扬子鳄Viperin基因的遗传衍化分析及表达量的测定

Cloning and evolution research and tissue expression detection of Chinese alligator Viperin gene

为研究扬子鳄Viperin基因的序列结构以及生物学功能,通过提取成年扬子鳄外周血液中的m RNA,经反转录得到c DNA,设计引物,对得到的c DNA进行PCR扩增,连接至p MD19-T载体上构建重组质粒,对PCR鉴定呈阳性的质粒进行序列测定,并进行进化树分析、同源性分析以及结构和功能预测,同时对扬子鳄不同组织中Viperin的表达水平进行测定和分析。结果显示,扬子鳄Viperin基因的完整ORF序列为1 011 bp,共编码336个氨基酸,前18个氨基酸为信号肽。成熟蛋白的分子质量为38 ku,等电点为8.72。三级结构显示,该蛋白主要由8段α螺旋,8段β折叠组成,与经典Viperin的结构一致。通过同源性和进化树分析,扬子鳄Viperin基因与湾鳄的同源性最高,为96.4%,在进化树中处于同一分支。Viperin在血液中表达量最高,在肌肉组织中表达量最低。本试验结果为扬子鳄Viperin基因的表达调控机制和功能研究奠定了基础。

In order to study the sequence structure and biological functions of Viperin gene,total RNAwas extracted from peripheral blood of an adult Chinese alligator and cDNA was synthesized with reverse transcription. The primers were designed and the cDNA was amplified according to PCR and ligated into pMDl9-Tvector. The sequence was analyzed by phylogenetie tree analysis,homology analysis, and structural prediction. The expression level of Viperin in different tissues of Alligator sinensis was detected and analyzed by qPCR. The results shows that the complete ORF of Chinese alligator Viperin gene was 1 011bp,encoding 336 amino acids and protein,the molecular weight was 38 ku and the the first 18 amino acids were signal peptides. In mature isoelectrie point was 8.72. The 3D structure showed that the protein consisted of 8 alpha helixes and 8 beta sheets. The Viperin gene shared 96.4~sequence identity with Crocodylus porosus. It was found that the highest expression level of Viperin was in the blood and the lowest was in the muscle. The results laid the foundation for further studies on the mecha- nism and function of Viperin gene expression regulation.