Cloning and Functional Identification of the 5' flanking Region of the aiiA Gene from Bacillus thuringiensis

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

Abstract

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

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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