Galectin-1枯草桿菌、阿拉伯芥與加洲苜蓿夜蛾核多角體病毒轉殖株的建立及殺蟲活性之研究

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簡勤益(Chin-I Chien) 查詢紙本館藏

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生命科學系

論文名稱

Galectin-1枯草桿菌、阿拉伯芥與加洲苜蓿夜蛾核多角體病毒轉殖株的建立及殺蟲活性之研究
(Establishment and Characterization of Insecticidal Galectin-1 Transgenic Strains of Bacillus subtilis, Arabidopsis thaliana and Autographa californica Multiple Nucleopolyhedrovirus)

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摘要(中)

先前，我們實驗室的研究結果已指出，Galectin-1 (GAL1) 是一個幾丁質結合蛋白，而且對小菜蛾具有殺蟲活性。在本篇研究中，將倉鼠卵巢細胞的GAL1基因序列，建構在pαHY300、pCAMBIA1390-35S和pBac-IR-GFP的表現載體上，並將載體分別命名為pαHY300-GAL1、p1390-35S-GAL和pBac-GAL1-IR-GFP，並且將他們分別送入枯草桿菌、阿拉伯芥和加洲苜蓿夜蛾核多角體病毒中，製成GAL1轉殖株，進行GAL1蛋白質的表現及其殺蟲活性的研究。而結果指出GAL1基因確實成功地送入枯草桿菌、阿拉伯芥和核多角體病毒中，並且都會表現GAL1蛋白質，在GAL1蛋白質表現量方面，阿拉伯芥和核多角體病毒遠高於枯草桿菌，在殺蟲活性的結果中，枯草桿菌GAL1轉殖株對小菜蛾及斜紋夜盜沒有明顯的殺蟲活性，而阿拉伯芥GAL1轉殖株對小菜蛾及斜紋夜盜則有明顯的殺蟲活性，核多角體病毒GAL1轉殖株的殺蟲活性，仍在進行當中。而以上的結果指出，以GAL1蛋白質做為新型的具有殺蟲活性的轉殖植物是一個很好的選擇。

摘要(英)

Previous studies in our laboratory have indicated that galectin-1 (GAL1) is a chitin binding protein and shows insecticidal activity toward Plutella xylostella. In these studies, GAL1 gene from Chinese hamster ovary cells was cloned into the plasmids pαHY300, pCAMBIA1390-35S and pBac-IR-GFP respectively. The resulting plasmids were designed as pαHY300-GAL1, p1390-35s-GAL1 and AcMNPV-GAL1 and introduced into Bacillus subtilis, Arabidopsis thaliana and Autographa californica multiple nucleopolyhedrovirus respectively for GAL1 expression and insecticidal activity investigation. The results showed that GAL1 gene was successfully introduced into B. subtilis, A. thaliana and AcMNPV for protein expression. The expression levels of GAL1 were highest in A. thaliana among the transformants. Though the GAL1-transgenic B. subtilis showed no significant insecticidal activity to S. litura and P. xylostella, GAL1-transgenic A. thaliana showed high insecticidal activity toward Spodoptera litura and Plutella xylostella. The insecticidal activities of GAL1-transfected AcMNPV will be under investigation. The results indicate that GAL1 would be a future candidate gene in transgenic plants.

關鍵字(中)

★ 枯草桿菌
★ 加洲苜蓿夜蛾核多角體病毒
★ 阿拉伯芥
★ 生物殺蟲劑

關鍵字(英)

★ Autographa californica Multiple Nucleopolyhedrov
★ bio-insecticide
★ Bacillus subtilis
★ Galectin-1
★ Arabidopsis thaliana

論文目次

Chinese abstract ……………………………………………… i
English abstract ……………………………………………… ii
Contents…………………………………………………………… iii
Figure Contents ……………………………………………… v
Abbreviation ……………………………………………… vii
Chapter 1 Introduction
1.1 Bio-insecticide………………………………………………1
1.2 Galectin-1 ……………………………………………………1
1.3 Bacillus subtilis transgenic bacterium ………………4
1.4 Arabidopsis thaliana transgenic plant…………………5
1.5 Baculovirus recombinant virus……………………………6
Chapter 2 Materials and Methods
2.1 Insect rearing ………………………………………………7
2.2 GAL1 cloning …………………………………………………7
2.3 Over-expression of GAL1 in Bacillus subtilis ………7
2.3.1 Construction of GAL1 expression vector for B. subtilis…7
2.3.2 Preparation of Bacillus subtilis ISW 1214 competent cell…8
2.3.3 he electroporation of Bacillus subtilis……………8
2.3.4 Detection of GAL1in Bacillus subtilis ISW-GAL1 by Western blotting…9
2.3.5 The insecticidal activity of Bacillus subtilis ISW-GAL1…10
2.3.5.1 Artificial diet feed assay…………………………10
2.3.5.2 Cabbages feed assay …………………………………10
2.4 Expression of GAL1 in Arabidopsis thaliana…………10
2.4.1 Construction of GAL1 expression vector in Arabidopsis thaliana…10
2.4.2 Preparation of Agrobacterium tumefaciens competent cell…11
2.4.3 DNA transferring into Agrobacterium tumefaciens…12
2.4.4 Agrobacterium-mediate transformation of Arabidopsis……12
2.4.5 Sectection of transgenic plant………………………13
2.4.6 Detection of GAL1 expression in Arabidopsis thaliana-GAL1…13
2.4.7 The insecticidal activity of Arabidopsis thaliana-GAL1…13
2.5 Expression of GAL1 in AcMNPV……………………………14
2.5.1 Construction of GAL1 expression vector in AcMNPV…14
2.5.2 Recombinant virus production…………………………15
2.5.3 GAL1 expression in AcMNPV-GAL1-infected sf-21 cell…15
2.5.4 The biological assays of AcMNPV-GAL1 virus………15
2.6 Statistical analysis………………………………………16
Chapter 3 Results
3.1 枯草桿菌GAL1轉殖株之構築及殺蟲活性之測試 …… 17
3.1.1 以限制酶BamHⅠ和HindⅢ分析選殖基因 …………… 17
3.1.2 GAL1基因於枯草桿菌系統的表現 …………………… 17
3.1.3 枯草桿菌轉殖株殺蟲活性的測試 …………………… 18
3.2 阿拉伯芥GAL1轉殖株之構築及殺蟲活性之測試 …… 19
3.2.1 以限制酶EcoRⅠ和SpeⅠ分析選殖基因……………… 20
3.2.2 GAL1基因於阿拉伯芥系統的表現 …………………… 20
3.2.3 阿拉伯芥轉殖株殺蟲活性的測試 …………………… 21
3.3 核多角體病毒GAL1轉殖株之構築 …………………… 21
3.3.1 以限制酶BglⅡ和EcoRⅠ分析選殖基因……………… 22
3.3.2 GAL1基因於核多角體病毒系統的表現 ……………… 22
Chapter 4 Discussion ……………………………………………24
Figures………………………………………………………………31
Reference……………………………………………………………44
Appendix ……………………………………………………………48

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指導教授

黃榮南(Rong-Nan Huang)

審核日期

2007-7-20

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