Identification Of Stress-Resistant Related Genes Of Group ?d WRKY Transcription Factor Family In Maize And Function Analysis Of ZmWRKY17

Maize is one of the crucial cereal crop,is the world recognized gold food.Environmental stress factors,such as drought and high salinity,can adversely affect maize growth,development and productivity.To study the mechanisms of maize response to environmental stresses and cultivate some new crop cultivars with better stress tolerance is one of the most effective measures to respond abiotic stress and increase maize yield.Many studies have indicated that transcription factors play vital roles in plant growth and tolerance to various environmental stresses.Overexpression of a specific transcription factor can regulate the expression of many downstream stress-related genes and consequently enhance comprehensive resistances of crops.WRKY transcription factor has become a hot spot in the gene function research.Among the WRKY proteins in plants,the molecular mechanisms of group ?d WRKY family is attracting the most attention at present.Members of this gene family are known to have diverse functions in regulating plant growth,development and response to biotic and abiotic stresses.In this study,we first investigate the molecular characterization of maize group ?d WRKY genes,including their expression patterns,transactivation activity,and calmodulin-binding ability.We subsequently study the functions of ZmWRKY17 gene in abiotic stress response by overexpressing this gene in Arabidopsis.We also analyze the transcriptome of WT and transgenic Arabidopsis under salt stress via high-throughput sequencing.The main results are as follows:1.Expression levels of the maize group ?d WRKY genes under drought and salt stresses were investigated by qRT-PCR.Results showed that of the 7 group ?d WRKY genes,the expressions of 6 genes were up-regulated,one gene was down-regulated.We subsequently isolate 6 group ?d WRKY genes from the maize inbred line B73.Transactivation assay revealed that these WRKY proteins showed no transcriptional activation activity.In addition,these WRKY proteins can interact with ZmCaM2 in the yeast assay system.2.According to the phylogenetic relationships and expression patterns,ZmWRKY17 is selected for further analysis.The full-length cDNA of ZmWRKY17 is 996 bp,which encodes a protein of 331 amino acids.Sequence alignments indicated that ZmWRKY17 shares 100% sequence identity with the predicted sequence in the maize B73 annotated database.Further expression analysis showed that ZmWRKY17 was also induced by exogenous ABA,and tissue-specific expression indicated that ZmWRKY17 was constitutively expressed in roots,stems,leaves,tassels,silks,ears and embryos,but ZmWRKY17 was expressed mainly in tassels.Transient expression showed that the GFP signal was detected just in the nucleus of the tobacco leaf epidermal cells for ZmWRKY17-GFP fusion protein through agroinfiltration.Yeast hybrid assays showed that ZmWRKY17 can specifically bind to W-box.3.We construct an overexpression vector pCAMBIA1301-ZmWRKY17 and transform it into Arabidopsis using Agrobacterium-mediated transformation.The transgenic plants and WT were disposed under high salt stress,and the malonaldehyde content and relative electrolyte leakage were analyzed.Results displayed that the malonaldehyde content and relative electrical conductivity in ZmWRKY17-overexpressing plants were dramaticlly higher than that in wild type plants under Nacl condition.Thus,overexpression of ZmWRKY17 enhances plant sensitivity to salt stress.4.ABA sensitivity assay indicated that ZmWRKY17 overexpression transgenic plants exhibit significantly decreased sensitivity to ABA.In addition,during the seed germination,when ABA biosynthesis inhibitor was added,the salt sensitivity phenotypes in the transgenic lines disappeared.5.Transcriptome analysis of WT and transgenic plant under treated with salt stress was conducted using RNA-Seq technology.The results show that the expression level of many well known stress-tolerant genes such as RD29 B,bHLH92,RAB18,KIN1 and DREB1 F were higher in wild type than that in trangenic line.However,the expression level of some ABA synthesis genes such as NCED5 were lower in WT than that in trangenic line.In conclusion,we investigate the molecular characterization of maize group ?d WRKY genes.According to the analysis of phylogenetic relationships and expression patterns,ZmWRKY17 was selected for further phenotypic analysis.Overexpression of ZmWRKY17 could significantly enhances plant sensitivity to salt stress.Moreover,the subdued sensitivity to ABA and up-regulated expression of some ABA synthesis genes of transgenic plants indicate that ZmWRKY17 might be participated in plant response to salt stress through an ABA-dependent pathway.