Study On SNP And DNA Methylation Of Genes Involoved In GH/IGF-I Axis Correlated To The Growth Of Male Half-smooth Tougue Sole (Cynoglossus Semilaevis)

1. Polymorghism in exons of GHR gene affecting its hormone level and growthtraits in male half-smooth tougue sole (Cynoglossus semilaevis)The growth hormone receptor (GHR) is the cell surface receptor for growthhormone (GH) and is required for GH to carry out its effects. The objectives of thepresent study were to estimate the GHR gene mutations and its assosiation withhormone level and growth traits. Through PCR-SSCP, two SNPs were found. SNP1[c.G1357A(p.Val376Ile)] located in exon8was named as L1locus and SNP2(c.G1479A) located in exon9was named as L2locus. The results analyzed byone-way ANOVA showed that only L1locus was significantly associated with gonadweight (P≤0.05), weight (P<0.01) and hormone level (P≤0.05). Though Ducanmultiple-range test, AA genotype and AG genotype had higher weight and T3levelthan GG genotype and AA and AG genotypes had lower gonad weight than GGgenotype. This indicated that mutations in GHR gene coding region might influncethe growth status of half-smooth tougue sole, and L1locus could be used a geneticmarker.2. DNA methylation status in exon8of GHR gene correlated with geneexpression level of male half-smooth tougue sole (Cynoglossus semilaevis)Epigenetic modification especially DNA methyaltion plays an important role inregulation of gene expression. In this study, we explored the methylation status ofexon8of GHR gene and its influence on gene expression. After bisulfite treatmentand because of the L1site, newly-created CpG site in GG genotype was highlymethlyated, while there were no difference of the other two CpG sites among threegenotypes. According to quantitative realtime PCR, gene expression of GG genotypewas significantly lower than AA and AG genotypes. Based on the present study, we proposed that the methylation status of exon8of GHR gene might influence theexpression level of GHR gene, which could provide epigenetic evidence for the studyof molecular mechanism of fish growth physiology.3. Polymorghism in promoter region and exons of GH gene affecting thehormone level and growth traits in male half-smooth tougue sole (Cynoglossussemilaevis)In this study, the polymorphism in promoter region and exons of GH gene andgenetic effect of genotypes on growth traits as well as hormone level were analyzed.Two SNPs were found in present study. SPN1[c.C382T (p.Phe106Leu)] located inexon4was named as L3locus. SNP2[c.C235T (p.Arg57Cys)] located in exon3wasnamed as L4locus. As the mathematical statistics requested and after tested byone-way ANOVA, L3locus was significantly associated with weight (P≤0.05), T3level (P<0.01) and weight without viscera (P≤0.05). Though Ducan multiple-rangetest, CC genotype had higher weight, T3level and weight without viscera than TTgenotype. The result of protein structure analysis showed that the L3mutationchanged the structure of GH protein. There results indicated that the mutations of GHgene could influence the growth status in male half-smooth tougue sole and the L3locus could be regarded as a candidate genetic marker in this kind of fish selection.Although there was no mutation in GH promoter region, the predicted transcriptionfactors involving CpG sites might lay the foundation of epigenetic marker of this fishspecies.In this study, we detected the SNPs of the coding region of GHR gene and GHgene and analyzed the methylation pattern of CpG site in exon of GHR gene. Theresults indicated that the mutations and methylation level of correlated gene ofGH/IGF-I axis had significant association with growth status. The research may havereference significance to the study of molecular mechanise of fish growth.