Regulation of Heat Stress Induced  CAF1H Promoter Expression in Rice

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弗琳娜(Afrilina Sinaga) 查詢紙本館藏

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生命科學系

論文名稱

(Regulation of Heat Stress Induced CAF1H Promoter Expression in Rice)

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摘要(中)

中文摘要

高等植物生長、發育與產量受環境影響極大，植物演化出能夠因應外在環境變化透過準確地調控體內基因表現，藉此改變胞內狀態適應環境變化。在真核生物中Poly(A) tail縮短又稱為deadenylation是mRNA降解的速率決定步驟，而CCR4-associated factor 1s（CAF1s）為真核生物中催化deadenylation的主要酵素之一。在我們實驗室先前的研究中指出，OsCAF1H為水稻中受到熱逆境誘導的基因。在45˚C熱處理一小時中，我們可以發現OsCAF1H 基因在水稻根部與地上部分別被快速誘導 9.9倍與4.2倍，並且當將植物移回室溫此基因表現會快速的下降。而在啟動子DNA序列分析中發現，OsCAF1H啟動子具有一個典型與非典型的熱休克片段，因此我們建立了OsCAF1H啟動子接上glucuronidase (GUS)報導基因的轉殖株。在GUS染色實驗中，我們發現在三天大的小苗經過熱處理(45˚C) 1到4小時，皆可在幼苗、芽、根、胚乳和胚中檢測到GUS活性。綜合以上結果，熱逆境可以直接調控OsCAF1H的啟動子。

摘要(英)

ABSTRACT

Various environmental stresses affect different growth stage and yield of the plants. Therefore, plants regulate the expression level of genes for adjusting cellular status in response to variable environment. Poly(A) tail shortening, also termed as deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. CCR4-associated factor 1 (CAF1) proteins are important enzymes for catalysis of mRNA deadenylation in the eukaryotes. This study investigated the role of a rice heat-induced CAF1, OsCAF1H, in the adaptation of rice plants to heat stress. Expression of OsCAF1H was closely linked with the high-temperature exposure. The expression level of OsCAF1H was increased 9.9-fold in roots and 4.2-fold in shoots after shifting to 45˚C for 1 hour, and dramatically decreased to the basal level after 3 hours of recovery at room temperature. The OsCAF1H promoter contains one canonical palindromic and one atypical Heat Shock Element. In order to investigate the OsCAF1H promoter activity, the transgenic seedlings of OsCAF1H promoter drive glucuronidase (GUS) reporter gene were generated and analysed. GUS staining was detected inshoots, roots, endosperm and embryos under heat treatment (45˚C) for 1h, 2h, 3h, and 4hours. Our data revealed that CAF1H promoter was positively regulated by heat stress in rice.

關鍵字(中)

★ Heat Stress
★ OsCAF1Hpromoter
★ Oryza sativa
★ GUS reporter gene
★ Luciferase

關鍵字(英)

★ Heat Stress
★ OsCAF1Hpromoter
★ Oryza sativa
★ GUS reporter gene
★ Luciferase

論文目次

TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………………...i
中文摘要………………………………………………………………………………………ii
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
FIGURE LIST...……………………………………………………………………………...vi
TABLE LIST vii
SUPPLEMENTARY LIST……………………………………………………………...…viii
1. INTRODUCTION 1
1.1 OsCAF1H Promoter 2
1.2 CCR4 and CAF1-required for Deadenylation 4
1.3 Plants Defense Responses (Heat Stress)……………………...…………….………5
1.4 Study Goal…………………………………...………………….…...…….…………8
2. MATERIALS AND METHODS 9
2.1. Plant materials, growth conditions and heat treatment……………..……………9
2.2. Primers…………………………………………………………………...….…….....9
2.3. Genotyping Analysis…………………………………………………….………….10
2.4. Plasmid Constructions…………………………………………….…..……...……10
2.5. RT-PCR………………………………………………………….……………...…..10
2.6. Plant Transformation………………………………………………………....…....11
2.7. Rice Embryo Transient Expression Assay………………………………….…….12
2.7.1. The fluorogenic assay for GUS activity………………………………………12
2.7.2. Luciferase assay……………………………………………..………………...12
3. RESULT and DISCUSSION 14
3.1 OsCAF1H is induced by heat stress 14
3.2 OsCAF1H promoter contains heat shock elements (HSEs) 14
3.3 Generation of transgenic lines harboring the chimeric OsCAF1H Promoter/β-Glucuronidase reporter gene 15
3.4 The putative OsCAF1H::GUS transgenic plants show normal phenotype under normal growth conditions 17
3.5 GUS activity was induced by heat stress in germination seeds of OsCAF1H::GUS transgenic lines 17
3.6 Testing of the heat response sequences in the OsCAF1H Promoter 18
CONCLUSION…………………………………………………………………...………….20
REFFERENCES 21
SUPPLEMENTARY DATA 41
APPENDIX 50
ACRONYM………………………………………………………………………………….60

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指導教授

陸重安(Chung-An Lu)

審核日期

2020-7-31

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