玫瑰单萜类花香成分的代谢调控研究

Rose (Rosa rugosa) is an excellent landscaping material and a precious natural perfume plants. Rose essential oil is known as 'liquid gold' and has high economic and health values. Citronellol, geraniol, and nerol in monoterpenes, as well as their acetic ester derivatives are the main fragrant components of rose flower and essential oil. The regulation and modification of the metabolic pathways of these monoterpenes by genetic and metabolic engineering aimed at improving the content of rose essential oil will have important theoretical and practical significance. Based on the partial genes related to rose monoterpene metabolism cloned at an early stage, RrDXR, RrGES, and RrAAT will be used as the controlling target genes. The cis-acting element sequence will then be identified and analyzed by genome walking to separate the promoter subsequence. With this sequence as bait, the transcription factors involved in rose monoterpene metabolism shall be screened using the yeast one-hybrid system, its temporal and spatial expression patterns and biological functions will then be analyzed. Subsequently, gene overexpression vectors shall be constructed based on agrobacterium-mediated genetics to transform petunias. The purpose is to verify the influence of overexpressing synthesis genes and transcription factor on the content of monoterpenes. Therefore, this study will be able to enrich knowledge on plant terpenoids metabolism and regulation theories. We also expect to be able to create petunias with the smell of rose and new species of Rosa rugosa with improved essential oil content. Genetic resources for the molecular improvement of the aroma quality of some other commercial flowers shall be provided as well.

玫瑰是优良的园林绿化材料和珍贵的天然香料植物，玫瑰精油被誉为"液体黄金"，经济和保健价值极高。单萜类化合物中的香茅醇、香叶醇、橙花醇及其乙酸酯类衍生物是玫瑰鲜花和精油的主要芳香成分，采用基因工程手段调控单萜类代谢途径，开展以提高玫瑰精油含量为目标的代谢工程研究具有重要的理论意义和应用价值。本项目拟在前期研究的基础上，选择与玫瑰单萜代谢相关的RrDXR、RrGES和RrAAT为调控靶基因，克隆其启动子，找出各自的顺式作用元件及其序列；以该序列为诱饵，利用酵母单杂交系统筛选调控玫瑰单萜代谢的转录因子，研究其时空表达模式和调控功能；分别构建三个靶基因及转录因子基因的过表达载体，农杆菌介导遗传转化矮牵牛，验证并比较过表达合成基因及转录因子对单萜类化合物含量的影响。从而丰富植物萜类代谢与调控理论，并有望获得具有玫瑰香味的矮牵牛，为接下来创制"高油"玫瑰新种质及其它商品花卉香气品质的分子改良奠定基础。

玫瑰（Rosa rugosa）是世界上重要的天然香料植物，单萜类化合物中的香茅醇、香叶醇、橙花醇及其乙酸酯类衍生物是玫瑰鲜花及其精油的主要香气成分。采用基因工程和代谢工程手段，调控玫瑰单萜类化合物的生物合成途径，促进下游代谢产物的积累，进而创制精油含量提高的玫瑰新种质，具有重要的理论和实践意义。本项目主要研究结果如下：.1. 以玫瑰主栽品种 ‘唐红’盛开期花瓣为试材，采用RACE法克隆获得了玫瑰Nudix水解酶基因家族成员RrNUDX1的全长cDNA序列。同时克隆获得了一个调控植物萜类代谢的重要转录因子RrMYB114-like。结合 ‘唐红’不同花发育时期和花器官不同部位香气成分的测定，我们发现，RrNUDX1基因表达水平的变化与玫瑰主要芳香成分含量的变化趋势一致，表明 RrNUDX1基因与玫瑰主要单萜类芳香成分的生物合成密切相关。.2. 以‘唐红’半开至盛开期的花瓣为试材，采用invitrogen系统，构建了玫瑰三框读码酵母单杂交文库，结果表明，库容量大于1X107CFU，质粒空载率小于5%，平均插入片段长度大于1.2Kbp，符合酵母文库的筛选要求。.3. 采用染色体步移技术，分别克隆获得了玫瑰RrNUDX1和RrDXR基因的启动子序列，结果表明，RrNUDX1基因共获得617 bp 的启动子序列，预测发现其中与花香代谢有关的元件2个。RrDXR基因共获得2213 bp 的启动子序列，预测发现其中与花香代谢有关的元件3个。将预测的5个顺式作用元件分别构建在报告基因的启动子之前，进行酵母单杂交筛选，发现这些元件与RrNUDX1和RrDXR不存在互作，无法进一步筛选出调控顺式作用元件的转录因子，后续研究需进一步扩大预测片段的筛选量，从而获得相应的转录因子。.4. 进一步构建了RrNUDX1、RrDXR和RrAAT基因的过表达载体，采用农杆菌介导法将过表达载体导入矮牵牛，结果发现，与野生型相比，转RrDXR和RrAAT基因的矮牵牛花朵挥发性成分变化不明显，而转RrNUDX1基因植株花中苯甲酸甲酯等主要香气成分的含量明显提高，且感官上能闻到浓郁的香气，说明过表达RrNUDX1基因具有增强矮牵牛花香的功能，这为接下来利用RrNUDX1基因提高玫瑰主要花香成分，培育高精油含量提高的玫瑰新种质奠定了突破性基础。