姜花沉香醇合成酶基因高效表达的转录调控机制解析

The flower scent is one of important ornamental character. Hedychium coronarium is a important fragrance cut flower in tropic and subtropic area. The linalool is main scent component in H. coronarium flower. The transcription regulations mechanisms of linalool or/and terpene biosynthesis are not clear. Screening and identification of their cis-acting elements and transcription factors controlling biosynthesis of linalool and terpenes are of important significance in the theory and practice. We have very good preperation for this research: two linalool synthases related with the production of linalool had been cloned from Hedychirum coronarium and their function identified by their prokaryotic expression in Escherichia coli, and also the gene expressin pattern had been investigated. In this study, we hope further carry out the following investigations: cloning of two linalool synthesis genes promoter sequence; screening and identifying of cis-acting elements of linalool synthase genes with flower tissue-specific expression by delelation and mutation method; screening of candidate transcription factors by yeast one-hybrid method. To investigate their transient expression in tobacco plant and linalool level in over-expression in Arabidopsis thaliana and BSMV challenged flower petal in Hedychium coronarium respectively to explore the function of the genes. The purpose of our research is to understand the molecular mechanism of the synthesis and release of flower scent by investigation of transcript regulation of linalool synthesis, and also to supply available genes for molecular breeding of new vareities with fragrance trait.

花香是花卉重要的观赏品质之一，姜花是热带亚热带地区重要的香型切花，沉香醇是其主要的萜类香气成分，然而沉香醇合成的转录调节机理尚不明确。筛选和鉴定控制沉香醇等萜类香气合成相关基因转录的顺式作用元件和反式作用因子，解析转录调控机理在理论和实践上都具有重要意义。申请者在已经获得两个经原核表达鉴定、花部特异表达和烟草转基因鉴定证明其与花香形成密切相关的姜花沉香醇合成酶基因的基础上，克隆出它们的启动子序列，采用缺失和突变的方法筛选和鉴定与花香基因花部特异表达密切相关的顺式作用元件；通过酵母单杂交方法筛选与之结合的候选转录因子；使用烟草植株的瞬时表达，拟南芥过表达、大麦条纹斑叶病毒VIGS沉默白姜花花瓣后测定沉香醇的挥发量等方法鉴定候选转录因子的功能。本研究旨在解析影响沉香醇合成的转录调节机制，为了解植物挥发性物质形成和释放的分子生物学机制奠定一定的理论基础，并为基因工程培育香花新种质提供理论指导。

花香是观赏植物的重要观赏性状，姜花是热带亚热带地区重要的香型切花，其花朵释放大量的萜类花香物质，沉香醇是其主要的萜类香气成分，课题组已通过原核表达体外酶反应证明了HcTPS1和HcTPS2是催化形成沉香醇的关键酶。本课题利用顺式元件筛选姜花花瓣的cDNA-AD 融合表达文库，获得与HcTPS1和HcTPS2启动子结合的HcMYC2-1、HcMYC2-2、WRKY、ZINC和NAC转录因子。5个转录因子都在有香味的白姜花和金姜花中表达，而在不香的红姜花中不表达，且基本在姜花的花瓣、花萼、雄蕊和雌蕊等花器官中表达，营养器官不表达，表达进程与开花释香进程相一致。基因定位实验结果表明HcMYC2-1、HcMYC2-2、WRKY、ZINC和NAC都定位于细胞核，在核中与HcTPS1和HcTPS2启动子结合，转录调控其表达。对HcTPS1和HcTPS2启动子的G-box 进行点突变后进行酵母单杂和凝胶阻滞分析，证明HcMYC2-1和HcMYC2-2与HcTPS1和HcTPS2启动子的G-box结合； pHcTPS1-gus与HcMYC2-1-pOx或HcMYC2-2-pOx农杆菌菌液共转后的本氏烟草叶片的GUS活性分别对照为的2.14和1.79倍，证明两个转录因子通过与HcTPS1启动子的G-box结合，激活其转录。构建HcMYC2-1 和HcMYC2-2的大麦条纹斑叶病毒载体分别VIGS白姜花花瓣后，沉香醇挥发量各减少了39%和56%。沉默HcMYC2-1后，HcMYC2-1和HcTPS1下降了95%，而沉默HcMYC2-2后，HcMYC2-2和HcTPS1分别降低了85%和90% 。通过染色质免疫共沉淀技术（ChIP）验证了HcNAC、HcZINC和HcWRKY能分别于HcTPS1和HcTPS2启动子在白姜花体内的进行结合，并确定了转录因子结合启动子的区域范围。通过该项研究，获得多个具有自主知识产权的重要花香转录因子，解析了其对沉香醇合成酶基因的调控模式，为培育具有香气的花卉品种打下了坚实的基础。