miR172及其靶基因调控甘蓝型油菜种皮色泽的功能研究

Yellow seed character is one of the important objectives in Brassica napus breeding. However, researches on miRNA regulating biosynthesis and accumulation of seed coat pigments have never been reported in rapeseed. Previously, we screened a number of differentially expressed miRNAs between yellow and black-seeded rapeseeds. We found miR172 was up-regulated in yellow seeds, and its target genes (e.g. AP2) were down-regulated in yellow seeds, indicating miR172 and the target genes might be related to seed coat pigmentation. In this research, we will perform functional verification of miR172 and the target genes, as well as the DEGs related to flavonoid biosynthesis that were screened by RNA-seq. Supporting with the variation of flavonoid contents, expressional pattern of target genes and flavonoid biosynthesis related genes, we will acknowledge the effects of miR172, target genes and candidate DEGs on pigment biosynthesis and accumulation. The downstream genes regulated by target genes will be analyzed using glucocorticoid receptor system, and the binding sites for AP2 will be confirmed with ChIP analysis. The interaction proteins of target genes will be screened using yeast two-hybrid system, and then confirmed with BiFC and Co-IP analysis. This work will decipher the mechanism of miR172 and its target genes in regulating genes participated in flavonoid biosynthesis, and explain the molecular mechanism of yellow seed character in rapeseed at the transcriptional and protein level.

黄籽性状是甘蓝型油菜遗传育种的重要目标之一，但关于miRNA调控油菜种皮色素合成和积累的研究还未见报道。本项目基于黄/褐籽甘蓝型油菜种子sRNA-seq筛选到大量差异表达miRNA，其中miR172在黄籽中上调表达，其靶基因AP2等在黄籽中下调表达，推测miR172及其靶基因可能与种皮色泽形成有关。本研究拟通过miR172及其靶基因的功能验证，结合RNA-seq筛选的与类黄酮合成相关候选DEG的功能验证，通过类黄酮类物质的含量变化、靶基因和类黄酮合成途径基因的表达分析，了解miR172及其靶基因、候选DEG对色素合成及积累的影响。通过糖皮质激素受体系统分析靶基因调控的下游基因，ChIP实验分析AP2等结合下游基因的靶位点，结合酵母双杂交筛选、BiFC和Co-IP验证靶基因的互作蛋白，解析miR172及其靶基因对类黄酮合成途径基因的调控机制，从转录和蛋白水平了解油菜黄籽性状形成的分子机制。

本研究通过黄、褐籽甘蓝型油菜的差异表达miRNA（miR172）和差异表达基因（BnTT2）的功能研究，表明miR172可以通过调控其靶基因AP2、TOE1/2的表达，抑制甘蓝型油菜下胚轴中类黄酮物质的积累；且过表达其靶基因可以促进低温条件下油菜下胚轴中类黄酮类物质的积累。我们发现BnTT2与拟南芥TT2的功能较为保守，且2个同源拷贝存在功能冗余，仅当2个拷贝均纯合突变时，油菜种子出现黄籽表型。组织切片和化学染色表明BnTT2突变体中原花青素的含量显著降低，表明BnTT2突变影响了种子中原花青素的积累，最终导致种皮颜色的变异。对多酚类物质的定性和定量分析发现BnTT2突变后种子中多酚类物质含量下降，其中异鼠李素和表儿茶素的含量显著降低，这两类物质的含量变化可能与黄籽性状的形成相关。RNA-seq结果显示BnTT2突变抑制了苯丙烷和类黄酮路径的基因表达。通过酵母双杂交、双分子荧光互补以及双荧光素酶报告实验，初步证明BnTT2与BnTT8、BnTTG1能够形成MBW复合体，通过直接结合下游靶基因的启动子从而调控苯丙烷和类黄酮路径的基因表达。此外，基于BnTT2突变体的RNA-seq分析，我们发现R3-MYB基因（BnCPC）在突变体中下调表达，且酵母双杂交、双分子荧光互补实验证明BnTT2可与BnCPC互作。本研究发现过表达BnCPC可以显著提高甘蓝型油菜种子中不溶性原花青素和可溶性原花青素的含量，抑制正常生长和低温诱导条件下油菜幼苗子叶和下胚轴中的花青素积累。本研究证明miR172、BnTT2和BnCPC可以调控甘蓝型油菜中类黄酮类物质的合成和积累，为油菜的品质性状改良奠定了基础。