Studies On The Autophagic Function Of BmAtg13 And The Difference Of Autophagy Level In Different Tissues Of Bombyx Mori

Autophagy is highly conservative biological process in eukaryotes,digesting and decomposing intracellular substances.The replacement of old tissue by new one is an important physiological event during insect metamorphosis and autophagy is requsite for this process.The Bombyx mori,as a representative of lepidopteran species under artificial selection and special evolution,exists high level of autophagy during the larvae-pupal metamorphosis stage in some tissues.The occurrence of autophagy involves a series of autophagy-related(Atg)proteins.ULK1/Atg1-ATG13/Atg13 is the key protein complex responsible for the initiation of autophagy.The mechanisms of ULK1/Atg1-ATG13/Atg13 complex in participating autophagy are mainly focused on yeast,fruit fly and mammals.However,the manners of Atg13 homologs in autophagy occurrence in the above three species are different,wich indicates that the functions and regulatory mechanisms of Atg13 protein are different in species with different evolutionary status.So far,we still know less about the Atg13 hmologs in lepidopteran insects.Thereforce,we studied the autophagic function of B.mori Bm Atg13 in this dissertation.Data showed that autophagy detected in different larval tissues varied differently at the same stage.In addition,the levels of autophagy in different tissues from Dazao,Liangguang No.2 and Yuecan No.6 strains were measured from the 5th instar to the prepupal stage.The main research results obtained are as follows:(1)Bm Atg13 harbors the conserved functional protein domainThe phylogenetic analysis of Bm Atg13 protein showed that Bm Atg13 was more conserved with the homologs from Galleria mellonella and other lepidopteran species.The structure of Bm Atg13 protein was predicted based on its amino acid sequence.Results showed that Bm Atg13 protein was composed of the HORMA domain at the N-terminal and the disordered region at the C-terminal.The HORMA domain contained the Bm Atg101 binding region and the C-terminal contained the Bm Atg1 binding region.(2)Developmental profiles of Bm Atg13 proteinDevelopmental profiles of Bm Atg13 protein from day 2 of 5th instar to day 2 of prepupal stage revealed that there were two bands of Bm Atg13 protein detected by western blotting.The upper band of Bm Atg13 was intensively decreased,while the lower band was significantly increased in accordance with its m RNA variation;and immunofluorescent staining indicated that Bm Atg13 was nucleo-cytoplasmic translocated during larval-pupal metamorphosis,when autophagy was dramatically induced.(3)The expression pattern of Bm Atg13 protein in different silkworm strains is conservedProtein levels of Bm Atg13 in fat body,posterior silk gland,midgut,malpighian tubule,trachea,ovary,and testis from Dazao,Liangguang NO.2,and Yuecan NO.6 strains at5L4 D and PP1 stages were detected by western blotting.The results showed that Bm Atg13 protein was highly expressed in the posterior silk gland,followed by fat body,midgut and malpighian tubule,and its variation was consistent in the same tissue from the three different strains.(4)Bm Atg13 protein is involved in the regulation of autophagyThe ds RNA of Bm Atg13 gene was injected into B.mori larvae at 12 h before initiation of wandering when autophagy occurred.After Bm Atg13 RNAi treatment for 24 h,the protein level of Bm Atg13 in the fat body was significantly decreased,the formation of Bm Atg8–PE conjugation was apparently inhibited,and lysosome acidification was significantly blocked,indicating inhibition of autophagy.Overexpression of Bm Atg13 gene in Bm N cells resulted in a decrease of Bm Atg8–PE formation,accumulation of Bm Sqstm1,and thus blockage of autophagy.Moreover,Bm Atg13 overexpression inhibited starvation-induced autophagy.(5)20E and starvation signals regulate Bm Atg13 to participate in autophagyThe 5L2 D larvae were injected with 20 E or the Bm N cells were treated with 20 E in vitro.Compared to the control,the m RNA level of Bm Atg13 gene increased significantly,while Bm Atg13 protein decreased significantly.20 E treatment in Bm N cells or starvation treatment in vivo all resulted in a significant decrease in the upper band of Bm Atg13 protein.Interfering of the 20 E receptor Bm Usp in vivo caused reduction of Bm Atg13 protein and autophagy.These results indicated that the autophagic function of Bm Atg13 protein is regulated by 20 E or starvation signals.(6)Autophagy levels differ in several tissues of silkwormThe Bm Atg8 protein levels in the fat body,posterior silk glands,midgut,malpighian tubule,trachea,ovary and testis of the Dazao,Liangguang No.2 and Yuecan No.6 at the stages of 5L4 D and PP1 were detected by western blotting.The results showed that the levels of autophagy in different tissues of the silkworm were different.The Bm Atg8–PE levels in the posterior silk gland,midgut,malpighian tubule and trachea of the three strains were higher than that in fat body at 5L4 D stage.And the Bm Atg8–PE levels in the midgut and trachea were still higher than that of fat body at PP1 stage.(7)The developmental variation of autophagy in different tissues are divergent in larvae from the 5th instar to the prepupal stageWestern blotting and Lyso Tracker-Red staining showed that the posterior silk glands of Dazao,Liangguang No.2,and Yuecan No.6 had strong Bm Atg8–PE during the 5th larval feeding stages,while Bm Atg8–PE conjugation decreased but lysosome acidification was stable at the PP2 stage.Bm Atg8–PE was stronger after initiation of wandering,but the lysosomal acidification suddenly decreased at the PP2 stage in midgut.Bm Atg8–PE formation was high during the whole developmental stages,but lysosome acidification was detected weak in the trachea.Bm Atg8–PE in the malpighian tubule,ovary and testis tissue were weak during the whole developmental stages,and there was no significant lysosome acidification.These results help to elucidate the precise molecular mechanism of autophagy involved in Bm Atg13,and obtain the specific or universal rules of Atg13 in participating autophagy in the species at the intermediate evolutionary scale.It helps to further clarify the molecular mechanism of autophagy in different tissues by providing a scientific basis for related research in the future,and provides new target sites and thinking directions for lepidopteran pest control.