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玫瑰孢链霉菌(Streptomyces roseosporus)NRRL11379是一种具有环脂肽结构抗生素-达托霉素(Daptomycin)的产生菌。建立高效稳定的遗传操作系统是研究该链霉菌各功能基因作用以及构建基因工程菌的基础。实验探索了通过电穿孔、接合转移向玫瑰孢链霉菌中转入外源DNA的可能性。以链霉菌(Streptomyces)噬菌体ΦC31所构建的整合型载体pSET152-dptP为出发质粒,玫瑰孢链霉菌NRRL11379新鲜菌丝体作为受体菌,在不同的电场强度下进行电穿孔实验,结果表明:以玫瑰孢链霉菌NRRL11379新鲜菌丝体为受体菌,未获得转化子。以ET12567(PUZ8002,pSET152-dptP)为供体,玫瑰孢链霉菌NRRL11379新鲜菌丝体为受体,选取MS培养基为接合转移培养基,利用属间接合转移将质粒pSET152-dptP转入菌株NRRL11379中。结果表明:当供体大肠杆菌ET12567细胞量和玫瑰孢链霉菌NRRL11379菌丝体量比例为1∶1,MS培养基中MgCl2的浓度为0.01 mol/L,培养20 h后覆盖阿泊拉霉素50μg/mL以及萘啶酮酸50μg/mL时,接合转移频率最高,可达1.75×10-4。通过PCR验证,质粒pSET152-dptP已整合至玫瑰孢链霉菌NRRL11379菌株的染色体上。确定了玫瑰孢链霉菌属间接合转移的最佳条件。
Streptomyces roseosporus NRRL 11379 is a producer of the Daptomycin antibiotic, a cycloaliphatic peptide peptide. Establishing efficient and stable genetic operation system is the basis for studying the function of each functional gene of Streptomyces and constructing genetic engineering bacteria. The experiment explored the possibility of transferring exogenous DNA into Streptomyces roseosporus via electroporation, conjugation transfer. The recombinant plasmid pSET152-dptP (Streptomyces phage ΦC31) was used as the starting plasmid and the mycelium of Streptomyces roseosporus NRRL11379 as the recipient strain. The electroporation was performed under different electric field strengths. The results showed that Streptomyces roseosporus NRRL11379 fresh mycelium as recipient bacteria, no transformants obtained. The recombinant plasmid pSET152-dptP was transformed into strain NRRL11379 by using the fusion gene of ET12567 (PUZ8002, pSET152-dptP) as donor, the mycelia of Streptomyces roseosporus NRRL11379 as receptor, and the MS medium as conjugate transfer medium. in. The results showed that when the amount of donor Escherichia coli ET12567 and Streptomyces roseus NRRL11379 mycelium was 1: 1, the concentration of MgCl2 in MS medium was 0.01 mol / L, and the concentration of apramycin 50μg / mL and nalidixic acid 50μg / mL, the highest frequency of conjugation and transfer, up to 1.75 × 10-4. The plasmid pSET152-dptP was integrated into the chromosome of Streptomyces roseosporus NRRL11379 by PCR. The optimal conditions for the indirect transfer of Streptomyces roseosporus were established.