Functional Analysis Of Osiris17 In The Wing Development Of Insects

The wings of insects are composed of ventral epidermis and dorsal epidermis,which attach to each other tightly and form a flat wing structure.In the process of wing development,the epidermis is secreted by epithelial cells,which are in close contact at their bottom.The adhesion of dorsal and ventral epithelial cells depends on the interaction between integrin and extracellular matrix.We have studied the function of Osiris17(Osi17)gene in the development of insect wings.It was found that Osi17 was involved in the location and function of integrins in Drosophila melanogaster and Locusta migratia,and then affected the shape of wings.The main findings are as follows:1 DmOsi17 expression characteristics.We used CRISPR repeat(CRISPR)/CRISPR-associated(Cas)technology to construct Osi17-Gal4 transgenic lines.After crossing with UAS-GFP lines,we observed the GFP signals of offspring.It was found that DmOsi17 was mainly expressed in the epidermis and intestines during the larval stage,mainly on the wings during the pupal stage,and DmOsi17 was detected in the wings 12 hours after pupation,with gradually increasing signal intensity.In addition,it is also expressed in the intestines and genitals of adult worms.The expression of DmOsi17 m RNA in pupal wings was detected by RT-q PCR,and it was found that the highest expression level was found on the second day of pupal wings,consistent with the expression trend reported by GFP signals.2 Interference with DmOsi17 results in Drosophila wing development defectThe Gal4/UAS system specifically silences the wing DmOsi17,and it is found that it exhibits a bubble wing phenotype after eclosion.However,overexpression of DmOsi17 has no visible phenotype.After interference,the permeability of the wings was significantly changed by eosin staining.Subsequently,we performed frozen sections and FB28 staining on the wings after DmOsi17 interference.Compared with the control group,there was a significant separation of the dorsal and ventral wing membranes of nub>Osi17-RNAi wings,but there was no significant change in FB28 signals,indicating that interference with DmOsi17 affected dorsal and ventral adhesion,but did not affect chitin content.The results of transmission electron microscopy showed that gaps appeared in the wing epidermis of adults with nub>Osi17-RNAi,and there were defects in the connection between the dorsal and ventral epidermis of the wings of adults with nub>Osi17-RNAi at 60 hours of pupal stage.3 Function of Drosophila DmOsi17 in wing developmentBased on previously observed interference phenotypes,we speculate that DmOsi17 is related to integrin function.We performed frozen sections and integrin staining of wings at different stages of pupal development(30 h,36 h,48 h,and 60 h).The staining results showed that compared with the control group,the integrin signal of nub>Osi17-RNAi decreased significantly at 48 h and 60 h of pupal stage,and almost disappeared.Further subcellular localization of DmOsi17 expression showed that DmOsi17 was colocalized with Rab5 and Rab11,respectively,in S2 cells and pupal wings.Therefore,we speculate that DmOsi17 affects the maintenance of integrins through Rab5 and Rab11 mediated transport pathways.In addition,overexpression of DmOsi17 to some extent saved the Drosophila wing blisters caused by integrin deficiency.This indicates that DmOsi17 functions by regulating integrin activity.4 Functional study of Locust Lm Osi17.According to the Drosophila DmOsi17 amino acid sequence,the Locust Lm Osi17 sequence was retrieved from the Locust Transcriptome Database.The length of the Lm Osi17 coding region is 2051 bp,encoding 627 amino acids.Similar to Drosophila melanogaster,Lm Osi17 has three domains,namely,two cysteine domains,Duf1676 domain,and transmembrane domain.Gene silencing was performed by injecting ds Lm Osi17 into Locusts at different instars,and it was found that Lm Osi17 may participate in the intestinal development of Locust nymphs at the third instar.Silence of Lm Osi17 in the fifth instar nymph can lead to wrinkles in its adult wings.The electron microscopic observation of the semithin section showed that the wing veins of the wing membrane were separated,while the frozen section FB28 staining showed that there was still no significant difference in chitin signals.This is similar to the phenotype that interferes with DmOsi17 on the wings of adult Drosophila.Using paraffin sections and HE staining to observe the morphology of the wings of Locusts with different developmental days in the fifth instar.On the sixth day of the fifth instar,compared with the control group,the distance between the dorsal and ventral compartments of the wing buds in the ds Lm Osi17 treatment group increased,while the integrin signal was significantly reduced by integrin antibody staining.In summary,Osi17 participates in the maintenance of integrins on the wing and is functionally conserved in diptera and orthoptera insects.