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)。

计量
- 文章访问数: 971
- HTML全文浏览量: 8
- PDF下载量: 544
- 被引次数: 0
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

HTML全文

参考文献(31)

庄启谦. 中国动物志, 软体动物门, 双壳纲, 帘蛤科[M]. 北京: 科学出版社, 2001: 230. Zhuang Qiqian. Fauna sinica, phylum mollusca, bivalvia, veneridae[M]. Beijing: Science Press, 2001: 230.

王如才, 王昭萍. 海水贝类养殖学[M]. 青岛: 中国海洋大学出版社, 2008: 396-408. Wang Rucai, Wang Zhaoping. Marine shellfish culture[M]. Qingdao: Ocean University of China Press, 2008: 396-408.

刘连生, 闫茂仓, 林志华, 等. 引起文蛤暴发性死亡病原菌的分离和鉴定[J]. 微生物学通报, 2009, 36(1): 71-77. Liu Liansheng, Yan Maocang, Lin Zhihua, et al. Pathogens isolation and identification of eruptive epidemic disease in Meretrix meretrix[J]. Microbiology, 2009, 36(1): 71-77.

刘连生, 闫茂仓, 赵海泉, 等. 文蛤疾病学研究进展[J]. 水产科学, 2009, 28(4): 234-237. Liu Liansheng, Yan Maocang, Zhao Haiquan, et al. Advancement of pathological researches on hard clam Meretrix meretrix[J]. Fisheries Science, 2009, 28(4): 234-237.

胥炜, 王昊, 宋林生, 等. 栉孔扇贝C型凝集素基因的克隆与表达研究[J]. 高技术通讯, 2005, 15(1): 83-88. Xu Wei, Wang Hao, Song Linsheng, et al. cDNA cloning and mRNA expression of C-type lectin from scallop Chlamys Farreri[J]. Chinese High Technology Letters, 2005, 15(1): 83-88.

Drickamer K. Engineering galactose-binding activity into a C-type mannose-binding protein[J]. Nature, 1992, 360(6400): 183-186.

Weis W I, Taylor M E, Drickamer K. The C-type lectin superfamily in the immune system[J]. Immunological Reviews, 1998, 163(1): 19-34.

胡钰婷, 张殿昌, 崔淑歌, 等. 合浦珠母贝C-型凝集素基因的序列特征和功能分析[J]. 水产学报, 2011, 35(9): 1327-1336. Hu Yuting, Zhang Dianchang, Cui Shuge, et al. Sequence features and functional analysis of the C-type lectin gene (PoLEC1) from pearl oyster Pinctada fucata[J]. Journal of Fisheries of China, 2011, 35(9): 1327-1336.

Zhu Ling, Song Linsheng, Xu Wei, et al. Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians[J]. Fish Shellfish Immunol, 2008, 25(3): 231-238.

Tunkijjanukij S, Olafsen J A. Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization[J]. Dev Comp Immunol, 1998, 22(2): 139-150.

Zhao Zhiying, Yin Zhixin, Xu Xiaopeng, et al. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity[J]. J Virol, 2009, 83(1): 347-356.

Luo Tian, Zhang Xiaobo, Shao Zongze, et al. PmAV, a novel gene involved in virus resistance of shrimp Penaeus monodon[J]. Febs Letters, 2003, 551(1/3): 53-57.

李猛, 周素明, 刘璐, 等. 文蛤(Meretrix meretrix)C型凝集素基因的分子克隆及表达分析[J]. 海洋与湖沼, 2015, 46(5): 1186-1192. Li Meng, Zhou Suming, Liu Lu, et al. Molecular clone and expression and expression of C-type lectin in Meretrix meretrix[J]. Oceanlogia et Limnologia Sinica, 2015, 46(5): 1186-1192.

Gerdol M, Venier P. An updated molecular basis for mussel immunity[J]. Fish Shellfish Immunol, 2015, 46(1): 17-38.

黄海宁. 栉孔扇贝C型凝集素基因的cDNA克隆和序列分析[J]. 福建水产, 2007(4): 6-10. Huang Haining. cDNA cloning and sequence analysis on C-type lectin gene of Chlamys farreri[J]. Journal of Fujian Fisheries, 2007(4): 6-10.

Wang Hao, Song Linsheng, Li Chenghua, et al. Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri[J]. Mol Immunol, 2007, 44(5): 722-731.

Zhang Huan, Song Xiaoyan, Wang Lingling, et al. AiCTL-6, a novel C-type lectin from bay scallop Argopecten irradians with a long C-type lectin-like domain[J]. Fish Shellfish Immunol, 2011, 30(1): 17-26.

宋小燕. 海湾扇贝凝集素基因和栉孔扇贝亲环素A基因的克隆与表达[D]. 咸阳: 西北农林科技大学, 2009. Song Xiaoyan. Molecular cloning, characterization and expression of Lectins from Argopecten irradians and cyclophilin a from Chlamys farreri[D]. Xianyang: Northwest A & F University, 2009.

胡钰婷, 张殿昌, 江世贵, 等. 合浦珠母贝C型凝集素2的序列特征和表达分析[J]. 湖北农业科学, 2011, 50(19): 4026-4030. Hu Yuting, Zhang Dianchang, Jiang Shigui, et al. Sequence and expression analysis of a novel C-type lectin gene from pearl oyster Pinctada fucata[J]. Hubei Agricultural Sciences, 2011, 50(19): 4026-4030.

Yamaura K, Takahashi K G, Suzuki T. Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas[J]. Comp Biochem Phys B, 2008, 149(1): 168-175.

Li Hui, Zhang Huan, Jiang Shuai, et al. A single-CRD C-type lectin from oyster Crassostrea gigas mediates immune recognition and pathogen elimination with a potential role in the activation of complement system[J]. Fish Shellfish Immunol, 2015, 44(2): 566-575.

Jing Xing, Espinosa E P, Perrigault M, et al. Identification, molecular characterization and expression analysis of a mucosal C-type lectin in the eastern oyster, Crassostrea virginica[J]. Fish Shellfish Immunol, 2011, 30(3): 851-858.

Wang Ning, Whang I, Lee J. A novel C-type lectin from abalone, Haliotis discus discus, agglutinates Vibrio alginolyticus[J]. Dev Comp Immunol, 2008, 32(9): 1034-1040.

Zhang Jian, Qiu Reng, Hu Yonghua. HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates Gram-negative bacterial pathogens[J]. Fish Shellfish Immunol, 2014, 41(2): 466-472.

高祥刚, 李云峰, 宋文涛, 等. 文蛤肠、外套膜和肝胰脏组织CDNA文库构建及其ESTs序列分析[J]. 中国水产科学, 2010, 17(2): 344-350. Gao Xianggang, Li Yunfeng, Song Wentao, et al. Construction of cDNA libraries from intestine, mantle and hepatopancreas of hard clam (Meretrix meretrix) and ESTs analysis[J]. Journal of Fishery Sciences of China, 2010, 17(2): 344-350.

Li Hongjun, Liu Weidong, Gao Xianggang, et al. Identification of host-defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix[J]. Mol Biol Rep, 2011, 38(2): 769-775.

Feinberg H, Taylor M E, Weis W I. Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewisx by a novel mechanism[J]. J Biol Chem, 2007, 282(23): 17250-17258.

Sonnhammer E L L, Von Heijne G, Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences[C]//Ismb. 1998, 6: 175-182.

Zhu Ling, Song Linsheng, Xu Wei, et al. Identification of a C-type lectin from the bay scallop Argopecten irradians[J]. Mol Biol Rep, 2009, 36(5): 1167-1173.

Zhang Huan, Wang Hao, Wang Lingling et al. Cflec-4, a multidomain C-type lectin involved in immune defense of Zhikong scallop Chlamys farreri[J]. Deve Comp immunol, 2008, 149(1): 168-175.

Zhang Huan, Wang Hao, Wang Lingling, et al. A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains[J]. Fish Shellfish Immunol, 2009, 26(5): 707-715.

施引文献

资源附件(0)

访问统计