玫瑰黄链霉菌men-myco-93-63接合转移体系及nsda基因的研究-study on the binding transfer system and nsda gene of streptomyces roseofluoensis men - myco - 93 - 63.docxVIP

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玫瑰黄链霉菌men-myco-93-63接合转移体系及nsda基因的研究-study on the binding transfer system and nsda gene of streptomyces roseofluoensis men - myco - 93 - 63.docx

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玫瑰黄链霉菌men-myco-93-63接合转移体系及nsda基因的研究-study on the binding transfer system and nsda gene of streptomyces roseofluoensis men - myco - 93 - 63

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. Key words: Streptomyces roseoflavus Men-myco-93-63; conjugal transfer; pKC1139; pSET152; nsdA gene; gene disruption 缩略语 (Abbreviation) nsdA negative regulator gene of streptomyces differentiation 链霉菌分化负调控基因 ORF open reading frame 开放阅读框 Ori origin of replication 复制起始点 OriT origin of transfer 接合转移起始位点 PCR polymerase chain reaction 聚合酶链式反应 resistance 抗性 sensitivity 敏感性 TE Tris-EDTA buffer Tris-EDTA 缓冲液 SDS sodium dodecyl sulfate 十二烷基磺酸钠 TES N-Tris(hydrosymethyl)methyl-2 -amino-ethanesulphonic acid N-三（羟甲基）甲基-2-氨基乙烷磺酸 s second 秒 min minute 分钟 d day 天 h hour 小时 G+C% percentage of G+C content G+C 百分含量 EDTA ethyl