草鱼胞质苹果酸脱氢酶(cMDH)的序列克隆及分析

Molecular Cloning and Characterization of Cytosolic Malate Dehydrogenase from Intestine of Ctenopharyngodon idellus

以实验室构建的草鱼(Ctenopharyngodon idellus)肠道cDNA文库苹果酸脱氢酶(cMDH)EST序列为基础,采用末端快速扩增法(RACE),从健康雌性草鱼(Ctenopharyngodon idellus)肠道细胞RNA中获得1391 bp的草鱼肠道cMDH的cDNA序列(NCBI登录号:EU569765)。结果显示:该序列包含62 bp的5′非编码区(5′-UTR),330 bp的3′非编码区(3′-UTR),典型加尾信号AATAA位于polyA起点上游16 bp。开放阅读框ORF长999 bp,共编码333个氨基酸,预测蛋白等电点为6.67,大小为36 kDa。多序列比对显示草鱼胞质苹果酸脱氢酶具有典型的苹果酸脱氢酶功能区保守序列,该酶与斑马鱼cMDH相似度最高达到95.5%,与模式植物拟兰介的相似度为57.8%,其预测三级结构具有典型cMDH功能区。Southern杂交结果表明草鱼cMDH属于多拷贝基因家族。

The full length cDNA of Ctenopharyngodon idellus intestinal cytosolic malate dehydrogenases （ GenBank accession number： EU569765） was cloned through the smart RACE technology. The cytosolic malate dehydrogenases （cMDH） EST sequence was picked out from the intestinal cDNA library of C. idellus that was constructed by our lab to design the RACE primers, which were used to clone the cDNA full length sequence of C. idellus＇s cMDH by using the smart RACE technology. The full length of this gene＇s cDNA was 1391 bp. It had a 62 bp 5＇-UTR and a 330 bp 3＇-UTR which included the typical polyadenylation signal AATAA just 16 bp upstream the polyA tail. The open reading frame（ORF） of this gene＇s cDNA was 999 bp which putatively coded 333 amino acids with the MW of 36 kDa and PI of 6. 67. The intestinal cytosolic malate dehydrogenase amino acid sequence of C. idellus had classical conservative domain of typical cytosolic malate dehydrogenase. Multiple alignments of cMDHs showed that the grass carp cMDH shared the highest identity with the cMDH of zebrafish and the lowest identity with the cMDH of thale cress, which were 95.5% and 57.8%, respectively. Southern blot analysis indicated that grass carp cMDH belonged to a multi-copy gene family.