PCR-寻靶体系构建棒状链霉菌lat基因插入突变的重组质粒pELA

本实验将PCR所得的1.8kb的lat基因片段插入到pUCm-T载体的多克隆位点,得到重组质粒pUCm-T-lat,经EcoRI-HindⅢ双酶切后得到lat基因片段,与经同样双酶切的穿梭质粒pGH112进行连接,得到重组质粒pGH112-lat。将其电转至大肠杆菌E.coli BW25113/pIJ790中得到E.coli BW25113/pIJ790/pGH112-lat。同时,根据棒状链霉菌lat基因的序列及质粒pIJ77(3 pIJ773为可提供阿泊拉抗性的模板质粒)中阿泊拉抗性基因(aac(3)iv)的序列设计一对长59nt及58nt的引物,两引物分别含有20nt及19nt的阿泊拉抗性基因的互补序列和39nt的lat基因两端的互补序列,以质粒pIJ773为模板,PCR扩增得到的产物为两端带有lat基因上下游同源序列的阿泊拉抗性基因,将此PCR产物称为阿泊拉抗性框,将此阿泊拉抗性框电转至E.coli BW25113/pIJ790/pGH112-lat转化子中。在质粒pIJ790的作用下,阿泊拉抗性框中两端的lat基因同源序列与pGH112-lat中的野生型lat基因发生同源双交换,使得阿泊拉抗性框插入lat基因中,得到lat基因中插入阿泊拉抗性基因(lat::apr)的重组质粒pELA。该质粒的构建为进一步构建棒状链霉菌lat基因插入阻断的突变株,从而实现克拉维酸产量的提高奠定了初步基础。

Construction of S.clavuligerus lat Gene Inserted Mutant Recombinant Plasmid pELA by PCR-targeting System

The 1.8kb PCR product lat was cloned into the MCS of pUCm-T vector.The lat gene was isolated as a EcoRI-BamHI fragment from plasmid pUCm-T-lat and ligated into the corresponding sites of pGH112 to give pGH112-lat.At the same time,two long PCR primers(59nt and 58 nt)were designed according to lat gene sequence and the apramycin resistant gene(aac(3)iv)sequence of pIJ773(pIJ773 is template plasmid which can provide apramycin resistant gene).Each primer has 39nt matching the Streptomycin clavuligerus sequence adjacent to both ends of lat,and a sequence(20nt or 19nt)matching the right or left end of the apramycin resistance gene.pIJ773 was used as the template to PCR amplify the apramycin resistant cassette on each end of which is the lat gene homology sequence.Both of pGH112-lat plasmid and the apramycin resistance cassette were introduced into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/pGH112-lat.The apramycin resistance cassette was integrated into the wild type lat gene of the plasmid pGH112-lat by homologous recombination in the presence of pIJ790,thus the recombinant plasmid pELA was obtained in which the middle fragment of the lat gene was replaced by an apramycin resistance marker.The study lays a primary foundation to get the lat gene disrupted S.clavuligerus mutant and to elevate the production of clavulanic acid.