Bioinformatics Analysis,Cloning And Expression Of One Antagonistic Gene Against Botrytis Cinerea

Tomato gray mold caused by Botrytis cinerea is an important plant diseases, people screened and improved biocontrol agents to control it in recent years. So biological control technology is becoming an important and effective way to control gray mold. Metagenomic technology can greatly enhance the range of biocontrol gene screening, and thus the antagonistic mechanism of antagonistic gene and its protein are in-depth study.9#clone was from rhizosphere soil metagenomic library of tomato plant infected gray mold. It had a strong antagonistic activity against Botrytis cinerea. Firstly, to research the antagonistic mechanism of9clone to Botrytis cinerea at molecular level, the antagonistic activity of9#clone existed after10subcultring by flat confrontation experiment. Recombinant pBluescript II KS+DNA was extracted and digested by restriction endonuclease PstI and HindⅢ, which proved a2215bp exogenous DNA in recombinant pBluescript Ⅱ KS+.Secondly, bioinformatics analysis was used in the2215bp exogenous DNA, including homology of DNA sequence, search of the biggest open reading frame (ORF), homology and structures of coding protein sequence. The results showed that the sequence may be a novel antagonistic gene because homology of it was only4%. One612bp ORF encode a22.2kDa protein, one small hydrophobic fat soluble protein and had a high homology with AMP nucleosidase. There were5protein kinase C phosphorylation sites,1casein kinase Ⅱ phosphorylation site and3N-myristoylation sites by searching motif. These sites were related to cell signaling, protein localization and adherence. So the gene may take part in cell signaling pathway of the resistance to Botrytis cinerea.Thirdly, in order to verify the interactions between612bp ORF gene fragment and antagonistic activity of9#clone to Botrytis cinerea,612bp ORF gene frgment was amplified by PCR, and subcloned into prokaryotic expression vector pET-32a(+), then the recombinant expression plasmid was successfully constructed. It was transformed into E.coli BL21(DE3). A fusion protein of42kDa was expressed after induced by IPTG induction. Which was identical to the theoretical value.Finally, the expression condition of the fusion protein was optimized in the recombinant bacterium. The majorized protein expression conditions were that recombinant bacteria obtained a higher fusion protein expression level at37℃, in50mL LB liquid medium (300mL flask), shaking culture for8h to obtain the seed solution, then the seed solution inoculated in LB liquid medium (containing100μg/mL Amp) with1%ratio, shaking culture for3.5h and induced by IPTG for4h.