除虫菊法尼烯（EβF）信号诱导瓢虫防御蚜虫的分子机制

Pyrethrum, an ornamental plant in Asteraceae, is famous for its natural pesticide pyrethrin. Previous research was mostly focused on the roles of the non-volatile pyrethrins to protect this plant against insect pests. However, the plant possesses another insect resistance pathway based on terpene volatiles which has not been reported until now. We have observed different intercropping ways between pyrethrum and fruit trees,vegetables and crops ,and pests on crops were reduced.Also in pyrethrum field ladybirds are attracted in large numbers to pyrethrum flower buds, despite the fact that hardly any aphids or other prey insects are present. Their presence however correlated with a very high content of E-β-Farnesene peaking in stage 2 flower (not fully opened buds). We hypothesized that pyrethrum has evolved a natural mechanism to enhance its reproductive success by recruiting ladybirds and dispersing aphids by E-β-Farnesene-based volatiles. Firstly, experiments to collect and detect headspace components from different pyrethrum organs during different developmental stages under both natural and stress conditions will be developed to analyse the emission of EβF. Moreover, insect behavior assays are combined to reveal the relationship among plant-aphid-ladybird beetle based on signal recognition of EβF in pyrethrum. Secondly, functional characterization of EβF Synthase is used to confirm the function of EβFS in vitro, combined with genetic engineering by silencing of EβFS in pyrethrum and overexpression of EβFS in chrysanthemum under control of the EβFS promoter. Thirdly, analysis of EβFS promoter activity is achieved in transgenic pyrethrum carrying ProEβFS::GUS constructs allowing the analysis of the subcellular localization of EβFS. It will help to determine precisely site of synthesis of EβF and the transcript regulation mechanism of EβFS. This work not only reveals a new insect resistance pathway based on volatiles in pyrethrum, but is also expected to reduce the usage of chemical pesticides by introducing such similar strategies into crop plants.

除虫菊是菊科观赏植物，以含天然杀虫剂除虫菊酯著称。前人研究主要集中于非挥发性的除虫菊酯，是否存在挥发物途径的抗虫尚无报道。我们前期调研到除虫菊与作物套种模式，发现可减少农作物害虫；田间除虫菊花期没有蚜虫但有大量瓢虫且检测到高丰度的挥发物法尼烯（EβF）。推测除虫菊存在基于EβF为主的挥发物引诱天敌防御害虫的新途径。本项目通过自然和胁迫下除虫菊不同发育阶段和组织的挥发物检测并结合昆虫行为实验，了解除虫菊基于EβF信号的植物-害虫-天敌关系；克隆除虫菊EβF合成酶基因，通过蛋白体外表达及除虫菊和菊花中沉默和超表达后挥发物检测和昆虫行为实验，明确其基因功能；对携带ProEβFS::GUS的除虫菊进行启动子活性分析并结合EβFS亚细胞定位明确EβF合成部位和响应特性。该研究对揭示除虫菊基于挥发物的新型抗虫机制有重要学术意义，且对开发利用植物自身防御能力，减少化学农药的施用等具有重要的产业环保意义。

除虫菊（Tanacetum cinerariifolium）花头不仅具有观赏价值，更是天然杀虫活性物质——除虫菊酯的植物源。前人研究多集中在除虫菊酯的提取工艺与合成代谢。课题组前期对除虫菊田间昆虫调查发现，瓢虫在没有食物源蚜虫的情况下，大量出现在除虫菊植株上。随后，花头挥发物测定发现花中含有高量的蚜虫报警素（E）-beta-farnesene（EβF）。本试验提出了除虫菊花头可能通过释放蚜虫报警素来吸引天敌，并趋避蚜虫的假设。通过除虫菊EbFS基因和启动子的克隆，表达分析和功能验证，克隆到两条除虫菊EβF合成酶同源基因序列EbFS1和EbFS2，分别编码575和577个氨基酸，属于萜类合酶家族，都催化FPP只产生EβF，并在细胞内的催化部位是细胞质。除虫菊EbFS基因在幼嫩的花器官表达量最高，且特异性地在花梗和花托部位高量表达。除虫菊EbFS基因2.2kb的启动子在靠近维管束的皮层细胞特异性表达。除虫菊EβF在自然条件和损伤处理后的释放规律表明，初花期除虫菊挥发物中EβF释放量最高，成为主要的萜类挥发物。除虫菊EβF释放受到发育阶段调控，并迅速地响应机械损伤。不同器官内含物提取和GC-MS分析发现，除虫菊EβF在花梗和花托大量合成，并暂时储存在皮层细胞。模拟含有EβF的蜜露对蚜虫行为的影响，利用含10 ng/μl EβF的人工蜜露，进行蚜虫报警行为实验，结果表明蜜露中EβF对蚜虫产生报警效果。本研究揭示了除虫菊-蚜虫-瓢虫三者生态关系， 首次揭示了除虫菊花器官中高量合成并释放蚜虫报警素EβF来吸引瓢虫，并趋避蚜虫的双重防御机制，有效地保护了除虫菊最重要的生殖器官花朵。该研究为以后利用植物源信息素进行生物防治和生态保护提供新的思路和手段。