草原红牛Acot2基因克隆及其在各组织中的表达差异研究

doi:10.16431/j.cnki.1671-7236.2019.11.003

摘要/Abstract

摘要： 本研究旨在探究草原红牛酰基辅酶A硫酯酶2（Acot2）的基因功能，并对其进行生物信息学分析，检测Acot2基因在草原红牛不同组织中的表达差异。根据GenBank中公布的牛Acot2基因序列（登录号：NM_001101938.1）设计引物，PCR扩增获得草原红牛Acot2基因的完整CDS并进行测序，利用分析软件进行序列同源性比对并构建系统进化树；获得对应的氨基酸序列并分析蛋白理化特性及蛋白亚细胞结构、亲疏水性和磷酸化位点，预测蛋白二级结构并构建蛋白质三级结构模型；利用实时荧光定量PCR方法检测Acot2基因在不同组织中的表达差异。结果显示，草原红牛Acot2基因CDS大小为1 395 bp，编码464个氨基酸，其核苷酸序列与亚洲水牛的同源性较高（98.3%），与猕猴和黑猩猩的同源性较低（80.5%和80.4%）。Acot2蛋白分子式为C₂₃₁₇H₃₆₀₆N₆₄₀O₆₂₈S₁₄，分子质量为50.924 ku，理论等电点为8.84。蛋白质不稳定指数为37.50，氨基酸残基多数为亲水性残基，总平均亲水性为-0.094。亚细胞定位分析表明，Acot2蛋白分布在内质网（30.4%）、线粒体（26.1%）、高尔基体（17.4%）、细胞质（17.4%）、液泡（4.3%）和细胞质（4.3%）中；磷酸化位点分析发现，Acot2蛋白存在20个磷酸化位点。二级结构主要形式有α-螺旋（21.8%）、β-转角（33.4%）、β-折叠（18.4%）和无规则卷曲（26.4%），三级结构预测结果与其相一致。实时荧光定量结果显示，Acot2基因在草原红牛胃中表达量最高，在肺脏中表达量极少。Acot2基因在生物进化过程中具有低保守性，其编码氨基酸组成的蛋白质结构稳定，属于水溶性蛋白，在线粒体和内质网中发挥作用，在草原红牛不同组织的表达量有明显差异。本研究结果为进一步探究Acot2基因对家畜脂代谢的影响和筛选草原红牛肉质候选基因提供资料。

关键词: 草原红牛; Acot2基因; 克隆; 生物信息学分析; 表达差异

Abstract: This experiment was aimed to study the gene function of Acot2 in Red steppe using bioinformatics,and investigate the mRNA expression of Acot2 gene in different tissues of Red steppe.Primers were designed according to the sequence of cattle Acot2 gene (accession No.:NM_001101938.1) in GenBank.The complete CDS region of Acot2 gene was cloned by RT-PCR and sequencing.Sequence homology alignment and phylogenetic tree of Acot2 gene constructed by software.The corresponding amino acid sequence was obtained.The physical and chemical property,subcellular structure,hydrophilicity and hydrophobicity and phosphorylation site were analyzed by online prediction software.Protein secondary and tertiary structures were predicted by DNAStar software and SWISS-MODEL.Real-time PCR was used to detect the mRNA expression of Acot2 gene in different tissues of Red steppe.The results showed that the length of Acot2 gene CDS was 1 395 bp,encoding 464 amino acids.Acot2 gene CDS in Red steppe had higher homology with Bubalus bubalis(98.3%),but lower homology with Macaca mulatta and Pan troglodytes (80.5% and 80.4%).The molecular formula of Acot2 protein was C₂₃₁₇H₃₆₀₆N₆₄₀O₆₂₈S₁₄,the molecular weight was 50.924 ku,the theoretical isoelectric point was 8.84.Most of the amino acid residues were hydrophilic residues,and the total average hydrophilicity was -0.094.The results of subcellular localization showed that Acot2 protein was located in the endoplasmic (30.4%),mitochondria (26.1%),golgi (17.4%),cytoplasmic (17.4%),vacuolar (4.3%) and nuclear (4.3%).There were 20 phosphorylation sites.The main forms of the secondary structure were alpha helix (21.8%),beta turn (33.4%),beta fold (18.4%) and random coil (26.4%),which was the same as the prediction of the tertiary structure.Real-time PCR result showed that the expression of Acot2 gene in Red steppe was the highest in stomach,it was rarely expressed in lung.The Acot2 gene was lowly conserved in the process of biological evolution.Acot2 protein was structurally stable,and it belonged to water soluble protein.The protein played a role in mitochondria and endoplasmic.The expression of Acot2 gene in different tissues of Red steppe was significant difference.This study provided the basis for further investigation of the effects of Acot2 gene on lipid metabolism in livestock and the screening of quality candidate genes in Red steppe.

Key words: Red steppe; Acot2 gene; cloning; bioinformatics analysis; expression difference

中图分类号:

刘理想, 高一, 吕阳, 薛佳佳, 胡忠昌, 张国梁. 草原红牛Acot2基因克隆及其在各组织中的表达差异研究[J]. 中国畜牧兽医, 2019, 46(11): 3154-3162.

LIU Lixiang, GAO Yi, LYU Yang, XUE Jiajia, HU Zhongchang, ZHANG Guoliang. Cloning of Acot2 Gene and Its Expression Difference of Different Tissues in Red Steppe[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3154-3162.

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