阎浩林,IKEDA Haruo,MURA Satoshi
YAN Hao lin 1; IKEDA Haruo 2; MURA Satoshi 2 (1. College of Pharmaceutical Engineering;

• 1:沈阳药科大学制药工程学院!辽宁沈阳110016
• 2:日本国北里研究所!日本东京591
• 3:日本国北里研究所!日本东京591

摘要(Abstract)：

经BamHI酶切的柯氏质粒pku4 0 3与MboⅠ部分消化并经碱性磷酸酶处理的Streptomycesgriseochromogenes染色体DNA片段相连接 ,体外包装后 ,转化大肠杆菌E .coliJM1 0 8,挑取转化子1 1 5 2个 ,构建Streptomycesgriseochromogenes的基因文库。分别以avermectin聚酮体合成酶 (PKS)的酰基转移酶基因 (AT)、酮基合成酶基因 (KS)及参与avermectin生物合成的内酯化酶基因(aveE)、C5 O 甲基转移酶基因 (aveD)为探针 ,利用菌落原位杂交、核酸杂交和以烯酰基还原酶基因(ER)引物的PCR扩增等手段 ,从文库中筛选出 2 4个阳性克隆。根据克隆DNA之间的重叠关系 ,将阳性克隆分为 7组 ,这 7组阳性克隆群可能覆盖控制milbemycin生物合成的基因簇
The arm parts of cosmid vector pku403 cut by Bam HI were ligated with 35 kb ～ 45 kb segments of chromosome of Streptomyces griseochromogenes digested by Mbo I partially and dephosporylated by alkaline phosphatase at 5' phosphoric acid ends. The constructed cosmids were transformed into the competent cells of E.coli JM108 after in vitro packed by packaging proteins. We chose 1152 transductants to construct the gene library of S.griseochromogenes. Acyltransferase(AT) and ketosynthase(KS) genes of avermectin polyketide synthase(PKSs) as well as aveE(determining the furan ring closure at C6 to C8a) and aveD(C5 O methylation: avermectin B O methyltransferase) genes involved in the biosynthesis of avermectin were used as probes respectively to screen the putative positive clones. 24 putative positive clones were selected from the library by colony hybridization, southern hybridization and PCR analysis and finally divided into 7 groups according to the relationships among them. The 7 groups of clones might contain the gene cluster involved in the biosynthesis of milbemycin.

关键词(KeyWords)： 柯氏质粒pku403;包装蛋白;转化;基因文库;聚酮体合成酶(PKS);菌落原位杂交;核酸杂交;PCR
cosmid pku403; packaging protein; transformation; gene library; PKS(polyketide synthase); colony hybridization; southern hybridization; PCR

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 阎浩林,IKEDA Haruo,MURA Satoshi
YAN Hao lin 1; IKEDA Haruo 2; MURA Satoshi 2 (1. College of Pharmaceutical Engineering;

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