| The silkworm(Bombyx mori)belongs to the family Lepidoptera,and is an important economic insect.The silk industry with the silkworm as the main body has always been dominant traditional industry of our country,and the sericulture industry has long held an important position in rural economy of our country.In addition,silkworm is the excellent research model of lepidopteran insects with the characteristics of short development cycle,moderate size,easy feeding and strong reproductive ability.It has a long research history,has accumulated a lot of research experience,and has retained precious mutants and breeding lines.Metamorphosis is an extremely complex developmental process involving a multi-level regulatory network involving Juvenile Hormone(JH)and Ecdysone(Ecd),and a large number of genes play a role in this process.Among them,the research on the regulatory network dominated by 20 E,adults of ecdysone,is relatively in-depth.Classical studies reveal that after 20 E enters the cell,it first binds to its nuclear receptor Ec R,and then binds to the invertebrate receptor USP to form a heterodimeric transcription complex,which is complexed by Ec R-USP.The primary response genes such as E93,E75 B,Br-C,etc.,are activated downstream,and the primary response genes further activate the secondary response genes and various effector genes,thereby promoting the process of insect metamorphosis.In recent years,some scholars have found that 20 E also has membrane receptors and membrane signaling pathways,and discovered transmembrane transport proteins and potential membrane receptor proteins,but the specific mechanism remains to be further studied.These new findings suggest that the 20 E signaling pathway may be more complex than current theories believe,and new technologies are urgently needed to more comprehensively reveal the genes involved in the 20 E signaling pathway and its detailed mechanism of action.Traditional 20 E research mainly starts from the mutant phenotype and single gene,and gradually analyzes the signaling pathway through interaction or functional association,which is not only time-consuming and labor-intensive,but also difficult to fully reveal the entire signaling pathway.With the advancement of biological multi-omics technologies such as genome,transcriptome,proteome,and metabolome,more and more studies are exploring from the genome level to comprehensively understand metamorphosis.In mammalian cells and a few classical model organisms,the genome-wide editing screen formed by the combination of CRISPR gene editing technology and high-throughput omics has become the trend of functional genome research on present.Genome-wide editing library screening technology can break through the limitations of traditional positional cloning technology and reference near-source species research methods,does not rely on existing gene function information and refined experimental design,and can conduct gene function research on the whole genome.On the basis of long-term research on insect genome editing,our laboratory has established a genome-wide editing library based on BmE cells of silkworm,and proved the library through various screenings such as essential gene identification,BmNPV resistance,and environmental pollutant tolerance.Efficiency in large-scale functional genomic have been studied.Therefore,in this paper,aiming at the problem that the silkworm 20 E signaling pathway has not been fully resolved,on the basis of systematically conducting genome editing research and establishing a genome-wide CRISPR library screening platform,this paper proposes to screen the related genes of the silkworm 20 E pathway through the genome-wide CRISPR library.We comprehensively used flow cytometry,CRISPR genome editing cell library screening,mutant identification and other technologies,combined with genetics,molecular biology,bioinformatics and other related experimental methods to analyze the role of key genes and their mechanisms in the action of silkworm 20 E pathway.The main results achieved are as follows:1.Explore the conditions for 20 E genome-wide screeningIn order to conduct effective genome-wide CRISPR library screening,it is necessary to explore the physiological effects of 20 E addition on silkworm BmE cells,and determine the corresponding screening conditions.According to existing literature reports,it is generally believed that the promotion of apoptosis is the main way for 20 E to play its role.In this regard,we verified the apoptosis induced by 20 E at the level of the silkworm embryonic cell line(Bombyx mori Embryonic,BmE).BmE cells were treated by adding different concentrations of 20 E,stained with propidium iodide(PI),and then detected cell apoptosis by flow cytometry.The results showed that in some treatment groups,with the increase of 20 E concentration and the prolongation of treatment time,the apoptosis of cells showed a gradually increasing trend,but the overall law was not obvious,and there were some nodes with large errors.It shows that20 E treatment can indeed induce apoptosis of BmE cells,but the apoptosis signal is not particularly strong,so it is not suitable to develop a screening strategy by detecting apoptosis.Since apoptosis cannot be directly used to reflect the state changes of BmE cells after 20 E treatment,we used MTS assay to measure cell viability to reflect the activity changes of BmE cells after 20 E treatment.BmE cells were treated with different concentrations of 20 E,cells were collected at one week,and cell viability was determined by MTS assay.The experimental results showed that when the concentration was 80 μM,the cell viability of the 20 E treatment group and the DMSO control group was significantly different.After increasing the concentration,DMSO also had cytotoxic effect,so we used 80 μM as the screening concentration of the cell bank.2.Library screening of 20 E pathway related genesIn the early stage,our laboratory constructed a CRISPR/Cas9-based genome-wide editing cell library in Bombyx mori,and successfully completed the screening of genes related to the response to(non)biotic stress factors in Bombyx mori,proving the feasibility of CRISPR library screening in Bombyx mori.So,we carried out the screening work based on this cell library.The BmE cell genome-wide editing library was treated with 20 E at a concentration of 80 μM,and the cells were collected when the cell confluency of the treated group reached 5%-10%.The sgRNA was amplified by PCR,and the product was subjected to high-throughput sequencing.After data filtering,splicing,screening results were obtained after alignment: we identified a total of 887 significantly enriched genes and876 significantly depleted genes.Among them is the known key gene E93 downstream of the 20 E pathway,which ranks third in our screening system,which proves the reliability of the screening results to a certain extent.However,the ecdysone transmembrane transporter-encoding gene ECI identified in Drosophila,BGIBMGA002723,the gene with the highest homology in Bombyx mori was not significantly enriched,suggesting that the transmembrane transport mechanism of 20 E is between Bombyx mori and Drosophila has a difference.There are also some genes that have been reported to be related to 20 E,but are ranked low or even not significantly enriched in the screening,such as BR-C,Ftz-f1,etc.We speculate that it may be due to the differences in their expression at the individual and cellular levels and some the efficiency of some gRNAs is too low.Our combined analysis of the library screening results and the transcriptome data of 20E-treated BmE cells found that significantly enriched and up-regulated genes accounted for the largest proportion,indicating that most of the genes involved in the20 E pathway were up-regulated by its induction.Through q RT-PCR verification of some of the top-ranked genes,it was further proved that they are indeed related to the20 E signaling pathway.Later,we performed GO classification on the screening results,and divided them into three categories according to biological processes,cellular components,and molecular functions.The two highest proportions are “binding” and“catalysis” in molecular functions.We speculate that 20 E mainly plays a regulatory role by affecting the functions of some specific proteins in Bombyx mori.We further classified the functions of genes according to their enrichment and depletion,and found that among the annotated genes,enzymes accounted for the highest proportion,which further indicated that 20 E affects the functions of some key enzymes in silkworm.In the results of subcellular localization,both enriched and depleted genes were mainly expressed in the cytoplasm and nucleus,and less in the cell membrane.This indicated that the signal transduction of Bombyx mori 20 E was mainly completed in the cytoplasm and nucleus,and there were not many genes involved in the transmembrane transport of Bombyx mori 20 E.In the cytoplasm,the distribution of enriched genes predominates,while in the nucleus it is dominated by depleted genes.This indicates that the activation process of the silkworm 20 E pathway is mainly completed in the cytoplasm,while the inhibition process of the pathway mainly occurs in the nucleus.Through the KEGG pathway enrichment analysis of the screening results,we found significantly enriched pathways: inositol phosphorylation metabolism,Toll and Imd,Gly,Ser,Thr metabolism and Hippo signaling pathways and significantly depleted pathways mTOR,Cys and Met Metabolism,autophagy and Toll and Imd signaling pathways.Through the analysis of the results,we identified the genes and pathways related to silkworm 20 E signal transduction,which can be used for subsequent verification work.3.Validation of core candidate genesThe innate immune signaling pathways of silkworm,Toll and Imd,ranked high in both enriched and depleted genes,indicating that 20 E may affect the innate immunity of silkworm.The depleting genes are mainly distributed in the Toll pathway,while the enriched genes are distributed in the Imd pathway.We speculate that the Imd pathway may be involved in the signal transduction of silkworm 20 E,and the Toll pathway has an inhibitory effect on it.Inositol phosphorylation metabolism ranked first in the enriched pathway,which included the enriched gene norp A for top23,which was annotated as the β4 subunit of phosphatase C.Knockout of norp A increases the tolerance of BmE cells to 20 E,indicating that norp A is indeed involved in the process of silkworm 20 E signal transduction.We detected the key molecule PI3 K in the phosphorylation pathway by Western blotting,and the results showed that the phosphorylation degree of PI3 K increased with the prolongation of 20 E treatment time,indicating that 20 E could induce the activation of phosphorylation signaling pathway.mTOR and autophagy are the first and third ranked pathways in the depletion pathway,respectively.The mTOR signaling pathway plays an important role in maintaining the balance of nutrient metabolism,and its consumption indicates that 20 E may interfere with the balance of nutrition and energy metabolism in silkworm cells.After treatment of cells with rapamycin,an inhibitor of the mTOR pathway,BmE cells were more sensitive to 20 E,further indicating that cells need a balanced nutrient metabolism to resist the effects of 20 E.Autophagy is a regulatory mechanism by which cells maintain survival by degrading their own non-essential cellular components when their own nutrients are deficient.Knockout of the autophagy pathway gene GH19770 will accelerate the process of 20 E killing cells,indicating that under the screening pressure of 20 E treatment,if the autophagy pathway of cells is damaged,it will be difficult to maintain survival,resulting in apoptosis.Overall,we screened related genes in the 20 E pathway using the Bombyx mori genome-edited cell bank,including 887 enriched genes and 876 depleted genes.The results of taxonomic annotation indicated that 20 E could affect the functions of some key proteins in Bombyx mori to play a regulatory role,and these processes were mainly completed in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20 E can activate the phosphorylation signaling pathway of silkworm cells,and may affect their innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.Cells became more tolerant to 20 E after knocking out the relevant gene.This paper starts with the unknown part of the silkworm 20 E signaling pathway,takes the silkworm as a model,and uses cutting-edge technologies such as CRISPR/Cas9 genome editing and library screening to reveal the key genes involved in the 20 E signaling pathway and their molecular mechanisms,which may be used for insects.The research on developmental metamorphosis provides important new insights and theoretical basis for the artificial regulation of insect populations,which has important theoretical and practical application value. |
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