TrMYB1介导的MYB/bHLH/WD40复合体调控V型紫斑白三叶叶片花青素合成的分子机理

Anthocyanins are responsible for the red, purple, and blue colors of many fruits, leaves, and flower. In addition, anthocyanins act as the precursors of proanthocyanidins (PAs) biosynthesis pathway. PAs, also known as condensed tannins, contribute to important forage legumes traits including disease resistance and forage quality. Most R2R3-MYB type TFs play key roles in the regulation of plant secondary metabolism including anthocyanin biosynthetic pathway. Some MYB factors recruit the bHLH (basic helix-loop-helix) transcription factors and the WD-repeat protein to form the MYB/bHLH/WD complex, which mediates anthocyanin biosynthesis mainly by activating the expression of late anthocyanin biosynthetic genes, such as DFR (dihydroflavonol 4-reductase). Using the methods of NMR based metabolomics, cDNA-AFLP and RNA-seq combined with co-expression data analysis, 10 MYB transcription factors full length genes were isolated from leaf V-markings region of Trifoluim repens L. Based on yeast two hybrid, protoplast trans-activation assays and transgenic plants analysis, one key R2R3 type MYB transcription factor TrMYB1 (KP861922) which regulated anthocyanin biosynthesis through binding the promoter of key enzyme gene DFR was identified. Therefore, understanding the transcriptional regulation mechanism of TrMYB1 contributes to develop a balanced concentration of PAs of transgenic Trifoluim repens L or forage crops. In this proposal, the objectives are: (1) Isolation and identification of TrMYB1 interacted partners, such as TrbHLHs, TrWD40s and other regulators, through proteomics analysis, yeast two hybrid (Y2H) screening, in vitro pull-down and in vivo bimolecular fluorescence complementation (BiFC); (2) Identification the TrMYB1 binding DNA sequence based on RNA-seq, yeast one hybrid (Y1H) and electrophoresis mobility shift assay (EMSA); (3) Understanding the TrMYB1 interacted partners, such as TrbHLH and TrWD40, how to regulate the transcriptional activity of TrMYB1, through protoplast trans-activation assay and functional analysis of transgenic plants; (4) Exploring the mechanism of TrMYB1 mediated TrMYB1-TrbHLH-TrWD40 complex regulates anthocyanins or PAs biosynthesis. This could provide an excellent opportunity to develop new cultivars which show balanced PAs concentration and expected leaf color of Trifoluim genus or forage plants in the future.

花青素不仅影响植物叶、花和果实颜色，而且为缩合单宁的生物合成提供了前体物质。MYB是调控花青素和缩合单宁生物合成的关键转录因子，它与bHLH、WD40互作形成MBW复合体调控基因表达。本研究以自主选育的白三叶新品种‘V型紫斑白三叶’为材料，筛选出了调控花青素合成的关键R2R3-MYB类转录因子TrMYB1 (KP861922)。拟在此基础上，利用蛋白质组学、酵母双杂交和双分子荧光互补等策略分离、鉴定与TrMYB1互作的TrbHLH和TrWD40类或其它类型蛋白；采用转录组测序、酵母单杂交结合EMSA技术分析TrMYB1调控的下游靶基因，并鉴定TrMYB1蛋白结合的顺式作用元件；运用瞬时转录激活方法，同时结合转基因植株功能分析阐明TrMYB1介导的MBW复合体转录调控活性，揭示MBW复合体调控花青素生物合成的分子机理，为白三叶或其它豆科牧草品质改良与分子育种提供理论与技术支持。

花青素是植物苯丙氨酸合成通路的衍生物，不仅影响植物叶、花和果实颜色，还影响到作物的品质和抗逆。MYB类转录因子是调控植物苯丙氨酸代谢物的关键转录因子，但是其调控分子机制仍然不是很明确。本项目以自主选育的白三叶新品种‘V型紫斑白三叶’为材料开展系列研究：通过差异转录组学，分离鉴定了调控植物黄酮类物质（花青素，芦丁等）合成的一类关键R2R3-MYB类转录抑制子TrMYB1/4，FtMYB11/13/15，MYB3/4/7；转录共表达分析发现，这类转录因子和PAL、DFR、C4H等苯丙氨酸代谢途径中关键酶基因有共表达模式，继而通过酵母单杂交和ChIP-PCR得到了蛋白质-DNA结合验证；通过酵母双杂交筛选文库，鉴定了TrMYB1/4与TrbHLH01互作，FtMYB11与SAD2和JAZ1互作，FtMYB13/15和MYB3/4/7都与SAD2互作，进一步通过体外pull-down实验、双分子荧光互补实验和瞬时转录激活进行了验证；茉莉酸JA是诱导植物次级代谢产物合成的重要激素，TrMYB1/4和FtMYB11/13/15，MYB3/4/7转录水平都明显受到JA诱导，蛋白水平研究发现JA能够通过26S蛋白酶体诱导JAZ1、TrMYB1/4、FtMYB11/13/15蛋白的降解；TrMYB1/4有多个同源基因TrMYB2/3，这些基因分别在紫斑白三叶的叶、茎和根中表达，我们同时发现荞麦中FtMYB11/13/15也在不同组织中特异性表达，JA的调控作用也存在组织特异性；TrMYB1/4，FtMYB13/15和MYB3/4/7类转录因子在蛋白水平和转录水平协同调控苯丙氨酸代谢生物合成，对植物叶片或是花的颜色分子育种提供了理论基础。.以本项目为第一支持取得的结果如下：发表学术论文7篇，其中在国际主流的植物学和农学杂志发表SCI论文4篇，分别是New Phytologist, Plant Physiology, Plant Journal 和Journal of Experimental Botany；出版学术著作2部，Elsevier出版社出版的英文著作，累计60万字；申请专利4项，获批3项；培养博士后1名，博士研究生3名，硕士研究生3名；项目成果获得省部级奖励2项，分别是广东省科技进步二等奖（2016）和农业农村部神农中华农业科技奖三等奖（2019）。