The Function Study Of SfAbd-B And SfHth Genes During Posterior Abdominal Development Of Spodoptera Frugiperda

Abdominal-B(Abd-B)gene,as the last member of the insect Hox gene family,mainly determines the morphological characteristics and appendage development of insect posterior somites.Homothorax(Hth)gene belongs to the TALE homeodomain protein subfamily,which not only binds to a specific domain to increase the regulatory activity of Hox protein,but also participates in the formation of insect appendages.Based on previous studies and literature accumulation,we speculate that Sf Hth and SfAbd-B genes are involved in regulating the development of the hind abdomen of Spodoptera frugiperda,but the specific regulatory mechanism is still unclear.This dissertation uses CRISPR/Cas9 technology to conduct targeted editing of Sf Hth and SfAbd-B genes,and combines RNA-seq technology and q RT-RCR technology to explore gene functions.The main research results are as follows:(1)Knockout of SfAbd-B: Microinjection of SfAbd-B-sgRNA and Cas9 protein into Spodoptera frugiperda embryos resulted in severe defects in postabdomen and abdominal feet in mutant embryos,and the embryonic lethality was extremely high.Mutant larvaes showed excess feet and fusion phenotype in the posterior somites,mutant pupaes showed pigment loss,developmental defects and somite deviation phenotype,and mutant adults showed defects such as hyperplasia,deviation or deformity of external genitalia.DNA sequencing showed that the SfAbd-B mutant had frameshift mutations caused by base insertions,deletions and substitutions at the target site.The expression of appendage development-related genes in the external genitalia of mutant adults was detected by q RT-PCR.The results showed that Dpp and Cp12 were down-regulated in the reproductive segments of mutant females and males,while Hth,Lab,En,and Exd were all up-regulated trend in females,with a downward trend in males.(2)Analysis of RNA-seq results of SfAbd-B mutant lethal embryos: Transcriptome sequencing and bioinformatics analysis and verification were performed on SfAbd-B mutant lethal embryos and wild-type embryos at the same stage.GO analysis showed that the effect of SfAbd-B gene on embryonic development mainly focused on molecular functions.The most significant enrichment GO term was the Structural constituent of cuticle,and cuticle protein genes under this GO term were all significantly down-regulated.KEGG analysis showed that the SfAbd-B gene was mainly related to the RNA polymerase pathway,and all genes annotated to this pathway were down-regulated.The differential genes were sorted by differential fold,and 7 of the 10 most significantly down-regulated differential genes were annotated to the epidermis gene family,indicating that the SfAbd-B gene was involved in embryogenesis by positively regulating the expression of epidermis genes.(3)Knockout of Sf Hth: Microinjection of Sf Hth-sgRNA and Cas9 protein into Spodoptera frugiperda embryos resulted in the failure of a large number of mutant embryos to hatch normally,and the dissection found that the posterior abdomen was severely deformed.Mutant larvae.Mutant larvae exhibit phenotypes with excess feet,degeneration of crochets,and difficulty in molting or pupation.The phenotypes of mutant pupae was defect of gonopore and defects and melanism epidermis in the 9th and 10 th abdominal segments.Sf Hth and SfAbd-B mutants have similar phenotypes.DNA sequencing showed that the Sf Hth mutants had frameshift mutations caused by base insertions,deletions and substitutions at the target site.The expression levels of somatogenesis-related genes in Sf Hth mutant lethal embryos were detected by q RTPCR,The results showed that Abd-B,Dac,En,Exd,Dpp and Lab genes were downregulated,and Dll and Ix genes were significantly up-regulated in appendage pattern genes.Epidermal protein genes Cp7,Cp12,Cp19,Cp18.6,Lcp A3 A and sg Abd-6 genes were significantly down-regulated,and some were nearly not expressed.