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研究生:王堂凱
研究生(外文):Tang-Kai Wang
論文名稱:幾丁質分解性桿菌屬細菌拮抗灰黴病菌活性之分析及幾丁質分解酵素基因之選殖
論文名稱(外文):Analysis of Antagonistic Activity on Botrytis spp. and Cloning of Chitinase Genes of Chitinolytic Bacillus spp.
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:2000
畢業學年度:88
語文別:中文
論文頁數:70
中文關鍵詞:分子選殖生物防治百合灰黴病菌桿菌屬細菌幾丁質分解酵素幾丁質分解酵素基因
外文關鍵詞:molecular cloningBacillus sp.biocontrollilyBotrytis ellipticaBotrytis cinereachitinasechitinase gene
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幾丁質(chitin)普遍地存在於自然環境中,許多植物病原真菌的細胞壁即含有幾丁質成份,故在植物病害防治上有許多利用幾丁質分解酵素(chitinase)的策略。本研究測試二株可以分解幾丁質的桿菌屬細菌Bacillus spp.菌株28-1及28-9對真菌病害的防治效果,發現二菌株對灰黴病菌(Botrytis elliptica及B. cinerea)的生長有很好的抑制效果。在百合切葉圓盤上測試二菌株對百合灰黴病病徵發展的影響,結果顯示在接種灰黴病菌時同時處理菌株28-1或28-9,均可降低灰黴病的發病程度。當菌株28-1或28-9的處理提前到接種灰黴病菌前一至三天時,可表現更好的防治效果。
以glycol chitin-SDS-聚丙烯醯胺膠體電泳分析菌株28-1及28-9的分泌性蛋白質,均可在分子量約為 45 kDa的位置呈現幾丁質分解酵素的活性。以不同螢光基質(4-MU-(GlcNAc)n)分析菌株28-1及28-9幾丁質分解酵素的作用方式,顯示二菌株可產生外切型幾丁質分解酵素,其最適作用溫度為37℃,最適作用酸鹼值為pH 4。
進一步以三種不同策略進行菌株28-1及28-9幾丁質分解酵素基因的選殖,在以質體pBluescript KS(+)建構的菌株28-1基因庫中篩選到一個可在幾丁質培養基上造成透明圈的選殖株,但此選殖株的幾丁質分解能力極不穩定。其次在以  ZAPII 載體系統建構的菌株28-9基因庫中,篩選到四個可能帶有幾丁質分解酵素基因的重組噬菌體,可在幾丁質培養基上造成較透明的溶菌斑。此外藉由引子設計及聚合酵素連鎖反應,已由菌株28-9的基因組DNA中擴增一段約400 bp的DNA片段,其核酸與氨基酸序列與許多幾丁質分解酵素的序列相似,與 Metarhizium anisopliae 的幾丁質分解酵素相似度高達98%。
Chitin, common in nature, is one of the major cell wall components in several pathogenic fungi. The application of chitinase has gained success in many cases of plant disease control. In this study, two chitinase-producing Bacillus spp., coded 28-1 and 28-9, were tested for their activity in the control of fungal disease. Both of bacteria showed inhibitory activity on the growth of Botrytis elliptica and B. cinerea. When the Bacillus spp. 28-1 and 28-9 were applied on the lily leaf discs at the time of inoculation of B. elliptica, a decrease in disease severity was observed. If Bacillus spp. 28-1 and 28-9 were applied before the inoculation of B. elliptica, more extents of decrease in disease severity were gained.
Analysis of proteins in the cultural supernatants of Bacillus spp. 28-1 and 28-9, a protein band with chitinase activity was observed in both bacteria at the size of about 45 kDa. The chitinases of Bacillus spp. 28-1 and 28-9 were identified as exo-type in the fluorescence assay with different substrates of 4-Mu-(GlcNac)n, showed highest activity at 37℃ and at the pH value of 4.
Three cloning methods were used to clone the chitinase genes from Bacillus spp. 28-1 and 28-9. Firstly, a genomic library of Bacillus sp. 28-1 was constructed in the plasmid vector, pBluescript KS(+), and screened a clone unstable forming clear zone on chitin medium. Secondly, a genomic library of Bacillus sp. 28-9 was constructed in the  ZAPII vector, and screened six putative chitinase gene-containing clones that forming clearer plaques on chitin medium. A further examination is proceeding. Finally, via primer design and polymerase chain reaction, a 400-bp DNA fragment was amplified from Bacillus sp. 28-9. The sequence of this DNA fragment showed high level of similarity to that of many chitinase genes,and showed 98% homology with that of a chitinsase gene of Metarhizium anisopliae.
壹、中文摘要………………………………………………………………….1
貳、英文摘要………………………………………………………………….2
參、前言……………………………………………………………………….4
肆、前人研究
一、微生物之幾丁質分解酵素及其特性………………..….………………6
二、微生物幾丁質分解酵素基因之選殖…………………………………….7
三、微生物幾丁質分解酵素在植物病害防治之應用……………………….9
四、幾丁質分解酵素之抑菌機制………………………………………....11
伍、材料與方法
一、供試菌株及質體……………………………………………………....13
二、桿菌屬細菌拮抗灰黴病菌之分析…………………………………....13
三、幾丁質分解酵素特性之分析………………………………………....15
1.基本步驟………………………………………..………………………..16
2.不同培養時間對桿菌屬細菌表現幾丁質分解酵素之影響試驗…..…..18
3.反應溫度及pH值對桿菌屬細菌幾丁質分解酵素活性之影響試驗......18
四、桿菌屬細菌基因組 DNA之抽取…………………………………………19
五、桿菌屬細菌基因組DNA之限制酵素不完全切割及DNA片段回收…....21
六、桿菌屬細菌基因庫之建構
(方法一)以質體系統建構基因庫……………………………………....22
(方法二)以λZAPII 載體系統建構基因庫…………………………....24
七、以聚合酵素連鎖反應釣取桿菌屬細菌之幾丁質分解酵素基因片段…26
八、選殖DNA之南方雜合分析……………………………………………….29
陸、結果
一、菌株28-1及28-9拮抗植物灰黴病菌之特性……………………………31
二、菌株28-1及28-9之幾丁質分解酵素特性………………………………32
三、菌株28-1及28-9幾丁質分解酵素基因之選殖…………………………34
柒、討論…………………………………………………………….……….36
捌、參考文獻…………………………………………………………………40
玖、圖表集……………………………………………………………………45
表一、供試菌株及質體
表二、依據胺基酸保守性序列分群之幾丁質分解酵素類似基因
表三、擴增幾丁質分解酵素基因之引子對序列
圖一、培養基上桿菌屬細菌抗菌試驗設計圖
圖二、桿菌屬細菌對百合灰黴病菌生長之抑制作用
圖三、桿菌屬細菌對灰黴病菌生長之抑制作用
圖四、百合灰黴病菌在百合切葉圓盤上造成之病徵等級
圖五、同時處理桿菌屬細菌對灰黴病菌感染百合葉片之影響
圖六、前或後處理桿菌屬細菌對灰黴病菌感染百合葉片之影響
圖七、桿菌屬細菌菌株幾丁質分解酵素之glycol chitin-SDS-聚丙烯醯胺膠體電泳
圖八、不同螢光基質下,桿菌屬細菌菌株胞外幾丁質分解酵素之活性
圖九、桿菌屬細菌菌株在不同培養時間之胞外幾丁質分解酵素活性
圖十、不同反應溫度下,桿菌屬細菌菌株胞外幾丁質分解酵素之活性
圖十一、不同pH值下,桿菌屬細菌菌株胞外幾丁質分解酵素之活性
圖十二、含有幾丁質分解酵素基因重組噬菌體之二次篩選
圖十三、可能含有菌株28-9幾丁質分解酵素基因 噬菌體之進一步篩選
圖十四、桿菌屬細菌菌株幾丁質分解酵素基因片段之擴增
圖十五、桿菌屬細菌菌株28-9之部份核酸序列與Metarhizium anisopliae幾丁質分解酵素基因部份核酸序列比對
圖十六、第四群幾丁質分解酵素胺基酸序列及Metarhizium anisopliae幾丁質分解酵素胺基酸序列比對
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