牦牛HYOU1基因克隆及组织表达分析

Molecular Cloning and Tissue Expression of Gene HYOU1

为探究HYOU1基因在牦牛中的组织表达谱,通过对西藏牦牛HYOU1基因的CDS区进行克隆测序,分析该基因的结构和功能,采用RT-PCR技术检测黄牛和牦牛肺脏、心脏、肝脏、乳腺、大脑及肌肉中HYOU1基因的相对表达量。结果表明:(1)HYOU1基因CDS区长度为3 006 bp,其中A、G、C和T含量分别为25.0%、31.1%、26.7%和17.1%,存在一定碱基偏好性;发现1个(ACG→ACA)SNP位点,为同义突变。(2)生物信息学分析表明该蛋白呈中性,为较不稳定、亲水性分泌信号蛋白;存在一个跨膜螺旋(TMhelix)区位于13-35氨基酸位置;二、三级蛋白结构分析发现其主要的空间构象为:无规则卷曲及α-螺旋。(3)聚类分析显示,西藏牦牛与野牦牛的遗传距离最近,与瘤牛及普通牛的遗传距离次之,与水牛最远。(4)RT-PCR结果显示HYOU1基因在黄牛和牦牛肝脏、乳腺、肺脏、大脑、心脏、肌肉6个组织中均有表达,且相对表达量依次递减;在相同组织中,黄牛组织的表达量均显著高于牦牛组织中的表达量(P<0.05),其中黄牛肝脏组织的表达量为牦牛表达量的4.4倍。

In order to understand the tissue expression profile of gene HYOU1,the CDS region of HYOU1 in Tibetan yak was cloned and sequenced,and the structure and function of gene HYOU1 were analyzed;furthermore,the mRNA expression profile of gene HYOU1 in the lung,heart,liver,mammary gland,brain and muscle of cattle and yak were detected by RT-PCR.The results were as followings.(1)The CDS region of gene HYOU1 was 3 006 bp,with the percentage of A,G,C and T being 25.0%,31.1%,26.7%and 17.1%,respectively,and there was base preference.One SNP site(ACG→ACA)was identified as a synonymous mutation.(2)Bioinformatics analysis showed that the protein was a neutral,relatively unstable and hydrophilic secretion signal one,and a transmembrane helix(TMhelix)region were obtained and located at the 13-35 amino acid sites.The second and tertiary structures analysis showed that the main spatial structure was random coil andα-helix.(3)Cluster analysis demonstrated that the gene HYOU1 of the Tibetan yak was the closest associated with wild yak,followed by zebu and cattle,and the most distant with buffalo.(4)RT-PCR results indicated that gene HYOU1 expressed in 6 tissues liver,mammary gland,lung,brain,heart,and muscle of cattle and yak at descending order.In addition,for the same tissue,the expression level of the cattle was significantly higher than that of the yak(P<0.05),i.e.,the expression in the liver of the cattle was 4.4 times higher than that of yak.