新西兰白兔SOD1基因编码区克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.08.006

摘要/Abstract

摘要：

试验旨在克隆新西兰白兔SOD1基因CDS区并对其进行生物信息学分析。根据GenBank中穴兔SOD1基因序列（登录号：NM_001082627.2）设计特异性引物，利用RT-PCR技术扩增并克隆新西兰白兔SOD1基因完整的CDS区序列，测序鉴定后与其他物种进行同源性比对并构建系统进化树，使用SOPMA、SWISS-MODEL、ExPASy、ProtParam等软件和在线工具对蛋白序列进行分析，并预测蛋白分子的亚细胞定位及互作网络。结果显示，新西兰白兔SOD1基因CDS区长度为459 bp，A、T、C、G碱基含量分别为24.84%、18.95%、23.09%和33.12%，GC含量大于AT含量，编码153个氨基酸。同源性比对结果发现，新西兰白兔与穴兔、卷尾猴、人、黑猩猩、普通狨、骆驼、家犬、猕猴、野猪、小鼠的同源性分别为99.8%、85.2%、85.0%、84.7%、84.1%、83.9%、83.9%、83.7%、83.2%和81.3%。系统进化树结果显示，新西兰白兔和穴兔进化关系最近，并单独聚成一个分支。新西兰白兔SOD1蛋白分子质量为15.7 ku，理论等电点（pI）为5.86，分子式为C₆₇₂H₁₀₈₄N₂₀₀O₂₂₁S₅，不稳定指数为23.79，脂肪系数为78.89，平均亲水指数（GRAVY）为-0.276；属水溶性蛋白，无信号肽及跨膜区。SOD1蛋白包含5个潜在的磷酸化修饰位点和3个潜在的糖基化位点；在45-120位氨基酸处存在1个保守的活性中心；二级结构以无规则卷曲为主，占53.59%；三级结构建模显示蛋白易形成同源二聚体。亚细胞定位结果显示，细胞质、细胞核和线粒体分别占65.2%、26.1%和8.7%；SOD1蛋白在体内能与SOD2、PRDX1、PRDX3、PRDX4、DERL1等分子形成互作网络。本试验结果为进一步深入研究SOD1基因的功能及探索其与相关疾病的关系提供了参考依据。

关键词: 新西兰白兔; SOD1基因; 克隆; 生物信息学; 预测

Abstract:

This study was aimed to clone and analyze the CDS region of SOD1 gene in New Zealand White rabbits.Specific primers were designed according to the SOD1 gene sequence of Oryctolagus cuniculus (accession No.:NM_001082627.2) in GenBank, then the complete CDS region of SOD1 gene in New Zealand White rabbits was amplified by RT-PCR and cloned into T vector.After sequencing and identification, the homology was compared with other species and phylogenetic tree was constructed.SOPMA, SWISS-MODEL, ExPASy, ProtParam and other software and online tools were used to analyze the protein sequence, the subcellular localization and interaction network of SOD1 protein were predicted.The results showed that the CDS region of SOD1 gene in New Zealand White rabbits was 459 bp in length, the contents of A, T, C and G bases were 24.84%, 18.95%, 23.09% and 33.12%, respectively, and the content of GC was higher than that of AT, which coding 153 amino acids.The homologies of SOD1 gene CDS between New Zealand White rabbit and Oryctolagus cuniculus, Cebus apella, Homo sapiens, Pan troglodytes, Callithrix jacchus, Camelus dromedarius, Canis lupus familiaris, Macaca mulatta, Sus scrofa and Mus musculus were 99.8%, 85.2%, 85.0%, 84.7%, 84.1%, 83.9%, 83.9%, 83.7%, 83.2% and 81.3%, respectively.The phylogenetic tree results showed that New Zealand White rabbits and Oryctolagus cuniculus had the closest evolutionary relationship, and clustered into a single branch.The molecular weight of SOD1 protein in New Zealand White rabbits was 15.7 ku, the theoretical isoelectric point (pI) was 5.86, the formula was C₆₇₂H₁₀₈₄N₂₀₀O₂₂₁S₅, the instability index, fat coefficient and average hydrophilic index (GRAVY) were 23.79, 78.89 and -0.276, respectively.SOD1 was a hydrophilic protein, with no signal peptide and transmembrane region.SOD1 protein contained 5 potential phosphorylation modification sites and 3 potential glycosylation sites, and there was a conserved active site at 45-120 amino acids.The secondary structure was mainly random coil, accounting for 53.59%.The tertiary structure modeling showed that the protein was easy to form homologous dimer.Subcellular localization results showed that the cytoplasm, nucleus and mitochondria accounted for 65.2%, 26.1% and 8.7%, respectively.SOD1 protein could interact with SOD2, PRDX1, PRDX3, PRDX4 and DERL1 molecules in vivo.The results provided a reference for further study of the function of SOD1 gene and the relationship between SOD1 gene and related diseases.

Key words: New Zealand White rabbit; SOD1 gene; cloning; bioinformatics; prediction

中图分类号:

邓小亮, 马文康, 洪玮, 付欣, 谭茵. 新西兰白兔SOD1基因编码区克隆及生物信息学分析[J]. 《中国畜牧兽医》, 2019, 46(8): 2236-2245.

DENG Xiaoliang, MA Wenkang, HONG Wei, FU Xin, TAN Yin. Cloning and Bioinformatics Analysis of SOD1 Gene in New Zealand White Rabbits[J]. , 2019, 46(8): 2236-2245.

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