| Spodoptera litura is an important agricultural pest of Lepidoptera.Spodoptera litura can harm more than 100 plants in 99 families,causing serious losses to our agricultural production every year.Therefore,efficient control of Spodoptera litura has always been the goal of scientific researchers.Traditional control methods are mainly chemical control.However,the long-term use of chemical agriculture has caused serious resistance to Spodoptera litura,and the problem of pesticide residues has always endangered people’s health.Therefore,it is urgent to develop a pollution-free method for controlling Spodoptera litura.In recent years,with the development of genome editing technology,a foundation has been laid for analyzing the gene functions of pests and for better control of pests.The purpose of this study is to use genome editing technology to develop a set of methods that can control Spodoptera litura.First,use CRISPR/Cas9 genome editing technology to build a Spodoptera litura genome editing platform for the gene function study of Spodoptera litura.Second,the key genes of sex determination pathway and reproductive development are found in Spodoptera litura.Third,the target gene function of Spodoptera litura genome editing platform is used to study the genetic control lines of Spodoptera litura pest control.Relevant research results can provide a theoretical basis for the control of Spodoptera litura pests.The specific research contents and results are as follows:1.Construction of Spodoptera litura genome editing platform based on CRISPR/Cas9technologyCRISPR/Cas9 technology is one of the more widely used and efficient technologies in genome editing systems.In this study,the melanin synthesis pathway Ebony gene was used as the targeted gene,Cas9 mRNA and Ebony gene-specific sgRNA were synthesized in vitro,and the Spodoptera litura embryo microinjection platform was used to construct the Spodoptera litura genome editing platform.The study found that the microscopic injection of Ebony in the pupal stage and the adult stage phenotype deepened in the body,the pupae shell blackened,and the adult body organs were significantly darker than the wild type.DNA extraction and PCR sequencing confirmed that sequence fragments were deleted near the Ebony targeted site.These results prove that we successfully knocked out the Ebony gene and constructed the Spodoptera litura genome editing platform.2.Study on the functions of key genes in sex determination pathway Masc and Doublesex in Spodoptera litura.Sex determination is the basis for the differentiation of male and female insects,and is the key to regulating pest populations.In this part of the study,we take the non-model organism Spodoptera litura as the research object,and use the Spodoptera litura CRISPR/Cas9 genome editing platform to study the key genes of the sex determination pathway Masc and Doublesex genes specific functions in Lepidoptera pest Spodoptera litura.Using CRISPR/Cas9 genome editing technology to knock out the Spodoptera litura Masc and Doublesex genes,we clarified that the SlMasc and SlDoublesex genes play an important role in the sex determination of Spodoptera litura males and females.Overexpression of SlMasc in silkworm BmN cells resulted in the emergence of a male-specific Bmdsx~M spliced isoform,and SlMasc mutations resulted in male embryonic lethality and male sterility.SlDoublesex mutations caused abnormal male and female genitalia,abnormal sperm development,and male and female sterility.Through these studies,we constructed male and female sterile lines of Spodoptera litura,which provided target genes for better control of Spodoptera litura,laying a theoretical foundation.3.Construction of male-female specific sterility lines in Spodoptera litura based on CRISPR/Cas9 technology.Insects’reproductive ability is the key to insect breeding offspring and expanding the population.This part of the study uses the CRISPR/Cas9 genome editing technology to study the functions of SlSer2 and SlOsp genes at the individual level.The study found that SlSer2 and SlOsp genes were specifically expressed in male and female adults,respectively.In vivo knocked-out of SlSer2 resulted in male sterility of the adult,but did not affect female reproduction.qPCR detection revealed that the loss of SlSer2function led to down-regulation of family genes such as actin,structural proteins and related enzymes,which proved that SlSer2 is an important Seminal Fluid Protein(SFP)component and participates in the process of Spodoptera litura fertilization.Disruption of the SlOsp gene function results in female adult sterility,the spawning rate and hatchability of the mutant are significantly lower than those of the wild type,and the loss of the SlOsp gene function does not affect male reproductive ability.These studies prove that we have identified the functions of the reproductive development genes SlSer2 and SlOsp genes in Spodoptera litura,and constructed SlSer2 male sterile lines and SlOsp female sterile lines of Spodoptera litura,laying a theoretical foundation for pest control. |
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