茉莉酸和乙烯协同调控植保素scopoletin生物合成的抗病分子机制研究

Alternaria alternata is a pathogen causing severe losses of many economic important plants, while hosts will usually produce phytoalexin to counterattack. Our early experiments have demonstrated that wild tobacco (Nicotiana attenuata) plants produce large amount of courmarin, scopoletin, to defend against Alternaria alternata，and the biosynthesis of this phytoalexin is regulated synergistically by phytohormone Jasmonic acid （JA）and ethylene. However, the mechanism behind is still not clear. We hypothesized that host plants activated JA and ethylene signalling pathways to activate an unknown transcription factor TFx protein to increase the transcripts of F6’H1, the key scopoletin biosynthetic gene, through bingding to its promoter region. We are trying to identify the key transcription factor TFx, and the detail mechanisms of how JA and ethylene function in the controlling of scopoletin biosynthesis through transcriptome analysis, transgenic plants, yeast-one-hybrid, virus-induced gene silencing techniques, gel shifting and chromatin immuno-precipitation. This work will provide molecular insights into the regulation of phytoalexin scopoletin biosynthesis and molecular targets for crop disease control.

链格孢菌是一种能够感染多种农作物并造成严重产量损失的病原菌，而合成植保素是植物抵御病原菌的重要手段。预实验表明野生烟草(Nicotiana attenuata)受链格孢菌侵染后会产生植保素scopoletin，该植保素合成被证明是受茉莉酸和乙烯协同调控的，但机理未知。申请人提出假说：野生烟草受链格孢菌侵染时，会同时激活茉莉酸和乙烯信号，二者协同诱导转录因子TFx表达，TFx可能通过与scopoletin合成酶基因F6’H1的启动子互作来促进其表达，从而调控scopoletin合成来抵御链格孢菌侵袭。本项目拟利用转录组测序、酵母单杂交、病毒介导的基因沉默和稳定转基因等手段筛选由茉莉酸和乙烯协同诱导表达并调控scopoletin合成的转录因子TFx，并利用凝胶阻滞和染色质免疫共沉淀解析TFx与F6’H1启动子的关系，为揭示scopoletin生物合成的分子调控机理和农作物病害防治奠定基础。

我们的早期研究结果表明野生烟草在感染链格孢菌后能够产生抗菌次生代谢物scopoletin，F6’H1是scopoletin生物合成过程中的关键酶。野生烟草在感染链格孢菌后，激活了植物体内的茉莉酸和乙烯信号途径，F6’H1的表达和scopoletin的合成依赖于植物体内的茉莉酸信号途径和乙烯信号途径，并且受到茉莉酸和乙烯的协同调控。但是茉莉酸和乙烯是如何协同调控scopoletin的合成还不清楚。我们通过分析转录组数据, 利用茉莉酸和乙烯缺陷材料和外施茉莉酸和乙烯筛选到受乙烯和茉莉酸协同调控的转录因子DOG1-like3和WRKY70。病毒所介导的基因沉默实验（VIGS实验）和RNAi遗传转化实验表明F6’H1的表达在DOG1-like3和WRKY70基因沉默的植株中下调了，并且启动子激活实验也表明了DOG1-like3和WRKY70能够激活F6’H1启动子所启动的Luciferase基因的表达。以上实验为进一步解析茉莉酸和乙烯协同调控scopoletin合成的分子机制和烟草赤星病的生物防控奠定了基础，也为其他的农作物病害防治提供一定的参考。