| 173 | 0 | 17 |
| 下载次数 | 被引频次 | 阅读次数 |
研究凡纳滨对虾Carcinin基因(简称Lvcar)的结构和功能,探究Lvcar基因是否参与副溶血弧菌、溶壁微球菌及白斑病毒(WSSV)诱导的免疫应答,为研究凡纳滨对虾的免疫机制提供参考。运用RACE扩增技术得到凡纳滨对虾Lvcar基因cDNA全长并进行生物信息学分析;通过实时荧光定量PCR检测Lvcar基因在不同组织中的表达情况及不同病源刺激后Lvcar基因的表达特征。克隆获得了凡纳滨对虾Lvcar基因(登录号:KY425707)的cDNA全长,Lvcar基因cDNA全长511 bp,其中包括48 bp的5'非编码区,82 bp的3'非编码区和完整开放阅读框(ORF)381 bp,编码126个氨基酸;对Lvcar蛋白结构分析表明,该蛋白含有1个信号肽、1个半胱胺酸富集区和1个WAP结构域,属于Ⅰ型Crustin。Lvcar基因在眼柄、肌肉和胃等11个组织中均有表达,其中在眼柄表达量最高;注射感染副溶血弧菌和WSSV后,Lvcar基因在凡纳滨对虾血细胞的表达量总体呈现出升高的变化趋势;而注射感染溶壁微球菌后,Lvcar基因的表达量呈现先上升后下降趋势。本研究克隆得到了凡纳滨对虾Lvcar基因的cDNA全长,并发现Lvcar基因表达量在感染病毒和细菌后发生明显变化,推测其参与了凡纳滨对虾的抗病免疫途径。
Abstract：The objective of this research was to study the structure and function of Carcinin(Lvcar) gene in Litopenaeus vannamei and explore whether Lvcar gene participate in the immune response of Vibrio parahaemolyticu, Micrococcus lysodeikticus and White spot syndrome virus(WSSV), aiming to provide a reference for the research of immune mechanism of L. vannamei. The full-length sequence of Lvcar gene of L. vannamei was obtained by RACE amplification technology and it was done with bioinformatics analysis. The m RNA expression pattern of Lvcar gene in different organizations and the expression characteristics of Lvcar gene stimulated by different etiology were detected by real-time quantitative PCR(q RT-PCR). The full length of c DNA of Lvcar gene in L. vannamei(accession number: KY425707) was cloned, which was 511 bp, including a 5'URT with 48 bp, a3'UTR with 82 bp and a complete open reading frame(ORF) with 381 bp, which encoded 126 amino acids; the analysis of Lvcar protein structure indicated that it contained 1 signal peptide, 1 Cysteine-rich region and 1 WAP domain, belonging to type Crustin Ⅰ. The Lvcar gene was expressed in the organization of eyestalk, muscle,stomach, and so on, and the expression in eyestalk was the highest among them; The expression of Lvcar gene in the hemocyte of L. vannamei generally presented an increasing trend after it was injected with Vibrio parahaemolyticus and WSSV; while the expression of Lvcar gene increased initially then decreased after it was injected with Micrococcus lysodeikticus. The research cloned full-length c DNA of Lvcar gene in L. vannamei.Also, we found that the expression of Lvcar gene changed obviously after viral and bacterial infection, so we speculated that Lvcar gene might participate in the disease-fighting immune way of L. vannamei.
Arocki araj J.,Gnanam A.J.,Muthukrishnan D.,Gudimella R.Milton J.,Singh A.,Muthupandian S.,Kasi M.,and Bhassu S.,2013,Crustin,a WAP domain containing antimicrobia peptide from freshwater prawn Macrobrachium rosenbergii Immune characterization,Fish&Shellfish Immunology,34(1):109-118
Balcazar J.L.,Rojas-Luna T.,and Cunningham D.P.,2007,Effec of the addition of four potential probiotic strains on the sur vival of pacific white shrimp(Litopenaeus vannamei)fol lowing immersion challenge with Vibrio parahaemolyticus Journal of Invertebrate Pathology,96(2):147-150
Bartlett T.C.,Cuthbertson B.J.,Shepard E.F.,Chapman R.W.Gross P.S.,and Warr G.W.,2002,Crustins,homologues o an 11.5-k D antibacterial peptide,from two species of pe naeid shrimp,Litopenaeus vannamei and Litopenaeus setiferus,Mar Biotechnol,4(3):278-293
Barracco M.A.,de Lorgeril J.,Gueguen Y.,and Bachère E.2005,Molecular characterization of penaeidins from two At lantic Brazilian shrimp species,Farfantepenaeus paulensis and Litopenaeus schmitti,FEMS Microbiol.Lett.,250(1)117-20
Bondad-Reantaso M.G.,Subasinghe R.P.,Josupeit H.,Cai J.,and Zhou X.,2012,The role of crustacean fisheries and aquacul ture in global food security:Past,present and future,Journa of Invertebrate Pathology,110(2):158-165
Brockton V.,Hammond J.A.,and Smith V.J.,2007,Gene charac teristics,isoforms and recombinant expression of carcinin an antibacterial protein from the shore crab,Carcinus maenas Mol.Immunol.,44(5):943-949
Brown K.L.,and Hancock R.E.,2006,Cationic host defense(an timicrobial)peptides,Curr.Opin.in Immunol.,18(1):24-30
Destoumieux-Garzon D.,Bulet P.,Loew D.,Van Dorsselaer A.Rodriguez J.,and Bachère E.,1997,Penaeidins,a new fami ly of antimicrobial peptides isolated from the shrimp Penaeus vannamei(Decapoda),J.Biol.Chem.,272(45)28398-28406
Donpudsa S.,Rimphanitchayakit V.,Tassanakajon A.,S觟derh覿l I.,and S觟derh覿ll K.,2010,Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus,J.Invert.Pathol.,104(3):234
Fagutao F.F.,Yasuike M.,Caipang C.M.,Kondo H.,Hirono I.Takahashi Y.,and Aoki T.,2008,Gene expression profile o hemocytes of kuruma shrimp,Marsupenaeus japonicas fol lowing peptidoglycan stimulation,Mar.Biotechnol.(NY)10(6):731-740
Grutter M.G.,Fendrich G.,Huber R.,and Bode W.,1988,The2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin,EMBO J.,7(2)345-351
Gui L.,2012,Recombinant expression and functional analysis of two proteins related to immunity of Chinese shrimp Fenneropenaeus chinensis,Dissertation for Ph.D.,Universi ty of Chinese Academy of Sciences,Supervisor:Xiang J.Hpp.19-20(桂朗,2012,中国明对虾两种免疫相关蛋白的表达与功能研究,博士学位论文,中国科学院研究生院,导师:相建海,pp.19-20)
Jia Y.P.,Sun Y.D.,Wang Z.H.,Wang Q.,Wang X.W.,Zhao X.Fand Wang J.X.,2008,A single whey acidic protein domain(SWD)-containing peptide from fleshy prawn with antimi crobial and proteinase inhibitory activities,Aquaculture,284(1-4):246-259
Jimenez-Vega F.,Yepiz-Plascencia G.,S觟derhall K.,and Var gas-Albores F.,2004,A single WAP domain-containing pro tein from Litopenaeus vannamei hemocytes,Biochem.Bio phys.Res.Commun.,314(3):681-687
Jiravanichpaisal P.,Lee S.Y.,Kim Y.A.,Andrén T.,and S觟derh覿ll I.,2007,Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacu leniusculus:characterization and expression pattern,Dev Comp.Immunol.,31(5):441-455
Krusong K.,Poolpipat P.,Supungul P.,and Tassanakajon A.2012,A comparative study of antimicrobial properties o crustin Pm1 and crustin Pm7 from the black tiger shrimp Penaeus monodon,Dev.Comp.Immunol.,36(1):208-215
Melena J.,Echeverria F.,Panchana F.,Betancourt I.,Santande R.,Candell J.,and Bonami J.R.,2015,Delayed mortality o juvenile shrimp Penaeus vannamei challenged to white spo syndrome virus(WSSV)previously exposed to infection hy podermal and haematopoietic necrosis virus(IHHNV)or in activated WSSV,Brazilian Journal of Veterinary Pathology8(2):51-57
Menz A.,and Blake B.F.,1980,Experiments on the growth o Penaeus vannamei boone,Journal of Experimental Marin Biology and Ecology,48(2):99-111
Okumura T.,2007,Effects of lipopolysaccharide on gene expres sion of antimicrobial peptides(penaeidins and crustin),ser ine proteinase and prophenoloxidase in haemocytes of th Pacific white shrimp,Litopenaeus vannamei,Fish Shellfish Immunol.,22(1-2):68-76
Ponce-Palafox J.,Martinez-Palacios C.A.,and Ross L.G.,1997The effects of salinity and temperature on the growth and survival rates of juvenile white shrimp,Penaeus vannamei boone,1931,Aquaculture,157(1-2):107-115
Relf J.M.,Chisholm J.R.,Kemp G.D.,and Smith V.J.,1999,Pu rification and characterization of a cysteine-rich 11.5-k Dantibacterial protein from the granular haemocytes of the shore crab,Carcinus maenas,Eur.J.Biochem.,264(2):350-357
Rojtinnakorn J.,Hirono I.,Itami T.,Takahashi Y.,and Aoki T.2002,Gene expression in haemocytes of kuruma prawn Penaeus japonicus,in response to infection with WSSV by EST approach,Fish Shellfish Immunol,13(1):69-83
Sallenave J.M.,2000,The role of secretory leukocyte proteinas inhibitor and elafin(elastase-specific inhibitor/skin-derived antileukoprotease)as alarm antiproteinases in inflammatory lung disease,Respir.Res.,1(2):87-92
Smith V.J.,Fernandes J.M.O.,Kemp G.D.,and Hauton C.,2008Crustins:enigmatic WAP domain-containing antibacteria proteins from crustaceans,Dev.Comp.Immunol.,32(7)758-772
Somboonwiwat K.,Marcos M.,Tassanakajon A.,Klinbunga S.Aumelas A.,Romestand B.,Gueguenc Y.,Bozeb H.Moulinb G.,and Bachère E.,2005,Recombinant expression and anti-microbial activity of antilipopolysaccharide facto(ALF)from the black tiger shrimp Penaeus monodon,Dev Comp.Immunol.,29(10):841-851
Song C.W.,2013,Cloning and expression analysis of antimicro bial peptide crustin of swimming crab Portunus trituberculatus,Thesis for M.S.,Chinese Academy of Science,Supervi sor:Cui Z.X.,pp.57-58(宋呈文,2013,三疣梭子蟹抗菌肽Crustin的基因克隆和表达分析,硕士学位论文,中国科学院海洋研究所,导师:崔朝霞,pp.57-58)
Sperstad S.V.,Haug T.,Paulsen V.,Rode T.M.,Strandskog G.Solem S.T.,Styrvold O.B.,and Stensv覽g K.,2009,Charac terization of crustins from the hemocytes of the spider crab Hyas araneus,and the red king crab,Paralithodes camtschaticus,Dev.Comp.Immunol.,33(4):583-591
Sun C.,Du X.J.,Xu W.T.,Zhang H.W.,Zhao X.F.,and Wang JX.,2010,Molecular cloning and characterization of three crustins from the Chinese white shrimp,Fenneropenaeu chinensis,Fish Shellfish Immunol.,28(4):517-524
Sun C.,2011,Molecular cloning and functional analysis of the crus tacean antimicrobial peptides and STAT(signal transducer and activator of transcription),Dissertation for Ph.D.,Shangdong University,Supervisor:Wang J.X.,pp.61-62(孙晨,2011,甲壳动物抗菌肤及信号转导和转录激活因子的基因克隆与功能分析,博士学位论文,山东大学,导师:王金星,pp.61-62)
Supungul P.,Tang S.,Maneeruttanarungroj C.,Rimphan itchayakit V.,Hirono I.,and Aoki T.,2008,Cloning,expres sion and antimicrobial activity of crustin Pm1,a major iso form of crustin,from the black tiger shrimp Penaeus monodon Dev.Comp.Immunol.,32(1):61-70
Wang L.U.,and Chen J.C.,2005,The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels,Fish&Shellfish Im munology,18(4):269-278
Wiedow O.,Harder J.,Bartels J.,Streit V.,and Christophers E.1998,Antileukoprotease in human skin:an antibiotic pep tide constitutively produced by keratinocytes,Biochem.Bio phys.Res.Commun.,248(3):904-909
Zhang J.,Li F.,Wang Z.,and Xiang J.,2007,Cloning and recom binant expression of a crustin-like gene from Chinese shrimp,Fenneropenaeus chinensis,J.Biotechnol.,127(4)605-614
Zheng Z.X.,2012,The research of the antimicrobial peptide crustin from Portunus trituberculatus,Thesis for M.S.,Zhe jiang Ocean University,Supervisor:Liu X.Z.,pp.23-24(郑兆祥,2012,三疣梭子蟹Crustin抗菌肽的研究,硕士学位论文,浙江海洋学院,导师:刘雪珠,pp.23-24)
基本信息:
DOI：10.13417/j.gab.036.003200
中图分类号:Q78;S917.4
引用信息:
[1]周婷婷,史黎黎,卓瑞群,等.凡纳滨对虾(Litopenaeus vannamei)Carcinin基因的克隆与表达分析[J].基因组学与应用生物学,2017,36(08):3200-3209.DOI:10.13417/j.gab.036.003200.
基金信息:
广东省自然科学基金-博士启动(2016A030310334)资助
2017-04-27
2017-04-27
2017-04-27