巴马小型猪ABCD4基因序列分析、真核表达载体构建及组织表达分析

doi:10.16431/j.cnki.1671-7236.2023.11.003

摘要/Abstract

摘要： 【目的】获取巴马小型猪ATP结合盒D亚科成员4（ATP binding cassette subfamily D member 4，ABCD4）基因CDS区序列并预测其编码蛋白的结构功能，构建真核表达载体，了解ABCD4基因在巴马小型猪中的组织分布情况，为探究ABCD4基因对巴马小型猪生长发育的作用提供理论和分子基础。【方法】以巴马小型猪皮下脂肪组织cDNA为模板，利用RT-PCR对ABCD4基因CDS区进行扩增、测序，利用生物信息学分析软件对巴马小型猪ABCD4基因CDS区序列与不同物种比对，构建系统进化树，并对ABCD4蛋白的理化性质、跨膜结构等进行预测。将获得的目的基因连接至pEGFP-N1载体并转染到IPEC-J2细胞，检测转染后荧光和基因表达量变化。通过实时荧光定量PCR检测巴马小型猪各组织ABCD4基因表达量。【结果】巴马小型猪ABCD4基因CDS区全长1 818 bp，编码605个氨基酸，与Ensembl上猪参考序列（ENSSSCT00000037529）的相似性为99.7%，存在6处碱基突变和1处碱基插入。相似性比对发现，猪和人、原鸡、倭黑猩猩、马、双峰驼、牛ABCD4序列的相似性分别为87.6%、72.7%、87.6%、91.7%、88.1%和88.6%。系统进化树显示，猪与马亲缘关系最近，与原鸡亲缘关系最远。生物信息学分析显示，ABCD4蛋白分子质量为68.66 ku，理论等电点（pI）为7.13，存在5个跨膜螺旋结构，无信号肽，属于跨膜蛋白；存在58个磷酸化位点和10个N-糖基化位点；二级结构中α-螺旋、β-转角、无规则卷曲、延伸链占比分别为50.91%、3.64%、28.43%和17.02%。细胞转染试验结果显示，转染30 h后重组质粒组和空载体对照组均有荧光出现，且转染重组质粒的细胞内ABCD4基因表达量极显著高于空载体对照组（P<0.01）。实时荧光定量PCR结果显示，ABCD4基因在巴马小型猪皮下脂肪中表达量最高，显著高于其他组织（P<0.05）；在心脏中表达量最低。【结论】本试验成功扩增获得巴马小型猪ABCD4基因CDS区序列，ABCD4蛋白是一种疏水性跨膜蛋白，主要在巴马小型猪皮下脂肪中表达。本试验结果为探究ABCD4基因对猪背膘厚的影响提供了分子依据。

关键词: 巴马小型猪; ABCD4基因; 序列分析; 真核表达载体; 组织表达

Abstract: 【Objective】 The purpose of this study was to obtain the CDS region sequence of ATP binding cassette subfamily D member 4 (ABCD4) gene in Bama Miniature pigs, predict the structure and function of ABCD4 protein, construct eukaryotic expression vectors, understand the tissue distribution of ABCD4 gene in Bama Miniature pigs, and provide a theoretical and molecular basis for exploring the effect of ABCD4 gene on the growth and development of Bama Miniature pigs.【Method】 Using cDNA of subcutaneous adipose in Bama Miniature pigs as the template, the CDS region of ABCD4 gene was amplified by RT-PCR and sequenced.The CDS region sequence of ABCD4 gene in Bama Miniature pigs was compared with different species by bioinformatics analysis software, and the phylogenetic tree was constructed.Meanwhile, the physicochemical properties and transmembrane structure of ABCD4 protein were predicted.The obtained target gene was ligated into pEGFP-N1 vector and transfected into IPEC-J2 cells, and then the fluorescence and gene expression changes were detected.The expression of ABCD4 gene in different tissues of Bama Miniature pigs were detected by Real-time quantitative PCR.【Result】 The CDS region of ABCD4 gene in Bama Miniature pigs was 1 818 bp in length, encoding 605 amino acids with 99.7% similarity to the porcine reference sequence on Ensembl(ENSSSCT00000037529).There were 6 base mutations and 1 base insertion.The similarity of ABCD4 sequence between Bama Miniature pigs and Homo sapiens, Gallus gallus, Pan troglodytes, Equus caballus, Camelus bactrianus and Bos taurus were 87.6%, 72.7%, 87.6%, 91.7%, 88.1% and 88.6%, respectively.The phylogenetic tree showed that the Bama Miniature pigs was closest distant to Equus caballus and the most distant to Gallus gallus.Bioinformatics analysis showed that the molecular weight of ABCD4 protein was 68.66 ku, the theoretical isoelectric point (pI) was 7.13.ABCD4 protein was predicted to have 5 transmembrane helical structures without signal peptide, which belonged to a transmembrane protein.The protein estimated 58 phosphorylation sites and 10 N-glycosylation sites.The secondary structure of ABCD4 protein was composed of 50.91% alpha helix, 3.64% beta turn, 28.43% random coil and 17.02% extended chain.The results of cell transfection showed that there were fluorescence in recombinant plasmid and empty plasmid control groups after transfection 30 h.The expression of ABCD4 gene in the cells transfected with the recombinant plasmid was extremely significantly higher than empty plasmid control group (P<0.01).Real-time quantitative PCR results showed that the expression of ABCD4 gene was the highest in subcutaneous adipose of Bama Miniature pigs, which was significantly higher than that in other tissues (P<0.05), the expression was the lowest in heart of Bama Miniature pigs.【Conclusion】 In this study, the CDS sequence of ABCD4 gene in Bama Miniature pigs was successfully amplified and obtained.ABCD4 protein was a hydrophobic transmembrane protein, which was mainly expressed in subcutaneous adipose.The results provided a molecular basis for exploring the effect of ABCD4 gene on backfat thickness of pigs.

Key words: Bama Miniature pigs; ABCD4 gene; sequence analysis; eukaryotic expression vector; tissue expression

中图分类号:

Q78
S828

邢天琦, 许迪, 颜港, 王钰滨, 王梦影, 梁晶, 兰干球. 巴马小型猪ABCD4基因序列分析、真核表达载体构建及组织表达分析[J]. 中国畜牧兽医, 2023, 50(11): 4338-4348.

XING Tianqi, XU Di, YAN Gang, WANG Yubin, WANG Mengying, LIANG Jing, LAN Ganqiu. Sequence Analysis, Eukaryotic Expression Vector Construction and Tissue Expression of ABCD4 Gene in Bama Miniature Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4338-4348.

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