The Application Of Crispr/Cas9 System In Diamondback Moth Cell Lines

Plutella xylostella is the most serious global pest that damages cruciferous vegetables,and its broad-spectrum resistance to pesticides leads to huge economic losses.The development of biotechnology tools has gradually become an ideal strategy for pest control,and the derivation of insect cell lines provides an efficient research platform for this.The application of the reverse genetics tool CRISPR/Cas system on this platform has opened up a new perspective for the study of insect gene function.Although the P.xylostella cell lines have been established,there are still issues with unstable transfection efficiency and missing expression elements in its application.In addition,there are still gaps in the application of CRISPR/Cas system in this cell line,such as report screening verification,genome editing verification,transgenic and gene knock in verification.Therefore,this study optimized the delivery effect,expression mode,editing effect,and editing method of the CRISPR/Cas system on the P.xylostella cell lines to improve the integrity of the application of the system.The main research content is as follows:1.By testing the transfection efficiency of four mainstream liposome transfection reagents with and without serum during the transfection process,it was confirmed that Roche’s Xtreme liposome transfection reagent can stably achieve a transfection efficiency of 50%-60% on the P.xylostella cell lines,which provides a guarantee for efficient delivery of the CRISPR/Cas9 system.2.The potential endogenous high expression genes of P.xylostella were obtained through gene conservation analysis,and a Pol II endogenous strong promoter(Pub promoter)was identified through double Luciferase report analysis,EGFP report analysis and promoter truncation analysis,which provides an efficient tool for the multi-element expression of CRISPR/Cas9 system.3.A multi-element expression plasmid containing Neo,EGFP,and Cas genes was constructed and applied to the CRISPR/Cas9 system.The targeted knockout of both endogenous and exogenous genes demonstrated the effectiveness of the CRISPR/Cas9 system in the P.xylostella cell lines.4.Using Piggybac transposon system and G418 antibiotic screening,a Cas9 P.xylostella cell line was generated,which laid the foundation for the development of diversified genome editing methods based on CRISPR/Cas9 system.5.A t RNA+si RNA expression vector was constructed using Oligo based multi fragment enzyme linkage method,and it was clarified that t RNA from foreign species and mitochondrial t RNA from P.xylostella cannot mediate g RNA and sh RNA expression to enhance the knockout effect of CRISPR/Cas9 system and RNAi.6.A donor plasmid based on the CRISPR/Cas9 system for HITI gene knock-in mode was designed.As the occurrence of this mode was not detected in the 9 endogenous genes of P.xylostella,the subsequent optimization direction was clarified.In summary,this study clarified the effectiveness of the CRISPR/Cas9 system in the P.xylostella cell lines,laying the foundation for further insect genetics and molecular research,especially for the analysis of gene function.