不同来源的苏云金芽孢杆菌aiiA基因表达及其抗病性分析

Expression and Antimicrobiotic Activity of aiiA Gene from Different Strains of Bacillus thuringiensis

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)aiiA基因所编码的AiiA蛋白是一种内酯酶,可以降解N-乙酰高丝氨酸内酯(N-acyl-homoserine lactones,AHLs),从而减弱致病菌产生的危害.本研究克隆了6株不同来源的苏云金芽孢杆菌aiiA基因,并对来自于苔藓(LLB15)和土壤(LLS9)的BtaiiA基因进行了生物信息学分析.结果表明,AiiA-B15分子量为27.97×103,等电点为4.59;AiiA-S9分子量为28.14×103,等电点为4.32,两者都是亲水性蛋白.AiiA蛋白具有很高的保守性,AiiA-B15和AiiA-S9同源性为90%.进化树结果表明,AiiA-B15与Bacillus thuringiensis subsp.kyushuensis进化距离最近,AiiA-S9与Bacillus cereus进化距离最近.将aiiA-B15基因克隆到pGEX-4T-3表达载体中,构建重组质粒pGEXaiiA-B15,IPTG诱导后可大量表达融合蛋白.对表达的条件进行优化.结果表明,0.6mmol/LIPTG,30℃下诱导3h为最佳诱导条件.致病性检测表明,该融合蛋白对胡萝卜欧文氏软腐病菌具有较强的抗病作用.

The enzyme encoded by aiiA gene was involved in the degradation of N-acyl-homoserine lactones （AHLs）, decreasing the virulence of bacterial pathogens. After the aliA genes from 6 different strains of Bacillus thuringiensis were cloned, the aliA genes from bryopsida （LLB15） and soil （LLS9） were analyzed. The deduced isoelectric point of AiiA-B15 was about 4.59 with a theoretical molecular weight of 27.97× 10^3 , while the deduced isoelectric point of AiiA-S9 was about 4. 32 with a theoretical molecular weight of 28.14 × 10^3. They were both hydrophilic proteins. AiiA proteins were highly conserved with the homology of 90 % between AiiA-B15 and AiiA-S9. Phylogenetic analysis showed AiiA-B15 was closest to the AiiA from B. thuringiensis subsp, kyushuensis, while AiiA-S9 was closest to those of B. cereus. The aliA-B15 genes from B. thuringiensis isolated from bryopsida were subcloned and inserted into the expression vector pGEX-4T-3. The recombinant plasmid pGEXaiiA-B15 was induced by IPTG. Large amounts of fusion protein were obtained by optimizing the expression conditions. The highest expression level of fusion protein was obtained by adding 0. 6 mmol/L IPTG and inducing for 3 hours at 30 ℃. The fusion protein greatly alleviated the pathogenesis of Eriwinia carotovora causing soft rot for carota. Fig 13, Tab 4, Ref 23