Identification Of The WRKY Gene Family And Functional Analysis Of Two CiWRKYs In Caragana Intermedia

WRKY transcription factors,one of the largest families of transcriptional regulators in plants,play key roles in plant signal pathway and regulate multiple plant biological processes,including biotic and abiotic stress responses.Fifty-three CiWRKYs with complete WRKY domain were identified from Caragana intermedia transcriptome data,based on this,we carried out the following studies:(1)According to the number of WRKY domains and the pattern of zinc finger structures,the 53 CiWRKY proteins were classified into three main Groups.Eleven,thirty-three and eight CiWRKYs were assigned to Group I,II and III,respectively.Additionally,group II was further classified into five sub-groups: IIa,IIb,IIc,IId,and IIe,which contains 5,6,13,4 and 6 CiWRKYs,respectively.(2)Twenty-eight CiWRKYs with complete open reading frames(ORFs)were obtained via transcriptome data or by RACE cloning,and the physicochemical properties,secondary structure and tertiary structure were further predicted.By comparing the Ci WRKYs with WRKYs from the other 8 leguminous plants,it revealed that WRKYs of Medicago truncatula may have the highest homology with CiWRKYs.Compared with the WRKYs from Arabidopsis,it was found that the Group III protein showed the lowest similarity,and the Group IIa and IId subgroups had higher similarity,while the protein similarity of Group I,IIb,IIc and IIe varied.(3)The expression patterns of the 28 CiWRKYs with complete ORFs were examined through quantitative real-time PCR(qRT-PCR)in different tissues and under different abiotic stresses(drought,cold,salt,high-pH and abscisic acid).The results showed that most of the 28 CiWRKYs genes are tissue-specific and each CiWRKY responded to at least one stress treatment.And the expression of CiWRKY75-1 were up-regulated under all these five treatments,and was significanty induced under drought,salt and high pH treatments.The expression of CiWRKY28-1 were up-regulated by drought and ABA,while down-regulated by salt,high pH and cold treatments.(4)The subcellular localization prediction of the 28 CiWRKYs by online software indicated that all of them localize to nuclei,among which,22 CiWRKYs had nuclear localization signals(NLSs).One of the proteins containing two WRKY domains(CiWRKY26)and one of the proteins containing one-WRKY-domain(CiWRKY28-1)were fused with green fluorescent protein(GFP)and transformed into Arabidopsis,the observation of GFP fused CiWRKY26 and CiWRKY28-1 indicated that these two proteins localized exclusively to nuclei,supporting their role as transcription factors.(5)Overexpression of CiWRKY75-1 in Arabidopsis thaliana suppressed salt stress tolerance of the transgenic plants,while increased ABA inhibited seed germination,suggesting that CiWRKY75-1 plays negative and positive roles in plants respossive to salt and ABA,respectively.(6)Overexpressed CiWRKY28-1 in A.thaliana decreased plant tolerance to drought stress and enhanced plant tolerance to ABA,suggesting that CiWRKY28-1 acts as a negative regulator in plants respossive to drought and ABA stresses.Taken together,these results indicate that different CiWRKYs play different roles in plant response to abiotic stresse and developmental processes.Hence,there is an urgent need for examination of the function of CiWRKYs against abiotic stresses in C.intermedia in future studies.