转座子挽救法克隆鞘氨醇单胞菌CDS-1中呋喃丹水解酶相关基因

用含Tn5转座子的自杀性质粒pSC123诱变呋喃丹降解菌Sphingomonas agrestis CDS-1,获得失去呋喃丹降解功能的突变株CDS-M1.以pMD18-T为载体在E.coli DH5α中构建了CDS-M1的基因组文库,采用转座子挽救法对Tn5插入位点两侧翼的序列进行克隆与测序,根据测序结果(共4 551个碱基)设计引物,从CDS-1的基因组中扩增到同样大小的片段,把该片断克隆到广宿主载体pPZP201上,得到重组质粒pCDZ1,通过三亲接合的方法把pCDZ1导入CDS-M1中进行功能互补实验,结果显示CDS-M1的呋喃丹水解功能得到了恢复,表明该片断中包含呋喃丹水解酶相关基因.图7表1参14

Cloning of a Gene Related to Carbofuran Hydrolyzing from Sphingomonas agrestis CDS-1 by Transposon Rescue

Carbofuran degrading mutant CDS-M1 was obtained by mutating Sphingomonas agrestis CDS-1 with transposon Tn5 carried on the suicide plasmid pSC123. Genomic DNA library was constructed in E. coli DH5α using pMD18-T as vector. By using transposon rescue, a 4 551 bp DNA sequence of S. agrestis CDS-1 flanking the Tn5 insertion site was obtained. A pair of PCR primers were synthesized according to the sequence adjacent to transposon. With these primers, a same size PCR product was amplified from the genomic DNA of CDS-1. The PCR product was ligated into broad host vector pPZP201 to construct recombined plasmid CDZ1. CDZ1 was then transferred into mutant CDS-M1 by triparental mating. The result showed that the transformant with CDZ1 had regained carbofuran degrading ability. The study indicated that the 4 551 bp DNA sequence was a gene fragment relative to carbofuran hydrolyzing. Fig 7, Tab 1, Ref 14