仿刺参超氧化物歧化酶基因cDNA全序列的克隆及其特征分析

Cloning and sequence analysis of superoxide dismutase gene in sea cucumber Apostichopus japonicus (Selenka)

中文关键词: 仿刺参  MnSOD  基因克隆  序列分析

英文关键词: Sea cucumber  MnSOD  Gene cloning  Sequence analysis

中文摘要:

      以仿刺参Apostichopus japonicus为材料，利用cDNA末端快速扩增(RACE)方法首次得到仿刺参MnSOD的全长cDNA。该全长cDNA大小为955bp，其中5′端非编码区67 bp，3′端非编码区216 bp，开放阅读框（ORF）672bp，在3′端有多聚腺苷酸信号序列ATTTA，并有polyA加尾，符合有效翻译的基因全长cDNA的特征。MnSOD全长cDNA可编码223个氨基酸的前体蛋白，预测蛋白分子质量为24.64kD，理论等电点是6.66，为亲水性蛋白。该蛋白序列没有信号肽和跨膜结构，仅有一段线粒体导肽。其二级结构主要以α-螺旋为主，无规则卷曲和延伸链构成了蛋白中量最大的结构元件，不含β-折叠。仿刺参SOD的氨基酸序列与目前已报道的其他物种如人、牛、斑马鱼、原鸡、非洲爪蟾、中国对虾、皱纹盘鲍、海湾扇贝、果蝇等的MnSOD氨基酸序列进行序列比对的结果表明，相似性均在61%～69%之间，说明该基因在各物种间具有一定的保守性。该段氨基酸序列无N-糖基化位点，含有3个蛋白激酶C磷酸化位点，1个酪氨酸蛋白激酶Ⅱ磷酸化位点，5个肉豆蔻酰基化位点，还发现了1个SOD家族的特征肽段DVWEHAYY。对磷酸化位点分析发现，有1个丝氨酸Ser，3个苏氨酸Tyr可能成为蛋白激酶磷酸化位点。二硫键分析表明，SOD含有3个半胱氨酸Cys，形成1个二硫键，连接着第17位和第107位的Cys。仿刺参MnSOD全长cDNA的克隆为更好地理解生物体内防御系统的催化O2-发生歧化反应、消除活性氧、维持机体活性氧代谢的平衡提供了理论基础。

英文摘要:

      One unique and highly compartmentalized sea cucumber superoxide dismutase gene MnSOD has been cloned and characterized. The full-length MnSOD cDNA of sea cucumber was comprised of 955bp, containing a 672bp open reading frame (ORF), 216bp 3′UTR and 67bp 5′UTR. The full-length MnSOD cDNA had an ATTTA deoxyadenosine monophosphate signal sequence in 3′ UTR and a polyA in the end, which is consistent with the characteristics of effective length of gene translation. Sequence comparison showed that the MnSOD shares 61%~69% similarity with MnSOD of Homo sapiens, Bos taurus, Gallus gallus,Xenopus laevis,Danio rerio,Haliotis discus,Argopecten irradians,Fenneropenaeus chinensis,and Drosophila melanogaster.The full-length MnSOD cDNA encoded 223 amino acids with protein molecule of 24.64 kD, andisoelectric of 6.66. The SOD had 17 alkaline amino acids, 20 strongly acidic amino acids, 83 hydrophobic amino acids, and 59 polar amino acids. The protein sequence had no signal peptide or transmembrane structure, only a mitochondrial leading peptide. The secondary structure was mainly comprised of alpha helix, with the largest number of irregular coiling and chain in the whole protein, without folding. The InterPro software analysis showed that the sequence of amino acids contains three protein kinase C phosphorylation sites, one casein kinaseⅡ phosphorylation site, 5 nutmeg acylation sites, and an SOD family signature peptide DVWEHAYY. The protein had 1 serine and 3 threonines, which were possibly protein kinase phosphorylation sites. Disulfide bond analysis showed that MnSOD contained three cysteines , forming a disulfide bond by connecting the 17th and 107th Cysteines. The cloning of MnSOD cDNAwould be helpful for better understanding of the body defense system to eliminate active oxygen O-2and maintain the metabolism balance.