Identification And Function Study Of The Cytokine Receptor BmDOME Gene In The Silkworm,Bombyx Mori

Insects are the earliest winged group in the animal kingdom,and they are also the only winged invertebrate group.As an important organ of insects,wing plays an important role in foraging,mate-finding,migration,expanding distribution,and avoiding enemies.Bombyx mori is an important economic insect and an important model organism of Lepidoptera,the main type of insect pests in agriculture and forestry.Research on wing development can not only enrich the wing development regulation theory of silkworm and other insects,but also provide theoretical support for the biological control of lepidopteran pests.There are many studies on the wing development mechanism of the model organism Drosophila,and it is now clear that EGFR signaling pathway,Notch signaling pathway,JAK/STAT signaling pathway,and morphogen genes such as Hedgehog(Hh),Decapentaplegic(Dpp)and Wingless(Wg)are involved in the development and formation of the wing.The JAK/STAT pathway is an important signal transduction pathway,and it is widely involved in the morphogenesis of Drosophila,the proliferation and differentiation of blood cells,and immunity processes.The JAK/STAT signaling pathway is highly conserved in evolution.Genes such as signal transducers and activator of transcription(STAT)Bm STAT,Janus(JAK)tyrosine kinase Bm HOP,and negative feedback regulator Bm SOCS,which are homologous to Drosophila,are found in silkworms.DOME,as the receptor of the signaling pathway,plays a vital role in the entire cascade kinase reaction in Drosophila,has not been reported in silkworm yet.In this study,cytokine receptor BmDOME was identified in Bombyx mori and its gene structure,temporal and spatial expression characteristics,tissue and subcellular localization were analyzed.Then its biological functions were explored at the cellular level and the individual level.The main findings are as follows:1.Cloning and bioinformatics analysis of BmDOME gene of Bombyx moriThrough amino acid comparison and analysis,we identified a gene in silkworm that has relatively high similarity with Drosophila DOME,and named it BmDOME.We isolated and cloned the ORF sequence of BmDOME c DNA.The sequence length is3765 bp,which encodes 1254 amino acids.Through chromosome location and genome comparison analysis,it was found that the gene is located on chromosome 17 of the silkworm,and it has only one exon and no introns.Conserved domain analysis showed that BmDOME has 2 transmembrane domains,2 cytokine binding domains(CBM)and3 Fn III domains,which are the same with Dm DOME.It is speculated that they may have similar functions.Then we constructed a phylogenetic tree of DOME from silkworm and other species,which showed that BmDOME were grouped with cytokine receptor of Manduca sexta while the relationship with mammals was relatively distant.2.Analysis of the expression characteristics and tissue localization of BmDOMEWe used q RT-PCR and Western Blot to detect the expression of BmDOME on the third day of the fifth instar in the silkworm,and the results showed that BmDOME was expressed in all tissues,with the highest expression in the fat body,and then in the wing disc and gonads.The expression characteristic of BmDOME in the wing was further analyzed by q RT-PCR.The results showed BmDOME was continuously expressed during silkworm wing development and the expression level was rapidly increased during the wandering period.This speculated that the BmDOME may involved in the development of the wing.The immunohistochemistry of fat body and wing disc showed that BmDOME was localized on the cell membrane of fat body cells and the entire wing disc cell membrane.It is consistent with the results of subcellular localization in the silkworm embryonic cell line Bm E.3.BmDOME involved in cell proliferation and apoptosisIn order to study the biological functions of BmDOME,we first carried out experiments at the cellular level.After overexpression of BmDOME in the silkworm embryonic cell,the expression level of JAK/STAT signaling pathway genes were detected.The results showed that the related genes of this pathway were all up-regulated.Using the Ed U staining kit to detect cell proliferation,it was found that the proliferation of the overexpressed cells was more vigorous than that of the control group.At the same time,we detected an increase in the expression of cycling genes.Then we studied the effect of overexpression of BmDOME on cell apoptosis.Using Hoechst staining to detect cell apoptosis,the results showed that the proportion of apoptotic cells overexpressing BmDOME was significantly lower than that of the control group.At the same time,the results of q RT-PCR showed that apoptosis related genes Caspase1,Caspase3,Caspase8 were all down-regulated,indicating that overexpression of BmDOME can inhibit cell apoptosis.We further verified its function by RNAi interference BmDOME.After the cellular level interfered with BmDOME,the RNA expression and protein expression of BmDOME decreased significantly,and the expression of JAK/STAT signaling pathway related genes were all down-regulated.After staining and observing the cells with the Ed U staining kit,it was found that compared with the control group,the number of fluorescent cells that interfere with BmDOME decreased significantly,cell proliferation was inhibited,and cell cycle-related genes were also down-regulated.Using Hoechst staining to detectcell apoptosis,the results showed that the proportion of apoptotic cells that interfered with BmDOME was significantly higher than that of the control group,and the apoptosis was significantly increased.The expression of apoptosis-related genes detected by q RT-PCR was up-regulated.4.Construction and phenotype analysis of transgenic silkworm lines knockout BmDOMEIn order to further study the function of BmDOME,we used the CRISPR/Cas9 system to knock out the BmDOME gene of the silkworm.First,the BmDOME transgenic knockout g RNA vector was constructed,and then microinjected into embryonic eggs.Fluorescence screening was performed in G1 generation individuals to obtain BmDOME transgenic g RNA-positive individuals,which were then crossed with a silkworm strain systemically overexpressing Cas9 protein.Fluorescence screening of hybrid offspring was used to obtain g RNA and Cas9 double-positive silkworms.Through genome PCR,we found that the BmDOME gene in the genome of double-positive individuals has different fragments missing compared with the wild type.Further Western Blot detection showed that compared with the wild type,the expression of double-positive silkworm BmDOME protein was significantly downregulated.The results showed that we successfully obtained the BmDOME knockout transgenic silkworm,we named it KO-BmDOME.Observing the transgenic knockout silkworms,we found that the wings of the pupa and moth stage have varying degrees of loss and shrinkage in transgenic silkworm.The pupa wings were vesicle-shaped and smaller compared with the wild-type silkworm.Some of them cannot extend their wing from the puparium after eclosion.At the same time,we found that the gonads of the knockout individuals were abnormally developed so they could not mate normally.Then we calculated the economic traits of the transgenic knockout silkworm,and the results showed that compared with the wild type,the cocoon layer of the male and female individuals did not change,while the pupal weight decreased.In order to explore the mechanism of BmDOME transgenic knockout of silkworm wing developmental abnormalities,we used q RT-PCR to detect the expression of cyclin genes and apoptosis-related genes in KO-BmDOME and wild-type silkworm wing disc.The results showed that the cyclin genes such as Cyclin A,Cyclin A,Cyclin B,Cyclin D,etc.are all down-regulated,while the apoptotic genes Caspase1,Caspase3 and Caspase8 are up-regulated.Based on the above results,we speculate that BmDOME can regulate cell proliferation and apoptosis by affecting the JAK/STAT signaling pathway,thereby affecting the development of wing disc.In summary,we have identified the receptor BmDOME of the silkworm JAK/STAT signaling pathway,and analyzed its gene sequence,temporal and spatial expression characteristics,and subcellular localization.It is verified at the cell level and individual level that BmDOME can regulate cell proliferation and apoptosis by regulating the activity of JAK/STAT signaling pathway,and further affect the development of silkworm wings.This study primarily elucidates the biological function of BmDOME gene in silkworm,which not only provides a new idea for the regulation of wing development,but also provides a reference for other Lidopteran insect function research of DOME,further verifying that the JAK/STAT signaling pathway is conserved in the regulation of wing development in silkworm.It is possible to control the number of pests,reduce the use of pesticides and protect the ecological environment by knocking out the DOME gene of the lepidopteran pest in subsequent studies.