Transcriptional Regulation And Function Analysis Of Maize Silk-Specific Expression ZmbZIP25 Gene

Each maize silk is a specialized stigma and style that directly participate in the process of pollination and fertilization.In order to facilitate the adhesion of pollen and provide enough water and nutrients for pollen germination,the silk surface do not have any protection,which gives an opportunity for the invasion of pathogens.Genes that are specifically expressed in maize silks in order to against pathogens.At present,there are few reports on the transcriptional regulation and function analysis of maize silk-specific expression genes.ZmbZIP25(Zea bZIP transcription factor 25)is a function-unknown protein that belongs to the group D of the bZIP transcription factor family.The bZIP transcription factor is involved in a variety of physiological processes and genes in group D are primarily involved in two different processes of defense against pathogens and plant development.RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks,and this specificity was confirmed by RT-PCR.In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks.A 5'RACE assay identified the transcription start site of the ZmbZIP25 gene.To characterize this silk-specific promoter,we isolated and analyzed a 2450 bp(from-2083 to+367)and a 2600 bp sequence of ZmbZIP25(from-2083 to+517,the transcription start site was denoted+1).Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas.Furthermore,transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues.However,no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays.A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants.GUS expression driven by the 1957 bp(-1590?+367),1879 bp(-1512?+367),1695 bp(-1328?+367)and 1484 bp(-1117?+367)exclusively in the papillae of Arabidopsis stigmas,respectively.When the fragment was truncated to 1324 bp(-957?+367),the histochemical assay showed GUS staining in many tissues,including seedling,rosette leaf,and whole floral organs.Therefore,the region from-1117 to-957 of the ZmbZIP25 5'-flanking sequence is necessary for silk-specific gene expression.And probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from-1117 to-957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.To study the function of ZmbZIP25,we cloned the ZmbZIP25 cDNA from Mo 17 maize silk.Subcellular localization showed that ZmbZIP25 was localized in the nucleus,consistent with its identity as a transcription factor.However,ZmbZIP25 does not show transcriptional activation activity and does not bind to G-box,C-box and A-box elements in yeast.The expression of ZmbZIP25 is not induced by PEG,ABA,NaCl,MeJA and SA in maize leaves.When ZmbZIP25 expressed in Arabidopsis,the transgenic Arabidopsis plants showed a small rosette and a dwarf phenotype,and PR genes up-regulated.In maize silk,ZmbZIP25 was up-regulated by MeJA and reached the highest level at 1 h after treatment.Overexpression of ZmbZIP25 in maize resulted in a dwarf phenotype.The height of the plant decreased with the increase of ZmbZIP25 expression level in both Arabidopsis and maize.Based on these results and previous studies,we speculated that ZmbZIP25 may be involved in the MeJA-regulated plant disease resistance pathway.