月季丁香酚合成酶基因RcEGS1的转录调控研究

Rose is not only one of the most important cut flowers in worldwide, but also serviced economically value as a source of natural fragrance and flavour. However, due to the limit of traditional cross-breeding, the fragrance of modern roses seems to have been largely lost during the later stages of breeding process. In our previous study, the eugenol synthase gene RcEGS1 in the pathway of phenylpropenes metalism was cloned and identified in rose. The overexpressing of RcEGS1 increases the relative content of eugenol in transgenic rose. The promoter of RcEGS1 was isolated from rose DNA by genome walking, and constructed 6 vectors of bait yeast with cis-transcription elements of the promoter and its fragments. According to the above achievements, the current study will identify the regulation gene of RcEGS1 using yeast one-hybrid system. “Point to point” validation was performed to further insure the transcript factors. Then, subcellular position of the transcript factors will be performed by combining the methods of fusion protein construction, infection, fluorescence microscope observation. The expressions of the transcript factors will be analyzed in different tissues and development stages in this study. Furthermore, transcriptional activity assay will be employed to verify whether the transcript genes possess trans-activation ability alone. Finally, the function of the transcript factors will be analyzed by the methods of VIGS and CRISPR/Cas9 system. These results can not only lay a foundation for understanding the upstream gene involved in regulation eugenol, but also provide new idea for rose-scent breeding through regulation the metabolic pathway.

月季不仅是世界第一大切花，而且因具有宜人的花香作为精油和香料生产的原材料具有重要的经济价值。由于传统育种的局限性，大多数现代月季品种缺乏明显的香味。项目在前期研究中克隆了苯丙烷类途径上丁香酚合成酶基因RcEGS1，获得了超表达RcEGS1丁香酚含量显著提高的转基因月季，克隆了RcEGS1的启动子序列，构建了诱饵酵母载体。在此基础上，本项目拟利用酵母单杂交技术，筛选调控丁香酚合成的转录因子，并分析其表达、亚细胞定位和转录激活活性；其次，采用VIGS技术对其录因子进行功能分析，对有功能的候选基因再分别构建超表达和CRISPR/Cas9敲除载体，并获得转基因月季，明确转录因子在花香代谢途径上的功能；分析转基因月季和对照的转录本表达的差异，探讨转录因子参与调控的基因网络。研究结果不仅有助于探明调控丁香酚合成的上游转录因子，而且为通过调控花香物质代谢流的合成培育具有浓郁花香的月季新品种提供候选基因。

月季是世界第一大切花，月季花香不仅是重要的观赏性状，而且花瓣作为精油和香料生产的原材料具有重要的经济价值。前期研究中克隆了苯丙烷类途径上丁香酚合成酶基因RcEGS1，本项目通过2年的研究，筛选了调控丁香酚合成的转录因子，主要取得如下结果，首先，以‘月月粉’盛开期的花朵为材料，构建了三框型读码酵母单杂交文库，其库容量为1.12×107，平均插入片段在1Kb-3Kb之间，符合酵母文库的筛选要求。其次，完成了中国古老月季 ‘月月粉’丁香酚合成酶基因RcEGS1的启动子克隆，获得了1776bp的启动子序列，分析发现该序列除包含大多数高等植物启动子具有的保守元件外，还包含有植物次生代谢、与次生代谢相关的MBS-box（TAACTG）、G-box（CACGTA）、TGACG-motif（TGACG）及3个与MYB core（CNGTTR）结合区。构建了2个元件诱饵载体MBS-box、G-box和1个片段诱饵载体；酵母单杂交初步筛选获得了R2R3型MYB家族基因RcMYB4和bHLH3类转录因子。通过亚细胞定位分析，发现RcMYB4和bHLH3分别均定位在细胞核内；酵母转录活性分析表明RcMYB4和bHLH3均具有独立转录激活活性分析；qPCR分析显示，RcMYB4在花瓣中特异表达，在花蕾和凋谢期表达量较低，而盛开期表达量较高，和已知花香基因的表达模式一致，表明RcMYB4可能参与了月季花香的转录调控。本项目研究结果不仅探明了调控丁香酚合成的上游转录因子，而且为通过调控花香物质代谢流的合成培育具有浓郁花香的月季新品种提供候选基因。. 项目发表论文3篇；培养研究生2名；2人晋升副研究员，项目负责人获得2020年云南省高层次人才培养支持计划“青年拔尖人才”专项。.