苏云金芽胞杆菌CT-43内生质粒pBMB0558上virD4基因的功能  

Function of virD4 gene in plasmid pBMB0558 in Bacillus thuringiensis CT-43

作　　者：陈慧杰[1] 刘晓艳[1] 曹诗云 阮丽芳[1] 孙明[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2011年第4期404-410,共7页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30970037)

摘　　要：苏云金芽胞杆菌CT-43高产苏云金素,其内生质粒pBMB0558上virD4基因编码的VirD4蛋白与Ⅳ型分泌系统中结合底物的偶合蛋白(T4CPs)高度同源。本研究利用插入缺失突变方法,构建同源双交换载体,敲除CT-43中的virD4基因,得到1株突变株BMB1122。HPLC检测结果显示,突变株BMB1122发酵上清液中未检测到苏云金素特征吸收峰;进一步通过LCMS-IT-TOF飞行质谱检测突变株胞内代谢物,结果显示其含有与苏云金素合成途径中的所有前体物分子质量相符的质荷比(m/z),这与出发菌株CT-43的结果一致。初步推测,virD4基因与苏云金素的分泌相关,virD4基因的敲除影响苏云金素的分泌。Bacillus thuringiensis CT-43 produces thuringiensin.The VirD4 protein encoded by the virD4 gene located on the plasmid pBMB0558 of CT-43 is highly homologous to the coupling proteins（T4CPs） of type IV secretion systems.In type IV secretion systems,T4CPs＇ basic function is to recruit substrates to the type IV secretion systems for secretion through the translocation channel.A homologous double-crossover vector was constructed through knocking out the virD4 gene in CT-43 and a mutant BMB1122 was obtained.HPLC result showed that the characteristic peak of thuringiensin was not detected in the fermentation supernatant of mutant BMB1122.The LCMS-IT-TOF result showed that the corresponding m/z with molecular weight of proposed precursors in Thu biosynthesis could be detected in intracellular metabolites of mutant BMB1122,consistent with that of CT-43.The knockout of virD4 gene interrupted the secretion of thuringiensin.Presumably,the virD4 was a critical gene for Thu biosynthesis.

关 键 词：Ⅳ型分泌系统 基因敲除 高效液相色谱 苏云金素 

分 类 号：Q754[生物学—分子生物学]