活性污泥宏基因组Fosmid文库的构建

Construction of Metagenomic Fosmid Library from Activated Sludge

大插入片段宏基因组文库的构建是开发大片段目的基因及分析其结构与功能的基础.文中分别采用十六烷基三甲基溴化铵(CTAB)法、试剂盒及琼脂糖包埋法提取活性污泥宏基因组DNA,其中琼脂糖包埋法获得的DNA片段大于23kbp,利用此DNA成功构建了以pCC1FOS为载体的Fosmid文库,该文库含有5280个克隆,平均插入片段长度为35～40 kbp,共包含约200 Mbp的宏基因组DNA.从此文库中随机挑选200个克隆,利用活性筛选方法快速筛选到了1个含有淀粉酶的阳性克隆,表明活性污泥Fosmid文库可用于功能基因的活性筛选,具有开发新基因的潜力.

The construction of large-fragment metagenomic library helps to discover large-fragment functional genes and analyze their organization and function.In this paper,first,three methods,namely the CTAB method,the kit and the agarose-embedding method,were used to extract metagenomic DNA from activated sludge,and DNA fragments larger than 23 kbp were extracted by the agarose-embedding method.Then,the metagenomic DNA was ligated into pCC1FOS vector to construct a Fosmid library consisting of 5 280 clones with an average insert fragment size of 35~40 kbp,which covered about 200 Mbp of metagenomic DNA.Finally,one clone exhibiting amylase activity was rapidly screened from 200 randomly-picked clones via the activity-based screening.It is thus concluded that the Fosmid library constructed from activated sludge is feasible in the screening of novel functional genes by activity-based methods.