二萜化合物在水稻抗虫中的功能

Terpenes are the biggest group of natural products in plants, and diterpenes show extensive and important bioactivities. Rice (Oryza sativa L.) produces 14 known diterpene phytoalexins. Intensive research has been conducted on roles of diterpene phytoalexins in rice resistance against pathogens. However, no study has been done on their role in rice defense against herbivore insects. This study will isolate diterpene phytoalexins from rice by chromatographic methods and identify them by spectral analysis and others. Insecticidal activities of the isolated diterpene phytoalexins against major rice herbivore insects will be evaluated in vitro. The effects of insect herbivory and treatments with jasmonic acid (JA) and other defense signaling compounds on phytoalexins accolumation and transcripts of biothethesis emzyme genes will be examined. Based on the obtained transgenic rice plants of phytoalexin biothethesis key emzyme genes such as CPS2, CPS4, KSL1, KSL4，KSL7，KSL8, KSL10 silenced and overexpressed by use of RNA interference and gene over-expression techniques, changes in insect resistance, phytoalexins accolumation and transcripts of biothethesis emzyme genes will be determined in transgenic rice plants. The study will characterize the functions of diterpenes in rice resistance against insect herbivores, and clarify the molecular mechanisms of regulation of diterpene production. The results shall provide a guideline for breeding for insect resistant rice varieties and genetic engineering of secondary metabolites.

萜类是最大的一类植物天然产物，二萜化合物具有广泛而重要的生物学活性。水稻产生的二萜类植保素已知有15个，它们在抗病中的作用已有大量研究。然而，它们在水稻抗虫中的功能一直缺乏研究。本项目拟利用现代色谱、光谱等技术分离鉴定水稻中的二萜植保素，离体测定这些化合物对水稻主要害虫（褐飞虱、稻纵卷叶螟和二化螟）的生物活性；研究主要水稻害虫为害和茉莉酸等防御信号物质处理对水稻二萜植保素的含量和合成关键酶基因表达的影响；利用已经构建的RNA干涉和基因过量表达材料，研究CPS2, CPS4, KSL1, KSL4，KSL7，KSL8, KSL10等二萜合成关键酶基因分别被沉默和过表达后，转基因水稻对主要害虫抗性、二萜植保素的含量和关键合成酶基因表达等的变化。研究将阐明水稻二萜植保素在水稻抗虫中的功能，为利用水稻自身化学防御开展抗性育种和次生代谢转基因工程提供理论依据

次生代谢物质在植物抗虫中发挥重要作用，萜类是最大的一类植物天然产物，二萜化合物具有广泛而重要的生物学活性。主要粮食作物水稻产生一系列特有的二萜植保素，而这些二萜化合物在水稻抗虫中的功能却不清楚。本项目利用现代色谱、光谱等技术从水稻中分离获得了七个二萜，其中三个为新的二萜化合物。稻壳酮A对褐飞虱有显著的抑制活性。褐飞虱和二化螟取食可以诱导稻壳酮合成关键基因OsCPS4、OsCYP99A2和OsCYP99A3的表达。通过Cas9基因编辑技术和基因过表达技术分别获得了二萜合成关键酶基因（OsCPS4, OsCPS2, OsKSL4）的敲除纯合突变体和基因过量表达水稻材料。生物测定表明，二萜合成关键酶基因OsCPS4和OsKSL4被敲除后，稻壳酮等二萜植保素合成显著减少，水稻对褐飞虱和二化螟的抗虫性明显下降。OsCPS2基因敲除对褐飞虱抗性没有显著影响。二萜合成关键酶基因过表达后，转基因水稻对主要害虫抗性增强，二萜植保素的含量增加。同时，研究鉴定到了一个在根部特异高表达的调控水稻二萜合成的WRKY家族的转录因子OsRMB1，它响应茉莉酸信号，正向调控水稻二萜类植保素的从头合成，负调控水稻对褐飞虱的抗性。研究成果阐明了二萜植保素在水稻抗虫中的功能，为利用水稻自身化学防御开展抗性育种和次生代谢转基因工程提供理论依据。