Zhao J L, Si Y F, He F, Wen H S, Li J F, Ren Y Y, Zhao M L, Huang Z J, Chen S L
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China.
Fish Physiol Biochem. 2015 Aug;41(4):853-65. doi: 10.1007/s10695-015-0052-y. Epub 2015 Apr 17.
The objectives of the present study were to estimate the GHR1 gene mutations and methylation status of CpGs, and whether those mutations and methylation were involved in the regulation of GHR1 gene expression, hormone level and growth traits in half-smooth tongue sole (Cynoglossus semilaevis). Identification of single-nucleotide polymorphisms was performed on 43 male fish. Through polymerase chain reaction-single-strand conformation polymorphism and sequencing, two SNPs were found. SNP1 [c.G1357A (p.Val376Ile)] creating one CpG site located in exon 8 was named L1 locus, and SNP2 (c.G1479A) located in exon 9 was named L2 locus. Individuals were divided into three genotypes, AA, AG and GG according to L1 locus (GG genotype had one more CpG site because of the mutation), and into two genotypes, AA- and GG-based on L2 locus. The results showed that only L1 locus was significantly associated with body weight (P < 0.01), gonad weight (P ≤ 0.05), triiodothyronine (T3) level (P ≤ 0.05) and mRNA expression (P < 0.01). At L1 locus, newly created CpG site in GG genotype was highly methylated (93.3 %), while there was no difference of methylation level in the other two CpG sites among three genotypes. AA genotype and AG genotype having higher T3 level were significantly different (P ≤ 0.05) from GG genotype. There were significant differences among body weights of AA, AG and GG genotypes (P < 0.01). Gonad weights of AA genotype and AG genotype were significantly lower than GG genotype. The GHR1 mRNA expression of GG genotype was significantly lower than AA and AG genotypes (P < 0.01). These implied that mutations and methylation status of GHR1 gene might influence the hormone level, growth traits and gene expression in male half-smooth tongue sole and the L1 locus could be regarded as a potential candidate genetic and epigenetic marker in half-smooth tongue sole selection.
本研究的目的是评估半滑舌鳎（Cynoglossus semilaevis）生长激素受体1（GHR1）基因突变情况及CpG位点的甲基化状态，以及这些突变和甲基化是否参与GHR1基因表达、激素水平和生长性状的调控。对43尾雄鱼进行单核苷酸多态性鉴定。通过聚合酶链反应-单链构象多态性分析和测序，发现了两个单核苷酸多态性位点。位于外显子8上产生一个CpG位点的SNP1 [c.G1357A (p.Val376Ile)] 被命名为L1位点，位于外显子9上的SNP2 (c.G1479A) 被命名为L2位点。根据L1位点，个体被分为三种基因型，AA、AG和GG（由于突变，GG基因型多一个CpG位点）；根据L2位点，分为两种基因型，AA和GG。结果表明，只有L1位点与体重（P < 0.01）、性腺重（P ≤ 0.05）、三碘甲状腺原氨酸（T3）水平（P ≤ 0.05）和mRNA表达（P < 0.01）显著相关。在L1位点，GG基因型中新产生的CpG位点高度甲基化（93.3%），而其他两个CpG位点在三种基因型中的甲基化水平没有差异。T3水平较高的AA基因型和AG基因型与GG基因型有显著差异（P ≤ 0.05）。AA、AG和GG基因型的体重存在显著差异（P < 0.01）。AA基因型和AG基因型的性腺重显著低于GG基因型。GG基因型的GHR1 mRNA表达显著低于AA和AG基因型（P < 0.01）。这些结果表明，GHR1基因的突变和甲基化状态可能影响雄性半滑舌鳎的激素水平、生长性状和基因表达，L1位点可被视为半滑舌鳎选育中潜在的候选遗传和表观遗传标记。
