Identification Of Grape WRKY Gene Family And The Function Analysis Of VlWRKY58 And VaWRKY10

Grape?Vitis vinifera L.?is one of the most important economic fruit worldwide,which can be used for fresh and dried,can make wine and juice,making it bring great commercial interests for people.However,the cultivated grapevines majority are European species,which exhibiting high susceptibility to variety biotic stresses.Exploring the grape resistant genes and expounding the regulation mechanism will provide valuable theoretical and practical significance for grape resistance improvement.WRKY is one of the plant specific transcription factor,which play important role in plant disease resistance.This research identified the grape WRKY genes according to the grape whole genome data,then analyzed the VvWRKY genes structure,evolution relationship and induced expression situation,preliminarily analyzed VvWRKYs biological function.Subsequently,cloned the grape resistance WRKY genes VlWRKY58 and VaWRKY10,and transformed to Arabidopsis and V.vinifera‘Thompson seedless',verified the function of these two genes.Furthermore,cloned the promoters of VlWRKY58 and VaWRKY10 and detected the induced activity of these two promoters.The main results are described as following:?1?59 VvWRKY were identified according to the grape whole genome,and then renamed VvWRKY1-VvWRKY59 according their chromosome location.59 VvWRKY proteins all have complete conserved domains.The phylogenetic tree was constructed among AtWRKY,SlWRKY and VvWRKY,which make the 59 VvWRKY genes classified to three groups?group?-??,containing 12,40 and 6 members each group,and a-e five subgroups in group?,containing 3,8,15,7 and 6 members each subgroup.4 tandem duplication events,16segregation duplication events and 66 synteny relationships were identified among 59VvWRKYs and with AtWRKYs,these events are useful for VvWRKYs evolution and function analysis.VvWRKY40 and VvWRKY45 expressed at high levels in grape root and VvWRKY42and VvWRKY52 expression were particularly high in leave,which showed the tissues specific expression of some VvWRKY genes.22 VvWRKYs were up-regulated after powdery mildew inoculation,18 and 14 VvWRKYs were up-regulated after SA and ABA treatment respectively,much more VvWRKYs showed up regulation after MeJA,ETH and drought treatment,indicating the close relationship of VvWRKYs with plant biotic stresses,abiotic stresses and phytohormones signaling.VvWRKY58 show significant up-regulation after grape powdery mildew inoculation and VvWRKY10 show obvious expression changing after MeJA and ETH treatments.?2?VlWRKY58 cDNA was cloned in V.labrusca×V.vinifera‘Kyoho'using the RT-PCR.The ORF is 2241bp,encoding 747 amino acids.Then constructed the 35S overexpression vector,following transformed to the model plant Arabidopsis.After inoculation with powdery mildew and grape Botrytis cinerea,the transgenic Arabidopsis lines showed obvious resistance to powdery mildew with spores number per gram fresh leaves significantly reduced,meanwhile showed obvious susceptibility to Botrytis cinerea.The SA signaling marker genes NPR1 and PR1 expressed high level in VlWRKY58 overexpression Arabidopsis lines after pathogen inoculation while the JA signaling marker gene PDF1.2 showed an inhibited expression.The VlWRKY58 transgenic Arabidopsis lines also showed susceptibility to bacteria Pst DC3000 with heavier yellow leaves and larger active bacteria numbers,meantime PR1 showed a reduced expression,which is different from powdery mildew and Botrytis cinerea inoculation.Furthermore,1952bp VlWRKY58 promoter was cloned in V.labrusca×V.vinifera‘Kyoho'by RT-PCR,following four deleted recombined vectors were constructed and transient transformed to grape leaves of V.vinifera‘Red globe'.GUS staining and activity assay indicated pVlWRKY58-608bp to-1034bp existing 2.1 times powdery mildew induction activity than control.Further analysis found 2 TCA-element,1 TGACG-motif,1P-box and 1 TC-rich repeats were located in these promoter region,possibly the TCA-element existing,which responding the SA signaling caused by powdery mildew and strengthen the powdery mildew resistance regulation.Additionally,five different grape proteins were screened by yeast two-hybrid screening,including V.vinifera glucan 1,3-beta-glucosidase,V.vinifera glyceraldehyde-3-phosphate dehydrogenase and V.vinifera oligopeptide transporter4.These proteins may beneficial for VlWRKY58 resistance regulation.?3?VaWRKY10 cDNA was cloned in V.amurensis‘Shuangyou'using the RT-PCR.The ORF is 954bp,encoding 318 amino acids.Then constructed the 35S overexpression vector,following transformed to Arabidopsis and V.vinifera‘Thompson seedless'.After inoculation with powdery mildew,Botrytis cinerea and Pst DC3000,we found that the transgenic Arabidopsis lines showed obvious resistance to necrotrophic pathogen Botrytis cinerea with lesions size significantly reduced,meanwhile showed obvious susceptibility to biotrophic pathogens powdery mildew and Pst DC3000 with a significantly increasing of powdery mildew spores and Pst DC3000 active numbers,also the O₂^-decreased after Pst DC3000inoculation.VaWRKY10 overexpression‘Thompson seedless'also showed obvious resistance to the pathogen Botrytis cinerea.The JA signaling marker genes LOX3 and PDF1.2 expressed high level in VaWRKY10 overexpression Arabidopsis lines after Botrytis cinerea inoculation,which may have the relationship with VaWRKY10 pathogen resistance regulation.Similarly,1597bp VaWRKY10 promoter was cloned in V.amurensis‘Shuangyou'by RT-PCR,following four deleted recombined vectors were constructed and transient transformed to grape leaves of V.vinifera‘Red globe'.GUS staining and activity assay indicated pVa WRKY10 0 to-280bp existing 4.2 times MeJA inducted activity than control.A TGACG-motif located in this region,which may respond JA signaling and helpful for VaWRKY10 Botrytis cinerea resistance regulation.