RsMYB158启动子变异导致萝卜肉质根花青苷积累功能缺失的分子调控机制

Radish cv. Xinlimei is a unique variety in Northern China which is rich in anthocyanins with a wide variety of health benefits. A mutant with white flesh was found in the populations of Xinlimei. Our previous studies showed that the inheritance of white flesh was controled by a single recessive gene and a key gene RsMYB158 for anthocyanin accumulation was detected based on BSA-MutMap strategy. By using of sequncing and qRT-PCR analysis, we concluded that promoter mutations in RsMYB158 gene led to the anthocyanin deficiency of the mutant. In this project, we will further verify the function of the promoter in RsMYB158 gene, the key mutation site and its up-stream regulation factors by promoter function element analysis, Arabidopsis transgene and Yeast one-Hybrid. Furthermore Chromatin Immunoprecipitation (ChIP), Pull-Down and Yeast Two-Hybrid experiments will also be performed to explore the target gene in the biosynthesis of anthocyanin and the interaction proteins of RsMYB158. This project will finally elucidate the molecular mechanisms of anthocyanin deficiency induced by promoter mutations in RsMYB158 gene. These research findings may lay a foundation for clearing the molecular mechanisms of flesh color formation, and provide guidance for breeding high-nutrition varieties in radish.

心里美萝卜是我国北方特有的品种类型，其肉质根中富含花青苷类物质，具有很高的营养和保健价值。项目组在心里美萝卜中发现并分离了肉质根白色突变体，证明该性状受隐性单基因控制；基于混池重测序和MutMap技术克隆了控制萝卜花青苷积累关键基因RsMYB158；基于序列变异和表达谱分析推断RsMYB158启动子变异是导致该突变体花青苷积累功能缺失的原因。本项目拟借助遗传转化和酵母单杂交技术，鉴定RsMYB158启动子关键功能元件及其调控蛋白，并确证导致RsMYB158启动子功能缺失的关键突变位点。利用染色质免疫共沉淀、Pull-Down和酵母双杂交技术，筛选与RsMYB158特异作用的靶标花青苷生物合成基因及其互作蛋白因子，初步阐明RsMYB158启动子变异导致心里美萝卜肉质根色突变体花青苷积累功能缺失的分子调控机制。该研究将为深入研究萝卜肉质根色形成的分子机制，培育高营养、高品质萝卜新品种奠定基础。

心里美萝卜是我国特有的萝卜类型，肉质根中富含对人体有益的花青苷。但是在心里美萝卜品种选育和生产中，频繁出现的肉质根白色突变体严重影响了商品的纯度和品质。目前引起突变的机制尚不清楚。本研究利用QTL-seq结合转录组分析获得，控制心里美萝卜肉质根花青苷积累的关键基因RsMYB1。序列分析表明在野生型心里美（MTH01）和白肉突变体（JC01）中，该基因没有核苷酸序列的差异，并且发现在RsMYB1启动子区域存在7,372bp的CACTA类型转座子。进一步基因甲基化分析表明，CACTA转座子区域在MTH01和JC01材料中均有较高的甲基化水平，但在RsMYB1基因启动子区域甲基化水平明显高于野生材料。因此，我们认为与RsMYB1紧邻的高度甲基化的转座子，外溢到RsMYB1启动子区域，抑制了该基因的表达，阻断了花青苷的合成，造成白肉突变体。我们利用萝卜愈伤组织过表达RsMYB1基因和VIGS互补验证了基因的功能，去甲基化试剂5-azaC处理白肉突变体材料能够部分恢复花青苷的积累。本研究初步揭示了心里美萝卜肉质根花青苷积累和白肉突变体出现的分子机制。