乌骨鸡黑色素皮质激素受体-1基因的克隆、序列分析与原核表达

Cloning and Analysis of MC1R Gene in Silky Fowl and Its Prokaryotic Expression

【目的】克隆乌骨鸡黑色素皮质激素受体-1(melanocortin 1-receptor,MC1R)基因,并对其进行生物学分析和原核表达。【方法】采集乌骨鸡的肌肉组织,提取其总RNA,根据GenBank上公布的原鸡(Gallus gullus)MC1R基因序列设计引物,利用反转录RT-PCR克隆乌骨鸡MC1R基因,对其进行生物学分析,并构建原核表达载体pET32a-MC1R,在大肠杆菌BL21(DE3)中进行表达。【结果】乌骨鸡MC1R序列由945个碱基组成,编码314个氨基酸。成功构建了重组质粒pET32a-MC1R的原核表达系统,并且体外诱导获得了MC1R蛋白。【结论】成功克隆了乌骨鸡MC1R基因并分析了其与乌骨鸡乌色性状的关系,其与原鸡的亲缘关系最近,达99.4%,经原核表达获得了乌骨鸡MC1R蛋白。

【Objective】 MC1R(melanocortin 1-receptor) gene was analyzed and expressed in prokaryotic expression system.【Method】The CDs of silky fowl melanocortin 1-receptor gene was amplified and cloned from total RNA of silky fowl by RT-PCR according to the corresponding sequence in Gallus gallus.The biological information analysis was performed with it.The prokaryotic expression vector pET32a-MC1R was constructed and expressed in E.coli BL21(DE3).【Result】The ORF of silky fowl MC1R gene consisted of 945 nucleotides and encoding 314 amino acids.The prokaryotic expression system of recombined vector pET32a-MC1R was constructed successfully and the protein MC1R was expressed in vitro by inducing with IPTG.【Conclusion】 The MC1R gene of silky fowl was closed successfully and its relationship with melanin traits and its genetic relationship with other kinds of chicken were analyzed by constructing phylogenetic tree.At the same time,the MC1R protein of silky fowl was optimizedcy expressed in E.coli.