摘要： 旨在根据GenBank 上公布的原鸡GnRHR基因序列设计2对引物,采用RT-PCR方法,从京海黄鸡肌肉组织中克隆GnRHR基因的编码区序列,并使用多种生物软件和在线工具对目的序列进行生物信息学分析,分析GnRHR基因与其他物种的同源性、蛋白质的理化性质、蛋白质序列的跨膜区、亚细胞定位、亲水性、潜在的磷酸化位点、保守结构域及该基因编码蛋白的二级结构、三级结构等。同时为GnRHR基因和β-actin基因各设计1对引物,采用RT-qPCR的方法研究GnRHR基因在京海黄鸡12个组织和器官中的表达情况。最终成功克隆了京海黄鸡GnRHR基因完整的编码区序列和部分侧翼序列,Blast结果显示,京海黄鸡GnRHR基因与原鸡、番鸭、藏羚羊、野猪、马、小鼠、大鼠、家兔、绵羊、人、牛、黑猩猩和斑马鱼的同源性分别为99.7%,86.7%,55.7%,54.6%,52%,51.6%,50.8%,50%,49.9%,49.6%,49.4%,47.4%和39.3%,并成功构建系统发育树。蛋白质结构分析显示,GnRHR蛋白分子量为45.432 kDa,理论等电点为9.55,包含20种氨基酸,其中,亮氨酸含量最高,占13.8%,赖氨酸含量最低,占0.7%;不稳定系数为65.35,显示该蛋白不稳定;脂溶指数为95.54,总平均疏水指数为0.312,为非水溶性蛋白;该蛋白不属于分泌型蛋白,主要存在于胞膜上,没有信号肽结构,存在16个潜在磷酸化位点和11个糖基化位点;保守结构域分析显示存在2个低复杂度序列和7段跨膜结构,跨膜分析显示该蛋白为7次跨膜蛋白,二级结构预测结果显示,在GnRHR二级结构中,α-螺旋占29.83%,β-折叠占17.18%,无规则卷曲占52.98%,GnRHR蛋白三级结构为复杂的7次跨膜螺旋结构,且为单链蛋白。组织表达分析显示,GnRHR基因主要在垂体中高表达,表达量显著高于其他组织,在其他11个组织和器官中表达量较低。

关键词: 京海黄鸡, GnRHR, 克隆, 生物信息学分析, 组织表达

Abstract: Based on the published mRNA nucleotide sequence of Gallus gallus GnRHR gene,two pair of primers were designed to clone the GnRHR gene coding sequence of Jinghai yellow chicken by RT-PCR.A variety of software and online tools were used to analyze the homology among different species,physical and chemical properties,transmembrane region,subcellular localization,hydrophilic,potential phosphorylation locus,conserved domain database,secondary structure and tertiary structure of GnRHR protein.Two pair of primers were designed to detect the tissue expression of GnRHR gene in twelve tissues of Jinghai yellow chicken by RT-qPCR.Finally,GnRHR gene was cloned which contained CDS region,part of promoter region and 3'region.Result of Blast showed that GnRHR gene of Jinghai yellow chicken shared 99.7%,86.7%,55.7%,54.6%,52%,51.6%,50.8%,50%,49.9%,49.6%,49.4%,47.4% and 39.3% identity with Gallus gallus,Cairina moschata,Pantholops hodgsoni,Sus scrofa,horse,mice,rats,rabbits,sheep,cows,chimpanzees and zebrafish.Phylogenetic tree was constructed.Analysis of GnRHR protein structure showed that the molecular weight was 45.432 kDa and pI was 9.55.GnRHR protein was consisted of twenty kinds of amino acids,in which leucine accounted for the highest content of 13.8% and Lysine accounted for the lowest content of 0.7%.The instability index was computed to be 65.35 which classified the protein as unstable.The grand average of hydropathicity was 0.312 which showed that the protein was Non-water-soluble protein.GnRHR protein did not belong to secreted protein,mainly presented on membrane,with no signal peptide and contained 16 phosphorylation sites and 11 glycosylation sites.Analysis of conserved domains showed that GnRHR protein included two low complexity sequences and seven transmembrane segments which agreed with the transmembrane analysis.The secondary structure of GnRHR was mainly composed of random coil.The tertiary structure of domain area of GnRHR protein showed a helix and single strand structure.The result of tissue expression showed that GnRHR was mainly expressed in pituitary.The expression quantity of GnRHR in other eleven tissues was low.

Key words: Jinghai yellow chicken, GnRHR, Cloning, Bioinformatics analysis, Tissue expression

中图分类号: 

• Q78