转录因子VvPIF1调控葡萄果实类胡萝卜素代谢的分子机制

Carotenoids are important components of grape fruit quality and it has become one of the important breeding targets of grape that improving the carotenoid contents in fruits. Light is an important environmental factor affecting the carotenoid metabolism in grape berries and the transcriptional regulation of VvPSY is a key factor determining the carotenoid biosynthesis in grape berries. Based on the previous studies, we found that VvPIF1, a core transcription factor involved in the light signal transduction pathway, could be an upstream regulator of VvPSY; however, the gene function and the regulatory mechanism remain unclear. This project intends to take VvPIF1 as the research object. Firstly, we verified the binding of VvPIF1 to the promoter of VvPSY through yeast one-hybrid, electrophoretic mobility shift assay and double-luciferase assay. Then, we overexpressed VvPIF1 in tomato to demonstrated its biological function in the carotenoid metabolism of fruits. The metabolic pathways regulated by VvPIF1 and interacting proteins were further analyzed by RNA-seq and yeast two-hybrid. This research will uncover the molecular mechanism of VvPIF1 regulating the carotenoid metabolism in grape berries. The research findings will provide some important theoretical basis for improving the carotenoid contents in grape berries by scientific light manipulation and molecular breeding.

类胡萝卜素是葡萄果实品质的重要组分，提高果实类胡萝卜素含量已成为葡萄重要的育种目标之一。光照是影响葡萄果实类胡萝卜素代谢的重要环境因子，VvPSY基因的转录调控是葡萄果实类胡萝卜素合成的关键因素。前期研究中，申请人发现光信号转导途径中的核心转录因子VvPIF1可能是VvPSY上游调控因子，但是VvPIF1基因功能及其调控机制还未清晰。本项目拟以VvPIF1为研究对象，首先通过酵母单杂交、凝胶阻滞电泳和双荧光素酶实验，验证VvPIF1与VvPSY启动子的互作关系；然后通过在番茄中超量表达VvPIF1，明确VvPIF1在果实类胡萝卜素代谢中的生物学功能；进一步利用RNA-seq和酵母双杂交等技术，分析VvPIF1调控的代谢通路和互作蛋白。最终揭示VvPIF1调控葡萄果实类胡萝卜素代谢的分子机制，为采用科学的光照处理措施和分子育种方法提高葡萄果实类胡萝卜素含量提供重要的理论依据。

光敏色素互作因子（PIFs）在光信号介导的次生代谢过程中发挥着重要作用，但PIFs在葡萄果实类胡萝卜素合成中的作用尚不清楚。本研究通过鉴定葡萄基因组中PIFs成员，探究了VvPIF1在葡萄类胡萝卜素代谢中的功能。随着果实发育，葡萄果皮中VvPIF1表达与类胡萝卜素含量及类胡萝卜素合成关键限速酶基因-八氢番茄红素合酶基因（VvPSY1/2）表达量呈负相关。光照显著抑制VvPIF1表达，但诱导VvPSY1/2等类胡萝卜素合成基因的表达。VvPIF1蛋白定位于细胞核，与光受体VvphyB相互作用。过表达VvPIF1导致烟草叶片内源PIF1基因的表达水平下调，进而造成类胡萝卜素含量和PSY表达水平增加。VvPIF1过表达的葡萄叶片和番茄果实中类胡萝卜素含量显著降低，PSY等类胡萝卜素合成基因的表达水平下调。进一步生化实验证明，VvPIF1直接与VvPSY1/2启动子结合并抑制其转录活性。综上所述，VvPIF1通过抑制VvPSY表达，负调控葡萄果实类胡萝卜素生物合成。研究结果揭示了PIFs在葡萄类胡萝卜素调控网络中的作用和作用方式。