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为了准确分析尼罗罗非鱼生长激素(growth hormone,GH)、生长激素受体(growth hormone receptors,GHRs)和胰岛素样生长因子I(Insulin-like growth factor-Ⅰ,IGF-Ⅰ)在早期发育阶段的作用,实验设计了尼罗罗非鱼GH、GHR、IGF-Ⅰ基因的特异性引物,提取垂体或肝脏总RNA并扩增出目的片段,将PCR产物克隆到pGEM-TEasy载体,经质粒PCR扩增、酶切和测序鉴定重组质粒,构建标准曲线等,成功建立了GH、GHR、IGF-Ⅰ基因荧光实时定量PCR检测方法。运用建立的荧光实时定量PCR检测了尼罗罗非鱼GH、GHR和IGF-Ⅰ基因表达的发育性变化。结果表明在早期发育阶段,尼罗罗非鱼GH与IGF-Ⅰ,GHR1与GHR2的mRNA表达存在一定的互补关系;GH的表达与GHR1的表达呈显著正相关,提示GH与IGF-Ⅰ,GHR1与GHR2在尼罗罗非鱼早期发育的不同阶段起主导作用,且GH可能主要通过与GHR1的结合起作用。
In order to analyze accurately the growth hormone (GH), growth hormone receptors (GHRs) and insulin-like growth factor-I (IGF-I) The experiment designed the specific primers of GH, GHR and IGF-Ⅰ gene of Nile tilapia to extract the total RNA of pituitary or liver and amplify the target fragment. The PCR product was cloned into pGEM-TEasy vector, PCR amplification, enzyme digestion and sequencing identification of recombinant plasmids, construction of a standard curve, the successful establishment of GH, GHR, IGF-Ⅰ gene real-time fluorescence quantitative PCR detection method. The established fluorescence quantitative real-time PCR was used to detect the developmental changes of GH, GHR and IGF-Ⅰ gene expression in Nile tilapia. The results showed that the mRNA expression of GH and IGF-Ⅰ, GHR1 and GHR2 in Nile tilapia had a certain degree of complementarity at the early stage of development. The expression of GH was positively correlated with the expression of GHR1, suggesting that GH and IGF-Ⅰ, GHR1 And GHR2 play a leading role in different stages of early development of Nile tilapia, and GH may play its role mainly through binding to GHR1.