山羊Ets-1基因cDNA的克隆与序列分析

Cloning and Sequence Analysis of Ets-1 Gene in Goats

根据GenBank已收录的牛(Bos taurus)、人(Homo sapiens)和小鼠(Mus musculus)等物种Ets-1基因序列的同源保守区域,设计特异性引物,采用RT-PCR和RACE技术,分离并克隆了西农萨能奶山羊(Capra hircus)Ets-1基因的cDNA序列。该序列全长2 263 bp(GenBank登录号HQ589338),包括5'UTR 331 bp,CDS 1 326bp和3'UTR 606 bp,编码441个氨基酸组成的蛋白质。核苷酸序列分析发现,山羊编码序列与牛、猪、人、小鼠等的相应序列同源性分别为98%、94%、92%和90%,3'UTR相应序列为96%、83%、81%和77%,5'UTR相应序列为98%、85%、82%和71%。氨基酸序列分析发现,山羊与牛、猪、人和小鼠的Ets-1的相似性较高,均在95%以上。蛋白质结构分析发现,其蛋白质分子量为50 340.8 D,等电点为5.08,具有典型的螺旋-转角-螺旋结构域,不存在跨膜结构,并且整个序列不含信号肽。

According to the highly conservative region of Ets-1 gene sequences from bovine（Bos taurus）,human（Homo sapiens） and mouse（Mus musculus） in GenBank,the specific primers were designed to clone the cDNA region of Ets-1 gene in mammary gland of Xinong Saanen dairy goats using RT-PCR and RACE（rapid amplification cDNA ends） methods,the sequence analyses of nucleotide sequence,and the deduced amino acid sequence of Ets-1 gene were conducted.The results showed that the full length of Ets-1 gene was 2 263 bp（GenBank accession No.HQ589338）,containing 331 bp of 5＇UTR,606 bp of 3＇UTR,and 1 326 bp of coding sequences（CDS）,which encoded a protein of 441 amino acid residues.The nucleotide sequence alignment indicated that the similarities of coding region of goat Ets-1 were 98 %,94 %,92 % and 90 %;the similarities of 5＇UTR were 98 %,85 %,82 % and 71 %;the similarities of 3＇UTR were 96 %,83 %,81 % and 77 %,compared to those of bovine,porcine,human and rat,respectively.The AA sequence alignment indicated that the protein sequence of goat Ets-1 shared high similarities（more than 95 %）with bovine,porcine,human and rat,respectively.The protein molecular weight was 50 340.8 D and the isoelectric point was 5.08.The protein of Ets-1 had a helix-turn-helix structure.No transmembrane structure was speculated and no signal peptide was found in the entire sequence.Thus,it can be concluded that the full length cDNA of goat Ets-1 gene has been successively cloned.