茉莉酸诱导的FtbHLH06转录因子调控苦荞芦丁降解酶基因表达的分子机制研究

Rutin is the main flavonoid compound in Fagopyrum tataricum. Rutin exerts protection on human health due to its antioxidative, anti-inflammatory and anticarcinogenic effect and its ability to reduce fragility of blood vessels. In F. tataricum, rutin can be degraded into poorly water soluble compound, quercetin, by rutin degrading enzyme (FtRDE), resulting in the bitter taste. So far, the gene regulation of FtRDE is still unkown. The bHLH transcription factors play key roles in the regulation of plant secondary metabolism, especially in the jasmonates (JAs)-induced secondary metabolites biosynthesis. Interestingly, rutin and quercetin contents can be accumulated by JAs treatment in F. tataricum, demonstrating that JAs is involved in regulation of FtRDE gene expression. The key enzyme gene FtRDE3 can degrade rutin into quercetin, it was identified based on HPLC and Polyamide-TLC. The 600 bp promoter of FtRDE3 was used as bait for yeast-one-hybrid (Y1H) screening, the JAs-responsive key bHLH type TF FtbHLH06 was isolated and identified from F. tataricum. Therefore, understanding the transcriptional regulation mechanism of FtbHLH06 on FtRDE gene expression contributes to balance the concentration of rutin and quercetin in buckwheat. In this proposal, the objectives are: (1) Isolation and identification of FtbHLH06 interacted partners through yeast two hybrid (Y2H) screening, in vitro pull-down and in vivo bimolecular fluorescence complementation (BiFC); (2) Identification of the FtbHLH06 binding DNA cis-elements by RNA-seq, Y1H, electrophoresis mobility shift assay (EMSA) combined with metabolomic analysis; (3) Understanding the regulation of FtbHLH06 transcriptional activity by its interacting partners through trans-activation assay and functional analysis of transgenic plants; (4) Exploring the regulatory mechanism of FtRDE gene expression by FtbHLH06. This could contribute to a better understanding of the ways to manipulate the content of rutin and bitter taste for further beneficial effects to consumers in buckwheat.

芦丁是苦荞中含量最丰富的一种重要黄酮类物质，具有抗氧化、保持及恢复毛细血管的正常弹性等功能。在苦荞中，芦丁由芦丁降解酶降解为具有苦味的槲皮素和芸香糖苷，从而导致口感较苦而不受大众喜爱。迄今为止，芦丁降解的分子机制尚未揭示。本项目以苦荞“川荞1号”为研究材料，前期工作已分离鉴定了受茉莉酸诱导的芦丁降解酶基因FtRDE3及其启动子，利用FtRDE3启动子筛选茉莉酸处理的cDNA文库，首次分离鉴定出参与调控芦丁降解酶基因表达的关键转录因子FtbHLH06。拟在此基础上，利用酵母双杂交和双分子荧光互补等实验鉴定与FtbHLH06互作的调控因子；采用转录组测序和EMSA等技术结合代谢组学系统分析FtbHLH06调控的下游靶基因；运用瞬时转录激活方法阐明FtbHLH06的转录调控活性。该项目旨在系统揭示FtbHLH06调控芦丁降解的分子机制，为苦荞高芦丁且口感好的性状改良提供基因资源和理论支持。

芦丁是苦荞中含量最丰富的一种重要黄酮类物质，具有抗氧化、保持及恢复毛细血管的正常弹性等功能。迄今为止，芦丁降解的分子机制尚未揭示。本项目是苦荞核心种质和重测序数据为基础，开展了对槲皮素等芦丁代谢相关物质含量的GWAS关联分析，鉴定到了与芦丁代谢密切相关的关键位点，其中包括RDE（Rutin degrading enzyme）基因，并分析了不同的单倍型以及启动子不同的SNP变异，FtbHLH06和FtMYB16是与RDE基因共表达的受茉莉酸（JAs）诱导表达的直接调控RDE表达的关键转录因子，单倍型分析发现RDE基因主要有Hap.T和Hap.G二种，高芦丁含量前体物的单倍型Hap.G和低芦丁含量前体物的单倍型Hap.T的差异主要是由于启动子-1395bp处的SNP位点变异，造成一个G-box（5’-CACGTC-3’）突变，从而使得FtbHLH06结合活性减弱而导致芦丁含量降低。进而研究发现FtHSFA1b2和FtHSFA1b3是与FtbHLH06互作的并且正调控FtbHLH06蛋白活性的调控蛋白，FtHSFA1b2和FtHSFA1b3是高温和JA诱导表达，进而调控FtbHLH06活性而促进芦丁代谢，FtbHLH06还调控芦丁代谢途径的其他关键酶基因，包括FtF3’H（Flavonoid 3'-hydroxylase）和FtF3’HL（Flavonoid 3'-hydroxylase like）等。此外我们在模式植物百脉根中鉴定到FtbHLH06同源蛋白LjbHLH7，LjbHLH7也可以通过结合G-box来调控JAs诱导的次生代谢物合成，比如生氰糖苷，而且该类bHLH转录因子都可以通过和JAZ蛋白互作来调控JAs诱导的植物次生代谢生物合成。研究结果为荞麦芦丁分子育种奠定了理论基础。.在本项目的支持下，取得如下主要成果：发表学术论文15篇，其中在国际主流的植物学和农学杂志发表SCI论文7篇，分别是Genome Biology, Plant Biotechnology Journal（2篇）、New Phytologist和Journal of Experimental Botany等杂志；出版学术著作1部，科学出版社，30万字；申请专利2项，获批1项；培养博士后 1 名，博士生 2 名，硕士研究生6 名；培育新品种1个；获得国际荞麦协会杰出青年科学家奖等荣誉。