臺灣博碩士論文加值系統

蛋白酶抑制子廣泛存在於生物體中，可降低或抑制蛋白酶的活性進而參與生理現象調控及抗逆境相關反應。本研究從刺角瓜 (Cucumis metuliferus) 中選殖出CmSPI基因可編碼出絲氨酸蛋白酶抑制子，屬於potato I type 蛋白酶家族。經序列分析發現CmSPI基因上游區域具有core abscisic acid response element、T/GBOXATPIN2 element以及wound response element，推測與逆境反應相關。為了瞭解CmSPI在植物中的功能性，因此在生物逆境方面，首先利用RNA interference (RNAi) 的方式靜默刺角瓜抗木瓜輪點病毒 (papaya ringspot virus, PRSV) 品系PI 292190之CmSPI基因表現。在CmSPI基因靜默刺角瓜品系H1和H2中發現，PRSV可突破植物的防禦機制並於接種病毒21天後開始產生病徵。此外，將刺角瓜CmSPI基因轉殖於圓葉菸草 (Nicotiana benthamiana) 中，得到的轉基因菸草品系F-21與F-51，對馬鈴薯病毒Y (potato virus Y, PVY ) 具有抗性。在非生物逆境方面發現，在鹽分逆境下，過表現CmSPI基因菸草品系Y1與Y2之種子發芽比率相較於未轉殖對照組可提高55.6-73.3%，而在Mannitol乾旱逆境處理下則可提高63-75%。此外在PEG乾旱逆境處理下，轉基因品系Y2之葉圓片總葉綠素含量相較於未轉殖對照組提高10%。綜言之，本研究證明CmSPI基因確實與刺角瓜抗PRSV之抗病反應相關，並可提供圓葉菸草對於PVY之抗性，同時CmSPI可增加圓葉菸草於鹽分及乾旱下的耐受性。本研究的結果將有助於瞭解刺角瓜CmSPI基因在生物及非生物逆境中的所扮演的腳色，在未來可進一步運用於作物抗病及環境逆境防治。

Proteinase inhibitors (PIs) is an ubiquitous protein involved in physiology reaction, biotic and abiotic stress defense responses via block the active center or allosteric interaction with specific proteinase. In this study, we cloned Cucumis metuliferus serine PI (CmSPI) gene, a potato I type PIs, from horned melon (C. metuliferus) papaya ringspot virus (PRSV) resistant line PI 292190. A core abscisic acid response element, a T/GBOXATPIN2 element, and a wound response element were identified on the promoter region of CmSPI indicating its role in plant stress responses. To identify CmSPI function, CmSPI RNA interference (RNAi) transgenic horned melon lines, H1 and H2 were generated, and showed broken down of the resistance after challenged with PRSV at 21 dpi. Transgenic tobacco (Nicotiana benthamiana) lines expressing a full length of CmSPI genomic fragment, F21 and F51, showed resistance to one of potyvirus, potato virus Y (PVY), but not Biden mottle virus (BiMoV) and Turnip mosaic virus (TuMV). In addition, CmSPI overexpression tobacco lines, Y1 and Y2, showed tolerance of salt and drought stresses. The germination rate of the two transgenic lines increased dramatically about 55.6% to 73.3% higher than wild type (WT) under NaCl salt stress and increase of 63% to 75% than WT under Mannitol drought stress. In addition, the chlorophyll content of leaf discs in transgenic lines, Y2, was 10% higher than WT under PEG drought stress. These results suggested that CmSPI can response to biotic and abiotic stresses. This study provided the evidence for a better understanding of CmSPI in interaction with pathogens and environment stresses. This can be applied to crop disease-resistance and stress-resistance breeding in the future.

目次/Contents
摘要 i
Abstract ii
目次/Content iii
表目次/Contents of table vi
圖目次/Comtents of figure vii

Chapter 1. Introduction and literature review 1
第一章、前言與前人研究
蛋白酶與蛋白酶抑制子 2
蛋白酶抑制子的種類及分群 3
蛋白酶抑制子調控植物生理作用 4
蛋白酶抑制子對於逆境之反應 5
蛋白酶抑制子轉基因植物的影響及發展 7
刺角瓜蛋白酶抑制子 8
總主旨 9
分章目標 9
參考文獻 10
Tables and figures 16

Chapter 2. Cloning and functional analysis of Cucumis metuliferus serine proteinase inhibitor (CmSPI) involve in Papaya ringspot virus (PRSV) resistance and against to Potato virus Y (PVY) in transgenic tobacco 21
第二章、選殖並分析刺角瓜蛋白酶抑制子對木瓜輪點病毒抗性影響及提供轉基因菸草對於馬鈴薯Y病毒之抗性
Abstract 22
Introduction 23
Plant materials and methods 25
Cloning CmSPI gene cDNA and full length genomic fragment of Cucumis metuliferus 25
Construction of phylogenetic tree 25
Construction of binary vector and bacterial strain 26
Plant material and gene transformation 26
Inoculation of plant viruses 27
Detection of gene expression 27
Results 30
Cloning and characterized of CmSPI 30
Generation of CmSPI RNAi transgenic plant 30
Alteration of the resistance in CmSPI RNAi C. metuliferus 31
Resistance to virus in transgenic tobacco 32
Discussion 33
CmSPI involved in plant physiological response and plant defence 33
Silencing of CmSPI altered the resistance to PRSV in horned melon 34
Expression CmSPI in tobacco plants can supply resistance for PVY 34
Conclusions 36
References 37
Tables and Figures 43

Chapter 3. Investigation of transgenic tobacco salt and drought tolerance in overexpression of horned melon serine proteinase inhibitor gene in tobacco plants
52
第三章、轉殖絲氨酸蛋白酶抑制基因之圓葉菸草對鹽分及乾旱逆境之耐性
摘要 53
Abstract 54
前言 55

材料與方法 57
試驗材料與載體構築 57
菸草基因轉殖與再生 57
基因表現及檢測 58
逆境發芽試驗 58
幼苗鹽害逆境處理 59
葉圓片非生物逆境處理 59
植株性狀檢測 59
試驗統計分析 60
結果 61
菸草轉殖株檢測與分析 61
菸草CmSPI轉殖株於鹽分逆境下的表現 61
菸草CmSPI轉殖株於乾旱逆境下的表現 62
討論 64
CmSPI基因提高種子於鹽害及乾旱逆境下的發芽率 64
CmSPI基因能維持植株在乾旱逆境下的葉綠素含量 65
轉基因套數與植株不同生長時期影響CmSPI基因表現及抗逆境能力 65
參考文獻 67
圖表 70

Chapter 4. Conclusions 76
第四章、結論

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