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研究生:郭雪
研究生(外文):Sheueh Kuo
論文名稱:台灣植物葉表本土蘇力菌之篩選
論文名稱(外文):Isolate indigenous Bacillus thuringiensis from phylloplane in Taiwan
指導教授:李瑞興 博士侯新龍 博士曾經洲 博士
指導教授(外文):Ruey-Shing Lee, Ph. D.Shin-Lon Ho, Ph. D.Ching-Chou Tzeng, Ph. D.
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農學研究所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:108
中文關鍵詞:聚合&;#37238;連鎖反應聚合&;#37238;連鎖反應聚合&;#37238;連鎖反應聚合&;#37238;連鎖反應聚合&;#37238;連鎖反應聚合&;#37238;連鎖反應
外文關鍵詞:TaiwanBacillus thuringiensisisolationphylloplanePCR (polymerase chain reaction)bioassay
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針對台灣台北植物園之木本科植物,進行植物葉表蘇力菌之分離、篩選,共自92.5%之樹種篩獲1,177個菌落,可產生孢子與結晶蛋白。挑選275株,利用四套專一性引子,進行已知基因之PCR,鑑定cry-型基因,只發現cry1及cry2基因。而每一菌株基因組合呈多樣性,一菌株內所含cry1類基因,以cry1Ab, 1Ac, 1D, 1E組合型態最常見,與源自穀倉之本土蘇力菌基因組合相似,有別於源自高山表土主要係cry1Aa, 1Ab, 1Ac基因組合之型態。以CA (Correspondence Analysis)對應分析,分析樹種與蘇力菌cry-型基因,在空間分佈之關係,發現有13種樹與cry1Ab、cry1C、cry1D、cry1E四個基因,呈現有關連性,其他9種樹與cry-型基因,相互間關連性較小。以vip3A基因特異性引子,鑑定源自植物葉表(275株)及穀倉(97株)之本土蘇力菌菌株,結果植物葉表菌株80%含vip3A基因,穀倉菌株72%含vip3A基因。選擇有別於市售商品蘇力菌cry-型基因的15菌株,以SDS-PAGE電泳分析菌株之毒蛋白分子量,並進行對小菜蛾(Plutella xylostella)、擬尺蠖(Trichoplusia ni)、甜菜夜蛾(Spodoptera litura)、斜紋夜蛾(Spodoptera exigua)幼蟲毒性測試,比較毒性測試結果,篩獲一菌株,對小菜蛾及夜蛾科(斜紋夜蛾、甜菜夜蛾) 具良好的防治效果。
Novel variants of Bacillus thuringiensis were isolated from the phylloplane of woody plants in Taipei Botanical Gardens, Taiwan. A total of 1,177 colonies, were obtained from 92.5% phylloplane samples, which produced spores and insecticidal crystal protein. Among them, 275 isolates presented cry1 and cry2 genes when tested by a multiplex PCR with general and specific primers. However, cry-type gene profiles are highly diverse, and the profile of cry1Ab, cry1Ac, cry1D, cry1E is most abundant. This profile is similar to those cry-type gene founded in granaries and different from the cry-type gene profile of cry1Aa, cry1Ab, cry1Ac founded in mountains. The PCR amplification method has been applied to analyze vip3A gene of Bacillus thuringiensis isolates. Chances of getting vip3A gene from phylloplane leaves was 80% but 72% from granaries was found. Fifteen predominant isolates of B. thuringiensis on the phylloplane harbored cry1 gene and were bioassayed against larvae of Plutella xylostella, Trichoplusia ni, Spodoptera litura and Spodoptera exigua. Data were analyzed by Correspondence analysis (CA) to obtain the relationship between phylloplane population and gene spatial distribution. The crystal protein molecular weight and activity were characterized by SDS-PAGE. Among them, two isolates are highly toxic to insects when compared with the Dipel and Xentari. LD50 value of one isolate is close to the LD50 value of Xentari against Plutella xylostella.
目 錄
中文摘要…………………………………………………………I
英文摘要…………………………………………………………II
目錄………………………………………………………………III
表目錄……………………………………………………………IV
圖目錄……………………………………………………………VI
附錄目錄…………………………………………………………VII
壹、前言....……………………………………………………1
貳、前人研究....………………………………………………3
參、材料與方法…………………………………………………16
肆、結果…………………………………………………………28
一、植物葉表蘇力菌的分離篩選………………………………28
二、蘇力菌殺蟲基因鑑定………………………………………28
三、蘇力菌分離株對鱗翅目昆蟲毒性表現測試………………31
伍、討論…………………………………………………………37
一、植物葉表蘇力菌的分離篩選………………………………37
二、蘇力菌殺蟲基因鑑定………………………………………38
三、蘇力菌分離株對鱗翅目昆蟲毒性表現測試………………42
陸、結論…………………………………………………………46
柒、參考文獻……………………………………………………48


表目錄
表一、採集植物葉片以四種熱處理後疑似蘇力菌的篩獲數量…64
表二、各科樹種葉面篩獲疑似蘇力菌之含有結晶菌落數………65
表三、以55℃熱處理5分鐘分離篩選得之植物葉表蘇力菌經四套PCR混合引子對鑑定cry型基因之結果…………………………………66
表四、以65℃熱處理30分鐘分離篩選得之植物葉表蘇力菌經四套PCR混合引子對鑑定cry型基因之結果…………………………………69
表五、以80℃熱處理3分鐘分離篩選得之植物葉表蘇力菌經四套PCR混合引子對鑑定cry型基因之結果……………………………………72
表六、以80℃熱處理30分鐘分離篩選得之植物葉表蘇力菌經四套PCR混合引子對鑑定cry型基因之結果…………………………………74
表七、275株植物葉面蘇力菌各菌株含cry1-型基因數之組合及其比率…………………………………………………………………………77
表八、275株台灣葉表蘇力菌以不同溫度時間熱處理之菌株含cry1型基因之組合株數………………………………………………………78
表九、275株源自植物葉表之蘇力菌PCR鑑定含vip3A基因之結果……………………………………………………………………79
表十、99株源自穀倉之蘇力菌PCR鑑定含vip 3A基因之結果…81
表十一、生物活性供試之植物葉表蘇力菌之cry-型基因組合…82
表十二、植物葉表蘇力菌對3齡初小菜蛾(Plutella xylostella)幼蟲生物活性測試………………………………………………………83
表十三、植物葉表蘇力菌菌株生物活性之多重比較(Multiple comparisons) LSD分析結果…………………………………………84
表十四、植物葉表蘇力菌對小菜蛾幼蟲之生物活性測試…………85
表十五、植物葉表蘇力菌對2齡初擬尺蠖(Trichoplusia ni)幼蟲之生物活性測試………………………………………………………………86
表十六、植物葉面蘇力菌對2齡初斜紋夜盜(Spodoptera litura)幼蟲之生物活性測試…………………………………………………………87
表十七、植物葉表蘇力菌對斜紋夜盜(Spodoptera litura)幼蟲之生物活性測試………………………………………………………………88
表十八、植物葉表蘇力菌對2齡初甜菜夜蛾(Spodoptera exigua)幼蟲之生物活性測試………………………………………………………89
表十九、植物葉表蘇力菌對甜菜夜蛾幼蟲(Spodoptera exigua)幼蟲之生物活性測試………………………………………………………90


圖目錄
圖一、以第一套混合引子,鑑定台灣植物葉表蘇力菌含cry1, 3, 4-型基因之電泳圖…………………………………………91
圖二、以第二套混合引子,鑑定台灣植物葉表蘇力菌含cry1-型基因之電泳圖……………………………………………………92
圖三、以第二套混合引子,鑑定275株植物葉表蘇力菌含 cry1-型基因之數量圖……………………………………………………93
圖四、275株本土分離之蘇力菌中各菌株含cry1-type基因數佔全部分離株之比率…………………………………………………94
圖五、採集自植物葉表蘇力菌之cry1-type gene 組合百分率分佈……………………………………………………………95
圖六、以第三套混合引子,鑑定台灣植物葉表蘇力菌含 cry1-型基因之電泳...........................................96
七、以第四套混合引子,鑑定台灣植物葉表蘇力菌含 cry-型基因之電泳圖………………………………………………………97
圖八、以第三、四套混合引子,鑑定275株植物葉表蘇力菌含 cry-型基因之數量圖………………………………………………98
圖九、各種cry型基因與各樹種之間的對應分析(Correspondance analysis)比較圖…………………………………………………99
圖十、以vip3A引子,鑑定台灣植物葉表蘇力菌含cry3A基因之電泳圖……………………………………………………………100
圖十一、以SDS-PAGE分析蘇力菌之Cry蛋白質圖譜…………101
圖十二、以SDS-PAGE分析蘇力菌之Cry蛋白質圖譜…………102


附錄目錄
附錄一、農委會林業試驗所台北植物園區植物分佈圖…………103
附錄二、PCR增幅cry1, cry3及cry4 基因之引子……………… 104
附錄三、cry1-型基因鑑定之PCR反應引子及預期產物片段…… 105
附錄四、cry1-型基因鑑定之PCR反應引子……………………… 106
附錄五、cry型基因鑑定之PCR反應引子及預期產物片段……… 107
附錄六、蒐集自臺灣穀倉之1024株本土蘇力菌的cry型基因組合分佈圖…………………………………………………………………108

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