Identification Of Autophagy Molecules And Their Roles In Patinopecten Yessoensis Under High Temperature Stress

Autophagy is a cellular metabolic process in which cells degrade their own components through lysosomes or vacuoles to maintain normal physiological activities and homeostasis in cells.When the body is faced with adverse conditions such as hunger stress,environmental stress and pathogen invasion,it will degrade some non-essential proteins and aging organelles in cells through autophagy process,so as to maintain the survival of cells.Patinopecten yessoensis is a kind of large cold-water aquaculture shellfish which has a large proportion in northern Liaoning province mariculture.In recent years,due to global warming and rising sea temperature,high temperature stress has been caused to floating raft cultured Patinopecten yessoensis,which has seriously affected the normal physiological metabolism level of Patinopecten yessoensis.High temperature stress can cause the imbalance of energy metabolism homeostasis,and autophagy,as a regulation of energy metabolism,plays an important role in the process of coping with high temperature stress in Patinopecten yessoensis.In this study,19 autophagy related molecules of Patinopecten yessoensis were selected from the National Center for Biotechnology Information(NCBI)database using bioinformatics,molecular biology,immunology and cell biology.The expression levels of 19 Autophagy related molecules(ATG)m RNA,4 autophagy related proteins and the changes of 2 phosphorylation sites under high temperature stress were detected,and the autophagy generation process of cells under high temperature stress was discussed.The results are as follows:From the NCBI database,the Patinopecten yessoensis(Py)UNC-51-like kinase 1(ULK1/ATG1),autophagy related 13(ATG13),ATG101,cytoplasmic type III phosphatidylinositol kinase(Vps34/PI3K),autophagy related 6(Beclin1/ATG6),Vps15(P150),Vps38(UVRAG),ATG9,ATG2,ATG5,ATG7,ATG10,ATG12,ATG16,ATG3,ATG4,microtubule a were selected.Associated protein 1 light chain 3(LC3/ATG3),SQSTM1(P62)NBR1 Autophagy Cargo Receiver(NBR1)has a total of 19 autophagy related molecules.The domains they contain are highly conserved compared to the domains of homologous proteins in higher animals.Multiple sequence alignment showed that the amino acid sequence of the Patinopecten yessoensis ULK1(Py ULK1 for short)was 52.17% similar to the amino acid sequence of the homologous protein of 15 species;The similarity between the Py PI3 K amino acid sequence and the amino acid sequences of 13 species’ homologous proteins is 56.75%;The similarity between Py Beclin1 amino acid sequence and 13 homologous protein amino acid sequences of species is 76.49%;The similarity between the Py Vps15 amino acid sequence and the amino acid sequences of 13 species’ homologous proteins is 68.84%;The similarity between the amino acid sequence of Py ATG9 and the amino acid sequence of 17 species’ homologous proteins is 60.69%;The similarity between the amino acid sequence of Py ATG7 and the amino acid sequences of 17 species’ homologous proteins is 60.39%;The similarity between the Py ATG12 amino acid sequence and the amino acid sequences of 13 species’ homologous proteins is 51.13%;The similarity between the amino acid sequence of Py ATG16 and the amino acid sequences of 13 homologous proteins in species is 62.74%;The similarity between the Py LC3 amino acid sequence and the amino acid sequences of 17 species’ homologous proteins is 70.40%.Under 25℃ acute heat stress,m RNA expression levels of 16 autophagy related molecules increased significantly in hemolymph cells,while m RNA expression levels of 3 autophagy related molecules decreased significantly.The m RNA expression level of Py ULK1 in blood lymphocytes was significantly increased after 1 h,3 h,6 h and 12 h under high temperature stress,which was6.82 – fold of that of control group(p < 0.05),14.28 – fold(p < 0.05),4.60 – fold p < 0.05),25.18– fold(p < 0.05),m RNA expression level of Py LC3 was significantly increased,which was 7.16– fold of that of control group(p < 0.05),7.04 – fold(p < 0.05),11.70 – fold(p < 0.05),7.76 –fold(p < 0.05).the expressions of Py ULK1,Py Beclin1,Py ATG16 and Py LC3 in hemolymph cells were up-regulated under high temperature stress.Both Py AMPK-Py Beclin1 and Py ULK1-Py Beclin1 autophagy signaling pathways are activated: Py Beclin1(ULK1 activation site)Py Beclin1-ser30 and Pybeclin1-Thr385(AMPK activation site)were activated,but Pybeclin1-ser30 activation was more intense.the expression levels of Py ULK1,Py Beclin1,Py ATG16 and Py LC3 in hemolymph cells increased significantly under high temperature stress.Both Py AMPKPy Beclin1 and Py ULK1-Py Beclin1 autophagy signaling pathways were activated.After treatment with ULK1 active inhibitor ULK101 and 25℃ acute heat stress for 3 h,no obvious bands were detected in hemolymph,and m RNA level of Py LC3 decreased significantly,which was 0.06 – fold of that of control group(p < 0.001).In conclusion,19 autophagy related molecules with conserved domains and high homology with autophagy related molecules in higher animals were identified in Patinopecten yessoensis.At the same time,they had relatively more conserved autophagy signaling pathways than higher animals.Under high temperature stress,19 autophagy related molecules responded to the regulation of autophagy signals,and the autophagy pathways of Py AMPK-Py Beclin1 and Py ULK1-Py Beclin1 were activated.No obvious bands of LC3-II were detected under high temperature stress after Py ULK1 activity was inhibited.The results of this study will lay a foundation for further research on the autophagy mechanism of Patinopecten yessoensis under high temperature stress,and also provide a certain reference for further research on the response mechanism of shellfish under high temperature stress.