VvCOP1介导高温抑制葡萄果实中花色苷合成的分子机理

Anthocyanins are important color compounds in grape berry and wine. High ambient temperature can inhibit synthesis of anthocyanins during grape ripening. The E3 ubiquitin ligase COP1 is a key negative regulator that represses light signaling such as synthesis of anthocyanins in darkness by targeting transcription factors involved in the light response for degradation. Here, we will demonstrate that COP1 also acts as an ambient temperature signaling to regulate anthocyanins accumulation, especially for the crops those grow under the high ambient temperature. In our previous study, we showed that overexpression of VvCOP1 in Arabidopsis cop1 mutant background can rescue the phenotype and showed less accumulation of anthocyanins. Herein, we intends to analyze the function of VvCOP1 negatively regulated the anthocyanins biotechnology, verify the effect of high ambient temperature on transition of VvCOP1 from nucleus to cytoplasm, .identify the interaction between VvCOP1 and transcription factors regulated positively anthocyanins biotechnology, such as VvHY5, VvMYBA1、VvMYBA2, analyze their degradation via ubiquitination VvCOP1, by using the virus induced gene silencing, bimolecular fluorescence complementation, genetic transformation of grape callus, et al. This program aim ultimately to illuminate the molecular mechanism of VvCOP1 mediated to regulated high ambient temperature to inhibit the anthocyanins accumulation in grape berry during reopening, and provide the theoretical basis for the molecular orientation reconstruction of grape genetics and molecular breeding heat-resisting grape variety.

花色苷是葡萄果实中最主要的呈色物质，葡萄成熟过程中高温会抑制其合成。作为E3泛素连接酶，COP1是植物光信号通路中的核心抑制因子，研究显示COP1也可能参与温度对植物花色苷的合成调控。基于此我们将葡萄COP1转入拟南芥，发现其花色苷合成能力降低。在此基础上，本研究拟进一步将VvCOP1在红花烟草中过表达，并利用病毒诱导的基因沉默技术瞬时转化葡萄果实，明确VvCOP1基因对花色苷形成的负调控作用；对拟南芥稳定转基因植株以及葡萄原生质体瞬时转化材料进行高温处理，验证高温能抑制光照下VvCOP1蛋白从细胞核向细胞质的转移而发挥生物功能；利用酵母双杂交、BiFC等技术研究VvCOP1与VvHY5等花色苷合成正调控因子的互作关系，分析高温对VvCOP1泛素化降解VvHY5等蛋白的影响，最终阐明VvCOP1介导高温抑制葡萄果实花色苷积累的分子机理，进而为分子选育优质耐热葡萄新品种提供研究基础。

生长季高温炎热是我国西部娶错葡萄产区影响葡萄生长发育的重要环境因子，高温可显著抑制葡萄果实呈色物质花色苷的积累。本项目中，通过葡萄果皮愈伤组织转化，获得了VvCOP1过表达和RNAi功能缺失材料，验证了VvCOP1在葡萄中负调控花色苷合成的生物学功能。通过不同温度处理，我们发现随着温度升高，花色苷含量减少，但在VvCOP1功能缺失突变材料中减少程度明显降低。通过Western-blotting检测VvCOP1蛋白在高温下的含量，发现细胞核中VvCOP1蛋白量随着温度升高而增加，揭示了高温促进光下VvCOP1蛋白在细胞核中的积累。为了进一步探明VvCOP1介导高温调控葡萄花色苷合成的分子机制，我们首先通过酵母双杂交（Y2H），双分子荧光互补实验证明了VvCOP1与VvHY5互作，进一步验证了VvCOP1可泛素化VvHY5蛋白促使其经26S蛋白酶体途径降解。对VvCOP1转基因愈伤材料进行高温处理后，检测到VvHY5蛋白含量减少，且其减少趋势和细胞核中VvCOP1蛋白的积累成相反的趋势。由此可知高温抑制了葡萄COP1由细胞核向细胞质的转移，使其在细胞核中得以积累，积累的COP1蛋白与花色苷合成转录调控因子VvHY5互作促进其泛素化降解，进而削弱了葡萄果实中花色苷的合成与积累。本研究的执行将为我国西部优质葡萄生产提供重要理论支撑。