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| 魁蚶C型凝集素基因cDNA的克隆及表达分析 |
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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
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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
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| 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 |
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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
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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
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| 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 |