| Insects have evolved various body patterns to adapt to the external environment,and one of the important features is the evolution of appendages.Appendages are an important part of the body structure of insects.The various appendages give them a better advantage for feeding and survival.Antp,Ubx and Abd-B of the Hox gene family play an important role in the formation of insect appendages.At present,the specific functions of these three genes in Spodoptera frugiperda have not been reported.Therefore,we chose Abd-B,Antp and Ubx as the research targets,and used CRISPR/Cas9 gene knockout technology to initially explore their biological functions in Spodoptera frugiperda.The specific research content is as follows:(1)Bioinformatics analysis of Antp,Ubx and Abd-B genes of Spodoptera frugiperda:The full-length open reading frame sequences of SfAntp,SfUbx and SfAbd-B genes are 912bp,765bp and 963bp,respectively,and the number of encoded amino acids are 303,254 and 320,respectively.They all contain 2 exons and 1 intron.According to the amino acid homology analysis and the phylogenetic tree,Antp,Ubx and Abd-B have high homology among lepidopteran insects,all reaching more than 90%,and SfAntp,SfUbx,and SfAbd-B genes are closely related to Spodoptera litura.(2)Analysis of the expression patterns of SfAntp,SfUbx and SfAbd-B:The expression profiles of SfAntp,SfUbx,and SfAbd-B were detected by QRT-PCR,and the results showed that these three genes were expressed throughout the life cycle of Spodoptera frugiperda.During embryonic development,the expression level of SfAntp is the lowest when the embryo develops to 6h,and has a high expression level from 12h to 48h;SfUbx expression level is low at the early stage of embryonic development,and the expression level gradually progresses Increase,and the expression level decreases in the later stage of embryonic development,and the overall trend is to increase first and then decrease.The expression pattern of SfAbd-B is similar to that of SfUbx,that is,the expression level is highest in the middle stage of embryonic development.During the different developmental stages from embryo to adult,SfAntp,SfUbx and SfAbd-B all had the highest expression levels in the egg stage.By detecting the expression levels of three genes in the adult head,thoracic segment,abdominal segment,external genitalia,antennae,wing and foot,the results showed that the expression level of SfAntp was the highest in the adult thoracic segment and foot,and SfUbx was highly expressed in the wing and foot and the expression of SfAbd-B is the highest in the genital section.(3)Gene knockout of SfAntp,SfUbx and SfAbd-B:Design sgRNA sites on the first exons of SfAntp,SfUbx and SfAbd-B to knock out the three genes by microinjection.The results showed that the lack of SfAntp produced thoracic fusion,thoracic foot defect and head There is a mutant phenotype of foot-like protrusions between the segment and thoracic segment;after knocking out SfUbx,foot-like protrusions in the first abdominal segment were observed in the dissected mutant embryos and hatched mutant larvae;knocking out SfAbd-B,a phenotype of severely defective somites and appendages appeared in the mutant embryos,and the hatched first-instar mutant larvae showed a mutant phenotype of excess feet and fusion of somites on the posterior abdomen.In addition,the deletion of each gene will lead to a high embryonic lethality rate.DNA sequencing found that the target sites of SfAntp,SfUbx and SfAbd-B mutants had different degrees of base frame shift mutations,indicating that the mutant phenotypes were indeed caused by the knockout of SfAntp,SfUbx and SfAbd-B genes.This study uses CRISPR/Cas9 gene editing technology to initially explore the functions of Antp,Ubx and Abd-B genes in the development of Spodoptera fugiperda’s appendages.The results showed that the knockout of SfAntp,SfUbx and SfAbd-B all resulted in abnormal foot development and severe embryo-lethal effect.It can be seen that SfAntp.SfUbx and SfAbd-B are essential to the development of Spodoptera frugiperda’s foot and are essential factors in the process of embryonic development.This study lays the foundation for the study of the regulation mechanism of other lepidopteran insects’ body development,and also provides candidate target genes for the construction of lethal genetic strains for the genetic regulation of the Spodoptera frugiperda population. |
