Positional Cloning Of Red Egg Lethal Mutation (re~l) And Functional Study Of Semaphorin And Plexin Genes In Silkworm

The rapid and complex developmental process of organisms is a streamlined mapping of the long evolutionary history of a species,which is precisely controlled by a regulatory network involving numerous genes.Some of these genes are essential for biological development,and their functional defects are lethal.With the emergence of genetic control technologies for pest populations,the mining of genes essential for insect development has become a very important task for pest control research.After a long period of adaptive evolution,organisms with defects in genes that are essential for development are often eliminated under natural selection.Therefore,for a long time,it was very difficult to discover genes that were essential for development.As a model organism for Lepidoptera research,silkworm has a deep genetic research foundation and abundant genetic mutation resource,which contains a large number of growth and developmental defects and lethality-related mutants,which provides valuable resources to explore the biological process of insect growth and development,and provides an opportunity to discover molecular targets for pest control.In this study,the molecular basis of the lethal phenotype of re~lwas elucidated through classical genetic localization,gene expression pattern analysis,sequence investigation,and knockdown and knockout of candidate genes.We found that the Bm Sema1a gene,a member of the Semaphorin family,was essential for the development of the silkworm embryos.To further explore the biological functions of the Semaphorin family in insects and to evaluate whether its family members can be used as universal molecular targets for pest control,we performed genome-wide identification and phylogenetic analysis of the Semaphorin gene family and its receptor gene,Plexin,in several insects.And the functions of several members of the Semaphorin and Plexin families were also investigated in silkworm.The main results of this study are as follows:Molecular basis analysis of the red egg lethal mutant(re~l)First,the phenotypic characteristics of re~l were determined based on anatomical and microscopic observations,i.e.,its eggs were dark red and the embryos died at about day six of development.Genetic hybridization experiments identified re~l as an allelic mutation to the published red egg(re)locus.Subsequently,gene sequencing,quantitative fluorescence PCR,and bioinformatic analysis demonstrated that the red egg phenotype of both re~l and re mutants was caused by a functional defect in Bm-re belonging to the major facilitator superfamily(MFS).To locate candidate genes for the re~l lethal phenotype(lethal,le),the genetic distance between le and re was determined using classical genetic hybridization experiments.By phenotypic observation and statistics of a large population revealed that the exchange value between le and re was2‰.That is the genetic distance between le and re was 0.2 c M.Based on these data,the expression patterns of genes within 300 kb upstream and downstream of Bm-re were investigated to screen the functional genes of le.It was found that the expression level of Bm Sema1a was substantially downregulated in the re~lmutant compared to the controls during embryonic development.Further sequence investigation of Bm Sema1a revealed a large amount of sequence variation in its genomic region in re~l mutants.Finally,functional studies of Bm Sema1a using gene interference and genome editing techniques showed that functional defects in Bm Sema1a lead to abnormal embryonic development and death.Results of q RT-PCR and in situ hybridization experiments further showed that Bm Sema1a may be involved in embryonic neural development and epidermal construction,which may be an important reason why Bm Sema1a defects lead to embryonic lethality in the silkworm.Genome-wide identification and evolutionary analysis of the Semaphorin and Plexin families in a variety of insectsSemaphorin genes encode a class of membrane or secretory proteins,and Plexin members often act as receptors for Semaphorin proteins,which are involved in a wide range of biological processes such as neurodevelopment and immune responses,all of which contain a Sema structural domain at their N termini.We found that Bm Sema1a in the silkworm Bombyx mori is critical for embryonic development,and to further investigate the distribution and conservation of the Semaphorin gene family and its receptors in multiple species.Sema domain-containing proteins were identified in a total of 34 insect species belonging to Lepidoptera,Hemiptera,Hymenoptera,Hymenoptera,Diptera,and Homoptera.A total of 148 Semaphorin protein sequences and 72 Plexin protein sequences were identified in these 34 species.Phylogenetic analysis of these genes using MEGA7.0 software showed that Semaphorin in insects clearly clustered into Sema1,Sema2 and Sema5.Plexin genes clearly clustered into Plexin A and Plexin B subclasses.Five Semaphorin genes(Bm Sema1a,Bm Sema1b,Bm Sema2a,Bm Sema2b and Bm Sema5B)and three Plexin genes(Plexin A,Plexin A3and Plexin B)are present in the silkworm.The Semaphorin and Plexin genes are highly conserved among 34 investigated insects,and the systematic elucidation of their functions in the silkworm will be an important reference for the functional analysis of Semaphorin and Plexin genes in other insects.Spatiotemporal expression patterns of Semaphorin and Plexin genes in silkwormExpression patterns of genes are important indicator of their function.To infer the functions of Semaphorin and Plexin in silkworm.The embryos developed at the egg stage of silkworm,the whole silkworm from the 4th instar to the emergence of silkworm moth,and the epidermis,wings and ovaries from the 5th instar to the emergence of silkworm moths were used as experimental materials to study the the tissue and period expression patterns of various genes by q RT-PCR.The results showed that they all had high expression levels in embryonic stage.In addition,it was found that Bm Sema1a and Bmplexin A genes had high expression levels in multiple tissues and stages of larvae and pupae,and their temporal expression patterns showed significant correlations during the stage of wing development.Functional analysis of semaphorin and Plexin family members in silkwormTo investigate the functions of other members of the Semaphorin family and Plexin in embryonic development,three knockout pure lines of Bm Sema2a,Bm Sema5B,and Bm Plexin B were constructed using CRISPR/Cas9 gene editing technology.The Bm Sema2a knockout heterozygotes were found to develop normally and the homozygote embryos were lethal,while the Bm Sema5B knockout heterozygotes and homozygotes both developed normally,and no abnormal phenotypes were observed;the Bm Plexin B knockout heterozygotes developed normally and the homozygotes embryos were lethal.The phenotypes of the Bm Sema2a knockout and the Bm Plexin B knockout were similar.That is,their embryos were lethal when they turn black,and the defect in embryo’s ability to creep did not allow it to bite through the eggshell and crawl out.Bm Sema1a is essential for silkworm embryonic development,and its expression level was high in both larval and pupal stages,so does it also play an important role in post-embryonic development?To investigate the function of Bm Sema1a in post-embryonic development of the silkworm,a heat-stimulation induced transgenic interference line of Bm Sema1a gene was constructed.Then,this transgenic line was treatmented with heat stress and it was found that knockdown of Bm Sema1a resulted in difficulty in silkworm larvae molting,failure of moth wings to extend properly,and difficulty in thoracic foot crawling.So,does Bm Sema1a regulate silkworm development through its receptor Bm Plexin A?We further constructed Bm Plexin A knockout lines based on the CRISPR/Cas9 system and found that both chimeric embryos and larvae developed normally,the moths had difficulty in thoracic foot crawling and thus could not mate properly or even break out of the cocoon,the wing extension area was significantly smaller,and the ovarian tube was shortened.In which the abnormal phenotypes of wings and thoracic foot were similar to the Bm Sema1a disrupted strain.These results suggested that Semaphorin and Plexin may be jointly involved in the embryonic and post-embryonic development of the silkworm.Transcriptome analysis of wing in pupal stage of Bm Plexin A transgenic knockout strainPhenotypic observations and statistics of the Bm Plexin A transgenic knockout strains showed that the phenotyp of wrinkled and small wings due to their functional defects was fully dominant.To investigate the signaling pathways involved in the involvement of Bm Plexin A in wing development,we performed a comparative transcriptome sequencing analysis of the wings of the Bm Plexin A transgenic knockout strain(experimental group)and the normal Dazao strain(control group)on day 5 of the pupal stage.Genes with significant expression differences between the experimental and control groups were functionally annotated and biologically enriched for signaling pathways.The differentially expressed genes between them were found to be mainly enriched in signaling pathways such as AMPK signaling,calcium signaling,and immune response,which are involved in neural development,ion pathways,and muscle structure formation.This provides a new perspective to further elucidate the molecular mechanism of Bm Plexin A involved in wing development.In summary,this study systematically investigated the functions of Semaphorin genes and their receptor,Plexin genes,in the domestic silkworm,and found that Semaphorin and Plexin play important roles in the embryonic and post-embryonic development of the domestic silkworm,and that Semaphorin and Plexin are highly conserved evolutionarily and may serve as important molecular pest control targets.