绵羊VDR基因CDS区克隆、生物信息学分析及组织表达研究

Cloning, bioinformatics and tissue expression analysis of the CDS region of VDR gene in sheep

为了克隆绵羊维生素D受体(vitamin D receptor,VDR)基因CDS区,并研究其序列特征和该基因的组织表达情况,试验采用克隆与测序技术获得绵羊VDR基因CDS区序列,使用多种在线软件对其进行生物信息学分析,并利用实时荧光定量PCR技术检测该基因在1月龄与12月龄的雄性和雌性绵羊各组织/器官内的差异表达情况。结果表明:绵羊VDR基因CDS区全长1278 bp,共编码425个氨基酸。VDR蛋白相对分子质量为48043.56,理论等电点为5.90,不稳定指数为55.77,平均亲水性分值为-0.483,属于不稳定酸性亲水性蛋白。VDR蛋白含有51个磷酸化位点,不存在跨膜螺旋结构与信号肽序列,主要定位于细胞核与细胞质内。VDR蛋白二级结构主要包含无规则卷曲、α-螺旋和β-折叠,三级结构中有一明显凹槽区域,该区域可能会与DNA结合并调控基因转录。VDR基因在1月龄和12月龄绵羊心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、卵巢/睾丸和小肠内均有表达,且均在小肠内的相对表达量最高。在12月龄绵羊的肝脏、脾脏、卵巢/睾丸与小肠中,VDR基因相对表达量极显著高于1月龄(P<0.01),而在心脏与背最长肌中1月龄VDR基因相对表达量极显著高于12月龄(P<0.01),但在肺脏与肾脏中1月龄VDR基因相对表达量与12月龄比较差异不显著(P>0.05)。说明VDR基因在绵羊体内广泛表达,且随着日龄增加,在各组织/器官中的表达趋势不同,提示其功能的广泛性与多重性,并且VDR基因在小肠内的相对表达量最高,提示其与肠道内物质的吸收息息相关。

In order to clone the CDS region of sheep vitamin D receptor(VDR) gene and explore its sequence characteristics and tissue expression, the CDS region of sheep VDR gene was obtained by cloning and sequencing technology, and bioinformatics analysis was carried out by softwares. Real-time PCR was used to detect the expression difference of VDR in the tissues of 1-month-old and 12-month-old male and female sheep. The results showed that the total length of CDS region of sheep VDR gene was 1 278 bp, encoding 425 amino acids. The molecular weight of VDR protein was 48 043.56, the theoretical pI was 5.90, the instability index was 55.77, and the grand average of hydropathicity was-0.483. Therefore, it belonged to unstable acidic hydrophilic protein. VDR protein contained 51 phosphorylation sites, which did not have transmembrane structure and signal peptide, and mainly existed in nucleus and cytoplasm. The secondary structure mainly included random coil, α-helix and β-sheet, there was an obvious groove region in the tertiary structure, which may bind to DNA and regulate gene transcription. VDR gene was expressed in the heart, liver, spleen, lung, kidney, longissimus dorsi muscle, ovary, testis and small intestine of male and female sheep at the age of 1 month and 12 months, and the highest expression was in the small intestine. The relative expression of VDR gene in the liver, spleen, ovary, testis and small intestine of sheep at 12 months of age was significantly higher than that at 1 month of age(P<0.01), while the relative expression of VDR gene in the heart and longissimus dorsi muscle of sheep at 1 month of age was significantly higher than that at 12 months of age(P<0.01), but there was no significant difference between 1 month old sheep and 12 months old sheep in lung and kidney(P>0.05). The results indicated that VDR gene was widely expressed in various tissues of sheep, and the expression trend in each tissue/organs was different with the increase of age, suggesting the universality and multiplicity of its function. In addition, the relative expression level of VDR gene in the small intestine was the highest, suggesting that VDR gene was closely related to intestinal material absorption.