| Insects are the most abundant and diverse animals in nature.Wings are one of the most important characteristics of insects,which play an important role in avoiding natural enemies,communicating with each other,long-distance migration,foraging and mating.Bombyx mori is one of the best model organisms for insect wing development based on its complete genome information,clear genetic background and mature genetic manipulation platform.In Bombyx mori,studies on wing not only reveal the regulation mechanism of insect wing development,but also provide a theoretical basis for biological control of lepidoptera pests in agriculture and forestry.The key signaling pathways that regulate insect wing development are Wnt/Wingless(Wg),Hippo,Hedgehog(Hh)and Decapentaplegic(Dpp),which act as morphogenetic proteins to form concentration gradients that induce signal transduction and activate gene expression in a concentrationdependent manner,thereby regulating wing development.Heparan sulphate proteoglycans(HSPGs)have been shown to play an important role in cell signaling and are conserved on the surface of various cell membranes.Heparan sulphate(HS)acts as a co-receptor to mediate the transmission of various signaling factors involved in tissue and organ development and normal physiological function,regulating metabolism,transport and information transfer in almost all organ systems.EXT1,EXT2 and EXTL3 of the human EXT(Exostosin)gene family encode glycosyltransferases involved in the synthesis of HS.The deletion of their homologs in Drosophila,tout velu(ttv),sister of tout velu(sotv)and brother of tout velu(botv),was found to cause wing defects in Drosophila.All lead to wing defects in Drosophila,but the detailed regulatory mechanisms are not yet clear.To further investigate the role of the EXT family in wing development,we investigated the EXT genes in the lepidopteran model insect,the silkworm.Firstly,we cloned the EXT2 homologous gene Sotv(BmSotv)of silkworm and analyzed its bioinformatics and expression characteristics.The BmSotv knockout mutants with obvious wing defect were obtained by CRISPR/Cas9 technique.The key period of BmSotv in silkworm wing development was determined.Finally,by comparing the proteome differences between BmSotv knockout mutants and wild type,and by cell related experiments,we preliminarily analyzed the regulatory mechanism of wing development.The main results obtained from this study are as follows:1.Cloning,identification and expression characterization of BmSotvBmSotv is located on chromosome 23 of the silkworm(Bombyx mori)and has the silk DB3.0 database number BMSK0013095.5945 bp of the gene contains four exons and three introns.Analysis of the predicted protein sequence revealed that BmSotv contains three structural domains,including a transmembrane domain,an Exostosin domain specific to the EXT protein family and a glycosyltransferase G64 domain,the latter two conserved domains being required for the synthesis of heparan sulfate.Homologous sequence comparison showed that BmSotv has 45% sequence similarity to EXT2 and Sotv.Evolutionary analysis showed that BmSotv clusters with other lepidopterans,suggesting that this gene and its homologues in lepidopterans may have evolved from a common ancestor and are highly conserved.The above results suggest that the conserved sequence of BmSotv may be involved in the regulation of wing development and is an important candidate gene for studying the regulation of insect wing development.To explore the function of BmSotv in the silkworm,we examined the expression of BmSotv in the midgut,wing primordia,epidermis,malpighian tubule,tracheal plexus,ovary,fat-body,head and silk gland of the 5L3 D larvae by q PCR,and the results showed that BmSotv was widely expressed in all tissues of the silkworm,with the highest expression in the midgut.To further explore the function of BmSotv in the development of silkworm wings,we analyzed the expression characteristics of the wing primordium of the 5th instar larvae.The results showed that the expression of BmSotv was stable and low in the 5th instar larvae stage,and began to increase in the migration stage.Subcellular localization of BmSotv showed that it was mainly located in cytoplasm and cell membrane.2.BmSotv is involved in the development of the wing primordium in the silkworm.Using CRISPR/Cas9 technology,we designed three sg RNAs on the first exon of BmSotv and constructed their sg RNA knockout vectors;after obtaining sg RNA-positive strains by embryo microinjection and fluorescence screening,they were crossed with Cas9 strains and F1 generation positive individuals were obtained by fluorescence screening;the knockout efficiency of F1 generation positive individuals was tested.The results showed that there were small fragment base deletions and single base insertions in the genomic knockout sites of the sg RNA-3 strain,with an overall editing efficiency of 85%.70% of the base deletions resulted in code-shifting mutations and premature termination of protein translation;another 15% of the mutations resulted in a reduction of amino acids,resulting in reduced expression of BmSotv.Compared to the wild type,the BmSotv-KO strain showed the following phenotypes:(1)malformed wing development during the pupal and moth stages,showing a distinctly wrinkled and smaller wing surface;(2)varying degrees of defective antennae and appendages,with shortened and upturned upright antennae during the pupal stage and a large number of feathery receptors on the antennae observed to be shed after mothification;malformed appendages,with severe cases of moths having difficulty achieving activities such as crawling;(3)At the moth stage,it was found that only 10.47% of the pupae could break the cocoon and moth,while the remaining 84.36% could not break the cocoon and moth.After cutting open the cocoon of the latter,it was found that 71.98% of them had difficulties in molting the upper part of the pupae and could only shed the pupae’s skin in the abdominal region,while the other 12.38% could not moth at all;(4)the moths could not mate properly and could not produce offspring.To further investigate the critical period when BmSotv plays a role in silkworm wing development,we observed the wing primordia of wild type and mutant silkworm from the 5th instar of larvae to the pupal stage of nesting,respectively.The results showed that the wing primordia of BmSotv mutants were only slightly smaller than those of the wild type at the 5th instar larvae;however,after the wandering stage,abnormal wing disc development was clearly observed in the knockout individuals,with disordered wing vein development and a much smaller wing disc shape than that of the wild type.Subsequently,RT-PCR was performed to detect known genes associated with wing development in the wing primordia of BmSotv mutants and wild type at the wandering stage,and the results showed that Wg,Hh,Dpp and Wnt were significantly down-regulated.The expression levels of BmSotv and Pangolin protein,a key protein in the Wnt signalling pathway,were significantly down-regulated using Western blot.The above results suggest that BmSotv is mainly involved in the development of wings during the wandering of the silkworm,and knockdown of this gene resulted in defective wing development.3.Investigation of the molecular mechanism of BmSotv affecting wing development in Bombyx mori.To investigate the molecular mechanisms by which BmSotv affects the development of the domestic silkworm wing,we performed proteomic sequencing of the wing primordia of the BmSotv mutants and the wild type wandering stage.Differential analysis showed that 30 proteins were significantly differentially expressed out of 4113 proteins shared between the knockout and control groups;32 proteins were significantly differentially expressed out of the proteins specific to each.The quantitative results were consistent with the proteomic sequencing results,indicating that the proteomic sequencing results were reliable.GO functional analysis of the above 62 significantly differential proteins showed that the differential proteins mainly functioned at the cell membrane and were mainly involved in metabolic,catalytic,and binding functions;KEGG enrichment analysis revealed that the differential proteins were significantly enriched in Glycosaminoglycan biosynthesis-keratan sulfate(KS),lysosomal and Hippo signaling pathways.We used RTPCR experiments to detect the genes BMSK0010499 and BMSK0005666 corresponding to significantly differentially expressed proteins in the KS pathway and lysosomal.The quantitative results showed that both were significantly up-regulated in expression,consistent with the histological sequencing results,indicating that the glycosaminoglycan pathway was significantly altered by knocking out BmSotv in the silkworm.suggesting that BmSotv may be involved in the synthesis of HS as a glycosyltransferase like to sotv/EXT2.Meanwhile,we examined the Hippo signaling pathway-related genes BMSK0006876(Dachsous,Ds),Hippo,Mats and Yki,of which Ds,Hippo and Mats all showed significant up-regulated expression in the mutant,while the negative regulatory Yki was significantly down-regulated,indicating that knockdown of BmSotv significantly inhibited the Hippo signaling pathway in the progenitors of the silkworm wandering wingsIn addition,we constructed BmSotv knockout cell lines at the cellular level,and genomic assays,immunofluorescence,and western blot assays all showed that BmSotv was successfully knocked out in the mutant cell lines,and that HS expression was significantly reduced.We then examined the expression levels of Wnt and Pangolin,key proteins in the Wnt signaling pathway,at the protein level and the results of grey-scale analysis showed that their expression levels were significantly down-regulated,which is consistent with the results obtained at the individual level,thus suggesting that BmSotv is also involved in the development of the silkworm wing primordium through the Wnt signaling pathway.In conclusion,BmSotv mainly functions in the wing disc of the silkworm during the wandering,and its involvement in the synthesis of HS has a similar function to that of the membrane receptor Ds in the Hippo signaling pathway,mediating the transduction of the Hippo signaling pathway and thus regulating the development of the silkworm wing.The combined effect of the deletion also leads to abnormal development of the wing primordia,resulting in a crumpled wing phenotype. |
