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研究生:李豐在
研究生(外文):Feng-Tsai Lee
論文名稱:芽孢桿菌屬細菌幾丁質分解酵素基因表現於螢光假單胞細菌及其抗菌活性
論文名稱(外文):Expression of Bacillus Chitinase in Pseudomonas Species and its Antifungal Activity
指導教授:陳昭瑩陳昭瑩引用關係
指導教授(外文):Chao-Ying Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:72
中文關鍵詞:幾丁質分解酵素芽孢桿菌屬細菌螢光假單胞細菌白絹病菌
外文關鍵詞:chitinaseBacillus circulansfluorescent pseudomonadsSclerotium rolfsii
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幾丁質是由N-acetylglucosamine以-1,4-鍵結而成的聚合物,廣泛地存在於許多生物體中,包括甲殼類生物、真菌、線蟲、昆蟲等,具有保護及支持生物體的功能。多種微生物能產生幾丁質分解酵素,能打斷N-acetylglucosamine間的-1,4-鍵結,使幾丁質分解成寡聚合體或單體。本研究擬篩選自植物根圈中對植物病原真菌具有拮抗性的螢光假單胞細菌,導入已知的B. circulans幾丁質分解酵素基因,希望藉幾丁質分解酵素與抗生物質的協力作用來增加拮抗性螢光假單胞細菌對土壤傳播性植物病原真菌的抗菌能力。為使芽孢桿菌屬細菌之幾丁質分解酵素能自螢光假單胞細菌細胞周質空間中轉移至菌體外側,首先利用聚合酵素連鎖反應,擴增大腸桿菌中ompA基因的分泌訊息及膜轉位序列DNA片段(ST-DNA),將ST-DNA構築於載體pCR2.1上。另外,以聚合酵素連鎖反應法自pNTU111擴增芽孢桿菌屬細菌的chiA基因,將chiA基因黏接於ST-DNA下游,於大腸桿菌TOP10F'轉形菌株所產生的幾丁質分解酵素,初步推測已轉移至細胞外膜。進一步,將ST-chiA DNA片段次選殖於載體pMMB6上,黏接於啟動子tac下游,於Pseudomonas putida轉形菌株產生可轉移至細菌體表的幾丁質分解酵素。自植物根圈分離出36株螢光假單胞細菌,在平板上分別與白絹病菌對峙培養,各菌株表現不同程度的抗生作用。在7個抗生能力較強的菌株中,有3個菌株能降低盆栽中菜豆受到白絹病菌感染的程度。將質體構築轉入其中一菌株FP-16進行盆栽生物防治試驗,結果顯示野生菌株FP-16與轉形菌株FP-16(pMMST2000)均能降低菜豆受到白絹病菌感染的程度,轉形菌株有稍強的防治效果,但與野生菌株FP-16的防治效果在統計上並無顯著差異。

Chitin, a -1,4-linked polymer of N-acetylglucosamine, is widely existed in many organisms, including crustances, fungi, nematodes and insect, and is able to protect and support the body of organisms. Many kinds of organisms produce chitinase to cleave -1,4-linkage between N-acetylglucosamines and hydrolyzes the chitin polymer to release N-acetylglucosamine oligomers or monomer. The goal of this study is to screen fluorescent Pseudomonas antagonists from rhizosphere and construct Bacillus chitinase gene-containing Pseudomonas strains. Based on the synergism of chitinase and antibioticts, the biocontrol activity can be enhanced. In order to make fluorescent Pseudomonas translocate Bacillus chitinase to the bacterial cell surface, firstly, the DNA sequence encoding signal peptide and transmembrane domain of OmpA (ST-DNA) was amplified from the genomic DNA of Escherichia coli and ligated with the vector pCR2.1. A Bacillus chiA-containing DNA was amplified from pNTU111 and placed downstream of ST-DNA. The chitinase translocated to the outer membrane of E. coli TOP10F' was assumed. Then, ST-chiA DNA was ligated with the shuttle vector, pMMB6, in the position downstream of tac promoter. In Pseudomonas putida, chitinase activity was apparently detected in membrane fraction, supporting that ompA ST-DNA could drive Bacillus chitinase to outer membrane. In addition, we have isolated thirty-six strains of fluorescent pseudomonads from plant rhizosphere. In the dual culture with Sclerotium rolfsii, different strains of fluorescent pseudomonads showed different antifungal activities. Among these antagonistic Pseudomonas strains, seven was performed biocontrol assay. Three Pseudomonas strains could decrease the diseased incidence on bean after seed-coating with Pseudomonas antagonist. Then, plasmid pMMST2000 was transferred into one of Pseudomonas strain, FP-16, the biocontrol activity of FP-16(pMMST2000) was higher than wild strain, FP-16, however, the biocontrol activity between wild and genetic engineered strain did not show significant difference.

目 錄
壹、中文摘要……………………………………………………………………..1
貳、英文摘要……………………………………………………………………..2
參、前言…………………………………………………………………………..4
肆、前人研究
一、幾丁質分解酵素……………………….………………………………….6
二、幾丁質分解酵素在植物病害防治上之應用…… ……………………….8
三、Bacillus circulans WL-12幾丁質分解酵素分泌系統及幾丁質分解酵素A1基因之功能……………………………………………………………….9
四、PGPR (plant growth-promoting rhizobacteria)……………………………10
五、幾丁質分解酵素表現在Pseudomonas根圈細菌及其生物防治效果…..11
六、大腸桿菌OmpA (outer membrane protein A)之特性及功能…….12
伍、材料與方法
一、OmpA分泌訊息序列與膜轉位功能區(transmembrane domain) DNA (ST-DNA)之選殖…………………………………………………………..14
1、ompA基因ST-DNA之擴增……………………………………….14
2、ST-DNA之回收…………………………………………………….14
3、ST-DNA與載體DNA之黏接……………………………………..15
4、大腸桿菌勝任細胞之轉形………………………………………….15
5、ST-DNA轉殖株之篩選…………………………………………….16
6、ST-DNA序列之確認……………………………………………….16
二、芽孢桿菌屬幾丁質分解酵素基因之次選殖………………..…………..17
1、幾丁質分解酵素基因chiA之擴增………………………………….17
2、幾丁質分解酵素基因chiA DNA片段之回收………………………17
3、幾丁質分解酵素基因chiA DNA片段與含ST-DNA載體構築之黏接…………………………….....……………………………………18
4、含ST-chiA DNA片段質體之篩選…………………………………..18
三、幾丁質分解酵素之分析…………………………………………………...19
1、幾丁質培養基分析…………………………………………………..19
2、轉形大腸桿菌全量蛋白質之抽取……………………...…………...20
3、SDS聚丙烯醯胺膠體之電泳分析………………………………20
4、膠體中幾丁質分解酵素之偵測………….……………………….20
5、蛋白質定量與螢光基質(4-MU-(GlcNAc)3)分析法…………...21
四、ST-chiA DNA片段之次選殖及表現………………………………….…21
1、載體pMMB6抽取及限制酵素之切割……………………….…….21
2、ST-chiA DNA片段之分離與回收………………………….………21
3、ST-chiA DNA與載體pMMB6之黏接與轉形…………….………22
4、轉形株之篩選及限制酵素之分析………………………….…..22
5、Trypan blue-glycol chitin培養基之分析…………………….…23
6、轉形株全量蛋白質之抽取………………………………………23
7、蛋白質定量與螢光基質(4-MU-(GlcNAc)3)分析法…………...23
五、Pseudomonas putida表現幾丁質分解酵素之分析……………………….23
1、以triparental mating法進行Ps. putida之轉形…………………23
2、Ps. putida轉形株之篩選…………………..……………………..24
3、Ps. putida轉形株之幾丁質分解酵素活性分析………..………24
4、大腸桿菌ST-DNA與B. circulans signal sequence功能之比較……24
六、螢光假單胞細菌幾丁質分解酵素基因之轉形及其抗菌效果分析..25
1、螢光假單胞細菌之分離…….………………………………….25
2、螢光假單胞細菌分離株之抗菌能力分析…………….………26
3、盆栽中螢光假單胞細菌抗S. rolfsii能力之分析……………26
4、拮抗性螢光假單胞細菌菌株在土壤中之纏聚能力……...27
5、拮抗性螢光假單胞細菌菌株之轉形…………………….27
6、拮抗性螢光假單胞細菌轉形菌株幾丁質分解酵素之表現分析……………………………………………….…...……….27
7、拮抗性螢光假單胞細菌轉形菌株之盆栽防治試驗…………28
陸、結果………………………………………………………………………….29
一、ompA基因ST-DNA之選殖……………………………………………..29
二、幾丁質分解酵素基因chiA之次選殖……………………………….…30
三、ST-chiA1200及ST-chiA2000轉形菌株之幾丁質分解酵素活性.…30
四、ST-chiA1200及ST-chiA2000之次選殖及幾丁質分解酵素活性……...31
五、ST-chiA DNA轉入Ps. putida與轉形菌株幾丁質分解酵素之表現分析………………………………………..……………………….…..32
六、幾丁質分解酵素基因導入拮抗性螢光假單胞細菌及其抗菌活性
…………………………………………………………………………34
柒、討論…………………………………………………………….…………….36
捌、引用文獻……………………………………………………………………..39
玖、圖表集………………………………………………………………………..49
表一、PCR引子
表二、以螢光分析法測定大腸桿菌及Pseudomonas putida轉形菌株之幾丁質分解酵素的活性
表三、以螢光分析法測定Pseudomonas putida轉形菌株細胞次級分佈幾丁質分解酵素的活性
表四、螢光假單胞細菌之抗菌能力分析
表五、螢光假單胞細菌分離株對Sclerotium rolfsii之生物防治效果分析
表六、野生型螢光假單胞細菌與轉形菌株對Sclerotium rolfsii之生物防治效果
圖一、質體pMMST2000之構築流程
圖二、擴增自大腸桿菌ompA基因之ST-DNA PCR產物
圖三、芽孢桿菌屬細菌幾丁質分解酵素基因之PCR產物
圖四、以質體快速檢定法篩選含ompA基因5'端序列及chiA催化功能區基因之選殖株
圖五、以質體快速檢定法篩選含ompA基因5'端序列及chiA催化功能區基因之選殖株
圖六、質體pST1200與pST2000之限制酵素切割分析
圖七、以trypan blue-glycol chitin平板培養基測定E. coli TOP10F'轉形菌株之幾丁質分解酵素活性
圖八、E. coli TOP10F'轉形菌株於幾丁質培養基上之生長
圖九、以含glycol chitin之膠體進行SDS-聚丙烯醯胺膠體電泳分析及活性染色偵測E. coli TOP10F'轉形菌株之幾丁質分解酵素活性
圖十、以PCR篩選含pMMST2000之E. coli TOP10F'轉形菌株
圖十一、pMMST1200與pMMST2000之限制酵素分析
圖十二、質體構築pMMST2000圖譜
圖十三、以trypan blue-glycol chitin平板培養基測定E. coli TOP10F'轉形菌株之幾丁質分解酵素活性
圖十四、以trypan blue-glycol chitin平板培養基測定Pseudomonas putida轉形菌株之幾丁質分解酵素活性
圖十五、pNTU118及pMMST2000插入DNA之遺傳結構
圖十六、拮抗性螢光假單胞細菌FP-16在土壤中的殘存能力分析
圖十七、E. coli TOP10F'及拮抗性螢光假單胞細菌轉形菌株於幾丁質培養基上之生長

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