| The symbiotic bacteria associated with insect pathogenic nematodes, which produce abundant secondary metabolite, are important resources of biopesticide and antibiotics, such as antifungal, antibacterial, insecticidal, nematicidal, antivirus and anticancer. Previous studies found that, there were three regulators-Lrp, Cpx R and Lrh A in X.nematophila, they regulated the expression of some genes that bacteria, nematode and isect needed in the interaction with each other. Cpx R negatively regulate the production of antibiotic, that is to say the inactivation of cpx R can enhance the antibacterial activity, but the mechanism is not clear. This research takes wild type YL001 as material, using gene knockout technology construct Δcpx R mutant strain. We analysed differences between mutant strain Δcpx R and wild strain YL001 in phenotype and growth by fermentation in flask and fermentation tank, detected mainly activity conpnent by HPLC-MS, antibiotic activity by bioassay. We researched the expression and transduction of some genes about in q RT-PCR and RT-PCR. The results list as follow:1. The upstream and downstream flanking DNA fragments of target gene cpx R and the kanamycin resistant gene Km were fused by PCR, and then cloned into the suicide vector p DM4, the recombinant plasmid was introduced into E.coli strain S17-1λpir and transferred into X.nematophila by conjugation, then we acquire a Δcpx R mutant and confirm it with inner primers and outer primers.2. We determined the antibiotic activity of mutant strain Δcpx R and wild strain YL001 on tested 15 plant pathogenic fungi, 5 kinds of bacteria. The results showed that the inhibition diameter of Bacillus subtilis was 30.89 mm, and the inhibition rate of B.Cinerea was 90.26%, it improved by 33.31% and 20.30% compared with the wild strain. Tissue experiments showed that the efficacy of therapeutic and protective of strain were 79.07%和77.24%, it improved by 13.74%和 14.67% compared with the wild strain.3. 5L fermenter experiments showed that Δcpx R grew slower than YL001 in the initial stage, after the logarithmic phase it became faster. The changing trend of glucose of two strains were similar, which decreased sharply at first and then kept stable, at the end of the fermentation, the residual glucose content of mutant strain Δcpx R and wild strain YL001 were 1.55 g/L and 1.67 g/L,the consumption of glucose were74.2% and 72.2% respectively.Research of antibiotic activity found that the activity of Δcpx R higher than YL001 in every period, the maximum antibacterial activity of Δcpx R and YL001 are 519.05 U/m L and 265.45U/m L.4. By means of RT-PCR and q RT-PCR,we analysed the main antibacterial substances biosynthesis gene xcn A-N, pax ABCT, xnd AB, isn A-C; Two-component signal transduction system Cpx RA major genes cpx A, cpx R and cpx P and Ack A-Pta approaches Ack A;The main transcriptional regulation genes related to antibacterial activity env Z,omp R, Lrp, rpo B, rpo E, rpo S and lrh A gene expression of wild strain YL001 and mutant strain Δcpx R.q RT-PCR results showed that the expression level of antibacterial substances biosynthesis gene xcn A increases 1.8 times as much as that of the wild strain,xcn M and xcn N,which were responsible for the transformation of XCN1 to XCN2, their expression level decreased 0.6 and 0.8 times respectively;The expression of cpx R was very low, about 0 because of its deleted; The expression level of cpx P and cpx A decreased 0.08 and 0.17 times than that of the wild strain respectively. The expression level of Env Z, Lrh A, Omp R, ack A were 0.35, 0.18, 0.2 and 0.32 times than that of the wild strain respectively.5. We detected the methanol extract of wild strain YL001 and mutant strain Δcpx R culture by HPLC-MS. The results indicated that in the activity components of methanol extract, XCN1, XCN2, PXCN3, PXCN4 and PXCN6 all increased compared with YL001 and XCN1 improved by 3.84 fold.6. When the strain Δcpx R was cultured under the condition of different initial p H(5.5,7.0,8.5),we researched the cell growth, nutrient consumption and antibacterial substances,the results indicated that 7.0 was better for cell growth and antimicrobial substances.The antimicrobial activity unit was 395.24 U/m L, which increased by 17.73% and 16.90% respectively,compared with p H8.5 and p H5.5. The relative contents of XCN1 Was 5.45 and 3.87 times higher than that of p H8.5 and p H5.5.Experiments of different culture mode on cell growth and antibiotic activity of strain Δcpx R showed that 70 L fermenter was optimum for the cell growth, however 5L fermenter was optimum for the antibiotic production with antibiotic activity of 473.3 U/m L, which improved by 29.27% and 31.40% than that of 70 L fermenter and flask. |
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