| The diamondback moth(DBM),Plutella xylostella(L.),is the global pest of crucifers.The DBM is notorious for rapid development ofresistence to pesticides,and selection for resisitance is accelerated by indiscriminate and unregulated pesticide application,which has become more difficult to control.The development of transgenic technology in pest control provides basic ideas for this study.This reseach was attempted to use the PiggyBac transposon for Trans-Cas9 insect construction,which can be used as a basis line,crossing with the sgRNA-expression insect for gene specificly knockout.Furthermore,functional indentification of development and pigamentation related genes in DBM by using CRISPR/Cas9.The following results were obtained:1.Transgenic DBM expressing Cas9 protein was constructed based on the PiggyBac transposon,and a well-developed Cas9 gene-carrying DBM mosaic was obtained.However,the G1 generation-positive individuals could not normally eclosion and finally die.This result indicates that PiggyBac can mediate the integration of exogenous genetic elements into genome,while systemic expression of Cas9 protein has a certain degree of fitness cost,resulting in the failure of eclosion.2.To indentificate the germline-specific promoter for gene overexprssion,the NanosO promoter was identified and characterized in DBM.The transcription activity of PxnosO was verified in DBM cells.The results showed that the PxnosO promoter can drive EGFP overexpression in vitro.Based on the transcriptome data,PxnosO was relatively high expressed in egg and female adult,exspecially in female adult.This results lay the foundation for the verification and analysis of PxnosO in vivo,and made basical application of PxnosO promoter in transgenic DBM construction.3.CRISPR/Cas9 mediate gene editing of Pxyellow.Direct injection of in vitro synthesized Cas9 mRNA and gene-specific sgRNA for gene knockout induced corresponding phenotypes:compared with wild-type animals(black),the pigmentation of 1st and 2nd instar larvae turn into yellow,while the 3rd instar larvae body color change to light yellow;body color of the 4th instar larvae and prepupae are consistent with wild type;pigmentation of pupa changed from black or dark brown to tan;adult body color changed from gray/black to yellow/tan.Development and reproduction of mutated DBM were consistent with the wild-type animals.The mutated phnotypes could be stably inherited to Gi generation.This results showed that knockout of Pxyellow can induce body color variation in DBM.The visible phenotype resulting from Pxyellow disruption can be used as a screening marker for transgenic DBM selection.4.PxASH2 disruption based on CRISPR/Cas9 system.The in vitro synthesized Cas9 mRNA and PxASH2 gene-specific sgRNA were directly injected into the eggs of DBM to achieve efficient knockout of PxASH2.The sequencing of Go and Gi individuals showed that the selected target sites contained different forms of insertions or deletions,but no predicted mutant phenotype was observed.From the results,PxASH2 potentially involved in wings development that needs further study.Based on the PiggyBac transposon,we try to construct transgenic DBM expressing Cas9 protein.Because the shortage of fitness cost of Cas9 systemic expression in DBM,we cloned and identified the PxnosO promoter,and verified the transcriptional activity of promoter in cells.This results presented the potential for the establishment of binary CRISPR/Cas9 system in DBM using a germline-specific promoter.In addition,CRISPR-mediated gene knockout showed that the phenotype resulting from the Pxyellow disruption can be further used as a marker gene for transgenic screening. |
