OsmiR1858靶向OsHI1D调控水稻白叶枯病抗性的分子机制研究

Rice bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo) is one of the most devastating diseases worldwide. In plants, miRNAs have been shown to play important roles in development, and responses to biotic and abiotic stresses. However, it is still unclear whether and how OsmiRNAs are directly involved in regulation of the resistance to Xoo in rice. miR1858a was firstly identified in rice and its targets and biological function were not reported. In our previous studies, the real-time PCR results showed that OsmiR1858a was induced in several resistant genotypes by different Xoo strains, whereas it did not chang in susceptible ones. OsmiR1858a cleavage site within OsHI1D, encoding a hypothetical　protein, was precisely determined by using 5’-RACE, and the expression of OsHI1D was anti-correlated with OsmiR1858a. Transgenic plants overexpressing OsHI1D showed a significant deacrease in Xoo-resistance. The results indicated that OsmiR1858a may play an important role in Xoo resistance. In this study, the generated transgenic rice plants will be used to analyze expression patterns and biological function of OsmiR1858a after challenge by Xoo, OsmiR1858a-mediated targeted downregulation of gene OsHI1D, and expression patterns and biological function of OsHI1D in response to Xoo infection. Therefore, we are to determine the biological functions of both OsmiR1858a and OsHI1D in Xoo-resistance in rice. The results will not only be useful to understand the new mechanism of Xoo-resistance, but also provide new ideas and genetic resources for crop disease-resistant breeding.

白叶枯病(Xoo)是水稻最严重的病害之一。尚不清楚miRNA是否及如何调控水稻对Xoo的抗性，未见OsmiR1858靶基因及生物学功能相关报道。我们发现，OsmiR1858a表达在抗病品种受不同Xoo小种诱导，而在感病品种无显著变化；初步确定OsmiR1858a剪切降解OsHI1D抑制其表达；过表达OsHI1D使抗病性下降。表明OsmiR1858a在调控水稻Xoo抗性中起重要作用。本项目拟分析OsmiR1858a响应Xoo的表达及生物学功能，OsmiR1858a靶调控OsHI1D表达，OsHI1D响应Xoo的表达与功能。期望明确OsmiR1858a和OsHI1D在Xoo抗性中生物学功能。本项目即有助于发现水稻抗Xoo的新机制，也为提高水稻抗病性提供新思路和有价值的基因资源。

白叶枯病（Xoo）是水稻最严重的病害之一。尚不清楚miRNA如何调控水稻对Xoo的抗性，未见OsmiR1858靶基因及生物学功能相关报道。我们前期研究发现，OsmiR1858a表达在抗病品种受不同Xoo小种诱导，而在感病品种无显著变化，推测OsmiR1858a与水稻的白叶枯病抗性相关。本项目利用RLM-5’RACE方法确定了预测靶基因OsHI1D的剪切位点。通过农杆菌介导法成功获得了OsmiR1858a启动子-GUS超表达、pre-OsmiR1858a超表达、OsHI1D超表达、OsHI1D同义突变超表达和OsHI1D基因编辑的转基因阳性株系。OsmiR1858a超表达株系中靶基因OsHI1D的表达量显著低于野生型，OsHI1D同义突变（其OsmiR1858a剪切位点突变）的超表达株系叶片OsHI1D表达水平比野生型的显著上调。此外，烟草叶片共表达实验也表明，OsHI1D的荧光强度随着OsmiR1858a的增加而下降。这些结果进一步证实OsHI1D是OsmiR1858a的靶基因，OsmiR1858a通过剪切负调控OsHI1D的表达。对各转基因2个株系与野生型SH5的叶片分别接种白叶枯病菌P3、P6小种，病斑面积及细菌生长情况显示，OsmiR1858a超表达株系的抗病性与其表达量呈正相关性；靶基因OsHI1D超表达株系的抗病性比野生型显著下降，OsHI1D沉默株系的抗病性则比野生型的显著增强，即OsHI1D与水稻白叶枯病抗性呈负相关性。OsHI1D在3个器官中的表达量为茎>叶>根，Xoo侵染显著下调OsHI1D在茎、叶中的表达；亚细胞定位于质膜上。本项目首次确定了OsmiR1858a的生物学功能，也是少数涉及调控水稻白叶枯病抗性的OsmiRNA功能及机制的研究；获得的转基因材料和实验结果为深入解析OsmiR1858靶向OsHI1D调控Xoo抗性的分子机制，奠定了扎实基础。