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WRKY蛋白是一类参与植物生物和非生物胁迫反应、代谢、发育等过程的转录调节因子.水稻WRKY89与其他的WRKY成员同源性较低,推测由263个氨基酸组成,其N端带1个可能的亮氨酸拉链结构.采用pET-29b载体原核表达了OsWRKY89和其3个N端缺失的衍生物,并采用Ni柱的方法对表达的蛋白进行了纯化.凝胶阻滞结果表明,OsWRKY89能与含顺式元件W盒的DNA序列特异结合,而缺失N端亮氨酸拉链的OsWRKY89与该元件的结合能力显著下降.酵母双杂交结果表明,亮氨酸拉链样结构参与OsWRKY89蛋白的同源相互作用.进一步缺失OsWRKY89至部分WRKY结构域导致表达的蛋白完全丧失与上述元件的结合活性,表明WRKY结构域是DNA结合中不可缺少的.这些结果说明亮氨酸拉链结构在蛋白质与蛋白质互作和DNA与蛋白质中起重要作用.
WRKY protein is a class of transcriptional regulators involved in plant biological and abiotic stress response, metabolism, development, etc. WRKY89 has low homology with other WRKY members, and is predicted to consist of 263 amino acids with 1 N-terminal Possible leucine zipper structure. OsWRKY89 and its three N-terminal deletion derivatives were prokaryoticly expressed in pET-29b vector and the expressed protein was purified by Ni column.The results of gel blocking showed that OsWRKY89 Could specifically bind to the DNA sequence containing the cis-element W box, whereas the ability of OsWRKY89 lacking the N-terminal leucine zipper significantly decreased its binding ability to this element.The yeast two-hybrid assay showed that the leucine zipper-like structure is involved in the OsWRKY89 protein Source interaction.The further deletion of OsWRKY89 to partial WRKY domain resulted in complete loss of the binding activity of the expressed protein with the above-mentioned elements, indicating that the WRKY domain is indispensable in DNA binding.These results indicate that the structure of leucine zipper is very important in the interaction between protein and protein It plays an important role in DNA and protein.