| Insects are the most abundant and abundant biological group on the earth.Their flight ability lays the foundation for their large-scale spread on the earth.In order to further adapt to the complex living environment of the earth,different insects have derived wings with different shapes,and there are great differences between the front and back wings of the same species.In recent years,with the continuous innovation of evolutionary developmental biology research strategy,it has been clear that the origin,occurrence and development of insect wings are closely related to homeotic gene（Hox）.Lweis first discovered Hox gene in Drosophila melanogaster in 1978.It is an important family of developmental regulatory genes that control the morphology of animal somites from front to back.Any mutation of Hox gene will cause abnormal development of corresponding somite organs and tissues.Except for fleas and Chlamys,most insects have two pairs of wings on the second and third thoracic segments.The Hox genes antennae（Antp）and Ultrabithorax（Ubx）mainly control these two somites,so it is speculated that wing development is related to these two genes.Studies have shown that Ubx deletion or overexpression in the fruit fly,beetle,and butterflies resulted in homeotic transformation between the hind wings（balance bars）,while the expression of Antp in the winged primordia was not affected by the down-regulation or heterotopic expression of the Ubx.Therefore,the reason for the specialization of the anterior and posterior wing shape is generally believed to be due to the regulation of a series of genes related to wing development in the hind wings.The forewing is not regulated by Hox gene Antp and is in the state of Antp-free.However,there are two functional deletion mutants of Antp gene in silkworm,NC and Wes.The phenotypes of these two homozygous mutants are similar to those of Drosophila Antp homozygous mutants at embryonic stage.They have the characteristics of embryonic death,thoracic segment healing and transition from chest foot to antennae.However,we observed wing type developmental defects in adults,which have not been mentioned in other species.Based on this,we speculate that Antp is involved in the development of insect wings.In this study,we take silkworm,Drosophila,Tribolium castaneum and Drosophila as the research objects to explore whether Antp affects the development of insect wings;we take silkworm hybrid mutant Wes as the research object to explore how Antp affects the development of insect wings.The main results are as follows:1.Overexpression of Antp in the wing discs of Bombyx mori does not affect wing developmentThe temporal and spatial expression pattern of Antp gene in the wings of Bombyx mori showed that Antp gene was expressed in both fore and aft wings,and the expression intensity in the wing primordium of the Antp mutant Wes(Antp+/-)was significantly lower than that in the wild-type wing primordium.We intercepted the upstream promoter of Bm Wcp4 gene,which was highly expressed in the pupal anaphase of silkworm wing primordium,and constructed a transgenic vector to drive the high expression of Antp gene in silkworm wing primordium.The vector was injected into fresh silkworm eggs,and the potential positive individuals were screened by red fluorescent tag in G1 generation.The results showed that:1.Reverse PCR showed that the insertion position of Antp overexpression cassette was intergenic region,so the structure of other genes was not destroyed;2.q RT-PCR and Western blot showed that the expression of Antp in wings was significantly up-regulated at the transcription level and protein level,so we considered that Antp overexpression strain in silkworm The construction is successful.Since the wing morphology of Antp transgenic overexpression line was not significantly different from that of wild type by naked eye observation,we expanded the overexpression line and measured the wing area and wing load accurately.The results showed that the wing area and wing load of overexpression line were not significantly different from that of wild type.2.Down regulation of Antp expression in wings leads to abnormal wingBecause the wing development of silkworm was not affected by overexpression of Antp in the wing primordium,we injected dsantp into the wandering wild type silkworm to down regulate the expression of Antp in the wing.The results showed that 48 hours after injection,the expression level of Antp interference individuals was significantly lower than that of the control group.At the same time,we observed the wing primordia of Antp interference individuals and the control group,and found that there was no significant difference in the shape and size of wing primordia,but the internal structure of wing primordia of Antp interference individuals was underdeveloped,which was consistent with the phenotype of Wes mutant(Antp+/-).After moth transformation,the wings of Antp interference individuals were curled and shrunk,and the wing type was significantly smaller.In order to further deter mine the effect of Antp loss on wing development,crispr-cas9 editing method was used to knock out Antp The sg RNA and Cas9 protein transcribed and synthesized in vitro were mixed and injected into fresh silkworm eggs.The abnormal wing phenotype of G0 generation adults was consistent with that of Wes(Antp+/-).The sequence of Antp gene of mutant individuals showed that there were large deletion and a small number of base insertion at the designed target site,which led to frameshift mutation Loss of function.These results indicate that Antp is necessary for wing development of silkworm.Antp,as a member of Hox family,is highly conserved in evolution.In order to elucidate the wide applicability of the Antp gene to the wing development,we have retrieved the full length sequence of the Antp protein from 7 orders and 49 insects from the NCBI database,and analyzed phylogenetic tree and protein two level structure.Among them,Hymenoptera,Lepidoptera,Coleoptera,Hemiptera and Orthoptera have complete wing structure in adult stage,while Collembola and lice do not have wings in the whole life cycle.The results showed that all the insects of the same order clustered in one branch,suggesting that the function of Antp in the same order was conservative.The results of protein secondary structure analysis showed that Antp of all species had homedomain domain domain,and Hox gene,as a transcription factor,regulated the downstream genes based on the homedomain region to bind to DNA,and then controlled the differentiation and specialization of cells at specific locations in embryos or animals.These results suggest that the function of Antp gene in controlling the development of thoracic tissues and organs is conserved in evolution.In order to show that the function of Antp gene is conserved during the development of insect wings,we took Diptera Drosophila and Coleoptera Tribolium castaneum as the research objects,and down regulated the expression of Antp in the wings.The results showed that the wings of Drosophila melanogaster and Tribolium castaneum adults were abnormal after the down-regulation of Antp expression.In conclusion,Antp is indispensable in the development of insect wings.3.Antp affects wing development by regulating 20E synthesisTo elucidate how Antp affects wing development,we investigated the expression of20E pathway genes in prothorax,hemolymph and wing primordia.q RT-PCR results showed that only the last gene shade in the 20E pathway was expressed in the wing primordium,and its expression in the wes(Antp+/-)mutant wing was significantly lower than that in the normal wing.ELISA measured the titers of ecdysone（precursor of 20E）and active 20E in mutant and normal wings of Wes（Antp+/-）.The results showed that there was no significant difference in the titers of ecdysone between the two strains,but the content of 20E in mutant wings of Wes(Antp+/-)was significantly lower than that in normal wings,which suggested that the reason of mutant wings might be due to the lack of 20E content in wings.Furthermore,dual luciferase assay,EMSA and chip PCR confirmed that Antp could directly bind to the upstream promoter region of shade gene to regulate the expression of shade.Based on the above,Antp can control 20E synthesis in insect wings by regulating the 20E synthesis gene shade.4.20E regulates the expression of Antp in wingsSince the expression of Antp in wings is consistent with the periodic change of 20E in insects,we suspect that 20E can regulate the expression of Antp during wing development.The results showed that there were three Ec R/USP binding motifs in Antp promoter.We constructed the Antp promoter into the firefly luciferase reporter vector（p GL3 basic）and co transfected it with the sea kidney luciferase reporter vector into Bm N cells.The cells added with 20E were used as the experimental group and the cells added with DMSO as the control group.After 48 hours,the cells were collected to detect the promoter activity of Antp promoter.The results showed that compared with the control group,the activity of Antp promoter was significantly enhanced after adding20E.In addition,the expression of Antp was significantly up regulated in Bm N cells and individuals after adding 20E.These results suggest that in insect wings,20E binds to Ec R/USP to form a complex,which binds to Antp promoter region and regulates the transcription of Antp.5.Antp affects wing development by regulating the expression of cuticular protein genesIn order to explore whether there are other genes besides shade that contribute to the abnormal phenotype of Wes(Antp+/-)mutant wings,comparative proteomic analysis of wings of the two strains was carried out based on data independent acquisition（DIA）proteomics technology.The results showed that 3993 proteins were identified,including370 differential proteins（p-value<0.01,|log2fc|>0.58）.Compared with normal wings,238 proteins were up-regulated and 132 proteins were down regulated in Wes(Antp+/-)mutant wings.We investigated the expression of three epidermal protein genes（CPH28,CPG24,CPG9）in the wing primordia of Bombyx mori with the same expression pattern as Antp in the mutant wings and normal wings of Wes(Antp+/-).The results showed that the expression of three epidermal protein genes in Wes(Antp+/-)mutant wing was significantly lower than that in normal wing.In Bm N cells,after overexpression of Antp,three epidermal protein genes were significantly up-regulated compared with the control group.Subsequently,we took CPH28 as an example,and proved that Antp protein could directly bind to CPH28 promoter region to regulate its expression through dual luciferase assay and in vitro and in vivo binding assay.After down regulating the expression of CPH28 in pupal stage,the wing type of adult was defective.These results indicate that epidermal proteins are very important in wing development.In conclusion,in this study,we found that knockdown or knockout of Antp in silkworm,Drosophila and Tribolium castaneum would lead to smaller wings and curl of adults.It was confirmed that Antp regulated the content of ecdysone 20E by directly regulating the expression of shade in the wing primordium;meanwhile,Antp further regulated the development of wings by combining with the homeostatic regulatory elements of wing epidermal protein gene.Studies have shown that forewing development is not Hox free,and Antp gene is indispensable for wing development.The comparative analysis of quantitative proteomics shows that epidermal proteins and epidermal lipids are equally important for wing development.We hope that these differential proteins obtained by differential proteomics can provide new targets for the control of lepidopteran pests. |
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