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

摘要/Abstract

摘要： 本研究旨在克隆山羊GATA3（GATA binding protein 3）基因，构建其真核表达载体，并对该基因进行生物信息学分析。以山羊GATA3基因编码区为种子序列（GenBank登录号：XM_018056969.1），通过SnapGene 4.1.9软件设计引物序列，应用RT-PCR方法扩增山羊GATA3基因完整编码区序列，测序鉴定后基于其核苷酸序列和蛋白质序列进行生物信息学分析，并构建pLVX-GATA3-IRES-ZsGreen1慢病毒重组质粒。利用脂质体转染方法将慢病毒重组质粒pLVX-GATA3-IRES-ZsGreen1与病毒包膜质粒pCMV-VSVG、包装质粒pNRF共转染HEK-293T细胞，进行慢病毒包装后收集病毒上清液，成功感染山羊耳尖成纤维细胞。结果显示，山羊GATA3基因编码区序列全长1 335 bp，编码444个氨基酸，分子质量为47.94 ku，分子式为C₂₀₉₃H₃₂₃₄N₆₂₆O₆₂₅S₂₄，等电点为9.47，其中丝氨酸含量最高（13.3%），色氨酸含量最低（1.1%）。山羊GATA3基因核苷酸序列与牛、猪、驴、马、小鼠及人的相似性分别为97.7%、94.2%、92.2%、92.4%、88.0%和90.1%，不同物种间相似性较高，进化过程中具有高度保守性。系统进化树分析表明，山羊与牛、猪、驴、马及人聚为一支，亲缘关系较近，与非洲蟾蜍、斑马鱼亲缘关系较远。跨膜结构域及亲/疏水性预测结果显示，山羊GATA3蛋白不存在跨膜结构域，为亲水性蛋白。信号肽预测结果表明，该蛋白定位于细胞质中。通过构建山羊GATA3基因慢病毒真核表达载体pLVX-GATA3-IRES-ZsGreen1，细胞水平上转染HEK-293T和山羊耳尖成纤维细胞，过表达GATA3基因，产生绿色荧光信号。本研究结果为山羊GATA3基因功能的研究提供了参考依据，为后续探究GATA3基因在山羊泌乳中的作用奠定了基础。

关键词: 山羊; GATA3基因; 克隆; 载体构建; 乳腺发育

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

中图分类号:

吴玉莲, 叶升, 王国栋, 刘权辉, 黄奔. 山羊GATA3基因克隆及其真核表达载体构建[J]. 中国畜牧兽医, 2021, 48(1): 12-21.

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.

参考文献

[1] PATIENT R K,MCGHEE J D.The GATA family (vertebrates and invertebrates)[J].Current Opinion in Genetics & Development,2002,12(4):416-422.
[2] CHEN C,XIANG H,PENG Y L,et al.Mature miR-183,negatively regulated by transcription factor GATA3,promotes 3T3-L1 adipogenesis through inhibition of the canonical Wnt/beta-catenin signaling pathway by targeting LRP6[J].Cellular Signalling,2014,26(6):1155-1165.
[3] FITCH S R,KAPENI C,TSITSOPOULOU A,et al.Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche[J].International Union of Bioche-mistry & Molecular Biology,2020,72(1):45-52.
[4] WANG S,LI F,LIU J,et al.Integrative analysis of methylome and transcriptome reveals the regulatory mechanisms of hair follicle morphogenesis in cashmere goat[J].Cells,2020,9(4):969.
[5] EMMANUEL N,LOFGREN K A,PETERSON E A,et al.Mutant GATA3 actively promotes the growth of normal and malignant mammary cells[J].Anticancer Research,2018,38(8):4435-4441.
[6] HAUGAS M,TIKKER L,ACHIM K,et al.Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe[J].Development,2016,143(23):4495-4508.
[7] NAVEH-MANY T,SILVER J.Transcription factors that determine parathyroid development power PTH expression[J].Kidney International,2018,93(1):7-9.
[8] LIU S,CHAN H L,BAI F,et al.Gata3 restrains B cell proliferation and cooperates with p18INK4c to repress B cell lymphomagenesis[J].Oncotarget,2016,7(39):64007-64020.
[9] VAN DE WALLE I,DOLENS A C,DURINCK K,et al.GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate[J].Nature Communications,2016,7:11171.
[10] XIAO L,MA L,WANG Z,et al.Deciphering a distinct regulatory network of TEAD4,CDX2 and GATA3 in humans for trophoblast transition from embryonic stem cells[J].Biochimica et Biophysica Acta (BBA)-Molecular Cell Research,2020,1867(9):118736.
[11] ZAIDAN N,OTTERSBACH K.The multi-faceted role of Gata3 in developmental haematopoiesis[J].Open Biology,2018,8(11):180152.
[12] PANDOLFI P P,ROTH M E,KARIS A,et al.Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis[J].Nature Genetics,1995,11(1):40-44.
[13] KOUROS-MEHR H,KIM J W,BECHIS S K,et al.GATA-3 and the regulation of the mammary luminal cell fate[J].Current Opinion in Cell Biology,2008,20(2):164-170.
[14] STEPHENS P J,TARPEY P S,DAVIES H,et al.The landscape of cancer genes and mutational processes in breast cancer[J].Nature,2012,486(7403):400.
[15] HOCH R V,THOMPSON D A,BAKER R J,et al.GATA-3 is expressed in association with estrogen receptor in breast cancer[J].International Journal of Cancer,1999,84(2):122-128.
[16] SHAHI P,WANG C Y,CHOU J,et al.GATA3 targets semaphorin 3B in mammary epithelial cells to suppress breast cancer progression and metastasis[J].Oncogene,2017,36(40):5567-5575.
[17] DOTY R T,YAN X,LAUSTED C,et al.Single-cell analyses demonstrate that a heme-GATA1 feedback loop regulates red cell differentiation[J].Blood,2019,133(5):457-469.
[18] MIMOTO M S,KWON S,GREEN Y S,et al.GATA2 regulates Wnt signaling to promote primitive red blood cell fate[J].Developmental Biology,2015,407(1):1-11.
[19] CASTAO J,ARANDA S,BUENO C,et al.GATA2 promotes hematopoietic development and represses cardiac differentiation of human mesoderm[J].Stem Cell Reports,2019,13(3):515-529.
[20] ZHONG C,ZHENG M,CUI K,et al.Differential expression of the transcription factor GATA3 specifies lineage and functions of innate lymphoid cells[J].Immunity,2020,52(1):83-95.
[21] WANG L,DI L.Wnt/β-catenin mediates AICAR effect to increase GATA3 expression and inhibit adipogenesis[J].Journal of Biological Chemistry,2015,290(32):19458-19468.
[22] GRIGORIEVA I V,OSZWALD A,GRIGORIEVA E F,et al.A novel role for GATA3 in mesangial cells in glomerular development and injury[J].Journal of the American Society of Nephrology,2019,30(9):1641-1658.
[23] ASSELIN-LABAT M L,SUTHERLAND K D,BARKER H,et al.Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation[J].Nature Cell Biology,2007,9(2):201-209.
[24] ZEITVOGEL J,JOKMIN N,RIEKER S,et al.GATA3 regulates FLG and FLG2 expression in human primary keratinocytes[J].Scientific Reports,2017,7(1):11811-11847.
[25] TSAROVINA K,REIFF T,STUBBUSCH J,et al.The Gata3 transcription factor is required for the survival of embryonic and adult sympathetic neurons[J].Journal of Neuroscience,2010,30(32):10833-10843.
[26] ANG Y,RIVAS R N,RIBEIRO A J S,et al.Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis[J].Cell,2016,167(7):1734-1749.
[27] LIU J,CHENG H,XIANG M,et al.Gata4 regulates hedgehog signaling and Gata6 expression for outflow tract development[J].PLoS Genetics,2019,15(5):e1007711.
[28] GHARIBEH L,KOMATI H,BOSSÉ Y,et al.GATA6 regulates aortic valve remodeling,and its haploinsufficiency leads to right-left type bicuspid aortic valve[J].Circulation,2018,138(10):1025-1038.
[29] BELAGULI N S,ZHANG M,GARCIA A H,et al.PIAS1 is a GATA4 SUMO ligase that regulates GATA4-dependent intestinal promoters independent of SUMO ligase activity and GATA4 sumoyla-tion[J].PLoS One,2012,7(4):e35717.
[30] KOUROS-MEHR H,SLORACH E M,STERNLICHT M D,et al.GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland[J].Cell,2006,127(5):1041-1055.
[31] ASSELIN-LABAT M,SUTHERLAND K D,BARKER H,et al.Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation[J].Nature Cell Biology,2007,9(2):103-201.
[32] KO L J,YAMAMOTO M,LEONARD M W,et al.Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer[J].Molecular and Cellular Biology,1991,11(5):2778-2784.