转录组学和代谢组学关联解析马铃薯块茎类黄酮生物合成的调控机制

Flavonoids are important secondary metabolites which are rich in pigmented potato tuber (Solanum Tuberosum L.). As natural antioxidants and pigments, they have been widely used in food, cosmetic industries, as well as pharmaceuticals due to their function of anti-cancer, cardioprotection and antioxidization. The main classes of the flavonoid pathway derivatives include flavonols, proanthocyanidins (PAs) and anthocyanins. 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 flavonoid biosynthesis in potato tuber. In this study, integrated transcriptomic and metabolomic analysis, 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 regulatory mechanism of flavonoid biosynthesis in potato tuber on four levels: (1) regulatory mechanism of transcription factors (TFs) on flavonoid biosynthetic genes; (2)interaction between MYB TFs and MBW complex; (3)interaction between MYB, bHLH and WD40 TFs at protein level; (4) obtain the model of the regulatory network of flavonoid biosynthesis in potato tuber. 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类黄酮。类黄酮不仅用于食品、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。类黄酮合成主要包括黄酮醇、原花青素、花色素苷等合成途径。我们已获得五个调控块茎花色素苷合成的MYB转录因子，其中三个为新发现的转录因子，并分析当MYB转录因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。关于块茎类黄酮合成的分子机制尚未见报道。本研究利用组学关联分析、Real-time PCR、Dual Luciferase Assay、酵母杂交及BiFC等技术从四个层次全面研究类黄酮生物合成的调控机制：（1）MYB、bHLH和WD40转录因子对结构基因的调控机理；（2）MYB与MBW复合体的调控关系；（3）MYB、bHLH和WD40转录因子在蛋白水平上的互作关系；（4）建立块茎类黄酮生物合成的调控模型。该项研究为实现人工调控马铃薯块茎类黄酮代谢产物和品种创新奠定基础。

彩色马铃薯块茎富含类黄酮。类黄酮不仅用于食品、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。类黄酮合成主要包括黄酮醇、原花青素、花色素苷等合成途径。关于块茎类黄酮合成的分子机制鲜有报道。本研究对三个颜色差异的马铃薯杂交子代在三个生育时期（块茎形成期、膨大期和成熟期）的薯肉（Y:白-浅黄-黄，R:白-浅黄-红，P:浅紫-紫-深紫）进行转录组和代谢组测序，通过转录组数据分析筛选出了和块茎类黄酮生物合成相关的结构基因和转录因子；基于筛选的转录因子，进一步从马铃薯全基因组水平鉴定出MYB-related、WD40、NF-Y和BBX转录因子家族，利用RNA-seq数据及qPCR验证，筛选出可能参与调控类黄酮代谢合成的关键转录因子；根据代谢组数据，共鉴定出72种黄酮类化合物并深入解析了块茎薯肉差异的类黄酮组分；利用加权基因网络共表达分析（WGCNA），确定了与类黄酮生物合成显著相关的5个基因共表达模块，在相关模块中筛选出了参与类黄酮生物合成的36个结构基因。更为重要的是在1个显著正相关的核心模块中获得了2个核心MYB转录因子—R2R3-MYB转录因子StMYB3和R3-MYB转录因子StMYBATV。通过烟草瞬时表达和稳定遗传转化实验发现StMYB3和StMYBATV均为转录抑制子，负向调控类黄酮合成途径中花色素苷的生物合成，通过酵母双杂交、双分子荧光互补技术BiFC发现StMYB3和StMYBATV均与花色素苷转录调控因子StbHLH1互作。综合以上结果说明在彩色块茎色泽积累过程中，转录激活子StAN1和StbHLH1协同正向调控块茎花色素苷生物合成，转录抑制子StMYB3和StMYBATV竞争性的与StbHLH1结合起反馈调节的作用，以避免花色素苷的过度积累。项目发表研究论文7篇（5篇SCI，2篇中文核心），中文核心期刊已接收1篇。培养博士研究生1名，硕士研究生2名。