跨膜转运蛋白MATE在柿原花色素合成代谢途径中的功能特性研究

Persimmon proanthocyanidin (known as PA) is rich in fruit, which exhibits important value in pharmacy, health care products, chemical engineering etc. It is significant to elucidating the pathway of proanthocyanidin biosynthesis for the development and utilization of Chinese local persimmon germplasm. The molecular mechanism of proanthocyanidins precursors transported into vacuole by membrane-assciated transporter protein is unclear. In this study, the content and structures of PA would be initially determined by HPLC -MS during the process of proanthocyanidin biosynthesis in persimmon fruits. Then to get the gene of multidrug and toxic compound extrusion (MATE) by homology-based cloning, and predict genes structure characteristics and function by bioinformatics analysis. Compare the expression pattern of MATE at different astringent stages, fruit parts in these three cultivars. Detect MATE subcellular localization in onion epidermal cells with transient expression. The gene function of transported proanthocyanidin precursors would be verified by yeast and E. coli functional complementation experiments, expression of persimmon MATE in transgenic Arabidopsis mutations tt12 plants. This research would contribute to fully illuminate the mechanism of proanthocyanidin biosynthesis in persimmon, and provide gene resources to breeding excellent persimmon varieties.

柿原花色素在医药、保健品、化工等方面有重要价值，了解柿原花色素合成代谢途径对开发和利用我国优良的特色柿种质具有重要意义，该代谢途径中在细胞质中合成的原花色素前体物质如何被转运进入液泡聚合成单宁的分子机制尚未知。本项目拟采用质谱法测定柿果实发育过程中原花色素成分和含量，通过同源克隆技术获取柿原花色素前体物质转运蛋白MATE基因全长，生物信息学分析基因结构和功能；比较该基因在不同脱涩类型柿果不同发育时期、不同组织中的表达模式；研究MATE基因亚细胞定位；通过酵母功能互补试验和大肠杆菌功能异源表达验证MATE基因转运原花色素前体物质的功能；将MATE基因转化拟南芥tt12突变体观测T2代种子种皮颜色及原花色素含量。从而阐明MATE基因在柿原花色素合成代谢途径中转运原花色素前体物质的调控机制。本项目将有助于全面揭示柿原花色素合成代谢途径的分子机制，为培育中国甜柿新品种奠定基因基础。

中国甜柿原花色素生物合成代谢途径是研究热点之一。该代谢途径的重要功能基因的克隆和功能鉴定有助于开发利用我国重要特色柿种质的基因资源。柿原花色素前体物质如何被转运蛋白转运到液泡的机理尚未明确。本项目以三种不同脱涩类型柿品种，‘鄂柿1号’，‘阳丰’，‘磨盘柿’为材料，筛选并克隆到与原花色素前体转运相关的3个MATE基因，DkMATE1-3；DkMATE1和DkMATE3亚细胞定位于液泡膜，DKMATE2定位于细胞质膜；qRT-PCR分析DkMATE1在果实发育前期大量表达，DkMATE2，DkMATE3均在果实发育后期大量表达。‘鄂柿1号’柿叶片瞬时表达DkMATE1验证了其对柿原花色素合成的促进作用，瞬时表达DkMATE3表明其对柿原花色素合成相关基因存在反馈调节，瞬时表达DkMAT2后单宁含量没有明显变化；启动子元件分析发现DkMATE1、DkMATE3启动子区域中存在多个MYB类蛋白的顺式作用元件，DkMATE1转录起始活性在0-900 bp处；免疫组织化学分析发现DkMATE1蛋白在液泡膜表达，并随着果实的发育，蛋白表达量逐渐降低，表达趋势和可溶性单宁含量变化相一致；拟南芥tt12突变体遗传转化DkMATE1，种子恢复了野生型拟南芥黄褐色种皮，表明该基因具有将原花青素前体物质转运至液泡的功能。以上结果明确MATE基因在柿原花色素合成代谢途径中转运原花色素前体物质的调控机制，为进一步揭示柿原花色素合成代谢途径的分子机制提供基础。本项目培养了5名研究生，相关结果已发表在Plant Physiology and Biochemistry（2016）, Scientific Reports（2017）杂志和《园艺学报》（2017）上。