油莎豆PEPCase基因ppc的克隆及初步分析

Cloning and sequence analysis of partial ppc gene for PEPCase from Cyperus esculentus L.

[目的]油莎豆磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的分子克隆及初步分析。[方法]通过对Gen Bank中其他植物PEPCase基因进行同源性比对,设计简并性引物,用PCR法克隆获得PEPCase编码基因ppc的两个片段S1和S2。[结果]S1、S2片段长度分别为577 bp和190 bp,生物信息学分析表明,S1片段全部位于ppc基因第8外显子区域,S2片段包含ppc基因第8外显子3’端序列,第9外显子5’端序列以及它们之间的内含子区域。[结论]S1、S2片段长度分别为577 bp和190 bp。Blast比对结果表明,油莎豆ppc基因S1S2全长片段与Cyperus ustulatus(PEPC-C4-1)、Cyperus longus、Cyperus iria和Cyperus papyrus的同源性均为97%,与Cyperus rotundus同源性为96%。进化树分析结果表明,油莎豆ppc基因S1和S2片段与莎草属Cyperus ustulatus、Cyperus longus、Cyperus iria等的ppc基因的进化亲缘关系最近。

[Objective]Partial ppc gene for phosphoenolpyruvate carboxylase（PEPCase） was cloned from Cyperus esculentus L.,and its DNA sequences were analyzed. [Methods]The ppc gene was cloned by PCR,using the program of Blast on NCBI Gen Bank database,the sequence presented a very high match with the genes from other plants. [Results]Two fragments for PEPCase was isolated from Cyperus esculentus L.,which displayed corresponding lengths of 577 and 190 bp,respectively. Bioinformatics analysis showed that S1 fragment was totally located in the eighth exon region of known ppc genes of plants,while S2 fragment consisted of tail end of the eighth exon,front part of ninth exon,as well as the intron region between them. [Conclusion]The length of S1 and S2 are of 577 and 190 bp,respectively. BLAST results revealed that S1 and S2 fragment of Cyperus esculentus L. ppc gene shared homology as high as 97% with Cyperus ustulatus（PEPC-C4-1）,Cyperus longus,Cyperus iria,and Cyperus papyrus,and as high as 96% with that from Cyperus rotundus. Phylogenetic analysis showed that the two fragments S1 and S2 of ppc gene from Cyperus esculentus L. have close genetic relationship with Cyperus longus,Cyperus iria and Cyperus ustulatus.