福氏2a志贺菌磷酸烯醇丙酮酸盐合成酶基因的克隆与序列分析

Cloning and sequencing of phosphoenolpyruvate synthase(pps) gene of Shigella flexneri 2a

目的 克隆福氏 2a志贺菌 30 1株染色体编码与代谢相关的酶类基因 ,分析结构特点并进行同源性比较 ,以期部分阐明其与大肠杆菌生化特性相似的分子生物学基础。方法 鸟枪法随机克隆福氏 2a志贺菌染色体DNA ,经探针杂交和末端核苷酸序列测定筛选出带有磷酸烯醇丙铜酸盐合成酶 (pps)完整基因的阳性克隆 ,利用exonucleaseⅢ制备嵌套缺失体亚克隆 ,采用双脱氧链末端终止法测定核苷酸序列。结果 福氏 2a志贺菌 30 1株pps基因 (全长 2 4 74bp)与大肠杆菌pps基因核苷酸及氨基酸序列的同源性分别为 98.8%和 97.6 %。编码区上游和下游存在较多变异。福氏 2a志贺菌 30 1株pps基因与GenBank中其它菌株pps基因的同源性均低于 5 5 %。结论 福氏

Objective Cloning and sequencing the enzyme gene of S.flexneri 2a, analysis its structure to identify the molecular biologic base of chemical characteristics of Shigella and E.coli . Methods The genome library of S.flexneri 2a was constructed by shotgun method; consequently choosing one clone, which possibly had pps in it by hybridization and sequencing both terminals. The subclones prepared by exonuclease Ⅲ were sequenced by Sanger′s method. Results The analysis of nucleotide sequence showed that the homology of pps between E.coli and S.flexneri 2a in this study was 98.8%, though there were many differences in UTR(un translation region). The amino acids sequence of these two proteins also showed high homology(97.6%). There was low homology(less than 55%) of pps in S.flexneri and other species offered by GenBank. Conclusion There is high homology of pps between S.flexneri 2a and E.coli . [