【作者】 朱自敏；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 苏云金芽胞杆菌华中亚种YBT978产生大分子量晶体蛋白Cry7Ba1,与其它典型晶体蛋白不同的是Cry7Bal需要在强碱性缓冲液中（pH11.5）才能溶解,常规杀虫晶体蛋白在弱碱性缓冲液中就能溶解,并且之前相关研究表明只有溶解后的Cry7Bal才能表现出对小菜蛾的毒力(LC₅₀:1.33μg/mL)。Cry1Ac和Cry1C是Cry1类晶体蛋白,能形成典型的菱形晶体,并且其晶体蛋白在弱碱性条件下（pH9.5）能有效溶解,从而发挥其杀虫活性。本试验为了找出晶体溶解性与蛋白结构的关系,分别用Cry1Ac和Cry1C晶体蛋白的N端和C端替换Cry7Ba1的N端和C端,结果发现替换后的4个重组蛋白都不能形成完好的晶体,只能形成包涵体。当Cry7Ba1的C端被Cry1Ac和Cry1C的C端替换后,重组蛋白0184,0185能像Cry1Ac和Cry1C等其它晶体蛋白一样在弱碱性条件下（pH9.5）有效溶解,而且其毒力没有发生明显变化(LC₅₀分别为:1.32μg/mL,1.39μg/mL);当Cry7Ba1的N端被Cry1Ac和Cry1C的N端替换后,其重组蛋白0181,0183溶解性没有发生明显变化,仍需要在强碱性缓冲液中才能溶解。本研究结果表明Cry7Ba1晶体蛋白的形成与C端结构相关,并且Cry7Ba1晶体蛋白难溶的特性是由其C端结构决定的,其弱碱性下难溶解的性质决定了它的杀虫活性难以发挥。本试验通过改变Cry7Ba1晶体蛋白C端结构来改善该晶体蛋白的溶解性,从而发挥其杀虫活性,为该晶体蛋白的生物防治应用提供了可能。更多还原

【Abstract】 Bacillus thuringiensis subsp.huazhongiensis YBT978 could produce a 130kDa- crystal protein Cry7Bal.Unlike some regular Insecticidal Crystal Proteins, the crystal protein of Bacillus thuringiensis subsp,huazhongiensis YBT978 could be dissolved only at pH values of≥12.5,and the regular Insecticidal Crystal Proteins could be dissolved in alkalescence buffer（pH9.5）.It was proved that the crystal protein Cry7Bal could exhibits toxicity to Plutella xylostella only while it was not dissolved(LC₅₀:1.33μg/mL).CrylAc and CrylC are the representative crystal protein of Cryl produced rhombus crystal,and they could exhibits toxicity while it was dissolved in alkalescence buffer（pH9.5）.In this study,the N-terminal and C-terminal of Cry7Bal were substituted by the N-terminal and C-terminal of CrylC and CrylAc. The 4 recombinants could not form the integrity crystals but only inclusion bodies. The results also indicated that,when the C-terminal of Cry7Ba1 was substituted,the recombinant crystal proteins（0184,0185）could be dissolved in alkalescence buffer （pH9.5） as Cry1Ac and Cry1C,and the toxicity has not be changed(LC₅₀:0184:1.32μg/mL;0185:1.39μg/mL);however,when the N-terminal fragment of Cry7Ba1 was substituted（0181,0183）,it is still insoluble in alkalescence buffer（pH9.5）.These data suggested that the property of insolubility of Cry7Ba1 was associated with the structure of C-terminal.This research could improve the solubility through swapping the C-terminal of Cry7Ba1,and supplied possibility for the crystal protein biocontrol application.更多还原