Genetic Variation Of HGF Gene And Its Expression Pattern In Qinchuan Cattle

HGF is a very important growth factor, which functions during development andregeneration of organs. It has no tissue specificity and species specificity. As a mitogen andmorphogen, HGF regulates the movement of myogenic precursors in embryo, and alsopromotes the proliferation of muscle satellite cell.In the paper, bovine HGF gene was selected as the candidate gene. Firstly, geneticvariation of HGF gene in Qinchuan cattle was investigated, and its association with growthtraits was performed. Secondly, the spatio-temporal expression of HGF mRNA was studied.The association between SNPs of HGF gene and its mRNA expression in61fetal cattle’sskeletal muscle was studied. Its mRNA expression level was also researched in bovinedifferentiated and undifferentiated muscle cell. Furthermore, the CDS of HGF in Qinchuancattle was cloned, which was analysed by bioinformatics methods. And the relative expressionof alternative splicing in different tissues was compared. This research would provide amolecular explanation to growth associated with SNP of HGF, and theoretical basis for thegenetic improment of Qinchuan cattle. It also might be a foundation for the further functionstudy of bovine HGF gene.1. The polymorphisms of HGF gene in Qinchuan cattleThough DNA pool sequencing,5novel SNPs of HGF gene in Qinchuan cattle werefound, L1(g.288T>C), L2(g.47944C>G), L3(g.72801G>A), L4(g.77172G>T) and L5(g.77408T>G). L2caused a missense mutaion,298Leu>Val and L3caused a silent mutation,492Gln>Gln. Then PCR-RFLP technology was used to detect individual genotype. The resultof statistical analysis showed that L1-C, L2-C, L3-A, L4-G and L5-T were dominant alleles.L1-CC, L2-CC, L3-AA, L4-GG and L5-TG were dominant genotypes. GTCAC wasdominant haplotype. In addition, Qinchuan cattle possessed low polymorphism at L1and L2locus and moderate polymorphism at L3, L4and L5locus. By chi-square test, Qinchuan cattlewas in Hardy-wenberg epuilibrium at L1, L3and L5(P>0.05). The association analysis showed that L1had a significant effect on chest circumference (P<0.05), L2was significantlyassociated with body length, chest circumference and hip width (P<0.05), and the relationshipbetween L3and chest depth, huchlebone width and hip width of Qinchuan cattle wassignificant (P<0.05). Consequently, it was supposed that the SNPs of HGF gene in Qinchuancattle would be DNA molecular markers for genetic improvement.2. The expression level of HGF mRNA in Qinchuan cattleThe spatio-temporal expression of HGF mRNA was studied using qPCR performedwith cDNA from6tissues in2stages (fetal cattle and adult cattle) of Qinchuan cattle. Theresult indicated that HGF expressed in all the tissues, which meaned it had no tissuespecificity. The mRNA expression level of HGF gene in tissues of fetal bovine were higherthan adult cattle excepet in heart. The genotype-phenotype association analysis between SNPsin HGF gene and mRNA expression in fetal cattle skeletal muscle indicated that L1locus hada significant effect on HGF mRNA expression level. What’s more, HGF showed a lowerexpression level in differentiated bovine muscle cells than in undifferentiated ones. It wasguessed that HGF inhibited differentiation of bovine muscle cell.3. The cloning of HGF gene in Qinchuan cattle and the comparison of AS expressionIn this research, the CDS sequence of HGF gene in Qinchuan cattle was obtained.As a result, T-HGF plasmid was constructed. The sequencing result indicated an AS, adeletion of15bp in exon. These two AS all expressed in tissues of fetal and adult cattle.Especially, the relative expression of HGF-M was lower than HGF-W in all tested organs.Bioinformatics analysis found HGF-M had little differences compared to HGF-W insecondary structure, pI and molecular weight of protein. And these two had no differences intransmembrane structure and protein domain. The result of evolution analysis displayed thatHGF in Qinchuan cattle had high similarity with others’, which suggested that HGF wasconservative. The evolutionary distance between HGF in Qinchuan cattle and bos mutus wasthe nearest, while the distance with Rattus norvegicus was the farthest.