鹅VNN1基因的克隆及组织表达分析

Molecular cloning and tissue expression analysis of VNN1 gene in goose

[目的]Vanin是一种泛酰巯基乙胺酶,在脂类代谢中发挥重要作用。本试验目的是扩增鹅Vanin基因亚型(VNN1)基因的c DNA序列,检测VNN1基因在鹅不同组织中m RNA水平的表达规律。[方法]以太湖鹅(Anser anser)肝脏总RNA为模板,采用RT-PCR和快速扩增c DNA末端(RACE)方法克隆鹅VNN1基因全长c DNA序列,并运用多种生物信息学软件对其进行分析,应用实时荧光定量PCR(q PCR)技术检测VNN1基因在不同组织的表达情况。[结果]序列分析表明:鹅VNN1基因(Gen Bank登录号:KY399733)c DNA全长1 924 bp,包含1 476 bp的开放阅读框,35 bp的5'UTR和417 bp的3'UTR,共编码491个氨基酸。经预测鹅VNN1基因编码的蛋白质由7 646个原子组成,相对分子质量为54 870.61,理论等电点为5.23,是不稳定蛋白。在线预测发现鹅与鸭的VNN1蛋白三级结构呈现高度相似的螺旋和折叠。序列分析表明,鹅与绿头鸭的VNN1基因核苷酸及氨基酸同源性较高。系统进化树分析表明,鹅与绿头鸭亲缘关系较近。q PCR结果表明,鹅VNN1基因在肝脏的表达量显著高于小肠、肾、脾和肺(P<0.01)。[结论]获得鹅VNN1基因的全长c DNA序列,该基因在肝脏中高表达。

[ Objectives ] Vanin is a class of pantetheinase and plays an important role in lipid metabolism. The purpose of this study is to amplify the cDNA sequence of goose vanin subgenotype （ VNN1 ） and detect the expression of VNN1 gene in different tissues. [ Methods ] In this study, the full-length cDNA of goose vanin subgenotype Vanin subgenotype （VNN1） gene was cloned by reverse transcription PCR and rapid amplification cDNA ends（RACE） with the total RNA from liver as template. The bioinformatics analysis and the expression detection of VNN1 mRNA in various tissues by real-time fluorescent quantitative PCR （qPCR）were performed, respectively. [ Results ] The resuhs showed that the full-length cDNA of goose VNN1 gene （GenBank No. KY399733）was 1 924 bp in length,which contained 1 476 bp open reading frame, 35 bp 5＇ UTR and 417 bp 3＇ UTR, encoding 491 amino acids. The VNN1 protein was predicted as a kind of unstable protein,which is composed of 7 646 atoms with the relative molecular weight of 54 870.61 and pI of 5.23. The three-dimensional structures of VNN1 protein prediction online between Anser anser and Anas platyrhynchos showed highly similar helix and fold. Sequence analysis showed that the nucleotide and amino acid homologies of VNN1 between Anser anser and Anas platyrhynchos were relatively high. Phylogenetic tree analysis showed the close affinity between Anser anser and Anas platyrhynchos. The qPCR results showed that the expression of goose VNN1 gene in the liver was significantly higher than that in the small intestine,kidney ,spleen and lung（ P〈0.O1 ）. [ Conclusions] This study has provided the full-length cDNA sequence of goose VNN1 gene, which is highly expressed in the liver.