| WRKY proteins are a type of inducible transcription factors in plants. They have highly conserved WRKYGQK amino acid sequences in their N-termini. It is commonly accepted that WRKY proteins specifically interact with the W-box [(C/T)TGAC(T/C)], which is found in the promoter region of numerous plant target genes, to accommodate the expressions of downstream target genes. To date, WRKY genes have been identified and characterized in model plants such as Arabidopsis and rice, but the research of WRKY gene family in ligneous species is still in its infancy. Survey and characterization of WRKY genes in a ligneous species would facilitate a better understanding of the evolutionary processes and functions of this gene family. Using the methods of bioinformatics，this study want to get complete WRKY genes in poplar based on analyzing the latest poplar (Populus trichocarpa) genome. Subsequently, phylogenetic analysis, chromosomal localization, motif analysis, exon-intron organization and expression patterns of WRKY genes were also investigated, which lay a solid foundation for further comparative genomics studies. The main results of this study are as follows：1. We performed search, comparison and selection in the poplar genome and protein sequences using various bioinformation softwares. The Pfam database was amplied for deciding if the candidates correlated with the features of WRKY proteins. Atotal of104WRKY genes were predicted. On the basis of the chromosomal location and triploid poplar’s naming rule, the WRKY genes were named of the Populus trichocarpa as PtWRKY1-PtWRKY104.2. Amultiple sequence alignment of126PtWRKY domains were performed. Three major groups (I, II, III) were categorized as described by Somssich. Additionally, several subgroups (Ia, Ib, IIa, IIb, IIc, IId, IIe, III) were clearly formed. To compare the two phylogenetic trees on the basis of PtWRKY domains and genes respectively, similar groups and subgroups were found.3. Based on a multiple sequence alignment of PtWRKY domains, structure modes of amino acid were obtained. Four kinds of variation were characterized which are WRKYGKK, WKKYGQK, WRKYGRK and FWRKYGQK in core sequences WRKYGQK. And the variation of FWRKYGQK was not found in other plants. The structure mode of zinc finger has five variations which are C-X4-C-X21-HXH, C-X4-C-X22-HXH, C-X4-C-X23-HXH, C-X5-C-X19-HXH and C-X5-C-X23-HXH. 4. MEME online program was employed as a method to analyse motif distribution and check the results of domain prediction. The conserved motifs1,2,3,5and11are characterized as WRKY domains, which are broadly distributed in the PtWRKY protein sequences.5. Approximately83%(86out of104) WRKY genes, participated in gene duplication events, including69%(29out of42) homologous pairs exhibited segmental duplication. To analysis of the WRKY protein sequences, we observed that numerous PtWRKY genes contained an intron in their WRKY domains.6. With PopGeneIE databases, we analysed expression characteristics of PtWRKY genes, and81genes have microarray data support. The expression pattern demonstrated that PtWRKY genes have different expression levels in various tissues. To explore the stress responses in subgroup III of the PtWRKY genes, we analyzed their expression profiles under various abiotic stress conditions (cold, drought and salinity) and SA treatment using semi-quantitative RT-PCR. The results indicate that almost all genes exhibited significant differences in their expression level in response to one or more stress treatment. PtWRKY76gene was remarkable responsive to SA and drought. |
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