苏云金芽胞杆菌aiiA的5'端侧翼序列的克隆与功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2018-0439

摘要/Abstract

摘要： 为了研究苏云金芽胞杆菌（Bacillus thuringiensis,Bt）N-酰基高丝氨酸内酯酶（N-acylhomoserine Lactonase,AiiA蛋白）表达调控机制,确定aiiA的启动子区域。本研究克隆了aiiA的5'端侧翼区（aP_-1930--1）,以gfp作为报告基因,分段克隆aiiA的5'侧翼区,鉴定启动子活性,并对aiiA启动子序列中的碱基对-150和-142之间的2个连续的TATA盒进行定点突变。结果显示,侧翼序列aP_-295--101和aP_-295--1可作为启动子,实现GFP在大肠杆菌中的异源表达,启动子序列aP_-295--101比aP_-295--1活性更强,aP_-101--1序列不能启动GFP表达。定点突变pET28a-aP_-295--101-gfp载体2个TATA盒显著降低GFP表达。表明-295--1bp为aiiA的启动子区,-429--296与-100--1序列是aiiA的负调控序列。-150--142 bp区域的2个TATA盒对aiiA的5'侧翼区域的启动子活性起关键的作用。

关键词: 苏云金芽胞杆菌, 群体感应, 启动子, N-酰基高丝氨酸内酯酶, 定点突变

Abstract: This work is to determine the promoter region in order to study the expression regulation mechanism of N-acylhomoserine lactonase（AiiA）in Bacillus thuringiensis. The 5'-flanking region of aiiA（aP_-1930--1）was cloned by sub-cloning the flanking region while having gfp as a reporter gene,and the promoter’s activity was identified. Site-directed mutagenesis on the two continuous TATA boxes between base pairs -150 and -142 in the aiiA promoter sequence was carried out. The results showed that the flanking region sequence aP_-295--101and aP_-295--1functioned as promoters to allow the heterologous expression in Escherichia coli. The promoter sequence aP_-295--101 was more active than aP_-295--1,but the aP_-101--1 sequence failed to initiate GFP expression. Two TATA box mutations in the site-directed mutant pET28a-aP_-295--101-gfp vector significantly reduced GFP expression. It is suggested that -295--1bp is the promoter region ofaiiA,-429--296 and -100--1are the negative regulatory sequences of aiiA. Two TATA boxes in the -150--142 bp region play a key role in the promoter activity of the 5' flanking region of aiiA.

Key words: Bacillus thuringiensis, quorum sensing, promoter, N-acylhomoserine lactonase, site-directed mutagenesis

陈少威, 吴程, 苏月华, 蔡斌斌, 谢盼盼, 杨梅. 苏云金芽胞杆菌aiiA的5'端侧翼序列的克隆与功能鉴定[J]. 生物技术通报, 2018, 34(11): 136-143.

CHEN Shao-wei, WU Cheng, SU Yue-hua, CAI Bin-bin, XIE Pan-pan, YANG Mei. Cloning and Functional Identification of the 5' flanking Region of the aiiA Gene from Bacillus thuringiensis[J]. Biotechnology Bulletin, 2018, 34(11): 136-143.

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