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

doi:10.16409/j.cnki.2095-039x.2018.03.019

Abstract

Abstract: 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.

Key words: Paenibacillus polymyxa, AiiA protein, gene expression

CLC Number:

S476

CHEN Junjun, LIU Xiaoyan, MIN Yong, YANG Ziwen. Construction of Gene Engineering Bacteria of Paenibacillus polymyxa NR1 and Functional Analysis for aiiA Gene Expression[J]. journal1, 2018, 34(3): 398-406.

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