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研究生:許瓊芳
研究生(外文):Chiung-Fang Hsu
論文名稱:利用DNAShuffling方法探討植物PR5重組變異基因之特性與功能
論文名稱(外文):Characterization and Functional analysis of mutated plant PR5 genes obtained from DNA shuffling method
指導教授:蘇睿智蘇睿智引用關係
指導教授(外文):Ruey-Chih Su
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:87
中文關鍵詞:PR-5蛋白DNA shufflingPR5基因pA13基因
外文關鍵詞:DNA shufflingPathogenesis-related group 5 proteinPR5pA13
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植物PR-5蛋白(Pathogenesis-related group 5 protein, PR-5 protein)包含了thaumatin、osmotin及其相似蛋白。PR-5蛋白在植物受到病原菌的侵襲或環境逆境時會被大量誘導表現,研究顯示此類蛋白具有抗真菌功能,雖然其確切的作用機制仍不清楚,但先前研究指出他們具有抑制真菌菌絲生長並或減少孢子萌發的能力。本研究將馬鈴薯pA13基因與水稻PR5基因等分屬兩種PR-5基因家族的同源基因,利用1994年WILLEM P. C. STEMMER設計的一套活體外分子演化技術-DNA shuffling進行無限可能的基因重組,並探討新重組基因的功能與演化意義。將兩個長度各約0.7 kb的PR5基因進行DNA shuffling之後,獲得了數個長度約為0.7~0.9 kb的重組基因,其中五個重組基因進行定序分析發現,有四個重組基因與馬鈴薯pA13基因的相同度高於86%,使用近鄰歸群法(Neighbor-Joining, NJ)分析其親緣關係發現,1301,51301,13502,51306與馬鈴薯的pA13胺基酸序列相近,而501則與水稻PR5基因相近。將這些重組基因送入大腸桿菌,使重組基因表現蛋白質產物,利用蛋白質電泳及西方墨點法觀察重組基因蛋白質的表現,顯示這些重組基因確實表現預期的蛋白質大小,約在46~48 kDa。將重組蛋白進行粗萃取後,在進行酵母菌Saccharomyces cerevisiae的抑菌生長試驗中發現,由大腸桿菌表現的重組蛋白51306在pH 7.5時可使酵母菌的平均菌落大小由2.0 mm縮小到0.5 mm,相對於pA13蛋白與PR5蛋白只能抑制酵母菌的平均菌落大小在0.8 mm左右,顯示重組蛋白對酵母菌的抑菌效果有增加的趨勢。但在紋枯病菌Rhizoctonia solani的抑菌試驗中,pA13蛋白、PR5蛋白與五種重組蛋白都觀察不到明顯的抑制效果。
Plant pathogenesis-related group 5(PR-5) proteins include thaumatin, osmotin, and their related proteins. The PR-5 proteins are known to accumulate in plant in response to pathogen invasion or environmental stress. Many PR-5 proteins have been shown to possess antifungal activity. Although the specific function of PR-5 proteins in plants is still largely unknown, they are reported to cause the inhibition of hyphal growth and reduction of fungal spore germination . In order to study the antifungal activity of PR-5 proteins, DNA shuffling technique were applied in this study to randomly recombind two homologous PR5 genes, namely, pA13 (Solanum commersonii) and PR5 (Oryza sativa) genes. We have obtained five (1301, 13502, 501, 51306, 51310) shuffled PR-5 proteins expressed in Escherichia coli. The phylogenic tree analysis illustrated that among the obtained shuffled proteins,all but except one 501, are close to pA13 (osmotin-like) protein. The shuffled proteins showed variable growth inhibition to Saccharomyces cerevisiae, and the protein 51306 had the best growth inhibition in pH7.5. However all of the proteins have no inhibitory effect to fungal pathogen Rhizoctonia solani in this study.
目錄………………………………………………………………………… Ⅰ-Ⅲ
表目錄……………………………………………………………………… Ⅳ
圖目錄……………………………………………………………………… Ⅴ-Ⅵ
中文摘要…………………………………………………………………… Ⅶ
英文摘要…………………………………………………………………… Ⅷ
前言………………………………………………………………………… 1-12
材料與方法
一、實驗材料……………………………………………………………. 13
菌種………………………………………………………………… 13
質體………………………………………………………………… 13
二、實驗方法…………………………………………………………… 13
1.大腸桿菌與酵母菌培養條件與保存方式 …………………………… 13
2. PR5與pA13基因之DNA Shuffling………………………………… 14
2.1 PR5與pA13基因的製備………………………………………… 14
2.2以DNaseⅠ水解PR5與pA13基因片段………………………… 15
2.3不加入引子的PCR反應…………………………………………… 15
2.4加入引子的PCR反應……………………………………………… 16
3. DNA電泳分析………………………………………………………… 16
4 . DNA之分離與回收…………………………………………………… 16
4.1 Electro-elutor (Bio-East co.)……………………………… 16
4.2 Viogene 膠體DNA條帶純化套組(Gel Extraction Kit, Viogene co.)………………………………………………………………… 17
4.3 QIAquick PCR Purification ……………………………………… 17
4.4少量純化細菌DNA質體…………………………………………… 17
5.大腸桿菌勝任細胞之製備 …………………………………………… 18
6.大腸桿菌之轉型作用 ……………………………………………….… 18
7.重組 PR5 基因(Shuffled PR5)的表現……………………………… 19
7.1 構築pET 32a-pA13……………………………………………….. 19
7.2構築Shuffled 基因501及1301於pET 32a表現載體…………… 19
7.3構築Shuffled 基因51306及51310與13502於pET 32a表現載體 …………………………………………………………………… 20
7.4 誘導重組基因的表現……………………………………………… 20
7.5大腸桿菌蛋白質濃縮粗萃取……………………………………… 21
8.蛋白質濃度測定(Bradford method)……………………………… 21
9.蛋白質電泳樣品的製備………………………………………………… 21
10.SDS-聚丙烯胺膠體(SDS-polyacrylamide)之製備………………… 21
11.SDS-聚丙烯胺膠體蛋白質電泳分析………………………………… 22
12.西方墨點分析法(Western blot)…………………………………… 22
13.DNA的酵素處理……………………………………………………… 23
13.1 DNA限制酵素的作用…………………………………………… 23
13.2 DNA去磷酸化 …………………………………………………… 23
14.DNA片段與質體的接合反應 ………………………………………… 23
14.1 T4 DNA 接合酵素(T4 DNA ligase)…………………………… 23
14.2 TOPO Cloning Vector System(Invitrogen Co.)……………… 24
14.3 yT&A Cloning Vector System(Yeastern Biotech Co.)………… 24
15.抗真菌活性試驗 ……………………………………………………… 24
15.1重組蛋白抗酵母菌 Saccharomyces cerevisiae活性測試 ……… 24
15.2重組蛋白抗紋枯病菌 Rhizoctonia solani活性測試 …………… 24
結果………………………………………………………………………… 26
1.以DNA shuffling的方式進行pA13基因與PR-5基因之重組…… 26
2.選殖及分析PR-5重組基因………………………………………… 27
2.1 重組基因的比對 ………………………………………………… 27
2.2 親緣關係樹分析 ……………………………………………… 28
3.大腸桿菌表現重組基因蛋白 ………………………………………… 28
3.1重組基因載體的構築…………………………………………… 28
3.2重組蛋白質的表現……………………………………………… 29
3.3西方墨點分析法分析重組蛋白………………………………… 29
4.重組蛋白的抗菌活性分析 ………………………………………… 30
4.1重組蛋白對酵母菌Saccharomyces cerevisiae的抗菌活性分析………………………………………………………………… 30
4.2重組蛋白對稻紋枯病菌Rhizoctonia solani抗菌活性分析…… 31
討論………………………………………………………………………… 32-36
DNA shuffling ……………………………………………………….. 32
重組基因之分析……………………………………………………… 33
大腸桿菌系統重組基因之表現……………………………………… 33
重組蛋白的抗菌活性分析……………………………………………… 34
參考文獻…………………………………………………………………… 37
附錄A……………………………………………………………………… 72
附錄B……………………………………………………………………… 75
附錄C……………………………………………………………………… 77
附錄D……………………………………………………………………… 84
附錄E……………………………………………………………………… 86
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