Molecular Cloning And Characterization Of Autophagy Related Gene ATG13 From Blood Clam Tegillarca Granosa

Autophagy is a highly conserved intracellular degradation system in cells,involved in many life processes,such as cell survival,aging and immune response,and plays a key role in the treatment of various diseases such as myocardial infarction,Parkinson ’s disease and type II diabetes.In the process of autophagy,mammalian autophagy-related gene(ATG13)is an important subunit of the autophagy initiation complex of ATG1,and plays an important role in autophagy.However,the structure of ATG13 in low invertebrates and its specific molecular mechanism in autophagy and the role of autophagy mediated by ATG13 in pathogen-resistant microbial infection remain unclear.This paper uses Tegillarca granosa,a common economic shellfish,as the research model,to explore the main function of ATG13 in its immune regulation.The main research contents include:1.The full-length cDNA sequence of T.granosa ATG13 was cloned by RACE technique and named TgATG13.The full-length cDNA of TgATG13 was 1918 bp in length,including 283 bp 5’UTR,252 bp 3’UTR and 1383 bp open reading frame(ORF),encoding 460 amino acids.Phylogenetic tree analysis showed that TgATG13 had the closest relationship with that of Crassostrea Virginica.2.The recombinant plasmid pcDNA3.1-EGFP-TgATG13 was constructed,and the confocal microscopy study showed that TgATG13 was located in the cytoplasm of 293T cells.3.Quantitative RT-PCR results showed that the mRNA of ATG13 was universally expressed in various tissues of blood clam and the expression level was the highest in hemocytes and mantle.At the same time,under the stimulation of LPS and Vibrio alginolyticus,the expression level of TgATG13 increased in 12 h;4.T.granosa hemocytes was exposed to Vibrio alginolyticus,and NH4Cl was used as an autophagy inhibitor.Agar plate coating and Quantitative RT-PCR results showed that inhibition of autophagy by NH4Cl increased the number of V alginolyticus,indicating that autophagy played an important role in pathogen defense of blood clam;5.Autophagy was traced by transient transfection of GFP-LC3 plasmid.Fluorescence confocal microscopy results showed that TgATG13 promoted the production of autophagosome.Western Blot showed that the amount of LC-3I band decreased while the amount of LC-3Ⅱ band increased significantly,resulting the increase of LC-3Ⅱ/Ⅰ ratio,indicating that TgATG13 played an important role in the formation of autophagosomes.In summary,this study shows that TgATG13 participates in the autophagy process of blood clam,and autophagy can improve the antibacterial activity of hemocytes of blood clam,that is,ATG13 is invovled in the immune regulation of blood clam.The results of this study will provide reference information for the molecular mechanism of autophagy in bivalves,help to further understand the innate immune mechanism of invertebrates,and provide theoretical basis for the prevention and treatment of blood clam diseases.