油菜素内酯合成基因CmDWFs参与糖信号调控菊花分枝形态建成的分子机制

Chrysanthemum, originated in China, as one of ten traditional famous flowers in China and one of four cut flowers in the world, is of high ornamental and economical values. Branching is an important ornamental trait which determines the cultivation type, producing cost and ornamental values of chrysanthemum. Recently, we found that sugar could regulate the growth of axillary buds. Additionally, the expression of CmDWFs, the key genes in Brassinolide (BR) biosynthesis, were induced by sucrose in the sugar induced outgrowing lateral buds, which prompted us to 1) elucidate the crosstalk nodes between the sugar signaling and BR signaling by isolating BR biosynthetic genes CmDWFs, isolation its upstream key transcription factors using yeast one hybridization, and confirming its regulatory roles using in vivo luciferase transient expression and in vitro EMSA assay, and functional analysis of the upstream candidate genes using reverse genetic approaches. 2) to clarify the molecular mechanisms of CmDWFs and discovery downstream gene networks in the regulation of chrysanthemum branching by characterizing the key genes' expression profiles, subcellular localization, and generating its overexpression or RNAi transgenic chrysanthemum or Arabidopsis, and mining downstream genes by transcriptome sequencing using transgenic lines of chrysanthemum. This work could not only enrich the molecular regulation networks of plant shoot branching, but also provide a theoretical basis and the elite genes for genetic engineering of branching traits of chrysanthemum.

菊花原产我国，是我国十大传统名花和世界四大切花之一，观赏和经济价值高。分枝性状是其重要观赏性状，直接决定其栽培方式、生产成本与观赏品质。菊花分枝分子机制研究滞后，本研究在前期发现糖信号促进菊花侧枝发育，且油菜素内酯（BR）合成关键基因CmDWFs受糖信号诱导表达的基础上，开展响应糖信号的CmDWFs的克隆与功能鉴定，通过酵母单杂交筛选其上游关键转录因子，采用荧光素酶瞬时表达、EMSA等体内体外实验加以验证，并对候选基因进行功能分析，阐明糖信号与油菜素内酯信号在分枝形态建成调控中交互对话的节点；通过表达特性、亚细胞定位分析，及利用超表达、RNAi技术获得关键基因的转基因菊花或转基因拟南芥，进一步对菊花转基因株系进行转录组表达分析，初步明确油菜素内酯合成关键基因调控菊花分枝的功能及下游调控网络。该项目的实施不仅可以完善植物侧枝发育的调控网络，还可为定向培育不同分枝类型菊花品种奠定理论基础。

菊花作为我国传统名花，具有极高的观赏价值和经济价值。分枝性状是其重要观赏性状，直接决定其栽培方式、生产成本与观赏品质。明确菊花分枝的遗传机制，是调控菊花分枝的重要前提。课题组前期研究发现前期发现糖信号调控菊花侧枝发育。在此基础上，本项目利用相关分子生物学方法，从基因层面解析了菊花分枝发育的分子机理，探究了蔗糖对菊花侧芽生长的影响并挖掘调控该过程的关键基因。结果表明，油菜素内酯（BR）可能参与蔗糖促进侧芽生长的调控过程。不同处理发现黑暗条件下外源施用BR合成抑制物BRZ处理则抑制蔗糖对侧芽生长的促进作用，而BR类似物eBL显著促进菊花茎段侧芽生长。同时，转录组分析发现，BR合成关键基因CmDWFs在蔗糖处理后显著上调。其中，BR合成路径关键基因CmDWF1和CmDWF4在菊花‘神马’摘心后的侧芽中上调表达。CmDWF1超表达菊花植株的株高增加、节间伸长、叶面积增大，摘心后侧枝生长变快；CmDWF1干扰植株表型相反。CmDWF4超表达菊花植株相对于野生型侧芽更长、侧芽数更多，在摘心后侧芽生长更快。利用CmDWF1启动子进行酵母单杂交筛库实验，筛选获得上游调控因子CmbZIP19；并进一步通过酵母单杂交验证、原生质体LUC和烟草LUC实验，表明CmbZIP19与CmDWF1启动子结合并抑制其表达。进一步对CmbZIP19超表达株系进行转录组分析，筛选到了BR合成途径、BR信号途径、分枝相关和细胞壁相关的差异基因，表明CmbZIP19通过抑制CmDWF1的表达来影响BR合成，进而抑制菊花分枝。项目结果揭示了糖信号与油菜素内酯信号在菊花分枝形态建成调控中交互对话的分子机制，完善了菊花侧枝发育的调控网络，为定向培育不同分枝类型菊花品种奠定理论基础，具有重要的科学意义和应用前景。