| Hox gene is one of the oldest and highly conserved genes in animals,and contains a conserved DNA-binding domain homeodomain,which plays a conserved role in controlling morphogenesis and organogenesis.The function of a Hox gene is closely related to its expression pattern,disruption of which leads to developmental defects and physiological diseases.During the development of the silkworm,more than 30 Hox gene mutants exhibit significant morphological changes,such as multiple or missing legs,abnormal silk glands,wing defects and abnormal tentacle development.In addition to regulating transcription factors or signaling molecules,Hox genes also repress or promote essential cellular processes.Autophagy is a process that involves the decomposition of its structure through lysosomes,which is tightly regulated by a series of genes that help the organism maintain a dynamic balance in the synthesis and degradation of organic molecules,and in the cycling of the internal environment.Autophagy plays a key role in maintaining the growth and development of the organism,disease development and other physiological activities.The silkworm is a completely metamorphic insect with high economic value,and its metamorphosis is regulated by several signaling pathways including ecdysis hormone(20E),juvenile hormone(JH)and nutrition.Autophagy plays an important role in the metamorphosis of the silkworm.Overexpression of Hox genes Abd-A,Abd-B,Ubx and Dfd in Drosophila effectively inhibited developmental autophagy of fat body,however,the regulatory mechanism remains unclear.In this study,we investigated the mechanisms by which the silkworm Hox gene Abd-B negatively regulates fat body autophagy at the cellular,tissue and individual levels to provide theoretical guidance for the regulation of insect fat body development.The main findings of this paper were as follows.1.Spatiotemporal expression profiles of Hox genes Abd-A,Abd-B,Ubx and Antp in the fat body of the silkwormqPCR showed that Abd-A,Abd-B,Ubx and Antp were expressed in different tissues on day 3 of the fifth larval instar(L5D3),and the expression of Abd-A and Abd-B was relatively high in the fat body.The expression profiles from the fourth instar to the moth stage showed that Abd-A and Abd-B was highly expressed in the feeding stage and lowly expressed in the molting and wandering stage in fat body.Their expression profiles were opposite to those of autophagy-related genes.The results implied that Abd-A and Abd-B may regulate the expression of autophagy-related genes in fat body.2.Injection of 20E decreased expression of Abd-A and Abd-B and autophagy of fat body in the silkwormThe expression of 20E downstream genes,Abd-A and Abd-B,and autophagy in fat body were examined at 3 h,6 h,12 h and 24 h post-injection of 20E into in the silkworm larvae on day 2 of the fifth larval instar(L5D2),respectively.The results showed that the expression of Abd-A and Abd-B at mRNA and protein levels were down-regulated after20E injection,while autophagy-related genes,Atg8 at protein level,acid phosphatase activity and lysosomal content were up-regulated to different degrees.The results suggest that Abd-A and Abd-B may be involved in the regulation of autophagy-related genes.3.Abd-B represses the transcription of Atg5 and Atg8 genesThe upstream regions of the key autophagy genes Atg5 and Atg8 promoters were predicted online to contain multiple Abd-A and Abd-B binding cis-regulatory elements(CREs).The promoters of Atg5 and Atg8 were cloned and ligated into pGL3 vector,and then co-transfected with Abd-A or Abd-B into BmE cells.Dual luciferase activity showed that Abd-A had no effect on Atg5 and Atg8 promoter activities,while Abd-B inhibited Atg5 and Atg8 promoter activities.Further,different truncations of Atg5 and Atg8 genes promoter were constructed and co-transfected with Abd-B into BmE cells.Dual luciferase activity showed that Abd-B significantly inhibited Atg5 promoter activity.While the-1542 element of Atg5 promoter was removed,there was no significant difference between Atg5 promoter and the control in luciferase activity.Unlike Atg5,Abd-B inhibited different Atg8 promoter activities.EMSA demonstrated that Abd-B bound to-1542 element upstream of Atg5 promoter and-267 element upstream of Atg8 promoter,respectively.In short,the results suggest that Abd-B binds directly to-1542 element upstream of Atg5 promoter and-267 element upstream of Atg8 promoter,thus repressing the transcription of Atg5 and Atg8 genes.4.βftz-F1 represses the transcription of Abd-B geneThe temporal expression profiles of Abd-B showed an opposite trend to 20E titer.The promoter region of Abd-B gene was predicted online using Jaspar database to contain multiple βftz-F1 binding CREs.Different truncations of the Abd-B promoter were connected to the dual luciferase reporter vector,and the 1180 overexpression vector ofβftz-F1 was co-transfected into BmE cell.Dual luciferase activity showed that βftz-F1 repressed Abd-B promoter activity.While the-2410 element upstream of Abd-B promoter was removed,there was no significant difference between Abd-B promoters and the control in luciferase activity.EMSA further demonstrated that βftz-F1 binds directly to the-2410 element upstream of Abd-B promoter,thereby inhibiting the transcription of Abd-B gene.5.Abd-B is involved in starvation-induced autophagy in fat body of the silkwormIt is known that starvation induces autophagy in fat body of the silkworm.After starvation on the silkworm larvae(L5D3),qPCR showed that the expression of autophagy-related genes was up-regulated,the lysosomal content in the fat body was significantly increased.After 9 h,acid phosphatase activity and the expression of Atg8 in the fat body were significantly increased,indicating that starvation induces fat body autophagy.Meanwhile,Abd-B was down-regulated at both mRNA and protein levels.Overexpression of Abd-B in BmN cells followed by starvation led to down-regulation of autophagy-related genes compared with the control,indicating that Abd-B was involved in starvation-induced fat body autophagy. |
PDF Full Text Request