Cloning,Expression And Functional Analysis Of Spodoptera Litura SlAtg1

Autophagy is a membrane compartment(mostly manifested as a double membrane,sometimes multi-storey or single)wrapped part cytoplasm,organelles and protein,finally fused with lysosomes to form autophagolysosome,which degradates its contents in order to achieve cells homeostasis and renewal of organelles.It is generally believed that autophagy is a defense and stress regulation mechanisms.It provides the necessary raw materials for the reconstruction,regeneration and cell repair,and achieves cells’ recycling and reuse.The cloning of autophagy gene(autophagy-related gene,atg)began in yeast.Atg 1 protein(autophagy-related protein 1)belongs to family of serine/threonine protein kinases,and plays a very critical role in autophagy.In the initial stage of autophagy,Atgl-Atg13 complex can raise other Atg proteins to induce autophagy.However,the interacting proteins of Atg1 were needed to be further studied,and regulation mechanisms of Atgl on autophagy are unclear.Atg8 is an ubiquitin protein which plays an important role in the extension of the autophagic vesicle membrane.It is commonly considered as a marker of autophagy.Spodoptera litura(Fabricius)belongs to the family of Noctuidae.It is a polyphagous pest,which causes serious damage to many crops,including soybean,cotton and vegetables.In this study,we designed degenerate primers based on homologous comparison of Atgl gene sequences from other lepidopteran insects,then cloned the open reading frame of SlAtgl by semi-nested PCR and carried out the bioinformatics analysis of the gene.The truncated Atgl genes(Atgl-N-terminal-600bp and Atgl-C-terminal-500bp)were amplified by PCR and inserted into prokaryotic expression vectors.The two expression vectors were transformed into the expression strain E.coli BL21,and the expression and purification of the truncated SlAtg1 proteins were carried out in order to prepare rabbit and mouse anti-Atgl antiserum.Meanwhile,we constructed SlAtgl eukaryotic expression vector.The eukaryotic expression plasmids with Atgl or other autophagy related genes such as Atg8,Atg6 and Atg5 were co-transfected into cells to observe the co-localization of Atg proteins.Sl-HP cells were treated by starvation or inhibitors of autophagy,respectively,to investigate the dgradation of Atgl protein during autophagy.Influence of RNAi and overexpression of SlAtgl on autophagy were also analyzed.Finally,the interaction between ATG1 and other Atg protein was tested by Co-IP analysis.We obtained the complete coding DNA sequence(CDS)of SlAtgl,the length of which is 2286 bp,coding 761 amino acids.The molecular mass of the putative SlAtg1 protein is about 82.6 kDa,and its pI is about 9.5.SlAtgl protein showed high conservation at N-terminal region including the catalytic domain and ATP-and Mg2+-binding sites.GFP-Atgl fused protein is localized in cytoplasm,not in nucleoplasm.SlAtgl was partially co-localized with Atg5 and Atg8.Autophagy induced by starvation resulted in the decrease of SlAtgl because of degradation by autolysosomes.Inhibition of autophagy by inhibitor can lead to Atg1 accumulation,indicating that SlAtg1 can be degradated through autophagy.The over-expression of SlAtgl accelerated the degradation of Atg8-PE,and the interference of SlAtgl resulted in the accumulaion of Atg8-PE,suggesting SlAtg1 promotes autophagy level.Co-IP analysis revealed that SlAtgl might interact with other Atg protein.These results provide important references for eclucidating the multiple regulation of SlAtg1 on autophagy at molecular level.