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

采用c DNA末端快速扩增(RACE)技术获得中国对虾(Fenneropenaeus chinensis)ATG5基因c DNA全长,命名为Fc ATG5。利用实时荧光定量技术分析了Fc ATG5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。基因分析显示,Fc ATG5c DNA全长为2225bp,开放阅读框为810bp,编码269个氨基酸,预测其编码的蛋白质分子量为31.103kDa,理论等电点为5.59,为疏水性蛋白,包含1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显示,Fc ATG5具有高度保守性,与凡纳滨对虾(Litopenaeus vannamei)同源性最高(98.14%)。组织表达分析显示,Fc ATG5在中国对虾各组织中均有表达,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,Fc ATG5在鳃中的表达量最低,为对照组的1.68倍;胁迫后96 h最高,为对照组的2.67倍。碳酸盐碱度胁迫后12 h,Fc ATG5在鳃中表达量最高,为对照组的2.77倍;胁迫后96 h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Fc ATG5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。实时定量结果表明,Fc ATG5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的调控。本研究结果对水生动物特别是甲壳动物中细胞自噬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。

The full-length cDNA of ATG5 in Fenneropenaeus chinensis was cloned using rapid amplification of cDNA end(RACE)technology and named FcATG5.The tissue expression of Fc ATG5and its expression characteristics under pH and carbonate alkalinity stress were analyzed using quantitative PCR.The function of ATG5 was verified using RNAi.Gene analysis showed that the Fc ATG5gene cDNA consisted of 2225 bp with an open reading frame of 810 bp,encoding 269 amino acids,and has a predicted protein molecular weight of 31.103 kDa 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.The homology and phylogenetic analysis showed that Fc ATG5 was highly conserved and had the highest homology with Litopenaeus vannamei,reaching 98.14%.The tissue expression analysis showed that Fc ATG5 was expressed in all tissues of F.chinensis,with the highest expression in muscle,and the lowest expression in blood lymphocyte(P<0.05).The expression level of Fc ATG5 in the gill tissue was the highest at 96 h after pH stress,2.67 times higher than that of the control group,and was the lowest at 48 h,which was 1.68 times higher than that of the control group.The expression level of Fc ATG5 in the gill tissue was the highest at 12 h after the carbonate alkalinity stress,2.77 times higher than that of the control group,and was the lowest at 96 h,which was 1.30 times higher than that of the control group.The results of the interference experiments showed that under pH and carbonate alkalinity stress,silencing the Fc ATG5 gene significantly increased the mortality of F.chinensis(P<0.05),indicating that the higher the expression of this gene,the higher the survival rate of F.chinensis.The results of the study showed that the expression of Fc ATG5 was significantly increased under pH and carbonate stress(P<0.05).It is speculated that autophagy may be involved in the regulation of F.chinensis in response to abiotic stress.The results of this study are an important reference for the study of autophagy in aquatic animals,especially crustaceans,and will help advance the research of Chinese shrimp in saline-alkaline aquaculture systems.