荧光实时定量PCR检测尼罗罗非鱼GH、GHR、IGF-I基因方法的建立及初步应用

 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-T Easy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。

Development and Application of Nile Tilapia (Oreochromis niloticus) Growth Hormone, Growth Hormone Receptors and Insulin-Like Growth Factor-I Gene Using Real-Time Quantitative PCR

To analyze accurately the roles of growth hormone (GH), two growth hormone receptors (GHR1 and GHR2) and insulin like growth factor -1 (IGF-I) during early development of Nile tilapia Oreochromis niloticus, methods of real-time quantitative PCR to examine GH, GHR and IGF-I gene expression were established. Total RNA was extracted from tilapia pituitary or liver tissue and the fragments of target gene were amplified by RT-PCR with special primers. PCR product was cloned into pGEM-T Easy vector. The success of recombinant plasmid was identified by PCR amplified and restriction enzyme analysis. Variation of mRNA levels of GH, GHR1, GHR2 and IGF-I during early development of Nile tilapiawas studied applying real-time quantitative PCR. The trends of GH and IGF-I expressions were compensatory, which was the same as that of GHR1 and GHR2. The expression of GH was significantly correlated with that of GHR1 instead of GHR2. The above results suggested that GH or IGF, GHR1 or GHR2 may play different roles during early development of Nile tilapia, furthermore, the actions of GH may mostly mediated through GHR1.