大额牛Myf-5基因克隆、序列分析及其分子系统进化研究

Cloning,Sequence Analysis and Molecular Evolution of the Myf-5 Gene in Gayal

对大额牛Myf-5基因进行PCR扩增、T-A克隆和序列分析,并应用DNAMAN4.0、BioEdit 4.8.10、DnaSP4.10、Mega3.1等生物信息学软件同普通牛、黑猩猩、恒河猴、人、狗、家鼠、软体贝壳、鸡、斑马鱼9个物种相应基因编码区核苷酸序列和氨基酸序列进行了比对分析,在此基础上采用NJ、ME和UPGMA法对其编码区核苷酸序列构建了分子系统进化树。结果表明:①大额牛Myf-5基因由3个外显子和2个内含子组成,其大小分别为660、76、1 226、852、407 bp;外显子和内含子数与其他9个物种相同,但大小存在较大的差异;外显子与内含子连接区遵循GT-AG基因组成规则。②大额牛与普通牛、黑猩猩、恒河猴、人、狗、家鼠、软体贝壳、鸡、斑马鱼的Myf-5基因编码区核苷酸序列同源性分别为99.0%、90.0%、90.5%、90.5%、87.2%、84.8%、72.9%、64.3%、51.8%,相应的氨基酸序列同源性分别为99.6%、94.9%、94.6%、94.9%、89.9%、88.7%、81.6%、70.4%、56.6%,这说明大额牛与其他9个物种在Myf-5基因的编码区核苷酸和相应氨基酸序列上具有较高的保守性。③用NJ、ME和UPGMA等3种方法聚类构建的分子系统进化树表明,3种方法的聚类结果基本一致,即大额牛与普通牛首先聚为一类,人、黑猩猩、恒河猴也先聚为一类,这两类相聚后再依次同其他物种聚在一起。这与mtDNA、其他功能基因和动物学分类的研究结果一致,表明Myf-5基因适合用于物种间系统进化关系的研究。

The My f-5 gene of the gayal （Bos frontalis） was cloned and sequenced by the T-A method, including the 5＇-flanking ragion, three exons, two introns and partial 3＇-flanking region, then based on the nucleotide and amino acid sequences compared with some species （cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish） by DNAMAN, BioEdit and DnaSP, bio-softs, and also constructed the molecular phylogenetic tree on nucleoside acid of the coding region of Myf-5 gene with method of NJ, ME and UPGMA. The results show that： ①The numbers of exons and introns of Myf-5 gene of gayal are the same with that of cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish, consisted by 3 exons and 2 introns, but the size had some differences. The size of 3 exons and 2 introns was 660,76,1 226,852,407 bp,respectively. The junction sequence of exons and introns of gayal＇s Myf-5 gene was accordance with gene composition of GT-AG rule. ② The homology rates of nucleotide sequences of the coding region of My f-5 gene between the gayal and cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish were 99.0%,90.0%,90.5 %,90. 5%,87. 2%,84. 8%,72. 9%,64. 3%,51. 8%,respectively; while the homology rates of amino acid sequences were 99.6%, 94.9%, 94.6%, 94.9%,89.9%,88.7%,81.6%,70.4%,56.6%,respectively. This result showed that the nucleotide sequence of the coding region and accordingly amino acid sequences of Myf-5 gene of gayal and cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish were quite conservative. ③The result was basically consistent with three methods which was NJ, ME, UPGMA constructed in molecular phylogenetic tree, the gayal was clustered together with cow firstly, chimpanzee, rhesus monkey and human clustered together, then together with each other, followed by other species. The clustering result was accordance with the results of mtDNA, other function genes and the zoological classification. This indicated that Myf-5 gene fit to construct molecular phylogenetic tree.