文章摘要
王 敏,吴 萍,叶元土,石桃丹,魏育红.秀丽白虾4种C型凝集素基因的克隆、组织表达与进化分析[J].水产学报,2013,37(11):1632~1644
秀丽白虾4种C型凝集素基因的克隆、组织表达与进化分析
Molecular cloning,expression characterization and phylogenetic analysis of four novel C-type lectin genes in white shrimp(Exopalaemon modestus)
投稿时间:2013-07-12  修订日期:2013-09-13
DOI:DOI:10.3724/SP.J.1231.2013.38807
中文关键词: 秀丽白虾  C型凝集素  克隆  组织分布  阳性选择
英文关键词: Exopalaemon modestus  C-type lectin  cloning  tissue expression pattern  positive selection
基金项目:国家自然科学基金项目(31101880/C190101);苏州市应用基础研究计划(SYN201103);2011苏州大学公派出国访学资助项目
作者单位
王 敏 苏州大学基础医学与生物科学学院 
吴 萍 苏州大学基础医学与生物科学学院 
叶元土 苏州大学基础医学与生物科学学院 
石桃丹 苏州大学基础医学与生物科学学院 
魏育红 苏州大学基础医学与生物科学学院 
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中文摘要:
      C型凝集素是一类Ca2 依赖活性的糖蛋白,在虾蟹类的免疫防御中发挥着重要作用。采用RT-PCR及RACE技术首次克隆了秀丽白虾C型凝集素家族4个基因( EmCTL-1、EmCTL-2、EmCTL-3、EmCTL-4)的cDNA全长。这4个C型凝集素基因的cDNA全长分别为1 390、1 119、1 364和1 299 bp,分别含996、969、 1 026和1 044 bp的开放阅读框,各编码332、322、341和347个氨基酸的蛋白。其氨基酸序列与罗氏沼虾相应的C型凝集素的相似性分别为40%、37%、69%和73%, 与其他海水虾类的相似性在30%~41%之间。阳性选择模型分析显示虾类的C型凝集素基因在进化过程中经历了阳性选择。进化树分析表明,秀丽白虾的 C型凝集素基因与罗氏沼虾的亲缘关系最近,并且除EmCTL-4属于十足类C型凝集素亚群A外,其他均属于亚群C。组织表达分析表明,EmCTL-1主要在血液中表 达,EmCTL-2主要在性腺、血液和胸神经节中表达,而EmCTL-3和EmCTL-4则主要在精巢中表达。研究表明,秀丽白虾的C型凝集素基因具有较快的 进化特点,同时又具备凝集素行使免疫功能时必需的保守氨基酸残基及钙离子结合位点,精巢和血液是秀丽白虾C型凝集素基因主要的表达器官。
英文摘要:
      Exopalaemon modestus is one of the main shrimps which has high economic value.But its genetic information and functional gene background are rarely known so far.In this study,the cDNAs of four novel C-type lectins(designated as EmCTL-1,EmCTL-2, EmCTL-3 and EmCTL-4)were cloned from the ovary of white shrimp,E.modestus,by means of PCR and 5′and 3′rapid amplification of cDNA ends (RACE).The full-length cDNAs consist of 1 390,1 119,1 364 and 1 299 bp with one open reading frame consisting of 996,969,1 026 and 1 044 bp,encoding 332,322,341 and 347 amino acids respectively.Their deduced amino acid sequences contained a putative signal peptide of 19,21,20 and 17 amino acid residues respectively.The four C-type lectin genes all contained two carbohydrate recognition domains,CRD1 and CRD2.The CRD1 was composed of 128 residues in EmCTL-2 and EmCTL-3,127 residues in EmCTL-1 and 126 residues in EmCTL-4. And the CRD2 was composed of 127 residues in EmCTL-1 and EmCTL-3,129 residues in EmCTL-2 and 130 residues in EmCTL-4.Both the CRD1 and CRD2 had 6 conserved cysteine residues and 3 conserved tryptophane residues.The motif of Ca2 binding sites 2 were EPA and FND in EmCTL-1,EPA and FHD in EmCTL-2,WHD and FDD in EmCTL-3 and EPD and FRD in EmCTL-4.So EmCTL-1,EmCTL-2 and EmCTL-4 can be deduced binding with mannose in immune reactions.And the four C-type lectin genes shared 40%,37%,69% and 73% identity with the parallel C-type lectin genes in Macrobrachium rosenbergii,existing in Palaemonidae.The CODEML program implemented in the PAML package was used to test for variations in selective pressures of shrimp and prawns C-type lectin genes with two CRDs,included the four genes acquired in this study.The result showed that the shrime and prawns lectin genes with two CRDs had undergone positive selection.According to the LTR test,3.8% of the sites were driven by positive selection in the M2a model,while 5.6% of the sites were driven by positive selection in the M8 model.Phylogenetic analysis showed that the lectins from the decapods could be divided into four clear clusters.Cluster C included the dual-CRD lectins while cluster D includ the single-CRD lectins,and cluster A included the dual-CRD lectins except the PmCTL-1 in tiger shrimp,cluster B included the single-CRD lectins except the FcCTL in Chinese shrimp.It indicated that the numbers of CRD played a key role in phyletic evolution.The results showed that EmCTL-1,EmCTL-2, EmCTL-3 belonged to cluster C and EmCTL-4 belonged to cluster A.Four novel C-type lectin genes in E.modestus had a close evolutionary relationship to the C-type lectins of giant freshwater prawn.Tissue distribution analysis using semi-quantitative RT-PCR methods showed that EmCTL-1 was main expressed in hemolymph,testis and ovary.EmCTL-3 and EmCTL-4 could be detected main in testis,ovary,hepatopancreas,hemolymph,heart and thoracic ganglion while EmCTL-2 was mainly expressed in ovary,testis,hemolymph,thoracic ganglion and heart.The results showed that the hemolymph was the mainly expression tissue of four C-type lectin genes in E.modestus.Interestingly,and the testis was the organ with highest expression level of C-type lectin transcripts in this study.It can be speculated that the C-type lectins in E.modestus may agglutinate microorganisms in the hemolymph for possessing signal peptides,conserved cysteine residues,conserved tryptophane residues,high expression level in hemolymph and observed positive selection.
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