巴马香猪CIDEa基因克隆及组织表达分析

doi:10.16431/j.cnki.1671-7236.2021.12.002

摘要/Abstract

摘要： 本试验旨在对巴马香猪CIDEa基因序列进行克隆,预测其蛋白结构和功能,并进行组织表达分析。根据NCBI已经公布猪的CIDEa基因序列(登录号:NM_001112696.2),利用Oligo 7.0软件设计引物,采用RT-PCR法扩增并克隆巴马香猪CIDEa基因序列,利用生物信息学软件分析其核苷酸序列及编码蛋白的疏水性、理化性质、跨膜结构域、二级结构、三级结构及互作蛋白,并采用实时荧光定量PCR方法检测CIDEa基因在巴马香猪皮下脂肪、肝脏、肾脏、肺脏、脾脏、背最长肌及心脏的表达规律。结果显示,试验成功获得巴马香猪CIDEa基因CDS区序列,全长660 bp,编码219个氨基酸,与GenBank中猪、牛、马、犬、人、猕猴和家鼠的核苷酸相似性分别为99.4%、84.7%、81.5%、80.9%、79.7%、78.9%和76.1%。与GenBank中野猪CIDEa基因核苷酸序列比对发现,发生4处碱基突变,其中A609G和T627C位点为同义突变,C173T(Pro→Leu)和C631T(Arg→Cys)位点为错义突变。巴马香猪CIDEa蛋白分子质量为24 483.57 u,等电点为9.48,不稳定指数为52.86,属于不稳定蛋白;该蛋白中亮氨酸(Leu)含量最高(13.2%),色氨酸(Trp)含量最低(0.5%)。巴马香猪CIDEa蛋白为膜外亲水性蛋白,包含1个超家族结构域(CIDE-N)。二级结构预测发现,巴马香猪CIDEa蛋白包含α-螺旋(45.66%)、β-转角(5.94%)、延伸链(14.61%)和无规则卷曲(33.79%),三级结构预测结果与二级结构一致。蛋白互作分析结果表明,巴马香猪CIDEa蛋白与PPARG、PPARGC-1、COX8H、PRDM16、DIO2、TMEM26、ELOVL3、COX7A1、CIDEC、DFFB蛋白存在相互作用。实时荧光定量PCR结果显示,CIDEa基因在巴马香猪皮下脂肪中表达量最高,且显著高于其他组织(P<0.05),心脏中表达量最低。研究结果为进一步探讨CIDEa基因对巴马香猪脂肪沉积的调控机制提供理论依据。

关键词: 巴马香猪; CIDEa基因; 克隆; 表达

Abstract: The purpose of this experiment was to clone the CIDEa gene sequence of Bama Xiang pig, predict its structure and function, and analyze its tissue expression.According to the CIDEa gene sequence of Sus scrofa published by NCBI (accession No.: NM_001112696.2), primers were designed with Oligo 7.0 software, and the CIDEa gene sequence of Bama Xiang pig was amplified and cloned by RT-PCR.The nucleotide sequence and the hydrophobicity, physicochemical properties, transmembrane domain, secondary structure, tertiary structure and interaction proteins of the encoded proteins were analyzed by bioinformatics software.The expression of CIDEa gene in subcutaneous fat, liver, kidney, lung, spleen, longissimus dorsi muscle and heart of Bama Xiang pigs were detected by Real-time quantitative PCR.The results showed that the CDS regions of CIDEa gene in Bama Xiang pig was successfully obtained, which was 660 bp in length, encoded 219 amino acid.The nucleotide similarity with Sus scrofa, Bos taurus, Equus caballus, Canis lupus familiaris, Homo sapiens, Macaca mulatta and Mus musculus in GenBank were 99.4%, 84.7%, 81.5%, 80.9%, 79.7%, 78.9%, and 76.1%, respectively.Compared with the nucleotide sequence of Sus scrofa CIDEa in GenBank, there were four base mutations, of which A609G and T627C were synonymous mutations, and C173T (Pro→Leu) and C631T (Arg→Cys) were missense mutations.The molecular weight of CIDEa protein in Bama Xiang pig was 24 483.57 u, the isoelectric point was 9.48, and the instability index was 52.86, indicating that the protein was unstable.The protein contained the highest Leu (13.2%) and the lowest Trp (0.5%).CIDEa protein in Bama Xiang pig was an extramembrane hydrophilic protein, which contains a superfamily domain (CIDE-N).The secondary structure prediction of the protein showed that the percentage of alpha helix, beta turn, extended chain, random coil of CIDEa protein in Bama Xiang pig was 45.66%, 5.94%, 14.61% and 33.79%, respectively.The predicted results of the tertiary structure were consistent with those of the secondary structure.Protein interaction analysis showed that CIDEA protein of Bama Xiang pig interacted with PPARG, PPARGC-1, COX8H, PRDM16, DIO2, TMEM26, ELOVL3, COX7A1, CIDEC, DFFB proteins.The results of Real-time quantitative PCR showed that the expression of the CIDEa gene was the highest in subcutaneous fat of Bama Xiang pig and significantly higher than that in other tissues (P<0.05), and the lowest expression in heart.The results provided a theoretical basis for further exploring the regulatory mechanism of CIDEa gene on fat deposition in Bama Xiang pig.

Key words: Bama Xiang pig; CIDEa gene; cloning; expression

中图分类号:

S828.8⁺9

罗云彦, 韦崇万, 刘春艳, 刘港怡, 蒋钦杨, 黄艳娜. 巴马香猪CIDEa基因克隆及组织表达分析[J]. 中国畜牧兽医, 2021, 48(12): 4339-4347.

LUO Yunyan, WEI Chongwan, LIU Chunyan, LIU Gangyi, JIANG Qinyang, HUANG Yanna. Cloning and Tissue Expression of CIDEa Gene in Bama Xiang Pig[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4339-4347.

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