重组枯草芽孢杆菌诱导表达AiiA及酶活性鉴定  

Expression and activity bioassary of aiiA gene with the promoter Pspac in Bacillus subtilis

作　　者：梁倩怡 明飞平 贾军皓 赵培静 李姣清[1] 邓锦波 张淑霞 曾敏 蔡海明[1] 马苗鹏[1] 张玲华[1] Qianyi Liang;Feiping Ming;Junhao Jia;Peijing Zhao;Jiaoqing Li;Jinbo Deng;Shuxia Zhang;Min Zeng;Haiming Cai;Miaopeng Ma;Linghua Zhang(Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms,College of Life Sciences,South China Agricultural University,Guangzhou 510642,China;Guangzhou Institute of Microbiology,Guangzhou 510663,China)

机构地区：[1]华南农业大学生命科学学院,广东省农业生物蛋白质功能与调控重点实验室,广东广州510642 [2]广州市微生物研究所,广东广州510663

出　　处：《生物技术》2018年第6期531-536,共6页Biotechnology

基　　金：广东省自然基金项目(“CRIP1通过调节Zn2+水平控制猪肠道沙门氏菌感染的机制研究”,No.2018A030313625);广州市科技攻关计划项目(“3-OH PAME淬灭酶分泌型高效青枯病复合生防菌剂研发及应用”,No.201803020001)

摘　　要：[目的]由于AiiA(Autoinducer inactivation,AiiA)蛋白能有效降解AHLs(N-acyl-homoserine,AHLs),导致病原菌由于群体淬灭(Quornm quenching,QQ)失去生存优势而抑制其致病能力;利用强启动子构建能高效表达AiiA蛋白的枯草芽孢杆菌,检验枯草芽孢杆菌中Pspac启动子表达AiiA蛋白的能力,寻求有效生物防治手段防治病原菌侵害。[方法]通过已知的aiiA基因序列和启动子Pspac序列设计引物,经搭桥PCR搭连启动子Pspac和aiiA基因,构建重组表达载体Pspac-aiiA-PHY300PLK,经酶活反应确定重组菌活性。[结果]成功克隆并搭连Pspac启动子和aiiA基因,AiiA蛋白成功在重组枯草芽孢杆菌C11中表达。经酶活检验,野生菌株和重组菌株Pspac-aiiA-C11有降解信号分子AHLs的能力,但重组菌降解能力更强。[结论]重组菌Pspac-aiiA-C11较野生菌株有更强的降解AHLs效力;要利用工程菌进行生物防治还需进一步提高工程菌表达AiiA蛋白的能力及稳定性。[Objective]Virulence of pathogens can be inhibited by quornm quenching(QQ)in the way of AiiA protein degradating AHLs.To construct Bacillus subtilis which is capable of efficiently expressing AiiA,in Bacillus subtilis experssion system,the ability of the Pspac promoter to express AiiA is needed to be tested.[Methods]Pspac and aiiA gene were ligated by SOE PCR.And the Pspac-aiiA fragment was inserted into PHY300PLK then transfer into Bacillus subtilis C11.The AHLs degradation ability was detected.[Results] Pspac-aiiA was amplified and Pspac-aiiA-pHY300PLK was transferred into C11 strain.Detection showed that the blue of the indicator bacteria in both experimental groups and positive groups become light in the middle of the strip culture medium,and all the lower colonies was white.Pspac-aiiA-C11 strain trun more blue colonies to be white than wild type,indicating that the Pspac-aiiA-C11 strain is capable of degrading signal molecules more effectively.[Conclusion]The ability of Pspac-aiiA-C11 strain degrade AHLs is more effective,compared with the wild type.And the stability and the ability to experss AiiA in this system is need further investigation to put into practice.

关 键 词：群体感应 AHLs信号分子 AIIA基因 Pspac启动子 桥式PCR 枯草芽孢杆菌 

分 类 号：Q81[生物学—生物工程]