一个转录因子调控水稻温敏雄性不育

Thermo-sensitive genic male sterile lines not only can be used for two-line hybrid breeding in rice (Oryza sativa L.), but also are important materials for studying temperature perception in plants. However, the molecular mechanism of thermo-sensitive genic male sterility still remains understudied. Previously, we have cloned a thermo-sensitive genic male sterile gene tms5 that is used in more than 95% of two-line hybrid rice in China. A transcription factor gene TF21 is up regulated in thermo-sensitive genic male sterile lines with the tms5 gene, and it is predicted to be regulated by a small RNA-miRX. The overexpression of TF21 in Zhonghua11 led to its sterility at high temperatures. This project aims at uncovering the molecular mechanism of TF21 controlling thermo-sensitive genic male sterility in rice. Firstly, the pollen fertility, abortion period, and abortion characteristics of TF21 overexpression plants and mutants at different temperatures will be observed. Secondly, the identification of the interacting protein of TF21 and its functional validation will be carried out by using yeast two-hybrid and Co-IP systems. The target gene under transcriptional regulation of TF21 will be identified through transcriptome profiling, ChIP, EMSA, and genetic transformation. The regulatory role of miRX will also be confirmed. Finally, the TF21 alleles will be evaluated for their applicability in tolerance to extreme temperatures of rice. The understanding of the thermo-sensitive genic male sterility mechanism will facilitate improving the hybrid seed production safety of two-line hybrid rice. Current project will also likely provide a better understanding of the regulation network of temperature perception in plants, as well as the genetic base for developing rice varieties with better tolerance to extreme temperatures.

温敏不育系既可用于两系杂交育种，也是研究植物感应温度的重要材料。但是，温敏不育的分子机理是知之甚少的。我们之前已经克隆了我国95%以上两系杂交水稻使用的温敏不育基因tms5。转录因子TF21在tms5温敏不育系中表达量升高，并且预测可能受小分子RNA－miRX调控。中花11中过量表达TF21导致其高温不育。本项目拟开展TF21调控的水稻温敏不育分子机理的研究。首先观察TF21过表达植株和突变体在不同温度下的花粉育性、败育时期和败育特征。其次通过酵母双杂交和Co-IP鉴定TF21的互作蛋白并进行功能验证；通过转录组、ChIP、EMSA和转基因鉴定TF21转录调控的靶基因；确认miRX对TF21的调控。最后评估TF21等位基因在耐极端温度中的应用价值。通过对温敏不育机理的解析，将有助于提高两系杂交水稻的制种安全 性，可能丰富植物感应温度的调控网络，提高水稻对极端温度的耐受性。

水稻是全世界50%以上人口的主粮。杂交水稻可以提高10-20%的产量，水稻温敏不育系既是两系杂交水稻育种的关键，也是开展植物感应温度的优良材料。前期，我们发现TMS5编码RNase ZS1，通过降解受温度诱导表达的UbL40的mRNA控制水稻温敏不育。本项目首先初步阐明了RNase ZS1切割靶RNA的结构基础。RNase ZS1的酶活中心或者其他保守氨基酸的突变引起蛋白三维结构的改变，能够导致RNase ZS1酶活的缺失或者弱化。RNase ZS1以同源二聚体起作用，其酶活依赖于底物RNA的类似三叶草结构。此外，我们发现RNase ZS1对TF21 mRNA并没有明显的切割活性。但是，前期研究发现过量表达TF21将导致高温下水稻雄性不育。本项目进一步解析了TF21调控水稻温敏不育的分子机理。TF21在小穗中表达较高，并且主要在花药的S5~S9的花粉母细胞、绒毡层和中层中表达。亚细胞定位显示TF21在细胞核中起作用。过量表达和敲除TF21都导致水稻温敏不育，在高于30℃或低于20℃条件下完全不育。半薄切片和TUNEL实验都显示TF21过量表达和敲除植株在高温或低温下的花药绒毡层提前开始降解，但是降解结束时间又延后。进一步的研究发现TF21受小分子RNA，miRX的调控。miRX的过量表达植株温敏不育，而敲除植株完全不育。通过转录组分析，筛选到一个TF21的候选靶基因ET1。ET1过表达植株无论在高温还是低温下都具有类似于高温下TF21过表达植株的细胞学表型。总之，本项目揭示了TF21调控水稻温敏不育的分子机理。这些工作有利于认识植物对温度的感应机制和促进两系杂交水稻的育种。课题执行期间，作为第二完成人获大北农科技奖1项，申请专利4项，授权专利1项，发表标注本项目的SCI论文6篇（Molecular Plant, 2022; Plant Cell and Environment, 2022; Plants, 2022; Plant Physiology and Biochemistry, 2021; Functional Plant Biology, 2021; New Phytologist, 2020）。