RsMYBL2在萝卜肉质根花青素积累过程中的功能及表达调控研究

The anthocyanins are the pigments for the red color of radish fleshy taproot and play an important role in plant growth and human health. But the biosynthesis and regulation mechanism of anthocyanins in radish is not clear, which hinders the improvement of taproot color in breeding. The applicant isolated RsMYBL2 from the red radish taproot by comparative transcriptome analysis, and one amino acid mutation was found at C-terminal of RsMYBL2. Preliminary research indicates that transient over-expression of RsMYBL2 increase anthocyanin accumulation in radish taproot significantly, shows completely different function with its homologous gene in Arabidopsis. In his study, firstly, we will analysis the expression characteristics of RsMYBL2 by qRT-PCR and histochemical analysis with β-glucuronidase (GUS). Secondly, the terminal motif of RsMYBL2 will be changed by PCR method. The transient over-expression, knockout and functionally complement to Arabidopsis AtMYBL2 mutant study will be conducted to clarify the function of RsMYBL2 and the effects of amino acid mutation in C-terminal on its function. The target gene and its interacting bHLH protein of RsMYBL2 will be predicted by qRT-PCR analysis of transient expression radish taproot tissue and RNA-Seq analysis of transgenic Arabidopsis and control. The yeast one- and two-hybrid methods will be used to verify the interaction of RsMYBL2 with predicted target gene and bHLH protein. The studies will provide new insights into the better understanding of the coloring mechanism of the radish taproot and theoretical basis for molecular design breeding of red flesh radish cultivars.

花青素是红肉萝卜的呈色色素，对植物生长和人类健康均有重要价值。萝卜肉质根花青素合成调控机制不明确制约了根肉色的遗传改良。申请者通过比较转录组分析筛选出萝卜RsMYBL2基因，初步证明该基因正调控花青素合成，与拟南芥同源基因功能相反；发现其C-端关键抑制基序一个氨基酸发生变异。在此基础上，本研究一方面利用qRT-PCR和GUS组织化学染色技术研究RsMYBL2的时空表达特性及对不同处理的响应。另一方面，采用PCR方法进行RsMYBL2变异位点的人工回复突变，通过同源瞬时过表达和沉默、拟南芥互补实验，明确该基因的功能及突变对功能的影响；通过对瞬时表达组织qRT-PCR分析和过表达拟南芥转录组测序预测RsMYBL2靶基因及互作bHLH蛋白；通过酵母单杂交和双杂交进行验证，明确该基因的作用机制。本研究将为解析萝卜肉质根花青素合成调控机制奠定基础，并为其根肉色的分子设计育种提供技术支撑。

‘心里美’类型萝卜为我国特有的红色根肉品种资源，花青素是其呈色色素，对植物生长和人类健康均有重要价值。萝卜肉质根花青素合成调控机制不明确制约了根肉色的遗传改良。对‘心里美’萝卜进行转录组测序，通过WGCNA、比较转录组和共表达分析最终获得78个可能参与了‘心里美’萝卜花青素生物合成的基因，其中RsMYBL2是一个关键的核心基因。系统进化分析发现，萝卜MYBL2.1与拟南芥MYBL2亲缘关系比较远，说明该基因在进化过程中变异较大，可能由于序列的变异引起了功能上的改变。qRT-PCR分析表明，RsMYBL2主要在‘心里美’萝卜肉质根积累花青素的组织（根肉、红皮期的根皮）表达，1.5% NaCl、2%葡萄糖及2%果糖处理萝卜幼苗显著增加其根中RsMYBL2表达量，而20% PEG-6000和2%蔗糖对其表达无显著影响；构建了RsMYBL2启动子驱动的GUS表达载体，并转化拟南芥，通过GUS染色分析了其时空表达特性。构建RsMYBL2及其人工回复突变基因RsMYBL2M过表达载体，稳定转化拟南芥、瞬时转化萝卜子叶，发现RsMYBL2和RsMYBL2M均显著促进花青素的积累。酵母双杂交结果表明，RsMYBL2及RsMYBL2M均能够与bHLH转录因子RsTT8互作，可能组成MBW-RsMYBL2蛋白复合体正向调控萝卜花青素的生物合成。此外，在本基金的资助下，我们还鉴定出了调控‘心里美’萝卜皮色转变的关键基因；发现‘心里美’和其它类型萝卜启动子InDel变异，并据此开发了有效用于红肉萝卜品种资源的筛选和辅助选择育种的分子标记；QTL定位获得了控制萝卜紫皮性状的关键基因RsMYB1.1，并开发了分子标记。上述研究结果为解析萝卜肉质根花青素合成调控机制及其根肉色的分子设计育种提供了理论依据和技术支撑。