Cloning of natural resistance associated macrophage protein2 (Sc-Nramp2)gene from sinonovacula constricta and the expression analysis under vibrio parahaemolyticus stimulation
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摘要: 克隆得到缢蛏天然抗性相关巨噬蛋白2(Sc-Nramp2)基因的cDNA全长序列3 681 bp,该基因的开放阅读框有1 776 bp,编码591个氨基酸,预测分子量为65.86 kDa;其结构具有Slc11蛋白家族的典型特征,包括有10个典型的跨膜结构和2个糖基化位点。Sc-Nramp2基因3'-UTR有2个类似于脊椎动物Nramp2中铁反应控制蛋白结合位点;同源性分析表明,Sc-Nramp2和太平洋牡蛎Nramp2-like的同源性最高为71.6%。实时荧光定量PCR结果表明,Sc-Nramp2基因在闭壳肌、外套膜、肝胰腺、斧足、水管和鳃6个组织中均有表达,其中肝胰腺中的表达量最高,其次是鳃,与其他组织均有极显著性差异(P<0.01);注射副溶血弧菌后,肝胰腺中Sc-Nramp2基因的表达量较对照组显著上调(P<0.01),且表达量呈现先上升后下降的趋势,在12 h时达到最大表达量,推测Sc-Nramp2基因参与了缢蛏非特异性免疫应答反应。Abstract: The cDNA sequence of natural resistance associated macrophage protein in Sinonovacula constricta (Sc-Nramp2) was cloned by SMART RACE techniques. The ORF of cDNA sequence, coding for a protein of amino acids, was 1 773 bp and the molecular weight was deduced for 65.86 kDa. Sc-Nramp2 protein has the typical structural features with Solute carrier family 11 member, with 10 typical transmembrane domains and 2 glycosylation site. In 3'-UTR, Sc-Nramp2 was the presence of two IRE which was similar to the vertebrate Nramp2. Homology analysis indicated that the identity of Nramp with Crassostrea gigas was 71.6%. Real-time quantitative RT-PCR analysis indicated that the mRNA was expressed in all the six tissues, including adductor muscle, mantle, hepatopancreas, foot, waterpipe and gill. Nramp was expressed with the highest expression level in the hepatopancreas, then the gill, which had extremely significant differences from other tissues(P<0.01). The expression of Nramp in the hepatopancreas injected with V. Parahaemolyticus increased significantly(P<0.01). It rose to maximum expression at the 12th hour and returned to the initial level. These results indicated that Sc-Nramp2 is involved in innate immunity of this species.
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Key words:
- Sinonovacula constricta /
- Sc-Nramp2 /
- gene cloning /
- Vibrio Parahaemolyticus /
- gene expression
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Skamene E, Pietrangeli C E. Genetics of the immune response to infectious pathogens[J]. Current Opinion in Immunology, 1991, 3(4):511-517. Blackwell J M, Goswami T, Evans C A W, et al. SLC11A1(formerly NRAMP1) and disease resistance[J]. Cellular Microbiology, 2001, 3(12):773-784. Feng J, Li Y, Hashad M, et al. Bovine natural resistance associated macrophage protein 1(Nramp1) gene[J]. Genome Research, 1996, 6(10):956-964. Gruenheid S, Cellier M, Vidal S, et al. Identification and characterization of a second mouse Nramp gene[J]. Genomics, 1995, 25(2):514-525. Bairoch A. The PROSITE dictionary of sites and patterns in proteins, its current status[J]. Nucleic Acids Research, 1993, 21(13):3097-3103. Bellamy R. NRAMP1 and susceptibility to tuberculosis[J]. The International Journal of Tuberculosis and Lung Disease, 2002, 6(9):747. Gros P, Skamene E, Forget A. Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice[J]. The Journal of Immunology, 1981, 127(6):2417-2421. Goto Y, Nakamura R M, Takahashi H, et al. Genetic control of resistance to Mycobacterium intracellulare infection in mice[J]. Infection and Immunity, 1984, 46(1):135-140. Govoni G, Vidal S, Gauthier S, et al. The Bcg/Ity/Lsh locus:genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1Gly169 allele[J]. Infection and Immunity, 1996, 64(8):2923-2929. Cellier M, Govoni G, Vidal S, et al. Human natural resistance-associated macrophage protein:cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression[J]. Journal of Experimental Medicine, 1994, 180(5):1741-1752. Sun H S, Wang L, Rothschild M F, et al. Mapping of the natural resistance-associated macrophage protein 1(NRAMP1) gene to pig chromosome 15[J]. Animal Genetics, 1998, 29(2):138-140. Dorschner M O, Phillips R B. Comparative analysis of two Nramp loci from rainbow trout[J]. DNA and Cell Biology, 1999, 18(7):573-583. Chen Songlin, Zhang Yuxi, Xu Jianyong, et al. Molecular cloning, characterization and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from turbot (Scophthalmus maximus)[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 2007, 147(1):29-37. 刘洋. 外源基因表达载体的构建以及向斑马鱼(Dania rerio)和鲈鱼(Lateolabrax japonicus)胚胎的转移[D]. 青岛:中国海洋大学, 2004. Liu Yang. Exogenous genes construction, transfer in to embryos of Zebrafish (Dania Rerio) and Sea Perch (Lateolabrax Japonicus)[D]. Qingdao:Ocean University of China, 2004. Chen Songlin, Xu Meiyu, Ji Xiangshan, et al. Cloning and characterisation of natural resistance associated macrophage protein (Nramp) cDNA from red sea bream (Pagrus major)[J]. Fish & Shellfish Immunology, 2004, 17(4):305-313. 范玉顶, 徐进, 罗晓松, 等. 草鱼天然抗性相关巨噬蛋白基因全长cDNA的克隆与表达分析[J]. 中国水产科学, 2011, 18(1):38-47. Fan Yuding, Xu Jin, Luo Xiaosong, et al. Molecular cloning, characterization and expression of natural resistance associated macrophage protein (Nramp) gene cDNA from grass carp (Ctenopharyngodon idella)[J]. Journal of Fishery Sciences of China, 2011, 18(1):38-47. Donovan A, Brownlie A, Dorschner M O, et al. The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1(DMT1)[J]. Blood, 2002, 100(13):4655-4659. 邱晴, 谢李珍, 傅兴隆, 等. 缢蛏副溶血弧菌的分离与鉴定[J]. 安徽农学通报, 2010, 16(12):46-47, 89. Qiu Qing, Xie Lizhen, Fu Xinglong, et al. Isolation and identification of Vibrio Parahaemolyticus from Sinonovacula constricta[J]. Anhui Agricultural Science Bulletin, 2010, 16(12):46-47, 89. Anderson R S. Effects of anthropogenic agents on bivalve cellular and humoral defense mechanism[J]. Special Publication. American Fisheries Society, 1988, 18:238-242. 冯冰冰, 牛东红, 钟玉民, 等. 缢蛏ScHsc70cDNA的分子特性和表达分析[J]. 中国水产科学, 2012, 19(1):33-44. Feng Bingbing, Niu Donghong, Zhong Yumin, et al. Molecular characteristics and expression analysis of ScHsc70 cDNA in agamaki clam (Sinonovacula constricta)[J]. Journal of Fishery Sciences of China, 2012, 19(1):33-44. 冯冰冰. 缢蛏肝胰脏cDNA文库构建和免疫相关基因的克隆与表达研究[D]. 上海:上海海洋大学, 2011. Feng Bingbing. cDNA library construction of hepatopancreas and immune-relevant genes cloning and expression analysis in Sinonovacula constricta[D]. Shanghai:Shanghai Ocean University, 2011. 冯冰冰, 钟玉民, 牛东红, 等. 缢蛏β-ACTIN 1基因的分子特性及其表达分析[J]. 水产学报, 2011, 35(5):650-659. Feng Bingbing, Zhong Yumin, Niu Donghong, et al. Molecular characteristics and expression analysis of β-ACTIN 1 gene from Sinonovacula constricta[D]. Journal of Fisheries of China, 2011, 35(5):650-659. 牛东红, 冯冰冰, 李家乐. 缢蛏铁蛋白基因的分子特性及其表达分析[J]. 上海海洋大学学报, 2012, 21(5):641-649. Niu Donghong, Feng Bingbing, Li Jiale. Molecular characteristics and expression analysis of ferritin gene from Sinonovacula constricta[J]. Journal of Shanghai Ocean University, 2012, 21(5):641-649. Niu Donghong, Jin Kai, Wang Lei, et al. Identification of cathepsin B in the razor clam Sinonovacula constricta and its role in innate immune responses[J]. Developmental & Comparative Immunology, 2013, 41(1):94-99. Kishi F, Tabuchi M. Complete nucleotide sequence of human NRAMP2 cDNA[J]. Molecular Immunology, 1997, 34(12/13):839-842. Klausner R D, Rouault T A, Harford J B. Regulating the fate of mRNA:the control of cellular iron metabolism[J]. Cell, 1993, 72(1):19-28. Gunshin H, Mackenzie B, Berger U V, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter[J]. Nature, 1997, 388(6641):482-488. Forbes J R, Gros P. Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions[J]. Trends in Microbiology, 2001, 9(8):397-403. Saeij J P J, Wiegertjes G F, Stet R J M. Identification and characterization of a fish natural resistance-associated macrophage protein (NRAMP) cDNA[J]. Immunogenetics, 1999, 50(1/2):60-66. Cellier M, Shustik C, Dalton W, et al. Expression of the human NRAMP1 gene in professional primary phagocytes:studies in blood cells and in HL-60 promyelocytic leukemia[J]. Journal of Leukocyte Biology, 1997, 61(1):96-105. Barthel R, Feng Jianwei, Piedrahita J A, et al. Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells:effect on Brucella abortus survival[J]. Infection and Immunity, 2001, 69(5):3110-3119. 金凯, 牛东红, 王劦, 等. 缢蛏丝氨酸蛋白酶基因的序列特征及其表达分析[J]. 上海海洋大学学报, 2013, 22(4):481-487. Jin Kai, Niu Donghong, Wang Lie, et al. Molecular characteristics and expression analysis of serine protease from Sinonovacula constricta[J]. Journal of Shanghai Ocean University, 2013, 22(4):481-487. 邢贺飞. 半滑舌鳎抗病相关SNP标记的筛选及两种免疫相关基因的克隆与初步分析[D]. 上海:上海海洋大学, 2015. Xing Hefei. Screening and identification of disease-resistant related single nucleotide polymorphism markers, and cloning and preliminary analysis of two immune-related genes in half smooth tongue sole (Cynoglossus semilaevis)[D]. Shanghai:Shanghai Ocean University, 2015. 李同明. 尼罗罗非鱼Nramp基因的克隆分析与表达研究[D]. 泰安:山东农业大学, 2013. Li Tongming. Molecular cloning and expression analysis of Nramp gene in Nile tilapia (Oreochromis niloticus)[D]. Tai'an:Shandong Agricultural University, 2013. Atkinson P G P, Blackwell J M, Barton C H. Nramp1 locus encodes a 65 kDa interferon-γ-inducible protein in murine macrophages[J]. Biochemical Journal, 1997, 325(3):779-786.
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