文章摘要
中国对虾ATG5基因的克隆及其在pH、碳酸盐碱度胁迫下的表达分析*
Cloning of ATG5 Gene of Fenneropenaeus chinensis And Expression Analysis under pH and Carbonate Alkalinity Stress
投稿时间:2021-03-06  修订日期:2021-04-04
DOI:
中文关键词: 中国对虾  FcATG5  pH胁迫  碳酸盐碱度胁迫  RNAi
英文关键词: Fenneropenaeus chinensis  FcATG5  pH stress  carbonate alkalinity stress
基金项目:国家自然科学基金项目;国家虾蟹产业技术体系;中国水产科学研究院基本科研业务费资助;山东省重点研发计划项目;中央提前下达渔业成品油价格改革财政补贴项目
作者单位邮编
魏威 青岛农业大学海洋科学与工程学院 青岛 266071
何玉英 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 青岛 
李朝霞 青岛农业大学海洋科学与工程学院 青岛 266071
周雨欣 水产科学国家级实验教学示范中心上海海洋大学 上海
水产科学国家级实验教学示范中心上海海洋大学 上海 
李健 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 青岛 
谢拥军 河北省水产良种与渔业环境监测保护总站 
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中文摘要:
      采用RACE技术克隆获得中国对虾(Fenneropenaeus chinensis) ATG5 基因cDNA全长,命名为FcATG5,利用实时荧光定量技术分析了FcATG5的组织表达及其在pH和碳酸盐碱度胁迫下的表达特征,并利用RNAi技术验证其功能。基因分析表明,FcATG5基因cDNA全长为2225bp,开放阅读框810bp,编码269个氨基酸,预测蛋白分子量为31.10347kD,理论等电点5.59,为疏水性蛋白,包含一个APG5自噬相关蛋白结构域,无跨膜结构,不包含信号肽。同源性和系统进化分析表明,FcATG5具有高度保守性,与凡纳滨对虾(Litopenaeus vannamei)同源性最高,达98.14%。组织表达分析表明,FcATG5在中国对虾的各个组织中均有表达,其中肌肉中表达量最高(P<0.05),血淋巴细胞中最低(P<0.05)。pH胁迫后48h FcATG5在中国对虾鳃组织的中表达量最低,为对照组的1.68倍,胁迫后96h最高,为对照组的2.67倍;碳酸盐碱度胁迫后12h,FcATG5在中国对虾鳃组织中的表达量最高,为对照组的2.77倍,胁迫后96h最低,为对照组的1.30倍。干扰实验结果显示, pH和碳酸盐碱度胁迫下,沉默FcATG5基因会使中国对虾死亡率显著增高(P<0.05),表明该基因的表达量越高,越有利于中国对虾存活。研究结果表明,FcATG5在pH、碳酸盐胁迫下的表达量均显著升高(P<0.05),推测自噬可能参与中国对虾应对非生物胁迫过程的调控机制。本研究结果对水生动物特别是甲壳动物中细胞自噬的研究具有重要的借鉴意义,有助于推进中国对虾盐碱水养殖的研究进程。
英文摘要:
      The full-length cDNA of ATG5 in Fenneropenaeus chinensis was cloned using RACE technology and named FcATG5. The tissue expression of FcATG5 and its expression characteristics under pH and carbonate alkalinity stress were analyzed using quantitative PCR. And the function of ATG5 was verified by RNAi. Gene analysis showed that FcATG5 gene cDNA consisted of 2225bp with an open reading frame of 810bp, encoding 269 amino acids, a predicted protein molecular weight of 31.10347kD, and a theoretical isoelectric point of 5.59. It is a hydrophobin, contains an autophagy-related protein domain APG5, has no transmembrane structure, and does not contain signal peptides. Homology and phylogenetic analysis showed that FcATG5 was highly conserved and had the highest homology with Litopenaeus vannamei, reaching 98.14%. Tissue expression analysis showed that FcATG5 was expressed in all tissues of Fenneropenaeus chinensis, with the highest expression in muscle (P<0.05) and the lowest expression in blood lymphocytes(P<0.05). The expression level of FcATG5 in the gill tissue was the highest at 96h after pH stress, 2.67 times higher than that of the control group, and was the lowest at 48h, which was 1.68 times higher than that of the control group; The expression level of FcATG5 in the gill tissue was the highest at 12h after the carbonate alkalinity stress, 2.77 times higher than that of the control group, and was the lowest at 96h, which was 1.30 times higher than that of the control group. The results of interference experiments showed that under the stress of pH and carbonate alkalinity, silencing the FcATG5 gene significantly increased the mortality of Fenneropenaeus chinensis (P<0.05), indicating that the higher expression of this gene, the higher survival rate of Fenneropenaeus chinensis. The results of the study showed that the expression of FcATG5 was significantly increased under pH and carbonate stress (P<0.05). It is speculated that autophagy may be involved in the regulation mechanism of Penaeus chinensis in response to abiotic stress. The results of this study have important reference significance for the study of autophagy in aquatic animals, especially crustaceans, and will help advance the research process of Chinese shrimp in saline-alkali aquaculture.
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