The Effect Of Simulated Space Environment On The Development Of Silkworm Embryo And Its Mechanism

The space environment including microgravity,high radiation,vibration,and noise,poses a serious threat to the health of astronauts.Especially the influence of space environment on embryonic development is currently poorly understood.In the space environment,silkworm embryos are affected by multiple extreme living environments such as radiation and microgravity,and more and more attention has been paid to the biological effect research mechanism.Bombyx mori embryos have many advantages such as small size,high reproduction rate,clear genetic background,active life activities during the embryonic period,and frequent occurrence of cellular events.They are ideal experimental materials for studying the influence of space environment on embryo development.In 2016,researcher Huang Yongping undertook the carrying experiment of silkworm embryos.China’s SJ-10 recoverable satellite carried silkworm embryos and monitored the embryo’s development status in the space environment in real time.It was observed that the development time of the larvae returning from space to the ground was shortened.The differentially expressed genes and signal pathways in the silkworm were screened out.Ground simulation has the advantages of low cost,low space occupancy rate,easy operation,more experimental opportunities,and easy to study the impact of a single space environment on the body.In this study,the ⁶⁰Go-γray simulated space radiation environment and the three-dimensional gyrator simulated space weightlessness environment were used to treat the silkworm embryos respectively,to study the effect of the simulated space environment on the embryonic development of the silkworm and its mechanism,This study provides a theoretical basis and experimental basis for the influence of the space environment on the embryonic development.The main content and work results of this paper are as follows:（1）Research on the effect of simulated space radiation on the development of silkworm embryos.⁶⁰Go-γrays were used to irradiate the silkworm embryos after 16 hours of egg laying with 7 dose gradients（0Gy,10Gy,20Gy,30Gy,40Gy,50Gy,100Gy）,and the silkworm eggs were cultivated until the ant silkworm hatched.The results showed that with the increase of ⁶⁰Go-γray radiation dose,the hatching rate of silkworm embryos gradually decreased and the embryo hatching time became longer.The silkworm larvae grew normally under low-dose radiation.The high-dose radiation of 40Gy affects the growth and development of the silkworm larvae,which shows that some individuals grow slowly and become smaller in size.The high-dose radiation of 50Gy seriously affected the growth and development of the silkworm larvae,which showed that the individuals developed unevenly,the epidermal markings disappeared,the irregular protrusions of the epidermis were formed,and the body segments were twisted.The silkworms embryos developed on the fifth day and ant silkworms of the control group and radiation groups were subjected to transcriptome sequencing.Sequencing results showed that the number of genes with significant differences in embryonic stage was greater than ant silkworm stage,indicating that embryonic stage gene expression was more sensitive to radiation.It was verified by q-RT-PCR that the expression of homer gene during embryonic stage of silkworm increased significantly with the increase of radiation dose,and the function of this gene in Drosophila is to regulate sleep and locomotor ability.It is speculated that the delayed development of silkworm eggs caused by radiation may be related to the homer gene.In addition,radiation can also cause multiple epidermal growth factor-like domian protein 8 and juvenile hormone binding protein gene expression changes.（2）Research on the effect of simulated spatial weightlessness on the development of silkworm embryos.The purpose of this study was to investigate the effects of simulated microgravity with a random position machine（RPM）on the silkworm embryo.Simulated weightlessness shortened the development time of silkworm eggs,but there was no difference in growth and development of larvae between the simulated weightlessness treatment group and the control group.Transcriptome sequencing was performed on the silkworm eggs and ant silkworm of the control group and the simulated weightlessness treatment group.Sequencing results show that with the extension of the weightlessness treatment time,the more the number of genes that are significantly different between the simulated weightlessness treatment group and the control group,the more pathways changed.Differential gene enrichment pathways mainly focus on leucine and isoleucine degradation,fructose and mannose metabolism,fatty acid degradation,focal adhesion signaling pathway,sphingolipid signaling pathway,tight junction and other pathways.It was further screened by q-RT-PCR that aminopeptidase N（Apn1）,collagen and calcium-binding EGF domain and other genes expression changed significantly.（3）The function study of silkworm embryo genes sensitive to simulated space environment.1)The functional study of Hox genes Bmbp and Bmscr and their cofactors sensitive to simulated weightlessness.The expression of Hox genes Bmpb and Bmscr was significantly reduced in embryos treated with simulated weightlessness.It is speculated that Bmpb and Bmscr genes are sensitive to weightlessness.Therefore,a detailed functional study of Bmpb,Bmscr genes and their related cofactors Bmexd and Bmhth has been carried out.During embryonic morphogenesis,CRISPR/Cas9-mediated mutagenesis of Bmpb induced transformation of spigot into thoracic leg and Bmscr mutation transformed spigot into maxilla.Mutagenesis of Hox-related cofactor extradenticle（Bmexd）and its nuclear localization factor homothorax（Bmhth）also induced distortion of embryonic spigot.Quantitative real-time PCR analysis further revealed that the transcriptional interaction among these genes.This research data provides a new theoretical basis for the Hox gene to regulate insect embryo development,and further speculates that the weightless environment affects the embryo development by affecting the expression of the important gene of embryo morphogenesis-Hox gene.In Drosophila,Teashirt（Tsh）protein is a cofactor of Hox and regulates the development of body segments.Tsh and tiptop（tio）homologous gene pairs exist in Drosophila,while only one homologous gene tsh/tio exists in silkworm.In the current study,we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B.mori.The result shows that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage.RNA-seq analysis and subsequent q-RT-PCR analysis reveal that depletion of tsh/tio significantly elevates expression of kynurenine 3-monooxygenase gene,as well as melanin synthase-related genes during the larval stage.Furthermore,ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality.These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage pattering,as well as the novel function of regulating larval pigmentation in B.mori,providing novel insights into how tsh/tio regulates insect growth and development.2)Study on the function of wnt6 gene sensitive to simulated radiation.With the increase of radiation dose,the expression of wnt6 gene in silkworm embryos gradually increased.This study shows that the ectopic expression of wnt6 gene in the epidermis can induce epidermal pigmentation and bulge formation.This phenotype is similar to that of silkworm larvae caused by ⁶⁰Go-γrays.It is speculated that wnt6 gene is a simulated radiation sensitive gene.The epidermis-specific promoter was used as a regulatory element to ectopically express the wnt6 gene in the silkworm epidermis.Twin-spot markings appeared on the epidermis of mutant larvae.Observation of the ultrastructure of twin-spot markings by electron microscopy revealed that the transgenic silkworm had bulges formed on the epidermis.The expression of genes related to pigment synthesis in mutant silkworms was detected by q-RT-PCR,and the expression of these genes was intermittently up-regulated with the molting of larvae.The up-regulation of wnt6 gene expression in the epidermis does not significantly affect the expression of other wnt genes in the wnt gene cluster.RNA-seq sequencing analysis found that compared with the WT silkworm epidermis,the expression of pigment synthesis genes,hormone regulatory genes,and tumor formation-related genes in the mutant silkworm epidermis changed.In summary,this thesis uses ⁶⁰Go-γ-ray to simulate the space radiation environment and the random positioning machine（RPM）to simulate the space weightless environment to treat silkworm embryos respectively,and observe the effects of simulated radiation and simulated microgravity conditions on the development of silkworm embryos.The results showed that simulated radiation reduced the hatching rate of silkworm embryos,prolonged embryonic development time,and affected embryonic gene expression;simulated weightlessness shortened the silkworm embryonic development time and affected embryonic gene expression.The expression of Hox genes Bmpb and Bmscr was screened to be sensitive to the simulated weightlessness environment.The functional analysis of Bmpb and Bmscr and the cofactors Bmexd and Bmhth found that these 4 genes are essential for silkworm embryonic spigot morphogenesis.Functional analysis of Bmtsh/tio found that Bmtsh/tio was involved in the regulation of larval epidermal pigmentation and the development of adult appendages.It was further screened that wnt6 gene expression was sensitive to simulated radiation environment.Functional analysis revealed that wnt6 gene was involved in regulating larval epidermal pigmentation and the formation of bulge.Studying the influence of simulated space environment on the embryonic development of silkworm will provide a reference basis for future space exploration and provide a theoretical basis for space life science.