SmBIC与SmCRY互作调控茄子花青素合成的分子机制研究

Solanum melongena (eggplant) is one of the few vegetables rich in anthocyanins. Light is key environmental factor in regulation of anthocyanin biosynthesis. However, we found that anthocyanin synthesis in some eggplant germplasm is not completely controlled by light, large amount of anthocyanin can be synthesized in dim light. But, the eggplant varieties, which most people like to eat belong to light-sensitive species, often show poor fruit coloring under low light, which seriously affects their quality. In the study of light-sensitive eggplant, we found the BIC protein which was involved in anthocyanin synthesis and interacted with CRY. But, it could not be detected in 10 samples of light-insensitive eggplant selected at random. So it is speculated that BIC may negatively regulate the synthesis of eggplant anthocyanins under low light. In order to clarify the regulation mechanism of the BIC protein involved in anthocyanin synthesis in eggplant, this project will identify the function of the BIC protein and its regulation on CRY by genetic transformation. And Western Blot will be used to reveal whether BIC could indirectly regulate HY5 and MYB1 and then affect the anthocyanin synthesis. Ultimately, through the comparative analysis of transcriptional, proteome and metabolic groups, the molecular network of BIC-regulated anthocyanin synthesis in eggplant will be constructed, and its regulatory mechanism will be clarified. The results of the project can further fill perfect the theoretical system of light-induced anthocyanin synthesis of eggplant, and provide new ideas and methods for molecular breeding of eggplant with weak light tolerance.

茄子是为数不多富含花青素的蔬菜，光是调控花青素合成的关键环境因子。但是，研究表明部分茄子种质的花青素合成不完全受光调控，在弱光下就可以合成大量花青素。然而，大多数市民喜食的茄子品种属于光敏感型，弱光常导致其果实着色不良，严重影响品质。我们在对光敏感型‘禾线茄’的进一步研究中，发现了参与花青素合成，并与CRY互作的BIC蛋白。但在随机抽检的10份非光敏茄子中检测不到BIC，推测其可能负调控弱光下茄子花青素的合成。为了明确其调控机理，本项目拟通过茄子遗传转化，鉴定BIC的功能及其对CRY的调控作用；利用Western杂交，进一步揭示BIC是否可间接调控HY5和MYB1，进而影响花青素的合成；最终通过转录组、蛋白组及代谢组水平的比较研究与分析，构建BIC调控茄子花青素合成的分子网络，阐明其调控机制。项目成果可进一步完善茄子光诱导花青素合成的理论体系，为耐弱光茄子的分子育种提供新思路、新方法。

光照是影响茄子花青素合成的主要环境因素，但植株密植与温室栽培等弱光条件常导致光敏感型茄子果实着色不良，严重影响茄果品质。本研究采用比较转录组研究方法，挖掘出一些参与光不敏感茄子花青素合成的调控因子，并研究了SmBIC1和SmBIC2对茄子花青素合成光敏性的调控机制。主要获得以下研究结果：.一、光敏感型茄子‘蓝山禾线’和光不敏感型茄子‘145’在光响应和花青素合成中存在显著差异，通过转录组比较分析，挖掘到一些参与光不敏感茄子花青素合成的基因。.对盛花期套袋，两周后开袋照光0h、0.5h、4h和8h的两个材料的果皮进行转录组测序和比较分析发现，花青素合成结构基因在光敏感茄子中，开袋后上调表达。根据结构基因的表达模式，挖掘到可能参与光不敏感茄子花青素合成的4个光信号转导因子SmBIC1、SmBIC2、SmSPA1和SmSPA2，以及22个转录因子。进一步通过转录因子靶基因预测、酵母单杂交和Luciferase实验验证发现，SmBIM1、SmAP2、SmHD、SmMYB94、SmMYB19、SmTT8、SmYABBY、SmTTG2和SmMYC2可以结合花青素合成结构基因的启动子并调控它们的表达，同时还构建了它们的互作网络。.二、通过茄子遗传转化和VIGS瞬时转化实验证明了SmBIC1和SmBIC2对茄子花青素合成的负调控功能，并进一步通过互作分析和蛋白降解实验证明，SmBIC1和SmBIC2可以通过抑制SmCRY2的光反应来调控茄子花青素合成的光敏性。.发现SmBICs可以结合SmCHS和SmF3H的光响应元件，并促进它们的表达。然而，在SmBICs沉默的茄子中，积累了更多的花青素，而过表达SmBIC1和SmBIC2的茄子和拟南芥对光更敏感，合成较少花青素，同时这些植株的下胚轴也显著伸长。QRT-PCR也表明，SmBIC1-OE和SmBIC2-OE转基因株系中下调表达的花青素结构基因，在SmBICs沉默的茄子中上调表达。此外，SmBIC1和SmBIC2过表达茄子的转录组测序分析也进一步证实，SmBICs的过表达显著抑制了光信号转导和花青素生物合成相关基因的表达。最终，互作分析和CRY2依赖于蓝光降解实验表明，SmBICs与光激发的SmCRY2相互作用抑制其光反应，从而抑制花青素生物合成相关基因的表达。

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