山羊GATA3基因克隆及其真核表达载体构建

doi:10.16431/j.cnki.1671-7236.2021.01.002

Abstract

Abstract: This study was aimed to clone GATA binding protein 3 (GATA3) gene in goats,construct the eukaryotic expression vector,and conduct bioinformatics analysis of GATA3 gene.The coding region(CDS) of GATA3 gene in goats was used as seed sequence (GenBank accession No.:XM_018056969.1).SnapGene 4.1.9 software was used to design primer sequence and RT-PCR method was used to amplify the complete CDS of GATA3 gene in goats.After sequencing and identification,bioinformatics analysis was conducted based on the nucleotide sequence and protein sequence,and pLVX-GATA3-IRES-ZsGreen1 lentivirus recombinant plasmid was constructed.The recombinant lentivirus plasmid pLVX-GATA3-IRES-ZsGreen1 was co-transfected with virus enveloped plasmid pCMV-VSVG and packaged plasmid pNRF into HEK-293T cells by liposome transfection method.The virus supernatant was collected after lentivirus packaging,and the ear fibroblasts in goats were successfully infected.The results of bioinformatics analysis showed that the total length of GATA3 gene CDS was 1 335 bp,444 amino acids were encoded,the molecular weight was 47.94 ku,the molecular formula was C₂₀₉₃H₃₂₃₄N₆₂₆O₆₂₅S₂₄,and the isoelectric point was 9.47,the contents of serine was the highest (13.3%),and tryptophan was the lowest (1.1%).The similarity of the nucleotide sequence of GATA3 gene in goats were 97.7%,94.2%,92.2%,92.4%,88.0% and 90.1% with Bos taurua,Sus scrofa,Equus asinus,Equus caballus,Mus musculus and Homo sapiens,respectively.The homology among different species was high and the evolution process was highly conservative.The phylogenetic tree results showed that goats were closely related to Bos taurua,Sus scrofa,Equus asinus,Equus caballus and Homo sapiens,but far related to Xenopus tropicalis and Danio rerio.Transmembrane domain and hydrophilic/hydrophobic prediction showed that GATA3 protein in goats did not exist transmembrane domain and was hydrophilic protein.The prediction of the signal peptide indicated that the protein was localized in the cytoplasm.GATA3 gene was overexpressed to produce green fluorescence signal by constructing the eukaryotic expression vector pLVX-GATA3-IRES-ZsGreen1 and transfecting with HEK-293T and ear fibroblasts in goats at the cell level.This results would provide a reference for the functional study of GATA3 gene in goats,and laid a foundation for further study on the role of GATA3 gene in lactation of goats.

Key words: goats; GATA3 gene; cloning; vector construction; breast development

CLC Number:

WU Yulian, YE Sheng, WANG Guodong, LIU Quanhui, HUANG Ben. Cloning and Construction of Eukaryotic Expression Vector of GATA3 Gene in Goats[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 12-21.

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Cloning and Construction of Eukaryotic Expression Vector of GATA3 Gene in Goats

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