Genetic Analysis Of MyoG, MSTN Gene And Construction Of MiR-143 Vector In Cattle

In this study, the gene polymorphism of MyoGenin (MyoG) and myostatin (MSTN) in Wandong cattle, Wannan cattle, Wagyu and Simmental cattle were investigated by PCR-RFLP method and PCR-SSCP method respectively, and the relationships between the gene polymorphism and body measurement traits in Wandong cattle and Wannan cattle were analyezed. And the interference vector and the expression vector of mir-143 of cattle were constructed successfully in this research. The results in details were as follows:1. Polymorphism of MyoG Gene and its association with body measurement traits106 Wandong cattle and 31 Wannan cattle were used to detect the polymorphism of MyoG gene by PCR-RFLP method, and to analyze the correlation between the SNPs (single nucleotide polymorphisms) and body measurement traits in Wandong cattle.The results shown that:(1) There was a G→C mutation on 466 base position of exon 1, and 2 types of genotypes (AA and AB) were found. The mutation had not caused the change of encoded amino acid. Genotypic frequencies of AB were 76.42% and 82.76% respectively in Wandong and Wannan cattle, and it could be concluded that the allele A was preponderant allele in the gene site. The polymorphism information content {PIC) was 0.3606 in Wandong and 0.3702 in Wannan, which were in situation of middle polymorphism. According to the results of x2 test, the genotype frequencies were not at Hardy-Weinberg equilibrium in Wandong (P<0.05) and at Hardy-Weinberg equilibrium in Wannan (P>0.05). (2) The associations between polymorphism of the MyoG gene locus with body measurement traits in Wandong cattle were analysed. It turned out that the body height of cattle with AA genotype were significantly higher than one of with AB genotype (P<0.05). The body length, heart girth, cannon circumference, hucklebone width and body weight of cattle with AA genotype cattle were higher than ones with AB genotype, but the differences were not remarkable (P>0.05).2. Polymorphism of MSTN Gene and its association with body measurement traitsPolymerase chain reaction single-stranded conformational polymorphism (PCR-SSCP) analysis was used to identify myostatin gene (MSTN) mutation in Wandong cattle, Wannan cattle, Wagyu and Simmental. The results show that:(1) An A→G synonymous mutation was occur at position 267 in exonl, and the mutation not caused encoded amino acid change.3 genotypes (AA, AG and GG) were detected in four cattle populations.The genotype AA was the dominant genotype in the four cattle population, which were 0.5755,0.6129,0.5667and 0.7321 in Wandong cattle, Wannan cattle, Wagyu and Simmental respectively. (2) The associations between polymorphism of the MSTN gene locus and body measurement traits in Wandong cattle were analysed. The results showed that the body height, heart girth, cannon circumference, hucklebone width and body weight of cattle with GG genotype were most significantly higher than these of cattle with AA genotype (P<0.01), and the body length of cattle with GG genotype was significantly longer than those of with AA genotype (P<0.05); the hucklebone width of cattle with GG genotype was significantly higher than those of with AA genotype (P<0.05); the heart girth index, cannon circumference index, cannon circumference index and body weight of cattle with AG genotype were most highly significant than these of cattle with AA genotype (P<0.01); there was no significant difference between the cattle with AG genotype and the cattle with AA genotype in body height and body length (P<0.05). Therefore, it was suggested that MSTN gene polymorphisms can be used asone of marker assisted selection genes in Wandong.3. Construction of cattle mir-143 vectorDesigned and synthesized 2 pairs of complementary oligo DNA according to the mir-143 mature sequence published in the miRBase database, recombinant cloning with the carrier kit, the sequencing results showed that the expression vector pcDNA6.2-143a and the interference vector pcDNA6.2-143b were successfully constructed. Gateway technology has been used to construct mir-143 lentivirus vector pLenti6.3-143a and pLenti6.3-143b. Lentiviral vector and packaging plasmids were co-transfected 293T cells, collected the virus dope, and determined the viral titer. The interference vector’s and expression vector’s titer are 6.5×108 TU/ml and 7.5×108 TU/ml.The results provided a theoretical basis for the work of molecular marker-assisted selection, laid a foundation work for study the superior traits formation mechanism of Anhui local cattle breeds in depth and provided a theoretical reference for the local cattle breeds resource protection and utilization.