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WRKY蛋白是植物中一类重要的转录因子,不仅参与植物生长发育的调控,还参与植物对各种生物和非生物胁迫的响应。本研究从水稻日本晴中分离OsWRKY7基因的编码序列(CDS),并克隆其启动子序列进行表达研究。首先通过实时定量PCR的方法检测不同组织中OsWRKY7基因的相对表达量,结果表明,OsWRKY7在叶片中的表达水平较高,且开花期的剑叶中表达量高于7d苗龄的幼叶。进一步将OsWRKY7启动子与GUS报告基因融合,构建了植物表达载体pOsWRKY7-GUS,并将此载体转化日本晴。转基因植株不同组织染色分析结果显示,该启动子在植株的主根尖、叶片、颖壳中有GUS活性,其中叶片上可见全叶范围分布的大量蓝色斑点,这些染色结果与实时定量PCR的结果一致。进一步的接菌和激素处理还显示,OsWRKY7启动子在根和叶中的表达均受水稻白叶枯病菌[Xanthomonas oryzae pv.Oryzae(Xoo)]P10生理小种侵染的诱导,同时还受外源施加的细胞分裂素和生长素诱导,而水杨酸则会抑制其在根和叶中的表达。此外,我们还将OsWRKY7基因的CDS序列分别与绿色荧光蛋白和酵母GAL4的DNA结合域融合,对该基因进行水稻茎原生质体亚细胞定位分析和酵母自激活检验,结果显示该基因定位于细胞核中并具有转录自激活活性。上述结果表明OsWRKY7具有明显的转录激活因子特征,其很可能参与了水稻对白叶枯菌的防御反应以及对多种激素信号的转导过程。
WRKY protein is a kind of important transcription factor in plants. It not only participates in the regulation of plant growth and development, but also participates in plant response to various biological and abiotic stresses. In this study, the coding sequence (CDS) of OsWRKY7 gene was isolated from rice Nipponbare and cloned its promoter sequence for expression study. First, the relative expression level of OsWRKY7 in different tissues was detected by real-time quantitative PCR. The results showed that the expression level of OsWRKY7 in leaves was higher, and the expression level of OsWRKY7 in leaves was higher than that of young leaves at 7 days. Further, the OsWRKY7 promoter was fused with the GUS reporter gene to construct the plant expression vector pOsWRKY7-GUS, and the vector was transformed into Nipponbare. The staining results of different tissues of transgenic plants showed that the promoter had GUS activity in the main apices, leaves and glumes of the plants, and a large number of blue spots distributed in the whole leaves were visible on the leaves. The results of these staining were in good agreement with the results of real-time quantitative PCR Consistent. Further inoculation and hormone treatment also showed that the expression of the OsWRKY7 promoter in roots and leaves was induced by the inoculum of Xanthomonas oryzae pv. Oryzae (Xoo)] P10, Cytokinin and auxin were applied by the source, whereas salicylic acid inhibited its expression in roots and leaves. In addition, we also fused the CDS sequence of OsWRKY7 gene with the DNA binding domain of GFP and yeast GAL4. The gene was subcloned into rice stem protoplasts and yeast self-activation assay. The results showed that the gene was located in the nucleus And has transcriptional self-activation activity. The above results indicate that OsWRKY7 has obvious characteristics of transcriptional activator, which is likely to be involved in the defense response against rice bacterial blight and the transduction of various hormone signals.