文蛤C型凝集素基因(Mm-Lec1)的克隆与表达分析

张晶晶; 李宏俊; 秦艳杰; 刘敏; 叶晟

doi:10.3969/j.issn.0253-4193.2016.06.012

文蛤C型凝集素基因(Mm-Lec1)的克隆与表达分析

doi: 10.3969/j.issn.0253-4193.2016.06.012

1.
大连海洋大学水产与生命学院, 辽宁大连 116023;国家海洋环境监测中心海洋生态室, 辽宁大连 116023
2.
国家海洋环境监测中心海洋生态室, 辽宁大连 116023
3.
大连海洋大学水产与生命学院, 辽宁大连 116023

基金项目: 国家自然科学基金(31101899,31572595);国家海洋局近岸海域生态环境重点实验室开放基金(201511)。

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出版历程
- 收稿日期: 2015-10-15

Cloning and expression analysis of a C-type lectin gene (Mm-Lec1) in hard clam Meretrix meretrix

1.
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China;Marine Ecology Department, National Marine Environmental Monitoring Center, Dalian 116023, China
2.
Marine Ecology Department, National Marine Environmental Monitoring Center, Dalian 116023, China
3.
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China

摘要

摘要: 文蛤(Meretrix meretrix)是我国重要的滩涂养殖贝类,病害严重影响文蛤增养殖业,研究文蛤的免疫机制有助于解决文蛤的病害问题。C型凝集素(C-type lectin)参与先天免疫,在识别病原相关分子模式和激活体液免疫因子等方面发挥重要作用。本研究检索已构建的文蛤全长cDNA 文库,经过Blast比对得到了文蛤C型凝集素1(Mm-Lec1)基因的全长cDNA序列。Mm-Lec1序列全长586 bp,5'和3'非翻译区(UTR)的长度分别为21 bp和79 bp,开放阅读框长度为486 bp,编码161个氨基酸,分子量为18.65 kD,理论等电点为4.98。预测的氨基酸序列中含有信号肽(Met1-Ser19)、糖识别结构域(CRD)和糖结合位点(QPN)。Mm-Lec1的三级结构是紧凑型,含有β片层结构。同源性分析结果表明,Mm-Lec1与其他物种C型凝集素相似度在20%~32%;邻接法(Neighbor-Joining, NJ)进化树分析结果表明,Mm-Lec1与紫贻贝CTL 6和栉孔扇贝CTL A聚为一支。实时荧光定量分析结果显示,Mm-Lec1在文蛤鳃、肝胰腺、闭壳肌、外套膜、性腺和血细胞中均表达,其中鳃表达量最高,血细胞次之,性腺中表达量最少;在鳗弧菌(Vibrio anguillarum)刺激实验中,6 h时Mm-Lec1在血细胞中的表达量最低,48 h表达量最高,暗示Mm-Lec1参与文蛤抵御细菌入侵的免疫过程。
- 文蛤 /
- C型凝集素 /
- 表达序列标签 /
- 三级结构 /
- 实时荧光定量PCR
Abstract: The hard clam (Meretrix meretrix) is an economically important bivalve species in China. Aquaculture of M. meretrix is seriously affected by the epidemic diseases. The study of the immune system of M. meretrix is an important approach to solve disease problems. C-type lectins (CTLs) play important roles in the identification of pathogen associated molecular patterns and activation of humoral immunity. In the present study, a C-type lectin gene of M. meretrix(denoted as Mm-Lec1) was obtained through sequencing full-length cDNA library. The full-length cDNA of Mm-Lec1 was 586 bp with a 486 bp open reading frame, encoding 161 amino acids. The length of 5' and 3' untranslated region was 21 and 79 bp, respectively. The molecular weight of predicted protein was 18.65 kD, and the theoretical isoelectric point was 4.98. The predicted amino acid sequence had a signal peptide (Met1-Ser19), a sugar-binding site (QPN) and a carbohydrate recognition domain (CRD). The tertiary structure of Mm-Lec1 was predicted as a compact type and had a β sheet structure with three beta sheet layers. The similarity between Mm-Lec1 and the other species mentioned in this study were 20%-32%. Mm-Lec1, Mytilus galloprovincialis CTL 6 and Chlamys farreri CTL A were clustered in one branch in the neighbor-joining (NJ) tree. Mm-Lec1 mRNA was expressed in all tested tissues, including gill, haemocytes, hepatopancreas, mantle, adductor muscle and gonad, with the highest expression level in gill, the second in haemocytes, and the least in gonad. In the bacteria exposure test, the mRNA expression level was lowest at 6 h, and highest at 48 h, suggesting that Mm-Lec1 play a role in defensing against bacterial invasion in M. meretrix.
- Meretrix meretrix /
- C-type lectin /
- EST /
- tertiary structure /
- real-time PCR

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