三角帆蚌CAT基因cDNA全长克隆及表达分析

Cloning and expression analysis of CAT gene from Hypriopsis cumingii

采用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)首次克隆了三角帆蚌过氧化氢酶(CAT)基因的cDNA全长序列,为2 804 bp,包含112 bp的5'非翻译区(untranslated region,UTR),1 303 bp的3'UTR和1 388 bp的开放阅读框(open reading frame,ORF)。ORF区共编码462个氨基酸,推算的分子量约为52.7 ku,理论等电点为6.35。多序列比对结果显示,有一段CAT氨基酸高度保守的催化位点序列FDRERIPERVVHAKGAG。三角帆蚌CAT基因有12个与还原型辅酶Ⅱ(NADPH)结合的氨基酸残基,分别是Asp107、His153、Phe157、Ser160、Arg162、Asn172、Try174、Lys196、Val261、Trp262、His264和Try317,其中第261位和第264位的氨基酸在不同物种间有所区别。比对结果得到的三角帆蚌CAT基因的氨基酸序列,与软体动物的CAT基因相似性高达99%,与虾类、鱼类、两栖类、哺乳类的CAT基因相似性也达到98%～99%,可推断属于CAT3。利用CAT基因推断得到的氨基酸序列构建NJ系统树,分析显示三角帆蚌首先与软体动物聚在一起,再与虾类聚在一起,然后依次与鱼类、两栖类和哺乳类聚在一起。荧光定量结果显示,CAT基因在三角帆蚌的7个组织均有表达,其中在肾中的表达量极低,在血液中表达呈上调趋势且明显区别于其他组织,在另外5个组织中总体上呈现不统一的先上调后下调的趋势。

A 2 804 bp full-length cDNA sequence of catalase（CAT）gene from Hypriopsis cumingii was obtained by rapid amplification of cDNA ends.It consists of a 112 bp 5′ UTR（untranslated region）,a 1 388 bp ORF（open reading frame）and a 1 303 bp 3′UTR,and the deduced protein is composed of 462 amino acids,with calculated molecular weight of 52.7 ku,and its isoelectric point was 6.35.Motif analysis showed that CAT deduced amino acid sequence contained a highly conserved catalytic site motif ＂23FDRERIPERVVHAKGAG39＂.Twelve amino acids（Asp107,His153,Phe157,Ser160,Arg162,Asn172,Try174,Lys196,Val261,Trp262,His264 and Try317）of CAT gene of H.cumingii were identified as putative residues involved in NADPH binding,and they were different in different species.The CAT amino acid residues of H.cumingii shared a high similarity with other molluscs（99%）,and shrimp,fish,amphibians,mammals（98%-99%）.The obtained CAT of H.cumingii was predicted as CAT3.NJ tree suggested that H.cumingii clustered with mollusca firstly,and then clustered with shrimp,fish,amphibians and mammals.Real-time quantitative RT-PCR results displayed that CAT gene was expressed in a wide range of seven organs,with the lowest level of transcripts found in kidney.Its expression was up-regulated in blood,which was significantly different from other organs.The expressions of other five organs were generally up-regulated first and then down-regulated.