Expression Of At7 Gene In Bacillus.cereus A-47 From Plant And Characterization Of The Engineered Strain On Biocontrol

Bacillus cereus A-47, isolated in the laboratory, is a kind of biocontrol agents which can enhance resistance to plant disease, increase the yield, improve the quality of crops, and promote tolerance successfully. At7 gene, isolated from Island Cotton with SSH, is induced expression by Vd-toxin (Verticillium dahliae toxin), and it was proved to be a LTP by sequence anglicized. pGFP4412 vector ,obtained through modified plasmid pBE2 in our laboratory, was an E.coli-Bacillus shuttle.In this experiment, 5'- of Atl gene connected with signal peptide sequence of Mn-SOD gene in Bacillus cereus by using the nested PCR, and the snippet had been inserted into the vector pGFP4412. And the recombinant vector (pSAt7-4412) was transformed into bacillus cereus A-47 via electroporation. The stability on heredity has been evaluated in this experiment; the result showed that the stability is 92.2%.The engineered strain had been studied in inhibitory activity to Verticillium dahliae V991 via indoor dish culture test; and in the same time, Vd-toxin yield had been studed through cultured together with Verticillium dahliae. It is suggested that the Vd-toxin yield by co-culture with engineered strain was slightly weaker than that of A-47 (wild strain) cultured with Verticillium dahliae. So it is showed that At7 gene was expressed in Bacillus cereus A-47 from plant, and had inhibitory activity. Furthermore, when the engineerd strain and wild strain had been studied on increasing the yield to wheat, it is indicated that they had difference in essence. Thus the At7 gene, transformed into the wild strain, had no effect to beneficial character of wild strain.The At7 gene was connected to the expression vector pET-28a. The recombinant plasmid pET-28a-At7 had been transformed into E. coli BL21 and then induced by IPTG to obtain a fusion protein. It was shown that the Atl gene was expressed at high level in 4h by SDS-PAGE analysis. An antiserum with high specificity was produced after the rabbit was immunized with purified fusion protein, and its titer was proved to be 1/1024 by micro-precipitation, or 1/16384 by ACP-ELISA. The result implied that the antiserum could be utilized to test the level of AT7 expressed in Bacillus cereus A-47.