三角帆蚌LRR基因的分子特征及表达分析
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摘要
近年来珠蚌养殖业一直饱受病害困扰,现有资料表明富亮氨酸重复序列(leucine-rich repeat,LRR)蛋白在无脊椎动物的蛋白互作、识别过程和免疫反应中都起到至关重要的作用,本实验通过RACE法克隆并表达了三角帆蚌LRR基因,全长为3 492 bp,其中开放阅读框2 142 bp,编码713个氨基酸,其中5个氨基酸组成信号肽,而其余688个氨基酸则形成成熟肽,氨基酸序列经相关软件预测存在跨膜结构。运用荧光定量PCR技术分析LRR在正常组织中及受到嗜水气单胞菌侵染后相关组织表达量的变化,分析发现,在非胁迫状态下,LRR基因在斧足中高表达,在其他组织中几乎不表达。嗜水气单胞菌刺激侵染后可以看到斧足和肝胰腺中LRR基因表达显著下调,而其他组织表达量在不同时间点均出现表达峰值,显示能被显著诱导,说明LRR基因表达可能与抵挡病原体入侵有关。
Infecti ous diseases have seriously inhibited the pearl oyster breeding in recent years. Available data showed that leucine-rich repeat(LRR) proteins play a crucial role in protein-protein interactions, recognition processes and immune reactions of invertebrates. In the present study, we cloned and characterized a LRR cDNA from the Hyriopsis cumingii by using the RACE strategy. Hyriopsis Cumingii LRR gene's full length was 3 492 bp,the open reading frame of which was 2 142 bp, encoding 713 amino acids, including the period of the 25 amino acid signal peptide and 688 amino acid mature peptide. Amino acid sequence analysis showed that the sequence contained a transmembrane structure. We used fluorescent quantitative PCR to examine the LRR expression changes in normal tissues and related tissues infected by Aeromonas hydrophila. The results showed that, under non-stress state, LRR gene highly expressed in foot, barely in other tissues. After been infected by Aeromonas hydrophila, it could be observed that the expression of LRR gene was significantly down-regulated in foot and hepatopancreas, while in other tissues the expression of LRR reached its peaked at different time points, indicating it could be significantly induced. It suggested the expression of LRR gene might be associated with the organism immunity against pathogens.
Infecti ous diseases have seriously inhibited the pearl oyster breeding in recent years. Available data showed that leucine-rich repeat(LRR) proteins play a crucial role in protein-protein interactions, recognition processes and immune reactions of invertebrates. In the present study, we cloned and characterized a LRR cDNA from the Hyriopsis cumingii by using the RACE strategy. Hyriopsis Cumingii LRR gene's full length was 3 492 bp,the open reading frame of which was 2 142 bp, encoding 713 amino acids, including the period of the 25 amino acid signal peptide and 688 amino acid mature peptide. Amino acid sequence analysis showed that the sequence contained a transmembrane structure. We used fluorescent quantitative PCR to examine the LRR expression changes in normal tissues and related tissues infected by Aeromonas hydrophila. The results showed that, under non-stress state, LRR gene highly expressed in foot, barely in other tissues. After been infected by Aeromonas hydrophila, it could be observed that the expression of LRR gene was significantly down-regulated in foot and hepatopancreas, while in other tissues the expression of LRR reached its peaked at different time points, indicating it could be significantly induced. It suggested the expression of LRR gene might be associated with the organism immunity against pathogens.
引文
Ao J.,Mu Y.,Wang K.,Sun M.,Wang X.,and Chen X.,2016,I-dentification and characterization of a novel Toll-like receptor 2 homologue in the large yellow croaker Larimichthys crocea,Fish&Shellfish Immunology,48(8):221-227
Bai Z.Y.,Wang G.L.,and Li J.L.,2011,Cloning and characterization of cathepsin L gene and evolution analysis in Hyriopsis cumingii,Shengwu Jishu Tongbao(Biotechnology Bulletin),6:104-111(白志毅,汪桂玲,李家乐,2011,三角帆蚌组织蛋白酶L基因的克隆和序列特征与进化分析,生物技术通报,6:104-111)
Bai Z.Y.,Yin Y.X.,Hu S.N.,Wang G.L.,Zhang X.W.,and Li J.L.,2010,Identification of genes potentially involved in pearl formation by expressed sequence tag analysis of mantle from freshwater pearl mussel(Hyriopsis cumingii Lea),Journal of Shellfish Research,29(2):527-534
Cao J.S.,Lin Z.Y.,Zhang Y.L.,Zhang X.Y.,Li S.K.,Zhang N.,Zou W.H.,Li Y.Y.,Chen J.H.,and Wang X.Y.,2013,Cloning and characterization of the Sp LRR c DNA from green mud crab,Scylla paramamosain,Fish&Shellfish Immunology,34(1):129-135
Dong S.J.,Wang G.L.,Bai Z.Y.,and Li J.L.,2011,Full-length c DNA cloning and molecular characteristic research of AMPgene in Hyiposis cumingii,Shanghai Haiyang Daxue Xuebao(Journal of Shanghai Ocean University),20(3):342-349(董姝君,汪桂玲,白志毅,李家乐,2011,三角帆蚌AMP基因c DNA全序列的克隆及分子特征研究,上海海洋大学学报,20(3):342-349)
Gao Q.X.,Xiao Y.P.,Zhang C.J.,Min M.H.,Peng S.M.,and Shi Z.H.,2016,Molecular characterization and expression analysis of toll-like re ceptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells,Fish&Shellfish Immunology,58:1-9
George R.A.,and Heringa J.,2002,Snap DRAGON:a method to delineate protein structural domains from sequence data,J.Mol.Biol.,316(3):839-851
Kelly L.A.,and Sterberg M.J.,2009,Protein structure prediction on the Web:a case study using the Phyre server,Nature Protocols,4(3):363-371
Kobe B.,and Kajava A.V.,2000,When protein folding is simplified to protein coiling:the continuum of solenoid protein structures,Trends Biochemistry Science,25(10):509-515
Kobe B.,and Kajava A.V.,2011,The leucine-rich repeat as a protein recognition motif,Current Opinion in Structural Biology,11(6):725-732
Lauren J.,Airaksinen M.S.,Saarma M.,and Timmusk T.,2003,A novel gene family encoding leucine-rich repeat transmembrane proteins differentially expressed in the nervous system,Genomics,81(4):411-421
Li X.L.,Wang G.L.,Li J.L.,and Yuan Y.M.,2010,Full-length c DNA cloning and encoding protein structure analysis of GPX in Hyriopsis cumingii,Yichuan(Hereditas),32(4):360-368(李西雷,汪桂玲,李家乐,袁一鸣,2010,三角帆蚌GPX基因c DNA全序列的克隆及其编码蛋白的结构分析,遗传,32(4):360-368)
Loimaranta V.,Hytonen J.,Pulliainen A.T.,Sharma A.,Tenovuo J.,Stromberg N.,and Finne J.,2009,Leucine-rich repeats of bacterial surface proteins serve as common pattern recognition motifs of human scavenger receptor gp340*,The Journal of Biological Chemistry,284(28):18614-18623
Matsushima N.,Miyashita H.,Mikami T.,and Kuroki Y.,2010,A nested leucine rich repeat(LRR)domain:the precursor of LRRs is a ten or eleven residue motif,BMC Microbiology,10:235-240
Nagasawa A.,Kubota R.,Imamura Y.,Nagamine K.,Wang Y.M.,Asakawa S.C.,Kudoh J.,Minoshima S.,Mashima Y.,Oguchi Y.,and Shimizu N.,1997,Cloning of the c DNA for a new member of the immunoglobulin superfamily(ISLR)containing leucine-rich repeat(LRR),Genomics,44(3):273-279
Sriphaijit T.,and Senapin S.,2007,High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon,Fish&Shellfish Immunology,22(3):264-271
Wang R.,and Sperry A.O.,2008,Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids,BMC Cell Biology,9(9):1-12
Yuan Y.M.,Li J.L.,Wang G.L.,Bai Z.Y.,and Li X.L.,2010,Cloning and expression analysis ofβ-actin gene from Hypriopsis cumingii,Shuichan Xuebao(Journal of Fisheries of China),34(6):691-700(袁一鸣,李家乐,汪桂玲,白志毅,李西雷,2010,三角帆蚌β-肌动蛋白基因的c DNA全长克隆及表达分析,水产学报,34(6):691-700)
Zhu Y.F.,Ragan E.J.,and Kanost M.R.,2010,Leureptin:a soluble,extracellular leucine-rich repeat protein from Manduca sexta that binds lipopolysaccharide,Insect Biochemistry and Molecular Biology,40(10):713-722
Bai Z.Y.,Wang G.L.,and Li J.L.,2011,Cloning and characterization of cathepsin L gene and evolution analysis in Hyriopsis cumingii,Shengwu Jishu Tongbao(Biotechnology Bulletin),6:104-111(白志毅,汪桂玲,李家乐,2011,三角帆蚌组织蛋白酶L基因的克隆和序列特征与进化分析,生物技术通报,6:104-111)
Bai Z.Y.,Yin Y.X.,Hu S.N.,Wang G.L.,Zhang X.W.,and Li J.L.,2010,Identification of genes potentially involved in pearl formation by expressed sequence tag analysis of mantle from freshwater pearl mussel(Hyriopsis cumingii Lea),Journal of Shellfish Research,29(2):527-534
Cao J.S.,Lin Z.Y.,Zhang Y.L.,Zhang X.Y.,Li S.K.,Zhang N.,Zou W.H.,Li Y.Y.,Chen J.H.,and Wang X.Y.,2013,Cloning and characterization of the Sp LRR c DNA from green mud crab,Scylla paramamosain,Fish&Shellfish Immunology,34(1):129-135
Dong S.J.,Wang G.L.,Bai Z.Y.,and Li J.L.,2011,Full-length c DNA cloning and molecular characteristic research of AMPgene in Hyiposis cumingii,Shanghai Haiyang Daxue Xuebao(Journal of Shanghai Ocean University),20(3):342-349(董姝君,汪桂玲,白志毅,李家乐,2011,三角帆蚌AMP基因c DNA全序列的克隆及分子特征研究,上海海洋大学学报,20(3):342-349)
Gao Q.X.,Xiao Y.P.,Zhang C.J.,Min M.H.,Peng S.M.,and Shi Z.H.,2016,Molecular characterization and expression analysis of toll-like re ceptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells,Fish&Shellfish Immunology,58:1-9
George R.A.,and Heringa J.,2002,Snap DRAGON:a method to delineate protein structural domains from sequence data,J.Mol.Biol.,316(3):839-851
Kelly L.A.,and Sterberg M.J.,2009,Protein structure prediction on the Web:a case study using the Phyre server,Nature Protocols,4(3):363-371
Kobe B.,and Kajava A.V.,2000,When protein folding is simplified to protein coiling:the continuum of solenoid protein structures,Trends Biochemistry Science,25(10):509-515
Kobe B.,and Kajava A.V.,2011,The leucine-rich repeat as a protein recognition motif,Current Opinion in Structural Biology,11(6):725-732
Lauren J.,Airaksinen M.S.,Saarma M.,and Timmusk T.,2003,A novel gene family encoding leucine-rich repeat transmembrane proteins differentially expressed in the nervous system,Genomics,81(4):411-421
Li X.L.,Wang G.L.,Li J.L.,and Yuan Y.M.,2010,Full-length c DNA cloning and encoding protein structure analysis of GPX in Hyriopsis cumingii,Yichuan(Hereditas),32(4):360-368(李西雷,汪桂玲,李家乐,袁一鸣,2010,三角帆蚌GPX基因c DNA全序列的克隆及其编码蛋白的结构分析,遗传,32(4):360-368)
Loimaranta V.,Hytonen J.,Pulliainen A.T.,Sharma A.,Tenovuo J.,Stromberg N.,and Finne J.,2009,Leucine-rich repeats of bacterial surface proteins serve as common pattern recognition motifs of human scavenger receptor gp340*,The Journal of Biological Chemistry,284(28):18614-18623
Matsushima N.,Miyashita H.,Mikami T.,and Kuroki Y.,2010,A nested leucine rich repeat(LRR)domain:the precursor of LRRs is a ten or eleven residue motif,BMC Microbiology,10:235-240
Nagasawa A.,Kubota R.,Imamura Y.,Nagamine K.,Wang Y.M.,Asakawa S.C.,Kudoh J.,Minoshima S.,Mashima Y.,Oguchi Y.,and Shimizu N.,1997,Cloning of the c DNA for a new member of the immunoglobulin superfamily(ISLR)containing leucine-rich repeat(LRR),Genomics,44(3):273-279
Sriphaijit T.,and Senapin S.,2007,High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon,Fish&Shellfish Immunology,22(3):264-271
Wang R.,and Sperry A.O.,2008,Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids,BMC Cell Biology,9(9):1-12
Yuan Y.M.,Li J.L.,Wang G.L.,Bai Z.Y.,and Li X.L.,2010,Cloning and expression analysis ofβ-actin gene from Hypriopsis cumingii,Shuichan Xuebao(Journal of Fisheries of China),34(6):691-700(袁一鸣,李家乐,汪桂玲,白志毅,李西雷,2010,三角帆蚌β-肌动蛋白基因的c DNA全长克隆及表达分析,水产学报,34(6):691-700)
Zhu Y.F.,Ragan E.J.,and Kanost M.R.,2010,Leureptin:a soluble,extracellular leucine-rich repeat protein from Manduca sexta that binds lipopolysaccharide,Insect Biochemistry and Molecular Biology,40(10):713-722