| Previous studies found that environment p H and Env Z/Omp R can exert an prominent effect on the growth metabolism and antibacterial activity of Xenorhabdus nematophila.In order to further clarify the influence of X. nematophila antibacterial activity by environment p H and Env Z/Omp R,we took the X. nematophila YL001 as our material to study its antibacterial activity,gene expression and metabolic product differences in different constant p H. At the same time we took homologous recombination technology to make env Z gene mutation to study the growth metabolism and antibacterial activity. The preliminary research results are as follows:1. The amplification and fusion of upstream and downstream flanking DNA fragments of target gene env Z and omp R. And linked the fusion flanking and suicide vector p K18, then we introduced the recombinant plasmid into E.coli S17-1λpir. In the final we transferred the fusion flanking into X. nematophila by conjugation acquired mutant strains Δenv Z.2. We determined the antibiotic activity of mutant strain Δenv Z and YL001 by growth rate method, agar diffusion method and vivo tissue method.The results showed that the antibiotic activity of mutant strain Δenv Z had different degrees of increase compared with the wild strain YL001. The inhibition diameter of B. subtilis was 24.9 mm, and the inhibition rate of P. capsici was 93.52%. The efficacy of therapeutic and protective effect on B. cinerea were 44.34% and 72.04%,improved by 27.71% and 37.59% compared with the wild strain.3. We analyzed the phenotype,growth metabolism,the expression of main antibacterial substances biosynthesis gene xcn,the transcriptional regulation genes cpx A、cpx R、omp R and lrp of strains Δenv Z and YL001. We also detected the methanol extract of wild strain YL001 and strain Δenv Z by LC-MS.The results are as follows:The study found that the growth characteristics, residual glucose content,the p H change of Δenv Z were consistent as YL001.In terms of antibacterial activity, mutant strain Δenv Z had different degrees of increase compared with the wild strain YL001.The expression of main antibacterial substances XCN1 biosynthesis gene xcn A increases 1.102 times as much as that of the wild strain, the expression level of xcn M and xcn N were 0.901 and 0.859 times of YL001,respectively. Two-component signal transduction system genes cpx A、cpx R and omp R decreased 0.129、0.199 and 0.393 times than that of the wild strain,respectively.The main transcriptional regulation genes lrp were improved 0.165 times than YL001.The relative content of main antibacterial material XCNs has improved 1.5 times than YL001.4. The analysis of antimicrobial activities, the metabolism difference of main metabolites(XCN1 and Nematophin),the expression of main biosynthetic gene and related transcriptional regulation gene of X.nematophila YL001 under different constant p H culture conditions showed that:The inhibitory effect of extracellular extracts on B. subtilis and B. cinerea were improved with the increase of constant p H values of the medium.The fermented liquid of X. nematophila YL001 exhibited highest inhibitory effect on B. subtilis and B. cinerea at p H7.0. At p H8.5, the inhibitory zone diameter of ethyl acetate extracts on B. subtilis had both increased by 94.6% compared with p H5.5 and p H7.0.The inhibition ratio on B. cinerea had increased by 64.85% and 26.48% compared with p H5.5 and p H7.0. The inhibitory zone diameter of methanol extracts on B. subtilis had increased by 41.75% and 252.37% compared with p H5.5 and p H7.0.The inhibition ratio of methanol extracts on B. cinerea had increased by 79.86% and 36.15%.The relative amount of XCN1 at p H8.5 were 1.59 and 19.3 times than p H5.5 and p H7.0. The relative amount of Nematophin at p H7.0 were 8.0 and 1.63 times than p H5.5 and p H8.5.The expression of main antibacterial substances XCN1 biosynthesis gene xcn A increased with the increase of constant p H values of the medium. At p H8.5,the expression level of xcn A were increased by 0.695 times than p H7.5,and xcn M and xcn N were decreased by 0.712 and 0.865 times than p H7.5,respectively.The biosynthetic gene pax ABCT had increased by 0.750、0.930、0.860 and 0.071 times than p H7.5.The main transcriptional regulation genes lrp were improved 2.369 times than p H7.5.The gene cpx R and omp R decreased by 0.570 and 0.595 times than p H7.5. |
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