黑暗链霉菌DNA同源重组系统的构建  

Construction of DNA Homologous Recombination System in Streptomyces tenebrarius

作　　者：朱碧银[1] 洪文荣[1] 严绍德[1] 朱宝泉[2] 林玉双 封成军 

机构地区：[1]福州大学生物科学与工程学院,福州350108 [2]上海医药工业研究院,上海200040 [3]常州方圆制药有限公司,常州213022

出　　处：《生物技术通报》2011年第4期162-166,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31070093/30801449);福建省教育厅科研项目(2007F5070);国家综合性新药研究开发技术大平台项目(2009ZX09301-007)

摘　　要：以黑暗链霉菌Tt-49基因组为模板,利用PCR方法,扩增安普霉素生物合成关键基因aprF-G的上、下游序列,作为同源交换臂,并将红霉素抗性基因筛选标记及其启动子插入两交换臂之间,以温敏型质粒pKC1139为基础,构建用于阻断黑暗链霉菌Tt-49安普霉素生物合成的重组质粒pFD8。该质粒通过E.coli ET12567/pUZ8002去甲基化修饰后,经接合转移进入黑暗链霉菌Tt-49,利用红霉素抗性筛选得到3株阳性转化子,分别命名为Tt-49 AG1、Tt-49 AG2和Tt-49 AG3。通过PCR鉴定,证明pFD8已插入黑暗链霉菌Tt-49基因组的目标位点。以亲株作对照,对3株工程菌进行红霉素抗性能力考察,发现3株工程菌的抗红霉素能力均高达1 000μg/mL以上。Two fragments（JHB1 and JHB2）in length of 2 165 bp and 1 853 bp,which are respectively located upstream and downstream of aprF-G,were amplified by PCR from S.tenebrarius Tt-49 genomic DNA and used as exchange arms.Erythromycin resistance gene（ermE,used as screening mark）and its promoter were inserted into the arms,and the recombinant plasmid pFD8,containing arms and ermE,for homologous recombination was constructed from thermal sensitive plasmid pKC1139.The plasmid pFD8,after demethylated by E.coli ET12567/pUZ8002,was transformed into S.tenebrarius Tt-49 by conjugation.Three positive transformants were selected on MS plate containing 25 μg/mL erythromycin,which are named Tt-49 AG1,Tt-49 AG2 and Tt-49 AG3,respectively.It was verified by PCR that the plasmid pFD8 has been inserted into the genomic DNA target site of S.tenebrarius Tt-49.The resistant ability of the three mutants strains to erythromycin are more than 1 000 μg/mL,control with parent strain S.tenebrarius Tt-49.

关 键 词：黑暗链霉菌 抗生素基因组学 同源重组 安普霉素 

分 类 号：TQ927[轻工技术与工程—发酵工程]