| 陈嘉蔚1，赵培静2，邓锦波1，李姣清1，明飞平2，张淑霞1，卢悄佳1，张玲华1.含 P43 启动子 aiiA 基因重组枯草芽孢杆菌的构建与表达…[J].广东农业科学,2018,45(11):21-27 |
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含 P43 启动子 aiiA 基因重组枯草芽孢杆菌的构建与表达… |
| Construction and expression of aiiA with P43 promoter in Bacillus subtilis |
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| DOI：10.16768/j.issn.1004-874X.2018.11.004 |
| 中文关键词: P43启动子 aiiA基因 群体感应 枯草芽孢杆菌 |
| 英文关键词: P43 promoter aiiA gene quorum sensing Bacillus subtilis |
| 基金项目:广东省自然科学基金（2018A030313625）；广州市民生科技攻关计划项目（201803020001） |
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| 中文摘要: |
| 细菌群体可以通过响应N-酰基高丝氨酸内酯（N-acyl-homoserine lactones, AHLs）激活某些致病基因的表达，而芽胞杆菌可以表达AiiA（Autoinducer inactivation）蛋白降解病原菌的AHLs从而减低损害。为构建外源高效表达AiiA蛋白的工程菌，利用AiiA蛋白降解AHLs分子来防治细菌性植物病害，通过搭桥PCR扩增P43-aiiA联合片段，连接pHY300PLK载体，构建P43-aiiA-C11枯草芽孢杆菌菌株，验证其AiiA酶活。结果成功克隆得到P43-aiiA片段，经鉴定P43-aiiA-pHY300PLK成功转入C11菌株，通过AiiA活性检测，野生菌株组指示菌全部变蓝，3个平行实验组的蓝色指示菌至条状培养基中间变浅，至下端全部为白色菌落，说明实验所构建的P43-aiiA-C11菌株具有高于野生型C11菌株的淬灭酶活性，能明显降解信号分子。与野生型菌株相比，构建菌株P43-aiiA-C11表达产物具有显著降解AHLs的能力，研究结果为推动植物病害防治提供了重要理论基础。 |
| 英文摘要: |
| Abstract：Some virulence genes in bacterial community would be activated responding to N-acyl-homoserine lactones (AHLs), while Bacillus sp. can express AiiA to degrade the AHLs and thus alleviate the damage of pathogenic bacteria. In order to construct an engineering strain which can efficiently express AiiA to degrade AHLs and prevent bacterial plant diseases, P43-aiiA fragment was amplified by SOE PCR and connected to the pHY300PLK vector. Bacillus subtilis strain P43-aiiA-C11 was constructed and its AiiA enzyme activity was detected. P43-aiiA was successfully amplified and P43-aiiA-pHY300PLK was successfully transferred into C11 strain. AiiA activity detection showed that all the indicator bacteria turned blue in wild strain group, the blue of the indicator bacteria in the three parallel experimental groups became light in the middle of the strip culture medium, and all the lower colonies was white, indicating that the P43-aiiA-C11 strain has higher quenching enzyme activity than the wild strain and could obviously degrade signal molecules. Compared with the wild strain, P43-aiiA-C11 strain has significant ability to degrade AHLs. The results of this study provide an important theoretical basis for promoting the plant disease biocontrol. |
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