Cloning, Expression And Transcriptional Activity Analysis Of Eight Wheat WRKY Transcription Factor

The WRKY proteins are one of the largest transcription factor family in plants. WRKY proteins comprise a DNA binding domain containing about 60 amino acid residues. It is defined by the conserved amino acid sequence WRKYGQK at its N-terminal end, together with a novel zinc-finger-like motif. WRKY transcription factors are named after these structural features. WRKY transcription factors are involved in a wide range of plants' response to biotic and abiotic stresses, as well as hormone, and in regulation of plant growth and developmental processes. However, studies on wheat WRKYs are limited,and systematic identification, expression analysis and functional studies of wheat WRKY gene family is still lacking.As an extension of our previous study on wheat WRKYs in this laboratory, we firstly searched wheat ESTs which have the invariant WRKY amino acid sequence in DFCI, NCBI and Ensembl database. The overlapped ESTs were assembled into contigs with putative open reading frames(ORFs). Then we cloned nine new cDNAs encoding WRKY transcription factors from wheat, and these nine wheat WRKY genes were designed Ta WRKY45-TaWRKY53, respectively. Bioinformatics analyses clearly showed that the nine members of the WRKYs that contain highly conserved WRKY structure domain and the zinc finger motif. The nine WRKY transcription factors were devided into three subgroups: TaWRKY45 belongs to group I; TaWRKY48, 51, 52, and 53 belong to subgroup II; and TaWRKY47, 49 and 50 belong to group III. Although we could not get full-length sequence of TaWRKY46, since it contains a conserved WRKY domain and C2H2 zinc finger structure, thus it may belong to subgroup I or subgroup II.To examine the expression profiles of those TaWRKYs, semi-quantitative RT-PCR was performed with the total RNA extracted from different organs of wheat(cv Chinese Spring). The results showed that all of them were ubiquitously expressed in all organs examined, including roots, stems, leaves and so on, but the expression levels of different genes showed differences in different organs. The expression levels of TaWRKY47, Ta WRKY49, TaWRKY50, TaWRKY51 and TaWRKY52 were higher in vegetative organs roots, stems and leaves compared to that of reproductive organ, but Ta WRKY45?TaWRKY46 and TaWRKY48 were higher in stamens and pistils. Besides Ta WRKY48 and Ta WRKY53, other WRKYs were upregulated by salt stress; all nine WRKY genes were upregulated to some extent by ABA; four genes including TaWRKY50, TaWRKY51, Ta WRKY52 and TaWRKY53 were upregulated by cold; four genes TaWRKY47, Ta WRKY50, TaWRKY52 and TaWRKY53 were upregulated by GA; Ta WRKY52 was upregulated by PEG, and TaWRKY53 was downregulated by H2O2. These results suggest that the expressions of TaWRKYs are differentially responsive to different abiotic stresses and signaling molecules.We also examined transcriptional activation activities of TaWRKY45?47-53 by Transcriptional activation experiment. Transcriptional activation activity analysis revealed that TaWRKY47, TaWRKY50, TaWRKY51 and TaWRKY52 had apparent transcriptional activity, transcriptional activation of TaWRKY50 was comparatively weaker, but TaWRKY45, TaWRKY48, TaWRKY49 and TaWRKY53 had no apparently transcriptional activity in this assay. Further analysis showed that, TaWRKY48, 49, 50, 51 and 53 could bind with the typical W-box. TaWRKY47 and TaWRKY52 coult not bind to the typical W-box, but was able to bind with W-box mutants, while mutant W-box base resulted in reduced or loss of binding activity of other TaWRKYs.Results of the present study provided with useful information for elucidating the biological function of these TaWRKYs.