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研究生:李信興
研究生(外文):Shin-Shing Li
論文名稱:綠豆防禦素VrCRP的突變與生物活性分析
論文名稱(外文):Mutation and biological activity of a mung bean
指導教授:陳慶三陳慶三引用關係
指導教授(外文):Ching-San Chen
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:定點突變植物防禦素
外文關鍵詞:site-directed mutagenesisplant defensin
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VrCRP是由46個胺基酸所組成的植物防禦素,自近等基因品系綠豆Vigna radiata VC 6089A (簡稱VC6089A)的種子中分離而來。我們以VrCRP胺基酸序列和其它具有抗真菌能力的植物防禦素序列比對的結果作為選擇突變點的依據,利用聚合連鎖反應 (PCR)的方式進行VrCRP的定點突變,再用大腸桿菌BL21(DE3) 作為蛋白質表現系統,並對突變種VrCRP進行活性分析。
由序列比對可發現,在這些序列中八個半胱胺酸均屬於保守性胺基酸,此外第35個位置的甘胺酸也為保守性胺基酸,植物防禦素藉由所謂的cysteine-stabilized αβ motif 穩定其結構。這些胺基酸於蛋白質結構穩定中扮演決定性的角色,因此本實驗並沒有改變這些保守性胺基酸。此外,有一些胺基酸殘基在植物防禦素上是保守性胺基酸,不過在VrCRP上卻出現不同胺基酸,這些胺基酸殘基分別為第8位置的離胺酸、第29位置的精胺酸,我們的實驗便是針對這些胺基酸殘基進行突變分析。藉由定點突變我們共得到三個突變株,包括兩個單一突變及一個雙重突變,針對這幾種突變種VrCRP進行生物活性分析。在胞外轉譯作用的抑制作用中,VrCRP/R29E濃度達200 μg/mL時只有65﹪的抑制效果,而VrCRP/Wild type則有80﹪以上。Fusarium oxysporum生長抑制試驗也顯示將第29位置的精胺酸突變成麩胺酸抑制效果會下降,而突變株VrCRP/K8S及VrCRP/K8S/R29E3所表現的蛋白質則會形成不溶性蛋白質沉澱。
VrCRP cDNA that encodes a forty-six-amino-acid plant defensin was isolated in our laboratory from a near isogenic line of mung bean Vigna radiata VC6089A (hereafter VC6089A). Comparison of the deduced amino acid sequence of VrCRP with other plant defensins revealed some unique amino acid residues in VrCRP molecule. In order to investigate if these unique amino acid residues correlate with function of VrCRP, site-directed mutagenesis of these amino acids was carried out using PCR method. The mutant cDNAs were transformed into E. coli BL21(DE3) and expressed.
Plant defensins have 8 conserved cysteines and a conserved glycine at position 35. These defensin molecules are stabilized by so called cysteine-stabilized αβ motif. The conserved amino acids that play a structural role in plant defensins were not the aim of this study. We focused on Lys8 and Arg29 of VrCRP because most of other plant defensins have Ser8 and Glu29. Two single mutants: K8S and R29E and one double mutant K8S/R29E therefore were created and their biological activities studied. The results indicated that VrCRP/R29E at 100 μg/mL inhibited 65﹪in vitro translation, while VrCRP/W showed 80﹪inhibition at the same concentration. Substitution of Arg29 by Glu resulted in less inhibitory effect on the growth of Fusarium oxysporum. However, the expressed proteins from broth VrCRP/K8S and VrCRP/K8S/R29E mutants tend to aggregate and become insoluble inclusion body.
縮寫表......................................................................................................Ⅰ
中文摘要..................................................................................................Ⅱ
英文摘要..................................................................................................Ⅳ
第一章 緒論…………………………………………………………….1
第一節 前言…………………………………………………………1
第二節 抗微生物胜………………………………………………2
第三節 植物防禦素…………………………………………………3
第四節 綠豆抗豆象品系VC6089A的由來與抗豆象因子之探討…4
第五節 VrCRP基因的篩選………………………………………… 5
第六節 大腸桿菌表現VrCRP蛋白質及活性分析…………………5
第七節 VrCRP蛋白質序列及特性分析…………………………… 6
第二章 材料與方法…………………………………………………….8
第一節 VrCRP大腸桿菌表現系統的構築…………………………10
第二節 VrCRP的定點突變…………………………………………15
第三節 蛋白質表現及純化分析…………………………………..19
第四節 VrCRP蛋白質的生物活性分析……………………………25
第三章 結果……………………………………………………………37
第一節 建構VrCRP的大腸桿菌表現系統…………..……………37
第二節 VrCRP定點突變的策略……………………………………37
三節 由E. coli中純化VrCRP蛋白質…………………………..38
第四節 蛋白質的生物活性分析與比較…………………………..40
第四章 討論與未來展望………………………………………………42
第一節 VrCRP蛋白質表現與純化…………………………………42
第二節 對黴菌的生長抑制試驗…………………………………..43
第三節 DNA shuffling與定點突變……………………………… 44
參考文獻………………………………………………………………..46
圖表...........................................................................
參考文獻
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