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Profiling and Characterization of the Micrornas and Their Targets in Important Vegetables of Solanaceae

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Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae PhD Thesis (Botany) Submitted By Muhammad Din PhD Scholar Department of Botany University of Balochistan, Quetta Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae PhD Thesis (Botany) Supervised by Dr. Muhammad Younas Khan Barozai Associate Professor Department of Botany University of Balochistan, Quetta Submitted by Muhammad Din PhD Scholar Department of Botany University of Balochistan, Quetta Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae Submitted by Muhammad Din PhD Scholar Department of Botany University of Balochistan, Quetta Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae Thesis submitted for the requirement of the degree of Doctor of Philosophy (PhD) in Botany, University of Balochistan, Quetta Supervised By Dr. Muhammad Younas Khan Barozai Associate Professor Department of Botany, University of Balochistan, Quetta Submitted by Muhammad Din PhD Scholar Department of Botany University of Balochistan, Quetta CERTIFICATE This is to certify that Mr. Muhammad Din who was registered in Doctor of Philosophy (PhD) (Registration No. 1993/UB-2013/R-215) in Botany, Department of Botany, University of Balochistan, Quetta, under the supervision of Dr. Muhammad Younas Khan Barozai, has successfully completed course work of eighteen (18) credit hours and research work under the title “Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae”. He may be allowed to submit the thesis on the above cited topic to the University of Balochistan for the fulfillment of PhD (Botany) degree. Dr. Muhammad Younas Khan Barozai Prof. Dr. Atta Muhammad Sarangzai Research Supervisor Chairperson Associate Professor Department of Botany, Department of Botany, University of Balochistan, Quetta University of Balochistan, Quetta . Dean External Examiner Faculty of Life Sciences University of Balochistan, Quetta I CERTIFICATE It is certified that the said dissertation is based on research work under the title “Profiling and Characterization of the MicroRNAs and Their Targets in Important Vegetables of Solanaceae” and its result carried out by me. This is written and compiled by me. This research work has not been submitted for higher studies in any other institution. MUHAMMAD DIN PhD Scholar Department of Botany, University of Balochistan, Quetta II ACKNOWLEDGEMENT First of all I am thankful to Almighty Allah for giving me such contentment and vision for accomplishment to my research work and compilation of this Thesis. I am sincerely obliged to my supervisor Dr. Muhammad Younas Khan Barozai, Associate Professor, Department of Botany, University of Balochistan, Quetta, for his precious guidance, proper supervision and appreciation during the period of research work. I wish to express my gratitude to Chairperson and all faculty members, Department of Botany, University of Balochistan, Dean, Faculty of Life Sciences, University of Balochistan, Quetta, for their significant guidance, encouragement, helpful suggestions and full cooperation. I would like to thank my humble colleagues for their moral support and helpful suggestions. III DEDICATION This thesis is dedicated to my beloved parents who have supported me all the way since the beginning of my studies and to my supervisor Dr. Muhammad Younas Khan Barozai who has been a great source of motivation and inspiration. Also, this thesis is dedicated to my wife and children (Ashfaq Ahmed and Irshad Ahmed). Finally, this thesis is dedicated to all those who believe in the richness of learning. IV INDEX CONTENT PAGE Certificate I Acknowledgment III Abbreviations X Abstract 2 1. Introduction 4 2. Review of Literature 9 2.1. Solanaceae 9 2.1.1. Tomato (Solanum lycopersicum L.) 9 2.1.2. Potato (Solanum tuberosum L.) 10 2.1.3. Eggplant (Solanum melongena L.) 10 2.1.4. Chilli (Capsicum annuum L.) 11 2.2. MicroRNA (miRNA) 12 2.2.1. History of miRNA 12 2.2.2. Biogenesis of miRNA 13 2.2.3. Function of miRNA 14 2.2.4. Mechanism of miRNA Action 15 2.2.4.1. Cleavage of mRNA directed by miRNA 15 2.2.4.2. Translational repression directed by miRNA 16 2.2.5. Cluster of miRNA 16 2.2.6. Secondary structure of miRNA 17 2.2.7. Convergence and divergence of miRNA in plants 18 2.2.8. Nomenclature of miRNA 19 2.3. Profiling and characterization strategies for miRNA 20 V 2.3.1. Computer-based (bioinformatics) 20 2.3.2. Experimental approach 22 2.4. Computer-based Databases (dbs) and tools 22 2.4.1. Databases (dbs) 22 2.4.1.1. The miRBase 22 2.4.1.2. Plant miRNA database (PMRD) 23 2.4.1.3. GenBank Database (dbGenBank) 24 2.4.1.4. Express Sequence Tags (ESTs)/Database (dbEST) 25 2.4.2. Computer-based tools 25 2.4.2.1. Mfold RNA folding server 25 2.4.2.2. RNAhybrid for targets prediction 26 2.4.2.3. BLAST (Basic Local Alignment Search Tool) 27 2.4.2.4. ClustalW 28 2.4.2.5. WebLogo (A Sequence Logo Generator) 29 3. Materials and Methods 31 3.1. Reference microRNA (miRNA) fetching 31 3.2. ESTs from EST Database (dbEST) 33 3. 3. Profiling initial potential miRNAs 33 3.4. Creation of single tone EST 33 3.5. Removal of protein coding sequences through BLASTx 34 3.6. Generation of stem-loop secondary structures 34 3.7. Physical scrutinization 35 3.8. Reverse Transcription PCR (RT-PCR) validation 35 VI 3.9. Gel visualization 35 3.10. Conservation analysis 38 3.11. Phylogenetic studies 38 3.12. Prediction of miRNA targets 39 3.12.1. A plant small RNA target analysis server (psRNATarget) 39 3.12.2. BLAST and RNAhybrid approach 40 3.13. Gene Ontology (GO) analysis 40 4. Results 42 4.1. Tomato miRNAs 42 4.1.1. Tomato new conserved miRNAs 42 4.1.2. Tomato miRNAs characterization 43 4.1.3. Tomato miRNAs experimental validation by RT-PCR 54 4.1.4. Tomato miRNAs convergence and phylogenetic investigation 54 4.1.5. Tomato potential miRNAs targets 55 4.2. Potato miRNAs 63 4.2.1. Potato new conserved miRNAs 63 4.2.2. Potato miRNAs characterization 68 4.2.3. Potato miRNAs experimental validation by RT-PCR 69 4.2.4. Potato sense/antisense miRNAs 73 4.2.5. Potato cluster miRNAs gene 74 4.2.6. Potato miRNAs convergence and phylogenetic investigation 75 4.2.7. Potato potential miRNAs targets 76 4.3. Eggplant miRNAs 82 VII 4.3.1. Eggplant new conserved miRNAs 82 4.3.2. Eggplant miRNAs characterization 82 4.3.3. Eggplant miRNAs experimental validation by RT-PCR 87 4.3.4. Eggplant miRNAs convergence and phylogenetic investigation 87 4.3.5. Eggplant potential miRNAs targets 87 4.4. Chilli miRNAs 98 4.4.1. Chilli new conserved miRNAs 98 4.2.2. Chilli miRNAs characterization 102 4.4.3. Chilli miRNAs validation through BLASTx 103 4.4.4. Chilli pre-miRNA cluster 108 4.4.5. Chilli miRNAs convergence and phylogenetic investigation 109 4.4.6. Chilli potential miRNAs targets 111 5. Discussion 118 6. Conclusion 133 7. References 135 8. Publications from the dissertation 151 TABLES Table 1. Solanum plant species miRNAs primers for RT-PCR expression analysis 36 Table 2. Tomato miRNAs characterization 44 Table 3. Tomato miRNAs targets 57 Table 4. Potato miRNAs characterization 64 Table 5. Potato miRNAs targets 77 Table 6. Eggplant miRNAs characterization 84 Table 7. Eggplant miRNAs targets 94 Table 8. Chilli miRNAs characterization 99 VIII Table 9. Chilli miRNAs targets 112 FIGURES Figure 1. Flow-chart for the computational identification of tomato, potato, eggplant 32 and chilli putative miRNAs using all known matures and precursors miRNAs in plants as a reference miRNAs Figure 2. Tomato pre-miRNA secondary structures 49 Figure 3. Tomato miRNA RT-PCR expressional validation 54 Figure 4. Tomato miRNA convergence investigation 56 Figure 5. Tomato miRNA phylogenetic investigation 56 Figure 6. Potato pre-miRNA secondary structures 70 Figure 7. Experimental validation of potato miRNAs by RT-PCR 73 Figure 8. Potato sense/antisense miRNA gene 73 Figure 9. Potato pre-miRNA cluster 74 Figure 10. Potato miRNA convergence study 75 Figure 11. Potato miRNA phylogenetic study 76 Figure 12. Eggplant pre-miRNA secondary structures 88 Figure 13. RT-PCR expressional validation for eggplant miRNAs 93 Figure 14. Eggplant miRNAs conservation studies 93 Figure 15. Eggplant miRNAs phylogenetic analysis 93 Figure 16. Chilli pre-miRNA secondary structures 105 Figure 17. Chilli pre-miRNA clusters 108 Figure 18. Chilli miRNA convergence study 110 Figure 19. Chilli miRNA phylogenetic study 110 IX ABBREVIATIONS Abscisic acid (ABA) Minimum Free Energy (MFE) Anthocyanin synthase (ANS) Myeloblastosis (MYB) Arabidopsis lyrata (aly) National Center for Biotechnology Information (NCBI) Arabidopsis thaliana (ath) Nicotiana tabacum (nta) ATP-binding cassette (ABC) Nucleotide-binding site-leucine rich repeat (NBS-LRR) Base pair (bp) Nucleotides (nt) Basic local alignment search tool Number of mismatches (NM) (BLAST) Brachypodium distachyon (bdi) Opposite strand of miRNA (miRNA*) Brassica rapa (bra) Organ of expression (OE) cAMP response element binding (CREB) Orthophosphate (Pi) Capsicum annuum (can) Oryza sativa (osa) Carotenoid cleavage dioxygenase (CCD) Phenyl ammonium lyase (PAL) Citrus sinensis (csi) Plant small RNA target (psRNATarget) Complementary DNA (cDNA) Populus trichocarpa (ptc) Database of EST (dbEST) Precursor miRNA length (PL) Dicer-like Enzyme1 (DCL1) Precursor of miRNAs (pre-miRNAs) Double-stranded RNA (dsRNA) Primary transcripts of mature miRNAs (pri-miRNAs) Expressed Sequence
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  • Technical Issues in Measuring Dropout in Primary Schools in Quetta Aamara Bibi Mrs Amir Bano Mr. Abdul Wahab Abstract

    Technical Issues in Measuring Dropout in Primary Schools in Quetta Aamara Bibi Mrs Amir Bano Mr. Abdul Wahab Abstract

    1 Bi-Annual Research Journal “Journal of Education & Humanities Research” ISSN: 2415-2366 Institute of Education and Research (IER), UOB, Quetta Pakistan Vol.6.NO 2, 2018 Technical Issues in Measuring Dropout In Primary Schools In Quetta Aamara Bibi M.Phil. Scholar. Balochistan Study Center, University of Balochistan, Quetta. Mrs Amir Bano Assistant Professor. IER, University of Balochistan, Quetta. Mr. Abdul Wahab Lecturer, IER, University of Balochistan, Quetta. Abstract: The issue of Primary school dropouts has produced expanded interest among Teachers, policymakers and Researchers especially in last few years. many technical issued are being faced in measuring this social phenomenon This Quantitative study on technical issues in measuring dropout looks at the technical issues faced into comprehend and understand this social and instructive issue which has direct impacts on society. To figure out the issue two areas have been focused for covering the data through self-constructed questionnaire , first random sampling with (50) fifty primary school Head teachers and second with stratified random sampling by educational administrators in Quetta. The data analysis tool based on Statistical Package for the Social Sciences (SPSS) which is assisted in measuring the data in descriptive and cluster form. Results of the study with discussion identified crucial finding especially for the future research and helpful paper for educational administration of Balochistan to revisit the policies regarding measuring method of dropout. The recommendations and implications are also discussed at the end of this study. Key Words. Technical issues. Dropout. Primary schools. Quetta. 2 Introduction Education with no doubt universally is considered as key for development in socio-economic conscious countries.