魁蚶C型凝集素基因cDNA的克隆及表达分析  

Molecular Cloning and Expression Analysis of C-Type Lectin from Scapharca broughtonii

作　　者：沈淑芳[1,2] 朱玲[2] 李加琦[2,3] 薛素燕 李阳[1,2] 陈琼琳 毛玉泽[2,3] 庄志猛[2] 方建光[2,3] SHEN Shufang;ZHU Ling;LI Jiaqi;XUE Suyan;LI Yang;CHEN Qionglin;MAO Yuze;ZHUANG Zhimeng;FANG Jianguang(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071;Laboratory for Marine Ecology and Environmental Science,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266071)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]农业部海洋渔业可持续发展重点实验室,中国水产科学研究院黄海水产研究所,青岛266071 [3]青岛海洋科学与技术国家实验室,海洋生态与环境科学功能实验室,青岛266071

出　　处：《渔业科学进展》2018年第1期128-136,共9页Progress in Fishery Sciences

基　　金：国家自然基金委员会-山东省人民政府联合资助海洋科学研究中心项目(U1406403)、国家海洋局海洋公益性行业科研专项经费项目(201205031;201305043)和国家贝类产业技术体系(CARS-48)共同资助

摘　　要：通过cDNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆得到魁蚶(Scapharca broughtonii)C型凝集素(C-type lectin,Sb-Lec1)基因,该基因全长为700 bp,其中,5′-UTR为29 bp,3′-UTR为167 bp,开放阅读框长度为504 bp,编码167个氨基酸,包括长度为23个氨基酸的信号肽序列、129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 k Da,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)和海湾扇贝(Argopecten irradians)C型凝集素的同源性分别为38%~40%、34%~35%和38%~39%,Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示,魁蚶先与贝类聚为一支,再与脊椎动物聚在一起,表明魁蚶Sb-Lec1基因在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术,检测了Sb-Lec1基因在组织中的表达情况,发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达,其中,肝胰腺表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示,与对照组相比,菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P<0.05),随着刺激时间的延长,表达量呈先升高后降低的趋势。本研究表明,魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。The current study cloned the full-length cDNA of C-type lectin(Sb-Lec1)using RACE(Rapid amplification of cDNA ends)method from Scapharca broughtonii with 700 bp that includes a 5′UTR of 29 bp and 3′UTR of 167 bp.The 504 bp open reading frame(ORF)encodes a polypeptide of 167 amino acids,including a signal peptide of 23 amino acids,one carbohydrate-recognition domain(CRD)motif of 129 amino acids and 6 cysteines involved in the formation of disulfide bond.The predicted protein molecular weight is 19.11 kDa,with a theory isoelectric point of 4.74.Multiple sequences alignment and phylogeny analysis showed that the identity of Sb-Lec1 gene shared with Crassostrea gigas,Mytilus galloprovincialis,and Argopecten irradians was 38%~40%,34%~35%,and 38%~39%,respectively.The amino acids of CRD motif had many similarities with other species such as 4 conserved Cys.Phylogenetic analysis revealed two main branches including all C-type lectin of molluscs and the C-type lectin of vertebrate,and that the deduced polypeptide of Sb-Lec1 had the characteristics of the C-type lectin family.Quantitative real-time PCR(qRT-PCR)was used to assess the mRNA expression in all tested tissues,including hemocytes,foot,adductor muscle,mantle,gill,and hepatopancreas.The highest and lowest Sb-Lec1 mRNA were in hepatopancreas and adductor muscle,respectively.Vibrio anguillarum challange induced Sb-Lec1 mRNA expression in all tested tissues(P<0.05).These results showed that Sb-Lec1 gene may play an important role in immune defense.

关 键 词：C型凝集素 魁蚶 鳗弧菌 基因克隆 基因表达 

分 类 号：S968.3[农业科学—水产养殖]