猪CuZnSOD基因的克隆、表达及功能分析

Cloning,expression and functional analysis of CuZnSOD gene in swine

为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Real-time PCR检测CuZnSOD基因的表达。结果表明,CuZnSOD基因cDNA序列全长658bp(GenBank登录号:GU944822),包含76bp的5′UTR和120bp的3′UTR序列。全部CDS序列462bp,编码153个氨基酸,分子量为15.9kDa,等电点为6.03。CuZnSOD基因编码的氨基酸序列中,第3氨基酸残基处存在1个O-糖基化位点,第86氨基酸残基处存在1个N-糖基化位点。二级结构中α螺旋仅占1.31%。在进化过程中高度保守,与人、牛、小鼠和褐鼠的编码区同源性分别为87.74%、87.66%、83.44%和83.23%;氨基酸序列同源性分别为90.26%、94.12%、92.21%和91.50%。CuZnSOD存在典型的金属结合配体结构域(GFHVHQFGDNT)。基于蛋白序列所构建的分子进化树表明猪与牛的亲缘关系最近。在mRNA水平上,CuZnSOD是一个广谱表达基因,在大脑、心脏、脾脏、肝脏、肾脏、肺、大肠、小肠、脊髓、肌肉、背膘和胃中都能检测到,其在肾脏、小肠和肺中表达量较高,在心脏和肌肉组织中表达量较低。

In order to understand the structure and function of CuZnSOD gene,reveal the effect of the anti-oxidant in swine,and find the molecule marker correlated with meat traits,the cDNA of CuZnSOD gene was cloned and sequenced from muscle of Laiwu black swine by RACE（rapid amplification of cDNA end） techniques.The structure and function of CuZnSOD were analyzed by bioinformatics,and the gene expression profile in different tissues was examined by real-time PCR.The results showed that the full sequence of CuZnSOD cDNA is 658 bp（GU944822）,containing 76 bp sequence of 5＇ UTR and 120 bp sequence of 3＇ UTR,and coding region（CDS,462 bp） encodes 153 amino acids.The isoelectric point（pI） of the protein is 6.03,and the molecular weight is 15.9 kDa.There were one Oglycosylation site at the third amino acid and one N-glycosylation site at the eighty-fourth amino acid.The percentage of alpha helix was 1.31%.The alignment similarities of the CDS sequence of swine CuZnSOD with those of cattle,human,rat,and mouse were 87.74%,87.66%,83.44%,and 83.23%,and the similarities of amino acid sequence were 90.26%,94.12%,92.21%,and 91.50%,respectively.CuZnSOD possesses the typical metal binding ligands（GFHVHQFGDNT）.The phylogenic tree based on CuZnSOD pro-tein sequence detected the closest relationship between swine and cattle.CuZnSOD mRNA is a broad-spectrum expression gene,which was detected in brain,heart,spleen,liver,kidney,lung,large intestine,small intestine,spinal cord,muscle,backfat,and stomach.In particular,high expression levels of CuZnSOD mRNA were detected in kidney,small intestine and lung,but low expressions were observed in heart and muscle tissues.