牦牛生长分化因子-9基因cDNA克隆及序列分析  

Molecular Cloning and Sequence Analysis of the Yak Growth Differentiation Factor 9 Gene

作　　者：尹荣华[1] 字向东[1] 马志杰[2] 陈绍威[1] 张大伟[1] 梁冠男[1] 

机构地区：[1]西南民族大学生命科学与技术学院,成都610041 [2]青海省畜牧兽医科学院畜牧研究所,西宁810016

出　　处：《畜牧兽医学报》2009年第7期999-1006,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省科技厅应用基础项目(07JY029-128)资助

摘　　要：根据GenBank中普通牛生长分化因子9(GDF-9)基因序列(AF 307092)设计1对引物,以麦洼牦牛卵母细胞总RNA为模板,通过RT-PCR技术对牦牛GDF-9基因cDNA进行克隆测序和序列分析。结果表明:所克隆的1399 bp片段为预期的牦牛GDF-9基因cDNA序列,包含由2个外显子组成的全编码区和3′-下游部分序列。牦牛GDF-9基因编码区核苷酸序列长度为1362 bp,编码453个氨基酸,与GenBank中报道的普通牛、水牛、绵羊、山羊相应序列一致,而与人和黑猩猩存在差异。和普通牛相比,牦牛GDF-9基因编码区存在1处碱基转换(C→T),导致相应的氨基酸由丙氨酸(A)转换为缬氨酸(V)。牦牛与普通牛、水牛、绵羊、山羊、人和黑猩猩的核苷酸同源性分别为99.9%、98.4%、97.0%、96.8%、85.6%和85.1%;氨基酸同源性分别为99.8%、97.1%、95.1%、95.4%、79.4%和79.5%。利用NJ法和MP法以该基因编码区核苷酸序列构建的物种间分子系统进化树结果基本一致,即牦牛与普通牛先聚为一类,再与水牛聚为一类,而后与绵羊和山羊聚为一类,最后与人和黑猩猩聚为一类。该聚类结果与物种间遗传距离大小一致,也与各物种在动物学上的分类相吻合,表明GDF-9基因编码区适用于构建物种间系统进化树。In order to amplify the cDNA of yak growth differentiation factor 9, the primers were designed according to the GenBank sequence of cow GDF-9 gene （AF 307092）. Total RNA was extracted from the oocytes of the Maiwa yak and the cDNA encoding GDF-9 was obtained by the reverse transcription PCR （RT-PCR）. The purified RT-PCR product was cloned into T vector, and then the sequence was analyzed. The results demonstrated that the 1 399 bp product was the yak GDF-9 cDNA, including the complete CDS and part of 3r noncoding region. The size of the yak GDF-9 gene coding region was 1 362 bp which encode 453 amino acids. Comparing GDF-9 nucleotide sequences and deduced amino acid sequences of coding region of the yak to those of other species including cow, buffalo, sheep, goat, human and chimpanzee retrieved from Gen- Bank, the size of the yak nucleotide and amino acid was the same with that of cow, buffalo, sheep and goat, and only one base was different, and this difference influenced polypeptide sequence after translation. The homologies of nucleotide sequences of the coding region of GDF-9 gene between the yak and cow, buffalo, sheep, goat, human and chimpanzee were 99.9%, 98.4%, 97.0%, 96.8%, 85.6% and 85.1%, respectively, and the homologies of the deduced amino acid sequences of the coding region were 99.8%, 97.1%, 95.1%, 95.4%, 79.4% and 79.5%, respectively. The molecular phylogenetic trees among species were constructed according to the nucleotide sequence of the coding region of GDF-9 gene. The result indicated that yak, cow, and buffalo, sheep and goat assembled separately, and then assembled to a genus with human and chimpanzee. This result of phylogenetic clustering was identical to the genetic distance and the zoological classification, which indicated that the GDF-9 gene was also fit to construct molecular phylogenetic tree among different species.

关 键 词：牦牛 GDF-9 克隆 序列分析 

分 类 号：S823.85[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]