RhNAC32调控月季单萜醇类花香物质合成的分子基础

The aroma of rose is one of the most important factors. Monoterpene alcohols is the main component of the scent. Though the monoterpene synthase genes have been identified, the study of its transcriptional regulation has not yet been reported. In previous study, we discovered that a new NAC transcription factor (RhNAC32) had the same expression pattern with a key enzyme called RhNUDX1 through coexpression analysis. Using yeast one-hybrid and bimolecular luciferase activity, RhNAC32 can specifically interact with NAC cis-acting elements and induce expression of RhNUDX1. Thus we speculate that RhNAC32 may regulate the expression of monoterpene alcohol synthase genes to synthesize monoterpene alcohols in rose. On this basis, further research is needed including the DNA biding activity of RhNAC32 analysis by EMSA，the correlation between RhNAC32 expression and monoterpene alcohols accumulation in rose, spatial and temporal expression analysis and subcellular localization of RhNAC32, RhNAC32 genetic transformation of tobacco and rose，screening the up stream regulators of RhNAC32 by yeast one hybrid. This study aims to demonstrate the molecular basis for the regulation of the synthesis of monoterpene alcohols by RhNAC32 in rose.

花香是月季的一个重要品质,单萜醇类物质是月季主要的香气成分之一。尽管已鉴定相关单萜醇合酶基因，但对其转录调控研究尚未报道。在前期研究中，通过月季共表达网络分析结合酵母单杂交、双分子荧光素酶活性检测，发现RhNAC32同单萜醇合酶关键基因RhNUDX1具有相似的表达模式，能够结合到RhNUDX1启动子上，激活RhNUDX1的表达，推测RhNAC32可能通过调控RhNUDX1的表达参与月季单萜醇类花香物质的合成。为了验证该猜想，拟采用EMSA鉴定RhNAC32与RhNUDX1及其他单萜醇合酶基因启动子重要顺式作用元件的相互作用；RhNAC32在不同香味月季品种间的表达分析及与单萜醇类物质积累的相关性分析；RhNAC32时空表达、亚细胞定位分析； RhNAC32转化烟草与月季，鉴定功能；酵母单杂交筛选RhNAC32上游调控因子并验证；最终解释RhNAC32调控月季单萜醇类花香物质合成的分子基础。

月季（Rosa hybrida），作为重要的一类切花和庭院花卉，素有“花中皇后”的美称，但是连续的杂交育种，导致很多月季失去了香味。因此阐明月季花香等次生代谢物质合成调控途径对月季品质改良具有重要的理论和实践意义。前期利用中国古老月季’月月粉’、现代月季品种‘紫罗兰’、‘金丝雀’、‘果汁阳台’花瓣、叶片组织器官转录组数据构建月季花瓣共表达网络。分别从‘紫罗兰’和’月月粉’中分离得到RhNAC032、RcMYBPA2，对其表达模式、基因功能及其次生代谢物进行检测进行分析。结果表明：RhNAC032主要在月季花中表达，其次为叶片；RhNAC032的表达与“月月粉”萜烯类物质的积累趋势一致；在烟草中过量表达RhNAC032,可以显著促进萜烯类物质的积累；同时qRT-PCR分析表明萜烯类合成途径相关基因在转基因烟草株系中显著上调表达；GUS活性结合荧光素酶活性检测，RhNAC032可以激活RhTPS1以及RhDXR1。此外我们对另一个共表达转录因子RcMYBPA2进行了研究，RcMYBPA2受到外源水杨酸、划伤、MV诱导；烟草中超量表达RcMYBPA2，导致萜烯类物质含量显著增加；同时我们还发现转基因烟草花瓣中原花青素含量增加，而花青素含量呈下降趋势；此外RcMYBPA2过量表达可以显著增强转基因植株的抗逆性。本研究为月季萜烯类物质的合成网络提供了新见解，获得的基因及转化材料将为我们下一步深入研究提供良好的基础。