日本沼虾细胞自噬基因ATG13和ATG101的克隆及其低氧胁迫下表达分析

为研究自噬相关基因(autophagy-related genes, ATG) ATG13和ATG101在甲壳动物应答低氧胁迫过程中的调节作用,实验采用RACE PCR技术通过克隆测序和基因序列拼接,首次克隆了日本沼虾的细胞自噬基因ATG13和ATG101的全长cDNA序列,日本沼虾的细胞自噬基因ATG13 cDNA全长2 043 bp (NCBI登录号为MT084347),包括211 bp的5′末端非翻译区(untranslated region,UTR),449 bp的3′UTR和1 383 bp的开放阅读框(open reading frame,ORF),开放阅读框编码460个氨基酸;ATG101 cDNA全长1 051 bp(NCBI登录号为MT084348),包括18 bp的5′末端非翻译区,373 bp的3′UTR和660 bp的开放阅读框,开放阅读框编码219个氨基酸。通过软件和生物信息网站对其序列进行分析,氨基酸相似度比对显示,日本沼虾的细胞自噬基因ATG13富含高度保守的LC3作用结构域(LIR);系统进化树分析显示,日本沼虾的细胞自噬基因ATG13与凡纳滨对虾ATG13具有最近的亲缘关系;通过实时荧光定量PCR技术(qRT-PCR)实验分析发现,日本沼虾ATG13和ATG101在其肝胰腺和脑组织表达量较高,而在肌肉中表达量较低;利用qRT-PCR追踪其在肝胰腺组织低氧胁迫过程中出现的表达差异情况,结果显示,实验组日本沼虾在低氧胁迫6和24 h时,其细胞自噬基因ATG13和ATG101表达量显著高于对照组,而在复氧12 h后,实验组与对照组的ATG13和ATG101表达量差异不显著;Western blot分析结果显示,日本沼虾ATG13和ATG101表达丰度基本与基因表达模式相似。透射电镜分析结果显示,在低氧胁迫6和24 h后,肝胰腺组织中的溶酶体开始出现自噬空泡,表明急性低氧胁迫会诱导自噬体的形成,本研究结果可为了解日本沼虾应对低氧胁迫下的调控机制提供理论参考。

Cloning and expression analysis of autophagy genes ATG13 and ATG101 in Macrobrachium nipponense under hypoxic stress

In order to study the regulation of autophagy-related gene 13 (ATG13) and ATG101 in Macrobrachium nipponense under hypoxia stress, the full-length cDNA sequences of ATG13 and ATG101 of M. nipponense were cloned for the first time by RACE PCR through cloning, expounds the cell autophagy genes ATG13 cDNA 2 043 bp (NCBI login id: MT084347), including 211 bp 5'' untranslated regions (UTR), 449 bp 3'' UTR and 1 383 bp open reading frame (ORF), which encodes 460 amino acids; ATG101 cDNA is 1 051 bp long (NCBI login id: MT084348), including the 5'' terminal non-translation region of 18 bp, the 3'' UTR of 373 bp and the open reading frame of 660 bp. The open reading frame encodes 219 amino acids. Based on bioinformatics analysis, the amino acid similarity ratio showed that the autophagy gene ATG13 of the biogas shrimp was rich in highly conserved LC3 functional domain (LIR). Phylogenetic tree analysis showed that the ATG13 gene of M. nipponense was closely related to the autophagy related genes of Penaeus vannamei. The results showed that ATG13 and ATG101 were highly expressed in hepatopancreas and brain tissues, but low in muscles. The difference of ATG13 and ATG101 expression in hepatopancreas tissues under hypoxia stress was tracked by real-time fluorescence quantitative PCR (qRT-PCR). The expression of ATG13 and ATG101 in the experimental group was significantly higher than that in the control group at 6h and 12h, but there was no significant difference between the experimental group and the control group at 12 h after the recovery of normoxia, which were similar with M. nipponense TAG13 and ATG101 protein expression abundance using western blot. The results of ultrastructure observation by transmission electron microscope (TEM) showed that autophagic vacuoles began to appear in lysosomes of hepatopancreas after 6 h and 24 h of hypoxia, suggesting that acute hypoxic stress could induce the formation of autophagosomes. The results of this study may provide a theoretical reference for understanding the regulatory mechanism of M. nipponense in response to hypoxic stress.