镉胁迫对河南华溪蟹两种C型凝集素免疫应答的影响
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摘要
C型凝集素是一类可以和糖类结合的蛋白质,是先天性免疫系统中重要的模式识别受体。其中,经典C型凝集素依赖Ca~(2+)对糖类进行识别。Ca~(2+)可作为细胞内第二信使,参与多种信息传递。而重金属镉可导致细胞钙稳态失调,干扰细胞内与Ca~(2+)相关的信息传递。研究旨在探明镉胁迫对河南华溪蟹(Sinopotamonhenanense) ShLec21和ShLec23两种C型凝集素免疫应答的影响。利用RACE方法,克隆了ShLec21和ShLec23,并进行了系统进化分析;利用实时荧光定量PCR的方法,研究了ShLec21和ShLec23的组织表达模式和镉联合嗜水气单胞菌(Aeromonas hydrophila)胁迫后肝胰腺和血淋巴中ShLec21和ShLec23表达模式。结果显示:ShLec21 cDNA全长863 bp,编码152个氨基酸残基;ShLec23 cDNA全长681 bp,编码164个氨基酸残基。ShLec21和ShLec23分别聚类为无脊椎动物的两个分支。ShLec21和ShLec23在血淋巴、鳃、肝胰腺、肠道、肌肉、卵巢和精巢中表达广泛,但二者均主要在肝胰腺中表达。在胁迫条件下,单独镉胁迫对肝胰腺和血淋巴中ShLec21和ShLec23表达量无显著影响;在单独嗜水气单胞菌感染后,肝胰腺中ShLec21和ShLec23表达量分别显著(P<0.05)与极显著(P<0.01)下调,血淋巴中ShLec23表达量显著(P<0.05)下调;而在镉胁迫后嗜水气单胞菌感染过程中,ShLec21和ShLec23表达量在肝胰腺和血淋巴中显著(P<0.05)或极显著(P<0.01)上调。研究结果表明,河南华溪蟹ShLec21和ShLec23在响应嗜水气单胞菌感染过程中的表达,在一定程度上能够被镉胁迫所上调。
C-type lectins are a type of proteins binding to carbohydrates and important pattern recognition receptors in the innate immune system.The classical C-type lectins recognize sugars in Ca~(2+)-dependent manners.It is highly acknowledged that Ca~(2+) acts as a second messenger in the cell and participates in a variety of physiological and biochemical process.Heavy metal cadmium can lead to dysregulation of cellular calcium homeostasis and interfere with intracellular Ca~(2+)-related information transmission.The aim of this study was to investigate the effects of cadmium stress on the immune responses of two types of lectins,ShLec21 and ShLec23,in the freshwater crab Sinopotamon henanense.The ShLec21 and ShLec23 cDNA were cloned by RACE method,and bioinformatic analysis was carried out.In addition to their constitutive expression in selected tissues,the stimulated expression of the two C-type lectins in hepatopancreas and hemolymph after the treatment of cadmium followed by Aeromonas hydrophila infection were detected.The results showed that ShLec21 cDNA was 863 bp in length that potentinally encoded 152 amino acid residues and ShLec23 cDNA was 681 bp in length that encoded 164 amino acid residues.ShLec21 and ShLec23 clustered into two branches of invertebrates.Both of ShLec21 and ShLec23 were widely expressed in hemolymph,gill,hepatopancreas,intestine,muscle,ovary and testis with highest level in hepatopancreas.Cadmium stress had no significant effect on the expression of ShLec21 and ShLec23 in hepatopancreas and hemolymph.Bacteria A.hydrophila infection significantly down-regulated the expression of ShLec21(P<0.05) and ShLec23(P<0.01) in the hepatopancreas,and significantly(P<0.05) reduced the expression of ShLec23 in hemolymph.However,in the course of infection with A.hydrophila after cadmium stress,the expression levels of ShLec21(P<0.05) and ShLec23(P<0.01) were significantly up-regulated in hepatopancreas and hemolymph.The results suggest that cadmium stress could upregulate the expression of ShLec21 and ShLec23 in response to A.hydrophila infection in a certain extent.
C-type lectins are a type of proteins binding to carbohydrates and important pattern recognition receptors in the innate immune system.The classical C-type lectins recognize sugars in Ca~(2+)-dependent manners.It is highly acknowledged that Ca~(2+) acts as a second messenger in the cell and participates in a variety of physiological and biochemical process.Heavy metal cadmium can lead to dysregulation of cellular calcium homeostasis and interfere with intracellular Ca~(2+)-related information transmission.The aim of this study was to investigate the effects of cadmium stress on the immune responses of two types of lectins,ShLec21 and ShLec23,in the freshwater crab Sinopotamon henanense.The ShLec21 and ShLec23 cDNA were cloned by RACE method,and bioinformatic analysis was carried out.In addition to their constitutive expression in selected tissues,the stimulated expression of the two C-type lectins in hepatopancreas and hemolymph after the treatment of cadmium followed by Aeromonas hydrophila infection were detected.The results showed that ShLec21 cDNA was 863 bp in length that potentinally encoded 152 amino acid residues and ShLec23 cDNA was 681 bp in length that encoded 164 amino acid residues.ShLec21 and ShLec23 clustered into two branches of invertebrates.Both of ShLec21 and ShLec23 were widely expressed in hemolymph,gill,hepatopancreas,intestine,muscle,ovary and testis with highest level in hepatopancreas.Cadmium stress had no significant effect on the expression of ShLec21 and ShLec23 in hepatopancreas and hemolymph.Bacteria A.hydrophila infection significantly down-regulated the expression of ShLec21(P<0.05) and ShLec23(P<0.01) in the hepatopancreas,and significantly(P<0.05) reduced the expression of ShLec23 in hemolymph.However,in the course of infection with A.hydrophila after cadmium stress,the expression levels of ShLec21(P<0.05) and ShLec23(P<0.01) were significantly up-regulated in hepatopancreas and hemolymph.The results suggest that cadmium stress could upregulate the expression of ShLec21 and ShLec23 in response to A.hydrophila infection in a certain extent.
引文
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[15]Duan L P,Huang B,Zhou L H,et al.Molecular cloning,characterization and expression of two novel lectins in mud crab,Scylla paramamosain[J].Acta Hydrobiologica Sinica,2015,39(2):321-330[段利朋,黄贝,周立红,等.拟穴青蟹两种新C-型凝集素基因的克隆与表达分析.水生生物学报,2015,39(2):321-330]
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[18]Zhan Q,Tang M.Research advances on apoptosis caused by quantum dots[J].Biological Trace Element Research,2014,161(1):3-12
[19]Hou Y H,Li N,Li D,et al.Bioaccumulation of cadmium and its effects on antioxidant enzyme activities,lipid,protein and dna in male accessory gland of the freshwater crab Sinopotamon henanense[J].Acta Hydrobiologica Sinica,2015,39(3):621-626[侯宇华,李娜,李丹,等.镉对河南华溪蟹副性腺抗氧化酶活性及脂质、蛋白质和DNA的影响.水生生物学报,2015,39(3):621-626]
[20]Yang J,Liu D M,He Y J,et al.Established of elisa method of vitellin from freshwater crab Sinopotamon henanense and effect of cadmium on vitellin accumulation in ovary[J].Acta Hydrobiologica Sinica,2015,39(2):287-293[杨健,刘冬梅,何永吉,等.镉对河南华溪蟹卵黄磷蛋白在卵巢中表达含量的影响及ELISA法的建立.水生生物学报,2015,39(2):287-293]
[21]Wang Q,Guo H F,Wang L.Effect of cadmium on the feeding capacity and physiological status of Daphnia magna[J].Acta Hydrobiologica Sinica,2018,42(3):616-621[王茜,郭鹄飞,王兰.镉对大型溞摄食能力和相关生理指标的影响.水生生物学报,2018,42(3):616-621]
[22]Qin Q,Qin S,Wang L,et al.Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium[J].Aquatic Toxicology,2012,(106-107):140-146
[23]Lang L,Zhang Z,Jing W,et al.Identification of a novel toll gene(Shtoll3)from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium followed by Aeromonas hydrophila infection[J].Fish&Shellfish Immunology,2017,71:177-190
[24]Li L,Zhao C P,Li H,et al.Establishment of the plasmid standard curve generation method for absolute quantification PCR[J].Journal of Agricultural Biotechnology,2011,19(6):1157-1162
[25]Lang X,Wang L,Zhang Z.Stability evaluation of reference genes for real-time PCR in zebrafish(Danio rerio)exposed to cadmium chloride and subsequently infected by bacteria Aeromonas hydrophila[J].Aquatic Toxicology,2016,170:240-250
[26]Robinson M J,Sancho D,Slack E C,et al.Myeloid C-type lectins in innate immunity[J].Nature Immunology,2006,7(12):1258-1265
[27]Runsaeng P,Thepnarong S,Rattanaporn O,et al.Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis in response to pathogenic inoculation[J].Molecular&Cellular Probes,2015,29(6):365-375
[28]Ren Q,Li M,Du J,et al.Immune response of four dualCRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii[J].Fish&Shellfish Immunology,2012,33(2):155-167
[29]Zelensky A N,Gready J E.The C-type lectin-like domain superfamily[J].FEBS Journal,2005,272(24):6179-217
[30]Wang L,Wang L,Huang M,et al.The immune role of C-type lectins in molluscs[J].Invertebrate Survival Journal,2011,8(2):241-246
[31]Li M,Li C,Ma C,et al.Identification of a C-type lectin with antiviral and antibacterial activity from pacific white shrimp Litopenaeus vannamei[J].Developmental&Comparative Immunology,2014,46(2):231-240
[32]Liu Y C,Li F H,Dong B,et al.Molecular cloning,characterization and expression analysis of a putative C-type lectin(Fclectin)gene in Chinese shrimp Fenneropenaeus chinensis[J].Molecular Immunology,2007,44(4):598-607
[33]Huang Y,Huang X,Wang Z,et al.Function of two novel single-CRD containing C-type lectins in innate immunity from Eriocheir sinensis[J].Fish&Shellfish Immunology,2014,37(2):313-321
[34]Ma T H,Tiu S H,He J G,et al.Molecular cloning of a C-type lectin(LvLT)from the shrimp Litopenaeus vannamei:early gene down-regulation after WSSV infection[J].Fish&Shellfish Immunology,2007,23(2):430-437
[2]Wang X W,Wang J X.Diversity and multiple functions of lectins in shrimp immunity[J].Developmental&Comparative Immunology,2013,39(1-2):27-38
[3]Wang X W,Wang J X.Pattern recognition receptors acting in innate immune system of shrimp against pathogen infections[J].Fish&Shellfish Immunology,2013,34(4):981-989
[4]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
[5]Chen D D,Meng X L,Xu J P,et al.PcLT,a novel C-type lectin from Procambarus clarkii,is involved in the innate defense against Vibrio alginolyticus and WSSV[J].Developmental&Comparative Immunology,2013,39(3):255-264
[6]Junkunlo K,Prachumwat A,Tangprasittipap A,et al.Anovel lectin domain-containing protein(LvC TLD)associated with response of the whiteleg shrimp Penaeus(Litopenaeus)vannamei to yellow head virus(YHV)[J].Developmental&Comparative Immunology,2012,37(3-4):334-341
[7]Luo T,Yang H,Li F,et al.Purification,characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon[J].Developmental&Comparative Immunology,2006,30(7):607-617
[8]Costa F H,Valenca N S,Silva A R,et al.Cloning and molecular modeling of Litopenaeus vannamei(Penaeidae)C-type lectin homologs with mutated mannose binding domain-2[J].Genetics&Molecular Research,2011,10(2):650-664
[9]Huang X,Li W,Jin M,et al.Single CRD containing lectin from Macrobrachium rosenbergii(MrLec)participates in innate immunity against pathogen infections[J].Fish&Shellfish Immunology,2016,51:282-290
[10]Jin X K,Li W W,Cheng L,et al.Two novel short C-type lectin from Chinese mitten crab,Eriocheir sinensis,are induced in response to LPS challenged[J].Fish&Shellfish Immunology,2012,33(5):1149-1158
[11]Fang Z Y,Li D,Li X J,et al.A single CRD C-type lectin from Eriocheir sinensis(EsLecB)with microbial-binding,antibacterial prophenoloxidase activation and hem-encapsulation activities[J].Fish&Shellfish Immunology,2016,50:175-190
[12]Kong H J,Park E M,Nam B H,et al.A C-type lectin like-domain(CTLD)-containing protein(PtLP)from the swimming crab Portunus trituberculatus[J].Fish&Shellfish Immunology,2008,25(3):311-314
[13]Guo X N,Jin X K,Li S,et al.A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity[J].Fish&Shellfish Immunology,2013,35(5):1554-1565
[14]Guo H Z,Zou P F,Fu J P,et al.Characterization of two C-type lectin-like domain(CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis[J].Fish&Shellfish Immunology,2011,30(2):515-524
[15]Duan L P,Huang B,Zhou L H,et al.Molecular cloning,characterization and expression of two novel lectins in mud crab,Scylla paramamosain[J].Acta Hydrobiologica Sinica,2015,39(2):321-330[段利朋,黄贝,周立红,等.拟穴青蟹两种新C-型凝集素基因的克隆与表达分析.水生生物学报,2015,39(2):321-330]
[16]Kumar S R,Agrawal M,Marshall F.Heavy metal contamination of soil and vegetables in suburban areas of Varanasi,India[J].Ecotoxicology&Environmental Safety,2007,66(2):258-266
[17]Valko M,Izakovic M,Mazur M,et al.Role of oxygen radicals in DNA damage and cancer incidence[J].Molecular and Cellular Biochemistry,2004,266(1/2):37-56
[18]Zhan Q,Tang M.Research advances on apoptosis caused by quantum dots[J].Biological Trace Element Research,2014,161(1):3-12
[19]Hou Y H,Li N,Li D,et al.Bioaccumulation of cadmium and its effects on antioxidant enzyme activities,lipid,protein and dna in male accessory gland of the freshwater crab Sinopotamon henanense[J].Acta Hydrobiologica Sinica,2015,39(3):621-626[侯宇华,李娜,李丹,等.镉对河南华溪蟹副性腺抗氧化酶活性及脂质、蛋白质和DNA的影响.水生生物学报,2015,39(3):621-626]
[20]Yang J,Liu D M,He Y J,et al.Established of elisa method of vitellin from freshwater crab Sinopotamon henanense and effect of cadmium on vitellin accumulation in ovary[J].Acta Hydrobiologica Sinica,2015,39(2):287-293[杨健,刘冬梅,何永吉,等.镉对河南华溪蟹卵黄磷蛋白在卵巢中表达含量的影响及ELISA法的建立.水生生物学报,2015,39(2):287-293]
[21]Wang Q,Guo H F,Wang L.Effect of cadmium on the feeding capacity and physiological status of Daphnia magna[J].Acta Hydrobiologica Sinica,2018,42(3):616-621[王茜,郭鹄飞,王兰.镉对大型溞摄食能力和相关生理指标的影响.水生生物学报,2018,42(3):616-621]
[22]Qin Q,Qin S,Wang L,et al.Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium[J].Aquatic Toxicology,2012,(106-107):140-146
[23]Lang L,Zhang Z,Jing W,et al.Identification of a novel toll gene(Shtoll3)from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium followed by Aeromonas hydrophila infection[J].Fish&Shellfish Immunology,2017,71:177-190
[24]Li L,Zhao C P,Li H,et al.Establishment of the plasmid standard curve generation method for absolute quantification PCR[J].Journal of Agricultural Biotechnology,2011,19(6):1157-1162
[25]Lang X,Wang L,Zhang Z.Stability evaluation of reference genes for real-time PCR in zebrafish(Danio rerio)exposed to cadmium chloride and subsequently infected by bacteria Aeromonas hydrophila[J].Aquatic Toxicology,2016,170:240-250
[26]Robinson M J,Sancho D,Slack E C,et al.Myeloid C-type lectins in innate immunity[J].Nature Immunology,2006,7(12):1258-1265
[27]Runsaeng P,Thepnarong S,Rattanaporn O,et al.Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis in response to pathogenic inoculation[J].Molecular&Cellular Probes,2015,29(6):365-375
[28]Ren Q,Li M,Du J,et al.Immune response of four dualCRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii[J].Fish&Shellfish Immunology,2012,33(2):155-167
[29]Zelensky A N,Gready J E.The C-type lectin-like domain superfamily[J].FEBS Journal,2005,272(24):6179-217
[30]Wang L,Wang L,Huang M,et al.The immune role of C-type lectins in molluscs[J].Invertebrate Survival Journal,2011,8(2):241-246
[31]Li M,Li C,Ma C,et al.Identification of a C-type lectin with antiviral and antibacterial activity from pacific white shrimp Litopenaeus vannamei[J].Developmental&Comparative Immunology,2014,46(2):231-240
[32]Liu Y C,Li F H,Dong B,et al.Molecular cloning,characterization and expression analysis of a putative C-type lectin(Fclectin)gene in Chinese shrimp Fenneropenaeus chinensis[J].Molecular Immunology,2007,44(4):598-607
[33]Huang Y,Huang X,Wang Z,et al.Function of two novel single-CRD containing C-type lectins in innate immunity from Eriocheir sinensis[J].Fish&Shellfish Immunology,2014,37(2):313-321
[34]Ma T H,Tiu S H,He J G,et al.Molecular cloning of a C-type lectin(LvLT)from the shrimp Litopenaeus vannamei:early gene down-regulation after WSSV infection[J].Fish&Shellfish Immunology,2007,23(2):430-437