光质调控植物黄酮甲基化反应的机理研究

Flavonoids are important secondary metabolites in plants and play an important role in the biological processes related to the adaptation of plants to the environment and stress resistance. In our previous study, the influence of light quality on the accumulation of flavonoids in tobacco was studied and the result indicated that the accumulation of flavonoid methyl derivatives was negatively correlated with the proportion of ultraviolet (UV-A; 350–400 nm) and positively correlated with the proportions of far-red light (FR; 716–810 nm), which was contrary to the mode of flavonoid and its glycoside derivatives regulated by light quality. The expression pattern of NtFOMT regulated by light quality was conflict with the main synthetic gene of flavonoid. To study the mechanism of flavonoid methylation reaction regulated by light quality, the promoter of NtFOMT was cloned from tobacco with the method of genome walking and five 5' truncated fragments of the promoter were used to construct a carrier containing YFP reporter gene. In this study, the site-directed mutagenesis technique will be employed to analyze cis-acting element response to the regulation of light quality and the transcription factor will be cloned by the yeast one hybrid system. The mechanism of flavonoid methylation regulated by light quality will be discussed by analyzing the transcription factor, expression profile of NtFOMT and accumulation of flavonoid methyl derivatives. The results will establish a theoretical basis for the study of plant secondary metabolism regulated by light quality.

黄酮化合物是植物重要的次生代谢物，与植物适应环境和抗逆有重要的关系。我们前期研究光质对烟草黄酮化合物积累的影响，发现高比例的UV-A能够抑制黄酮甲基衍生物的积累，而远红光促进其积累，与黄酮及黄酮糖苷衍生物受光质调控的模式相反，并且黄酮甲基转移酶基因（NtFOMT）与主要黄酮合成基因受光质调控的表达模式相矛盾。为了研究光质调控黄酮甲基化反应的机理，本研究利用基因组步移技术，从烟草基因组中克隆到NtFOMT基因的启动子。并进行启动子5’端连续缺失片段切割，构建融合YFP的表达载体，明确了响应光质调控的启动子片段。后续拟通过定点突变分析确定响应光质调控的顺式作用元件，然后利用酵母单杂交方法克隆介导光质调控的转录因子。通过综合分析不同光质条件下转录因子和NtFOMT的表达规律以及黄酮甲基衍生物的积累规律，明确光质调控黄酮甲基化反应的机理，为光质调控植物次生代谢提供理论基础。

前期研究发现黄酮甲基衍生物受远红光（FR）的正调控，而受紫外光（UV-A）波段的负调控，与黄酮醇（山奈酚）和黄酮糖苷衍生物受光质调控的模式相反。为了研究黄酮甲基化反应受光质的调控的机理，本项目克隆NtFOMT基因的启动子，分析该启动子中能够响应光质调控的顺式作用元件，并分析受光质调控的转录因子，探讨光质调控黄酮甲基衍生物积累的机理。结果显示NtFOMT 基因启动子受NIR光的正调控，受UV的负调控。启动子968-1467bp的片段中含有响应UV-A的顺式作用元件，而635-968bp片段中含有响应FR的作用元件。验证发现该启动子中ACE和MYB顺式作用元件能够响应光质的调控。通过对转录因子的分析发现MYB、UNE10、EREBP-15和PIF5可以响应光质的调控，其中MYB转录因子受远红光和近红外光的正调控，而UNE10和PIF5受远红光和近红外光的负调控。项目的研究对解释植物适应环境具有重要意义。