| S. roseoflavus Men-myco-93-63 was isolated from potato scab (S. scabies) decline soil and its fermentation can inhibit many phytopathogenic fungi and control related important plant diseases such as cotton verticillium wilt (V.dahliae) and cucumber powdery mildew (Sphaerotheca fuliginea Poll).To study the functional genes in S.roseoflavus Men-myco-93-63 and achieve some engineering strain which producers of some important antibiotics, in this study, the conjugative transfer system of Men-myco-93-63 was developed and optimized. The genetic integrate plasmid pSET152 and genetic damage plasmid pKC1139 used as the starting plasmid, ET12567 (pUZ8002, pSET152) and ET12567 (pUZ8002, pKC1139) used as the donor, Men-myco-93-63 spores and hyphae used as the receptor, respectively. The system for the conjugal transfer of pSET152 or pKC1139 from Escherichia coli in to Men-myco-93-63 has been developed. Different medium, such as MS, PDA, TSB agar, oats medium were selected for conjugal transfer, respectively. The results showed that MS medium was fit for conjugal transfer between Men-myco-93-63 and ET12567 (pUZ8002 ), and it was confirmed that plasmids of pSET152 and pKC1139 had been successfully transferred to the Men-myco-93-63, respectively. When spores were used as the receptor, the preconditions of the germination of spores were 50℃heat shock 10 min, 37℃incubated 2.5 h in MS medium, and the conversion efficiency was 10-7 to 10-6. If the mycelium were uesd as the receptor, the conversion efficiency was 10-7 to 10-6, but the mycelium pollution can not be avoided. In addition, the coverage time on antibiotics effected the joint transfer efficiency significantly, 16 to18 hours was best. Therefore, the final choice conjugal transfer system as follows: Men-myco-93-63 spores were 50℃heat shock 10 min, 37℃and incubated 2.5 h in MS medium, the time of antibiotic coverage for 17 h.nsdA (negative regulator of Streptomyces differentiation ) is globally negative regulator gene, which is first found in S. coelicolor. In this study, nsdA gene of S. roseoflavus strains Men-myco-93-63 was amplified by the primer sequences designed from S. coelicolor A3(2). After sequencing, Blastn, and Blastx, it showed that the cloned fragment from Men-myco-93-63 with the primer of nsdA-2R and nsdA-2L compairing with reported nsdA conservative nucleotide sequence homology on the region reached 99 %. The full length gene sequence of Men-myco-93-63 nsdA gene amplification with primers of nsdA WZ-R and nsdA WZ-L have a complete open reading frame encoding 369 amino acids, with the S. lividans and S. coelicolor A3 (2) nsdA gene at nucleotide sequence 100 %, and amino acid sequence homology up to 99 %. The full length nsdA gene of Men-myco-93-63 was named nsdAmgh, and Dot-blot hybridization was used to verify the correctness of PCR amplification results. To further study function of the gene, I used the gene disruption plasmid pKC1139 to build the homologous recombination vector pSRNA2500 (pKC1139::1.5kb nsdAmgh::1.0 kb KmR) and pSRNA800(pKC1139::800bp nsdAmgh), which will promote the obtaintion of high-yielding antibiotic strains of Men-myco-93-63 through interrupting the negative regulator. I put gene disruption recombinant plasmid pSRNA2500 and pSRNA800 into ET12567 (pUZ8002), and obtain conjugative transfer donor strains ET12567 (pUZ8002,pSRNA800/pSRNA2500) through conjugative transfer into Men-myco-93-63. Transformants were verified to right. Under the high temperature and antibiotic selection pressure, transformants Men-myco-93-63 (pSRNA800) had not get mutant with gene disruption, and the Men-myco-93-63 (pSRNA2500) isolated phenotype for AmSKmR of the mutant with nsdAmgh gene disruption and was confirmed by dot blot hybrid. Compared with the original strain, nsdAmgh gene mutant against V. dahliae V41 of inhibition doubled in the level of flask.
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