虫害诱导的玉米单萜合成相关基因ZmGPPS和Zm-monoTPS的功能解析

Monoterpenes are major constituents of herbivore-induced plant volatiles, which involved in plant defense against pest insects. Geranyl diphosphate synthase (GPPS) and monoterpene synthase (monoTPS) are key enzymes involved in monoterpene biosynthesis. Previous study showed that the co-expression of GPPS with monoTPS genes can enhance monoterpene production in dicots, but few reports in monocots. In order to understand the functions of maize ZmGPPS and Zm-monoTPS in monoterpene biosynthesis and defense against Ostrinia furnacalis (Guenée), we will clone the O. furnacalis-specific induced genes, candidate ZmGPPS and Zm-monoTPS genes, among which O. furnacalis-specific induced Zm-monoTPS genes were screened and one candidate Zm-monoTPS gene was already cloned, and characterize their enzyme activities by catalytic activity analyses in vitro. We also overexpress and co-express candidate ZmGPPS and Zm-monoTPS genes in transgenic maize plants and compare the monoterpene production. In addition, we will investigate the direct defense of maize monoterpenes to O. furnacalis, as well as their functions in regulating the behavior of the larval parasitoid, Macrocentrus cingulum (Brischke), based on a model tritrophic system of maize-O. furnacalis-M. cingulum. Knowledge gained from this research will help to understand the regulation of monoterpene accumulation in maize and their functional roles in maize defense against insect pests.

单萜是重要的虫害诱导植物挥发物，参与植物对害虫的防御反应。牻牛儿基焦磷酸合成酶GPPS和单萜合成酶mono-TPS是单萜生物合成过程中的关键酶。二者在双子叶植物体内共表达可显著提高植物单萜释放量，但在单子叶植物玉米中鲜有报道。为明确GPPS和monoTPS基因在玉米单萜生物合成及对亚洲玉米螟抗性中的作用，本项目拟在前期筛选到了玉米螟特异诱导的玉米Zm-monoTPS基因并克隆到了1个Zm-monoTPS基因基础上，克隆ZmGPPS和其它Zm-monoTPS，通过体外酶催化反应，明确ZmGPPS和Zm-monoTPS候选基因的体外酶活性；进一步用转基因手段在玉米中单独及共同超表达ZmGPPS和Zm-monoTPS，比较单萜释放量；以玉米-玉米螟-腰带长体茧蜂三级营养关系为模式，研究单萜对玉米螟的直接防御和对天敌行为的调控作用。研究结果将有助于阐明玉米单萜的合成调控机制及其在玉米抗虫中的作用。

单萜类化合物是植物体内的一类重要的次生代谢化合物，可作为植物-昆虫间的信号物质来吸引害虫天敌，也可作为植物源杀虫活性物质抑制害虫取食及生长发育，因此通过基因工程的方法培养高单萜释放量的玉米可以看做是一种低成本可持续的害虫防治策略。.本项目全面鉴定了玉米单萜合成过程中的两个关键基因GPPS和TPS，通过对单萜合成酶基因monoTPS在杂交种京科968以及其父本京92、母本京724进行基因克隆和进化分析，发现该基因在三个基因型中高度保守，可能与芳樟醇在植物中广泛分布及其重要功能有关；通过对monoTPS蛋白进行原核表达和生化功能测定，发现该蛋白能催化GPP生成芳樟醇、但不能催化FPP生成倍半萜烯，明确了该蛋白是一个单萜合成酶；通过在烟草中瞬时表达monoTPS蛋白，发现该蛋白在叶绿体中定位；通过在烟草对monoTPS基因过表达，以及在玉米中利用基因编辑技术对其进行沉默，进一步发现该基因的过表达与芳樟醇的合成有关；通过昆虫生测明确了高浓度芳樟醇抑制玉米螟生长发育，低浓度促进其发育；分离克隆了2个ZmGPPS基因，通过体外酶活试验时发现GPPS重组蛋白不能催化GPP的产生，但在烟草体内过表达可显著增加烟草柠檬烯等单萜的合成，推测GPPS1基因为小亚基基因；利用基因家族分析结合转录调控预测分析，筛选获得了2个参与玉米萜烯合成的代谢通路的关键的WRKY转录因子。.本项目对玉米ZmGPPS基因和monoTPS基因进行了系统研究，阐述了monoTPS基因催化芳樟醇合成的核心单萜合成酶的生化功能，获得了玉米GPPS.SSU基因，构建了单独沉默monoTPS基因的玉米植株，以及GPPS和monoTPS基因单独及二者共表达的烟草植株，为后续深入探索其生态功能及在病虫害防治中的潜在应用提供了基础。研究结果将有助于促进玉米害虫的可持续治理。