灰红链霉菌AL7基因组文库及异源表达文库的构建

灰红链霉菌AL7对多种植物病原真菌和细菌都具有明显的抑菌活性。本研究在通用的大肠杆菌克隆宿主基础上,构建获得Dcm甲基化缺陷菌株,再引入接合转移辅助质粒pUZ8002得到菌株LP3。以LP3为宿主构建灰红链霉菌AL7的基因组粘粒文库,平均插入片段大小40kb。用高通量接合转移的方法将文库转到变铅青链霉菌TK24中,得到了异源表达文库,并从表达文库中初筛获得一些表型特异的克隆,如光秃表型、产素提前表型、产孢提前表型等。

Construction of genomic library and heterologous expression library of Streptomyces griseoruber AL7

Streptomyces griseoruber AL7 has been shown to display strong bioactivity against various plant pathogenic fungi and bacteria.The Escherichia coli was modified to clone host XL1-Blue MR to yield LP3,a new cloning host strain holding a Dcm DNA methylation deficient mutation and the conjugation helper plasmid pUZ8002.LP3 was used to construct the S.griseoruber AL7 genomic cosmid library with an average insert size of 40 kb.The cosmid library was then transformed into S.lividans TK24 via high-throughput conjugation to yield a heterologous expression library.A few expression clones with interesting phenotype were obtained,such as bald phenotype,precocious antibiotic production phenotype,precocious sporulation phenotype and so on.