臺灣博碩士論文加值系統

中文摘要
為了研究植物體因應逆境所產生的變化，我們使用單子葉植物的水稻(Oryza sativa)為研究材料，觀察水稻懸浮細胞在缺乏碳素源的營養逆境下的基因表現，並利用mRNA differential display的方法分離出此一逆境下基因表現量增加的脂肪運輸蛋白(lipid transfer protein，LTP)基因。LTP是一種小分子量、鹼性的蛋白質，結構上具有高保留性的八個cysteine形成四個雙硫鍵。由分子量的不同可分為LTPI (分子量約9 kDa)與LTPII (分子量約7 kDa)。LTP普遍存在植物體內，並在不同的組織中各有獨特的表現型態與功能。我們發現此一水稻LTPI (Osltp)基因在正常生長狀態的水稻植株中，表現在發育中與新鮮成熟種子以及成熟植株根部；並於與水份有關的逆境：高鹽、脫水、低溫與ABA (abscisic acid)處理等狀況下，在根部的表現增加。進一步觀察雙子葉植物綠豆(Vigna radiata)LTPI基因的表現，分離出兩個綠豆LTPI基因，Vrltp1與Vrltp2。Vrltp1表現於花芽、發育中的種子，而Vrltp2則只表現於花芽。但Vrltp1與Vrltp2在綠豆植株中的表現部位是在地上部的莖與葉，與Osltp在水稻中的表現部位在根部極為不同。在高鹽、脫水與ABA (abscisic acid)處理等的葉子，Vrltp1與Vrltp2表現亦增加。我們推測這些LTPI基因不僅具有組織表現的特異性，並皆為與水份有關的逆境，如高鹽、脫水、低溫與ABA (abscisic acid)處理等增加表現，可能與對抗逆境有關；而詳細機轉，則需要更進一步的研究。

ABSTRACT
To explore the changes in response to environmental stresses, we isolated a lipid transfer protein (LTP) gene in rice (Oryza sativa) suspension cells in the presence or absence of sucrose by using mRNA differential display. LTPs are small and basic proteins, which consist of eight highly conserved cysteines forming four disulfide bonds. Two main families of LTPs, LTPI and LTPII, are identified with molecular mass of 9 kDa and 7 kDa. LTPs exist in various plants, and the expression of plant LTPs shows temporal and spatial patterns. We found this rice ltp (Osltp) mRNA expressed in developing and fresh seeds, as well as roots in mature plants; and the level of Osltp mRNA increased under water stress, such as high salt, dehydration, low temperature, and abscisic acid (ABA) treatments. Furthermore, we isolated two novel mungbean (Vigna radiata) ltp genes, Vrltp1 and Vrltp2, by screening cDNA library. Both Vrltp1 and Vrltp2 mRNAs expressed in floral buds and only Vrltp1 mRNA expressed in immature seeds. In the vegetative tissues, the Vrltp1 and Vrltp2 mRNAs display their shoot specificity. The levels of Vrltp1 and Vrltp2 mRNAs also increase in response to salt, dehydration and ABA treatments. We assume that these ltp genes participate in the protection from damage under water stress, and the regulatory mechanisms are needed to be elucidated.

Table of Contents
Page
Acknowledgements-------------------------------------------------------------------------- iv
List of Figures--------------------------------------------------------------------------------- v
Abbreviation---------------------------------------------------------------------------------- vii
Abstract (in Chinese)------------------------------------------------------------------------ ix
Abstract (in English)------------------------------------------------------------------------- x
Background Study------------------------------------------------------------------------ xi
CHAPTER I: Cloning and Characterization of a Water Stress-responsive Lipid Transfer Protein I Gene in Rice (Oryza sativa)------------------------------------------- 1
1. Abstract------------------------------------------------------------------------------------ 2
2. Introduction------------------------------------------------------------------------------- 4
3. Materials and Methods------------------------------------------------------------------- 6
Plant Materials---------------------------------------------------------------------------- 6
Isolation of Sucrose Starvation Induced Genes by Differential Display---------- 7
Isolation of a Rice Ltp Gene------------------------------------------------------------ 8
Primer Extension Analysis-------------------------------------------------------------- 8
Isolation of Genomic DNA and Southern Blot Analyses-------------------------- 9
Stress Treatments------------------------------------------------------------------------- 9
Northern Blot Analyses----------------------------------------------------------------- 10
4. Results and Discussion------------------------------------------------------------------- 12
Sequence Analysis of the Osltp Gene------------------------------------------------ 12
Analysis of the Transcription Start Site of Osltp------------------------------------ 14
Osltp Is a Member of a Small Multigene Family----------------------------------- 15
Developmental and Spatial Expression Patterns of Osltp------------------------- 15
Osltp Gene Is Induced under Stress Conditions and ABA Treatments----------- 16
5. Figures------------------------------------------------------------------------------------- 20
6. References--------------------------------------------------------------------------------- 32
CHAPTER II: Cloning and Characterization of Two Novel Lipid Transfer Protein I Genes in Mungbean (Vigna radiata)------------------------------------------------------ 37
1. Abstract------------------------------------------------------------------------------------ 38
2. Introduction------------------------------------------------------------------------------- 39
3. Materials and Methods------------------------------------------------------------------- 41
Plant Materials and Growth------------------------------------------------------------ 41
Isolation of Mungbean Ltp Genes----------------------------------------------------- 41
Stress Treatments------------------------------------------------------------------------ 42
Northern Blot Analysis----------------------------------------------------------------- 43
4. Results and Discussion------------------------------------------------------------------- 44
Sequence Analysis of the Vrltp Genes------------------------------------------------ 44
Developmental and Spatial Expression Patterns of Vrltps------------------------- 45
Vrltp Genes Are Induced under Stress Conditions and ABA Treatments--------- 47
5. Figures------------------------------------------------------------------------------------- 50
6. References--------------------------------------------------------------------------------- 56
Conclusion------------------------------------------------------------------------------------ 61
APPENDEX--------------------------------------------------------------------------------- 62
(1) Plant Gene Register PGR98-058
Cloning and Sequencing of a Rice cDNA Encoding Translation Elongation Factor-1 Alpha (Accession No. AF030517)---------------------------------------------- 63
(2) Plant Gene Register PGR98-085
Cloning of a cDNA Encoding a Lipid Transfer Protein from Rice (Accession No. AF051369)----------------------------------------------------------------------------- 72

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