臺灣博碩士論文加值系統

FIN219啟動子功能的研究中，利用農桿菌轉殖P219::FIN219-GUS載體到fin219 突變體。結果發現，T2世代在遠紅光的處理下，0802品系呈現非常短的下胚軸，而50個transgenic lines之中只有此品系 (0802) 具有rescued fin219 下胚軸的外表型。但轉移到白光生長卻具有非常矮小的植株，與野生型顯著不同。另外，0802品系是一個隱性突變外表型，因為T2種子在Hygromycin篩選之下，具有1/4比例為非常短小的外表型，且只有這1/4的短小幼苗對於光處理呈現hypersensitive現象，不像是正常互補之後應該有3/4比例是正常外表型。因此0802品系，很可能是T-DNA插入某一個基因造成抑制fin219突變體的外表型。而此基因很可能是FIN219的一個基因外抑制者。因此本篇論文藉由功能性研究的方式，來探討FIN219與其可能的抑制者之間的關係。經由不同光源的處理，發現0802這個品系在遠紅光中的下胚軸長度有回復成野生型的現象，並且0802品系在白光、藍光、紅光、與黑暗中亦具有hypersensitive 的短下胚軸外表型。所以0802這個品系乃是因T-DNA插入某個基因突變之後造成在遠紅光下抑制fin219突變體的外表型。利用Inverse PCR的方式將T-DNA插入位置的旁邊DNA片段分離出來，並且經由定序以及序列的比對發現，T-DNA插入在阿拉伯芥Brassinosteroid (BR) 生合成途徑CPD (Constitutive Photomorphogenesis and Dwarfism) (CYP90A1)基因的第三個intron。利用PCR與Genomic Southern Blot 方式進一步確認T-DNA的確是插入在這個位置；藉由RT-PCR的方式檢測CPD基因在0802品系中的表現發現，CPD mRNA在homozygous突變體中完全偵測不到，而在heterozygous突變體中的表現則較野生型中低。此結果顯示，很可能CPD基因的失去功能而造成0802品系在不同光源下的短小外表型。此外，利用CPD anti-sense 轉殖株的分析發現，CPD anti-sense 轉殖均可以部分的抑制fin219、fin219-null、phyA抽長的下胚軸；暗示，CPD 可能是FIN219的抑制者。另外利用FIN219啟動子與GUS報導基因的轉殖株，經由BR的處理結果發現，在黑暗處理2天轉移到遠紅光2天，BR的處理會抑制FIN219的表現。RT-PCR與Northern blot分析發現受光所調控的基因，CHS (Chalcone synthase)、RBCS (Ribulose Bisphosphate Carboxylase/oxygenase Small subunit)等，在0802品系中也會明顯的受到改變。綜合以上結果顯示CPD在光與BR訊息交互作用之中可能扮演重要角色。

A putative suppressor line 0802 of fin219 was identified from studies of FIN219 promoter activities that investigated expression patterns of fin219 transgenic seedlings harboring the construct P219：：FIN219-GUS. The T2 line of 0802 exhibited a rescued hypocotyl phenotype under far-red light, had a very short (dwarf) phenotype under white light. In addition, under hygromycin selection, 1/4 hypersensitive segregation ratio is resistant and homozygous, leading to the possibility that the phenotype of 0802 was due to a T-DNA inserted in an unknown gene to suppress the long hypocotyl phenotype of fin219. To confirm this hypothesis, we focus on the cloning of the putative suppressor gene and address the relationship between FIN219 (Far-red Insensitive 219) and its putative suppressor by a functional study approach. This putative suppressor line 0802 showed a fin219-rescued phenotype under far red light, and it also exhibited a hypersensitive phenotype under white light、red light、blue light and darkness. The flanking sequence of T-DNA was amplified by an inverse-PCR approach and sequenced. It was found that the T-DNA was inserted in the 3rd intron (1784 nt) of Arabidopsis CPD (Constitutive Photomorphogenesis and Dwarfism) gene. CPD (CYP90A1) was reported as Arabidopsis brassinosteroid biosynthetic gene and belonged to cytochrome P450 super gene family. This result was confirmed by RT-PCR and genomic southern. Besides, CPD anti-sense lines could partially suppress the long hypocotyl of fin219, fin219 -null and phyA under continuous far-red light (cFR), which indicates that CPD is the suppressor of FIN219. Furthermore, FIN219 promoter activities could be reduced by Brassinosteroid (BR), 24-epi-BL. The expression analysis show that the expression of light regulated genes such as CHS (Chalcone synthase) and RBCS (Ribulose Bisphosphate Carboxylase/oxygenase Small subunit) could be altered in 0802. Taken together, CPD might play a vital role to crosstalk between BR and light signaling.

ABSTRACT……………………………………………………………………………….1
中文摘要…………………………………………………………………………………..3
INTRODUCTION…………………………………………………………………………5
1. Light and Plants………………………………………………………………….5
2. Phytochromes…………………………………………………………………..5
3. Light Signaling Pathway……………………………………………………….7
4. Brassinosteroids………………………………………………………………..8
5. Cytochrome P450………………………………………………………………9
6. The BR Biosynthetic Genes and Light Signaling…….……………………..10
7. Specific Aims……………………………………………………………………12
METHODS………………………………………………………………………………..13
1. Plant Growth Condition………………………………………………………..13
2. Inversed PCR............................................................................................13
3. Genomic Southern blot..............................................................................14
4. Plasmid Construction…………………………………………………………..15
5. Histo chemical GUS Staining………………………………………………….16
6. GUS Activity Assay……………………………………………………………..16
7. RT-PCR…………………………………………………………………………..17
8. Northern blot……………………………………………………………………..18
RESULTS………………………………………………………………………………….19
1. The Putative Suppressor Line 0802 Was Obtained From The……………19
P219：：FIN219-GUS Transgenic Plants
2. 0802 Could Rescue fin219 Under Far-Red Light……………………………20
3. Molecular Cloning of the Putative Suppressor of fin219 by RT-PCR……...20
4. 0802 Exhibited Pleiotropic Dwarf Phenotypes……………………………….21
5. FIN219 Promoter Can be Repressed by BR…………………………………22
6. Suppressor Line 0802 Can Recover Target Gene Expression to the…….23
Wild-type Level
7. Continuous Red Light and Far-red Light Could down-regulated CPD……24 Expression
8. CPD is a Suppressor of FIN219………………………………………………25
DISSCUSSION…………………………………………………………………………….26
I. Definition of a Suppressor…………………………………………………………26
II. Crosstalk between light signaling and BR………………………………………28
III. The relationship between FIN219 and CPD…………………………………...29
FIGURES……………………………………………………………………………………32
REFERENCE……………………………………………………………………………….57
APPENDIX I…………………………………………………………………………………65
APPENDIX II……………………………………………………………………………..…97

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