玉米EIN3转录因子调控对玉米螟抗性机理研究

Maize is an important grain crop and benzoxazinoids (Bxs) are the most important chemicals defensing against a broad range of herbivores in maize. Yet, little is known how maize regulates Bxs biosynthesis against herbivores. We created transgenic maize lines which are insensitive to ethylene (ETR1) and these plants showed increased defense against Ostrinia furnacalis, and Bxs induced by O. furnacalis is higher in the ETR1 lines than that in wild-type plants. By phylogenetic and expression analysis and protoplast transformation, we identified the candidate EIN3 gene in maize, which is the key regulator in ethylene signaling, and we found that it negatively regulates Bxs biosynthesis. In this project, we aim to identify proteins and genes regulated by EIN3 via yeast two-hybrid and chromatin-immunoprecipitation sequencing (ChIP-seq) methods. How the above-mentioned proteins and genes regulate Bxs will be investigated by Co-immunoprecipitation assays and promoter-GUS activity assays, respectively. We will further overexpress and down-regulate EIN3 in maize. Using wild-type and these transgenic maize, Bxs biosynthesis and volatile release and the performance of O. furnacalis will be determined, and the behavior of Trichogramma ostriniae in the lab and field condition will be analyzed. Taken together, this work will provide knowledge on the biochemical molecular mechanisms and chemical ecology of how EIN3 and ethylene signaling regulate direct and indirect defense response of maize against O. furnacalis.

玉米是重要的粮食作物，丁布类物质是玉米中的广谱抗虫化合物，而玉米如何调控丁布的合成以应对虫害胁迫尚缺乏深入研究。我们发现乙烯途径受阻转基因玉米（ETR1）增强了对玉米螟的抗性，且玉米螟诱导的丁布含量在ETR1玉米中高于野生型。通过基因表达、进化分析及原生质体转化技术，我们初步确定了乙烯信号途径核心转录因子EIN3，发现其负调控丁布的合成。我们拟使用酵母双杂交、ChIP-seq结合RNA-seq技术，获得EIN3调控的蛋白及基因网络，并通过免疫共沉淀技术及启动子激活GUS活性分析，解析EIN3调控丁布合成的生化机理。此外，我们将制备EIN3过表达和基因沉默的转基因玉米，与野生型玉米比较分析：1）丁布含量及挥发物释放的变化，2）对玉米螟抗性，3）田间和实验室条件下对玉米螟赤眼蜂的吸引力，进而从生化分子机理和化学生态学层面上剖析乙烯信号途径中转录因子EIN3如何调控玉米对玉米螟的直接和间接抗性。

通过CRISPR-Cas9技术，敲除了EIN3两个同源基因，获得了突变体植株，其对乙烯诱导的三重反应不敏感。ein3突变体在本底水平，负调控丁布主要合成酶的表达和丁布类化合物积累，和对玉米螟的抗性。通过ein3突变体的转录组筛选，原生质验证，我们发现EIN3正向调控转录因子ERF87，而ERF87负调控丁布主要合成酶的表达和丁布类化合物的积累。我们还发现在原生质体中，EIN3在调控主要丁布合成酶BX10的活性时，和茉莉酸信号途径核心转录因子MYC2有协同作用，且通过酵母双杂和双分子荧光发现EIN3和MYC2存在直接蛋白互作。以突变体为材料，发现EIN3也调控吲哚上游合成基因IGPS1的表达。通过ChiP-PCR发现EIN3可以直接结合IGPS1启动子区域，调控其基因表达，进而介导对丁布的正向调控。以ein3突变体为材料，发现ein3在诱导后正向调控EREB58、TPS10等基因，与之对应的是，ein3突变体玉米受虫害诱导后的重要萜类挥发物，如芳樟醇、DMNT的释放量较野生型下降，同时也降低了对天敌寄生蜂的吸引。.我们还发现玉米中的ZmMPK6通过正调控乙烯的合成，负调控主要丁布的合成，进而负调控玉米的诱导抗虫性。综上，玉米乙烯信号调控的诱导抗虫性，在本底水平或者较低乙烯浓度下，通过上游的ZmMPK6和EIN3调控的下游转录因子ERF87负调控丁布的合成积累，而在高浓度乙烯或者虫害诱导后，则通过和MYC2转录因子及吲哚上游关键基因IGPS互作，正向调控丁布的积累和对害虫的抗性反应，表现出浓度依赖型的调控模式。本研究丰富了乙烯信号途径调控的诱导抗虫分子机理。我们还参与研究了我国主栽商品化玉米在田间对玉米螟的间接诱导抗性，发现其在田间表现出较好的直接和间接诱导抗虫性，为玉米分子育种，提供理论支撑。