金草鱼Serpinc1基因克隆及其序列分析

Cloning and sequence analysis of the goldgrass carp Serpinc1 gene

获取金草鱼Serpinc1基因序列,研究金草鱼与草鱼Serpinc1基因的差异,并预测金草鱼Serpinc1基因序列的特征及其编码蛋白的高级结构。本研究利用RT-PCR克隆测序技术获得金草鱼Serpinc1基因序列,分析其与草鱼Serpinc1基因的差异,并通过生物信息学方法预测金草鱼Serpinc1基因序列的特征及其编码蛋白的高级结构。结果显示,金草鱼Serpinc1基因与草鱼相比共有4个核苷酸位点发生改变(c.1091T>A、c.1116A>T、c.1176T>G和c.1179C>T),其中仅有1个位点(c.1091T>A)为错义突变,导致第364位缬氨酸(Val)转变为谷氨酸(Glu)。生物信息学预测结果表明,金草鱼Serpinc1基因开放阅读框长度为1353 bp,共编码450个氨基酸,分子量为50.89 kDa,理论等电点为6.58,不稳定指数为34.55,属于不稳定蛋白;编码氨基酸包含20种标准氨基酸,其中亮氨酸最多(10.2%),而色氨酸(Trp)含量最少(1.3%);整条肽链的亲疏水平均值为-0.213,表现为亲水性;包含4个糖基化位点和37个磷酸化位点;蛋白质二、三级结构以α螺旋和无规则卷曲为主。研究结果可为深入研究金草鱼Serpinc1分子结构和功能及其发挥该功能的活性位点的验证奠定了基础,为金草鱼出血性疾病相关分子标记的筛选提供了理论基础。

In this study,the sequence of Serpinc1 gene of gold grass carp was obtained by RTPCR cloning and sequencing,the difference of Serpinc1 gene between the gold grass carp and grass carp was analyzed,further more,characteristics of the squid Serpinc1 gene and the high-level structure of the encoded protein were predicted by bioinformatics tools.The results showed that Serpinc1 gene of the gold grass carp had a total of 4 nucleotide mutations compared with grass carp(c.1091 T>A,c.1116 A>T,c.1176 T>G and c.1179 C>T),of which only One site(c.1091 T>A)was missense mutation,resulting in the conversion of the 364 th proline(Val)to glutamic acid(Glu).The results of bioinformatics prediction showed that the open reading frame of Serpinc1 gene of gold grass carp was 1353 bp in length,encoding 450 amino acids,with a molecular weight of 50.89 kDa,a theoretical isoelectric point of 6.58,and an instability index of 34.55,which is an unstable protein.20 standard amino acids were detected in Serpinc1 gene,of which leucine is the most(10.2%)and tryptophan(Trp)is the least(1.3%),the average cross-hydrophobicity of the entire peptide chain is-0.213,which is hydrophilic,and 4 glycosylation site and 37 phosphorylation sites were observed.Second and tertiary structures of Serpinc1 protein were mainly composed of alpha helices and random coils.The results can lay a foundation for further study on the molecular structure and function of the golden grass squid Serpinc1 and the verification of the active sites of this function,and provide a theoretical basis for the screening of molecular markers related to hemorrhagic diseases of the gold grass carp.