Gene Pyramiding of Peptidase Inhibitors Enhances Plant Resistance to the Spider Mite Tetranychus urticae 英文参考文献.docVIP

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Gene Pyramiding of Peptidase Inhibitors Enhances Plant Resistance to the Spider Mite Tetranychus urticae 英文参考文献.doc

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Gene Pyramiding of Peptidase Inhibitors Enhances Plant Resistance to the Spider Mite Tetranychus urticae 英文参考文献

GenePyramidingofPeptidaseInhibitorsEnhancesPlant ResistancetotheSpiderMiteTetranychusurticae MariaEstrellaSantamaria1,2,3,Ine′sCambra,ManuelMartinez1,ClaraPozancos1,PabloGonza′lez- Melendi1,VojislavaGrbic2,PedroCastan?era3,FelixOrtego3,IsabelDiaz1* 1Centro de Biotecnolog?′a y Geno′mica de Plantas (UPM-INIA). Campus Montegancedo Universidad Polite′cnica de Madrid, Autopista M40 (km 38), Madrid, Spain, 2DepartmentofBiologyWesternUniversity,Ontario,Canada,3Dpto.BiologiaMedioambiental,CentrodeInvestigacionesBiolo′gicas,CSIC,Madrid,Spain Abstract Thetwo-spottedspidermiteTetranychusurticaeisadamagingpestworldwidewithawiderangeofhostplantsandan extreme record of pesticide resistance. Recently, the complete T. urticae genome has been published and showed aproliferationofgenefamiliesassociatedwithdigestionanddetoxificationofplantsecondarycompoundswhichsupports itspolyphagousbehaviour.Toovercomespidermiteadaptabilityagenepyramidingapproachhasbeendevelopedbyco- expressing twobarley proteases inhibitors,thecystatin Icy6andthetrypsin inhibitor Itr1genesinArabidopsis plantsby Agrobacterium-mediatedtransformation.Thepresenceandexpressionofbothtransgeneswasstudiedbyconventionaland quantitativerealtimeRT-PCRassaysandbyindirectELISAassays.Theinhibitoryactivityofcystatinandtrypsininhibitorwas invitroanalysedusingspecificsubstrates.Singleanddoubletransformantswereusedtoassesstheeffectsofspidermite infestation.Doubletransformedlinesshowedthelowestdamagedleafareaincomparisontosingletransformantsandnon- transformed controls and different accumulation of H2O2 as defence response in the leaf feeding site, detected by diaminobenzidine staining. Additionally, an impact on endogenous mite cathepsin B- and L-like activities was observed afterfeedingonArabidopsislines,whichcorrelateswithasignificantincreaseinthemortalityofmitesfedontransformed plants. These effects were analysed in view of the expression levels of the target mite protease genes, C1A cysteine peptidaseandS1serinepeptidase,ide