Construction Of Resolution Vector Based On Tn5401 And Efficient, Stable Expression Of AiiA Protein In Bacillus Thuringiensis

Protein AiiA widely present in Bacillus species is capable of inactivating the N-acyl-homoserine lactones(AHLs) which are involved in the inducement of the express of many virulence genes of bacterial pathogens.By degrading the AHLs and abolishing its accumulation in quorum-sensing systems of bacterial pathgengs, the diseases dependent on the density of AHLs can be attenuated. However, AiiA protein has a low expression amount in Bt and it is the insecretion of AiiA protein that make it unable to degrade the AHLs in environment. The two disadvantages greatly limit the potential of Bt against the diseases caused by by Envinia caroto-vora. In order to make great use of AiiA protein, two ways were used to rebuild AiiA gene. First, AiiA express amount was substantially enhanced by replacing its promoter with the one of cry3Aa gene and secondly AiiA protein was exposed on the cell surface to work againat AHLs directly by using s-layer protein as a carrier protein .So two fused genes were obtained, one pro3Aa-aiiA and the other slh-aiiA, through fusion the Xba\ site of etc gene. In order to improve the express stability of AiiA protein and eliminate the undesired fragments such as restriction marker and pUC19, resolution vector pBMB5401 was constructed based on Tn5401.The two fused genes referred as above were cloned into pBMB5401 respectively and simultaneously, obtaining three plasmids pBMBcaiiA, pBMB3aiiA and pBMB3439 which harbored fused gene slh-aiiA, pro3A-aiiA and both of them respectively. Then the three plasmids were introduced into 8MB 171 respectively and so was the helper plasmid pEG922 containing tnpl gene sequentially. The site-specific recombination event occurs within two copies of IRS, thus allows for the selective elimination of undesired fragments on pBMB3439, pBMBcaiiA and pBMB3aiiA, resulting in the expected strain BMB3439R. BMBcaiiAR and BMB3aiiAR. In this study, the ability AHLs inactivation was compared among the three strains .The result showed that the two different kinds of promoters can be simultaneously used to express AiiA protein without obvious side-effect on the products of fusion gene. Moreover the result indicated that exposure of AiiA protein to cell surface can improve the ability of AHLs inactivation because of its direct contact with AHLs. The expected strain BMB3439R revealed stronger ability of AHLs inactivation and more effective restraint to the potato's soft rot disease caused by Erwinia carotovora than BMBcaiiAR and BMB3aiiAR.