水稻OsVHA-A基因参与类黄酮响应UV-B辐射胁迫代谢的互作机制

The enhanced UV-B radiation from atmospheric ozone layer depletion has seriously endangered the crop production. The previous studies have revealed that OsVHA-A mutant rice is extremely sensitive to UV-B radiation, and its flavonoids mechanism induced by UV-B radiation is also blocked. In order to study the involvement of OsVHA-A in flavonoids mechanism, the present study will screen the proteins that interacted with OsVHA-A, especially the functional proteins involved in flavonoids metabolism, in the tisues of rice plant responding to UV-B radiation throught the yeast two hybrid techonology, and then the interaction relationships between proteins will be tested by GST-pull down and BiFC techniques. Meanwhile, the western blotting and real-time PCR technologies will also be performed to investigate the expression profiles of OsVHA-A and its interacting proteins for rice plant responding to UV-B radiation. Subsequently, the differential flavonoids mechanism will be analysed in the two rice genotypes under the UV-B radiation stress. In addition, the interaction relationships of OsVHA-A with its interacting proteins, and the flavonoids component of various tisues will be tested for the OsVHA-A over-expression rice plant which suffered from supplementary UV-B radiation. The expected results will provide theoretical basis and technological support for enhancing rice UV-B tolerance by cultivation measures and molecular improvement.

大气臭氧层破坏导致地表UV-B辐射增强，严重影响作物生产。前期研究发现OsVHA-A基因突变水稻对UV-B辐射极其敏感，且响应UV-B辐射的类黄酮代谢受到抑制。本研究拟通过酵母双杂交技术，筛选与OsVHA-A蛋白存在互作的类黄酮代谢调控蛋白，并采用Pull-down和BiFC技术验证蛋白间的互作关系，同时，采用Western blotting和qRT-PCR技术，检测OsVHA-A与关键互作蛋白响应UV-B辐射胁迫的表达模式，并分析不同基因型水稻响应UV-B辐射的类黄酮代谢差异。此外，构建OsVHA-A基因超表达转基因水稻株系，检验OsVHA-A蛋白与类黄酮代谢互作蛋白在超表达植株中响应UV-B辐射的表达模式、以及类黄酮代谢变化，以期阐明OsVHA-A基因参与水稻类黄酮响应UV-B辐射胁迫代谢的作用机制，预期研究结果可为耐UV-B辐射水稻的栽培调控和分子改良提供理论与技术支持。

大气臭氧层破坏导致地表UV-B辐射增强，严重影响农作物生产。本项目以对UV-B辐射敏感的突变体水稻及其野生型为实验材料，调查其在UV-B胁迫下的色素物质和光合代谢指标、以及产量形成变化，并通过RNA-seq技术分析了2个基因型水稻在UV-B辐射胁迫下的转录组代谢情况，同时分析研究OsVHA-A蛋白的互作蛋白，结果表明，突变体水稻在UV-B辐射胁迫下，色素类物质合成减弱，叶片对光能利用下降，光合作用速率减缓，水稻籽粒的千粒重和结实率等产量指标变差。转录组实验结果表明，突变体水稻在UV-B辐射胁迫下叶片细胞的次生代谢减弱，光合器官的碳固定能力和光合作用代谢下调，叶片衰老代谢的转录因子表达增强，生长素、细胞分裂素、茉莉酸甲酯等促进生长的激素代谢减弱，脱落酸等促进衰老脱落的激素代谢增强，衰老叶片中的抗氧化代谢和碳水化合物代谢变弱，并导致UV-B敏感突变体水稻叶片的衰老加剧，黄酮类物质等次生代谢关键基因表达下调。此外，筛选到一个与OsVHA-A互作的关键蛋白，蛋白ID编号为A2YM05_ORYSI，编码WAT1-related protein，含364个氨基酸，属于类EamA转运蛋白家族，具有跨膜转运活性，位于液泡膜。