Study Of Genetic Variation Of SH2B1 Gene And Expression Of MYOG Gene In Chinese Cattle

SH2B1 is a member of the SH2B family (SH2-B/SH2B1, APS/SH2B2, Lnk/SH2B3). As an adaptor protein, SH2B1 binds, via its SH2 domain, to a variety of cytokines and growth factor receptors, which were involved in many signaling methods such as leptin and insulin, and take part in the regulation of appetite, body weight, growth, energy balance, and glucose metabolism, and so on.MYOG, also the myogenin gene, is a member of the MRFs family. It regulates muscle differentiation by controlling the fusion of muscle cells and the formation of muscle fiber.To obtain some molecular markers important for cattle growth and development, which will contribute to the moderate breeding program, genetic variation of SH2B1 gene in 1028 samples of five cattle breeds (297 for Jiaxian, 240 for Caoyuanhong, 204 for Nanyang, 183 for Qinchuan and 104 for Luxi) was identified by CRS-PCR-RFLP and sequencing, their correlation with growth traits of Jiaxian, Nanyang and Qinchuan were also estimated. The coding sequence of MYOG gene in Nanyang was cloned by Overlap-PCR and constructed to the prokaryotic expression vector. The high level of MYOG protein expressed in our study laid the foundation of purification and identification of biological activity of MYOG protein in future.The results of the present study are as follows:1. Genetic variation of SH2B1 gene and their correlation with cattle growthFive SNPs in eight loci of SH2B1 gene, namely, g.796G>A, g.823G>T, g.1537C>A, g.2017A>G and g.4368A>G were identified, among which g.796G>A and g.823G>T led to the corresponding amino acids change from Ala to Thr and from Ala to Ser respectively, while g.4368A>G was a synonymous mutation, and g.1537C>A and g.2017A>G occurred in introns one and three. Genetics analysis, Linkage analysis, haplotype analysis, and correlation analysis were conducted for the three SNPs in the coding region. At g.796G>A locus, Hardy–Weinberg equilibrium was observed in Caoyuanhong and Qinchuan (P>0.05), but not in Jiaxian, Nanyang and Luxi (P<0.05). All populations were in Hardy–Weinberg disequilibrium at g.823G>T locus (P<0.05) and Hardy–Weinberg equilibrium at g.4368A>G locus (P>0.05). At g.796G>A locus, Nanyang and Luxi had a moderate genetic diversity (0.25<PIC<0.5) while Jiaxian, Caoyuanhong and Qinchuan were of low level (PIC<0.25). At g.823G>T locus, all populations possessed a moderate genetic diversity (0.25<PIC<0.5) except Caoyuanhong (PIC<0.25). At g.4368A>G locus, all populations belonged to a poor genetic diversity (PIC<0.25). There was a strong linkage between g.796G>A and g.823G>T sites in Jiaxian, Nanyang, Qinchuan and Luxi(r~2>0.33), other linkages whose pair-wise r~2 <0.33 were of weak kind. A total of eight haplotypes were constructed, out of which haplotype GGA, GTA, ATA were the major ones and accounted for 67.9%, 14.5%, 12.7% respectively.The association analysis showed that in Jiaxian, g.796G>A locus was correlated with the height of hip cross (P<0.05), g.823G>T locus was correlated wth body height (P<0.01) and the height of hip cross (P<0.05). This indicated that g.796G>A and g.823G>T loci can be molecular markers of body measurements in Jiaxia. In Nanyang, g.796G>A locus was correlated with 12-month body height (P<0.05), g.823G>T locus was correlated with 12-month body height, body length and hucklebone width (P<0.05), g.4368A>G locus was correlated with 6-month hucklebone width (P<0.05). These results indicated that g.796G>A, g.823G>T and g.4368A>G can be considered as molecular markers of Nanyang growth in the early age, and used for Marker-assisted selection. In Qinchuan, g.796G>A and g.823G>T loci were associated with body length (P<0.05), so they could be molecular markers of growth traits.2. Clone and prokaryotic expression of MYOG geneThe coding sequence of MYOG gene in Nanyang was obtained by Overlap-PCR and constructed to pET28a vector. Optimization on the condition of expression of MYOG protein were conducted in three aspects, including inducing time and tempreture together with IPTG density. A high level of MYOG protein will be available when pET28a-MYOG/BL21(DE3) was induced by 0.01 mmol/L IPTG for six hours at 37℃. Western blot was used to identify the MYOG protein. These results supplied evidence for the function study of MYOG gene.