柚果实采后粒化过程中木质素合成的转录调控机制研究

Pummelo postharvest granulation is an important physiological disorder which directly impacts fruit flavor and commodity value, thus intensive study of the molecular regulation mechanism during pummelo granulation process will play significant roles for pummelo production and fresh-keeping storage. According to our previous researches, the secondary cell wall thickening caused by lignin deposition is a major reason for juice sac granulation, however, the granulation symptoms were relieved in seeded fruit after cross pollination (rich in endogenous hormones) or after spraying of a low concentration gibberellin at late fruit development period, which made us to speculate that the lignin deposition during pummelo granulation process was associated with gibberellin response sensitivity. MYB is an important transcription factor both in gibberellin signal transduction (GAMYB) and lignin biosynthesis pathways. In the present study, we will first analyze the phylogenetic relationship between reported GAMYB and lignin biosynthesis related MYB transcription factors, predicting the MYB which is simultaneously involved in gibberellin signal transduction and lignin biosynthesis, and then to clone its homologous gene CgMYB in pummelo based on sweet orange and Clementine genomes. After analysis of its gene structure, subcellular localization and transcriptional activity, its target genes in lignin biosynthesis pathway will be identified both by yeast one-hybrid and electrophoretic mobility shift assay (EMSA), finally the response of CgMYB to GA and its transcriptional regulation to targent genes in lignin biosynthesis will be verified using Arabidopsis mutants. The present research will enrich our understanding of pummelo postharvest granulation .

柚枯水粒化是影响果实风味和商品性的重要生理病害，研究汁胞粒化的调控机理对柚果实生产及贮藏保鲜有重要意义。我们前期发现木质素沉积引起的次生细胞壁加厚是柚果实汁胞粒化的主要原因，而产生大量內源激素的授粉果实或在果实发育后期喷施赤霉素后粒化症状有所减轻。我们认为柚粒化中的木质素沉积与赤霉素应答途径有关。在赤霉素信号转导途径中，受GA诱导的MYB（GAMYB）是重要的转录因子，而MYB也是直接调控木质素合成的转录因子。本项目分析GAMYB与木质素合成调控相关MYB的聚类关系，预测同时调控GA信号转导和木质素合成的MYB家族成员，基于甜橙基因组克隆柚同源基因CgMYB，通过亚细胞定位和转录活性分析确定其转录因子功能后，利用酵母单杂交和EMSA技术鉴定木质素合成途径中的靶标基因，在拟南芥突变体中验证CgMYB对GA的响应以及对木质素合成的转录调控。研究结果对解析柚果实粒化发生原因有重要意义。

柚枯水粒化是影响果实风味和商品性的重要生理病害，研究汁胞粒化的调控机理对柚果实生产及贮藏保鲜有重要意义。本项目以‘早香柚’‘琯溪蜜柚’和‘麻步文旦’为试材研究异花授粉和赤霉素处理对柚果实生长发育及采后枯水粒化的影响，首先利用透射电镜观察柚粒化果实的细胞超微结构，发现粒化果实细胞扭曲、次生细胞壁加厚等形态学变化，然后利用气相色谱-质谱连用、高效液相色谱、紫外分光光度等方法检测果实内源激素、木质素、可溶性糖和有机酸等果实品质指标，分析异花授粉和赤霉素处理对柚果实生长发育及采后枯水粒化的影响，评价异花授粉产生的大量种子在果实内裂及采后枯水粒化过程中的作用，明确柚果实内裂与采后枯水粒化的关系；在研究柚果实木质素合成的转录调控机制时，克隆了参与赤霉素信号转导和木质素生物合成的MYB家族成员CgMYB86以及木质素合成途径中的关键基因Cg4CL，发现CgMYB86具有典型的R2R3和抑制木质素合成的EAR结构域，在Cg4CL基因启动子区域发现MYB结合元件和GA响应元件，初步分析了柚果实采后粒化过程中CgMYB86转录因子对木质素合成关键基因Cg4CL的调控作用。在异花授粉减轻柚果实内裂及采后枯水粒化的实际生产中，利用轻便型电动授粉枪进行人工授粉，研制柚机械授粉花粉稀释剂，以干粉喷授代替传统的毛笔点授授粉方式，形成机械授粉减轻柚果实内裂及采后枯水粒化的省力化栽培技术，显著提高人工授粉效率。本项目研究结果为柚果实枯水粒化机理研究以及异花授粉在实际生产中的推广应用提供了依据。