核转录因子NF-YB1调控番茄果实色泽形成的机制研究

Color character, determined by fruit peel and fresh, is an important aspect of quality of tomato (Solanum lycopersium) fruit. According to the content of Naringenin Chalcone, the color of peel can be divided into two types: yellow and colorless. Our previous research result showed that the NF-YB1 gene involves in tomato fruit color regulation. Suppressed the expression of NF-YB1 could transfer the red tomato to the pink tomato, and the fruit peel always kept colorless comparing the yellow peel of wild type fruit at red ripening stage. Gene expression analysis showed that the transcripts of CHS, CHS2 and CHI-like which involved in flavonoid biosynthesis pathway were obvious suppressed in NF-YB1 RNAi transgenic lines, and the expression level of these genes were positively related to the expression of NF-YB1 gene. At the same time, we found that the expression level of MYB12, an important transcription factor involved in flavonoid biosynthesis in tomato, was not corresponding change in NF-YB1 RNAi transgenic lines. Based on the previous research, we want to make clear which metabolisms products changing are response for the fruit color changing of NF-YB1 RNAi transgenic lines at first. And then, an Y2H assay will be performed to discovery the proteins which can interact with NF-YB1 to form the NF-Y complex. Furthermore, the downstream genes regulated by NF-Y complex will be found through ChIP-Seq method, and then the regulated genes which role on flavonoid biosynthetic pathway will be screened out by bioinformatics analysis. Finally, combining with the metabonomics and transcriptional data of transgenic lines, we want to clarify the molecules mechanism of NF-YB1 gene leads to tomato fruit peel color.

色泽是番茄果实品质的重要方面，由果皮和果肉颜色共同决定。根据柚皮素查尔酮含量的多少，番茄果皮分为黄色和无色两类。前期研究中，项目组通过转基因功能分析发现NF-YB1影响番茄果实的色泽形成，抑制该基因的表达导致红果番茄果实转变为粉色，果皮无色。基因表达分析结果表明，在NF-YB1-RNAi转基因株系中，CHS1、CHS2、CHI-like等黄酮类合成代谢途径中的关键基因的表达显著被抑制。本项目拟在前期工作的基础上，明确NF-YB1调控番茄果皮颜色的物质代谢基础，利用酵母双杂交等方法获得NF-YB1互作蛋白，解析调控番茄果皮类黄酮代谢的NF-Y复合体的组成及其亚基间的相互作用，应用ChIP-Seq技术发掘NF-Y复合体调控的下游基因，分析下游基因对黄酮类合成代谢途径的影响，并结合代谢组学和转录组学研究，最终阐明NF-YB1基因影响番茄果实果皮色泽形成的分子机制。

番茄果实色泽主要由果肉中的类胡萝卜色和果皮中的类黄酮类化合物决定。柚皮素查尔酮是番茄果实果皮中主要的显色物质，柚皮素查尔酮缺失会导致果皮无色，果实表现为粉色。SlMYB12能够调控番茄果实果皮中类黄酮类化合物的积累，是唯一已知的能够调控粉果发育的转录因子。本研究中，我们发现转录因子NF-Y复合体在调控番茄果实果皮类黄酮生物合成过程中发挥重要作用。同一分枝上的SlNF-YB8a，SlNF-YB8b和SlNF-YB8c三个NF-YB蛋白能够与处于同一分枝上的SlNF-YC1b、SlNF-YC1e和SlNF-YC9三个NF-YC蛋白两两互作，形成9种NF-YB-YC异源二聚体。其中，NF-YB8c-YC1e二聚体能够招募至少两个NF-YA蛋白形成稳定的NF-Y异源三聚复合体，并结合到SlCHS1启动子上游的CCAAT作用元件。同时抑制SlNF-YB8a，SlNF-YB8b和SlNF-YB8c的表达能够提高SlCHS1和SlCHS2位点的H3K27me3水平，进而明显的抑制SlCHS1和SlCHS2的表达，减少果皮中柚皮素查尔酮类代谢物的积累，最终导致番茄果皮无色，果实呈现为粉色。转录组分析及RT-PCR实验表明三干涉果实果皮中的SlMYB12的表达完全没有受到抑制。同时，蛋白互作实验表明NF-Y复合体和SlMYB12之间不存在蛋白互作关系。综上所述，本研究表明NF-Y复合体通过调节SlCHS1和SlCHS2位点的H3K27me3水平，进而影响SlMYB12激活其表达的能力，从而以一种“开关”的形式调控SlCHS1和SlCHS2的表达。本研究首次报道了NF-Y复合体在调控类黄酮生物合成方面的功能，并深度解析了粉果发育形成的分子机理，具有重要的应用价值。