黔北麻羊Tβ4基因多态性及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.08.026

摘要/Abstract

摘要：

本研究旨在对黔北麻羊胸腺素β4（thymosin beta 4，Tβ4）基因的多态位点进行筛选及生物信息学分析。根据GenBank中山羊Tβ4基因序列（登录号：NC_030810和NW_017189516），应用Primer Premier 5.0软件设计引物，运用混合DNA池结合PCR产物直接测序获得黔北麻羊Tβ4基因X染色体第1、2、3外显子和3号染色体第1外显子序列进行多态性分析，利用生物信息学分析软件对黔北麻羊Tβ4基因进行同源性、系统进化树、理化性质、疏水性、卷曲螺旋、跨膜结构、信号肽、亚细胞定位、结构域、磷酸化位点、糖基化位点、二级结构和三级结构分析。结果显示，共筛选出10个SNPs，分别为：G55C、C82T、G112A、C129T、G216A、G431A、G551T、T579A、A609G和A619G。同源性比对分析结果显示，黔北麻羊与黄牛、马、野猪、人、裸滨鼠、褐家鼠、小家鼠和原鸡Tβ4基因核苷酸序列同源性分别为97.8%、96.3%、95.6%、94.1%、94.8%、91.1%、92.6%和86.7%。蛋白理化性质分析显示，黔北麻羊Tβ4蛋白理论等电点为5.02，亲水值最小为-3.011，无疏水性氨基酸，无卷曲螺旋，不是跨膜蛋白，无信号肽存在，分布在细胞核（60.9%）、线粒体（4.3%）、细胞骨架（17.4%）和细胞质（17.4%），有1个THY结构域，有1个苏氨酸磷酸化位点，无N糖基化位点，有4个O糖基化位点。二级结构分析显示，C129T突变使其蛋白质二级结构中α-螺旋增加，无规则卷曲减少。本研究成功筛选出黔北麻羊Tβ4基因多态位点，为研究黔北麻羊育种的分子遗传标记辅助选择提供了参考依据。

关键词: 黔北麻羊; Tβ 4基因; 生物信息学

Abstract:

This study was aimed to screen the polymorphism of thymosin beta 4 (Tβ4) gene in Qianbei Ma goat, and analyze the genetic structure by bioinformatic method.Primers were designed according to predicted sequence of Capra hircus Tβ4 gene in GenBank (accession No.:NC_030810 and NW_017189516) by Primer Premier 5.0.The mixed DNA pool and PCR products combined with direct sequencing were used to obtain the polymorphisms of exons 1, 2 and 3 in chromosome X and exon 1 in chromosome 3 of Tβ4 gene.The homology, phylogenetic tree, physicochemical properties, hydrophobicity, coiled coil, transmembrane structure, signal peptide, subcelluar localization, structure domain, phosphorylation site, glycosylation site, secondary structure and tertiary structure of Tβ4 gene in Qianbei Ma goat were analyzed using bioinformatic analysis softwares.The results showed that a total of 10 SNPs (G55C, C82T, G112A, C129T, G216A, G431A, G551T, T579A, A609G and A619G) were screened.The homology analysis results showed that the identity of Tβ4 gene between Qianbei Ma goat and Bos taurus, Equus caballus, Sus scrofa, Homo sapiens, Heterocephalus glaber, Rattus norvegicus, Mus musculus and Gallus gallus were 97.8%, 96.3%, 95.6%, 94.1%, 94.8%, 91.1%, 92.6% and 86.7%, respectively.Protein physical and chemical properties analysis showed that the theoretical isoelectric point of Tβ4 protein was about 5.02, the minimum value of hydrophilicity was -3.011, no hydrophobic amino acid and coiled coil existed, which was not a transmembrane protein and no signal peptide existed, was located in the nucleus (60.9%), mitochondria (4.3%), cytoskeleton (17.4%) and cytoplasm (17.4%).There was 1 THY domain, 1 threonine phosphorylation site, no N-glycosylation site and 4 O-glycosylation sites.The analysis of secondary structure showed that C129T mutation in Qianbei Ma goat made the increase of α-helix and the decrease of random coil.This results indicated that the polymorphism of Tβ4 gene in Qianbei Ma goat was successfully screened, which provided a theoretical foundation for the study of molecular genetic marker-assisted selection in breeding of Qianbei Ma goat.

Key words: Qianbei Ma goat; Tβ4 gene; bioinformatics

中图分类号:

S852.65⁺9.1

罗铮, 翁吉梅, 艾锦新, 龙安炬, 罗卫星, 蔡惠芬. 黔北麻羊Tβ4基因多态性及生物信息学分析[J]. 《中国畜牧兽医》, 2019, 46(8): 2406-2414.

LUO Zheng, WENG Jimei, AI Jinxin, LONG Anju, LUO Weixing, CAI Huifen. Polymorphism and Bioinformatics Analysis of Tβ4 Gene in Qianbei Ma Goat[J]. , 2019, 46(8): 2406-2414.

参考文献 25

[1]	GOLDSTEIN A L,SLATER F D,WHITE A.Preparation,assay and partial purification of a thymic lymphocytopoietic factor (thymosin)[J]. Proceedings of the National Academy of Sciences of the United States of America,1966,56(3):1010-1017.
[2]	HUFF T,CHRISTIAN S G,OTTO A M,et al.β thymosins,small acidic peptides with multiple functions[J]. The International Journal of Biochemistry & Cell Biology,2001,33:205-220.
[3]	LOW T L,HU S K,GOLDSTEIN A L,et al.Complete amino acid sequence of bovine thymosin β4:A thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations[J]. Proceedings of the National Academy of Sciences of the United States of America,1981,78(2):1162-1166.
[4]	ZIEGLER T,KRAUS M,HUSADA W,et al.Steerable induction of the thymosin β4/MRTF-A pathway via AAV-based overexpression induces therapeutic neovascularization[J]. Human Gene Therapy,2017,Epub ahead of print.
[5]	KIM S,KWON J.Thymosin β4 has a major role in dermal burn wound healing that involves actin cytoskeletal remodelling via heat-shock protein 70[J]. Journal of Tissue Engineering & Regenerative Medicine,2017,11(4):1262-1273.
[6]	GAO X Y,HOU F,ZHANG Z P,et al.Role of thymosin beta 4 in hair growth[J]. Molecular Genetics and Genomics,2016,291(4):1639-1646.
[7]	YUAN J,SHEN Y,YANG X,et al.Thymosin β4 alleviates renal fibrosis and tubular cell apoptosis through TGF-β pathway inhibition in UUO rat models[J]. BMC Nephrology,2017,18(1):314.
[8]	ZHENG X Y,LV Y F,LI S,et al.Recombinant adeno-associated virus carrying thymosin β4 suppresses experimental colitis in mice[J]. World Journal of Gastroenterology,2017,23(2):242-255.
[9]	SAHIN S,EKINCI O,SECKIN S,et al.Thymosin beta-4 overexpression correlates with high-risk groups in gastric gastrointestinal stromal tumors:A retrospective analysis by immunohistochemistry[J]. Pathology Research and Practice, 2017,213(9):1139-1143.
[10]	RUCHI S,KARINA R G,MARCOS R.Thymosin β4 inhibits PDGF-BB induced activation,proliferation,and migration of human hepatic stellate cells via its actin-binding domain[J]. Expert Opinion on Biological Therapy,2018,18(sup1):177-184.
[11]	WANG L,CHOPP M,SZALAD A,et al.Angiopoietin-1/Tie2 signaling pathway contributes to the therapeutic effect of thymosin β4 on diabetic peripheral neuropathy[J]. Neuroscience Research,2018,Epub ahead of print.
[12]	LEVER M,THEISS C,MOROSAN-PUOPOLO G,et al.Thymosin β4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum[J]. Histochemistry and Cell Biology,2017,147(5):555-564.
[13]	HAO Q,HE L,ZHOU J,et al.A dimeric thymosin beta 4 with novel bio-activity protects post-ischemic cardiac function by accelerating vascular endothelial cell proliferation[J]. International Journal of Cardiology,2018,261:146-154.
[14]	SKRUBER K,READ T A,VITRIOL E A.Reconsidering an active role for G-actin in cytoskeletal regulation[J]. Journal of Cell Science,2018,131(1):jcs203760.
[15]	SHAH R,REYES-GORDILLO K,CHENG Y,et al.Thymosin β4 prevents oxidative stress,inflammation,and fibrosis in ethanol-and LPS-induced liver injury in mice[J]. Oxidative Medicine & Cellular Longevity,2018,2018:9630175.
[16]	JIANG Y,HAN T,ZHANG Z G,et al.Serum thymosin beta 4 as a noninvasive biomarker in patients with nonalcoholic steatohepatitis[J]. Revista Espanola de Enfermedades Digestivas,2017,110(1):19-24.
[17]	罗卫星,史忠辉,刘贞德,等.黔北麻羊种质特性研究初报[J].山地农业生物学报,2010,29(5):466-470. LUO W X,SHI Z H,LIU Z D,et al.The preliminary investigation in characteristics of Qianbei Ma goat[J]. Journal of Mountain Agriculture and Biology,2010,29(5):466-470.(in Chinese)
[18]	罗卫星,张启林,穆林,等.黔北麻羊的肉用性能和肉质特性研究[J].西南农业学报,2010,23(5):1706-1710. LUO W X,ZHANG Q L,MU L,et al.Study on mutton performance and quality of Qianbei Ma goat[J]. Southwest China Journal of Agricultural Sciences,2010,23(5):1706-1710.(in Chinese)
[19]	ZHAO Y,SONG J,BI X,et al.Thymosin β4 promotes endothelial progenitor cell angiogenesis via a vascular endothelial growth factor dependent mechanism[J]. Molecular Medicine Reports,2018,18(2):2314-2320.
[20]	WANG X,YANG G,LI S,et al.The Escherichia coli-derived thymosin β4 concatemer promotescell proliferation and healing wound in mice[J]. Biomed Research International,2013,2013:241721.
[21]	JANARTHⅡNI R,WANG X,CHEN L,et al.A tobacco-derived thymosin β4 concatemer promotes cell proliferation and wound healing in mice[J]. Biomed Research International,2016,2016:1973413.
[22]	KLEINMAN H K,SOSNE G.Thymosin β4 promotes dermal healing[J]. Vitamins and Hormones,2016,102:251-275.
[23]	SHI B,DING Q,HE X,et al.Tβ4-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics[J]. Transgenic Research, 2017,26(1):77-85.
[24]	郭栋东.Tβ4转基因绒山羊中目的基因拷贝数及表达量与产绒性能关系的研究[D].呼和浩特:内蒙古大学,2014. GUO D D.The study of target gene copy number/expression level and production performance in Tβ4 transgenic cashmere goats[D].Hohhot:Inner Mongolia University,2014.(in Chinese)
[25]	桓蕾.阿尔巴斯白线山羊胸腺素β4毛囊发育相关信号通路的研究[D].呼和浩特:内蒙古大学,2015. HUAN L.The research of hair follicle developmental related signal pathways of thymosin beta 4 in Arbas cashmere goat[D].Hohhot:Inner Mongolia University,2015.(in Chinese)