沼泽红假单胞菌GroE基因高保守区DNA片段的克隆,测序和特性分析

Cloning,Sequencing,and Analysis of the High Conserved Region in GroE of Rhodopseudomonas palustris Y6

利用原核微生物GroE基因特定区域高保守性设计了简并引物 ,以RhodopseudomonaspalustrisY6的染色体为模板进行PCR扩增 ,得到片段 5 94bp的产物 .将该DNA片段连接到pUC -T载体多克隆位点上 ,转化大肠杆菌DH5α菌株 ,得到阳性重组子 .经Southern杂交验证 ,已克隆的 5 94bpDNA片段来自Rhodopseudomonaspalustris.测序结果分析表明该片段是细菌groE基因高保守区 .

A set of degenerate PCR primers,deduced from the conserved region in a number of bacteria groEs,were synthesized and used to amplify an 594bp fragment.The PCR fragment was ligated into pUC-T to construct a recombinant plasmid pUY600 that was transformed into E.coli DH5a.The transformant was analyzed by probing Southern blot of 594bp fragment recovered from the pUY600 with Rhodopseudomonsa palustris Y6 genomic DNA digested with HindⅢ,EcoRⅠ,BamHⅠ,respectively.Sequencing and alignment analysis of 594bp fragment revealed it was the best conserved area of bacteria groE,and the degenerate PCR primers can be used to study groEs from another bacteria.