蜡梅花香挥发物生物合成关键基因的鉴定及分子调控网络

Floral volatiles of plants are very useful for propagation and extension of their population and protection of themselves from injury. Whereas from mankind opinion, floral fragrance can improve the quality of flowers, and can be used as materials of essential oil extraction and cosmetics industry. Chimonanthus praecox is a famous plant and loved by Chinese people because of its fragrant flowers blooming in winter. The studies showed that the floral volatiles of the plant were mainly controlled by β-ocimene, linalool, benzyl acetate, benzylalcohol etc.. A new clone with flowers of no fragrant odor was recently discovered and the last three components didn’t exist in it. In this project, the transcriptome, proteome and metabonome of flowers of the two clones respectively with and without fragrant odor were sequenced and established, and their databases were then compared between two clones. On the basis of their differences between the two clones, we can speculate the mechanism that the substrate and the floral volatile organic compounds (VOCs) accumulate, are consumed and divided into different paths, and finally are synthesized to final floral volatils. Then the key synthase genes encoding the floral volatiles were determined, and their qPCR expression and function were studied furthermore. The transcription factors (TF) involved in regulating the key synthase genes were identified using library construction. Then the function and interaction among the TFs and the genes were studied at last. Finally the molecular networks regulating the biosynthesis of final floral volatile were probed out from the above studies. It will play a foundation for that finally revealling the biosynthesis mechanism of VOCs and breeding the new flower with pleasing floral volatiles using molecular manipulation.

花香挥发物对植物种群的繁衍与外源伤害的防御具有重要作用，而对于人类来说，花香挥发物不仅改善花卉品质，还能用作香精提取与化妆品加工的原料。蜡梅是我国著名的香花植物，花开冬季，深受人们喜爱。前期研究表明，蜡梅花香挥发物主要为β-罗勒烯、芳樟醇、乙酸苄酯和苯甲醇等成分，而新近发现的无香品系‘WS001’缺乏后三个组分。本项目以无香与有香品系为材料，建立转录组—蛋白组—代谢组数据库，通过不同品系间的差异比较，从基因转录—蛋白合成到代谢产物三个层面分析，了解花香代谢途径关键底物以及花香挥发有机物（VOCs）的积累、消耗以及途径支路竞争到最后花香产物形成的调控机制，确定主要挥发产物合成的酶基因，并进一步对候选基因进行功能验证与时空表达分析，通过构建文库筛选与其互作的转录因子，对之进行功能鉴定与调控研究，从而明确蜡梅花香最终产物合成的分子调控网络，为揭示蜡梅花香挥发物合成机理及花香分子育种提供依据。

项目尝试阐述蜡梅花香形成与调控的分子机制。以芳香的‘H29’和无香的‘SW01’品系为研究材料，发现‘H29’花香为单萜类和苯环型化合物占主导地位，主成分是芳樟醇、乙酸苄酯、水杨酸甲酯等。‘SW01’则是单萜类，主成分有反式-β-罗勒烯、别罗勒烯、水杨酸甲酯等。采用转录组和蛋白质组测序，结合RT-PCR表达验证，挖掘到参与萜烯类和苯环类合成途径差异表达的候选基因，包括CpDXS、CpGPPS、CpTPS、CpPAL和CpBEAT等。得到16个差异表达的萜烯类合成酶基因CpTPSs，以及转录因子。.聚焦于萜类化合物，分析表明，CpTPS1的表达量与反式-β-罗勒烯的释放量一致，CpTPS5与芳樟醇释放趋势基本一致。体外酶活实验证实，CpTPS1催化GPP 产生罗勒烯；CpTPS5催化GPP 产生芳樟醇。转化早花烟草，转CpTPS5幼苗产生了大量挥发性芳樟醇。分析CpTPS5基因启动子片段，序列包含光应答、非生物胁迫响应以及与生长发育相关的元件，还预测CpTPS5启动子中花香相关的调控元件分布。表明，CpTPS1和CpTPS5共同负责有香和无香株系芳樟醇和罗勒烯的合成。.克隆了‘H29’和‘SW01’差异表达基因CpTPS10和CpTPS14，发现Cp TPS10启动子有差异，‘H29’中特有顺式反应元件ABRE3a、ABRE4和chs-CMA2a，可能与脱落酸响应和光响应有关；‘SW01’中特有的MBS和O2-site分别为参与干旱诱导的MYB位点和参与玉米醇溶蛋白代谢调控的顺式作用调控因子。CpTPS14启动子，‘SW01’较‘H29’多2个TATA-box。构建双元表达载体转化烟草，发现CpTPS10的转化植株中倍半萜-石竹烯和4,5-di-epi-马兜铃烯的含量显著提高，CpTPS14的转化植株芳樟醇和4,5-di-epi-马兜铃烯均显著提升，芳樟醇含量提升最显著。推测启动子的序列差异可能是导致CpTPS10、CpTPS14在不同基因型间表型差异的原因。.筛选bHLH家族转录因子CpMYC2和CpbHLH13，实时定量表达及在拟南芥和烟草中过表达，分别促进芳樟醇和-ß-石竹烯的合成。茉莉酸与赤霉素促进了MYC2转录因子的表达。可见，启动子差异是导致基因型间萜类化合物差异的主要原因，这为明确调控因子与启动子互作调控萜烯类挥发物的合成机理奠定了基础。