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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 BamHI-cut Koz plasmid pku4 0 3 was ligated with a DNA fragment of the Streptomyces griseochromogenes chromosomal DNA partially digested with Mbo I and treated with alkaline phosphatase, packaged in vitro, and then transformed into E. coli E. coli JM108 to select transformants 115 2, to construct a gene library of Streptomyces griseochromogenes. Avermectin polyketide synthase (PKS) acyltransferase gene (AT), keto-synthase gene (KS) and avermectin biosynthesis of the lactonase gene (aveE), C5 O methyltransferase gene (aveD) as probes, 24 positive clones were screened out from the library by means of colony in situ hybridization, nucleic acid hybridization and PCR amplification with enoyl reductase gene (ER) primers. Based on the overlapping relationship between the cloned DNA, the positive clones were divided into 7 groups, which may cover the gene cluster that controls the milbemycin biosynthesis