茉莉花JsMYB305转录因子调控香气合成的分子机理

Jasmine (Jasminum sambac) is a well-known aromatic plant, which has important economic value due to its abundant use in jasmine tea, essential oils, cosmetic industry, food and garden applications. However, degradations often happen in character and aroma during production.The regulation mechanism of aroma formation in Jasmine is not clear, which restricts the development and application of agricultural technology and biotechnology aimed at improving floral quality. linalool, alpha-farnesene and benzyl acetate dominated the volatile aroma of jasmine. However, little is known about the transcription factors involved in the regulation of their metabolic pathways and their potential functions. A transcription factor, JsMYB305, expressed simultaneously with aroma release was cloned from jasmine based on previous functional studies of linalool synthase (JsLIS), α-farnesene synthase (JsTPS1) and benzylalcohol acetyltransferase (JsBEAT) gene. JsMYB305 is highly homologous to petunia hybrida PhEOBII and could activate the transcription of JsLIS and JsTPS1. In present project, we intend to analysis the regulation of JsMYB305 in benzenoid/phenylpropanoid and terpenoid synthesis pathway, as well as the regulation network and potential molecular mechanism of aroma substances synthesis through EMSA (Electrophoretic Mobility Shift Assay), yeast one hybrid, trans-activation experiment, yeast two hybrid, BiFC (bimolecular fluorescence complementation) ,petunia and Arabidopsis transgenic research. The results will provide a theoretical basis for the breed of jasmine variety with high fragrance.

茉莉花是著名的芳香植物，广泛应用于茉莉花茶窨制及化妆品、食品和园林应用等方面，具重要经济价值。然而生产上常出现性状退化和花香减弱现象。茉莉花香气形成的调控机理尚不清楚，制约了旨在提高花香品质的农业技术和生物技术的开发和应用。芳樟醇、α-法呢烯和乙酸苄酯为茉莉主要挥发物质，但调控它们代谢途径的转录因子及其功能知之甚少。在前期研究茉莉芳樟醇合成酶（JsLIS）、α-法呢烯合成酶（JsTPS1）以及苯甲醇乙酰基转移酶（JsBEAT）基因功能的基础上，克隆到与香气释放同步表达的JsMYB305转录因子，它与矮牵牛PhEOBII高度同源且能激活JsLIS和JsTPS1表达。本项目拟通过EMSA、酵母单杂、转录激活实验、酵母双杂、BiFC、矮牵牛和拟南芥转基因等研究手段分析JsMYB305对苯环/苯丙烷类和萜类合成途径的调控方式，以及调控香气合成的网络和潜在分子机理。为高香型茉莉花的创新提供理论基础。

夜间开花的茉莉花是一种受欢迎的观赏植物，在香水、医药、饮料和食品工业中具有传统用途。法尼烯、芳樟醇以及乙酸苄酯在茉莉花中的挥发性物质中占重要组成。JsMYB305是基于前期茉莉花叶转录组筛选到转录因子，分析它对萜烯合成酶基因JsTPS1、JsLIS、苯甲醇乙酰基转移酶基因JsBEAT1和JsBEAT2的调控，以及对萜烯合成途径和苯环类/苯丙烷合成途径的影响。JsMYB305可显著提高茉莉愈伤组织中JsTPS1、JsLIS、JsBEAT1基因的转录水平；烟草的转录激活实验发现JsMYB305可显著提高JsTPS1、JsLIS、JsBEAT1启动子活性；酵母单杂结果表明JsMYB305 转录因子与JsTPS1、JsLIS启动子序列能结合。将JsMYB305在大肠杆菌中表达并纯化，大小约49kDa。通过酵母双杂系统，从茉莉花蛋白库中筛选获得一个互作蛋白水杨酸甲酯转移酶（SAMT），SAMT 属于苯环类/苯丙烷生物合成途径，说明JsMYB305 转录因子参与了茉莉花苯环类/苯丙环类香气物质合成过程。此外，通过定向酵母双杂、共表达实验发现一个bHLH类转录因子JsMYC2与JsMYB305存在互作，可协同调控JsTPS1、JsLIS的表达水平。JsMYB305转基因拟南芥出现提早抽薹、植株簇生、植株发红、叶片表皮毛增多增大、叶片细长变多、挥发物质中一些萜类物质含量升高等现象。相对应的，转基因株系中差异表达的基因主要集中的路径为萜类合成相关的代谢途径，包括萜类合成途径、苯丙氨酸合成途径、类黄酮合成途径、花色苷合成途径等。此外，JsMYB305转基因矮牵牛植株花朵中苯甲酸甲酯、已烯醛、十六醇三种香气物质含量显著提高。综上所述，JsMYB305可通过直接作用于JsTPS1、JsLIS启动子，单独或与JsMYC2互作协同提高JsTPS1、JsLIS启动子活性，提升它们的转录本水平，调控萜类合成途径；JsMYB305可以提高JsBEAT1启动子活性、提高其转录水平，并与茉莉SAMT互作，调控茉莉苯环类/苯丙环类香气物质合成途径。