土壤宏基因组文库的构建及格尔德霉素类PKS基因的初步筛选  

Construction of a soil metagenomic library and preliminary screening of geldanamycin-like type-I polyketide synthase gene

作　　者：林灵[1,2] 陈菲菲[2,3] 王以光[2] 赫卫清[1,2] 王相晶[1] 

机构地区：[1]东北农业大学,哈尔滨150030 [2]中国医学科学院/北京协和医学院医药生物技术研究所卫生部抗生素生物工程重点实验室,北京100050 [3]中国医药集团总公司四川抗菌素工业研究所,成都610052

出　　处：《中国抗生素杂志》2011年第6期426-430,共5页Chinese Journal of Antibiotics

基　　金：中央级公益性科研院所基本科研业务费专项(IMBF20060202)

摘　　要：目的构建土壤宏基因组文库并对格尔德霉素类I型聚酮合酶(polyketide synthase,PKS)基因进行初步筛选研究。方法直接提取土壤样品中宏基因组DNA,以Fosmid为载体,构建宏基因组文库。根据格尔德霉素的I型PKS基因的保守序列设计引物,使用菌落PCR方法直接筛选所获得的宏基因组文库。结果成功构建了土壤宏基因组文库,获得约6800个克隆子,其平均插入片段在25kb以上,覆盖至少170Mb的基因组信息。通过PKS基因筛选,获得了新的PKS基因片段。结论本文报道了土壤宏基因组文库的成功构建,并为利用宏基因组技术寻找新的聚酮类次级代谢产物的生物合成基因,以便于其异源表达或组合生物合成新的聚酮类次级代谢产物奠定基础。Objective Construction of a soil metagenomic library and preliminary screening of geldanamycinlike type-Ⅰ polyketide synthase genes. Methods A metagenomic library was constructed by inserting the metagenomic DNA directly extracted from soil into Fosmid vector, and then transduced into E. coli EPI300. The primers for screening polyketide synthase （PKS） genes were designed according to the conserved region of geldanamycin type Ⅰ PKS genes. Colony-PCR was performed for screening the metagenomic library. Results A soil metagenomic library was constructed successfully, which was consisted of about 6800 clones containing over 25kb inserted exogenous DNA on average, and covered almost 170Mb genomic information. And a new PKS gene fragment was obtained by screening from metagenomic library. Conclusion The strategy for finding novel PKS gene using metagenomic-technology was reported in this study and could facilitate to heterologous expressing or combinatorial biosynthesis of novel polyketide secondary metabolites.

关 键 词：宏基因组文库 PKS 土壤 

分 类 号：Q933[生物学—微生物学]