一株高产杀虫蛋白苏云金杆菌的生物学特性研究以及其aiiA基因的克隆和生物信息学分析

Biological Characteristics of Bacillus thuringiensis Strain BRC-PC6 and Cloning, Bioinformatics Analysis of its aiiA Gene

在细菌群体密度(quorum-sensing,简称QS)系统中由苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)的aiiA基因所编码的胞内解酯酶AiiA蛋白对AHLs信号分子N-酰基高丝氨酸内酯(N-acyl-homoserine lactones)的浓度响应,从而决定了许多动、植物病原菌致病基因的表达。本研究由生物农药厂周边环境中分离得到一批Bt菌株,其中有1株BRC-PC6在发酵过程中可产生大量杀虫晶体蛋白。以库斯塔克亚种HD-1为对照,对该菌株生理生化反应、杀虫基因、蛋白质图谱等生物学特性进行了研究,并对其aiiA基因进行了检测、克隆和比对测序,以期更好地研究aiiA基因在不同来源的苏云金芽胞杆菌中的分布,并对其进行分子改造。结果表明,该菌株含有cry1Aa、cry1Ac等活性基因,主要对鳞翅目幼虫具有较高毒力。所克隆的PC6-aiiA序列全长均为753bp,目前已知的aiiA基因与其同源性较高,达88%～97%。生物信息学分析表明,该基因编码蛋白为不稳定疏水蛋白,含有1个结构域和信号肽序列。以已知结构的AiiA蛋白为模板,模拟构建了PC6-AiiA的三级结构,二者结构高度相似,这些结果为aiiA基因的深入研究提供了参考数据。

In the QS system, the AiiA enzyme can decide the expression of pathogenic genes in several plant and human pathogenic bacteria, which is encoded by alia gene from Bacillus thuringiensis, involving in the degradation of N-acyl-homoserine lactones （AHLs）. BRC-PC6 was isolated from the samples collected from the surroundings of a pesticide factory, and it can produce several insecticidal crystal proteins （ICPs） during the fermentation. Comparing with Bt subsp, kurstaki （HD-1）, strain BRC-PC6 was characterized with biochemical characteristics, ICPs gene, ICPs, etc. The PC6-aiiA gene from the strain BRC-PC6 was identified, cloned and sequenced in this study. It was shown that crylAa and crylAc were detected in this novel strain. Basing on the bioassay, BRC-PC6 was toxic to the larva of Lepidoptera. Sequencing analysis was shown that the gene of aiiA was 753 bp, and it was 88% - 97% homology with other known aiiA genes. For further research of this novel aiiA gene, bioinformatic analysis was carried ont. An instability hydrophobie protein with one structural domain was predicted for Bt BRC- PC6 strain predicted, and AiiA had the signal predicted peptide.