文章摘要
日本沼虾血清淀粉样蛋白A(SAA)基因的克隆及其免疫功能分析
Molecular cloning and characterization of serum amyloid A gene and the analysis of its immune function in Macrobrachium nipponense
投稿时间:2018-12-17  修订日期:2019-04-14
DOI:
中文关键词: 日本沼虾  血清淀粉样蛋白A  基因克隆  组织表达  病原体感染  RNA 干扰
英文关键词: Macrobrachium nipponense  serum amyloid A  gene cloning  tissue expression  pathogen infection  RNA interference
基金项目:河南省教育厅科学技术重点研究项目(13A240509),河南师范大学博士科研启动费支持项目(qd17142);河南省重点科技攻关项目(192102110081)
作者单位E-mail
江红霞 河南师范大学 水产学院 jianghongxia2007@126.com 
林展畅 河南师范大学 水产学院  
林鑫辉 河南师范大学 水产学院  
李学军 河南师范大学 水产学院 xjli@htu.cn 
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中文摘要:
      为探索血清淀粉样蛋白A(SAA)在日本沼虾免疫防御系统中的作用,利用RACE方法克隆日本沼虾SAA(MnSAA)基因cDNA全长序列,采用实时荧光定量PCR(QPCR)分析其在日本沼虾不同组织、不同发育时期的表达情况,以及溶壁微球菌(Micrococcus lysoleikticus)、嗜水气单胞菌(Aeromonas hydrophila) 和白斑综合症病毒(WSSV)感染后MnSAA在血淋巴细胞和肝胰腺中的表达情况,同时还利用RNA干扰(RNAi)技术检测了MnSAA基因沉默后,日本沼虾再感染嗜水气单胞菌后其肝胰腺中激活子蛋白-1(AP-1)和B类清道夫受体(CD36)基因表达变化和虾的累积死亡率变化。结果显示,MnSAA基因cDNA全长649 bp (GenBank登录号: MK292888),包含21 bp 5′非编码区,232 bp 3′非编码区,369 bp开放读码框,编码131个氨基酸残基;氨基酸序列比对及系统进化分析结果显示,MnSAA蛋白属于急性期血清淀粉样蛋白A蛋白(A-SAA),在进化上与无脊椎动物香港巨牡蛎(Crassostrea hongkongensis)的亲缘关系最近;mRNA表达分析显示,MnSAA基因在日本沼虾的各组织和各生长阶段均有表达,分别在肝胰腺和成虾阶段中的表达量最高;病原体感染实验表明,在日本沼虾感染溶壁微球菌、嗜水气单胞菌和白斑综合症病毒后12-72 h其血淋巴和肝胰腺中的MnSAA基因表达量与对照组相比均显著升高(P<0.05);RNAi实验显示,MnSAA基因沉默后,日本沼虾肝胰腺中AP-1和CD36基因分别在其感染嗜水气单胞菌后 12-72 h和6-72 h与对照组相比显著下降(P<0.05),日本沼虾累积死亡率在感染嗜水气单胞菌后与对照组相比明显升高。研究表明,MnSAA参与日本沼虾的抗病原体感染的反应,在日本沼虾的免疫防御体系中具有重要的作用。
英文摘要:
      In order to study the function of serum amyloid A (SAA) in the immune defense of Macrobrachium nipponense, the full-length cDNA sequence of the SAA gene in M.nipponense, named MnSAA, was cloned using rapid amplification of cDNA ends (RACE) method. The expression level of MnSAA in different tissues and different development stages of M. nipponense, and the expression profile of MnSAA in hemocytes and hepatopancreas of M. nipponense after Micrococcus lysoleikticus, Aeromonas hydrophila and white spot syndrome virus (WSSV) infections were also examined by quantitative real-time PCR (QPCR). At the same time, the expression changes of activator protein-1 (AP-1) and class B scavenger receptor (CD36) genes in hepatopancreas and cumulative mortality of prawn after MnSAA gene silencing plus A. hydrophila infection were further investigated using RNA interference (RNAi) technology. The results showed that the full-length cDNA of MnSAA was 649 bp, including a 21 bp 5′-untranslated region (UTR), a 232 bp 3′UTR, and a 369 bp open reading frame (ORF) encoding a deduced protein with 131 amino acids. Sequence and phylogenic analyses revealed that MnSAA belongs to acute-serum amyloid A (A-SAA) and had a close relationship with the invertebrate Crassostrea hongkongensis. mRNA expression analysis revealed that MnSAA gene was ubiquitously expressed in various tissues and growth stages of M. nipponense, and abundantly expressed in the hepatopancreas and adult prawn stage. Pathogen infection experiment showed that the expressions of MnSAA in hemocytes and hepatopancreas were all significantly increased compared with the control group after M. lysoleikticus, A. hydrophilahe and WSSV infections for 12-72 h (P<0.05). The RNAi experiment showed that after MnSAA silencing, AP-1 and CD36 gene expressions in hepatopancreas of M. nipponense were all significantly decreased compared with the control group after 12-72 h and 6-72 h, respectively, of A. hydrophila infection (P<0.05), and the cumulative mortality of prawn was increased compared with the control group after A. hydrophila infection. These results suggested that MnSAA is involved in the activity of anti-pathogen infection in M. nipponense and plays an important role in the immune defense system of M. nipponense.
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