水稻AP2/ERF转录因子基因OsERF096启动子克隆及活性分析
Cloning and activity analysis of promoter of an AP2/ERF transcription factor gene OsERF096 in rice
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摘要: 提高水稻耐冷性对保障水稻高产、稳产具有重要意义。AP2/ERF转录因子能够抵抗外界逆境胁迫，课题组前期研究发现水稻OsERF096基因显著受冷胁迫诱导表达。本研究克隆了OsERF096启动子序列(-1~-1 215 bp)，通过软件预测发现OsERF096启动子中含有多个与低温及激素响应相关的顺式作用元件。根据元件分布特征进行了5′端片段缺失设计，构建了GUS表达载体POsERF096-1215-GUS、POsERF096-1102-GUS、POsERF096-827-GUS、POsERF096-501-GUS，并转化拟南芥。通过GUS染色和GUS基因qRT-PCR检测发现，正常生长条件下，4个不同长度的启动子中POsERF096-1215启动子活性最高，POsERF096-1102最低。与前期qRT-PCR检测不同，冷处理下OsERF096启动子活性未发生显著变化。这些结果为后续OsERF096基因的表达调控及生物学功能研究奠定重要基础。Abstract:
Enhancing chilling resistance is of significance to high and stable yield in rice.AP2/ERF transcription factor can resist the cold stress.Our previous study found that rice OsERF096 gene expression was significantly induced by cold stress.In this study, we cloned the OsERF096 promoter (-1~-1 215 bp) and found several cis-elements related to cold and hormone responses.Based on the distribution of cis-elements in OsERF096 promoter, four 5′-truncated fragments were designed and cloned into the pCAMBIA3301 vector, resulting in POsERF096-1215-GUS, POsERF096-1102-GUS, POsERF096-827-GUS, POsERF096-501-GUS constructs, which were then transformed into Arabidopsis.By comparing the GUS staining and GUS gene expression analyzed by qRT-PCR, we found that under control conditions, among the four OsERF096 promoter fragments, POsERF096-1215 and POsERF096-1102 displayed the highest and lowest activity, respectively.Unexpectedly, no significant difference was observed in the activity of four OsERF096 promoter fragments under cold stress, which was contradictory to previous qRT-PCR result showing that OsERF096 expression was greatly induced by cold stress.The findings will facilitate further research on the regulatory mechanism of OsERF096 expression and biological function.
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