SPECIAL PROBLEM TITLE Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp. BY Miss Chanthra Timclub PROGRAM IN AGRICULTURAL BIOTECHNOLOGY FACULTY OF AGRICULTURE KAMPHAENGSAEN KASETSART UNIVERSITY 2004 Bachelor’s Degree Special Problem Program in Agriculture Biotechnology TITLE Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp. BY Miss Chanthra Timclub This special problem has been accepted Date……...March 2005 (Mrs. Parichart Burns, Ph. D.) Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp. Miss Chanthra Timclub Abstract The plant hormone ethylene is known to stimulate a wide variety of plant response to flower senescence and fruit ripening. A gene in ethylene synthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene, was identified from flower of Cattleya spp. Lucky Strike variety. In this study, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were used to amplify the ACC oxidase sequences. The cDNA of ACC oxidase (ACO) was cloned and characterized. The cDNA from Cattleya-ACOs, open reading frame (ORF) and 3’untranslated region (3’UTR), were 1,081 nucleotides in length. The sequence was further analysed by comparing with other ACC oxidase genes in GenBank Database. The results show that major difference between Cattleya spp. located at 3’UTR while the amino acid sequence alignment indicated high percent identity at 96.7%. In addition, the amino acid sequence of Cattleya-ACC oxidase was compared with 11 ACC oxidase genes of other plants from Genbank database. Ascorbate binding domains, ACC binding domain, ferrous ion binding domain, bicarbonate binding domain, leucine zipper and basic zipper domain were found in all ACOs. Keyword : Cattleya spp., Cattleya, Ethylene, ACC oxidase genes, senescence. Degree : Bachelor of Science, Program in Agricultural Biotechnology, Faculty of Agriculture Kamphaeng saen, Kasetsart University Advisor : Parichart Burns, Ph.D Year : 2005 Page : 54 การหาลาดํ ับเบส และวิเคราะหยีน 1-aminocyclopropane-1-carboxylate (ACC) oxidase ในกลวยไมสก ลแคทลุ ียา นางสาวจันทรา ทิมคลับ บทคัดยอ เอทิลีนเปนฮอรโมนพชทื าหนํ ากระตนการเสุ ื่อมสภาพของดอกไมและการสุกของผลไม ใน การทดลองครงนั้ ี้ไดศึกษา ACC oxidase ซึ่งเปนยีนที่เกยวขี่ องในกระบวนการสราง เอทธีลนี โดย ใชหลักการ Reverse Transcription Polymerase Chain Reaction (RT-PCR) และ Rapid Amplification of cDNA Ends (RACE) ในการ Amplify ลําดับนิวคลโอไทดี ของยีน ACC oxidase (ACO)ในกลวยไมสกุลแคทลียาพนธั ุ Lucky Strike จากการโคลนยีน และหาลาดํ ับนิวคลีโอไทด นํามาวิเคราะห พบวาล ําดบนั ิวคลีโอไทดในบร ิเวณ 3’untranslated region (3’UTR) มีความ แตกตางที่เหนได็ อยางชัดเจน เมื่อนาไปเปรํ ียบเทียบกบลั ําดับนวคลิ ีไทดของแคทลียาพนธั ุ Bicolor และแคทลียาพันธ ุ Intermedia ซึ่งมีรายงานใน GenBank แตเมื่อเปรยบเที ียบลําดบกรดอะมั ิโนใน สวน Open Reading Frame (ORF) พบวาม ีความเหมือนถึง 96.7% นอกจากนยี้ ังพบ Ascorbate binding domain, ACC binding domain, ferrous ion binding domain, bicarbonate binding domain, leucine zipper and basic zipper domain เมื่อนํามาเปรียบเทียบกับยนี ACC oxidase ของพืชชนิดอื่นๆที่มีรายงานไวในฐานขอมลู Genbank คําสําคัญ : Cattleya spp., แคทลียา, เอทธีลนี , ยีน ACC oxidase, การเหี่ยว. ปญหาพิเศษ : ปริญญาตรี สาขาวิชาเทคโนโลยีชวภาพทางการเกษตรี คณะเกษตร กาแพงแสนํ มหาวิทยาลยเกษตรศาสตรั อาจารยท ี่ปรึกษา : ดร.ปาริชาติ เบิรนส ป : 2548 จํานวนหนา : 54 ACKNOWLEDGEMENTS I am highly thankful to my supervisor, Mrs. Parichart Burns, Ph.D., for her kindness, generous help, suggestion and criticism of my special problem. I would like to thank the members of my supervisor committee, Ms. Orawan Kumdee, Ms. Suwanna Bandee, Ms. Saifon Cheuabunmee and Ms. Yada Mukjang, for their helpful advices and criticisms of the manuscript. Thanks are also expressed to the Plant Genetic Engineering Unit, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom province for making available research facilities. I would like to express special appreciation and gratitude to all of my friends for their help and fellowship during the period of my study. Finally, I gratefully and respectively thank to my family for their understanding, consolation and encouragement. Chanthra Timclum March, 2005 i TABLE OF CONTENTS Page TABLE OF CONTENTS…………………………………………………………………………….i LISTS OF TABLE…………………………………………………………………………………...ii LISTS OF FIGURES……………………………………………………………………………….iii INTRODUCTION…………………………………………………………………………………..1 LITERATURE REVIEW Cattleya……………………………………………………………………………………2 Ethylene…………………………………………………………………………………...6 Senescence……………………………………………………………………………….9 MATERIALS AND METHODS Plant Material……………………………………………………………………………14 Plant Sample…………………………………………………………………………….14 Cloning, Sequencing and Characterization of Cattleya-ACO……………………..14 Identification of functional domains in ACO putative proteins…………………….22 RESULTS AND DISCUSSIONS Cloning, Sequencing and Characterization of Cattleya-ACO..……………………24 Identification of functional domains in ACO putative proteins…………………….37 CONCLUSION……………………………………………………………………………...........41 LITERATURE CITED……………………………………………………………………………..42 APPENDIX………………………………………………………………………………………...46 ii LISTS OF TABLE Table Page 1. The nucleotie sequence alignment indicated percent Identity between Cattleya spp. Variety……………………………………………....33 2. The amino acid sequence alignment indicated Percent Identity between Cattleya spp. Variety……………………………………………….34 Appendix Table 1. Abbreviations of amino acids………………………………………………...53 2. The genetic code………………………………………………………………54 iii LISTS OF FIGURE Figure Page 1. The ethylene biosynthetic pathway of higher plants………………………..7 2. Schematic illustration of morphological changes in mesophyll cells……11 3. Hypothetical scheme for the action of ethylene in inducing flower senescence…………………………………………………………….12 4. Flow diagram outlining the strategy used for the cloning, sequencing and characterization of Cattleya-ACO gene……………………………......14 5. Flow diagram outlining the strategy used for the identification of functional domains in Cattleya-ACO putative proteins…………………….22 6. Diagramatic representation of the Cattleya-ACO cDNA………………..…23 7. Ethidium bromide stained 1% agarose gel of Cattleya flower total RNA ……………………………………………………..25 8. Ethidium bromide stained 1% agarose gel of open reading frame Cattleya- ACO genes……………………………………......26 9. Identification the recombinant clones by amplifying the recombinant plasmids with M13F and M13R primers………………...27 10. Ethidium bromide stained 1% agarose gel of 3’untranslated region of Cattleya- ACO genes ACO genes……………..28 11. The colony hybridization analysis of clone 3’UTR Cattleya-ACO……..…29 12. Identification the recombinant clone by amplifying the recombinant plasmids with with 3’ end Cattleya-ACO specific primer and VFO2.……………………………………………………30 13. Nucleotide and amino acid sequence of Cattleya-ACO …………………31 14. The sequence comparison of Cattleya spp ………………………..……...32 15. Phylogenetic tree of ACO nucleotide sequences………………………….35 16. Phylogenetic tree of ACO amino acid sequences…………………………36 iV LISTS OF FIGURE (cont’d) Figure Page 17. Alignment of ACC binding domain and ascorbate binding domain in ACO sequences……………………………………………………………….38 18. Alignment of basic zipper domain and leucine zipper domain in ACO sequences………………………………………………….39 19. Alignment of Fe2+ binding domains and bicarbonate binding domain in ACO sequences………………………………………..40 Appendix Figure 1. Disection of Cattleya flower…………………………………………………..47 2. pDrive Cloning Vector……………………………………………………….48 1 INTRODUCTION Cattleya Orchid (Cattleya spp.), is native to Central and South America. With its beauty, large flower, fragrance and colorful flowers, it is widely known as Queen of the orchid (Buranachonnabud, 1991; Papp, 2004). Although well adapted and cultivated in various areas around the world including Thailand, Cattleya flower has short vast life. This limits its market and popularity. There is little known about its senescence and the role of ethylene in the process. Therefore, a gene in ethylene synthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene is identified and characterized. This study will provide a basic knowledge of Cattleya senescence. Objective 1. Isolate ACC oxidase (ACO) gene from Cattleya spp. flower 2. Characterize ACC oxidase (ACO) gene from Cattleya spp. flower Place and Duration All experiment was done at Plant Genetic Engineering Unit, Kasetsart University Kamphaengsaen Campus, Nakhorn Pathom. The experiment began in November, 2003 and completed in January, 2005. 2 LITERATURE REVIEW Cattleya Cattleyas were among the first tropical orchids to come into cultivation. Their culture is often used as the basis for comparison with other types of orchids, being highly prized for their large blooms. They have well-adaptation to their habitat organs include thickened stems for food storage called pseudo-bulbs, roots that cling to the substrate to hold the plant in place, and thick, leathery leaves that transpire little water (Rittershausen, 1999; Ombrello, no date). 1. Origin Most of the Cattleyas have been developed from plants native of the America’s, especially Central and South America. The species range through tropical across the Panama Isthmus as far north as the rainforests of Mexico, but the best are found in the steamy Brazilian rainforests. The epiphytic or air plants grow on the trunks and stout branches of the forest trees, in their wild state existing for