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

Abstract

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

CLC Number:

Q78
S828

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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Sequence Analysis, Eukaryotic Expression Vector Construction and Tissue Expression of ABCD4 Gene in Bama Miniature Pigs

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