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, and the cell autophagy genes ATG13 cDNA had 2 043 bp (NCBI 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 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 Litopenaeus 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 6 h and 12 h, 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 to M. nipponense ATG13 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.