简介：RiceisanimportantfoodcropinChina,andthedevelopmentofhybridriceisacrucialwaytoincreasegrainyield.Thecreationofdual-purposenuclear-sterilelinesfortwo-linehybridbreedinghasbecomevitalforcommercialricebreeding.WeconstructedthepC1300-2x35S::Cas9-sgRNAPTGMS2-1expressionvectorforeditingthemalefertilitygenePTGMS2-1intwowidelycompatiblericevarieties,93-11andHuazhan,byusingtheCRISPR/Cas9system.Weobtainedthemarker-freephotoperiod-/thermo-sensitivegenicmale-sterile(P/TGMS)linesinT1generation.Accordingtotheexperimentsinphytotronwithfourtemperatureandphotoperiodtreatments,wefoundthetemperatureisthemainfactorforrestoringthepollenfertilityofptgms2-1mutantsin93-11andHuazhan,andthephotoperiodalsohassomeeffectsonpollenfertilityintwodifferentricebackgrounds.Theapplicationofcultivatingnewmale-sterilelinesbygenomeeditingsystemwillsignificantlyacceleratethericebreedingprocess.

简介：Pheromone-bindingproteins(PBPs)arethoughttobindandtransportsexpheromonesontotheolfactoryreceptorsonthedendritemembraneofolfactoryneurons,andthusplayavitalroleinsexpheromoneperception.However,thefunctionofPBPshasrarelybeendemonstratedinvivo.Inthisstudy,twoPBPs(PBP1andPBP3)ofChilosuppressalis,oneofthemostnotoriouspyralidpests,wereinvivofunctionallycharacterizedusinginsectswiththePBPgeneknockedoutbytheCRISPR/Cas9system.First,throughdirectinjectionofPBP-singleguideRNA(sgRNA)/Cas9messengerRNAintonewlylaideggs,ahighrateoftarget-geneediting(checkedwithpolledeggs)wasinducedat24hafterinjection,21.3%forPBPl-sgRNAinjectedeggsand19.5%forPBP3-sgRNAinjectedeggs.Second,byanin-crossingstrategy,insectswithmutantPBP1orPBP3(bothwithaprematurestopcodon)werescreenedandhomozygousmutantswereobtainedintheG3generation.Third,themutantinsectsweremeasuredforelectroantennogram(EAG)responsetofemalesexpheromones.Asaresult,bothPBPmutantmalesdisplayedsignificantreductioninEAGresponse,andthisreductioninPBP1mutantswashigherthanthatinPBP3mutants,indicatingamoreimportantroleofPBP1.Finally,therelativeimportanceoftwoPBPsandthepossibleofftargeteffectinducedbysgRNA-injectionarediscussed.Takentogether,ourstudyprovidesadeeperinsightintothefunctionofandinteractionbetweendifferentPBPgenesinsexpheromoneperceptionofC.suppressalis,aswellasavaluablereferenceinmethodologyforgenefunctionalstudyinothergenesandothermothspecies.