菊花转录因子CmTCP9 参与花瓣生长调控的分子机制研究

Chrysanthemum (Chrysanthemum morifolium), one of the ten traditional famous flower and one of four important cut flowers over the world, is of high ornamental and economic values. The TCP transcription factor family is unique to plants, and is closely related with plant growth and flower petal development. We have previously cloned 18 candidate TCP genes, among these, CmTCP9 shown high transcript abundance in petals. Overexpression of CmTCP9 shown larger and longer petals in A. thaliana, however, the mechanism involved in regulating petal growth in chrysanthemum remained unclear. The present proposal aimed to identify its function by analyzing the expression pattern of the CmTCP9, its subcellular localization and transcriptional activation activity; to obtain the CmTCP9 overexpressed and silenced gene function transgenic plant lines; to screen and verify genes regulated downstream by CmTCP9 using bioinformatics analysis; to screen its interaction proteins using yeast two-hybrid, to confirm the interaction in vitro and in vivo using bimolecular fluorescence complementation, and GST Pull-down and Co-Immunoprecipitation. Taken together, the present proposal will disclose the mechanism of CmTCP9 in regulating petal of chrysanthemum, which will provide theoretical basis for molecular breeding. Therefore, the present proposal is of both theoretical and practical significance.

菊花是我国十大传统名花和世界四大切花之一，有很高的观赏和经济价值。TCP 转录因子家族参与植物的生长发育，与花器官形态建成有着密切关系，我们先前获得了18个菊花TCP 转录因子，发现CmTCP9 在切花菊‘神马’花瓣中表达量最高。超表达CmTCP9 能使拟南芥花瓣明显变长、变宽，但其参与菊花花瓣生长调控的分子机制尚不清楚。本项目拟分析CmTCP9 表达特性、亚细胞定位、转录激活活性，阐述其在花瓣生长发育过程中的基本功能；构建超表达和功能沉默载体获得转基因材料，分析转基因植株与对照株系转录本表达差异，找出CmTCP9 的候选下游基因；通过酵母双杂交筛选CmTCP9 的互作蛋白，并使用双分子荧光互补、Pull-down和免疫共沉淀等手段进行体内、外验证，最终阐释CmTCP9 参与菊花花瓣生长的作用，解析其在花瓣生长调控中的具体机制，为菊花花器官的分子育种奠定基础，具有重要科学意义和应用前景。

菊花是我国十大传统名花和世界四大切花之一，有很高的观赏和经济价值。TCP转录因子家族参与植物的生长发育，与花器官形态建成有着密切关系，通过对花瓣伸长生长过程进行标记与观察，发现花瓣基部的生长速率最高，且花瓣基部表皮细胞的伸长最为明显。转录组测序结果分析显示，在花发育的4个时期中，属于生长素信号转导路径的差异基因远多于其他激素信号转导路径。CmTCP9基因定位于细胞核中，没有转录激活活性，在舌状花中表达量最高，其次是管状花。表达量在整个花瓣和花瓣基部中均表现为下降趋势。超表达CmTCP9的拟南芥中与野生型相比，花蕾、花瓣变长，莲座叶长度、角果数目明显增多，整个植株的生长量变大。CmTCP9的菊花超表达株系与野生型相比，具有破蕾提前，花序直径变大，花瓣伸长的表型。anti-CmTCP9表型表现出跟野生型类似的性状，花序直径和花瓣长度与野生型一致。利用酵母双杂交技术对CmTCP9进行筛库，得到其互作蛋白CmJAZ1-like，进一步通过酵母双杂、烟草BiFC技术和pull-down验证了其互作真实性，JAZ是JA信号传递中的关键因子，推测CmTCP9可能通过参与JA路径影响菊花花瓣的发育。同时发现CmTCP7与CmFDL2有互作，CmTCP9与CmFDL2、CmSEP3分别有互作，且CmTCP7的启动子能与受生长素响应因子CmARF3结合，几方通过生长素途径共同调控菊花花瓣生长。根据菊花花发育转录组库，克隆得到差异基因CmBPEp，选取CmBPEp最高表达的初绽期，对超表达CmTCP9转基因株系进行定量分析，发现超表达CmTCP9后，CmBPEp下调，而已有报道拟南芥中CmBPEp的同源基因AtBPEp可以通过响应JA路径在花发育后期抑制花瓣伸长，由此可以推测，CmTCP9可能通过参与JA路径，抑制CmBPEp的表达进而使花瓣变长。研究结果为菊花花器官的分子育种奠定基础，具有重要科学意义和应用前景。