表达aiiA基因多黏芽胞杆菌工程菌构建与功能分析

Construction of Gene Engineering Bacteria of Paenibacillus polymyxa NR1 and Functional Analysis for aiiA Gene Expression

AiiA蛋白能够专一性地水解革兰氏阴性菌分泌的信号分子,从而抑制其致病基因的表达,减弱致病性。根据NCBI上公布的蜡状芽胞杆菌ATCC14579的aiiA序列设计引物,从苏云金芽胞杆菌Bacillus thuringiensis NBIN-861中扩增得到aiiA基因(GenBank登录号:MG704113),全长基因约750 bp。将aiiA基因插入大肠杆菌表达载体pET-28a和芽胞杆菌表达载体pBMB1A中,构建重组表达载体pETaiiA和pBMaiiA,pETaiiA转化大肠杆菌BL21(DE3),pBMaiiA转化多黏类芽胞杆菌NR1,获得重组工程菌BLaiiA和BPaiiA。SDS-PAGE和蛋白质谱鉴定表明AiiA蛋白均成功表达,利用指示菌紫色杆菌CV026检测发现目的蛋白具有水解N-酰基高丝氨酸内酯活性。其中纯化的AiiA蛋白(1.13 mg/mL)和重组菌BPaiiA浓缩的发酵上清液中的AiiA蛋白活性较高(透明圈直径分别为16和18 mm),马铃薯致病性试验和抑菌试验结果表明该蛋白对胡萝卜欧文氏菌具有一定的抗病活性,但不具有抑菌活性。多黏芽胞杆菌NR1和重组菌BPaiiA发酵产生的抑菌物质含量均为1.2%～1.3%,具有良好的抑菌效果。本研究为构建更有效的生物防治菌株奠定了理论基础。

AiiA protein can specially hydrolyze signal molecular secreted by Gram-negative bacteria, thereby restrains the expression of pathogenic genes and attenuates the virulence caused by pathogens. A pair of primers was designed based on aiiA gene sequence of Bacillus cereus ATCC14579 reported in NCBI, the 750 bp entire coding region of aiiA gene （GenBank No. MG704113） was successfully amplified from Bacillus thuringiensis NBIN-861. The aiiA gene was inserted into multiple cloning sites of the Escherichia coli expression vector pET-28a and Bacillus expression vector pBMB1A to generate the recombinant expression vector pETaiiA, pBMaiiA. The pETaiiA were transformed into E. coli BL21 （DE3）, and pBMaiiA was electrotrans formed into Paenibacillus polymyxa NR1 strains. SDS-PAGE and protein Mass Spectrum analysis suggested that the AiiA protein was successfully expressed. The recombinant AiiA protein showed the activity of degrading the N-acylhomoserine lactones when using Chromobacterium violaceum CV026 as reporter strain. The activity of purification AiiA protein and AiiA protein in the concentration fermentation supernatant fluid of engineering bacteria BPaiiA were significantly high with transparent circle diameter of 16 mm and 18 mm. Potato disease resistance experiment and antimicrobial experiment showed that AiiA protein had strong disease-resistant activity for Erwinia carotovora with no antimicrobial activity. Anmicrobial substances yield of P. polymyxa NR1 and BPaiiA reached the values of 1.2%-1.3% with the high antimicrobial activity through fermentation. This work could lay the foundation for constructing more effective biocontrol strains.