, it is tentatively indicated downstream of the DB fragment. In addition, YE rapidly induces the accumulation of mRNAs encoding ZCT proteins, which can repress gene expression via binding to the DB fragment of the TDC promoter and the BA and, to a lesser extent, the RV fragments of the STR promoter. Also, YE induces accumulation of mRNA encoding CrBPF1, which is putatively involved in regulation of STR via interaction with the BA region. CrGBF transcription factors can repress STR promoter activity via binding to the NR region.
Bea et al., (2004). In Catharanthus roseas cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidinesynthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one- hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of
28 ______Review of Literature
the Cys2/His2-type (transcription factor IIIA-type) zinc finger protein family from C. roseas, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene responsefactor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseas. Elizabeta et al., (2004). Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached 2 mg g−1 dry wt after 27 d in culture. Maximal vindoline accumulation coincided with maximum activities of deacetoxyvindoline 4-hydroxylase, deacetylvindoline acetyl-CoA acetyl transferase and tryptophan decarboxylase. Shoot exposure to jasmonate shortened the time required for the maximal vindoline accumulation to 14 d.
29 ______Review of Literature
Fig. (8). Model for elicitor signal transduction leading to Str expression. The model shows the position of CrBPF-1 in a JA-independent elicitor signal transduction pathway. Protein phosphorylation and calcium influx are required for the activation of the ODA pathway, as well as for the induction of CrBPF-1. The position of the TATA box and the jasmonate- and elicitor- responsive element (JERE) are indicated.
II.5. Effect of radiation on Proteins banding patterns. El-Shebawi (1984). reported that irradiation with doses of
Gamma rays 0.05, 0.25, 0.5, 1.0 and 2.0 KGy. caused significant in some protein bands of broad bean globulin fraction. He observed the presence of additional bands in electrophoretic diagram of broad bean protein. Afify and Shousha (1988). investigated changes in the protein patterns of five soybean cultivar, separated by sodium dodecyle sulphate play acrylamide gel electrophoresis (SDS- PAGE), after exposure to Gamma irradiation, and they attributed changes in protein patterns to partial protein decemination, scission of peptide and disulfide bands and addition to aromatic and heterocyclic amino acid residues. Novak et al., (1990). found that protein banding pattern of the original Grandnain and the mutant were different. Probably
30 ______Review of Literature
the most prominent differences was in the intensity quantity and mobility of a major protein raving a molecular might of about 33 Kda. The original clone showed a densely stained band which migrated faster (Rf = 0.44) than that of the mutant Gn = 60 Gy/A. in addition three other bands were nat observed in the mutant, but only in the original grand nain. Such band (i.e. proteins) were less densely stained with an Rf value of 0.19, 0.31 and 0.64 and molecular weight of about 94 and 26 Kda, respectively. Cambecedes et al., (1991). found that among 20 regeneration plants from irradiation tests on Lonicera nitida Maigrum, only one very slender mutants was characterized by the lock of a 52 Kda. Band in the banding pattern of denaturated soluble protein. Kazuyuki et al., (2001). A peptidase (GICP) that cleaves the Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a sequence in phenylalanine oxidase, was purified from bovine pancreas. The purified enzyme had an Mr of approximately 29,000, as determined by SDS-PAGE, and its N-terminal sequence was identical to that of bovine pancreatic elastase II. The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1 (Km 5 8.3 mM kcat 5 2.1 s21) and the catalytic efficiency (2.6 3 105 M21s21) was comparable to those of elastase II from porcine pancreas and rat mesenteric arterial bed perfusate. The P1 site specificity of GICP toward oxidized insulin A and B chains suggested that major cleavage sites were the peptide bond at the C-terminal side of Gln, Leu, His, and Tyr residues.
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Rashed et al., (1997). treated high and low yielding soybean plants with 15 Krad. of Gamma rays and analyzed then for protein electrophoresis patterns (SDS – PAGE). The low and high yielding treated plants were characterized by appearance and / or the disappearance of some minor bands, which confirmed the association between these bands and the gene expression of high yielding trait. II.6. Randomly amplification 0f polymorphic DNA (RAPD) Annemarie et al., (1993) reported that cytochrome P-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. An efficient PCR strategy for isolating cytochrome P-450 cDNA clones from plant cDNA libraries is described. A set of degenerate primers for PCR amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome P-450 proteins. Using this primer set and a non- specific primer, complementary to either the poly (A) tail of the cDNA clones or a phage vector sequence, the others isolated 16 different cytochrome P-450 cDNA sequences from a cDNA library of Catharanthus roseus. Maria and Matgorzata (1999) infected Alstroemeria seedlings with naturally infected lily 'Casablanca' with stunting and flower bud deficiency phytoplasma resulted 3-4 weeks after top grafting in chlorotic and/or necrotic stripes, whitening of the leaves, shoot necrosis and die back. Flower discoloration or
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malformation was not observed. Attempts to transmit phytoplasma from naturally infected lily and experimentally infected Alstroemeria to Catharanthus roseus by top grafting resulted in stunted growth, dull yellowing and malformation of the leaves in 4-6 weeks. Some plants were temporary entirely vegetative and did not produce flowers. The periwinkle plants that were bridged by Cuscuta odorata from the diseased lilies and Alstroemerias showed similar symptoms as top-grafted ones. With the universal primer pairs rU3/fU5 specific PCR product with expected length -900 was amplified from samples collected from lilies with severe symptoms and top grafted test plants. All PCR products used for RFLP analysis after digestion with Alu I showed the same restriction profiles. Position of three obtained bands corresponded to the lengths of the DNA fragments of American aster yellows (AAY) phytoplasma group. A significant limitation of classical loss-of function screens designed to dissect genetic pathways is that they rarely uncover genes that function redundantly, are compensated by Helen et al., (2004) alternative metabolic or regulatory circuits, or which have an additional role in early embryo or gametophyte development. Activation T-DNA tagging is one approach that has emerged in plants to help circumvent these potential problems. This technique utilises a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus (CaMV) 35S enhancer sequence. This element enhances the expression of neighbouring genes either side of the randomly integrated T-DNA tag, resulting in gain-of-
33 ______Review of Literature function phenotypes. Activation tagging has identified a number of genes fundamental to plant development, metabolism and disease resistance in Arabidopsis. This review provides selected examples of these discoveries to highlight the utility of this technology. The recent development of activation tagging strategies for other model plant systems and the construction of new more sophisticated vectors for the generation of conditional alleles are also discussed. These recent advances have significantly expanded the horizons for gain-of-function genetics in plants.
Fig. (9). a, b Molecular architecture of the activation tagging vectors pVICE n4HPT and pSKI015. apVICE n4HPT contains four copies of the CaMV 35S enhancer sequence adjacent to the right T-DNA border sequence (RB), which potentially enhance expression of genes neighbouring both the left border (LB) and RB sequences. The construct harbours an ampicillin gene (Amp) and an origin of replication (ori) which allows the isolation of flanking sequences by plasmid rescue in Escherichia coli. A hygromycin resistance gene (HPT) fused with the nopaline synthase promoter (PNOS) and the poly A sequences of gene 4 of the A. tumefaciens T-DNA (g4pA) provides a selectable marker for successful transformation. b The activation tagging vector pSKI015 (Weigel et al., 2000). derived from pVICE n4HPT. BAR: glufosinate resistance gene. The construct harbours an ampicillin gene (Amp) and an origin of replication (ori) which allows the isolation of flanking sequences by plasmid rescue in E. coli. The restriction enzyme sites indicated can be employed to rescue pUC19 and adjacent plant sequences from transformed plants. The restriction sites NotI, SpeI and BamHI can be used to rescue plant sequences adjacent to the left T-DNA border. In a similar
34 ______Review of Literature fashion, KpnI, PstI, EcoRI and HindIII sites can be used to rescue plant sequences adjacent to the right T-DNA border
Fig. (10). a–c Recently developed activation tagging vectors. a The pGA2715 vector (Jeong et al., 2002). developed for activation tagging in rice contains four copies of the CaMV 35S enhancer (4x35S) adjacent to the left border (LB); the OsTubA1intron 2 carrying three putative splicing donor sites (I2); the GUSreporter gene; the NOS terminator (TNOS) sequence; the terminator of the OsTubA1 gene (TT); the hygromycin resistance gene (HPT); and the first OsTubA1 intron (I1), to increase gene expression. b The pER16 vector (Zuo et al., 2002). designed for 17-β-estradiol inducible expression of adjacent genes possesses a G10-90 promoter fused to the XVE transcription factor gene and ribulose diphosphate carboxylase E9 poly A addition sequence (TE9). The G10-90promoter is a tetramer of the G-box motif; this synthetic promoter drives constitutive non-tissue specific expression in both dicots and monocots (Ishige et al., 1999). The chimeric XVE protein consists of the DNA-binding domain of the bacterial repressor LEXA (X), the acidic transactivation domain of VP16 (V) and the regulatory domain of the human estrogen receptor (Zuo et al., 2000). A second transcriptional unit is comprised of the NOS promoter (PNOS), a kanamycin resistance gene (NPTII) and NOS terminator (TNOS). Eight copies of the LexA operator sequence (OLexA), a binding site for the XVE transcription factor, are fused to the minimal 46-bp CaMV 35S promoter sequence, which is adjacent to the LB. Addition of 17-β-estradiol induces XVE binding to the OLexA sites, which promotes expression of the gene(s) adjacent to the left border. c The heat-shock tagging vector pTT101 which possesses the promoter sequence of the ArabidopsisHSP18.2gene (Matsuhara et al., 2000)., 93 bp from the LB sequence. This promoter contains a TATA sequence and transcription initiation site and promotes the expression of neighbouring genes following 2 h of heat-shock at 37°C (RBright border).
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Ajaswrata et al., (2007) a. Plants produce secondary metabolites in response to various external signals. Coordinated transcriptional control of biosynthetic genes emerges as a major mechanism dictating the accumulation of secondary metabolites in plant cells. However, information about stress regulation of secondary metabolites and the molecular mechanisms regulating these specialized pathways are poorly understood. Here, we show that terpenoid indolealkaloid (TIA) biosynthetic pathway is differentially regulated in response to different abiotic stresses in Catharanthus roseus a model medicinal plant producing important anticancer and antihypertensive drugs. Semi quantitative RT-PCR analysis of TIA and related primary pathway genes in response to dehydration, low temperature, salinity, UV-light and wounding revealed their negative regulation in response to low temperature. HPLC analysis further supports the notion that TIA biosynthetic pathway is negatively controlled by low temperature stress. Furthermore, we report the cloning of a C-repeat binding transcription factor from C. roseus (CrCbf), belonging to AP2 class of transcription factor and possessed the NLS and CBF signatures equence characteristic of CBFs. CrCbf was found to be similar to Brassica Cbfs, whereas it was distant to monocot Cbfs. Southern analysis of CrCbf revealed the presence of more than one copy of CrCbf gene or other Cbf homologues in C. roseus genome. The transcription of CrCbf was found to be constitutive in response to low temperature but it showed differential distribution. The need for identifying novel
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transcription factors in understanding secondary metabolite biosynthesis is discussed. The understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid Indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseas, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseas cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol- 4-phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macroarray, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseas comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseas cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseas cell cultures. Ajaswrata et al., (2007). b.
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Sarika et al., (2007) an integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F2 population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseas.
F
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igure (11). Linkage map of Catharanthus roseas in the F
2 population of the cross ‘Pink Delhi’ × gsr8. Loci names and cumulative genetic distances, in cM are indicated on the right and left side of the vertical bars, respectively. RAPD and ISSR markers are represented as the primer name and number is followed by band size in subscript. Other PCR based markers (designed and designed forward/reverse + RAPD) are also represented by the primer name (SGD and SSG, respectively) and number followed by band size in subscript. The parental origin of each band is shown in the figure by the letter P for ‘Pink Delhi’ and letter G for gsr8 placed next to the values of band size in brackets. The acronyms of morphological markers have been used as shown.
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III-Materials and methods III.1-Materials: III.1.1-plant materials: This study was carried out by the cooperation between genetic unit (Botany Dept., Faculty of Agriculture., AL-Azhar University) and (Genetic Engineering Laboratory, Department of Natural Products Research, National Center for Radiation Research and Technology, Atomic Energy Authority, Nasr city, Cairo, Egypt). Catharanthus roseus seeds of tow varieties (LM & CP3) were obtained from Desert research center (D.R.C.), Almatarea, Cairo, Egypt. III.1.2- Cesium irradiation source: 137Ce. was used a source of gamma rays with dose rate 1K rad / 7.35 min. Catharanthus roseus cultivars were exposed to gamma irradiation at National center for radiation research and technology, Nasr city, Cairo, Egypt. III.1.3-Media: Water agar, free hormones were used in this work. III.2-Methods: Preparation seeds of tow varieties LM & CP3 of Catharanthus roseas and culture initiation: Seeds were washed with running after wards seeds tap surface stabilized by soaking for 15 min in 30 % Clorox.
٤١ ______Materials and methods
Then they were thoroughly washed to assure that any residues of Clorox had been removed. They were placed in jars contained Water agar free hormones. The cultured jars were incubated in growth chamber at (29.5o + 2o C) under photoperiod of 16 h. of 1000 LUX intensity. III.2.1-Gamma radiation treatment: Catharanthus roseas seedling were irradiated after four weeks from culture on the media by gamma radiation doses 0 as a control and 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 Krad. Cultures were incubated at the previous conditions. Sub culturing was carried out after 4 weeks.
III.2.2. Alkaloids determination for LM & CP3 varieties:
For the determinations of terpenoid Indole alkaloids and fed precursor(s) in the leaf, Pollets of cell were leaf ground in a mortar with glass beads and centrifuged at 14000 g for 5 min and 25 µl supernatant was injected directly into the HPLC system Lucumi et al., (2001). Terpenoid Indole alkaloids, Catharanthine, Vindoline, Vallesiachotamine(a), Vallesiachotamine (b) Ajmalicine, Horhammericine (a), Horhammericine (b), Vindolinine, 19-Epivindolineine, Strrictosidinelactam, Serpentine and Vinamidine were extracted from 100 mg freeze dried cell material with 15 ml methanol using an Ultraturrax. After centrifugation at 3500 g
٤٢ ______Materials and methods for 30 min, the methanolic extract was evaporated under reduced pressure, 1 ml of the Mobile phase A was added to the dry extract and the suspension was homogenized using a vortex mixer. After centrifugation of the alkaloid solution at 14 000 g for 5 min, 25 µl of the supernatant was injected into the HPLC system. The identity of the analyses was established by photodiode array detection of their UV spectra and ESP LC-MS. ESP LC-MS was performed on a Finnegan MAT TSQ–700 triple-stage quadropole mass spectrometer equipped with ESI–MS electro spray ionization. A gradient system was developed for the determination of the iridous and the alkaloids present in the sample and in the medium. The solvent A consisted of water/ acetonitrile / tri fluoro acetic acid (95:5:0.01, by vol.) (pH = 3) and the solvent B of water/acetonitrile / tri fluoro acetic acid (5:95:0.01, by vol.) (pH = 3), linear gradient of 18.5% acetonitrile over 20 min at 1 ml min−1. The column used was a Vydac C18 4.6, 25 cm for peptide analysis with a C-18 pre-column. 1693 Because of lack of sufficient pure reference compounds, quantification was based on calibration curves with alkaloids with similar UV-spectra: Strrictosidine was quantified using the Ajmalicine calibration curve and vallesamine, O- acetylvallesamine, voaphylline and others above using a tryptamine calibration curve (detection at 280 nm).
٤٣ ______Materials and methods
III.2.3. Isozymes banding patterns analysis for LM variety.
Native-polyacrylamide gel electrophoresis (Native- PAGE) was conducted to identify isozyme variation among studied taxa the using two isozymes systems.
Table (2) stock solution for isozymes analysis
Stock Solution 8% gel Acrlyamide (30%) 28 ml gel buffer pH 8.6 26 ml 10 % Ammonium per sulfate 3 ml TEMED 50.75 μl Fresh and young leaf samples for each variety and leaf position were used separately for isozymes extraction. The utilized isozymes are Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) separated in 12 % polyacrylamide gel electrophoresis according to Stegemann et al., (1985).
III.2.3.1. Stock Solutions a- Extraction buffer for TDC enzyme: 200 mM Tris-Cl (pH 7.6):
Solution was prepared by dissolving 200 mMTris-Cl pH 7.6, 10 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol. Tris was dissolved in about 50 ml distilled
٤٤ ______Materials and methods water and pH was adjusted to7.6 by HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC. b- Equilibrated buffer to TDC enzyme for columns:
Solution was prepared by dissolving 50 mM Tris-Cl pH 7.5 and 28 mM ß-mercaptoethanol. About 50 ml distilled water and pH was adjusted to7.5 by HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC. c- Extraction buffer for STR enzyme: 200 mM Tris-Cl (pH 7.6):
Solution was prepared by dissolving 1 m Tris-Cl pH 7.5, 4 mM dithiothreitol and 2 mM ethylenediaminetetra acetic acid (EDTA), 5 mM dithiothreitol. Tris in about 50 ml distilled water and pH was adjusted to7.6 by HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC. d- 30 % acrylamide stock:
The solution was prepared by dissolving 29.2 g acrylamide and 0.8 g N, N, methylene bis–acrylamide in about 70 ml distilled water, then the volume was completed to 100 ml by distilled water. The stock solution was kept in dark bottle at 4º C.
٤٥ ______Materials and methods e- Electrode buffer (pH 8.65):
Electrode buffer was prepared by dissolving 18.55 g boric acid and 2.5 g sodium hydroxide in 500 ml distilled water and mixed well with magnetic stirrer, then pH was adjusted into 8.6 by distilled water, and then kept at 4º C. f- Gel buffers:
Separating gel buffer (1.5 M Tris – HCl, pH 8.8):
Tris 18.15 g
H2O (distilled water) up to 100 ml
Stacking gel buffer (0.5 M Tris – HCl pH 6.8):
Tris 6.05 g
H2O (distilled water) up to 100 ml
Ammonium persulfate solution (APS 10 %):
Ammonium persulfate 0.1 g
H2O (distilled water) up to 10 ml
III.2.3.2. Extraction of enzymes: a- TDC enzyme:
Enzymes were extracted from the different C. roseas taxa by homogenizing 1g fresh leaves samples in 2.5 ml extraction buffer using a mortar and pestle. The extract was then transferred into clean eppendorf tubes and centrifuged at
٤٦ ______Materials and methods
10000 rpm for 5 minutes. The supernatant was transferred to new clean eppendorf tubes. Supernatant was desalted on PD- 10 columns (Pharmacia), previously equilibrated with the equilibrated buffer. For TDC assays, the protein extract was mixed with 50 nCi of L-[14C] H3 tryptophan (Amersham), 0.5 mM pyridoxal phosphate, and 50 mM Tris-Cl pH 7.5 in a final volume of 120 µL. The mixtures were incubated at 37°C for 20 min and the reaction was stopped with 1.0 M NaOH. The unreacted substrate (Rf 0.52) was separated from the product (Rf 0.22) by thin layer chromatography (TLC) (silica plates 0.2 mm, Merck) using 10% methanol in ethyl acetate as solvent. The chromatograms were visualized under UV light (366 nm) and the spots corresponding to the product were scraped from the plates, counted and kept at – 20o C until use for the electrophoretic analysis. Felipe et al., (2002).
b- STR Enzyme
Enzymes were extracted from the different C. roseas taxa by homogenizing 1g fresh leaves samples in 2.5 ml extraction buffer using a mortar and pestle. In the presence of polyvinylpyrrolidone (50 mg/g fresh weight). To the frozen material one volume of extraction buffer. The material was allowed to thaw and the extracts were clarified by
٤٧ ______Materials and methods centrifugation for 30 min at 10000 rpm. The supernatant was desalted on Sephadex G-25 (PD-10 columns, Pharmacia, Uppsala, Sweden) equilibrated buffer. Incubation mixture for SSS or STR activity determination contained 25 μl of desalted protein extract and 62.5 μl of a cocktail containing 1.6 mM tryptamine-HCL and 8.0 mM secologanin in 0.1 mM sodium phosphate buffer pH 6.8, together with 12.5 μl of a freshly prepared solution of 0.8 mg D(+)-gluconic acid-δ- lactone in 0.8 mg Tris. After incubation at 30 oC for 60 min. the reaction was stopped by adding 75 μl of 5 % trichloroacetic acid. Blanks were made by adding trichloroacetic acid prior to the 60-min incubation period. After addition of 14.8 μl of internal standard (8.0 mM cold- HCL) and centrifugation, samples were analyzed on acrylamide gel electrophoresis.
III.2.3.3. Gel preparation:
Vertical slab gel electrophoresis apparatus was used. All glass plates were washed with tap and distilled water, then surface sterilized with ethanol. Spacers of 1.5 mm were used. Separating gel was prepared by mixing the chemical components listed in Table (2). The prepared gel solution was poured immediately between the two glass plates and overlaid with isopropanol and left to polymerize for at least one hour. After polymerization, isopropanol was removed.
٤٨ ______Materials and methods
Stacking gel was similarly, prepared by mixing the chemical ingredients listed in Table (2) and then poured over the separating gel. The comp was placed immediately. The gel was left to polymerize. Table (2) showed the chemical ingredients and their concentrations that used for preparing both separating gel (resolving gel) and stacking gel.
III.2.3.4. Application of samples
A volume of 40 μl extract of each sample was mixed with 20 μl sucrose and 10 μl bromophenol blue, then a volume of 50 μl from this mixture was applied to each well.
III.2.3.5. Electrophoresis conditions:
The gel glasses were fixed to both lower and upper tanks of the electrophoresis apparatus. The run (electrode) buffer was added to both lower and upper tanks. The apparatus was connected to the power supply. The run was performed at 30 volt until the bromophenol blue dye has reached the separating gel and then the voltage was increased to 70 volt. Electrophoresis apparatus was placed inside a refrigerator during running duration.
III.2.4. Protein banding patterns analysis for LM variety: III.2.4.1. Stock Solutions SDS polyacrlamid gel electrophoresis (SDS PAGE):
٤٩ ______Materials and methods
Sodium dodecyl sulphate polyacrlamid gel electrophoresis (SDS PAGE) was performed according to the method described by Laemmli, U. K. (1970). and modified by Studier et al., (1973). a- The monomer solution: 29.2g Acrylamide and 0.8 g Bis-acrylamide were dissolved in 50 ml distilled water and completed to 100 ml, filtered and kept at 4oC in a dark bottle to be used within three months. b- Resolving gel buffer (1.5 m tris-base, pH 8.8): 18.2g Tris-base was dissolved in 50 ml distilled water and completed to 100 ml. the pH was adjusted at 8.8 with analar Hcl. The solution was filtered and kept at 4oC. c- Stacking gel buffer (0.5 m tris- base, pH 6.8): 6 g Tris-base were dissolved in 50 ml distilled water and completed to 100 ml. The pH was adjusted at 6.8 with anal as Hcl. The solution was filtered kept in a dark bottle at 4oC. d- 10 % Sodium dodecyle sulfate (SDS): 10 g SDS were dissolved in 50 ml distilled water and completed to 100 ml. This solution should be kept at room temperature to avoid precipitation. e- Ammonium persulphata (10 %) initiator: 0.5 g Ammonium persulphate was dissolved in 2 ml distilled water. This solution must be freshly prepared to
٥٠ ______Materials and methods avoid disactivtion of the ammonium persulphate as a polymerization catalyst. f- TEMED: N,N Tetramethylenediamine: TEMED is a strong polymerizing agent. The volume required to carry out polymerization depends on the quality of the used TEMED. In the present work, 50 µl and 30 μl of TEMED were added for stacking and resolving geles, respectively. g- Overlay isopropyl: 50 ml Isopropyl alcohol and 5 ml distilled water. h- Tank buffer: 6 g Tris-base, 28.8 g glycine and 20 ml of solution were dissolved in 500 ml distilled water and completed to 2 liters. i- Staining – Solution (1% COBB): 2g Coomassie brilliant blue (COBB, R250) were dissolved in 200 ml distilled water, filtered and kept as a stock solution. The staining solution was prepared as follows: 62.5 ml of stain stock, 250 ml of methyl alcohol and 50 ml of acetic acid glacial were added, then completed to 500 ml with distilled water. j- Destining solution: 45 ml Methyl alcohol, 10 ml acetic acid glacial and 45 ml distilled water were added from the destining solution. 3.2.4.2. Extraction of proteins:
٥١ ______Materials and methods
2g from leaf of the Catharanthus roseus plants ground were to a final powder using a pestle and mortar in liquid nitrogen. Total soluble proteins were then extracted supernatant was taken as the total proteins extract. a- Simple protein Determination: Simple protein were analyzed by PAGE technology according to Laemmi, U. K. (1970). SDS-PAGE is currently the most commnly used electrophoretic technique in protein analysis due to the ability of the strong anionic detergent SDS, when used in the presence of disulfide band cleaving reagents, to solubilize, denature and dissociate most proteins to produce single polypeptide chains. Here, the protein migration will be according to the molecular mass size only. b- Preparation of protein samples: Protein samples were prepared by mixing clear supernatant with the sample buffer (treatment buffer) {tris- Hcl, pH 6.8, 2% (W/V) SDS, 10% (W/V) sucrose, 0.1 % (V/V) 2,β. Mercaptoethanol, 0.5 % (W/V) bromophenol blue} in the ratio of 1:1 and denatured by heating in a boiling water for 4 min, then loaded in equal amounts. For the native gel, protein samples were prepared by mixing clear supernatant with the treatment buffer which in this case contains all components of denatured gel treatment buffer except SDS.
٥٢ ______Materials and methods
III.2.4.3. Gel preparation: a- Resolving gel: 10 ml of monomer, 7.5 ml of resolving Gel buffer 0.3 ml of 10% SDS, and 12 ml distilled water were mixed and shaked well.300 μl of freshly prepared ammonium persulphate (soln.5) were added and shacked well. Finally, 30μ l TEMED was added just before gel casting. b- Stacking gel: 1.33 ml of monomer solution (soln.1), 2.5 ml of stacking gel buffer (soln.3), 0.1 ml of 10% SDS (soln.4), 100 μl ammonium persulphate (solen.5) and 6.1 ml distilled water were added, respectively and shaked just before gel casting, 50 μl TEMED were added. c- Gel casting: Biorad multigel-long is the device which was used in protein electrophoresis. It is a complete system with two sets of (12.5 x 11) Cm glass plates. Straight edge while, the other is with fixed 1.0 mm spacers. A silicon rubber seal, 1 mm was placed around the periphery of the fixed spacers, then the other notched glass plate was carefully placed upon it. The two plates were together by the mean of 6 clips. Two cobbined plates were prepared as above to carry out gel casting.
٥٣ ______Materials and methods
Resolving gel was poured in. between the two plates leaving 2 Cm benath the plates end. Alayer of 90% isopropyl alcohol was added over resolving gel to prevent corrugation of the gel surface. Polymerization of the resolving gel took from half to one hour after gel solidification, which can be signicant by the interphase line between the alcohols arid the solid gel, the alcohol was poured off. Stacking gel mixture was added over the solid resolving gel till the top of the glass plates. A comp consisted of 12 wells and 1.0 mm derision was used. The comb was placed very gently into the liquid gel to avoid any air bubbles. Stacking gel needed a pit longer time then resolving to be solididfied. Once the solidification took place, the comb, clips and the silicon rubber were removed. The two plate’s acre installed in the electrophoresis chamber. The tank buffer was added to immerse the wells completely. The protein samples were pipetted in wells by automatic variable micropipette (2-200) μ l. III.2.4.4. Electrophoresis condition: The run was earned out at 30 V till to loaded samples passed the stacking gel and entered the resolving gel, tracking dye (Bromophenol blue) reached the gel bottom.
٥٤ ______Materials and methods
III.2.4.5. Staining and de staining of gels: The gels were stained for 24 hours in the prepared staining solution (1% COBB, R-250). To obtain a clear background, the gels were de stained by the prepared de staining solution. III.2.4.6. Photo graphing and gel scaning: Destained gels were photographed and the results were analyzed by the GDS (Gel docurnentahon systems) – Biorad 2005.
III.2.5. Randomly amplified polymorphic DNA (RAPD). a- First variety (LE): In this study, RAPD was used for the identification of markers associated with 11 radiation treatments taxa genotypes after four weeks according to Shilpa and Jayabaskaran (2007). Five primers random decamers were used in this study; their names and nucleotides sequences were presented in table (3). Table (3): List of primer names and their nucleotide sequences used in this study (RAPD) NO. Name Sequence Tm 1 OP-B01 5' CTGTCGTCGT 3' 32 2 OP-B07 5' AGGTGACCGT 3' 32 3 OP-B11 5' CAGCACTGCT 3` 32 4 OP-B12 5' CCTTGACGCA 3' 32 5 OP-F06 5' AGGTGCGTCC 3' 34 Tm : annealing temperature
٥٥ ______Materials and methods b- Scand variety (CP3):
In this study, RAPD was used for the identification of markers associated with 8 times (con., 0, 2, 4, 8, 16, 48 and 186) hours which were treated by using
gamma radiation at 16 Krad taxa genotypes after four weeks according to Ajaswrata et al., (2007). Ten random decqwers primers were used in this study, their names and nucleotides sequences were presented in table (4). Table (4): List of primer names and their nucleotide sequences used in this study (RAPD) No. Name Sequence Tm 1 OP-C09 5' CTCACCGTCC 3' 34 2 OP-C10 5` TGTCTGGGTG 3` 32 3 OP-C13 5` AAGCCTCGTC 3` 32 4 OP-C15 5` GACGGATCAG 3` 32 5 OP-G17 5' CAGCAGCAGG 3` 34 6 OP-L12 5` GGGCGGTACT 3` 34 7 OP-L13 5` ACCGCCTGCT 3` 34 8 OP-L16 5` AGGTTGCAGG 3` 32 9 OP-L20 5` TGGTGGACCA 3` 32 10 OP-Z03 5' CAGCACCCCA 3' 32 Tm : annealing temperature III.2.5.1. DNA isolation
Young and fresh leaf samples were collected separately from C. roseas seedlings for each radiation treatments. Then bulked DNA extraction was performed using DNeasy plant Mini Kit (QIAGEN). Isolation protocol of DNA was as follows:
٥٦ ______Materials and methods
1- Plant tissues were ground in liquid nitrogen to a fine powder using a mortar and pestle. Then, the powder was transferred to an appropriately sized tube and the liquid nitrogen was allowed to evaporate.
2- Then, 400 µl of buffer AP1 and 4 µl of RNase a stock solution (100 mg/ml) were added to a maximum of 100 mg of ground plant tissues and vortexed vigorously.
3- Mixture was incubated for 10 min at 65oC and mixed 2-3 times during incubation by inverting tube.
4- Then, 130 µl of buffer AP2 was added to the lysate, mixed and incubated for 5 min on ice.
5- Lysate was applied to the QIA shredder spin column sitting in a 2 ml collection tube and centrifuged for 2 min at 12000 rpm
6-Flow-through friction from step 5 was transferred to a new tube without disturbing the cell-debris pellet. Typically, 450 µl of lysate was recovered.
7- Then, 0.5 volume of buffer AP3 and 1 volume of ethanol (96- 100%) were added to the cleared lysate and mixed by pipetting.
8- Then, 650 µl of the mixture from step 7 was applied through DNeasy Mini spin column setting in a 2 ml collection tube.
٥٧ ______Materials and methods
Then, centrifuged for 1 min at 8000 rpm and flow-through was then discarded.
9- DNeasy column was then placed in a new 2 ml collection tube. Then, 500 µl buffer AW was added onto the DNeasy column and centrifuged for 1 min at 8000 rpm. Flow- through was discarded and reuse the collection tube in step 10 was reused.
10-Then, 500 µl buffer AW was added to DNeasy column and centrifuged for 2 min at maximum speed to dry the column membrane.
11-DNeasy column was then transferred to a 1.5 ml microfuge tube and 100 µl of preheated (65oC) buffer AE was pipetted directly onto the DNeasy column membrane. Then, incubated for 5 min at room temperature and centrifuged for 1 min at 8000 rpm to elute.
12-Elution (step11) was repeated once as described. A new microfuge can be used for first elute. Alternatively, the microfuge tube can be reused for the second elution step to combine the elutes.
III.2.5.2. Randomly amplified polymorphic DNA (RAPD) procedure PCR reactions were conducted using 15 arbitrary 10-mer primers.
٥٨ ______Materials and methods
Polymerase chain reaction (PCR) condition Stock solutions a- 5X Tris-borate (TBE), pH 8.0 Tris-base 5.40 g Boric acid 2.75 g 500 mM EDTA, 8.0 0.29 g
H2O (d.w) up to 100.00 ml
b- Ethidium bromide
The stock solution was prepared by dissolving 1 g of ethidium bromide in 100 ml distilled water and mixed well with magnet-ic stirrer. It was transferred to a dark bottle and stored at room temperature. c- Sample loading dye Na-EDTA, pH 8.0 (500 mM) 2.00 ml Glycerol (100%) 5.00 ml Bromophenol blue (2%) 0.75 ml Xylene cyanole (2%) 0.75 ml
H2O (d.w.) 1.50 ml PCR was performed in 30-µl volume tubes according to Williams et al., (1990). that contained the following: DNTPs (2.5 mM) 3.00 µl
MgCl2 (25 mM) 3.00 µl Buffer (10 x) 3.00 µl Primer (10 pmol) 2.00 µl Taq DNA polymerse (5U/µl) 0.20 µl
٥٩ ______Materials and methods
Template DNA (25 ng) 2.00 µl
H2O (d.w.) 16.80 µl The amplification was carried out in a DNA thermocycler (MWG-BIO TECH Primuse) Programmed as follows: One cycle 94oC for 5 min 45 cycles each of
94 oC for 1 min 4GC + 2AT each primer as shown in tables 3 & 4 for 90 sec 72 oC for 2 min One cycle 72 oC for 7 min, then 4 oC infinit.
III.2.5.3. Sample preparation
PCR product 15.00 µl Loading dye 5.00 µl
III.2.5.4. Gel preparation
Agarose 1.40 g TBE (1 x) buffer 100.00 ml Ethidium bromide 5.00 µl
Agarose was mixed with l x TBE buffer and boiled in water bath. Ethidium bromide was added to the melted gel after the temperature became 5 oC.
The melted gel was poured in the tray of mini-gel apparatus and comb was inserted immediately, then comb
٦٠ ______Materials and methods was removed when the gel become hardened. The gel was covered by the electrophoretic buffer (1 x TBE). Fifteen µl of DNA amplified product was loaded in each well. DNA ladder mix was used as standard DNA with size as follows:
a- For variety LM
12000, 11000, 10500, 10000, 9500, 9000, 8800, 8500, 6000, 3300, 2300, 1900, 1500, 1200, 1000, 800 600, 400, 300, 100 and 80 bp.
b- for variety CP3
10000, 9000, 8000, 7000, 6000, 5000, 4500, 4000, 2500, 1500 and 900 bp.
The run was performed for about 1 hour at 80 V in Pharmacia submarine (20 cm X 20 cm). III.2.5.5. Gel analysis 1-Gels were photographed and scanned with Bio-Rad video densitometer Model 620, at a wave length of 577. III.2.5.6. Data analysis The similarity matrices were done using Gel works ID advanced softwere UVP-England Program. The relationships among rootstock genotypes as revealed by dendrograms were done using SPSS windows (Version 10) program.
٦١ ______Results and discussion
ІV-Results and discussion
IV.1. Radiation treatments:
Table (5) presents the survivor of plants Catharanthus roseus varieties (LM and CP3) which were treated by using Gamma radiation doses; 0, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20 and 22 Krad. The doses from 0 to 20 Krad. gave 100 % survivor, on the other hand, the survivor percentage following dose 22 Krad. was found to be a lethal dose (20 %). Table (5): Effect of the radiation treatments on the Catharanthus roseus plants (LM & CP3) varieties survivor.
Radiation treatments by No. of plants after radiation Survivor No. Krad. treatments percentage after 2 days after 14 days 01 00 10 10 100 % 02 02 10 10 100 % 03 04 10 10 100 % 04 06 10 10 100 % 05 08 10 10 100 % 06 10 10 10 100 % 07 12 10 10 100 % 08 14 10 10 100 % 09 16 10 10 100 % 10 18 10 10 100 % 11 20 10 10 100 % 12 22 10 02 *020 % *lethal doses
Our results are in agreement with Shilpa and Jayabaskaran (2007). How studded UV-B-induced signal leading to enhance production of Catharanthine in Catharanthus roseus cell suspension cultures. Medium alkalinization an early event occurring in elicitor-treated
٦٣ ______Results and discussion plant cell cultures, has been used as a marker of elicitor responses in studying elicitor-binding sites in plant cells.
IV.2. Effect of radiation on Indole alkaloids biosynthesis
IV.2.1. assay in variety. a- First variety (LM): The TDC and STR in C. roseas variety (LM) and over expression appeared gave a result in an increased alkaloid accumulation but only enhanced tryptamine levels. Therefore, in the present study we irradiated of C. roseas variety (LM). Total alkaloids, Catharanthine, Vindoline, Vallesiachotamine (a), Vallesiachotamine (b) Ajmalicine, Horhammericine (a), Horhammericine (b), Vindolinine, 19- Epivindolineine, Strrictosidinelactam, Serpentine and Vinamidine of were assayed and compared to their relative percentage with those of the intact plant [Fig. (12) and Table (6)] the highest relative percentages 6.190, 7.200, 5.940, 5.860, 7.790, 6.000, 6.672, 2.720, 7.000, 7.920, 7.230 and 6.400 μg of alkaloids respectively above , in radiation treatments 18, 12 & 16, 14, 14, 18, 18, 18, 12, 12, 8, 12 and
16 Krad. respectively compared with other treatments. However the Indolealkaloids of Catharanthine, Ajmalicine, Horhammericine (a) and Horhammericine (b) were increased
٦٤ ______Results and discussion commensurate with increasing of the radiation doses up to 18 Krad the production produced of the alkaloids of Vindoline, Vallesiachotamine (a) and Vallesiachotamine (b) [Fig. (12) and Table (6)] were increasing up to radiation doses 12 & 16,
14 and 14 Krad respectively. On the other hands the highest values of alkaloids, Vindolinine, 19-Epivindolineine, Strrictosidinelactam, Serpentine and Vinamidine as a relative percentage with those of C. roseas variety (LM) intact plant were recorded with 12, 12, 8, 12 and 16 Krad respectively Fig. (12). Our results un agreed with those of Carolyn et al., (2004) Growth and alkaloid production with and without MJ A typical growth and alkaloid production curve for C. roseas suspensions with and without methyl jasmonate (MJ). In elicited cultures, MJ was added on the inoculation day (day 0) at 100 µM; rapid increases in Ajmalicine and serpentine occurred after 3 day, which continued up to 7 and 11 day, respectively. Ajmalicine and serpentine production in MJ-elicited cultures maximized at 5.4 ± 0.4 mg l−1 and 3.7 ± 0.7 mg l−1, respectively; this represents a 165% and 78% increase over the maximum Ajmalicine and serpentine content of controls. Optimum growth stage for MJ elicitation The optimum growth stage for inducing alkaloid production with MJ in C. roseas suspensions was investigated by adding MJ at 0, 10, 100, or 1000 µM on either day 0, 3, 6, 9, 12, or 15. Resin bags containing XAD-7HP were also added to
٦٥ ______Results and discussion promote the adsorption and extra cellular recovery of alkaloids and to potentially reduce Feedback inhibition and product metabolism Asada and Shuler (1989) & Lee- Parsons and Shuler (2002). Resin bags were added to suspensions 3 d after elicitation and then exchanged every 3 d to minimize the saturation of resins. Resin was added while metabolite production was believed to be most rapiday, i.e. 3 d after elicitation. Expression of genes involved in the C. roseas TIA pathway reached a maximum between 4 to 24 h after MJ elicitation Collu et al., (2001). Activity of enzymes associated with secondary metabolism in Glycine max and Lithospermum erythrorhizon suspensions peaked between 24 to 72 h after MJ elicitation Gundlach, et al., (1992) & Mizukami, et al., (1993). Rapid secondary metabolite accumulation was presumed to follow the maximum expression of mRNA and enzymes associated with secondary metabolism, i.e. within 72 h after elicitation. For this reason, the experiment was designed with resin added 3 d after elicitation when TIA accumulation was believed to be most rapid. This variation in Indolealkaloids production in the C. roseas cells which were treated by Gamma rays to the doses
0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 Krad. my be stable mutation in the DNA of plant.
٦٦ ______Results and discussion
No. 1 8 7 6 5 4
Concentratin 3 2 1 0
Vindoline -EpivindolinineSerpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam Indole alkaloids C0ntrol
2 8
7 6
5 4
Concentration(un 3 2
1
0
Epivindolinine Vindoline - Serpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam
Indolealkaloids 2 K rad
3
8
7
6 n 0 5
4
Concentration 3
2
1
0
Epivindolinine Vindoline - Serpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam
Indolealkaloids 4 K rad
Fig. (12) Indolealkaloids determination by HPLC in Catharanthus roseas variety (LM) which is treated by gamma radiation Concentration by (μg).
٦٧ ______Results and discussion
Fig. (12) Continue 4 8 7 6 5 4
Concentration 3 2 1 0
Vindoline Serpentine Vinamidine Vindolinine Ajmalicine -Epivindolinine Catharanthine 19 Horhammericine Horhammericine Strrictosidine lactam Vallesiachotamine Vallesiachotamine
Indolealkaloids 6 K rad
5 9 8 7 6 5 4 Concentration 3 2 1 0
Epivindolinine Vindoline - Serpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam
Indolealkaloids 8 K rad
6 8
7
6 5
4
Concentration 3
2
1
0
Vindoline Serpentine Vinamidine Vindolinine Ajmalicine -Epivindolinine Catharanthine 19 Horhammericine Horhammericine Strrictosidine lactam Vallesiachotamine Vallesiachotamine
Indolealkaloids 10 K rad
٦٨ ______Results and discussion
Fig. (12) Continue
7 8
7
6 5
4
Concentration 3 2
1
0
Epivindolinine Vindoline - Serpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam
Indolealkaloids 12 K rad
8 8
7
6
5
4
Concentration 3
2
1
0
Vindoline Serpentine Vinamidine Vindolinine Ajmalicine -Epivindolinine Catharanthine 19 Horhammericine Horhammericine Strrictosidine lactam Vallesiachotamine Vallesiachotamine
Indolealkaloids 14 K rad
9
8
7
6
5
4
3
Concentration 2 1
0
SerpentineVindoline Ajmalicine Epivindolinine Vinamidine Vindolinine - Catharanthine 19 HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam Indolealkaloids 16 K rad
٦٩ ______Results and discussion
Fig. (12) Continue 10 9 8 7 6 5 4 3 Concentration 2 1 0
Vindoline -EpivindolinineSerpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam Indolealkaloids 18 K rad
11 4 3.5 3 2.5 2 1.5
Concentration 1 0.5 0
Vindoline -EpivindolinineSerpentine Vinamidine Vindolinine Ajmalicine 19 Catharanthine HorhammericineHorhammericine VallesiachotamineVallesiachotamine Strrictosidine lactam Indolealkaloids 20 K rad
٧٠ ______Results and discussion 20 3.130 3.328 1.300 1.100 3.470 2.890 2.990 1.000 2.250 2.910 1.200 1.760 18 6.190 6.550 4.550 4.690 7.790 6.000 6.672 1.000 5.000 5.200 3.760 5.983 200 16 . 5.810 7 5.660 5.660 7.400 5.750 6.110 1.440 5.811 6.000 5.200 6.400 14 5.000 7.000 5.940 5.860 6.370 5.100 5.730 2.400 6.319 6.330 6.440 6.000 720 12 . 2.910 7.200 5.670 5.621 5.821 3.900 4.530 2 7.000 6.900 7.230 5.220 10 1.370 5.240 3.120 3.000 2.820 1.920 2.530 0.930 5.270 7.400 5.520 5.000 8 1.200 5.180 2.110 2.000 1.970 1.520 2.010 0.410 5.110 7.920 5.610 4.900 Radiation treatments (K rad) 6 970 . 1.100 5.180 2.000 1.960 1.830 1.310 1.890 0.320 4.990 7.490 5.580 3 4 1.000 5.130 1.860 1.880 1.620 0.920 1.720 0.420 4.710 7.520 5.400 3.800 2 510 . 0.900 5.100 1.500 1.520 1.340 0.120 1.310 0.210 4 7.490 5.010 3.410 0 0.900 4.890 1.430 1.520 1.110 0.120 1.310 0.210 4.220 7.510 4.880 3.210 mine a mine ) ) GammaEffect of treatment radiation on biosynthesis alkaloids concentration Indole of by (µg). 6 Vindoline Serpentine Ajmalicine Vinamidine Vindolinine Epivindolineine Catharanthine - Horhammericine Horhammericine a Horhammericine Horhammericine b 19 Strrictosidinelactam Vallesiachota Vallesiachotamine b Vallesiachotamine Type of IndoleType alkaloids Table No. ( No. Table
٧١ ______Results and discussion b- Second variety CP3 Depended on the previous assay results in 4.5 and (table 7 & fig. 13) we are design the experiment to study the effect of the gamma radiation stress on the Ajmalicene production on the Development of time in another C. rouses. Sterna to understanding the gene exprassion behavior in the cells. Chosen the Ajmalicine especially because it is a gate of Indole alkaloids production in a pathway system. And chose dose rate 16 Krad. because this dose is equilibrium dose among all Indole alkaloids components above. In (table 7 and fig. 13) revealed that Gamma irradiation doses were effective on reducing the content of Ajmalicine, as compared with the control. In all cases, raising the doses of Gamma irradiation decreased the content of Ajmalicine production from 0 times to 48 hours but after one week level Ajmalicine production increasing suddenly. The occur of the biosynthesis of the indole alkaloids, and Ajmalicine specially was reduced in C. roseus seedling as reported previously seedling cultures of C. roseus typically exhibit a growth-dissociated accumulation of secondary products. As shown in (Fig. 13 and table 7), Ajmalicine levels began to decrease directly after irradiated on the other hand increase during the deceleration of linear
٧٢ ______Results and discussion growth and reached plateau levels early in the stationary phase. The accumulation kinetics of the Ajmalicine-derived alkaloids succeeded the increase in into cellular, Ajmalicine concentration (table 7 & Fig. 13 and 14). In contrast, product levels failed to increase significantly in seedling during the exponential growth period. For example, the maximum level of Ajmalicine in cells was approximately 2.1801 µg, compared with a maximum concentration of greater than 2.8991 µg after 168 hour. Alkaloids were not detected in the culture medium of either system indicating that product release was not responsible for low intracellular concentrations. The accumulation kinetics suggest that alkaloid biosynthesis is reduced in cells, but not completely inhibited, since the intracellular product levels, expressed as l µg / g (fresh weight), remained relatively constant during the growth cycle. These results are consistent with those reported for the effects of other non-alginate immobilization methods on Indole alkaloid accumulation in C. roseus cells. The decreased biosynthesis of Ajmalicine was initially suggested as responsible for the reduced accumulation of indole alkaloids in cells of C. roseus. In order to test this hypothesis, invitro assays were performed to determine the specific activity of enzymes involved in Ajmalicine biosynthesis in cells. Preliminary evidence from these experiments suggested
٧٣ ______Results and discussion that the biosynthesis of total extractable proteins was enhanced in immobilized cells relative to cells in suspension. Since this phenomenon was in contrast to the otherwise reduced primary metabolism of immobilized cells, manifested as a decreased growth rate, formal experiments were performed to determine the total extractable protein level in both systems. The kinetics of total extractable Ajmalicine biosynthesis established that irradiated cells were synthesizing more extractable Ajmalicine than control cells. Ajmalicine levels per gram (fresh weight) of cells increased rapidly in both systems reaching peak values within 7 days of inoculation into plant. The synthesis of total proteins decreased by day 7 as seedling entered the linear growth phase. In combination with a rapidly increasing age. Irradiated seedling may stimulate increased Ajmalicine biosynthesis, at least in some species. Maximum specific activity of TDC was similar in seedling cells Fig. (15) and (table 8). The specific activity of TDC in seedling cells was greatest on dose rate 16 Krad and declined continuously to undetectable levels by dose rate 20 Krad The increased accumulation of Ajmalicine beginning after day 7 at dose rate 16 Krad (Fig. 13 & 14) is consistent with high TDC activity during this period. Although the time-course of the specific activity for TDC during the growth of seedling in 4.6
٧٤ ______Results and discussion was altered relative to that of seedling in 4.5 maximum activity levels were similar in both systems. However, increased accumulation of Ajmalicine was not observed in seedling in other times suggesting that the limited availability of an Ajmalicine precursor was responsible for the reduced Ajmalicine pool. TDC has been characterized and purified to homogeneity from cell seedling of C. roseus. The relationship between the expression of enzyme activity under certain conditions and the accumulation of Ajmalicine implicated TDC expression as the controlling factor for alkaloid production in C. roseus seedling. Workers in several laboratories have observed the presence in a number of tissues of enzyme systems which are active in the synthesis of Indole alkaloids. The result agreement to the result in the assay in (LM). Our result agreement with Shilpa et al., (2007) who irradiated C. roseas by UV and studied enzyme (TDC) and (STR) Since the UV-B-induced early cellular responses, medium alkalinization and ROS production were inhibited by suramin, we investigated whether. The effect of UV-B irradiation on expression of TIA biosynthetic genes, Tdc and Str, and Indole alkaloids production has been reported previously in C. roseus leaves. The transcription factor GT-1 binds to the promoter region of Tdc in vitro. The functional
٧٥ ______Results and discussion importance of GT-1 in the induction of Tdc expression by UV light has been demonstrated by point mutations in the GT-1 binding site. However, the molecular basis of UV-B signaling cascades leading to the induction of expression of Tdc and Str genes and the production of TIAs is largely unknown. It has been observed that the polypeptide wound signal, systemin- specific cell surface receptors initiate a signal transduction cascade upon UV-B irradiation in L. peruvianum cell suspension cultures. In the present study, the signaling pathways mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. UV-B induced alkalinization of the culture medium, generation of hydrogen peroxide, activation of CDPK and MBPK as well as accumulation of catharanthine and stimulation of transcription of Tdc and Str genes were studied. Inhibitors of binding of ligand-cell surface receptors, protein kinases and phosphatases, calcium fluxes and H2O2 were used to dissect the UV-B signaling cascade. The oxidative burst, a rapid consumption of oxygen and production of reactive oxygen species (ROS) such as
H2O2, is a typical early event in plant defense responses with
5 min of UV-B irradiation of C. roseus cells H2O2 production increased six-fold compared to control cells. We next examined effects of suramin, an inhibitor of G-protein
٧٦ ______Results and discussion inhibitor, N-acetyl cysteine, a putative ROS scavanger, verapamil, a calcium channel blocker and staurosporine, a serine-threonine kinase inhibitor, SB 203580, a P38 MAPK inhibitor, PD 98059, an ERKK inhibitor and SB 600125 JNK inhibitor. The UV-B induced H2O2 production was suppressed by all the inhibitors except the MAPK cascade inhibitors. This indicated that upon receiving the UV-B signal by a putative receptor in C. roseus cells, calcium influx and activation of serine/threoine kinases are required to induce H2O2 production. However, activation of the
MAPK cascade occurs downstream of H2O2 production. Table No. (7) Effect of gamma radiation on the Ajmalicine content in C. roseus. Parameters Time Area Height Con. µg/g tissue Standard 14.42 0864 3.8961 2.0000 Control 14.39 1410 1.9974 2.1801 000 time 14.22 0727 1.4011 2.0001 002 hour 14.22 0926 1.5388 1.8777 004 hour 14.00 0701 1.0001 1.4062 008 hour 14.42 0682 1.0116 1.2001 016 hour 14.21 0600 0.7101 1.0006 048 hour 14.32 0612 0.7147 0.9963 168 hour 14.40 1993 4.9691 2.8991
٧٧ ______Results and discussion
4
3.5 3 2.5 2
Concen./ug 1.5 1
0.5 0 0 2 4 8 16 48 168 Time / hoars
Fig. 13 effect of time development on the Ajmalicine prodction in C. ruses. under radiation doas 16 Krad.
VI.2.2. compare of Ajmalicine production in LM & CP3 variety in Catharanthus rouses. Data obtained from HPLC experiment in LM & CP3 variety are similarity at dose 16 Krad., in LM variety we are see concentration of Ajmalicine 7.4 µg after four weeks compare the control 1.11 µg (table 6). on the other hand seen concentration of Ajmalicine in CP3 variety which are treated by Gamma radiation at 16 Krad after 168 hours (one week) were 2.899 µg compare standard 2 µg table (7) so we can see compatibly in gene expiration in Ajmalicine production in both variety LM & CP3.
٧٨ ______Results and discussion
control 0 hour
2 hour 4 hour
8 hour 16 hour
48 hour 168 hour Fig. 14 HPLC analysis of time development on the Ajmalicine production in C. ruses. under radiation dose 16 Krad.
٧٩ ______Results and discussion
This system provided alternating conditions of cellular growth and proliferation and thus allowed to study the transcriptional changes of TIA pathway genes and regulators simultaneously under changing culture conditions. The results were compared with TIA production data by HPLC. This study demonstrates the complexities of molecular regulation of TIA metabolism in C. roseus cell culture and indicates the probable bottleneck for scale-up production of TIAs in this system. Understanding the Terpenoid indole alkaloids (TIAs), an important group of secondary metabolites are produced by C. roseus (L.) G. Don (Madagascar periwinkle), a member of the apocynaceae family. Some of these alkaloids have high therapeutic values such as, the antihypertensive alkaloids, ajmalicine, serpentine and anticancer alkaloids, vincristine, vinblastine. The monoterpenoid moiety of TIAs originates from the 2-C methyl erythritol phosphate (MEP) pathway whereas the indole skeleton of TIAs is derived from the shikimate pathway. The biosynthesis of TIAs in C. roseus is reported to be under strict regulation. Their metabolism has been found to be restricted to certain tissues and it is modulated by developmental and environmental mechanisms. Despite significant efforts, TIA biosynthesis in plant cell cultures remains poorly characterized. Regulation
٨٠ ______Results and discussion of two important TIA biosynthetic pathway genes, namely, strictosidine synthase Str and tryptophan decarboxylase Tdc has been studied in great detail, which revealed thereof coordinate regulation and elicitor-mediated induction in cell cultures. The regulatory mechanisms controlling many of the remaining steps of the TIA pathway are yet unknown. Mustafa and Verpoorte (2007). the understanding of which may reveal the complex molecular program that governs TIA metabolism in cell culture. Functional analysis of Str and Tdc gene promoters led to the identification of cisregulatory sequences involved in elicitor and Me JAinduced responses. Octadecanoid- derivative responsive Catharanthus AP2-domain (ORCA3) transcription factor was found to be jasmonateresponsive transcriptional regulator of C. roseus primary and secondary metabolism. Furthermore, G-box binding factors (GBF1, GBF2 and GBF3) and zinc finger proteins (ZBF1, ZBF2 and ZBF3) were shown to act as repressors in the regulation of elicitor-induced TIA metabolism in C. roseus. Two downstream vindoline pathway genes, desacetoxyvindoline 4-hydroxylase (D4h) and deacetoxyvindoline 4-O- acetyltransferase (Dat) are known to be transcriptionally blocked in C. roseus in vitro grown cultures. Many aspects of such key regulatory events have been recently explored, but
٨١ ______Results and discussion understanding of the regulation of complete TIA pathway in cell cultures is still lacking for which it has not yet become a viable alternative for improved production of TIAs. The differential expression of genes in different state of cultures is expected to be different and will also be associated with different levels of secondary metabolites. In order to demonstrate the status of similar changes in expression of TIA pathway genes and regulators in C. roseus cell culture. The expressions of two feeder primary (MEP, shikimate) and committed TIA metabolite pathway genes were analyzed in C. roseus rotation culture system along with TIA pathway regulators using a Gamma radation stress. The database search for C. roseus genome revealed six genes encoding selected enzymes for each of the primary (MEP and shikimate) and TIA pathways along with eight TIA pathway regulator genes. A single amplified product obtained for each target gene was radiated and identities of putative RAPD- PCR which showed 95–99% homology with the available sequences in database. The genes were studding and analysis with CP3 variety RAPD-PCR (Table 23). Consequently, 495 fragments were spotted in agarose gel to facilitate the expression analysis and their expressions were monitored in the tissues originating from cultures. Each membrane was hybridized simultaneously under radiation stress sample.
٨٢ ______Results and discussion
There were significant changes in the expression levels of primary as well as TIA pathway genes and the regulators in the samples originating. TIA pathway repressor genes were, however, relatively highly expressed in the same tissues. This system revealed a high degree of reproducibility. Gene, activator and repressor transcripts of TIA and related primary pathways in seedling culture system We known the transcript profiles of three TIA pathway repressors each of G-box binding factor type, namely, Gbf1, Gbf2 and Gbf3, and zinc finger protein type, namely, Zct1, Zct2 and Zct3, one master regulator, Orca3, one MYB transcription factor, CrBpf1 and TIA and related primary pathway genes in cells initially. The appropriate number of cycles for PCR amplification of each target gene was determined so that it can be quantified and that the amplification lies in the exponential range before reaching the plateau. PCR amplification of 30 cycles was found to be optimum for all the target genes studied and their amplification was found to be in the exponential range. The mRNA levels of primary pathway genes, TDC and STR were higher in cells-induced by Gamma radiation in cultured seedling, compared to that of the control as below. The transcript levels of the latter two genes, TDC and STR remained undetected in seedling cultured. The mRNA levels
٨٣ ______Results and discussion of early TIA pathway genes, Tdc and Str, showed changes with changing conditions of irradiated and thus were higher in 16 Krad but low in other doses. Ajmalicine level showed low or undetected level of expression under similar conditions. The levels of expressions of late TIA pathway transcripts, TDC and STR, Data changed from high to low with changing culture irradiated. Some of these show restoration of expression in seedling cultures. Similar study was carried out using UV irradiated cell cultures, which revealed similar pattern of expression for specific TIA as well as primary pathway genes except G10h, which was faintly detected in this system. One gene from each pathway, namely G10h (MEP pathway), Tdc (shikimate pathway), Str (early TIA pathway) and Dat (late TIA pathway) were used as probes for northern analysis, which revealed biphasic nature of expression of Tdc and Str transcripts in cell cultures noted by a reduction in the level of expression in equilibrium doses but again the level of expression was restored in high doses. The transcript level of TDC must be confirmed to be low in seedling cultured without any treatments and Data transcript remained undetected by HPLC analysis. There was significant induction of the repressor Ajmalicene in seedling cultured. Some of these transcripts, Gbf1, Gbf3, Zct1 were
٨٤ ______Results and discussion upregulated in seedling cultured. Similarly, the TDC levels, which play a role in elicitor responsive signal transduction pathway of TIA biosynthesis, were high in 16 Krad. dose. Under similar conditions, the STR, the master regulator for TIA pathway biosynthesis, were relatively high. However, in seedling cultures, the expression of this STR was reverse in
10 Krad. The steady state TDC levels were confirmed by gel electrophoresis analysis in seedling cultures, but STR remained undetected because this enzyme is very low in molecular Weight.
IV.3. Effect of radiation on Isozymes banding patterns.
VI.3.1. Tryptophandecarpoxylase enzyme (TDC):
The results of electrophoretic patterns of Tryptophan decarpoxylase isozyme extracted from the seedling variety (LM) bush of the different doses 0, 2, 4, 6, 8, 10, 12, 14, 16,
18 and 20 Krad in the radiation treatments taxa, Catharanthus roseas are shown in Figure (15) and are diagrammatically illustrated in Figure (15) and table (8).The Figures showed a maximum number of eleven bands.
In taxa with (bands 0 to 18) showed that one bands, the 20 exhibited bands (4) with lower intensity than the treated samples, the radiation effects on gene expression. Band 1 presents and very faint in the control untreated but was
٨٥ ______Results and discussion contradictory in the intensity and molecular Weght in the other doses treatments. Therefore, irradiation modified gene expression in taxa.
In 20 Krad (lanes 1 to 4) showed that, the enzyme degradation while complete in control and all doses, so that should be considered as a conferring modification of gene expression by irradiation. As kown Irradiation induces the expression of the Kinase proteins genes via posttranslational modification which further interacts with the Tdc and Str promoter enhancing the gene expression, many protein kinases are known to respond to both biotic and abiotic stresses. Two kinases, MAPKs and CDPKs, have been implicated to play pivotal roles in response to diverse stimuli. Previous studies have demonstrated that C. roseas variety (LM) cells also respond to UV-B irradiation by expressing biosynthetic genes and production of TIAs. To establish a functional link between these processes, we first examined the possible activation of MAPK and CDPK in cells irradiated with UV- B. MBP is known to be a conventional MAPK substrate and MAPK homologs also have MBP kinase activity. To determine if a MAPK is associated with the UV-B signaling the activation of MBP kinase was investigate Shilpa et al., (2007).
٨٦ ______Results and discussion
The obtained results are in agreement with Shilpa et al., (2007). Who analyzed a C. roseas which are treated by UV throw isoenzyme (TDC) The enzyme was estimated to be approximately 49 kDa. The 49-kDa MBPK activity increased by UV-B irradiation in cells compared with that of the un- irradiated control.
25 118
116 20
114
15 112
110
Intenesity 10 108 M. W. K. Da. K. W. M. 106 5 104
102 0 0 1 2 3 4 5 6 7 8 9 10 11 12 100 Bands 0 1 2 3 4 5 6 7 8 9 10 11 12 Tryptophan decarboxylase (TDC) Intenesity Bands Tryptophan decarboxylase (TDC) M. W. K. Da.
Figure (15): Tryptophan decarpoxylase histogram for the Catharanthus roseus variety (LM) which is treatments by Gamma radiation.
٨٧ ______Results and discussion
The maximum MBPK activity was observed at 10 min after UV-B treatment. In all the in vitro experiments carried out with MBP as substrate, the phosphorylation peaked at 10 min; these results were consistently obtained when the experiments were repeated with different batches of cells. Therefore, in all further experiments the MBPK activity was assayed at 10 min after irradiation. To further characterize the MBPK activity induced by UVB, immunoprecipitation and in-gel kinase assays were used. The protein extracts were incubated with anti-phosphotyrosine monoclonal antibody and immunoprecipitated with protein A-agarose. The immunoprecipitated proteins were separated on a SDS- polyacrylamide gel containing MBP as a substrate and MBPK activity was assayed in the gel in the presence of 32P- ATP. As shown in Figure 3c, a 49 kDa protein kinase was again detected in the immunoprecipitated from UV-B- irradiated cells. Co-incubation with phosphotyrosine prevented immunoprecipitation of the 49 kDa protein kinase with antiphosphotyrosine antibody, but co-incubation with phosphothreonine did not. These results indicate that only phosphotyrosine and not phosphothreonine could act as a competitor during immunoprecipitation, showing that MBP phosphorylating kinase was specifically phosphorylated on a tyrosine residue. Till date MAPK are the only known plant
٨٨ ______Results and discussion kinases to be phosphorylated on tyrosine residues. Calcium dependent protein kinases (CDPKs) belong to the unique family of calcium-regulated kinases and histone IIIS was one of the best exogenous substrates for assaying CDPKs to characterize the kinase (s) induced by UV-B, the activities were assayed using histone IIIS as a substrate in protein extracts from cells irradiated with UV-B, as well as the controls. The protein extracts from 5-min UV-B irradiated cells, assayed in the presence of calcium using histone IIIS as substrate showed that, the kinase activity increased significantly peaking at 4 min after UV-B irradiation and remained high even at 20 min after UV-B irradiation. The protein extracts from 5-min UV-B irradiated cells assayed by in- gel kinase assay in the absence and presence of calcium using histone IIIS as substrate demonstrated that the phosphorylation of histone IIIS was calcium dependent in both UV-B irradiated and un-irradiated cells. CDPK activities were identified at two positions with an apparent molecular weight of 55 kDa and 40 kDa. One of the CDPK activated had an apparent molecular weight of 40 kDa and was constitutive, as it was observed to phosphorylate histone IIIS to a similar extent in both un-irradiated and irradiated cells whereas the 55 kDa kinase activity showed UV-B dependence and peaked at 4 min. Therefore, the
٨٩ ______Results and discussion phosphorylation of histone IIIS observed in vitro experiments was both due to the activities of the 55 and assayed by in-gel kinase assay containing histone IIIS as substrate. Figure 4c shows that the 55 and 40 kDa kinases identified by in-gel kinase assay were both phosphorylated on serine residues and that the activity of 40 kDa kinase was constitutive in our cell cultures. In all the in vitro experiments carried out with histone IIIS as substrate, the phosphorylation peaked at 4 min. These results were consistently obtained when the experiments were repeated with different batches of cells. Therefore, in all further experiments the CDPK activity was assayed at min after irradiation. IV.3.2. Strrictosidinesynthase enzyme (STR). Zymograms of Strrictosidinesynthase enzyme (STR) for the Catharanthus roseas variety (LM) taxa are shown in Figure (16) and Table (8) and are histogrammatically illustrated in Figure (16).
In taxa with (lanes 0 to 18) showed that one bands, the 20 exhibited no bands with lower intensity than the treated samples, the radiation effects on gene expression. Bands from control to 10 Krad. presents and very dark but was contradictory in the intensity and molecular Weght in the other doses treatments. Therefore, irradiation modified gene expression in taxa. On the other hand radiation treatments 12,
٩٠ ______Results and discussion
14, 16, and 18 Krad. were feint or very feint and contradictory in the molecular Weght so that should be considered as a conferring modification of gene expression by irradiation. Our results agreed with those of Shilpa et al., (2007) who irradiated C. roseas by UV and studied enzyme (TDC) and (STR) since the UV-B-induced early cellular responses viz., medium alkalinization and ROS production were inhibited by suramin, we investigated whether
25 40
20 30
15 20
10 Intenesity
M. W. K. Da. K. W. M. 10
5
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 Bands Bands Strictosidine synthase(SSS) Strictosidine Synthase(SSS) M. W. K. Da.
Figure (16): Strictosidine synthase histogram for the Catharanthus roseus variety (LM) which is treatments by Gamma radiation.
٩١ ______Results and discussion suramin could inhibit the UV-B induced other cellular responses related to synthesis of TIAs. When the cells were pretreated for 10 min with 0.1 and 1 mM suramin concentrations and subsequently irradiated with UV-B for 5 min, the UV-Binduced MBPK and CDPK activities, accumulation of Tdc and STR transcripts and Catharanthine was strongly inhibited. However, the UV-B-induced MBPK activity could not be completely inhibited by suramin. To rule out the possibility that the inhibitory effects of suramin on responses triggered by UV-B are not due to the unspecific binding to cell surface components, we used heparin a structurally similar molecule viz., heparin that possesses sulfonic acid groups similar to that of suramin for inhibition of UV-B responses.
٩٢ ______Results and discussion
Table (8) M. W. and Intensity of Tryptophan decarpoxylase (TDC) and Strictosidine synthase (STR) Enzymes in Catharanthus roseus variety (LM) which is treatment by Gamma radiation.
Symbol Radiation TDC STR No. treatment M. W. KDa Intensity M. W. KDa Intensity 1 0 101 5 31 6 2 2 113 8 32 8 3 4 105 9 37 9 4 6 115 10 37 10 5 8 109 9 36 10 6 10 110 9 38 10 7 12 112 8 30 7 8 14 112 7 29 6 9 16 116 8 28 2 10 18 107 3 34 1 11 20 113 2 00 0
IV.4. Effect of radiation on Protein banding patterns.
The results of electrophoretic patterns of protein extracted from the seedling of Catharanthus roseus variety (LM) bush of the different doses of γ-rays control, 2, 4, 6, 8,
10, 12, 14, 16, 18 and 20 Krad in the taxa, the obtained of results show in table (9) and figure (16) and are histogrammatically illustrated in Figure (16).The Figures showed a maximum number of twelve bands.
In taxa with lanes 1 showed that two bands, the control exhibited band 1 with lower intensity than the treated samples, the radiation effects on gene expression. Bands 1 and 2 presents in the control and sample 2 Krad treated but
٩٣ ______Results and discussion was absent in the other doses treatment. Therefore, irradiation modified gene expression in these taxa.
In the radiation treatments 2 Krad lanes 2 showed that, band 3, 4, 5 and 6 was absent in control while present in 6 and 8 Krad doses, so that should be considered as a conferring modification of gene expression by irradiation.
The pattern of 4 Krad lanes 3 exhibited five bands, band 7 to 12 were present. Indicating the effect of 6 Krad irradiation treatments on modification of gene expression in C. roseas variety (LM) Bands 3 and 5 were presents only in the 6 Krad treatment but were absent in control and 4, 12 and
14 Krad treatments. Therefore, irradiation treatments effected on modification of gene expression in C. roseas variety (LE). treated.
The protein Zymograms of lanes 5 to 11 showed nine bands, which are absent in the some treatments and were present in the others irradiation treatments affected on gene expression in C. roseas variety (LM). Our results agreed with those of Kazuyuki et al., (2001) in the preliminary experiments, we found that a TPCK-treated bovine trypsin (Sigma, type XIII) was contaminated with GICP. Therefore, we tried to purify GICP from 116 mg trypsin. Using a combination of several
٩٤ ______Results and discussion chromatographic procedures, we obtained 0.09 mg enzyme (the specific activity of 12 n molls/min/mg proteins). SDS- PAGE pattern of the enzyme showed a major band with a Mr of approximately 29,000. The N-terminal sequence of major band was homologous to that of chymotrypsinogen, starting from Ile-16 Meloun et al., (1966). Therefore, we tested whether chymotrypsin isozymes, a-, d-, and p-chymotrypsins have the activity to cleave T-1 in the presence of TPCK. However, none of these chymotrypsins had such activity. These results suggest that the amount of GICP is very small in the commercial preparations of trypsin. Thus, we decided to purify GICP from bovine pancreas. Seedling to affect on both Tdc and STR genes, where STR enzyme wills condensate tryptamine and secologanin to produce Strrictosidine. The obtained results showed that the following experiments of C. roseas were used as seedling bush for extra Tryptophan decarpoxylase and Strrictosidinesynthase genes
٩٥ ______Results and discussion
7 80
70 6
60 5 50
4 40
3 Da. K. W. M. 30 Intenesity
2 20
10 1
0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 Bands Bands M. W. of protein bands Intenesity of protein bands
Figure (16): Protein histogram for the Catharanthus roseus variety (LM) which are treated by Gamma radiation
٩٦ ______Results and discussion
Table (9): SDS – Page protein analysis of the Catharanthus roseas variety (LM) which is treated by Gamma radiation.
M. W. of bands Total number of 70 65 44 40 37 36 35 27 25 20 18 15 10 bands7 5 00 M + + ------+ + + + - - + 7 I V.f d ------v.f v.f v.f v.f - - d 02 M + + + + + + - - + + + + + - + 12 I d f d f v.f v.f - - d d f d d - vd 04 M ------+ + + + - - + 5 I ------f f f f - - Vd 06 M - - - + - + - - + + + + + - + 8 I - - - d - d - - vd d d d d - Vd . rad 08 M - - + - + - + - + - + + + + + 9 I - - f - f - f - vd - f f f f Vd 10 M - - - + - + - - + - + + + - + 7 I - - - vf - vf - - vd - f f f - Vd 12 M ------+ - + + + - + 5
Radiation treatments K I ------f - f f f - Vd 14 M ------+ - + + - - + 4 I ------f - f f - - Vd 16 M - - - - - + + + + - + + - - - 6 I - - - - - d d d d - vf vf - - - 18 M - - - - + - + - + - + - - + + 6 I - - - - d - d - vd - d - - d vd 20 M - - - - + - + - + - + - + - + 6 I - - - - f - f - vd - f - f - vd
M: molecular Weght by K da. I: intensity
٩٧ ______Results and discussion
IV.5. DNA finger print analysis Random amplified polymorphic DNA (RAPD). a- First variety (LM): Result obtained from RAPD PCR analysis with 5 primers were used for the identification of markers associated with 11 radiation treatments taxa genotypes after four weeks presented as follows; 1- Primer OP-B01 (Table 10) shows the effect of gamma radiation doses on Catharanthus roseas genome throw PCR techniques, Primer OP-B01 gave 2 monomorphic fragments with molecular sizes ranging from 80 to 12000 bp. (Figure 17). with 17 polymorphic fragments (80.9 %) with numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20 and 21 with corresponding molecular sizes of 11000, 10500, 10000, 9500, 9000, 8800, 8500, 6000, 3300, 2300, 1900, 1200, 1000, 800, 600, 400, 300, 100 and 80 bp. were observed. On the other hand unique bands were 11000 bp. whereas, monomorphic were at 1 and 13 with corresponding 12000 and 4500 bp. radiation treatment 16 and 18 Krad. exhibited the maximum number 12 of fragments, while the lowest number
6 appeared in radiation treatment 0 and 2 Krad.
This primer showed that a fragments with 11000 and 10500 bp. appeared exclusively in radiation treatment20
٩٨ ______Results and discussion
Krad., the fragments with 9500 bp. appeared exclusively in
radiation treatment 4 and 6 Krad. but was absent in all other taxa. So it could be used as molecular marker for both above tretments. the fragments with 300 bp. appeared exclusively
in radiation treatment16 and 18 Krad., The fragments with 80
bp. appeared exclusively in radiation tretments 12 or 14 Krad. respectively, so the fragments could be used as molecular markers for both above tretments.
Table (10): RAPD profiles of the Catharanthus roseas which were treated by Gamma radiation amplified with primer OP-B01.
Band size Radiation treatments Krad No. (bp.) M. 0 2 4 6 8 10 12 14 16 18 20 1 12000 ------2 11000 + 3 10500 + 4 10000 + + + + + + 5 9500 + + 6 9000 + + + + + + 7 8800 + + + + + + 8 8500 + + + + + + + + 9 6000 + + + + + + + 10 3300 + + + + + + + + 11 2300 + + + + + + 12 1900 + + + + + + + + + 13 1500 ------14 1200 + + + + + + + + + + 15 1000 + + + + + + + 16 800 + + + + + + + + + 17 600 + + + + + 18 400 + + + + 19 300 + + 20 100 + + + 21 80 + + Unique bands: 2 (9.5) % Polymorphic: 17 (80.9) % Monomorphic: 3 (14.3) % Total bands / column 5 6 7 8 11 11 12 9 12 12 9 Total bands: 102
٩٩ ______Results and discussion
2- Primer OP-B07 Table (11) Figure (17) were presented the results obtained by using primer OP-B07 gave 4 monomorphic fragments with molecular sizes ranging from 80 to 12000 bp. with 16 polymorphic fragments (76.2 %) with numbers 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 18 and 21 with corresponding molecular sizes of 12000, 11000, 10500, 10000, 9500, 9000, 8800, 8500, 6000, 3300, 2300, 1900, 1500, 1200, 1000, 800, 600, 400, 300, 100 and 80 bp. were observed on the other hand unique band with numbers 20 corresponding 2000 bp. (4.8 %). Specific markers were 6 polymorphic fragments (8 %) and unique band fragments on the other hand unspecific markers were 10 polymorphic fragments (13 %). Radiation treatment 16 and 18 Krad exhibited the maximum number 11 of fragment; while the lowest number 4 appeared in radiation treatment 10 and 20 Krad., So it could be used as molecular markers for both above tretments. The fragment with 300 bp. appeared in radaition treatment4 Krad. only but was absent in all other taxa. So we could consider this primer as molecular marker for radaition tretments 4, 10, 16, 18 and 20 Krad.
١٠٠ ______Results and discussion
Fig. (17) Continue
PO-B01
PO-B07
PO-B11
١٠١ ______Results and discussion
PO-B12
PO-F06 Figure (17): RAPD profiles of the Catharanthus roseas which are treatment by Gamma radiation amplified with 5 primers (PO-B01, PO-B07, PO-B11, PO-B12, PO- F06)
١٠٢ ______Results and discussion
Table (11): RAPD profiles of the Catharanthus roseas which were treated by Gamma radiation amplified with primer OP-B07. size Band Radiation treatments Krad (bp.) No. M. 0 2 4 6 8 10 12 14 16 18 20 1 12000 + + 2 11000 + + 3 10500 + + + 4 10000 + + + + 5 9500 + + + + 6 9000 + + + + 7 8800 + + + + + + 8 8500 + + + + + + + 9 6000 + + + 10 3300 + + + + + + + 11 2300 + + + 12 1900 + + + + + + + + 13 1500 + + + + + + + + + + 14 1200 + + + + 15 1000 ------16 800 ------17 600 ------18 400 + + + + + 19 300 + 20 100 ------21 80 + + Unique bands: 1 (4.8) % Polymorphic: 16 (76.2) % Monomorphic: 4 (19) % Total bands / columns 5 5 7 5 5 4 9 9 11 11 4 Total bands: 75 3- Primer OP-B11 Result obtained from RAPD PCR analysis was presented in Table (12) Primer OP-B11 gave 9 (42.9) monomorphic fragments with molecular sizes ranging from 100 to 12000 bp. (9 %) figure (17). With 11 polymorphic fragments (52.4 %) with numbers 5, 6, 7, 8, 10, 11, 12, 13, 18, 19 and 20 with corresponding molecular sizes of 9500, 9000, 8800, 8500, 3300, 2300, 1900, 1500, 400, 300 and 100 bp. were observed. Unique band was number 21 at 80 bp. on the other hand specific markers didn’t observed, unspecific
١٠٣ ______Results and discussion markers were 54 polymorphic fragments (98.2 %). Whereas, the numbers 1, 2, 3, 4, 14, 15, 16 and 17 were monomorphic.
Radiation treatment 20 Krad. exhibited the maximum number 9 of fragments, while the lowest number 2 appeared in radiation treatments 0 and 2 Krad.
This primer showed that a fragment with 80 bp. appeared exclusively in radiation treatment18 Krad., only but was absent in all other taxa so the fragment could be used as molecular markers for this doas.
Table (12): RAPD profiles of the Catharanthus roseas which were treated by Gamma radiation amplified with primer OP-B11. size Band Radiation treatments Krad. (bp.) No. M. 0 2 4 6 8 10 12 14 16 18 20 1 12000 ------2 11000 ------3 10500 ------4 10000 ------5 9500 + + + 6 9000 + + 7 8800 + + + + 8 8500 + + + + + + 9 6000 ------10 3300 + + + 11 2300 + + + 12 1900 + + 13 1500 + + + + + + 14 1200 ------15 1000 ------16 800 ------17 600 ------18 400 + + + + + + + + + 19 300 + + + + + + + + + 20 100 + + + + + + + 21 80 + Unique bands: 1 (4.8) % Polymorphic: 11 (52.4) % Monomorphic: 9 (42.9) % Total bands / columns 2 2 8 8 7 3 2 2 7 5 9 Total bands: 55
١٠٤ ______Results and discussion
4- Primer OP-B12 As shown in Table (13) and fig. (17) Primer OP- B12 gave 9 monomorphic fragments with molecular sizes ranging from 80 to 12000 bp. with 11 polymorphic fragments (52.4 %) with numbers 8, 9, 10, 11, 12, 13, 14, 15, 18, 19 and 20 with corresponding molecular sizes of 8500, 6000, 3300, 2300, 1900,1500,1200, 1000, 400, 300 and 100 bp. were observed. Unique bands was 80 bp. at 20 Krad. On the other hand specific markers were one polymorphic fragments (9.1 %) and unspecific markers were 10 polymorphic fragments
(99.9 %). Radiation treatment 20 Krad exhibited the maximum number 12 of fragments, while the lowest number
1 appeared in radiation treatments 2 Krad while radiation treatments 0 Krad didn’t give any fingerprint. This primer showed that a fragment with 6000 bp. appeared in 2 and exclusively in radiation treatment20Krad., so the fragment could be used as molecular markers for thes tretment. 5- Primer OP-F06
The effect of different doses on the Genomic DNA was cleared in Primer OP-F06, it was gave 7 monomorphic fragments with molecular sizes ranging from 80 to 12000 bp. (7 %) (figure 17) and (table 14). with 11 polymorphic fragments (52.4 %) with numbers 6, 8, 11, 12, 13, 14, 15, 16,
١٠٥ ______Results and discussion
Table (14): RAPD profiles of the Catharanthus roseas which were treated by Gamma radiation amplified with primer OP-B12.
size Radiation treatments Krad Band No. (bp.) M. 0 2 4 6 8 10 12 14 16 18 20 1 12000 ------2 11000 ------3 10500 ------4 10000 ------5 9500 ------6 9000 ------7 8800 ------8 8500 + + + 9 6000 + + 10 3300 + + + + 11 2300 + + + + + + + 12 1900 + + + + + + + 13 1500 + + + + + + + + + 14 1200 + + + + + + + + + 15 1000 + + + + + + + + + 16 800 ------17 600 ------18 400 + + + + + + + + + 19 300 + + + + + + + + + 20 100 + + + + + 21 80 + Unique bands: 1 (4.8) % Polymorphic: 11 (52.4) % Monomorphic: 9 (42.6) % Total bands / columns 0 1 7 7 7 7 7 7 10 9 12 Total bands: 74
17, 18, 19, 20 and 21 with corresponding molecular sizes of 9000, 8500, 2300, 1900, 1500, 1200, 1000, 800, 600 and 400 bp. unique band was 3 fragments at 2, 3 and 9 corresponding 11000, 10500 and 6000 bp. (14.3 %) were observed. On the other hand specific markers were 1 polymorphic fragments (9.1 %) and unspecific markers were 10 polymorphic fragments (99.1 %). radiation treatments 18
Krad. exhibited the maximum number 11 of fragments, while
the lowest number 3 appeared in radiation treatments 0 Krad.
١٠٦ ______Results and discussion
This primer showed that a fragment with 100 bp.
appeared exclusively in radiation treatment10 and 14 Krad., So the fragment could be used as molecular marker for both above tretments.
Table (15): RAPD profiles of the Catharanthus roseas which were treated by Gamma radiation amplified with primer OP-F06. Band size (bp.) Radiation treatments Krad No. M. 0 2 4 6 8 10 12 14 16 18 20 1 12000 ------2 11000 + 3 10500 + 4 10000 ------5 9500 ------6 9000 + + + 7 8800 ------8 8500 + + + 9 6000 + 10 3300 ------11 2300 + + + + + + + + + + + 12 1900 + + + 13 1500 + + + + + + + + + 14 1200 + + + + 15 1000 + + + + + + 16 800 + + + + + + 17 600 + + + + + + + 18 400 + + + + + + 19 300 + + + + + + + + + + + 20 100 + + + + + 21 80 + + Unique bands: 3 (14.3) % Polymorphic: 11 (52.4) % Monomorphic: 7 (33.3) % Total bands / columns 2 4 5 4 5 9 9 9 10 11 10 Total bands: 78
VI.5.1.1. RAPD markers of the 11 radiation treatments with 5 RAPD primers: Data of the amplified fragments using the aforementioned five 10-mer arbitrary primers OPB-01, OP- B07, OP-B11, OP-B12, and OP-F06 for the 11 radiation
١٠٧ ______Results and discussion
treatments controlled with 0 Krad; 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 Krad indicated successful amplification of PCR products. Polymorphism levels differed from one primer to the other .The main results was as following table (16). Data in Table (16) showed that the five primers showed polymorphic differences among the radiation treatments, while some primers exhibited high polymorphism such as OPB-01 (80.9 %) and OP-B07 (76.2 %) on the other hand, some primers exhibited medium levels of polymorphism such as OP-B07, OP-F06 and OP-B01 (52.4 %).
The Randomly Amplified Polymorphic DNA (RAPD) technique is suitable for developing the molecular markers because of its technical simplicity and the modest cost of generating a large number of markers. Furthermore, the technique dose not require knowledge of the genome and RAPD marker have been adopted as a convenient means of tagging genes of interest in Catharanthus roseas Quarta et al., (2001).
PCR-based multi-locus DNA fingerprints represent one of the most informative and cost-effective measures of genetic diversity and are useful population-level biomarkers of toxicological and other anthropogenic impacts. However, Concerns about reproducibility of DNA fingerprints have
١٠٨ ______Results and discussion
limited their wider use in environmental biology. We assessed polymorphism and reproducibility of common fingerprinting techniques, RAPD (randomly amplified polymorphic DNA).
The results that by excluding bands that comprised less than 1% of total intensity, and by excluding the largest and smallest 10% of the bands, we could achieve nearly 100% reproducibility of RAPD fingerprints. Similar application of band exclusion criteria to RAPD fingerprints did not significantly enhance their reproducibility, and at least 15% of RAPD bands were not fully repeat table, heir table, or transmit table. Our results are in agreement with Sarika et al., (2007) whom analyzed a C. roseas by isoenzyme and RAPD markers in order to assess their genetic relationships. They stated that RAPD technique, through discriminating among all the species and distinguishing among 144 F2 plants of C. roseas, developed by crossing the accession ‘Pink Delhi’ (pink colored flower petals and stem, dark green leaf lamina bearing elliptic apex and borne on small petiole, long pods, tall habit, less salt and drought sensitivity, and high in alkaloid yield).
١٠٩ ______Results and discussion
Table (16): RAPD markers of the 11- radiation treatment with 5 RAPD primers Radiation Primers Total treatments Krad. OPB- OP-B07 OPB-11 OP-B12 OP-F06 amplified 01 fragments 0 Uniq.* 0 0 0 0 0 0 Poly. ** 5 5 2 0 3 15 Mono*** - - - + - 0 2 Uniq.* 0 0 0 1 0 1 Poly. ** 6 5 2 0 4 17 Mono*** 0 0 0 0 0 0 4 Uniq.* - 0 0 0 0 0 Poly. ** 7 7 8 7 5 34 Mono*** 0 0 0 0 0 0 6 Uniq.* 0 0 0 0 0 0 Poly. ** 8 5 8 7 4 32 Mono*** 0 0 0 0 0 0 8 Uniq.* 0 0 0 0 0 0 Poly. ** 11 5 7 7 5 35 Mono*** 0 0 0 0 0 0 10 Uniq.* 0 0 0 0 0 0 Poly. ** 11 4 7 7 9 38 Mono*** 0 0 0 0 0 0 12 Uniq.* 0 0 0 0 0 0 Poly. ** 11 9 2 7 9 38 Mono*** 0 0 0 0 0 0 14 Uniq.* 0 0 0 0 0 0 Poly. ** 9 9 2 7 9 36 Mono*** 0 0 0 0 0 0 16 Uniq.* 0 0 0 0 0 0 Poly. ** 12 11 7 10 10 50 Mono*** 0 0 0 0 0 0 18 Uniq.* 0 0 0 0 0 0 Poly. ** 12 11 6 10 11 50 Mono*** 0 0 0 0 0 0 20 Uniq.* 0 0 0 0 0 0 Poly. ** 9 4 9 12 10 44 Mono*** 0 0 0 0 0 0 *Unique bands: 1 **Polymorphic: 389 ***Monomorphic: 0 Total bands: 390 VI.5.1.2 Genetic similarity and cluster analysis based on RAPDs markers
The RAPD data were used to estimate the genetic similarity among 11 radiation treatments of seedling taxa by
١١٠ ______Results and discussion using Biorad softwere computer analysis was shown in (table 17). The highest similarity index recorded was 0.998, which was observed between the two taxa control and radiation treatment 20 Krad while the lowest similarity index recorded was 0.91, which was observed between radiation treatments
2 and 6 Krad
A dendrogram for the genetic relationships among the control and 10 radiation treatments was carried out as in Fig. (18). The 11 taxa were separated into two clusters; cluster one included 0, 20, 12, 16, 18, 8, 2 and 4 Krad., while the cluster two included 10, 14 and 6 Krad.
Within the cluster one, three sub clusters were found; the first one contained 2 Krad. and 4 Krad., the second sub clusters was divided into sub-sub-sub clusters the first contain 0 and 20 Krad. The while the third sub clusters contain
12, 16 and 18 Krad. (in the first division), and 8 Krad. (in the second division). The second cluster divided into two sub clusters the first one contained 6 and 14 Krad. , while the second sub cluster contained 10 Krad. only.
١١١ ______Results and discussion
Table (17): Similarity indices among the 11 radiation treatment Taxa Based on RAPD-PCR using 5 primers
Radiation treatment 0 2 4 6 8 10 12 14 16 18 20 Krad 0 2 143 4 500 286 6 636 091 318 8 286 500 571 182 10 737 105 368 864 211 12 875 125 438 727 250 842 14 667 095 333 955 190 905 762 16 824 118 412 773 235 895 441 810 18 929 154 538 591 308 684 813 619 765 20 998 143 500 636 286 737 875 667 824 929
Figiure No. (18 )
١١٢ ______Results and discussion b- Second variety (CP3): Depended on the above results, chosen the dose 16
Krad. because this dose appears of change on the Indole alkaloids production, the result obtained from RAPD PCR analysis with 10 primers were used for the identification of markers associated with 8 time hours (con, 0, 2, 4, 8, 16, 48 and 168) hours which were radiated at 16 Krad. taxa genotypes after four weeks presented as follows: 1- Primer OP-C09 Figure (19) and Table (18) showed that primer OP-C09 gave 11 polymorphic fragments with molecular sizes ranging from 10000 to 900 bp. (100%) with numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 with corresponding 10000, 9000, 8000, 7000, 6000, 5000, 4500, 4000, 2500, 1500 and 900 bp. were observed. Specific markers were 3 polymorphic fragments (27%) and unspecific markers were 8 polymorphic fragments (73%) this primers showed that a fragment with 10000, 9000 and 8000 bp. appeared exclusively after 168 hours on the other hand no monomorphism. So we can concider these fragments as molecular marker for radiation treatment 16
Krad. after 186 hours.
١١٣ ______Results and discussion
Table (18): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-C09. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 0 0 0 0 0 0 0 1 2 9000 0 0 0 0 0 0 0 1 3 8000 1 1 1 1 1 1 1 0 4 7000 1 1 0 1 0 0 0 1 5 6000 0 1 0 0 0 0 0 0 6 5000 0 1 0 1 0 0 0 1 7 4500 0 1 0 1 0 0 0 0 8 4000 0 1 0 1 0 0 0 0 9 2500 1 1 0 1 1 1 1 0 10 1500 1 1 0 1 0 1 0 1 11 900 0 1 0 1 0 1 1 1 Total bands 4 9 1 8 2 4 3 6 1= present 0= absent Table (19): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-C10. 2- Primer OP-C10
Primer OP-C10 exhibited eleven DNA fragments ranging in molecular sizes from 10000 to 900 bp. (Figure 16 and Table 19). Five polymorphic fragments (45%) with numbers 7, 8, 9, 10 and 11 with corresponding molecular sizes of 4500, 4000, 2500, 1500 and 900 bp., respectively, were observed. Whereas, the remaining bands were monomorphic (55%).
١١٤ ______Results and discussion
Time by hours 3- Band No. M.W (bp) Con. 0 2 4 8 16 48 168 Primer 1 10000 0 0 0 0 0 0 0 0 2 9000 0 0 0 0 0 0 0 0 3 8000 0 0 0 0 0 0 0 0 OP-C13 4 7000 0 0 0 0 0 0 0 0 5 6000 0 0 0 0 0 0 0 0 Pri 6 5000 0 0 0 0 0 0 0 0 7 4500 1 1 0 1 0 1 0 1 mer OP- 8 4000 1 1 0 1 0 1 1 1 9 2500 1 1 1 1 1 1 1 1 C13 10 1500 1 1 0 1 0 1 0 1 11 900 1 1 0 0 0 1 0 1 exhibite Total bands 5 5 1 4 1 5 2 5 1= present 0= absent d 11 DNA fragments ranging in molecular sizes 10000 to 900 bp. (Figure 19 and Table 19). Sex fragments were polymorphic with number 1, 2, 3, 4, 6 and 9 with corresponding molecular sizes of 1000, 9000, 8000, 7000, 5000 and 2500 bp. (82%) meanwhile bands number (5, 7, 8, 10 and 11) were monomorphic(18%). This primer showed that a fragment with 9000, 5000 bp. after 168 hours and 2500 bp. after 8 hours appeared exclusively so we can concider these fragments as molecular marker for radiation treatment 16
Krad. after 8 and 186 hours.
Table (20): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-C13. Band No. M.W (bp) Time by hours
١١٥ ______Results and discussion
Con. 0 2 4 8 16 48 168 1 10000 0 0 0 0 1 0 0 0 4- 2 9000 1 1 1 1 1 1 1 0 Prime 3 8000 0 0 0 0 1 1 1 1 4 7000 0 0 0 0 1 1 1 1 r OP- 5 6000 1 1 1 1 1 1 1 1 C15 6 5000 0 0 0 0 0 0 0 1 7 4500 0 0 0 0 0 0 0 0 Pr 8 4000 0 0 0 0 0 0 0 0 9 2500 1 1 1 1 0 1 1 1 imer 10 1500 0 0 0 0 0 0 0 0 11 900 0 0 0 0 0 0 0 0 OP- Total bands 3 3 3 3 5 5 5 5 1= present 0= absent C15 resulted in 11 DNA fragments with molecular sizes from 10000 to 900 bp. (Figure 19 and Table 20). Four polymorphic fragments (36%) with numbers 1, 2, 3 and 4 with corresponding molecular sizes of 10000, 9000, 8000 and 7000 bp. were observed, while the other bands were monomorphic. This primer showed that a fragment with 7000 bp. appear in 4 time hour. but was absent in all other taxa. So the fragment could be used as molecular marker for 4 time hour.
Table (20): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-C15. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168
١١٦ ______Results and discussion
1 10000 1 1 1 1 0 0 0 0 2 9000 1 1 1 1 0 0 0 0 3 8000 1 1 1 1 0 0 0 0 4 7000 0 0 0 1 0 0 0 0 5 6000 1 1 1 1 1 1 1 1 6 5000 0 0 0 0 0 0 0 0 7 4500 0 0 0 0 0 0 0 0 8 4000 1 1 1 1 1 1 1 1 9 2500 0 0 0 0 0 0 0 0 10 1500 0 0 0 0 0 0 0 0 11 900 0 0 0 0 0 0 0 0 Total bands 5 5 5 6 2 2 2 2 1= present 0= absent
5- Primer OP-G17 Primer OP-G17 gave 4 monomorphic fragments with molecular sizes 7000, 6000, 5000 and 4000 bp. (36%) and 7 polymorphism fragments (64%) (Figure 19 and Table 21). The fragment with 4500 and 900 bp. appeareds exclusively in 4 and 0 time hours fluctuated but was absent in other taxa. So it could be used as molecular marker for 4 and 0 time hours.
Table (21): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-G17. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 0 0 0 1 0 0 0 0
١١٧ ______Results and discussion
2 9000 0 0 1 1 0 1 0 0 3 8000 1 1 1 1 0 1 0 0 4 7000 1 1 1 1 1 1 1 1 5 6000 1 1 1 1 1 1 1 1 6 5000 1 1 1 1 1 1 1 1 7 4500 1 1 1 0 1 1 1 1 8 4000 1 1 1 1 1 1 1 1 9 2500 0 1 1 1 0 1 0 1 10 1500 1 0 1 0 1 0 0 1 11 900 0 1 0 0 0 0 0 0 Total bands 7 8 9 8 6 8 5 7 1= present 0= absent
6- Primer OP-L12 Primer OP-L12 resulted polymorph in 6 DNA fragments with molecular sizes 1000, 9000, 8000, 7000, 6000 and 900 bp. (Figure 19 and Table 22) (55%). Three polymorphic fragments (Number 3, 4 and 5) with corresponding molecular size of 8000, 7000 and 6000 bp were observed exclusively in control, while the other bands were monomorphic So, it could be used as molecular marker for control.
7- Primer OP- L13 Primer OP-L13 gave 7 monomorphic fragments with molecular sizes 10000, 9000, 7000, 5000, 4500, 4000 and 2500 bp. and 4 polymorphism with molecular size 8000, 6000, 1500 and 900 bp. (Figure 19 and Table 22).
Table (22): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-L12. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 0 1 1 1 1 0 1 1
١١٨ ______Results and discussion
2 9000 0 1 1 1 1 0 1 1 3 8000 0 1 1 1 1 1 1 1 4 7000 0 1 1 1 1 1 1 1 5 6000 0 1 1 1 1 1 1 1 6 5000 1 1 1 1 1 1 1 1 7 4500 1 1 1 1 1 1 1 1 8 4000 1 1 1 1 1 1 1 1 9 2500 1 1 1 1 1 1 1 1 10 1500 1 1 1 1 1 1 1 1 11 900 1 0 1 0 0 0 0 0 Total bands 6 10 11 10 10 8 10 10 1= present 0= absent
The fragments 8000 and 6000 bp. were observed exclusively in control and 48 time hours fluctuated, but were absent in other taxa. So it could be used as molecular marker for control and 48 time hours.
Table (23): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-L13. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 0 0 0 0 0 0 0 0 2 9000 0 0 0 0 0 0 0 0 3 8000 0 1 1 1 1 1 1 1 4 7000 1 1 1 1 1 1 1 1 5 6000 0 0 0 0 0 0 1 0 6 5000 1 1 1 1 1 1 1 1 7 4500 0 0 0 0 0 0 0 0 8 4000 1 1 1 1 1 1 1 1 9 2500 1 1 1 1 1 1 1 1 10 1500 0 1 0 0 0 1 0 0 11 900 1 0 0 0 1 1 0 0 Total bands 5 6 5 5 6 7 6 5 1= present 0= absent
8- Primer OP- L16
١١٩ ______Results and discussion
Primer OP-L16 showed 2 monomorphic fragments with molecular sizes 10000 and 4000 bp. with 9 polymorphism corresponding molecular size of 9000, 8000, 7000, 6000, 5000, 4500, 2500, 1500 and 900 (Figure 19 and Table 24).The fragments 7000, 6000, 5000, 4500 and 900bp. were observed in 0 time hour, on the other hands the 1500 bp. were observed exclusively in control but this fragments were absent in other taxa. So it could be used as molecular marker for control and 0 time hours.
9- Primer OP-L20
Table (24): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-L16. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 0 0 0 0 0 0 0 0 2 9000 1 0 1 1 1 0 1 1 3 8000 1 0 1 1 1 0 1 1 4 7000 1 0 1 1 1 1 1 1 5 6000 1 0 1 1 1 1 1 1 6 5000 1 0 1 1 1 1 1 1 7 4500 1 0 1 1 1 1 1 1 8 4000 1 1 1 1 1 1 1 1 9 2500 1 1 1 1 0 1 1 1 10 1500 0 1 1 1 0 1 0 0 11 900 0 1 0 0 0 0 0 0 Total bands 8 4 9 9 7 7 8 8 1= present 0= absent Primer OP-L20 resulted in 6 monomorphic DNA fragments with molecular sizes from 6000 to 1500 bp. (Figure 19 and Table 25). On the other hands their fife polymorphic fragments (45%) with numbers1, 2, 3, 4 and 11
١٢٠ ______Results and discussion with corresponding molecular size of 10000, 9000, 8000, 7000 and 900 bp. were observed, while the band of 7000 was appeared exclusively in 48 time hours but this fragments was absent in other taxa. So it could be used as molecular marker for 48 time hours.
Table (25): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-L20. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 1 1 1 1 1 0 0 1 2 9000 1 1 1 1 1 0 0 1 3 8000 1 1 1 1 1 0 0 1 4 7000 1 1 1 1 1 1 0 1 5 6000 1 1 1 1 1 1 1 1 6 5000 1 1 1 1 1 1 1 1 7 4500 1 1 1 1 1 1 1 1 8 4000 1 1 1 1 1 1 1 1 9 2500 1 1 1 1 1 1 1 1 10 1500 1 1 1 1 1 1 1 1 11 900 1 0 0 0 0 1 1 0 Total bands 11 10 10 10 10 8 7 10 1= present 0= absent 10- Primer OP-Z03 The results of Primer OP-Z03 gave 11 DNA fragments ranging in molecular sizes from 10000 to 900 bp. (Figure 19 and Table 26). All fragment were polymorphic (100 %) were observed. Whereas, on the other hands was no monomorphic (0 %). This primer showed that the fragment with 8000, 7000 and 4500 bp. appeared in 16, 8 and control time hours fluctuated exclusively only, but it were absent in all other taxa. So it could be used as molecular marker for 16, 8 and control time hours.
١٢١ ______Results and discussion
Table (26): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad. with primer OP-Z03. Time by hours Band No. M.W (bp) Con. 0 2 4 8 16 48 168 1 10000 1 1 1 1 0 0 1 1 2 9000 1 1 1 1 0 0 1 1 3 8000 1 1 1 1 1 0 1 1 4 7000 1 1 1 1 0 1 1 1 5 6000 0 1 1 1 1 1 1 1 6 5000 0 1 1 1 1 1 0 1 7 4500 0 1 1 1 1 1 1 1 8 4000 0 1 1 1 1 1 0 0 9 2500 1 1 0 1 0 1 0 0 10 1500 1 1 0 1 0 1 0 0 11 900 0 1 0 1 0 1 0 0 Total bands 6 11 8 11 4 8 6 7 1= present 0= absent
OP-C09
Figure (19): RAPD profiles of the 8 times treatment which are Gamma Irradiated at 16 Krad. amplified with primers (OP-C09, OP-C10, OP-C13, OP-C15, OP-G17, OP-L12, OP-L13, OP-L16, OP-L20 and OP-Z03).
Figure 19 continue,
١٢٢ ______Results and discussion
OP-C10
OP-C13
OP-C15
Figure 19 continue,
١٢٣ ______Results and discussion
OP-G17
OP-L12
OP-L13
Figure 19 continue,
١٢٤ ______Results and discussion
OP-L16
OP-L20
OP-Z03
١٢٥ ______Results and discussion
VI.5.2.1. RAPD markers of the 16 Krad radiation treatments with 10 RAPD primers Data of the amplified fragments using the aforementioned five 10-mer arbitrary primers OP-C09, OP- C10, OP-C13, OP-C15, OP-G17, OP-L12, OP-L13, OP-L16, OP-L20 and OP-Z03 for 8 times (con., 0, 2, 4, 8, 16, 48 and 168) which are treated by using gamma radiation on the dose 16 Krad. controlled with con. Which is none treated by gamma ray; 0, 2, 4, 6, 8, 10, 12, 14, 16, 48 and 168 hours indicated successful amplification of PCR products. Polymorphism levels differed from one primer to the other .The main results was as following table (27). Data in Table (27) showed that the tine primers showed polymorphic differences among the development time by hours, while some primers exhibited high polymorphism such as OP-C09 (73 %), OP-C13 (82 %), OP- L13 (82%), OP-L16 (82%) and OP-Z03 (100 %) on the other hand, some primers exhibited medium levels of polymorphism such as OP-C10 (45 %), OP-C15 (63 %), OP- G17 (64%), OP-L12 (55) and OP-L20 (45%).
١٢٦ ______Results and discussion
Table (27): Cultivars-specific RAPD markers of the 16 Krad with 10 RAPD primers
Time by hours Primers Total amplified fragments 17 09 10 13 15 12 13 16 20 03 L L L L Z C C C C G ------OP OP OP OP OP OP OP OP OP OP
Con. AF 4 5 3 5 7 6 5 8 11 6 60 SM - - - - 1 - - - - - 1 0 AF 9 5 3 5 8 10 6 4 10 11 71 SM ------1 - - 1 2 AF 1 1 3 5 9 11 5 9 10 8 59 SM ------4 AF 8 4 3 6 8 10 5 9 10 11 64 SM - - - 1 ------1 8 AF 2 1 5 2 6 10 6 7 10 4 53 SM ------16 AF 4 5 5 2 8 8 7 7 8 8 62 SM ------48 AF 3 2 5 2 5 10 6 8 7 6 54 SM ------1 - - - 1 168 AF 6 5 5 2 7 10 5 8 10 7 65 SM 2 - 1 ------3 SM: specific markers AF: amplified fragments
Our result agreement with Ajaswrata et al., (2007) who’s studying the terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in Catharanthus roseus cell cultures. Since TIA biosynthetic pathway is reported to be stress induced in C. roseus, we wanted to know the response of different TIA and related pathway genes to different a biotic stressors. The expression profiles of twelve genes that encode enzymes for
١٢٧ ______Results and discussion
TIA and related primary (MEP and Shikimate) pathways were monitored in leaf tissue separately exposed to 4_C, dehydration, 200 mM NaCl, UV-light and wounding conditions by semi quantitative RT-PCR analysis and compared with the untreated control after 6 h treatment.
VI.5.2.2. Genetic similarity and cluster analysis based on RAPDs markers
The RAPD data were used to estimate the genetic similarity among 8 time hours which are treated by using Gamma radiation bush taxa by using Concort softwere computer analysis was shown in table (28). The highest similarity index recorded was 0.906, which was observed between the two taxa 8 hour and 48 hour while the lowest similarity index recorded was 0.341, which was observed between 0 hour and 8 hour.
A dendrogram for the genetic relationships among the 8 different times was carried out as in Fig. (20). The 8 taxa were separated into two clusters; cluster one included 2, 4, 8, 16 and 168 hours while the cluster two included con., 0 and 48 hours.
Within the cluster one, four sub clusters were found; the first one contained 2 and 8 hours, the second sub clusters
١٢٨ ______Results and discussion
8 and 168 hours, the three sub clusters was 186 and 16 hours the four sub clusters was 16 and 4 hours.
The second cluster divided into two sub clusters the first one contained 4 and 48 hours, while the second sub cluster contained 0 and control.
Table (28): Similarity indices among the 8 different time Taxa Based on RAPD- PCR using 10 primers
Time by hours Con. 0 2 4 8 16 48 168 Con. 0 397 2 729 461 4 642 802 742 8 652 342 838 507 16 586 517 738 580 743 48 474 341 812 582 906 781 168 679 595 719 721 821 818 819
Figure (20): Dendrogram for the genetic distances relationships among the8 different time taxa based on similarity indices data of RAPD analysis.
١٢٩ ______Results and discussion
VI.6. Similarity and unsimilarity between (LM) & (CP3) Catharanthus roseus Varieties in Genomic under radiation stress. The results obtained from RAPD-DNA in two C. roseus variety (LM) & (CP3) appeared complementary in both it especially at treatment 16 Krad we are shown in variety (LM) primers OP-B11 & OP-B12 appeared low specific markers one fragment only on the other hand variety (CP3) which are treated by using gamma radiation at 16 Krad after 168 hours given the same result low specific markers two and one fragments in primers OP-C09 and OP-C13 respectively. But all the remaining primers don’t give any specific fragments in both two variety LM (at 16 Krad) & CP3 (at 168 hours). There is suggested between both genomes LM & CP3 varieties after radiation treatment especially at 16 Krad. and complimentary in gene expression to indole alkaloids production, this are appeared clearly Suffix in HPLC results. This results agreement with Sarika et al., (2007) that analyzed a C. roseas by isoenzyme and RAPD markers in order to assess their genetic relationships. The molecular marker mapped in this study was of the RAPD types. The fingerprinting RAPD markers are known to be useful in increasing the map density in both eu chromatic and hetero
١٣٠ ______Results and discussion chromatic regions. Indeed, 86 (68.8%) of the 125 DNA markers placed on the C. roseus map are of RAPD kinds. The sequence specific and EST-SSR markers are known to cover the euchromatic regions of genome/map. A total of 12 such markers were placed on the map. Thirty markers have their origin in RAPD combinations. The combinations apparently gave markers by promoting new amplifications in regions not covered by a single RAPD. RAPD markers are also expected to have covered the chromatic regions of C. roseus map. This study showed that combining of RAPD with anther kinds of primers can be a reliable means to generate new markers.
١٣١ ______Results and discussion
١٣٢ ______Summary
Summary: This work is a cooperation between Agriculture Botany Department; Faculty of Agriculture, Al-Azhar University, Nasr city, Cairo, Egypt; and Natural products department; National center for radiation research and technology, Atomic Energy Authority, Nasr city, Cairo, Egypt. During the period (2007 – 2012), to study the gamma irradiated effects on the Indole alkaloids production, protein marker, TDC & STR enzymes, and DNA content in Catharanthus roseus.
The present work was aimed also to obtain the maximum values of Indole alkaloids content using ionizing radiation in tow variety (LM & CP3). Quantity & quality of Indole alkaloids, protein, enzymes TDC & STR and DNA determinations during expression of Indole alkaloids which helped to achievement of these objectives.
I- Indole alkaloids determination by HPLC:
a- variety (LM): Gamma rays effects on Catharanthus roseus (L.) were genetically engineered to over express the two enzymes Tryptophandecarboxylase and Strrictosidinesynthase, which catalyze key steps in the biosynthesis of terpenoid Indole alkaloids, using gamma radiation effect with the two
١٣٣ ______Summary corresponding genes. The percentages of total alkaloids, Catharanthine, Vindoline, Vallesiachotamine a (a), Vallesiachotamine b (b) Ajmalicine, Horhammericine (a), Horhammericine (b), Vindolinine, 19-Epivindolineine, Strrictosidinelactam, Serpentine and Vinamidine were recorded as relative to C. roseus intact plant. The highest values of alkaloids were 6.190, 7.200, 5.940, 5.860, 7.790, 6.000, 6.672, 2.720, 7.000, 7.920, 7.230 and 6.400 μg of alkaloids respectively above , in radiation treatments 18, 12 & 16, 14, 14, 18, 18, 18, 12, 12, 8, 12 and 16 K rad respectively compared with other treatments. b- Variety (CP3):
Gamma irradiation at 16 Krad was effective on reducing the content of Ajmalicine, as compared with the control. In all cases, raising the doses of Gamma irradiation decreased the content of Ajmalicine production from 0 times to 48 hour this content were 2.1801, 2.0001, 1.8777, 1.4062, 1.2001, 1.0006 and 0.9963 µg / g leaf tissue fluctuated. But after one week 168 hours, Ajmalicine level production increasing suddenly 208991 µg / g leaf tissue. ; this results corresponds rationalize the cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in Gamma ray signaling leading to stimulation of Tdc and Str genes and the accumulation of
١٣٤ ______Summary catharanthine in C. roseus seedling cultures. Based on these findings, a model for signal transduction cascade has been proposed. II- Enzyme analysis verity (LM) a- TDC enzyme:
The radiation with Gamma rays effects on TDC. In taxa with (lanes 0 to 18) showed that one bands, the 20 exhibited bands (4) with lower intensity than the treated samples, the radiation effects on gene expression. Band 1 presents and very faint in the control untreated but was contradictory in the intensity and molecular Wight in the other doses treatments. Therefore, irradiation modified gene expression in taxa.
In 20 K rad (lanes 1 to 4) showed that, the enzyme de dig ration while complete in control and all doses, so that should be considered as a conferring modification of gene expression by irradiation. That we could be considered as a positive molecular marker associated with irradiation treatments.
b- STR or SSS enzyme:
STR or SSS, enzyme activity. In taxa with (lanes 0 to 18) showed that one bands, the 20 exhibited no bands with lower intensity than the treated samples, the radiation effects
١٣٥ ______Summary
on gene expression. Bands from control to 10 Krad presents and very dark but was contradictory in the intensity and molecular Wight in the other doses treatments. Therefore, irradiation modified gene expression in taxa. On the other hand radiation treatments 12, 14, 16, and 18 Krad were feint or very feint and contradictory in the molecular Wight so that should be considered as a conferring modification of gene expression by irradiation. That we could be considered as a positive molecular marker associated with irradiation treatments.
III- SDS- protein Page electrophoresis in variety (LM):
The effect of radiation treatments on the protein banding were 2 Krad. lanes 2 showed that, band 3, 4, 5 and 6 was absent in control while present in 6 and 8 K rad. doses, so that should be considered as a conferring modification of gene expression by irradiation. The pattern of 4 Krad. lanes 3 exhibited five bands, band 7 to 12 were present. Indicating the effect of 6 K rad. irradiation treatments on modification of gene expression in C. roseas Bands 3 and 5 were presents only in the 6 K rad treatment but were absent in control and
4, 12 and 14 Krad. treatments. Therefore, irradiation treatments effected on modification of gene expression in C. roseas treated.
١٣٦ ______Summary
IV-RAPD-PCR analysis showed that:
a- first verity (LM):
All the 5 primers successfully exhibited DNA fragments with 11 radiation treatments taxa genotypes after four weeks presented as follows; a total number of 72 fragments were visualized across the taxa. Different levels of polymorphism were observed from one primer to the other. Some primers exhibited high levels of polymorphism as:
OB-P01 primer showed that, fragment with 5500 bp could be used as molecular marker for 6 Krad. Fragment with 4500 bp could be used as molecular marker for 8 K rad. Fragment with 400 bp. could be used as molecular marker for
14 Krad.
OB-P07 primer showed that, fragment with 10000 bp could be used as molecular marker for 12 Krad. Fragment with 250 bp. could be used as molecular marker for 20 Krad.
OB-P11 primer showed that, fragment with 8500 bp could be used as molecular marker for16 Krad. Fragment with
5000 bp. could be used as molecular marker for 2 Krad. Fragment with 500 bp. could be used as molecular marker for
10 Krad. Fragment with 300 bp. could be used as molecular marker for 4 Krad. Fragment with 100 bp. could be used as molecular marker for 18 Krad.
١٣٧ ______Summary
OB-P12 primer showed that, fragment with 8500 bp. could be used as molecular marker for 16 Krad. Fragment with 4600 bp. could be used as molecular marker for 2 K rad. Fragment with 400 bp. could be used as molecular marker for
12 Krad.
OP- F06 primer showed that, fragment with 11000 bp. could be used as molecular marker for 20 Krad. Fragment with 10000 bp. could be used as molecular marker for 18
Krad.
b- Second verity (CP3): the result obtained from RAPD PCR analysis with 10 primers were used for the identification of markers associated with 8 time hours deferens (con, 0, 2, 4, 8, 16, 48 and 168) hours which are radiated at 16 Krad taxa genotypes after time above presented as follows; a total number of 495 fragments were visualized across the taxa. while some primers exhibited high polymorphism such as OP-C09 (73 %), OP-C13 (82 %), OP-L13 (82%), OP-L16 (82%) and OP- Z03 (100 %) on the other hand, some primers exhibited medium levels of polymorphism such as OP-C10 (45 %), OP-C15 (63 %), OP-G17 (64%), OP-L12 (55%) and OP-L20 (45%). Primer OP-C09 this primer showed that a fragment with 10000, 9000 and 8000 bp. appeared exclusively after
١٣٨ ______Summary
168 hours So we can concider these fragments as molecular marker for radiation treatment 16 K rad. after 186 hours.
Primer OP-C13 This primer showed that a fragment with 9000, 5000 bp. after 168 hours and 2500 bp. after 8 hours appeared exclusively so we can concider these fragments as molecular marker for radiation treatment 16Krad. After 8 and 186 hours.
Primer OP-C15 This primer showed that a fragment with 7000 bp. appear in 4 time hour. but was absent in all other taxa. So the fragment could be used as molecular marker for 4 time hour. Primer OP-G17 The fragment with 4500 and 900 bp appeareds exclusively in 4 and 0 time hours flectuated but was absent in other taxa. So it could be used as molecular marker for 4 and 0 time hours. Primer OP-L12 The fragments 8000 and 6000 bp. were observed exclusively in control and 48 time hours flectuated, but were absent in other taxa. So it could be used as molecular marker for control and 48 time hours. Primer OP- L16 this primer showed that a fragment with 900 bp was observed in 0 time hour So it could be used as molecular marker for control and 0 time hours. On the other hands Primers OP-C10, OP- L13, OP-L20 and OP-Z03 didn’t gave any molecular markers with samples above.
١٣٩ ______Reference
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151 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ
أﺟﺮﯾﺖ ھﺬه اﻟﺪراﺳﺔ ﺑﺎﻟﺘﻌﺎون ﺑﯿﻦ ﻗﺴﻢ اﻟﻨﺒﺎت اﻟﺰراﻋﻲ– ﻛﻠﯿﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ اﻻزھﺮ– ﺑﻤﺪﯾﻨﺔ ﻧﺼﺮ وﻣﻌﻤﻞ ﺑﺤﻮث اﻟﮭﻨﺪﺳﺔ اﻟﻮراﺛﯿﺔ ﺑﻘﺴﻢ ﺑﺤﻮث اﻟﻤﻨﺘﺠﺎت اﻟﻄﺒﯿﻌﯿﺔ ﺑﺎﻟﻤﺮﻛﺰ اﻟﻘﻮﻣﻲ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﯿﺎ اﻻﺷﻌﺎع - ﺑﮭﯿﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﯾﺔ ﺑﻤﺪﯾﻨﺔ ﻧﺼﺮ ﺑﺎﻟﻘﺎھﺮة ﺧﻼل اﻟﻔﺘﺮة ﻣﻦ ٢٠٠٧ - ٢٠١٢ وذﻟﻚ ﻟﺪراﺳﺔ ﺗﺄﺛﯿﺮ اﻻﺷﻌﺎع اﻟﺠﺎﻣﻲ ﻋﻠﻰ أﻧﺘﺎﺟﯿﺔ ﻗﻠﻮﯾﺪات اﻻﻧﺪول و ﺣﺰم اﻟﺒﺮوﺗﯿﻦ و أﻧﺰﯾﻤﺎت (اﻟﺘﺮﺑﺘﻮﻓﺎن دي ﻛﺎرﺑﻮﻛﺴﯿﻠﯿﺰ و ﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ) وﻛﺬﻟﻚ اﻟﻤﺤﺘﻮى ﻣﻦ اﻟﻤﺎدة اﻟﻮراﺛﯿﺔ ﻓﻲ ﻧﺒﺎت اﻟﻮﻧﻜﺎ.
واﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ ﺗﮭﺪف اﻟﻲ اﻟﺤﺼﻮل ﻋﻠﻲ ﻗﯿﻢ ﻋﺎﻟﯿﺔ ﻣﻦ ﻗﻠﻮﯾﺪات اﻻﻧﺪول ﺑﺄﺳﺘﺨﺪام اﻻﺷﻌﺎع اﻟﻤﺆﯾﻦ ﻓﻲ ﺻﻨﻔﯿﻦ ﻣﻦ ﻧﺒﺎت اﻟﻮﻧﻜﺎ وﺗﻢ أﺟﺮاء ﺗﺤﺎﻟﯿﻞ ﻛﻤﯿﺔ وﻛﯿﻔﯿﺔ ﻟﻘﻠﻮﯾﺪات اﻻﻧﺪول واﻟﺒﺮوﺗﯿﻦ وأﻧﺰﯾﻤﻲ (اﻟﺘﺮﺑﺘﻮﻓﺎن دي ﻛﺎرﺑﻮﻛﺴﯿﻠﯿﺰ و ﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ) وﻛﺬﻟﻚ اﻟﻤﺎدة اﻟﻮراﺛﯿﺔ وذﻟﻚ أﺛﻨﺎء اﻟﺘﻌﺒﯿﯿﺮ اﻟﺠﯿﻨﻲ ﻻﻧﺘﺎج ﻗﻠﻮﯾﺪات اﻻﻧﺪول وذﻟﻚ ﻟﺘﺤﻘﯿﻖ اﻟﮭﺪف ﻣﻦ اﻟﺪراﺳﺔ.
I - ﺗﺤﻠﯿﻞ ﻗﻠﻮﯾﺪات اﻻﻧﺪول ﺑﺎﺳﺘﺨﺪام ﺟﮭﺎزHPLC :
ا- اﻟﺼﻨﻒ (LM) :
أﻇﮭﺮت اﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎﻋﯿﺔ ﻋﻠﻲ ﻧﺒﺎت اﻟﻮﻧﻜﺎ ﺗﻐﯿﺮات وراﺛﯿﺔ وﺑﺎﻟﺘﺎﻟﻲ ﺗﻐﯿﺮات ﻋﻠﻲ اﻟﺘﻌﺒﯿﯿﺮ اﻟﻔﺎﺋﻖ ﻻﻧﺰﯾﻤﯿﯿﻦ ھﻤﺎ اﻟﺘﺮﺑﺘﻮﻓﺎن دي ﻛﺎرﺑﻮﻛﺴﯿﻠﯿﺰ واﻟﻤﻌﺮوف أﺧﺘﺼﺎرا (TDC) و ﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ واﻟﻤﻌﺮوف اﺧﺘﺼﺎرا (SSS or STR) وھﻤﺎ اﻻﻧﺰﯾﻤﺎن اﻻﺳﺎﺳﯿﺎ ن اﻟﻼذﻣﺎن ﻟﺤﻔﺰ ﺧﻄﻮات ﻋﻤﻠﯿﺔ اﻟﺘﺨﻠﯿﻖ اﻟﺤﯿﻮي ﻟﻘﻠﻮﯾﺪات اﻻﻧﺪول و ﺑﺄﺳﺘﻌﻤﺎل اﻻﺷﻌﺎع اﻟﺠﺎﻣﻲ ﻛﺎن ھﻨﺎك ﺗﺄﺛﯿﺮ ﻋﻠﻲ ﺗﺪاﺧﻞ ﻓﻌﻞ ﻛﻼ اﻟﺠﯿﻨﯿﻦ اﻟﻤﺴﺆﻟﯿﻦ ﻋﻦ اﻧﺘﺎج ﻛﻼ ﻣﻦ اﻻﻧﺰﯾﻤﯿﻦ اﻟﺴﺎﺑﻘﺘﯿﻦ.
وﻛﺎﻧﺖ اﻟﻘﻠﻮﯾﺪات اﻟﺘﻲ ﻗﺪرت ھﻲ:
1 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
Catharanthine, Vindoline, Vallesiachotamine a (a), Vallesiachotamine b (b) Ajmalicine, Horhammericine (a), Horhammericine (b), Vindolinine, 19-Epivindolineine, Strrictosidinelactam, Serpentine and Vinamidine
وﻧﺴﺒﺔ ھﺬة اﻟﻘﻠﻮﯾﺪات اﻟﺘﻲ ﺳﺠﻠﺖ ﻗﯿﻢ ﻋﺎﻟﯿﺔ ھﻲ ﻋﻠﻲ اﻟﺘﻮاﻟﻲ :
6.190و7.200 و5.940 و5.860 و7.790 و6.000 و6.672 و2.720 و ٧٫٠٠٠و7.920 و7.230 و 6.400 ﻣﯿﻜﺮو ﺟﺮام / ﺟﺮام ﻧﺴﯿﺞ ورﻗﺔ.وذﻟﻚ ﻟﻠﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ:
18, 12 & 16, 14, 14, 18, 18, 18, 12, 12, 8, 12, 16 K rad وذﻟﻚ ﻣﻘﺎرﻧﺔ ﺑﺒﺎﻗﻲ اﻟﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ. ب- اﻟﺼﻨﻒ (CP3) :
أﻇﮭﺮ ﺗﺄﺛﯿﺮ اﻹﺷﻌﺎع اﻟﺠﺎﻣﻲ ﺑﺠﺮﻋﺔ Krad 16 ﺗﺄﺛﯿﺮا ﻣﻠﺤﻮﻇﺎ ﻓﻲ اﻟﻤﺤﺘﻮى ﻣﻦ ﻗﻠﻮﯾﺪ اﻻﺟﻤﺎﻻﯾﺴﻦ وذﻟﻚ ﻣﻘﺎرﻧﺔ ﺑﺎﻟﻜﻨﺘﺮول ﻓﻘﺪ ﻟﻮﺣﻆ أن اﻟﻤﺤﺘﻮى ﻣﻦ اﻟﻘﻠﻮﯾﺪات ﺑﺪاﯾﺔ ﻣﻦ ﻋﻘﺐ اﻟﺘﺸﻌﯿﻊ ﻣﺒﺎﺷﺮة وﺣﺘﻰ ٤٨ ﺳﺎﻋﺔ ﻓﻲ ﺗﺪھﻮر ﻓﻲ إﻧﺘﺎﺟﯿﺔ اﻻﺟﻤﺎﻻﯾﺴﯿﻦ ﻓﻜﺎﻧﺖ اﻟﻘﯿﻢ
2.1801و2.0001 و1.8777 و1.4062 و1.2001 و 1.0006 0.9963ﻣﯿﻜﺮو ﺟﺮام / ﺟﺮام ﻧﺴﯿﺞ ورﻗﺔ. وﻟﻜﻦ ﺑﻌﺪ ﻣﺮور ١٩٦ ﺳﺎﻋﺔ أو أﺳﺒﻮع ﻓﺄن ﻣﺴﺘﻮى إﻧﺘﺎج اﻻﺟﻤﺎﻻﯾﺴﯿﻦ ﻗﺪ أرﺗﻔﻊ ﻓﺠﺄة ﻟﯿﺼﺒﺢ 2,08991 ﻣﯿﻜﺮو ﺟﺮام / ﺟﺮام ﻧﺴﯿﺞ ورﻗﺔ. ، ھﺬه اﻟﻨﺘﺎﺋﺞ ﺗﺘﻤﺎﺷﻰ ﻣﻊ اﻟﺘﻔﺴﯿﺮ ﺑﺄن اﻟﻤﺴﺘﻘﺒﻞ (S) ﻋﻠﻰ ﺳﻄﺢ اﻟﺨﻠﯿﺔ و وﺗﺪﻓﻖ أﯾﻮﻧﺎت اﻟﻜﺎﻟﺴﯿﻮم وﻗﻠﻮﯾﺔ اﻟﺒﯿﺌﺔ وﻓﻮق أﻛﺴﯿﺪ اﻟﮭﯿﺪروﺟﯿﻦ وﺑﺮوﺗﯿﻨﯿﺎت اﻟﻜﯿﻨﯿﺰ ﺗﻠﻌﺐ دور ﻣﻠﻤﻮس ﻣﻊ اﻹﺷﻌﺎع اﻟﺠﺎﻣﻲ ﻓﻲ ﺗﺤﻔﯿﺰ ﻛﻼ ﻣﻦ ﺟﯿﻨﻲ إﻧﺰﯾﻤﻲ ﻧﺎزع اﻟﻜﺮﺑﻮﻛﺴﯿﻞ وﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ ﻛﻤﺎ
2 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
ﯾﺴﺎﻋﺪ ﻋﻠﻰ ﺗﺮاﻛﻢ وﺗﺠﻤﯿﻊ ﻣﺎدة اﻟﻜﺰاراﻧﺴﯿﻦ ﻓﻲ ﺑﺎدرات ﻧﺒﺎت اﻟﻮﻧﻜﺎ و اﻋﺘﻤﺎدا ﻋﻠﻰ اﻟﺪراﺳﺎت ﯾﻌﺘﺒﺮ ﻣﻮد ﯾﻞ ﻧﻘﻞ اﻹﺷﺎرة ﺑﺎﻧﺘﻈﺎم ھﻮ اﻟﻨﻤﻮذج اﻟﻤﺜﺎﻟﻲ.
II- ﺗﺤﻠﯿﻞ اﻷﻧﺰﯾﻤﺎت ﻟﻠﺼﻨﻒ (LM) :
ا- إﻧﺰﯾﻢ ﻧﺎزع ﻣﺠﻤﻮﻋﺔ اﻟﻜﺮﺑﻮﻛﺴﯿﻞ ﻣﻦ اﻟﺘﺮﺑﺘﻮﻓﺎن:
أﻇﮭﺮ ﺗﺤﻠﯿﻞ ﻧﺸﺎط اﻧﺰﯾﻢ اﻟﺘﺮﺑﺘﻮﻓﺎن دي ﻛﺎرﺑﻮﻛﺴﯿﻠﯿﺰ واﻟﻤﻌﺮوف أﺧﺘﺼﺎرا (TDC) وذﻟﻚ ﺑﻌﺪ اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻻﺷﻌﺎع اﻟﺠﺎﻣﻲ ان اﻟﻤﻌﺎﻣﻼت ﻣﻦ ٠ اﻟﻲ ١٨ اﺣﺘﻮت ﻋﻠﻲ ﺑﻨﺪ واﺣﺪة ﺑﯿﻨﻤﺎ اﻟﻤﻌﺎﻣﻠﺔ اﻻﺷﻌﺎﻋﯿﺔ ٢٠ ﻛﺎﻧﺖ ﺑﮭﺎ ٤ ﺣﺰم ذات ﻛﺜﺎﻓﺔ ﺧﻔﯿﻔﺔ ﻣﻘﺎرﻧﺔ ﻣﻊ ﺑﺎﻗﻲ اﻟﻤﻌﺎﻣﻼت وﻛﺎن ھﻨﺎك ﺗﺄﺛﯿﺮ ﻟﻼ ﺷﻌﺎع ﻋﻠﻲ اﻟﺘﻌﺒﯿﯿﺮ اﻟﺠﯿﻨﻲ ﻣﺘﻤﺜﻼ ﺑﻮﺟﻮد ﺑﻨﺪ واﺣﺪة ﺧﻔﯿﻔﺔ ﻓﻲ اﻟﻤﻌﺎﻣﻠﺔ ٠ ﻛﯿﻠﻮ راد وﻟﻜﻦ ﻛﺎﻧﺖ ﻋﻠﻲ اﻟﻨﻘﯿﯿﺾ ﻣﻦ ذﻟﻚ ﻓﻲ اﻟﻜﺜﺎﻓﺔ واﻟﻮذن اﻟﺠﺰﯾﺌﻲ ﻓﻲ ﺑﺎﻗﻲ اﻟﻤﻌﺎﻣﻼت وﻟﺬﻟﻚ ﯾﻤﻜﻦ ﻣﻼﺣﻈﺔ وﺟﻮد ﺗﺤﻮﯾﺮ ﻓﻲ اﻟﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ ﻧﺘﯿﺠﺔ اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻻﺷﻌﺎع اﻟﺠﺎﻣﻲ. واﻻرﺑﻊ ﺣﺰم ﻓﻲ اﻟﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﺎﻋﯿﺔ ٢٠ ﻛﯿﻠﻮ راد اﻇﮭﺮت ان اﻻﻧﺰﯾﻢ ﻣﺘﺤﻠﻞ ﻣﻘﺎرﻧﺔ ﻣﻊ اﻟﻜﻮﻧﺘﺮول وﺑﺎﻗﻲ اﻟﺠﺮﻋﺎت اﻻﺷﻌﺎﻋﯿﺔ وﻟﺬﻟﻚ ﻣﻦ اﻟﻤﻤﻜﻦ اﻋﺘﺒﺎر ھﺬة اﻟﺤﺰم ﻣﻌﻠﻤﺎت ﻣﻮﺟﺒﺔ ﻟﻠﻤﻌﺎﻣﻠﺔ ﺑﺎﻹﺷﻌﺎع.
ب- إﻧﺰﯾﻢ ﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ:
أﻇﮭﺮ ﺗﺤﻠﯿﻞ ﻧﺸﺎط اﻧﺰﯾﻢ ﻣﺨﻠﻖ اﻻﺳﺘﺮﻛﺘﻮﺳﯿﺪﯾﻦ واﻟﻤﻌﺮوف اﺧﺘﺼﺎرا (SSS or STR) ان ﻣﺨﺘﻠﻒ اﻟﺠﺮﻋﺎت اﻻﺷﻌﺎﻋﯿﺔ ﻣﻦ (٠) وﺣﺘﻲ (١٨) ﻛﯿﻠﻮ راد اﻋﻄﺖ ﺣﺰﻣﺔ واﺣﺪة اﻣﺎ اﻟﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ (٢٠) ﻛﯿﻠﻮ راد ﻻ ﺗﻌﻄﻲ اى ﺣﺰم ﻣﻊ اﻧﺨﻔﺎض ﻣﻠﺤﻮظ ﻓﻲ اﻟﻜﺜﺎﻓﺔ ﻣﻘﺎرﻧﺔ ﺑﺒﺎﻗﻲ اﻟﻤﻌﺎﻣﻼت.
اﻇﮭﺮ ﺗﺄﺛﯿﺮ اﻻﺷﻌﺎع ﻋﻠﻰ اﻟﺘﻌﺒﯿﯿﺮ اﻟﺠﯿﻨﻲ ان اﻟﺤﺰم ﻣﻦ (٠) ﻛﯿﻠﻮ راد وﺣﺘﻲ (١٠) ﻛﯿﻠﻮ راد ﻛﺎﻧﺖ ﻣﻮﺟﻮدة وذو ﻛﺜﺎﻓﺔ ﻛﺒﯿﺮة ﻣﻘﺎرﻧﺔ ﻣﻊ اﻟﻜﺜﺎﻓﺔ واﻻوزان اﻟﺠﺰﯾﺌﯿﺔ ﻓﻲ اﻟﺠﺮﻋﺎت اﻻﺧﺮي وﻟﺬﻟﻚ ﺣﺪث ﺗﺤﻮﯾﺮ ﻓﻲ اﻟﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ. وﻣﻦ ﺟﮭﺔ اﺧﺮي ﻛﺎﻧﺖ اﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎﻋﯿﺔ ١٢، ١٤، ١٦، ١٨ ﻛﯿﻠﻮ راد ﺧﻔﯿﻔﺔ او
3 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
ﺧﻔﯿﻔﺔ ﺟﺪا ﻣﺘﻨﺎﻗﻀﺎ ﻣﻊ اﻻوزان اﻟﺠﺰﯾﺌﯿﺔ ﻟﻨﻔﺲ اﻟﺤﺰم وﻟﺬﻟﻚ اﻟﻤﻌﻄﯿﺎت اﻟﻤﺪروﺳﺔ دﻟﺖ ﻋﻠﻲ ان ھﻨﺎك ﺗﺤﻮﯾﺮ ﻓﻲ اﻟﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ ﺑﻮاﺳﻄﺔ اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻻﺷﻌﺎع اﻟﺠﺎﻣﻲ وﻟﺬﻟﻚ ﻧﺴﺘﻄﯿﻊ اﻟﻘﻮل ﺑﺄن ھﻨﺎك ﺗﻼذم واﺿﺢ ﺑﯿﻦ اﻟﻤﻌﻼﻣﺎت اﻟﺠﺰﯾﺌﯿﺔ اﻟﻤﻮﺟﺒﺔ واﻟﻤﻌﺎﻣﻼت اﻹﺷﻌﺎﻋﯿﺔ.
III - ﺗﺤﻠﯿﻞ اﻟﺤﺰم اﻟﺒﺮوﺗﯿﻨﯿﺔ ﺑﺎﺳﺘﺨﺪام ﺗﻜﻨﯿﻚ اﻻﻟﻜﺘﺮوﻓﻮرﯾﺴﯿﺲ ﻓﻲ اﻟﺼﻨﻒ .(LM)
ﻛﺎن ﺗﺄﺛﯿﺮ اﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎﻋﯿﺔ ﻋﻠﻲ اﻟﺤﺰم اﻟﺒﺮوﺗﯿﻨﯿﺔ ﻇﺎھﺮا ﻓﻲ
اﻟﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ .K rad 2 ﻓﻈﮭﺮت ﺣﺰم 6 ,5 ,4 ,3 ﺑﯿﻨﻤﺎ ﻛﺎﻧﺖ ھﺬة اﻟﺤﺰم
اﻟﺒﺮوﺗﯿﻨﯿﺔ ﻏﺎﺋﺒﺔ ﻓﻰ اﻟﻤﻌﺎﻣﻠﺔ .K rad 0 ﺑﯿﻨﻤﺎ ھﺬة اﻟﺤﺰم ﻛﺎﻧﺖ ﻣﻮﺟﻮدة ﻓﻲ
اﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎﻋﯿﺔ .K rad 8 ,6 وﻟﺬﻟﻚ ﻣﻦ اﻟﻤﻤﻜﻦ اﺳﺘﻌﻤﺎل ﺗﺤﻠﯿﻞ اﻟﺤﺰم اﻟﺒﺮوﺗﯿﻨﯿﺔ ﺑﺄﺳﺘﺨﺪام ﺗﻜﻨﯿﻚ اﻻﻟﻜﺘﺮوﻓﻮرﯾﺴﯿﺰ ﻟﻼﺳﺘﺪﻻل ﻋﻠﻲ اﻟﺘﻐﯿﯿﺮ ﻓﻲ اﻟﺘﻌﺒﯿﺮ
اﻟﺠﯿﻨﯿﻲ اﻣﺎ ﻓﻲ اﻟﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ .K rad 4 ﻇﮭﺮت ﺧﻤﺲ ﺣﺰم ھﻲ ﻣﻦ ٥ اﻟﻲ
٧ واﺳﺘﺤﺪﺛﺖ اﻟﻤﻌﺎﻣﻠﺔ اﻻﺷﻌﺎﻋﯿﺔ .K rad 6 ﺗﻐﯿﯿﺮ ﻓﻲ اﻟﺘﻌﺒﯿﯿﺮ اﻟﺠﯿﻨﻲ ﻓﻲ ﻧﺒﺎت اﻟﻮﻧﻜﺎ ﻓﻈﮭﺮت اﻟﺤﺰم ٣ و ٥ ﺑﯿﻨﻤﺎ ﻛﺎﻧﺖ ﻏﺎﺋﺒﺔ ﻓﻲ اﻟﻤﻌﺎﻣﻼت ٠ و٤ و ١٢ و ١٤ ﻛﯿﻠﻮ راد وﻟﺬﻟﻚ ﯾﻤﻜﻦ اﻟﻘﻮل ﺑﺄن اﻟﻤﻌﺎﻣﻼت اﻟﺸﻌﺎﻋﯿﺔ أﺣﺪﺛﺖ ﺗﻐﯿﯿﺮ ﻓﻲ اﻟﺘﻌﺒﯿﯿﺮ اﻟﺠﯿﻨﻲ ﻓﻲ ﻧﺒﺎت اﻟﻮﻧﻜﺎ.
ΙV- أﻇﮭﺮ ﺗﺤﻠﯿﻞ ﺗﻔﺎﻋﻞ اﻟﺒﻠﻤﺮة اﻟﻤﺘﺴﻠﺴﻞ ﺑﺎﺳﺘﺨﺪام اﻟﺒﺎدﺋﺎت اﻟﻌﺸﻮاﺋﯿﺔ آﻻﺗﻲ:
ا- اﻟﺼﻨﻒ اﻷول (LM):
أﻇﮭﺮ اﻟﺘﺤﻠﯿﻞ ﺑﺎﺳﺘﺨﺪام ﺗﻘﻨﯿﺔ اﻟـ RAPD – PCR وﻋﺪد ٥ ﻣﻦ اﻟﺒﺎدﺋﺎت اﻟﻌﺸﻮاﺋﯿﺔ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ ٧٢ ﺣﺰﻣﺔ ، وﻗﺪ اﺧﺘﻠﻒ ﻣﺴﺘﻮى اﻟﺘﺒﺎﯾﻦ ﻣﻦ ﺑﺎدئ ﻻﺧﺮ . ﺣﯿﺚ أﻇﮭﺮت ﺑﻌﺾ اﻟﺒﺎدﺋﺎت ﻣﺴﺘﻮى ﻋﺎﻟﻲ ﻣﻦ اﻟﺘﺒﺎﯾﻦ وﻛﺎن ذﻟﻚ ﻛﻤﺎ ﯾﻠﻲ:
4 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
أﻇﮭﺮ اﻟﺒﺎدئ OP-BO1 ﺣﺰﻣﺔ .pb 5500 وﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻲ
ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ Krad 6. وأﻇﮭﺮ ﺣﺰﻣﺔ .pb 4500 ﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻲ
ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ Krad 8. وﻛﺬﻟﻚ أﻇﮭﺮ ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻲ.pb 400 ﺗﻌﺘﺒﺮ
ﻛﺸﺎف ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ .Krad 14
أوﺿﺢ اﻟﺒﺎدئ OP-B07 ﺣﺰﻣﺔ .pb 10000 واﻟﺘﻲ ﺗﻌﺘﺒﺮ ﻛﺸﺎف
ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ Krad 12 واﻇﮭﺮ ﺣﺰﻣﺔ .pb 250 ﻣﻦ اﻟﻤﻤﻜﻦ
اﻋﺘﺒﺎرھﺎ ﻛﺸﺎف ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ Krad 20 .
أوﺿﺢ اﻟﺒﺎدئ OP-B11 ﺣﺰﻣﺔ.pb 8500 واﻟﺘﻰ ﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ
ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ .Krad 16 واﻇﮭﺮ ﺣﺰﻣﺔ .pb 5000 ﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻲ
ﻟﻠﺠﺮﻋﺔ .Krad 2 واﻇﮭﺮ ﺣﺰﻣﺔ.pb 500 ﻣﻦ اﻟﻤﻤﻜﻦ اﻋﺘﺒﺎرھﺎ ﻛﺸﺎف ﺟﺰﯾﺌﻲ
ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ .Krad 10 وﻇﮭﺮت ﺣﺰﻣﺔ .pb 300 واﻟﺘﻲ ﻣﻦ اﻟﻤﻤﻜﻦ
اﻋﺒﺎرھﺎ ﻛﺸﺎف ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ .Krad 4 وﻇﮭﺮت ﺣﺰﻣﺔ .pb 100
ﻣﻦ اﻟﻤﻤﻜﻦ اﻋﺘﺒﺎرھﺎ ﻛﺸﺎف ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ .Krad 18 .
أﻇﮭﺮ اﻟﺒﺎدئ OP-B12 ﺣﺰﻣﺔ .pb 8500 واﻟﺘﻰ ﺗﻌﺘﺒﺮ ﻛﺸﺎف
ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ Krad 16. واﻇﮭﺮ ﺣﺰﻣﺔ .pb 4600 وﺗﻌﺘﺒﺮ ﻛﺸﺎف
ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ Krad 2 ﻛﻤﺎ ﻇﮭﺮت ﺣﺰﻣﺔ .pb 400 ﯾﻤﻜﻦ اﻋﺘﺒﺎرھﺎ ﻛﺸﺎف
ﺟﺰﯾﺌﻲ ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ Krad 12 .
ﺑﯿﻦ اﻟﺒﺎدئ OP-F06 ﺣﺰﻣﺔ .pb 11000 وﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ
ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ Krad 20 ﻛﺬﻟﻚ أﻇﮭﺮ ﺣﺰﻣﺔ .pb 10000 واﻟﺘﻰ ﺗﻌﺘﺒﺮ
ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﻠﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ Krad 18 .
ب- اﻟﺼﻨﻒ اﻟﺜﺎﻧﻲ (CP3):
أﻇﮭﺮت اﻟﻨﺘﺎﺋﺞ اﻟﻤﺘﺤﺼﻞ ﻋﻠﯿﮭﺎ ﻣﻦ ﺗﺤﻠﯿﻞ RAPD – PCR ﻟﻌﺪد ١٠ ﻣﻦ اﻟﺒﺎدﺋﺎت اﻟﻌﺸﻮاﺋﯿﺔ ﻟﻠﺘﻌﺮﯾﻒ ﻛﻤﻌﻠﻢ وذﻟﻚ ﻟﺜﻤﺎﻧﻲ ﻋﯿﻨﺎت ﺗﻤﺜﻞ ٨ أوﻗﺎت ﻣﺨﺘﻠﻔﺔ
5 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
(ﻛﻨﺘﺮول و ٠ و ٢ و ٤ و ٨ و ١٦ و ٤٨ و ١٦٨) ﺳﺎﻋﺔ واﻟﺘﻲ ﻋﻮﻣﻠﺖ ﺑﺎﻟﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ ١٦ ﻛﯿﻠﻮ راد وﻛﺎن اﻟﻤﻈﮭﺮ اﻟﻌﺎم ﻟﻠﺤﺰم ﻋﻠﻰ اﻟﺠﻞ ﺑﻌﺪ اﻷوﻗﺎت اﻟﺴﺎﺑﻖ ذﻛﺮھﺎ ﻇﮭﺮت ﻛﻤﺎ ﯾﻠﻲ:
ﻛﺎن اﻟﻤﺠﻤﻮع اﻟﻜﻠﻲ ﻟﻠﺤﺰم اﻟﻤﺘﺤﺼﻞ ﻋﻠﯿﮭﺎ ٤٩٥ ﺣﺰﻣﺔ ﻣﻨﻈﻮرة ، وﻛﺎن ھﻨﺎك ﺑﻌﺾ اﻟﺒﺎدﺋﺎت اﻟﺘﻲ أﻋﻄﺖ ﺗﺒﺎﯾﻦ ﻋﺎﻟﻲ ﻣﺜﻞ OP-C09 (٧٣%) و OP-C13 (٨٢%) و OP-L13 (٨٢%) و OP-Z3 (١٠٠%) وﻣﻦ ﺟﮭﺔ أﺧﺮى ﻧﺠﺪ أن ﻧﻌﺾ اﻟﺒﺎدﺋﺎت أﻋﻄﺖ ﻣﺴﺘﻮى ﻣﺘﻮﺳﻂ ﻣﻦ اﻟﺘﺒﺎﯾﻦ ﻣﺜﻞ: OP-C10 (٤٥%) و OP-C15 (٦٣%) و OP-G17 (٦٤%) و OP-L12 (٥٥%) و OP-L20 (٤٥%).
أﻇﮭﺮ اﻟﺒﺎدئ OP-C09 ﺣﺰم وزﻧﮭﺎ اﻟﺠﺰﯾﺌﻲ ١٠٠٠٠ و ٩٠٠٠ و ٨٠٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻓﻘﻂ ﻣﻊ اﻟﻤﻌﺎﻣﻠﺔ اﻹﺷﻌﺎﻋﯿﺔ ١٦ ﻛﯿﻠﻮ راد ﺑﻌﺪ ١٦٨ ﺳﺎﻋﺔ وﻟﺬﻟﻚ ﯾﻤﻜﻦ اﻋﺘﺒﺎرھﺎ ﻣﻌﻠﻤﺎت ﺟﺰﯾﺌﯿﮫ.
أﻇﮭﺮ اﻟﺒﺎدئ OP-C13 ﺣﺰم وزﻧﮭﺎ اﻟﺠﺰﯾﺌﻲ ٩٠٠٠ و ٥٠٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻓﻘﻂ ﻣﻊ اﻟﻤﻌﺎﻣﻠﺔ اﻹﺷﻌﺎﻋﯿﺔ ١٦ ﻛﯿﻠﻮ راد ﺑﻌﺪ ١٦٨ وﻛﺬﻟﻚ أﻇﮭﺮ ﺣﺰﻣﺔ واﺣﺪة ﺑﻮزن ٢٥٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻓﻲ اﻟﻌﯿﻨﺔ ﺑﻌﺪ ٨ ﺳﺎﻋﺎت وﻟﺬﻟﻚ ﯾﻤﻜﻦ اﻋﺘﺒﺎر ھﺬا اﻟﺒﺎدئ ﻣﻌﻠﻢ ﺟﺰﯾﺌﻲ ﻣﻊ اﻟﻤﻌﺎﻣﻠﺔ اﻹﺷﻌﺎﻋﯿﺔ ﺑﻌﺪ ھﺬا اﻟﻮﻗﺖ.
أﻇﮭﺮ اﻟﺒﺎدئ OP-C15 ﺣﺰﻣﺔ واﺣﺪة ﺑﻮزن ٧٠٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻣﻊ اﻟﻌﯿﻨﺔ اﻟﻤﺄﺧﻮذة ﺑﻌﺪ ٤ ﺳﺎﻋﺎت وﻟﺬﻟﻚ ﯾﻤﻜﻦ اﻋﺘﺒﺎر ھﺬه اﻟﺤﺰﻣﺔ ﻣﻌﻠﻢ ﺟﺰﯾﺌﻲ.
اﻟﺒﺎدئ OP-G17 أﻋﻄﻰ ﺣﺰم وزﻧﮭﺎ اﻟﺠﺰﯾﺌﻲ ٤٥٠٠ و ٩٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻣﻊ اﻟﻤﻌﺎﻣﻼت اﻟﻮﻗﺘﯿﺔ ﺻﻔﺮ و ٤ ﺳﺎﻋﺔ وﻗﺪ اﺧﺘﻔﺖ ھﺬه اﻟﺤﺰم ﻓﻲ ﺑﺎﻗﻲ اﻟﻤﻌﺎﻣﻼت وﻟﺬﻟﻚ ﺗﻌﺘﺒﺮ ﻣﻌﻠﻤﺎت ﺟﺰﯾﺌﯿﮫ ﻟﮭﺬه اﻟﻤﻌﺎﻣﻼت.
6 ______اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ
اﻟﺒﺎدئ OP-L12 أﻋﻄﻰ ﺣﺰم وزﻧﮭﺎ اﻟﺠﺰﯾﺌﻲ ٨٠٠٠ و ٦٠٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﺣﺼﺮﯾﺎ ﻓﻲ اﻟﻜﻨﺘﺮول و ٤٨ ﺳﺎﻋﺔ ﺑﺎﻟﺘﺮﺗﯿﺐ وﻟﺬﻟﻚ ﯾﻤﻜﻦ اﻋﺘﺒﺎر ھﺬه اﻟﺤﺰم ﻣﻌﻠﻤﺎت ﺟﺰﯾﺌﯿﮫ ﺧﺎﺻﺔ ﺑﮭﺬه اﻟﻤﻌﺎﻣﻼت.
اﻟﺒﺎدئ OP-L16 أﻋﻄﻰ ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻲ ٩٠٠ زوج ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪي ﻓﻲ اﻟﻤﻌﺎﻣﻠﺔ ﺻﻔﺮ ﺳﺎﻋﺔ (ﻋﻘﺐ اﻟﺘﺸﻌﯿﻊ ﻣﺒﺎﺷﺮة) وﻟﺬﻟﻚ ﺗﻌﺘﺒﺮ ھﺬه اﻟﺤﺰﻣﺔ ﻣﻌﻠﻢ ﺟﺰﯾﺌﻲ ﻟﮭﺬه اﻟﻤﻌﺎﻣﻠﺔ.
وﻣﻦ ﺟﮭﺔ أﺧﺮى اﻟﺒﺎدﺋﺎت OP-C10, OP- L13, OP-L20 and OP-Z03 ﻟﻢ ﺗﻌﻄﻲ أي ﻣﻌﻠﻤﺎت ﺟﺰﯾﺌﯿﮫ ﻣﻤﯿﺰة ﻣﻊ اﻟﻤﻌﺎﻣﻼت اﻹﺷﻌﺎﻋﯿﺔ.
7 ( ) ( - ) ﺠﺎﻤﻌﺔ ٢٠٠١ ( ) ﻴﺔ ( - ) ﺠﺎﻤﻌﺔ ٢٠٠٦
( ) ( - )
–
١٤٣٤ﻫـ ٢٠١٣ ﻋﻴﺔ
(ﺒﻜﺎﻟ ) ( - ) ﺠﺎﻤﻌﺔ ٢٠٠١ ( ) ( - ﻟ ) ﺠﺎﻤﻌﺔ ٢٠٠٦
( ) ( - )
–
١٤٣٤ﻫـ ٢٠١٣
: ...... / . . ﺒ - ﺠﺎﻤﻌﺔ ...... / . . . - ( ) ( - ) ﺠﺎﻤﻌﺔ ٢٠٠١ ( ) ( - ) ﺠﺎﻤﻌﺔ ٢٠٠٦
( ) ( - )
– ﺠﺎﻤﻌﺔ
١٤٣٤ ﻫـ ٢٠١٣
: ...... / . . - ...... / . . - ...... / . . - ...... / . . -
: ٢٣ / ١ / ٢٠١٣