MYB转录抑制子和激活子对马铃薯块茎花色素苷合成的共同调控机理研究

Anthocyanins are important secondary metabolites in pigmented potato tuber(Solanum Tuberosum L.). As natural pigments, they have been widely used in food, cosmetic industries, as well as pharmaceuticals due to their function of anti-cancer, cardioprotection and antioxidization. Five MYB anthocyanin activators have been obtained from previous study, three of them are new discovered activators. Previous study also showed that five MYB anthocyanin activators have different activating abilities, combined with expression analysis in potato tuber, results suggest that bHLH TFs are key co-regulators of anthocyanin biosynthesis, while the transcripts of MYB activator variants are well expressed, even in the absence of pigmentation. However, little is known on the underlying molecular mechanisms of MYB anthocyanin repressors and activators on anthocyanin biosynthesis in potato tuber. In this study, RNA-Seq, Real-time PCR, dual luciferase assay of transiently transformed tobacco leaves, yeast two-hybrid , yeast three-hybrid, BiFC assay will be employed to deeply elucidate the co-regulatory mechanism of MYB anthocyanin repressors and activators on anthocyanin biosynthesis in pigmented tetraploid potato cultivars on four levels: (1) co-regulatory mechanism of MYB anthocyanin repressors and activators on anthocyanin biosynthetic genes; (2)interaction between MYB repressors, activators and MBW complex; (3)interaction between MYB repressors, activators and bHLH TFs at protein level; (4) obtain the model of the anthocyanin gene regulation network in potato. This work will provide the genetic resources for breeding and developing a new field for utilization of potato.

彩色马铃薯块茎富含花色素苷。花色素苷不仅用于食品，化妆品等行业，更因其抗癌，抗氧化等作用，广泛应用于医药行业。我们已获得五个调控块茎花色素苷能力各不同的MYB转录激活子，其中三个为新发现的转录因子，并分析当MYB激活子在白色和彩色块茎中都高表达时，bHLH才是关键的转录因子。关于MYB抑制子和激活子共同调控块茎花色素苷合成的分子机制尚未见报道。本研究利用RNA-Seq、Real-time PCR、Dual Luciferase Assay、酵母杂交及BiFC等技术从四个层次研究MYB抑制子和激活子的共同调控机制：（1）MYB抑制子和激活子对结构基因的共同调控机理；（2） MYB抑制子和激活子与MBW复合体的调控关系；（3）MYB抑制子和激活子与bHLH转录因子在蛋白水平上的互作关系；（4）建立MYB抑制子和激活子的共同调控模型。该项目为实现人工调控马铃薯块茎花色素苷合成和品种创新奠定基础。

彩色马铃薯块茎富含花色素苷。花色素苷不仅应用于食品，化妆品等行业，更因其抗癌，抗氧化等作用，广泛应用于医药行业。MYB类转录因子调控花色素苷合成，对植物颜色的形成具有重要作用，但关于MYB抑制子和激活子共同调控块茎花色素苷合成的分子机制未见报道。本研究利用RNA-Seq、Real-time PCR、Dual Luciferase Assay、及BiFC等技术研究了MYB抑制子和激活子的共同调控机制：（1）利用生物信息学方法对马铃薯MYB转录因子家族进行了全基因组分析，并通过对白色和彩色马铃薯的RNA-seq转录组分析，筛选出了与花色苷生物合成相关的MYB转录因子。此外，在全基因组范围内筛选出了21个含有抑制功能EAR序列的StR2R3-MYB基因，并构建了这些MYB蛋白与其它马铃薯蛋白之间的互作网络；（2）对马铃薯块茎花色素苷合成相关R2R3 MYB 蛋白StAN1-R0，StAN1-R1和StAN1-R3的功能进一步进行验证，确定其正向调控烟草和马铃薯块茎花色素苷合成；（3）为深入了解MYB转录因子共同调控花色素苷的机制，对彩色品种进行了温度处理，结果发现高温下紫色薯肉维管束周围区域变为白色，通过RNA-seq数据分析和qPCR实验筛选出了白肉中高表达的关键转录抑制子StMYB44-1和StMYB44-2，两种抑制子蛋白都含有抑制相关的EAR序列，但在StMYB44-2中该序列发生变异导致其抑制能力显著低于StMYB44-1，它们不与bHLH形成转录复合体，可直接通过抑制二氢黄酮醇还原酶（DFR）启动子的活性来抑制烟草叶片中花色素苷的积累。这类抑制子的增强转录会下调转录激活子StAN1和StbHLH1及花色素苷结构基因的表达，导致花色素苷代谢途径分支受到抑制后，苯丙烷代谢途径通入绿原酸或木质素生物合成途径分支中。该项研究揭示了与已报道的花色素苷抑制子不同的抑制机理。