水稻反温敏雄性不育基因RTMS3的功能及其作用机理研究

Reverse thermo-sensitive male sterility is an important genetic resource for breeding two-line hybrid rice. The mode of its fertility conversion is opposite to that of thermo-sensitive male sterility, behaving as fertile under high temperature and sterile under low temperature. We obtained two reverse thermo-sensitive male sterile mutants with similar phenotypes. Genetic analysis and gene mapping showed that the two mutants were two different allelic mutations of the nuclear gene RTMS3. The results of bulked segregant sequencing analysis and genetic complementary experiments confirmed that RTMS3 protein is probably a key enzyme involved in the metabolism of branched amino acids. It is preliminarily speculated that RTMS3 protein probably interacts with thermo-sensitive factors to regulate the amino acid content and its metabolites during spikelet development, so as to regulate anther and pollen development. Based on these results, the present project aims to detect the genes related to rice fertility, screen the proteins interacting with RTMS3, and analyze the causes of fertility changes in rtms3 mutant due to different temperature conditions, by comprehensively using the related techniques and methods of genetics, proteomics and genomics, so as to elucidate the function and mechanism of RTMS3 gene in regulating the fertility of rice. The results of this study will provide new insights into the molecular regulation mechanism of male sterility in rice, and lay the foundation for widening the application of two-line hybrid rice in the future.

反温敏雄性不育是培育两系杂交水稻的重要遗传资源，其育性转换模式与温敏雄性不育相反，表现为高温可育、低温不育。我们获得了2种表型相似的水稻反温敏雄性不育突变体。遗传分析与定位结果表明，这两种突变体是核基因RTMS3的不同等位突变。利用混合分离测序分析和遗传互补实验验证，确定RTMS3蛋白可能是一种与分支氨基酸代谢有关的酶。初步推测，RTMS3蛋白可能与温度敏感因子互作，调节水稻小穗发育过程中的氨基酸含量及其代谢产物，从而影响花药和花粉的发育。在此基础上，本项目拟综合利用遗传学、蛋白组学、基因组学等相关的技术、方法，检测与水稻育性相关的基因，筛选与RTMS3互作的蛋白质，分析不同温度条件引起rtms3突变体育性改变的原因，阐明RTMS3基因调节水稻育性的功能与作用机制，为揭示水稻雄性不育的分子调控机制提供新的见解，也为利用该基因拓宽两系杂交水稻的应用途径奠定基础。

反温敏雄性不育是培育两系杂交水稻的重要遗传资源，但目前有关基因的发掘、利用与机理研究尚未取得重要进展。本课题组前期鉴定了2种具有反温敏雄性不育特性的水稻等位突变体，确定了该性状由编码一种酰基辅酶A脱氢酶的RTMS3基因突变引起。在此基础上，本项目综合采用遗传学、蛋白组学、基因组学等有关的技术方法，深入研究了RTMS3基因及其调节的2个基因TSD1/SPD1和OsLUX/LJP1的功能和机制，取得了以下重要进展：(1)明确了低温影响rtms3花药绒毡层的形成，是导致其花粉败育的主要原；而孕穗期30-32℃的日均温对突变体花粉育性的影响至关重要。(2)RTMS3基因在成熟的雄蕊和花粉粒中高表达，其编码的蛋白质定位于细胞核、线粒体和内质网中。(3)利用RNA-seq方法，筛选到487个受RTMS3调节的基因，其功能涉及ROS的积累与降解。(4)利用酵母双杂交、亲和纯化-质谱分析技术，鉴定了包括OsCAT在内的8个RTMS3的互作蛋白（已利用多种方法进行验证），而OsCAT的功能涉及ROS水平的调节。(5)综合上述结果，提出了水稻花药发育和花粉形成过程中，RTMS3通过调节ROS的动态平衡，促进绒毡层形成、提高花粉活力，从而影响水稻育性的分子机制。在rtms3的花粉发育过程中，降低温度增加了突变体花药的ROS积累、引起育性下降，而提高温度则减少其ROS的积累、导致育性提高甚至完全恢复。(6)TSD1/SPD1编码一种FHY3/FAR1转录因子，通过直接调节水稻YABBY1和YABBY3的转录以及与YABBY1、3、4和5的物理相互作用，增强小穗适应高环境温度的能力。(7)OsLUX/LJP1是水稻夜晚合体的关键组分，与OsELF4a和OsELF3a结合形成三聚体，通过调节生物钟和光周期调控途径有关基因的表达，促进水开花。这些研究结果，为全面揭示水稻反温敏雄性不育的调控机制、小穗适应高环境温度以及光周期开花调控等，提供了新的见解，也为利用这些基因改良水稻奠定了基础。.已在“Plant Physiol.”和“Front. Plant Sci.”上发表论文2篇，获得授权专利2项，已毕业博士1名、硕士1名，在读硕士生4名。另有一篇论文拟于近期提交 “Plant Cell”。