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

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 3′ 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 GenBank,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.