| Bacillus thuringiensis(Bt)is a pervasive gram-positive soil bacterium that generates biodegradable insecticidal toxins during metabolic process,which is pathogenic to pest insects.Plutella xylostella is a member of Lepidoptera and Plutellidae.Alterations of midgut receptors such as cadherin(CAD),alkaline phosphatase(ALP),aminopeptidase N(APN)and ABC transporters(e.g.ABCC2,ABCC3)disrupt toxin binding and are generally associated with high-level resistance to Bt Cry toxins in insects.CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats CRISPR and the CRISPR-associated gene Cas9)mediated genome editing technology has been applied to many organisms for genetic modification or transformation.In recent years,this technology has also been applied to arthropods,this paper provides a comprehensive overview of the progress of the CRISPR/Cas9 system in different arthropods,also discuss their future prospects for becoming crucial technologies in arthropods.Subsequently,CRISPR/Cas9-mediated knockout of the PxABCC2,PxABCC3,PxAPN1 and PxAPN3 a midgut genes were conducted in diamondback moth,and four knockout strains exhibited high levels of resistance to the Cry1 Ac protoxin.Our results provide conclusive in vivo functional evidence that all the PxABCC2,PxABCC3,PxAPN1 and PxAPN3 a proteins c an serve as midgut functional receptors of Cry1 Ac toxin,and provides an excellent platform for the exploration of other receptor genes,also provides a new insight for the development of new pest management strategies.The main research contents of this paper are as follows:(1)Progress and prospects of CRISPR/Cas systems in arthropodsClustered regularly interspaced short palindromic repeats(CRISPR)and the CRISPR-associated gene Cas9 represent an invaluable system for the precise editing of genes in diverse species.Thus,this paper seeks to provide a comprehensive and impartial overview of the progress of the CRISPR/Cas9 system in different arthropods.(2)Construction of the CRISPR/Cas9 gene editing system of P.xylostellaThe sgRNAs of PxABCC2 and PxABCC3 genes were designed via CRISPR Cas-Designer software,the potential off-target effects of both the selected sgRNA target sequences were eliminated by searching the P.xylostella genome database and Gen Bank database combined with the software Cas-OFFinder.Microinjection of eggs from the susceptible strain(DBM1Ac-S)were carried out.Subsequently,we used germline transformation and mutation screening strategy to successfully to construct stable homozygous mutant strains of PxABCC2 and PxABCC3 genes designated ABCC2 KO and ABCC3 KO,respectively.(3)The bioassay and interstrain allelic complementation tests in ABCC2 KO and ABCC3 KO strainsBy using Leaf-dip method,susceptibility to the Cry1 Ac protoxin was tested in ABCC2 KO and ABCC3 KO strains,the results indicated that the resistance level of the ABCC2 KO and ABCC3 KO strains to the Cry1 Ac protoxin were approximately724 and 413-fold higher,respectively,than that of the susceptible DBM1Ac-S strain.To determine the mode of inheritance of Cry1 Ac resista nce in both knockout strains,interstrain crossing was performed between pairs of strains from susceptible(DBM1Ac-S),ABCC2 KO,ABCC3KO and near isogenic resistance(NIL-R),and the responses of these four strains and their F1 progeny were examined with a diagnostic dose of the Cry1 Ac protoxin.(4)Binding analysis of BBMV proteins and Bt Cry1 Ac toxin in midgut of ABCC2 KO and ABCC3 KO strainsThe midgut BBMV proteins of the susceptible strain(DBM1Ac-S),two knockout strains(ABCC2KO and ABCC3KO)and the near isogenic resistance strain(NIL-R)were extracted,respectively.The binding between trypsin-activited Cry1 Ac toxin and the midgut BBMV proteins were analyzed by Western Blot.The result showed that Cry1 Ac toxin could bind to the midgut BBMV proteins in the susceptible DBM1Ac-S strain,while nearly complete loss of binding of the toxin to the midgut BBMV proteins was observed in the resistant NIL-R strain.The significantly reduced binding of the Cry1 Ac toxin to midgut BBMV proteins was detected in both resistant knockout strains,the Cry1 Ac toxin binding assays demonstrated that disruption of both the PxABCC2 and PxABCC3 genes resulted in decreased Cry1 Ac binding,thereby conferring high levels of Cry1 Ac toxin resistance to P.xylostella.(5)Heterologous expression of different APN genes in P.xylostellaWhen the constitutive midgut transcription profiles of these genes in fourth-instar larvae from both Bt-susceptible and resistant strains were compared,it was revealed that both PxAPN1 and PxAPN3 a genes were substantially decreased in all of the Bt-resistant strains,whereas PxAPN5 and PxAPN6 genes were drastically increased.To determine the role of these four APN genes in the mechanism of Bt toxin action,the PxAPN1,PxAPN3 a,PxAPN5 and PxAPN6 genes were transiently expressed in Sf9 cells.The successful ectopic expression of these GFP-fused PxAPN proteins was further verified by Western Blot.Immunofluorescent localization detection illustrated that all four PxAPN proteins were located on the cell surface,but that only PxAPN1 and PxAPN3 a proteins could bind Cry1 Ac toxin,it is clear that PxAPN1 and PxAPN3 a are functional receptors of the Bt Cry1 Ac toxin in P.xylostella.(6)CRISPR/Cas9-mediated knockout of PxAPN1 and PxAPN3 a genesThe specific sgRNAs sequences of PxAPN1 and PxAPN3 a genes were designed and mixed with Cas9 protein.The eggs of P.xylostella were microinjected by our mature CRISPR/Cas9 platform.We used an optimized germline transformation and mutation screening strategy to successfully to construct homozygous mutant strains of PxAPN1 and PxAPN3 a genes designated APN1 KO and APN3 a KO,respectively.Subsequent bioassays showed that APN1 KO and APN3 a KO larvae exhibited approximately 463-fold and 346-fold levels of resistance to Cry1 Ac protoxin,respectively.At the same time,PxAPN1 or PxAPN3 a were proved to be midgut functional receptors Cry1 Ac toxins and confers high-level Cry1 Ac resistance in P.xylostella. |
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