Cloning And Identification Of 5’flanking Region Of AiiA Gene From Bacillus Thuringiensis

The AiiA protein expressed by aiiA gene from Bacillus thuringiensis can inactivate the N-acylhomoserine lactone (AHL) which is a critical signaling molecule in the gram-negative bacteria quorum-sensing (QS) system, but the expression level of AiiA protein in Bacillus thuringiensis is low. In order to explore the regulation mechanism of aiiA gene transcriptional in Bacillus thuringiensis, the 5’flanking region aP-1930--1 of aiiA gene was cloned and sequenced (GenBank Accession Number:KP690195), then it was analyzed by various on-line softwares. The results suggested that the flanking region of aP-295--101,aP-295--1 and aP-1930--1429 may be the promoter function areas. The small fragment DNA cloning of—the flanking region of aP-1930--1, were carried out, a serious of recombinant expression vectors pET28a-aP-x-GFP with GFP(green fluorescent protein) as a report gene were constructed and transformed into E. coli BL21(DE3) respectively. The GFP fluorescence intensities in the bacteria with different insert DNA fragments were analyzed qualitatively and quantitatively by using fluorescence microscope and fluorescene spectrophotometer. The results showed that GFP gene expression was presented in E. coli BL21(DE3) with the flanking sequence of aP-295-101,aP-295--1 and aP-1930--1429.The promoter function of Sequence aP-295-101 was stronger than that of Sequence aP-295--1, and the fact that the Sequence aP-100--1 didn’t activate the GFP expression in E. coli BL21(DE3) suggested that-100～-1bp area of aiiA gene promoter was the negative regulatory region. Circular site-directed mutagenesis on the vector pET28a-aP-295--101-GFP was carried out, point mutations was produced in the two continuous TATA box located between -150 and -142bp in aiiA gene promoter. Single point mutation strains E. coli BL21(DE3)-pET28a-aP-295--101(-144Mu)-GFP and E. coli BL21(DE3)-pET28a-aP-295--101(-147 Mu)-GFP, and double point mutation strain E. coli BL21(DE3)-pET28a-aP-295--101(-144,-147 Mu)-GFP, and deletion mutation strain E. coli BL21 (DE3)-pET28a-ap-295--101(-162～-151 dMu)-GFP were constructed. The results of analysis with fluorescent microscope and fluorescent spectrophotometer suggested that the amount of GFP expressed by the point mutant strains were significantly reduced comparing with the strain E. coli BL21(DE3)-pET28a-aP-295--101-GFP, and the decrease of the promoter function of the double point mutant strain and deletion mutation strain were the most notable. The results showed that the two continuous TATA box affected the GFP expression in E. coli BL21(DE3) to a large extent, and it revealed that the two continuous TATA box located between -150 an d-142bp in aiiA gene promoter of Bacillus thuringiensis could determine the expression of aiiA gene.