Functional Analysis Of Rice WRKY10 Gene

The WRKY proteins, mainly present in plants, constitute a superfamily of transcription factors. They are extensively involved in some developmental and metabolic processes, as well as in biotic and abiotic stress responses in plants. In this paper, employing a PCR strategy, a WRKY gene, designated OsWRKY10, was cloned from rice cDNA generated from total RNA of shoots of rice plants at 5 d after germination. The two splicing modes, the structure and biochemical characteristics and expression patterns under various hormone treatments and abiotic stresses were analyzed. The putative functions in vivo of this gene were explored here as well. Results are summarized as follows:OsWRKY10 encodes a peptide harboring only one WRKY domain and a zinc finger of CX4CX23HXH, falling into Ib subgroup of rice WRKY proteins. It shares low identity with other WRKY proteins, indicating a new member of WRKY family.Interestingly, two splice variants, named WRKY10 (full-length transcript) and WRKY 10-2 respectively, were found in this gene, verified by RT-PCR and Northern blot analysis. Alternative splicing events occurred in the 125th nucleotide (nt) of the third exon of the full-length transcript, which resulted in the deletion of 161 nts and a stop codon introduced in advance, thus the open reading frame (ORF) of WRKY 10-2 was 327 nt shorter than that of WRKY10. WRKY10 and WRKY10-2 encode peptides with sizes of 379 and 270 amino acids (aa), respectively.OsWRKY10-GST fusion protein was expressed in E. coli and purified by affinity chromatography on a glutathione sepharose 4 B column. The recombinant OsWRKY10 was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays (EMSAs).A Ubiv.OsWRKY10-GFP gene fusion construct, as well as a Ubiv.GFP as acontrol, was bombarded into the onion epidermal cells. The results showed the onion epidermis cells transformed with the control construct showed GFP fluorescence diffused randomly in both the cytoplasm and the nucleus. By contrast, the onion epidermis cells transformed with the construct encoding the OsWRKY10-GFP fusion protein showed GFP fluorescence exclusively in the nucleus, suggesting that OsWRKY10 is a nuclear-targeting protein. This observation, together with nuclear localization of OsWRKY10 mentioned above, indicates it possessing common features of most WRKY proteins investigated.The coding region of the two transcripts of OsWRKY10 and a series of deletion mutants of the full-length transcript were fused in frame to yeast GAL4 binding domain vector and transformed into yeast strain AH 109. The results indicated that the two transcripts possessed transactivating activity and the N-terminal region covering 61-120 aa, enriched in acidic acids, containing a putative activating motif LXXLL, was responsible for the activity.OsWRKY10 mRNA abundance was observed in roots, stems, leaves and flowers. Moreover, the expression of OsWRKY10 was regulated upon developmental stages ofrice plants. Under hormone treatments and abiotic stresses, it showed different expression patterns. The expression levels of OsWRKY10 were inhibited significantly by PEG, NaCl treatments and ABA application, suggesting that it might be involved inresponses to osmotic stress and salinity stress, and ABA signaling pathways might mediate these processes. However, mechanical wounding, cold (4℃), ethephon ,GA3(gibberellin) and JA( jasmonic acid) had no evident effects on its expression.