Cloning Of AiiA Gene From Bacillus Thuringiensis And Its Expression Product Against Plant Bacterial Disease

Plant pathogens are mostly gram-negative bacteria, their metabolites, N-acyl-homoserine lactones (AHL), are critical signaling molecules in bacterial quorum-sensing systems. When AHL reach a threshold concentration, they can activate the expression of pathogenic genes and induce diseases. Therefore reducing the concentration of AHLs is a key point of the diseases'control in plants.In this study, aiiA genes, designated as aiiA-B15, aiiA-B19, aiiA-S2, aiiA-S9, aiiA-P4 and aiiA-P28, were cloned respectively from six different strains of Bacillus thuringiensis from Bryophyte, soil and lamina. Bioinfromatical methods were utilized to analyze AHL-Lactonase, the products of these genes. The deduced AHL-Lactonase were weak hydrophilic proteins and all included three typical conserved domains belonging to theβ-lactamase superfamily. It was found that His-104, His-106, Asp-108, His-109, His-169, Asp-191, Tyr-194 and His-235 in AHL-Lactonase closed up on the spatial structure and formed an important activity center according to the simulative three-dimensional structure of AHL-Lactonase. Sixα-helices surrounded the activity center to keep its stabilization. By comparison of the phylogenetic tree of the cloned aiiA genes with that of 16S rRNA genes from the cloning stains, it was suggested that evolution of Bt species and that of aiiA genes are asynchronous. The differentiation of aiiA gene may be earlier than that of Bt species . Thus aiiA gene could not be used as a hereditary marker for Bt species evolution. These data provide important information for further study on aiiA gene expression and protein purification and also provide the valuable reference for the study of the enzyme character and antiviral ability of the AHL-Lactonase.pGEX-4T-3, an expression vector of AHL-Lactonase, was constructed and transformed into bacteria strain of E.coli BL21(DE3) for construction of genetically engineeringed bacterium strain. After 3h induction with 0.6 mmol/L IPTG at 30℃in the shaking flask culture, the expression of AHL-Lactonase protein reached to maximal level in the form of inclusion bodies. In order to obtain soluble expression of AHL-Lactonase protein, the expression vector of pET29a-aiiA was further constructed and transformed into E.coli BL21(DE3) for construction of soluble expression strain of BL21(DE3)-pET29a-aiiA. After 25h induction with 0.8 mmol/L IPTG at 20℃in the shaking flask culture, the AHL-Lactonase protein expressed in soluble form by this strain reached about 54.4μg/mL. The recombinant protein was purified with Ni-affinity chromatography. Biological activity analysis showed that AHL-Lactonase protein had a capability to hydrolyze AHL, and strong antimicrobial activity for Eriwinia carotovora. It was interesting to note that the supernatant of BL21(DE3)-pET29a-aiiA after breaking-up cell wall also had an AHL-hydrolyzing activity. This meant that rencombinant AHL-lactonase-B15 was able to hydrolyze AHL at low concentration under the contition of presenting many other native proteins and implied the potential for recombinant AHL-lactonase as a prospective biologic pesticide.The results of enzyme dynamic study demonstrated that the optimum temperature of AHL-lactonase was 40℃and the optimum pH value was 8.This study might provide theoretical support for protein engineering, large-scale production and application of AHL-Lactonase. Development of biological pesticides will reduce the pollution of chemical pesticides and be of economic,social and ecological benefits to our society.