调控香雪兰挥发性萜类合成的转录因子克隆及其对萜类合酶基因表达和花香释放的影响

Transcription factors (TFs) play a predominant role in regulating the expression of genes involved in various physiological and developmental processes, including plant secondary metabolism. As an important part of secondary metabolites, volatile terpenoids biosynthesis is also regulated by several TFs, among them, MYC2 and WRKY1 have been proved to activate sesquiterpene synthase gene expression to enhance relevant terpenes biosynthesis and emission. However, their roles in monoterpenes biosynthesis has not been researched. Flowers of Freesia hybrida can emit sorts of monoterpenes and sesquiterpenes in floral scents, and the emission of terpenoid compounds is organized and coordinated. Therefore, it is reasonable to hypothesize that same TFs exist in both two kind of volatile terpenes biosynthesis process. Furthermore, the number of monoterpene and sesquiterpene and their ratio varies between some cultivars which suggests that monoterpene and sesquiterpene biosynthesis is controled by special TFs respectively. In this research, we will utilize transcriptome sequencing, Homologous gene cloning and yeast one-hybrid method to clone MYC2, WRKY1 homologous gene sequence and search differential expression TFs in red and white Freesia hybrida, their expression pattern in different flower development stage, tissues, cultivars and environment factors will also been analyzed. After clarifying the coordinated relation between TFs, terpenoid synthase gene expression and volatile terpenoid emission, the regulation mechanisms of transcription factors in Freesia volatile terpenoid metabolism will be preliminary revealed. At present, transcriptome sequencing has been completed; several transcription factor and terpene synthase gene sequences were separated, which laid the foundation for the implementation of the project.

转录因子在植物很多生理和发育过程中发挥着主导作用，挥发性萜类作为次生代谢产物的重要成员，其生物合成同样受多种转录因子调控，其中MYC2和WRKY1被证明可以直接激活倍半萜合酶基因转录，进而控制倍半萜的合成和释放，但是二者如何调控单萜合成还未见报道。 香雪兰花香成分中包含大量的单萜和倍半萜，且各成分之间的释放规律呈现一致性，说明萜类合酶基因的转录调控中可能存在共同的转录因子，但是品种之间单萜和倍半萜的差异很大，暗示可能存在特异性转录因子分别控制两种萜类化合物的合成。 本研究综合利用转录组测序，同源基因克隆，酵母单杂交和挥发代谢谱分析等技术手段，克隆MYC2和WRKY1同源序列并寻找红花和白花香雪兰之间差异表达的转录因子，通过对比它们与萜类合酶基因及代谢产物之间的关系，初步揭示转录因子对香雪兰挥发性萜类物质合成的调控作用。 目前，已经获得多个转录因子和萜类合酶基因序列，为项目实施奠定了基础。

植物花香成分主要由挥发性萜类、苯丙环类和脂肪酸衍生物三类物质组成，其中挥发性萜类物质是形成花香的主要成分。. 本课题组利用固相顶空微萃取和气相色谱质谱联用技术分析了自然条件下，香雪兰品种（Red River○R、Ambiance）花朵发育各时期和花朵各组织挥发性萜类物质的释放规律，结果显示萜类物质的释放与花朵的发育具有一定相关性，随着花朵的发育萜类物质的释放量逐渐增加。同时，香雪兰花朵中释放的挥发性萜类主要在花朵的花瓣、雄蕊和雌蕊中释放。. 同时，基于香雪兰花朵转录组结果，本课题组成功克隆得到8条萜类合酶基因，并对部分基因进行了基因功能和表达量分析。体外酶活实验证明，萜类合酶蛋白可以催化相应酶活反应。而且基因表达分析表明，萜类合酶基因的表达与挥发性萜类物质的合成和释放之间具有一定的相关性。. 另外基于香雪兰挥发性萜类释放规律和萜类合酶基因表达模式，结合香雪兰花朵发育各时期和花朵各组织的基因数字表达谱结果，筛选出90条与萜类物质释放和萜类合酶基因表达相关的转录因子。通过拟南芥原生质体瞬时转化实验，筛选到6条具有调控萜类合酶基因表达能力的转录因子基因。. 综上，课题组完成了研究计划，并根据阶段研究结果完善了原有研究方案。发表SCI研究论文4篇，申请国家专利1项。部分研究成果处于投稿和整理阶段。