三种单子叶植物叶组织中GWSF诱导转录特性

Transcriptional Properties of GWSF-induced in Leaf Tissue of Three Monocotyledonous Plants

理想病原诱导型启动子的应用在植物抗病基因工程中非常重要。本研究以高粱、玉米和小麦为材料,检测人工病原诱导型启动子GWSF在单子叶植物叶组织中的诱导转录特性,寻找理想病原诱导型启动子。用GWSF替代pBI121中调控gus基因的CaMV35S启动子构建重组质粒,导入农杆菌(Agrobacterium tumefaciens) GV3101;通过农杆菌渗入法转化高粱、玉米和小麦12 h后,用水杨酸,青枯菌,疫霉菌孢子诱导处理12 h,本底表达用无菌水处理12 h;最后用GUS染色法和实时荧光定量PCR (real-time quantitative PCR, qPCR)检测GWSF的转录特性。GUS染色结果显示:GWSF在三种植物中都具有本底低、受水杨酸,青枯菌,疫霉菌孢子诱导的特性。设CaMV35S的转录活性为1,qPCR结果为:GWSF在高粱中本底水平为0.67,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性明显升高,分别为:2.53、0.87、7.33;玉米中本底水平为0.11,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性分别为:1.92、0.19、2.06;小麦中本底水平为0.13,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性分别为:0.69、0.45、1.16。结果表明,GWSF在三种单子叶植物叶组织中具有本底低、诱导因子广、诱导活性高的特性,是较理想的人工病原诱导型启动子,可应用于单子叶植物转基因抗病育种。

The application of ideal pathogen inducible promoter is very important in plant disease resistance genetic engineering. In this study, sorghum, maize and wheat were used as materials to evaluate the induced transcriptional properties of GWSF promoter in monocotyledonous leaf tissues, and to find the ideal pathogen-inducible promoter. GWSF was used to replace the CaMV35 S promoter which regulated gus gene in the pBI121 in order to construct recombinant plasmid. Then, the recombinant plasmid was introduced into Agrobacterium tumefaciensus GV3101. The sorghum, maize and wheat were transformed by agroinfiltration for 12 hours. Then, the salicylic acid, Ralstonia solanacearum and Phytophthora spores were induced for 12 hours, and the background expression was treated with sterile water for 12 hours. Finally, the transcriptional properties of GWSF were evaluated by GUS staining and real-time quantitative quantitative(qPCR). GUS staining showed that GWSF had the properties of low background and induction by salicylic acid, Ralstonia solanacearum and Phytophthora spores. With the transcriptional activity of the CaMV35 S be normalized to one, the qPCR results showed that the background level of GWSF in sorghum was 0.67, and the relative transcriptal activity significantly increased to 2.53, 0.87, 7.33 after being induced by salicylic acid, Ralstonia solanacearum and Phytophthora spores, respectively. Meanwhile, the background level of GWSF in maize was 0.11, and relative transcriptal activity were 1.92, 0.19 and 2.06. And the background level was 0.13 and relative transcriptal activity were 0.69, 0.45 and 1.16 in wheat. The results indicated that GWSF had the characteristics of low background, wide inducing factors and high induced activity in the leaf tissues of three monocotyledons. It might be an ideal promoter for artificial pathogen induction and could be apply to transgenic disease resistance breeding in monocotyledonous plants.