AHO诱导miR172在植物抗病中的作用机制研究

3-acetonyl-3-hydroxyoxindole (AHO), induces plant disease resistance through salicylic acid pathway-mediated defense responses. However, the role of AHO in the molecular mechanism of plant disease resistance is not yet fully understood. Our previous research found that miR172 and c56968.g_c0 genes were significantly up-regulated expressed in AHO-treated tobacco plants, speculating that AHO can induce plant disease resistance through regulating miRNA expression. In this project, miR172 sequence and secondary structure will be analyzed by bioinformatics method, and GUS reporter gene is used to determine whether AHO induces miR172 expression. Subsequently, RT-qPCR method is used to detect expression of miR172 and c56968 genes after AHO treatment, and c56968.g_c0 protein expression is detected by western blot method. Thus, the regulating relationship between miR172 and c56968.g_c0 gene is determined. To determine whether functional miR172 confers resistance to TMV, the content of TMV coat protein in over-expression of miR172 plants is determined. Furthermore, AHO is applied to plants whose functional miR172 gene is silenced by siRNA to evaluate the resilience degree of plant disease resistance. The completion of this project is not only to reveal the mechanism of plant disease resistance from the miRNAs level, but also to provide a practical basis for the use of AHO to prevent plant diseases.

3-丙酮基-3-羟基羟吲哚（AHO）通过作用于水杨酸途径介导的防御反应而诱导植物抗病能力，但其诱导作用的分子机制尚不清楚。前期发现，miR172和c56968.g_c0基因在AHO处理后均表达升高，推测AHO通过调控miR172而诱导了植物抗病性。本项目拟利用生物信息学分析miR172的序列和二级结构，并用GUS报告基因确定miR172是否由AHO诱导表达；采用RT-qPCR和Western Blot方法，检测AHO诱导后miR172和c56968.g_c0基因的表达情况，探究miR172对c56968.g_c0基因的调控方式；测定过表达miR172植株内TMV外壳蛋白含量，明确miR172是否使植株产生抗病性；对miR172基因沉默植株施用AHO，分析其对植株抗病能力的回复程度。本项目的完成，不仅从miRNAs层面揭示植物抗病性产生的机制，而且为更好利用AHO防控植物病害提供实验依据。

3-丙酮基-3-羟基羟吲哚（AHO）通过作用于水杨酸途径介导的防御反应而诱导植物抗病能力，但其诱导作用的分子机制尚不清楚。该项目比对了miR172f和miR172g的前体及成熟体序列的同源性，发现虽然miR172f和miR172g前体序列的相似度只有64.75%，但它们的成熟序列完全一致，均由21个碱基组成；预测miR172f和miR172g前体序列的二级结构，发现miR172f和miR172g前体序列具有典型的茎环二级结构，均形成6个茎环，但位置不同，形成成熟体的位置均靠近5’端；分析了miR172f和miR172g基因上游启动子区顺式作用元件，发现miR172基因上游0.67 ～ 2.0 kb区域存在潜在的启动子，长度为1120～1330 bp，启动子区域含有类似TATA 盒、起始位点和CAAT框等核心启动子元件以及光响应元件、生物胁迫响应元件、生长素响应元件、激素响应元件以及机械损伤响应元件等其他响应元件；成功构建了由miR172f和miR172g启动子驱动的GUS报告质粒并利用GUS报告基因明确了miR172受AHO特异性诱导表达。AHO处理后烟草中miR172f和miR172g的表达分别升高了2.89倍和2.01倍，它们的靶基因c56968.g_c0的表达升高了3.3倍；把AP2-like基因的全长序列克隆到原核表达载体上诱导表达，但未获得目标蛋白；综合现有结果，推测miR172调控AP2-like基因发生在翻译水平。 获得了pre-miR172过表达转化植株并用RT-qPCR方法加以确证；对转化植株强制接毒后，TAS-ELISA检测发现过表达pre-miR172植株中TMV含量减少，RT-PCR和WB检测发现过表达pre-miR172植株中TMV CP的mRNA和蛋白表达量均减少。由此判断，过表达pre-miR172能抑制TMV基因表达，增强植株的抗病性。构建了2对miR172基因的互补RNA（siRNA）导入烟苗后获得了siRNA: miR172转化苗，但RT-qPCR检测发现转化苗中的siRNA未能抑制植株中miR172的表达。因此，转化苗强制接毒后CP蛋白的检测以及再次喷施AHO后植株抗病性回复实验有待后续完成。本项目的完成，不仅从miRNAs层面揭示植物抗病性产生的机制，而且为农作物的抗病育种工作提供理论基础。