| Autophagy,as an intracellular degradation system,delivers cytoplasmic constituents to the lysosome.Recent progress in recent years has shown that Autophagy has variously complex physiologicai and pathophysiological effects.Several consecutive procedures of Autophagy include sequestration,transport to lysosomes,degradation,and utilization of degradation products and each step may play different roles.p62,a multifunctional ubiquitin-binding protein,is involved in two protein degradation processes:ubiquitin proteasome system(UPS)and autophagy lysosomal system.As a scaffold and adaptor protein in signal transduction pathway,p62 has multiple functional domains in its molecular structure that can interact with other proteins and mediate a variety of cellular functions,especially in the selective autophagy and antioxidant reactions of cells.p62 connects autophagy mechanism with ubiquitinated protein substrates through LC3 interaction region and c-terminal ubiquitin-associated domain to promote the selective degradation of these molecules.In this study,the open reading frame of p62 was firstly cloned by PCR from the Spodoptera litura.The p62 gene was constructed by PCR and inserted into prokaryotic expression vectors.The expression vector was transformed into the expression strain E.coli BL21,in order to prepare rabbit anti-p62 antibodies,the expression and purification of p62 protein was carried out.Meanwhile,we constructed pET-28a-p62 prokaryotic expression vector.After transfection into Sl-HP,Hi5 and Sf9 cells,the sublocation of the overexpressed p62 protein was determined..In order to exclude the effect of label on the localization of p62 protein,the subcellular localization of endogenous expression of p62 protein and the difference of endogenous expression level in different cell lines were detected by p62 antibody.In addition,the bioinformatics analysis of the p62 was carried out to compare the expression level of p62 in different organs of the larvae of the fourth instar.In order to explore the possible regulatory mechanism of p62 in autophagy,IF,Pull down,BIFC and other technologies were used to explore its interaction with autophagy related genes such as Atg8,and Atgl.Based on laboratory proves that the induction of autophagy by toxins requires the toxin receptor to mediate and is positively correlated with the sensitivity of cells to toxins.We also use the method for Helicoverpa armigera.Technologies were used to investigate the relationship between the expression of p62 protein and the level of autophagy.The experimental results show the complete coding DNA sequence(CDS)of Sl-p62,the length of which is 1719 bp,coding 572 amino acids.There are six main domain in the p62 protein including the PB1 domain,the central zinc finger ZZ domain and the TRAF6-binding domain,the UBA domain,the LIR domain and the KIR domain,which are necessary for its mutual interaction with.The full-length protein expressed in E.coli was in the form of inclusion body,and anti-p62 antibody was successfully prepared.p62-GFP fused protein is localized in cytoplasm,and the expression in Sf9 cells was the least.This result is the same as that of endogenous expression.The expression of midgut was higher than that of other parts of larva.Slp62 was co-localized with Atg8 and Atg1,what’s more,it showed that p62 and Atg8 had strong interaction signals,and it showed that p62 and Atgl had possibly interaction signals.When the autophagy level of sl-hp cells was increased,there was no significant change in the content of p62 protein in the cells.In cotton bollworm larvae,the level of autophagy was increased,and the WB results showed that the expression of p62 was slightly decreased,but the tissue immunofluorescence results still showed no significant change in the content of p62 protein.Since more antibodies are required for the systematic study of autophagy in the laboratory,polyclonal antibodies of three autophagy related proteins have been prepared for the laboratory study. |
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