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魁蚶C型凝集素基因cDNA的克隆及表达分析
沈淑芳1,2, 朱 玲2, 李加琦2,3, 薛素燕2,3, 李 阳1,2, 陈琼琳1,2, 毛玉泽2,3, 庄志猛2, 方建光2,3
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
摘要:
通过cDNA末端快速扩增(Rapid amplification of cDNA 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 kDa,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。
关键词:  C型凝集素  魁蚶  鳗弧菌  基因克隆  基因表达
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Molecular Cloning and Expression Analysis of C-Type Lectin from Scapharca broughtonii
SHEN Shufang1,2, ZHU Ling2, LI Jiaqi2,3, XUE Suyan2,3, LI Yang1,2, CHEN Qionglin1,2, MAO Yuze2,3, ZHUANG Zhimeng2, FANG Jianguang2,3
1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071
Abstract:
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.
Key words:  C-type lectin  Scapharca broughtonii  Vibrio anguillarum  Gene clone  Gene expression