摘要
WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.
WRKY proteins are transcriptional regulators involved in plant responses tobiotie and abiotic stresses, metabolisms, and developmental processes. In the present study, weisolated a WRKY cBNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 aminoacid residues with a potential leucine zipper structure in its N-terminus, sharing low identity withother known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal wereexpressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein,and purified by employing one-step affinity ehromatography on a Ni-NTA column. The recombinantGsWRKY89 protein was found to biud specially to sequences harboring W box cis elements by usingelectrophoretic mobility shift assays. This binding activity was decreased significantly by deletionof the leucine zipper-like structure in the N-terminal of Os-WRKY89. Using a yeast two-hybrid.assaysystem, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-proteininteraction. Further deletion to remove partial WRKY domain abolished completely the interactionbetween the expressed protein and the W boxes, indicating that the WRKY domain is essential to theDNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays asignificant role in the protein-protein and DMA-protein interactions.
基金
This work was supported by the State Basic Research and Development Plan(G200001 6203)
the National Natural Science Foundation of China(Grant Nos.30370139&30471122).
关键词
亮氨酸
DNA
水稻
种植技术
转基因技术
DNA-binding
leucine zipper
Oryza saliva
transcription factor
WRKY protein
