Full-length CDNA Cloning Of Insulin-like Growth Factor-2 Gene(IGF-2), Expression And Association Of Gene Polymorphisms With Production Traits In TianZhu Yak

T In order to study the associated genes affected growth traits of white yak, the IGF-2 gene was used as the research object in this paper.The full-length c DNA of IGF-2 gene in Tianzhu white yak was cloned by RT-PCR and RACEand the sequence of IGF-2 gene and encoded protein were analyzed with bioinformatics softwares. Using homology modeling to predict the three-dimensional structure of Tianzhu white yak IGF-2 protein. Specific primers were designed for the conserved regions according to the cloned IGF-2 gene c DNA sequence. Cattle GAPDH gene was the reference gene. Its expression in different tissues was also studied byreal-time fluorescent quantitative PCR. The polymorphism of exon 8 and exon 10 of Tianzhu white yak IGF-2 gene were detected by DNA mixing tank, direct sequencing and high resolution melting curve. In addition, the correlation of the polymorphism and the weight and body size traits of Tianzhu white yak were also analyzed.The results were as follows:1. A 1060-bp c DNA encoding Tianzhu white yak IGF-2 gene was cloned from liver tissues of Tianzhu white yak for the first time successfully. The 540-bp coding sequence, which encodes a polypeptide of 179 amino acids, shares 99% identity with Bos taurus; nucleotide sequence shares 98% identity with Bos taurus and Ovis aries. The results showed that IGF-2 gene kept highly conservertive in the evolutionary process. The protein has helix of 36.31% strand and loop of 63.69% in secondary structure with the detection of Predict Protein。The 3D structure of IGF-2 protein of Tianzhu white yak was predicted by using the human 3D structure in PBD database as the template, and the result showed the protein structure was high similar with human. 2. The m RNA expression level of IGF2 in nine yak tissues was like this: testis had the highest expression of IGF2, followed by liver, muscle, spleen, ovary, brain and kidney. And the expression of IGF-2 m RNA was not found in heart and lung.3. Two SNP sites were found in exon8 and exon10 by DNA mixing tank and direct sequencing. With the polymorphic analysis of the two SNP sites by HRM, three genotype(TT, CC, TC) were detected at exon8, and the dominant genotype was TT. There were three genotype(GG, AA, GA) at exon10, and the dominant genotype was GG. Through population genetics analysis, the results showed that exon 8 locals was at moderate polymorphic status(PIC=0.25), and exon10 locals was at low polymorphic status(PIC=0.192). The χ2 tests showed that the two locals were in the status of Hardy-Weinberg equilibrium(P>0.05). Analysis of association of polymorphic sites with growth traits showed that exon8 genotype had significant differences in herrt girth, body length, weight, and there was no effects on tube girth and body height. Exon10 genotype had significant difference in herrt girth, and there was no effects on other growth traits.