绵羊磷脂酶C-γ1基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.06.001

摘要/Abstract

摘要： 本研究旨对绵羊磷脂酶C-γ1（PLC-γ1）基因预测序列进行筛选鉴定，获取PLC-γ1基因序列并对其生物信息学进行分析。根据GeneBank （登录号：NC_040264.1）及Ensembl （登录号：ENSOARG00000001700.1）给出的绵羊PLC-γ1的4种不同预测的转录本的编码区（CDS）区进行BLAST对比，在非同源区内两端设计引物，从绵羊卵巢提取总RNA并转录成cDNA，运用RT-PCR方法扩增该特异性片段，胶回收后测序，经BLAST比对筛选出和与该片段一致基因序列。根据筛选序列的引物，运用LA Taq（GC Buffer）髙保真酶对绵羊PLC-γ1基因进行PCR扩增，胶回收产物连接PUC57-T载体并转化大肠杆菌DH5α感受态细胞，挑选单克隆进行测序。利用生物信息学技术对该基因及编码的氨基酸序列进行分析。结果显示，试验成功扩增出非同源片段330 bp，经BLAST对比分析，预测的转录本X3预测序列与其一致，连接PUC57-T载体，经测序成功扩增3 870 bp的绵羊PLC-γ1基因。绵羊PLC-γ1基因与山羊关系紧密，与牛、马、猪等家畜具有较强的进化关系，蛋白分子式为C₆₅₈₂H₁₀₁₆₀N₁₈₁₂O₁₉₆₂S₅₈。理论分子质量为147.9 ku，存在于细胞质内，且不具备跨膜性，无信号肽，为亲水性弱酸不稳定蛋白。该基因N、C端GC含量偏高，分别达80%、70%以上，具备SH2、SH3、C2等高度保守结构域，二级结构中α-螺旋、延伸链、β-转角、无规则卷曲占比分别为36.46%、16.14%、5.04%和42.36%，磷酸化位点有112个，Y428、Y509和S514为关键磷酸化位点。本研究结果为绵羊PLC-γ1蛋白表达研究提供了理论依据，对研究新疆地方品种羊的胚胎发育、提高受精率等具有重要意义。

关键词: 绵羊; 磷脂酶C-γ1(PLC-γ1)基因; 生物信息学分析

Abstract: The purpose of this study was to screen and identify the predicted sequence of phospholipase C-γ1 (PLC-γ1) gene in sheep,obtain the gene sequence and analyze its bioinformatics.Four different transcripts given by GenBank (accession No.:NC_040264.1) and Ensembl (accession No.:ENSOARG00000001700.1) were compared by BLAST.Specific primers were designed in the non-homologous region.The total RNA was extracted from sheep ovary and transcribed into cDNA,to amplify the specific fragment by RT-PCR.After gel recovery,the sample was sequenced,and the gene sequence was screened and consistent with the fragment by BLAST comparison.According to the selected primers,the sheep PLC-γ1 gene was amplified by PCR with LA Taq (GC Buffer) fidelity enzyme.The gel recovery product was ligated to PUC57-T vector and transformed into Escherichia coli DH5α competent cells,and the single clone was sequenced.According to the obtained sequence,the gene and the encoded amino acid sequence were analyzed by bioinformatics technology.The results showed that the non-homologous fragment of 330 bp,was successfully amplified,and the predicted sequence of transcript X3 was consistent with it by BLAST analysis.The PLC-γ1 gene fragment of 3 870 bp in sheep was successfully amplified by sequencing.PLC-γ1 gene in sheep was closely related to Capra hircus,and had a particularly strong evolutionary relationship with Bos taurus, Equus caballus, Sus scrofa and other livestock animals.The protein molecular formula was C₆₅₈₂H₁₀₁₆₀N₁₈₁₂O₁₉₆₂S₅₈,and the molecular weight was 147.9 ku.It was a hydrophilic weak acid unstable protein,which existed in cytoplasm and had no transmembrane and signal peptide.The N- and C-terminal GC content of this gene was relatively high,reaching 80%,70% or more.It had highly conserved domains such as SH2,SH3,and C2.The ratio of α-helix,extended chain,β-turn and random coli in the secondary structure was 36.46%,16.14%,5.04% and 42.36%,respectively.There were 112 phosphorylation sites.Y428,Y509 and S514 were the key phosphorylation sites.It provided a theoretical basis for the study of PLC-γ1 protein expression in sheep,and was of great significance for studying the embryonic development of Xinjiang local breed sheep and improving the fertilization rate.

Key words: Ovis aries; phospholipase C-γ1 (PLC-γ1) gene; bioinformatics analysis

中图分类号:

Q781

袁利明, 胡广东, 刘欣杰, 吴晓雪, 刘素平, 陈宁, 赛务加甫. 绵羊磷脂酶C-γ1基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(6): 1883-1893.

YUAN Liming, HU Guangdong, LIU Xinjie, WU Xiaoxue, LIU Suping, CHEN Ning, Saiwujiafu. Cloning and Bioinformatics Analysis of PLC-γ1 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1883-1893.

参考文献

[1] REBECCHI M J,PENTYALA S N.Structure,function,and control of phosphoinositide-specific phospholipase C[J].Physiological Reviews,2000,80(4):1291.
[2] JI Q S,ERMINI S,BAULIDA J,et al.Epidermal growth factor signaling and mitogenesis in PLC-γ1 null mouse embryonic fibroBlasts[J].Molecular Biology of the Cell,1998,9(4):749-757.
[3] PAPAKONSTANTI E A,EMMANOUEL D S,GRAVANIS A,et al.PLC-γ1 signaling pathway and villin activation are involved in actin cytoskeleton reorganization induced by Na⁺/pi cotransport up-regulation[J].Molecular Medicine,2000,6(4):303-319.
[4] SATO K I,TOKMAKOV A A,IWASAKI T,et al.Tyrosine kinase-dependent activation of phospholipase cgamma is required for calcium transient in xenopus egg fertilization[J].Developmental Biology,2000,224(2):453-469.
[5] KASSIS J,LAUFFENBURGER D,TURNER T,et al.Tumor invasion as dysregulated cell motility[J].Seminars in Cancer Biology,2001,11(9):105-119.
[6] LIAO H J,KUME T,MCKAY C,et al.Absence of erythrogenesis and vasculogenesis in PLC-γ1-deficient mice[J].Journal of Biological Chemistry,2002,277(11):9335.
[7] WING M R,BOURDON D M,HARDEN T K.Plc-epsilon:A shared effector protein in Ras-,Rho-,and G alpha beta gamma-mediated signaling[J].Molecular Interventions,2003,3(5):273-280.
[8] JI Q,WINNIER G E,NISWENDER K D,et al.Essential role of the tyrosine kinase substrate phospholipase C-γ1 in mammalian growth and development[J].Proceedings of the National Academy of Sciences,1997,94(7):2999-3003.
[9] LAWSON N D,MUGFORD J W,DIAMOND B A,et al.Phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development[J].Genes Development,2003,17(11):1346-1351.
[10] ZHANG Q,YU C,PENG S,et al.Autocrine VEGF signaling promotes proliferation of neoplastic Barrett’s epithelial cells through a PLC-dependent pathway[J].Gastroenterology,2014,146(2):461-472.
[11] MA A C H,LIANG R,LEUNG A Y H.The role of phospholipase C gamma 1 in primitive hematopoiesis during zebrafish development[J].Experimental Hema-tology,2007,35(3):368.
[12] JI Q S,WINNIER G E,NISWENDER K D,et al.Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(7):2999-3003.
[13] SCHNÖDER T M,ARREBA-TUTUSAUS P,GRIEHL I,et al.Epo-induced erythroid maturation is dependent on PLC-γ1 signaling[J].Cell Death and Differentiation,2015,22(6):974-985.
[14] COVASSIN L D,SIEKMANN A F,KACERGIS M C,et al.A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/PLC-γ1 signaling during artery development[J].Developmental Biology,2009,329(2):212-226.
[15] JING C B,CHEN Y,DONG M,et al.Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish[J].Developmental Biology,2013,374(1):24-31.
[16] FRANCESCO M,FRANCESCO L,SILVIA D,et al.Ca²⁺ signalling in endothelial progenitor cells:A novel means to improve cell-based therapy and impair tumour vascularisation[J].Current Vascular Pharma-cology,2014,12(1):87-105.
[17] STITH B J.Phospholipase C and D regulation of Src,calcium release and membrane fusion during Xenopus laevis development[J].Development Biology,2015,401(2):188-205.
[18] XU X H,JIN T.The novel functions of the PLC/PKC/PKD signaling axis in g protein-coupled receptor-mediated chemotaxis of neutrophils[J].Journal of Immunology Research,2015,2015(10):817604.
[19] LLAVERO F,ARTASO A,LACERDA H M,et al.Lck/PLCγ control migration and proliferation of interleukin (IL)-2-stimulated T cells via the Rac1 GTPase/glycogen phosphorylase pathway[J].Cellular Signalling,2016,28(11):1713-1724.
[20] 潘耀谦,李瑞珍,唐海蓉,等.家兔脑炎原虫性脑肉芽肿形成与PLCγ/PKC信号转导通路的关系[J].中国兽医科学,2017,47(12):1588-1594. PAN Y Q,LI R Z,TANG H R,et al.Relationship between cerebral granulomatous formation caused by Encephalitozoon cuniculi and PLCγ/PKC signal transduction pathway[J].Chinese Veterinary Science,2017,47(12):1588-1594.(in Chinese)
[21] YANAGIHARA M,OKADA K,NOZAKI M,et al.Cephem antibiotics and alcohol metabolism:(1) Disulfiram-like reaction resulting from intravenous administration of cephem antibiotics[J].Nihon Yakurigaku Zasshi Folia Pharmacologica Japonica,1982,79(6):551.
[22] PEREZ-QUINTERO L A,RONCAGALLI R,GUO H,et al.EAT-2,a SAP-like adaptor,controls NK cell activation through phospholipase Cγ,Ca⁺⁺,and Erk,leading to granule polarization[J].Journal of Experimental Medicine,2014,211(4):727-742.
[23] 李秀梅,邓凡,曾位森,等.人全长PLCγ1基因真核表达载体的构建及鉴定[J].第一军医大学学报,2004,2(8):849-853. LI X M,DENG F,ZENG W S,et al.Construction and identification of eukaryotic expression vector of human PLCγ1 gene[J].Journal of Southern Medical University,2004,2(8):849-853.(in Chinese)