Molecular Cloning and Expression Analysis of PfCLEC17A in Pearl Oyster Pinctada fucata

doi:10.7668/hbnxb.20193071

Abstract

Abstract:

To explore the immune function and expression pattern of C-type lectin family members in Pinctada fucata,a cDNA sequence of C-type lectin gene PfCLEC17A in P.fucata was cloned through RACE technique.The molecular characteristics of PfCLEC17A were determined by bioinformatics analysis and its mRNA expression level was detected in different tissues including gill,mantle,adductor muscle,gonad,blood,foot and hepatopancreas,as well as expression level in hepatopancreas after Vibrio alginolyticus challenged using the Real-time fluorescent quantitative PCR.The results showed that a new C-type lectin gene PfCLEC17A was successfully cloned from P.fucata,the full cDNA was 699 bp in length with an open reading frame(ORF)of 534 bp,which encoded 178 amino acids.The deduced amino acid sequence was predicated containing a CTL domain.Phylogenetic tree analysis showed that the PfCLEC17A of P.fucata and other shellfish proteins were clustered together in a branch.The Real-time fluorescent quantitative PCR results showed that PfCLEC17A was expressed in all tissues detected in P.fucata,and the highest was found in hepatopancreas,which was significantly higher than the other tissues.The high expression level in hepatopancreas,one of the most important immune tissues in mollusc,suggested that PfCLEC17A gene might be involved in the body's immune reaction.The expression level of PfCLEC17A increased significantly and reached the peak at 1 h after V.alginolyticus challenge,indicating that the gene expression was induced by microbial stimulation at early stage.Therefore,based on the above findings,it could be concluded that the PfCLEC17A gene participates in the immune activation response of P.fucata and functions in immune system.

Key words: Pinctada fucata, C-type lectin, PfCLEC17A, Gene cloning, Gene expression

ZHENG Yusi, WANG Pei, GUO Ying, BAI Lirong, YU Dahui, ZHAO Sen. Molecular Cloning and Expression Analysis of PfCLEC17A in Pearl Oyster Pinctada fucata[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 230-238. doi: 10.7668/hbnxb.20193071.

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Figures/Tables 8

Tab.1 Primers used in amplification and expression of PfCLEC17A

引物 Primer	引物序列(5'—3') Prime sequence(5'—3')	用途 Application
PfCLEC17A-F	ATGATTTCTGCAGATAAGAGAACGTG	RT-PCR
PfCLEC17A-R	TTTTGCCGAAGACTACATCACCAGTG
PfCLEC17A 3'-GSP	GATTACGCCAAGCTTTCTGCAGATAAGAGAACGTGGTATAG	RACE
PfCLEC17A 3'-NGSP	GATTACGCCAAGCTTTTGGATTGGTGGAAGGGACAGCGTAA
PfCLEC17A 5'-GSP	GATTACGCCAAGCTTCAAAAGTTCCTGTAGAAGTCCATTCG
PfCLEC17A 5'-NGSP	GATTACGCCAAGCTTCGAATCTACCCCAAATTACGCTGTCC
UPM	CATGGCTACATGCTGACAGCCTA
UPM-Short	CGCGGATCCACAGCCTACTGA TGATCAGTCGATG
PfCLEC17A-q-F	GCAGCAGAATGTCAGTCTTGGA	qRT-PCR
PfCLEC17A-q-R	CTAAGCTTACCTGAAGATGTCC
18S-F	GAGAAACGGCTACCACATCC
18S-R	CACCAGACTTGCCCTCCAA

Fig.1 Electrophoresis result of amplified product of PfCLEC17A in P.fucata The DNA Marker is DL2000.

Fig.2 cDNA sequence and deduced amino acid sequence of PfCLEC17A in P.fucata Double underlined.CLECT domain;Shaded part.Signal peptide region;Yellow shaded part.Disulfide bond;Blue shaded part.Glycosylation sites.

Fig.3 PfCLEC17A bioinformatics prediction of Pinctada fucata A.Protein signal peptide prediction;B.Protein domain prediction;C.Protein tertiary structure prediction;D.Protein hydrophilicity/hydrophobicity prediction.

Fig.4 PfCLEC17A amino acid sequence alignment

Fig.5 Phylogenetic tree analysis of CLEC17A

Fig.6 Relative expression of pfCLEC17A among tissues in P.fucata M.Mantle;B.Blood cell;GO.Gonad;F.Foot;A.Adduct muscle;GI.Gills;HE.Hepatopancreas.Different letters indicate significant differences in relative expression among tissues (P<0.05).

Fig.7 Relative expression of PfCLEC17A of P.fucata in hepatopancreas after challenged with V.alginolyticus Different letters indicate significant differences in relative expression at different time after challenged with V.alginolyticus(P<0.05).

References 41

[1]	严雪瑜, 张斌, 李选积, 钟方杰. 浅谈广西钦州南珠产业发展策略[J]. 水产养殖, 2020, 41(11): 66-67.doi: 10.3969/j.issn.1004-2091.2020.11.020. doi: 10.3969/j.issn.1004-2091.2020.11.020
	Yan X Y, Zhang B, Li X J, Zhong F J. Talking about the development strategy of Nanzhu industry in Qinzhou,Guangxi[J]. Journal of Aquaculture, 2020, 41(11): 66-67.
[2]	Chi C, Zhang C Y, Liu J D, Zheng X C. Effects of marine toxin domoic acid on innate immune responses in bay scallop Argopecten iradians[J]. Journal of Marine Science and Engineering, 2019, 7(11):407. doi: 10.3390/jmse7110407. doi: 10.3390/jmse7110407 URL
[3]	葛海亮, 李素, 于少雄, 李淑红, 李连峰, 周萍萍, 杨玉莹, 仇华吉. 病毒感染引起的炎症反应:宿主对抗病毒的"双刃剑"[J]. 微生物学报, 2022, 62(5):1571-1586.doi: 10.13343/j.cnki.wsxb.20210543. doi: 10.13343/j.cnki.wsxb.20210543
	Ge H L, Li S, Yu S X, Li S H, Li L F, Zhou P P, Yang Y Y, Qiu H J. Inflammatory responses to viral infections:a double-edged sword in the antiviral defense of host[J]. Acta Microbiologica Sinica, 2022, 62(5):1571-1586.
[4]	Du J, Yue K D, Peng Y X, Ning Q J. Crucial roles of a novel exoskeletal-derived lectin in innate immunity of the oriental river prawn,Macrobrachium nipponense[J]. Journal of Fish Diseases, 2022, 45(5): 717-728. doi: 10.1111/jfd.13597. doi: 10.1111/jfd.13597 URL
[5]	Morales-Moya V, Stambuk F, Farlora R, Guzmán F, Mercado L, Brokordt K, Schmitt P. Molecular characterization and cellular localization of a transmembrane C-type lectin receptor in hemocytes from the scallop Argopecten purpuratus[J]. Aquaculture, 2022, 546: 737293. doi: 10.1016/j.aquaculture.2021.737293. doi: 10.1016/j.aquaculture.2021.737293 URL
[6]	Lu Y Q, Zhang H K, Cheng D W, Liu H X, Li S K, Ma H Y, Zheng H P. A multi-CRD C-type lectin gene Cnlec-1 enhance the immunity response in noble scallop Chlamys nobilis with higher carotenoids contents through up-regulating under different immunostimulants[J]. Fish & Shellfish Immunology, 2018, 83:37-44. doi: 10.1016/j.fsi.2018.09.014. doi: 10.1016/j.fsi.2018.09.014
[7]	Zhang J N, Zhang Y F, Chen L Z, Yang J M, Wei Q Y, Yang B W, Liu X L, Yang D L. Two C-type lectins from Venerupis philippinarum: Possible roles in immune recognition and opsonization[J]. Fish & Shellfish Immunology, 2019, 94:230-238. doi: 10.1016/j.fsi.2019.09.009. doi: 10.1016/j.fsi.2019.09.009
[8]	Lin J L, Ning J H, Lu X, Chen M, Cao W A, Wang C D. Transcriptomic analysis and expression of C-type lectins in response to Vibrio parahaemolyticus challenge in Scapharca subcrenata[J]. Fish & Shellfish Immunology, 2020, 106:365-373. doi: 10.1016/j.fsi.2020.08.016. doi: 10.1016/j.fsi.2020.08.016
[9]	He J J, Shen C H, Liang H Y, Fang X C, Lu J Z. Antimicrobial properties and immune-related gene expression of a C-type lectin isolated from Pinctada fucata martensii[J]. Fish & Shellfish Immunology, 2020, 105:330-340. doi: 10.1016/j.fsi.2020.07.017. doi: 10.1016/j.fsi.2020.07.017
[10]	Nagae M, Yamaguchi Y. Structural aspects of carbohydrate recognition mechanisms of C-type lectins[J]. Current Topics in Microbiology and Immunology, 2020, 429: 147-176. doi: 10.1007/82_2019_181. doi: 10.1007/82_2019_181
[11]	Zelensky A N, Gready J E. The C-type lectin-like domain superfamily[J]. The FEBS Journal, 2005, 272(24): 6179-6217. doi: 10.1111/j.1742-4658.2005.05031.x. doi: 10.1111/j.1742-4658.2005.05031.x URL
[12]	李红岩, 张祥敏, 杨帅奇. 海洋生物C型凝集素研究进展[J]. 中国海洋大学学报(自然科学版), 2020, 50(9): 113-119. doi: 10.16441/j.cnki.hdxb.20200030. doi: 10.16441/j.cnki.hdxb.20200030
	Li H Y, Zhang X M, Yang S Q. Research progress of C-type-lectin in marine organisms[J]. Periodical of Ocean University of China, 2020, 50(9),113-119.
[13]	Wang L, Huang M, Zhang H, Song L. The immune role of C-type lectins in molluscs[J]. ISJ-Invertebrate Survival Journal, 2011, 8(2):241-246.
[14]	Li D D, Nie H T, Jahan K, Yan X W. Expression analyses of C-type lectins(CTLs)in manila clam under cold stress provide insights for its potential function in cold resistance of Ruditapes philippinarum[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2020, 230:108708.doi: 10.1016/j.cbpc.2020.108708. doi: 10.1016/j.cbpc.2020.108708
[15]	Li H, Zhang H, Jiang S, Wang W L, Xin L S, Wang H, Wang L L, Song L S. 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 Immunology, 2015, 44(2):566-575.doi: 10.1016/j.fsi.2015.03.011. doi: 10.1016/j.fsi.2015.03.011
[16]	Mu C K, Chen L L, Zhao J M, Wang C L. Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum[J]. Molecular Biology Reports, 2014, 41(1):139-144.doi: 10.1007/s11033-013-2846-2. doi: 10.1007/s11033-013-2846-2
[17]	Xin Z F, Yu D D, Yang B W, Chen L Z, Hayouka Z, Chen X F, Gong Y T, Dai H Y, Wang L, Zhao Y C, Liu X L, Yang D L. Molecular characterization,expression and immune functions of two C-type lectin from Venerupis philippinarum[J]. Fish & Shellfish Immunology, 2020, 107:260-268.doi: 10.1016/j.fsi.2020.10.006. doi: 10.1016/j.fsi.2020.10.006
[18]	Mizgina T O, Chikalovets I V, Molchanova V I, Ziganshin R H, Chernikov O V. Identification and characterization of a novel lectin from the clam Glycymeris yessoensis and its functional characterization under microbial stimulation and environmental stress[J]. Marine Drugs, 2021, 19(9):474. doi: 10.3390/md19090474. doi: 10.3390/md19090474
[19]	Xu X R, Liao K, Shi P, Xu J L, Ran Z S, Zhou C X, Zhang L, Cao J Y, Yan X J. Involvement of a novel Ca²⁺-independent C-type lectin from Sinonovacula constricta in food recognition and innate immunity[J]. Fish & Shellfish Immunology, 2020, 104: 374-382. doi: 10.1016/j.fsi.2020.05.074. doi: 10.1016/j.fsi.2020.05.074
[20]	胡钰婷, 张殿昌, 崔淑歌, 郭华阳, 陈明强, 江世贵. 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.doi: 10.1631/jzus.B1000171. doi: 10.1631/jzus.B1000171
	Hu Y T, Zhang D C, Cui S G, Guo H Y, Chen M Q, Jiang S G. 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.
[21]	胡钰婷, 张殿昌, 江世贵, 苏天凤, 崔淑歌, 郭华阳, 陈明强. 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.doi: 10.14088/j.cnki.issn0439-8114.2011.19.062. doi: 10.14088/j.cnki.issn0439-8114.2011.19.062
	Hu Y T, Zhang D C, Jiang S G, Su T F, Cui S G, Guo H Y, Chen M Q. 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.
[22]	吴羽媛, 郭志颖, 梁海鹰, 王庆恒, 邓岳文, 杜晓东. 马氏珠母贝PmLec-8基因的克隆与表达分析[J]. 广东海洋大学学报, 2017, 37(4):1-7.doi: 10.3969/j.issn.1673-9159.2017.04.001. doi: 10.3969/j.issn.1673-9159.2017.04.001
	Wu Y Y, Guo Z Y, Liang H Y, Wang Q H, Deng Y W, Du X D. Clone and expression analysis of PmLec-8 gene from Pinctada fucata martensii[J]. Journal of Guangdong Ocean University, 2017, 37(4):1-7.
[23]	Rauta P R, Samanta M, Dash H R, Nayak B, Das S. Toll-like receptors(TLRs)in aquatic animals:signaling pathways,expressions and immune responses[J]. Immunology Letters, 2014, 158(1/2):14-24.doi: 10.1016/j.imlet.2013.11.013. doi: 10.1016/j.imlet.2013.11.013 URL
[24]	Monteiro J T, Lepenies B. Myeloid C-type lectin receptors in viral recognition and antiviral immunity[J]. Viruses, 2017, 9(3):59.doi: 10.3390/v9030059. doi: 10.3390/v9030059 URL
[25]	Robinson M J, Sancho D, Slack E C, LeibundGut-Landmann S, Sousa C R. Myeloid C-type lectins in innate immunity[J]. Nature Immunology, 2006, 7(12):1258-1265.doi: 10.1038/ni1417. doi: 10.1038/ni1417 pmid: 17110942
[26]	Van Kooyk Y, Rabinovich G A. Protein-glycan interactions in the control of innate and adaptive immune responses[J]. Nature Immunology, 2008, 9(6):593-601.doi: 10.1038/ni.f.203. doi: 10.1038/ni.f.203 pmid: 18490910
[27]	Koh G, Low A, Poh D, Yao Y J, Ng S K, Wong V V T, Vagenende V, Lam K P, Lee D Y. Integrative analysis workflow for the structural and functional classification of C-type lectins[J]. BMC Bioinformatics, 2011, 12(S14):S5.doi: 10.1186/1471-2105-12-S14-S5. doi: 10.1186/1471-2105-12-S14-S5
[28]	陈政良. 哺乳类C型凝集素超级家族[J]. 生物化学与生物物理进展, 1997, 24(6):491-496.
	Chen Z L. Mammalian C-type lectin superfamily[J]. Progress in Biochemistry and Biophysics, 1997, 24(6):491-496.
[29]	王昊. 栉孔扇贝凝集素家族基因的研究[D]. 青岛: 中国科学院研究生院(海洋研究所), 2006.
	Wang H. Research on lectin family genes of Chlamys farreri[D]. Qingdao: Institute of Oceanology(Chinese Academy of Sciences), 2006.
[30]	Drickamer K. Engineering galactose-binding activity into a C-type mannose-binding protein[J]. Nature, 1992, 360:183-186.doi: 10.1038/360183a0. doi: 10.1038/360183a0 URL
[31]	Iobst S T, Drickamer K. Binding of sugar ligands to Ca²⁺-dependent animal lectins.Ⅱ.Generation of high-affinity galactose binding by site-directed mutagenesis[J]. Journal of Biological Chemistry, 1994, 269(22):15512-15519. pmid: 8195195
[32]	彭扣, 陈永玲, 王军花, 邱齐骏, 洪一江. 池蝶蚌HsCTL的分子特征及表达分析[J]. 生物学杂志, 2018, 35(6):31-36.doi: 10.3969/j.issn.2095-1736.2018.06.031. doi: 10.3969/j.issn.2095-1736.2018.06.031
	Peng K, Chen Y L, Wang J H, Qiu Q J, Hong Y J. Molecular characterization and expression analysis of the C-type lectin gene(HsCTL)from the freshwater pearl mussel,Hyriopsis schlegelii[J]. Journal of Biology, 2018, 35(6):31-36.
[33]	赵玲玲. 三角帆蚌凝集素和C1qDC蛋白在先天免疫中的功能研究[D]. 南京: 南京师范大学, 2017.
	Zhao L L. The functional study of lectin and C1qDC protein of Hyriopsis cumingii in the innate immunity[D]. Nanjing: Nanjing Normal University, 2017.
[34]	张峘. 扇贝补体样成分的基因及其功能研究[D]. 青岛: 中国科学院研究生院(海洋研究所), 2010.
	Zhang H. Study on the genes and their functions of the complement-like components from scallops[D]. Qingdao: Institute of Oceanology,Chinese Academy of Sciences, 2010.
[35]	Zhang H, Song X Y, Wang L L, Kong P F, Yang J L, Liu L, Qiu L M, Zhang Y, Qiu L H, Song L S. AiCTL-6,a novel C-type lectin from bay scallop Argopecten irradians with a long C-type lectin-like domain[J]. Fish and Shellfish Immunology, 2011, 30(1):17-26.doi: 10.1016/j.fsi.2009.12.019. doi: 10.1016/j.fsi.2009.12.019 pmid: 20045059
[36]	李东东. 菲律宾蛤仔C型凝集素免疫功能及分子识别机制的研究[D]. 大连: 大连海洋大学, 2019.doi: 27821/d.cnki.gdlhy.2019.000015. doi: 27821/d.cnki.gdlhy.2019.000015
	Li D D. study of immune function and molecular recognition mechanism of Manila clam Ruditapes philippinarum C-type lectins[D]. Dalian: Dalian Ocean University, 2019.
[37]	Kong H J, Park E M, Nam B H, Kim Y O, Kim W J, Park H J, Lee C H, Lee S J. A C-type lectin like-domain(CTLD)-containing protein(PtLP)from the swimming crab Portunus trituberculatus[J]. Fish and Shellfish Immunology, 2008, 25(3):311-314.doi: 10.1016/j.fsi.2008.05.003. doi: 10.1016/j.fsi.2008.05.003 URL
[38]	王志新, 梁海鹰, 杜晓东, 黄荣莲, 邓岳文, 王庆恒, 焦钰. 马氏珠母贝热休克蛋白HSP60基因的克隆与表达分析[J]. 广东海洋大学学报, 2013, 33(6):14-23.doi: 10.3969/j.issn.1673-9159.2013.06.003. doi: 10.3969/j.issn.1673-9159.2013.06.003
	Wang Z X, Liang H Y, Du X D, Huang R L, Deng Y W, Wang Q H, Jiao Y. Cloning and express characters of HSP60 gene from Pinctada martensii[J]. Journal of Guangdong Ocean University, 2013, 33(6):14-23.
[39]	张英霞, 邹瑗徽, 满初日嘎, 周永灿. 马氏珠母贝鳃组织抗菌活性的研究[J]. 水产科学, 2010, 29(8):465-468.doi: 10.16378/j.cnki.1003-1111.2010.08.006. doi: 10.16378/j.cnki.1003-1111.2010.08.006
	Zhang Y X, Zou A H, Manchu R G, Zhou Y C. Antimicrobial activity in gills of shellfish Pinctada martensii[J]. Fisheries Science, 2010, 29(8):465-468.
[40]	沈淑芳, 朱玲, 李加琦, 薛素燕, 李阳, 陈琼琳, 毛玉泽, 庄志猛, 方建光. 魁蚶C 型凝集素基因cDNA的克隆及表达分析[J]. 渔业科学进展, 2018, 39(1):128-136.doi: 10.11758/yykxjz.20161221001. doi: 10.11758/yykxjz.20161221001
	Shen S F, Zhu L, Li J Q, Xue S Y, Li Y, Chen Q L, Mao Y Z, Zhuang Z M, Fang J G. Molecular cloning and expression analysis of C-type lectin from Scapharca broughtonii[J]. Progress in Fishery Sciences, 2018, 39(1):128-136.
[41]	李猛, 周素明, 刘璐, 彭頔, 王国良. 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.doi: 10.11693/hyhz20150100017. doi: 10.11693/hyhz20150100017
	Li M, Zhou S M, Liu L, Peng D, Wang G L. Molecular clone and expression of C-type lectin in Meretrix meretrix[J]. Oceanologia et Limnologia Sinica, 2015, 46(5):1186-1192.

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