苏云金芽胞杆菌XL6^-候选生物被膜调控基因00940序列分析及基因敲除突变体构建  

Sequence Analysis of A Putative Biofilm Gene 00940 from Bacillus thuringiensis XL6^- and Construction of Its Gene Knockout Mutant

作　　者：姚俊敏 张韶芮 金鑫 凡肖 束长龙[3] 李清禄[1] 黄天培[1,2] 关雄 YAO Jun-Min;ZHANG Shao-Rui;JIN Xin;FAN Xiao;SHU Chang-Long;LI Qing-Lu;HUANG Tian-Pei;GUAN Xiong(State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops/Key Laboratory of Biopesticide and Chemical Biology（Ministry of Education）,College of Life Sciences and College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian-Taiwan Joint Center for Ecological Control of Crop Pests,Fuzhou 350002,China;State Key Laboratory of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]福建农林大学生命科学学院/植物保护学院/闽台作物有害生物生态防控国家重点实验室/生物农药与化学生物学教育部重点实验室,福州350002 [2]闽台特色作物病虫生态防控协同创新中心,福州350002 [3]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100093

出　　处：《农业生物技术学报》2018年第8期1401-1409,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(No.2017YFD0200400);国家自然科学基金(No.31672084和No.31201574);高校领军人才项目(No.k8012012a);福建省出国留学基金(资助黄天培);福建农林大学2017年度(第二批)科技创新专项基金(No.CXZX2017266和No.CXZX2017214)

摘　　要：细菌生物被膜(bacterial biofilm,BBF)可以提高细菌的紫外线抗逆性,调控细菌对环境胁迫的适应能力。本研究以课题组前期比较基因组工作中筛选出的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)XL6-(Gen Bank No.CP013000.1)的调控生物被膜形成的候选基因00940为基础,分析其基因功能,并构建基因敲除突变株。通过生物信息学分析发现00940基因可能编码谷氨酰胺合成酶,并受到转录因子Sig L、CcpA、Deg U和Lex A的调控。在枯草芽胞杆菌(Bacillus subtilis)中,Sig L是一种增强子,位于枯草芽孢杆菌的谷氨酸脱氢酶基因的下游,负责转录编码谷氨酸脱氢酶的roc G基因;CcpA转录因子参与代谢产物的分解;Deg U控制鞭毛形成和生物被膜形成的基因表达;Lex A蛋白在DNA损伤的情况下被诱导,是细菌SOS DNA修复系统的转录抑制因子,推测这些转录因子在Bt中也发挥相似的作用。通过PCR获得00940基因上、下游同源臂和卡那霉素(kan)抗性基因,利用重叠PCR获得完整的基因敲除片段。根据酶切位点将基因敲除片段和p MAD温敏载体进行重组反应,得到重组质粒。将重组质粒电击转化Bt XL6-,筛选获得了00940基因敲除突变株。以Bt Xl6-作为对照,对Bt Xl6-00940基因突变株进行表型分析,包括生长曲线测定、群游能力测定以及生物被膜形成能力测定。结果表明,00940基因的敲除对菌株的生长趋势没有影响,但是群游能力和生物被膜形成能力提高,初步确定00940基因的敲除提高Bt Xl6-菌株的生物被膜形成能力。基因敲除突变株的获得为进一步分析相关调控基因的功能提供科学依据和基础资料。Bacillus thuringiensis （Bt） has become one of the most widely uses and most successful microbial insecticides in the world because of its insecticidal effect, specificity to the target organism, harmlessness to humans and animals, non-polluting environment, no harm to the natural enemies of pests, and high efficiency. However, there is a serious problem that Bt and its ICPs sprayed on crops have a short survival period under sunlight, which seriously hinders the further utilization of Bt. Therefore, in order to enhance the insecticidal effect of Bt, it is a key measure to prolong the persistence time of Bt. Bacterial biofilm （BBF） can improve the UV resistance of bacteria and regulate the adaptability of bacteria to environmental stress. Based on a previous comparative genomic analysis of our lab, a putative biofilm gene 00940 from Bt XL6-was identified （GenBank No. CP013000.1）. According to bioinformatics analysis, 00940 gene is likely to be a glutamine synthase, which is regulated by SigL, CcpA, DegU and LexA. In Bacillus subtilis, SigL is an enhancer located downstream of the B. subtilis glutamate dehydrogenase gene and transcribes the rocG gene encoding glutamate dehydrogenase;CcpA transcription factor is involved in the breakdown of metabolites; DegU controls the gene expression of flagella formation and biofilm formation; LexA protein is induced in the case of DNA damage and is transcription of the bacterial SOS DNA repair system inhibitor. It is speculated that these transcription factors also play a similar role in Bt. The 1 kb upstream sequence and 1 kb downstream sequence of 00940 gene were obtained using PCR, as well as the kanamycin resistant gene. The 00940 gene knockout box containing the above three sequences was inserted into the thermosensitive vector pMAD, and the resulting recombinant plasmid was transformed into Bt XL6-. A 00940 gene knockout mutant Bt XL6-Δ00940ΩKm was then obtained. Bt XL6- was used as a control for the phenotypic analysis of the knockout mutants. Growth curve

关 键 词：苏云金芽胞杆菌 生物被膜 基因敲除突变株 序列分析 谷氨酰胺合成酶 

分 类 号：S182[农业科学—农业基础科学]