紫茄果实特异基因SmCOP1调控果皮花色苷生物合成的分子机理研究

Fruit color is one of the decisive factors in relation to the commercial value of eggplant. In addition, light is the most important environmental factor regulating the biosynthesis of anthocyanins in the fruit of purple eggplant. In previous studies, a fruit specific photomorphogenesis suppressors, SmCOP1 was cloned from the purple eggplant cultivar (sanyueqie). It was found that the overexpression of SmCOP1 can significantly inhibit the accumulation of anthocyanins in the fruit peel of purple eggplants. qRT-PCR analysis showed that the expression of anthocyanin biosynthetic genes at transcriptional level in the transgenic eggplant peels was significantly lower than that of the control materials, while expression of anthocyanin biosynthetic associated regulatory genes remain stable in transgenic eggplants. These results suggest that SmCOP1 may regulate the transcription of anthocyanin biosynthetic genes by regulating the stability of anthocyanin biosynthesis regulatory proteins. To test the hypothesis, we intend to use protein two-dimensional electrophoresis (2-DE) combined with mass spectrometry technology to screen proteins with differential expression between transgenic and control eggplants. Furthermore, interaction between candidates proteins and SmCOP1 will be verified by yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and immuno co precipitation (CoIP) in vivo and in vitro. Finally, all these results will facilitate the studies on revealing molecular mechanisms underlying the anthocyanin accumulation in fruit of purple eggplant controled by the light-regulated SmCOP1.

果皮中花色苷所呈现的色泽是影响茄子果实商品价值的决定性因素之一，而光照是影响紫茄果皮花色苷合成最重要的环境因素。在前期研究中，申请人从紫茄中克隆到一个果实特异表达的光形态建成基因SmCOP1，发现该基因过表达能够显著地抑制紫茄果皮花色苷的积累。qRT-PCR研究表明转基因茄子果皮中花色苷生物合成结构基因在转录水平的表达显著低于对照，而参与调控结构基因表达的转录因子在转录水平却未发生明显变化，这些结果表明SmCOP1可能通过调节花色苷合成调控蛋白的稳定性进而调控花色苷合成基因的转录表达。为验证该推测，本研究拟利用双向电泳（2-DE）结合质谱技术寻找差异表达蛋白，进一步通过酵母双杂交（Yeast two-hybrid），双分子荧光互补(BiFC)和免疫共沉淀(CoIP)从体内、体外分析SmCOP1与靶蛋白的互作，进而探明光信号通过SmCOP1调控茄子果实花色苷合成代谢的分子机理。

茄子是世界范围内的重要蔬菜，被联合国粮食及农业组织列为第四大蔬菜作物。2016年我国茄子生产达到317亿公斤，约占当年全球总产量的60%，可见茄子在我国蔬菜生产中的重要战略地位。色泽是影响消费者购买水果蔬菜的重要外观因素，而紫茄果实的色泽主要来源于花色苷类物质的合成与积累。花色苷不仅能够赋予植物器官靓丽的色泽和抗逆特性，还能够显著降低人类罹患心血管和肿瘤等疾病的风险。因此，培育富含花色苷的种质资源对茄子产业发展具有重要的意义。.作为光形态建成的核心调节因子，COP1参与了拟南芥子叶与下胚轴花色苷的积累调控，但COP1在果实发育中的调控功能尚不清楚。为了探索SmCOP1在果实发育和花色苷积累中的功能，该研究创制了目标基因特异沉默和过表达的转基因茄子株系。该研究着重以SmCOP1基因特异性沉默的转基因茄子为材料，探索了SmCOP1调控茄子果实花色苷合成积累的代谢与分子机理。通过比较野生材料和转基因材料的表型差异，研究发现SmCOP1的表达能够抑制茄子果实花色苷的积累。此外，SmCOP1还参与了茄子果实形态和种子发育的调控过程。该研究采用qRT-PCR、Western-blotting、酵母双杂交以及高效液相色谱等技术方法，取得了以下研究结果。（1）SmCOP1基因沉默能够显著增加花色苷在茄子果实和叶中的积累；（2）SmCOP1基因沉默能够显著增强花色苷合成结构基因在茄子果实中的表达；（3）SmMYB1能够有效激活花色苷在茄子中的合成积累；（4）SmCOP1基因沉默能够显著增强花色苷调节蛋白SmMYB1在茄子果实中的表达；（5）SmCOP1蛋白的WD40结构域和SmMYB1蛋白的R3结构域介导了两个生物大分子的互作。综合以上结果，研究证实光形态建成因子SmCOP1主要通过泛素化途径抑制花色苷激活蛋白SmMYB1的稳定性，最终实现对茄子果实花色苷生物合成的负向调节。该研究重点阐明了光信号传递核心因子SmCOP1参与茄子果实花色苷生物合成调控的分子机理，为全面阐明光信号调控果实花色苷积累的遗传分子机理奠定了坚实的基础。光调控茄子果实着色机理的阐明则能够有效指导耐弱光茄子种质资源的开发和新品种的培育。