Functional Analysis Of The Genes Regulating The Synthesis Of Lipopeptides And Construction Of Multifunctional Biocontrol Genetically Modified Strains

Bacillus subtilis have significant advantages such as growing fast, producing abundant metabolites and high food safety, etc. It is widely used in biocontrol of the plant disease. Therefore, it is significant to isolate and identify more Bacillus subtilis which can be applied in agriculture.The B. subtilis strains B3,38,43,49,55,85were isolated from soil of the plant root and identified using biochemical, physiological methods previously in our lab. In this article, these strains were identified using16S rDNA sequence analysis methods. It showed that16S rDNA sequences of the isolate were highly homologous to the Bacillus subtilis. B3,38,43,49,55,85showed99.4%,99.5%,99.7%,93.8%,96.8%,99.4%similarity to Bacillus subtilis168respectively. The16S rDNA sequence of the B3had been deposited in the GenBank database, under the accession number EF492885. The phylogenetic tree was constructed using DNAstar software basing on their16S rDNA sequence. From the phylogenetic tree, B3,38,43,49,55,85and Bacillus subtilis168got together on close genetic relationship.An innovative method was developed for rapid sensitive detection and efficient structural characterization of compound of the bacteria by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. In this article,the isolate Bacillus subtilis strains B3,38,43,49,55,85were also identified by MALDI-TOF-MS technique, and standard strains were used in this experiment, Bacillus subtilis168, ATCC6633, ATCC13915, ATCC21332were present by American Bacillus Genetic Stock Center, and A1/3, b213, C1were present by Dr.Joahim Vater from Technical University of Berlin. The results showed that each strain usually had three to four bands, among these characteristics bands, the bands with m/z379,493,618,714,843and969were regular and stable which represent the secondary metabolites of the Bacillus subtilis. Bacillus subtilis strains, identified in molecular methods, were also used to inhibit the growth of the Xanthomanas oryzae pv.oryzal and Sclerotinia sclerotiorum. The results had showed that the B3,38,43,49,55,85strains had obviously inhibitory effect on the Sclerotinia sclerotiorum. MALDI-TOF-MS analysis had showed that these strains can produced antifungal lipopeptides fengycin and iturin family(Bacillomycins D, Iturin and Mycosubtilins). Moreover, due to produce the antibacterial surfactin, the38,43,55,85strains also had obvious inhibitory effect on the Xanthomanas oryzae pv.oryzal.In order to improve the yield of the lipopepetides, we study the regulation mechanisms of the lipopepetides. In this article, the two putative regulator genes ycxD and aspB3were studied. The E. coli expression system was used to express the recombinant YcxD and AspB3protein, and Ni-NTA was used to purify the recombinant protein. In vitro analysis of these two proteins had showed that the YcxD protein have no aminotransferase activity, we presume that YcxD may belong to GntR transcriptional regulator family. The AspB3protein have aminotransferase activity. The studies of the properties of the AspB3aminotransferase had showed:the optimum temperature was40℃; the optimum pH was8.2; the optimum substrate was L-Asp. The amino acid sequence alignment and the phylogenetic tree had showed that the AspB3aminotransferase is a novel thermostable aspartate aminotransferase and blong to the AspAT Ⅰα subfamily. Site-directed mutagenesis of the conserved amino had showed:The D232residue enhances the function of the enzyme-bound coenzyme pyridoxal5’-phosphate(PLP); The function of the K270residue is responsible for binding the PLP coenzyme; The R403residue is responsible for binding the substrate α-carboxylate.HpaGXooc, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret HpaGxooc protein in Bacillus subtilis, we constructed three recombinant expression vectors pMSF, pM43SF and pM43HF. The pMSF expression vector carried HpaⅡ promoter and signal peptide element nprB; pM43SF and pM43HF carried P43and signal peptide element nprB, the difference is the recombinant HpaGxooc expressing by pM43HF was fused with6×histidines in its C-terminal. The SDS-PAGE and western blot analysis demonstrated the expression of the protein HpaGxooc in B. subtilis. The ELISA analysis the expression level of HpaGXooc showed that the activity of the P43promoter is stronger than the Hpa Ⅱ, and the amount of HpaGXooc expressing by B. subtilis OKBHF is lower than the B. subtilis WBHF. The ELISA analysis also determined the optimum condition for HpaGXooc expression in B. subtilis WBHF. The biological function analysis indicated that the protein HpaGXooc from B. subtilis WBHF elicits hypersensitive response (HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that HpaGXooc induces expression of the pathogenesis-related genes PR-1α and PR-1b in plant defense response.