菊花CmWRKY51参与分枝调控的分子机理

Chrysanthemum is one of ten traditional famous flowers in China and a leading ornamental species, second only to the rose in terms of its market value. Branching is an important horticultural traits of chrysanthemum, which determined the cultural style, production costs, and ornamental quality. With the increasing cost of human resource, manual control on cut-flower chrysanthemum branching characteristics is becoming the main costs of production. In recent years, some progresses have been achieved in physiological, genetic and biotechnology research of chrysanthemum branching development, but the molecular mechanism is still not clear. Based on the previous research, CmWRKY51 gene was suggested as a critical regulator of plant branching. Through the subcellular localization, expression pattern analysis, and creating CmWRKY51 over-expression/ chimeric repressor gene-silencing transgenic chrysanthemum, basic functions of CmWRKY51 will be clarified. Cloning and analyzing the downstream genes and their promoters, which will clear the transcriptional regulatory networks of CmWRKY51. The yeast-two-hybrid assay will be applied to screen CmWRKY51 interacting proteins, then using the BiFC and GST Pull-down to validate both in vivo and in vitro. These evidences will reveal the post-translational regulation mechanism of CmWRKY51. Finally, we will clarify molecular mechanism of CmWRKY51 involved in the regulation of chrysanthemum branching. Our proposal can provide a theoretical basis and the elite gene pool for molecular assisted breeding of chrysanthemum branching traits.

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值极高。分枝是切花菊最重要的园艺性状之一，决定其栽培方式、生产成本与观赏品质。目前，菊花分枝的生理机理与基因工程改良已有比较深入研究，但分枝调控的分子机制仍不明确。我们前期研究发现菊花CmWRKY51在调控植物分枝中发挥着重要作用，本项目拟通过亚细胞定位、表达模式分析及利用超表达和融合抑制技术获得CmWRKY51转基因菊花，明确CmWRKY51基本功能；克隆CmWRKY51下游直接调控基因及其启动子，分析CmWRKY51的结合元件并验证，明确CmWRKY51的转录调控机制；通过酵母双杂交筛选CmWRKY51的互作蛋白，并运用BiFC和GST Pull-down进行体内体外验证，揭示CmWRKY51的翻译后调控机制，最终阐明CmWRKY51参与菊花分枝调控的分子机制，为菊花分枝性状的分子设计育种提供理论基础和优异基因储备。

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值极高。分枝是切花菊最重要的园艺性状之一，决定其栽培方式、生产成本与观赏品质。目前，菊花分枝的生理机理与基因工程改良已有比较深入研究，但分枝调控的分子机制仍不明确。我们前期研究发现菊花CmWRKY51在调控植物分枝中发挥着重要作用，本项目通过实时定量PCR对CmWRKY51的组织特异性表达进行了分析，结果表明CmWRKY51在不同器官中的表达水平差异显著，在茎中的表达量较高。构建载体pMDC43-CmWRKY51，通过基因枪转化洋葱表皮细胞，结果证明CmWRKY51蛋白定位在细胞核中。酵母单杂交实验证明CmWRKY51具有转录激活活性。构建pENTR1A-CmWRKY51入门载体，通过重组构建植物超表达载体pMDC32-CmWRKY51与功能沉默载体pDEST_35S_SRDX_BCKH-CmWRKY51，通过农杆菌介导的叶盘侵染法获得转基因菊花。转基因株系SRDX51-21、SRDX51-78和SRDX51-80相对于野生型，分枝更为紧凑，分枝量更多。进一步通过生物信息学筛选并验证了转基因植株中CmWRKY51下游直接调控基因CmBRC，明确了CmWRKY51的转录调控机制。通过三代Pacbio全长转录测序及二代转录组测序，找到了调控菊花分枝发育过程中每个阶段的核心调控模块（Module）及核心调控基因（Hub gene），阐明了菊花侧枝发育调控网络。在切花菊‘神马’侧枝发育不同阶段进行取样，混合提取RNA，构建菊花侧枝发育酵母文库，通过酵母双杂交技术从文库中筛选CmWRKY51的互作蛋白，结果表明CmWRKY51能够与核转录因子NF-YC、COP9信号复合体、MYB9A、SNF1类蛋白激酶互作，明确了CmWRKY51在翻译后和哪些蛋白互作共同调控菊花的分枝性状。从而阐明CmWRKY51参与菊花分枝调控的分子机制，为菊花分枝性状的分子设计育种提供理论基础和优异基因储备。