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研究生:李彥鋒
研究生(外文):Yan-Fong Li
論文名稱:重組蛋白AiiA對蝴蝶蘭細菌性病原之抑菌效果分析
論文名稱(外文):The inhibition effect of recombinant protein AiiA on phytobacteria isolated from phalaenopsis
指導教授:朱木貴
指導教授(外文):Mu-Kuei Chu
口試委員:陳瑞祥蔡志濃
口試委員(外文):Ruey-Shyang ChenJyh-Nong Tsai
口試日期:2011-07-13
學位類別:碩士
校院名稱:中華醫事科技大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:85
中文關鍵詞:蝴蝶蘭AHL 內酯酶
外文關鍵詞:AiiAphalaenopsisAHL-lactonase
相關次數:
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近年來蝴蝶蘭的經濟栽培面積大幅擴增,目前已成為台灣主要外銷花卉。由於溫室栽培之環境高溫多濕,若種植過程管理不當,經常會助長蝴蝶蘭病害之發生與蔓延,造成蘭園重大之損失。病害之發生通常都為蝴蝶蘭生產的限制因子,本研究將就細菌性病害之防治進行探討,期能提供解決細菌性病害之可行性方案。本研究利用由蘇力菌 (Bacillus thuringiensis)中選殖出AHL內酯酶基因,測試其抑制或破壞病原細菌群體密度感應系統的效果,分析此蛋白是否可降低植物病原細菌的致病性,並達到防治蝴蝶蘭細菌性病害之效果。研究中將可表現AHL內酯酶基因選殖菌株之全蛋白萃取液分別對不同病原進行抑菌分析,結果發現此酵素對蝴蝶蘭的3種細菌性病害皆有抑制效果。進一步測試長期保存於不同溫度下之全蛋白萃取液之抑菌效果,結果顯示於4℃之存放效果最佳,在長期保存後仍可對褐斑病、葉班病及軟腐病菌等病原細菌具有抑制效果。本研究結果亦顯示AHL內酯酶確實可於pQE表現系統中產生,且具抑菌效果,但進行AHL內酯酶蛋白質純化後,卻發現純化後之蛋白不具抑菌之效果。AHL內酯酶在本研究之分析中具有抑制蝴蝶蘭細菌性病原菌之效果,爾後將進一步開發可大量純化出具有抑制效果之AHL內酯酶之方法,未來對於蝴蝶蘭細菌性病害之防治將有極大之助益。而如何快速且正確的鑑別感染原,係病害管理上另一個重要的課題。台灣蝴蝶蘭細菌性病害之主要病原有三種,分別為 Acidovorax avenae subsp. Cattleyae (AAC)、Burkholderia gladioli (BG)及Pectobacterium chrysanthemi (PCH),蝴蝶蘭細菌性病害之檢測可利用不同菌株間Internal transcribe spacer的特異性序列設計專一性引子組,再進行AAC、BG及PCH之單引子組及多引子組PCR鑑定,但於實際田間檢測時,存在於植株及栽培環境中的抑制因子常影響PCR檢測之敏感度及正確性。本研究利用過濾及磁顆粒分離技術,成功去除大部分抑制因子,確實提昇單引子組及多引子組PCR檢測之正確性;另於PCR敏感度測定結果得知,對AAC、BG及PCH分別可達104 CFU/mL、102 CFU/mL及104 CFU/mL,此結果優於ELISA之敏感度檢測,未來將可應用此技術於田間病害之檢測。
Phalaenopsis became the most important product in the industry of flowers for export, as the cultivation quantity of orchid increased rapidly. The high desnisty cultivation of orchids in the greenhouse caused the increasing of the temperature and humidity and this environment would cause the occurrence of plant diseases, which caused enormous losses of orchid growers. Acidovorax avenae subsp. cattleyae (AAC), Burkholderia gladioli (BG) and Pectobacterium chrysanthemi (PCH) caused infectious diseases of Phalaenopsis in Taiwan. The additional issue of Disease Management was the prevention of phytobacterial diseases. First of all, we cloned the AHL-lactonase gene from Bacillus thuringiensis. Then we constructed lactonase gene to pQE expression system to express lactonase in the E. coli (pQE-aiiA). The extraction of the total protein from E. coli (pQE-aiiA) would be used to test its inhibition-ability to phytobacteria on the media. The results of this antibacterial analysis revealed the total protein could inhibit the growth of three kinds of phytobacteria. In the further testing, the total protein would be test by the same method above to understand the inhibition-ability after long-term storage at different temperatures. The results shown the activity of total protein extracts which stored at 4 ℃ would be better than other temperatures. In order to understand more about lactonase, we try to purify lactonase with his-tag column from total protein. Unfortunately, the purified proteins couldn’t emerge the inhibition-ability. This study would need to modify the methods of protein purification to extract the active lactonase. Then, we would have more powerful opportunity to apply lactonase on the management of field in the future. For detecting these pathogens, we used an in-house database of bacterial sequences, which were designed primers based on the results of multiple sequence alignments. We could utilize these specific primer-pairs to detect different kinds of phytobacteria from Orchids by PCR reactions. In the field of orchids, the PCR reactions were influenced by some limited factors which existed in the cultural environment of field. The factors could decrease the specificity and sensitivity of uniplex and multiplex PCR reactions. In order to resolve this problem, we used filtration and magnetic separation to avoid these limited factors. The testing results revealed these treatments could increase the sensitivity and specificity. The sensitivity of above-mentioned technique was better than ELISA technique. In addition, the detection of each PCR reaction cost about NT $ 5 dollars, would enhance this technology to be apply in the fields.
口試委員審定書
授權書...................................................Ⅱ
中文摘要.................................................Ⅲ
英文摘要.................................................Ⅴ
致謝.....................................................Ⅶ
壹、 前人研究.............................................1
一、蝴蝶蘭簡介..........................................1
二、蝴蝶蘭產業發展狀況..................................1
三、蝴蝶蘭作物細菌性病害之種類..........................2
(一). 蝴蝶蘭細菌性褐斑病..........................3
(二). 蝴蝶蘭細菌性葉斑病..........................4
(三). 蝴蝶蘭細菌性軟腐病..........................4
四、細菌性病害之防治....................................6
(一).蘭花細菌性病害之防治現況.....................6
(二).群體密度感應系統.............................7
(三).群體密度感應系統之訊號分子...................8
1. AHL內酯酶(AHL-lactonase).................8
2. AHL內酯醯化酶(AHL-acylase)...............9
(四). 群體密度感應系統之作用機制............... ...9
五、細菌性病害之檢測...................................10
(一).鑑別型培養基................................11
(二).酵素連結抗體檢定法..........................11
(三).磁顆粒分離法................................12
(四).微生物之PCR檢測............................12 (五).多引子組PCR................................13
六、研究目標...........................................14
貳、 材料與方法..........................................16
一、材料.............................................16
(一).植物材料...................................16
(二).菌株.......................................16
(三).培養基.....................................16
(四).奈米磁顆粒.................................17
(五).質體.......................................17
二、 方法.............................................17
(一).質體製備...................................17
(二).大腸桿菌勝任細胞之製備.....................18
(三).大腸桿菌勝任細胞之轉形作用.................18
(四).質體構築...................................18
(五).質體之定序分析.............................19
(六).生物資訊分析...............................19
(七).基因之蛋白質表現及純化.....................19
(八).蛋白質表現之分析...........................20
1. SDS-PAGE分析...........................20
2.膠體染色.................................20
3.西方墨點法(Western Blotting)................21
(九).濾紙圓盤擴散法(paper disc diffusion method) ....21
(十).初步過濾...................................22
(十一).植株材料中干擾PCR反應之分析.............22
(十二).磁顆粒分離技術...........................22
(十三).聚合酵素鏈鎖反應.........................23
(十四). 人工接種後發病之磁顆粒Uniplex PCR檢測分析..............................................23
(十五). 人工接種後發病材料之磁顆粒Multiplex PCR檢測分析............................................24
(十六).PCR產物之膠體電泳分析...................24
參、 結果................................................26
一、AHL內酯酶(aiiA)基因之序列分析...................26
二、利用表現載體來分析及純化AHL lactonase(aiiA)基因之表現..................................................27
三、aiiA 蛋白質之表現及純化..........................27
四、pQE30-aiiA之抑菌效果分析.........................28
五、pQE30-aiiA萃取液進行三種保存時間之抑菌效果.......29
六、檢測.............................................30
(一). 過濾處理與磁顆粒分離對檢測褐斑病菌之PCR敏感度之分析........................................30
(二). 過濾處理與磁顆粒分離對檢測葉斑病菌之PCR敏感度之分析........................................30
(三). 過濾處理與磁顆粒分離對檢測軟腐病菌之PCR敏感度之分析........................................31
七、人工接種蘭花之磁顆粒Uniplex PCR檢測分析.........32
八、人工接種蘭花之磁顆粒Multiplex PCR檢測分析........33
肆、 討論................................................34
伍、 參考文獻............................................37
陸、 圖表................................................53
表1 本研究所用褐斑病菌、葉斑病菌及軟腐病菌之菌株表......54
表2 本研究所使用之引子..................................55
表3 25℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表.....................................56
表4 4℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表.......................................57
表5 負20℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表...................................58
圖1 蘇力菌之AHL內酯酶基因之核苷酸序列及選殖本基因之引子組序列...................................................59
圖2  AHL內酯酶胺基酸(aiiA)之多重序列比對結果............60
圖3  pQE30-aiiA之構築示意圖.............................61
圖4  選殖株pQE30-aiiA之基因片段分析....................62
圖5 pQE30-aiiA蛋白質之表現分析..........................63
圖6 選殖株之蛋白在pQE30/M15表現系統中之Western blotting 分析.......................................................64
圖7 選殖株pQE30-aiiA可溶及不可溶蛋白萃取物之抑菌效果分析.......................................................65
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對褐斑病菌之抑菌效果分析(A) ......................66
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對葉斑病菌之抑菌效果分析(B) .....................67
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對軟腐病菌之抑菌效果分析(C) .....................68
圖9 過濾處理與磁顆粒分離對檢測褐斑病菌之PCR敏感度之分析.......................................................69
圖10 過濾處理與磁顆粒分離對檢測葉斑病菌之PCR敏感度之分析.......................................................70
圖11 過濾處理與磁顆粒分離對檢測軟腐病菌之PCR敏感度之分析.......................................................71
圖12 人工接種後發病材料之磁顆粒Uniplex PCR檢測分析.....72
圖13 人工接種後發病材料之磁顆粒Multiplex PCR檢測分析....73

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