植物激素调控绿原酸合成关键基因HQT分子机制研究

Chlorogenic acid (CGA) in vitro has anti-cancer, anti-inflammatory, anti-bacterial, anti-virus and other extensive pharmacological effects. Tobacco is one of the plants with high chlorogenic acid content, which can extract large amount of CGA. It has been demonstrate that HQT gene regulates plant CGA synthesis, and the HQT gene expression is regulated by a variety of plant hormones. In order to further study the mechanism of CGA synthesis in tobacco, in this study, based on our former cloned HQT gene, the promoter of HQT gene would be cloned. The expression model of HQT and CGA content in tobacco were analyzed after different plant hormone treatments. By using bioinformatics analysis, the corresponding cis-element of the promoter related to plant hormone would also be characterized. Meanwhile, the significant differences between control and transgenic tobacco overexpressing HQT would be observed with the parameters of plant height and chlorophyll contents under different plant hormone treatments. Additionally, combining with RNA-Seq analysis of the differences between control and plant hormone-treated tobacco plants, the significant induction of genes involved in CGA synthesis would also be identified. These results would clarify the positive roles of HQT in CGA synthesis under plant hormone treatments and provide some new theoretic basis for the abiotic stress study.

绿原酸在体外具有抗癌、抗炎、抗菌、抗病毒等广泛的药理学作用。烟草是绿原酸的含量较高的植物之一，是大批量提取绿原酸的理想植物。目前已知，HQT基因可调控植物绿原酸合成，并且推测HQT基因表达受多种植物激素调控。为了进一步研究烟草绿原酸合成代谢相关机理，本项目拟以新克隆到的烟草HQT基因为基础，克隆其启动子序列，分析在不同植物激素处理下，HQT基因的表达模式以及绿原酸含量的变化；构建HQT基因过表达以及启动子截短的转基因烟草；并比较在不同激素处理下表型差异；结合RNA-Seq等技术，分析鉴定激素调控HQT基因表达信号转导途径关键基因及调控元件；探查植物激素调控烟草绿原酸合成的相关分子机制，为烟草绿原酸的合成调控研究提供新的思路。

烟草是绿原酸的含量较高的植物之一，HQT基因是绿原酸合成的关键基因。本项目在前期开展NtHQT1基因克隆的基础上，进一步完成了NtHQT1基因的亚细胞定位，发现NtHQT1基因定位在细胞质中。通过分析不同生长发育时期基因的表达模式，发现NtHQT1基因在不同生育期的叶片中表达量较高。另外还发现NtHQT1基因表达受MeJA、IAA、GA和ABA诱导。在此基础上结合转基因载体构建、启动子克隆和烟草的转基因功能验证等，重点对NtHQT1过表达的转基因植株进行分析，发现过表达烟草叶片中的绿原酸含量明显升高。利用 PCR 的方法对 NtHQT1基因上游约2000 bp的片段进行了克隆，得到了NtHQT1基因的启动子序列。序列分析发现，NtHQT1基因启动子区含有一些响应植物激素（GA、IAA、MeJA和ABA）诱导的重要顺式作用元件。通过对照、转NtHQT1基因过表达植株进行转录组学分析，初步明确了NtHQT1基因过表达植株可能通过下调木质素合成基因CCR和类黄酮合成基因CHS，使更多的木质素中间产物流向绿原酸合成支路，从而使绿原酸的合成增加。本项目研究内容的完成，初步明确了NtHQT1基因在烟草绿原酸合成中的作用，解析了NtHQT1基因受诱导的作用机制，为深入研究烟草绿原酸的合成提供了理论基础。