在苏云金芽胞杆菌中高效和稳定表达AiiA蛋白

High Efficient and Stable Expression of AiiA Protein in Bacillus thuringiensis

【目的】摸索提高AiiA蛋白对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力的方法。【方法】苏云金芽胞杆菌的AiiA蛋白是一种胞内蛋白,能降解参与诱导调控多种植物病原菌致病基因表达的N-乙酰高丝氨酸内酯信号分子。本文采用两种方式来提高AiiA蛋白的活性,即利用苏云金芽胞杆菌S-层蛋白在细胞表面表达该蛋白以及利用苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa启动子来提高aiiA基因的表达量。为此构建该蛋白基因与细胞表面S-层蛋白的锚定区结合而成的融合蛋白基因slh-aiiA以及带有基因cry3Aa启动子的融合基因pro3A-aiiA。为了提高表达的稳定性以及去掉重组菌中非苏云金芽胞杆菌片段,本文构建了解离载体pBMB5401,并将上述两个融合基因单独或同时装入解离载体pBMB5401,分别得到重组质粒pBMBcaiiA,pBMB3aiiA和pBMB3439。转化苏云金芽胞杆菌无晶体突变株BMB171,随后导入温度敏感型辅助质粒pEG922,重组质粒在整合酶的作用下发生体内重组,消除了抗性基因等非必需片段。【结果】得到3个重组菌BMBcaiiAR,BMB3aiiAR和BMB3439R,在无抗性选择压力下的稳定性均在90%以上。融合蛋白SLH-AiiA及pro3A-AiiA在解离后的重组菌中得到表达,具有对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力。【结论】在苏云金芽胞杆菌中高效和稳定表达AiiA蛋白,可增强其对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力,当同时结合两种方式来表达AiiA蛋白时效果最好。

[ Objective ] The aim of this study is to grope associated methods in improving the protein AiiA ability of AHLs inactivation and restraint to the potato soft rot disease caused by Erwinia carotovora. [ Method ] In this study, AiiA was displayed on cell surface by means of S-layer protein and its expression was boosted by promoter of cry3Aa gene, then aiiA gene was fused into XbaI site of ctc gene and Spel site of cry3Aa gene respectively, obtaining fusion genes slh-aiiA and pro3A-aiiA. To improve the stability of AiiA expression, resolution vector pBMB5401 was constructed based on Tn5401. Fusion genes slh-aiiA and pro3A-aiiA were cloned into pBMB5401 by different ways, resulting in pBMBcaiiA, pBMB3aiiA and pBMB3439, which harbor slh-aiiA, pro3A-aiiA and both of them, respectively, and introduced into Bacillus thuringiensis acrystalliferous strain BMB171. The site-specific recombination event occurs within two copies of IRS, thus allows for the selective elimination of undesired fragments on pBMB3439, pBMBcaiiA and pBMB3aiiA. [Result] The expected strains named BMB3439R, BMBcaiiAR and BMB3aiiAR, respectively, and they are stable after 40 generation and reveal ability of AHLs inactivation and effective restraint to the potato soft rot disease caused by E. carotovora. [ Conclusion ] To improve the protein Alia ability of AHLs inactivation and its restraint to the potato soft rot disease caused by E. carotovora, it can be achieved by the method of high efficient and stable expression of AiiA protein in B. thuringiensis.