ZmEREB58转录因子在玉米虫害胁迫响应中的调控机制研究

Plants damaged by herbivorous insect emit a complex volatile blend that can attract natural enemies of herbivores and it is called indirect defense. The majority of the volatile compounds are sesquiterpene that are produced by terpene synthase. AP2/ ERF family is a large gene family of transcription factor involved in regulating plant development and stress response. The AP2/ERF transcription factor is an important defense response factor involved in different phytohormone regulation pathway. In our previous study we found ZmEREB58, an AP2/ERF transcription factor, regulated the transcript of tps10 gene and was induced by hormone jasmonic acid (JA) and ethylene (ET). However, little to nothing is known about ZmEREB58 role involved in JA- and ET-induced defense pathway and its function in indirect defense. In this project we will use the promoter-reporter system to determine the cis-element of ZmEREB58 promoter and then screen the candidate gene that regulates the transcript of ZmEREB58 using Yeast One Hybrid (Y1H), and the functions of both ZmEREB58 and upstream regulator gene in JA-induced pathway would be further studied. Based on the understanding of endogenous mechanism of herbivore resistance and the biotic stress defense regulation network, we hope to explore a new pest control way to combine the endogenous and exogenous stress defense method to achieve comprehensive plant protection.

植物在受到虫咬后会产生以倍半萜为主的挥发性物质，该类物质能够吸引害虫的天敌来杀灭害虫，被称为间接防御。萜类合成酶是催化倍半萜产生的关键酶。AP2/ERF转录因子通过不同激素途径参与植物虫害防御，是重要的逆境胁迫应答因子。在前期研究中发现玉米AP2/ERF类转录因子ZmEREB58调控玉米萜类合成酶基因tps10的表达，并受茉莉酸（JA）和乙烯（ET）的诱导。本项目将对ZmEREB58转录因子参与JA途径的虫害胁迫响应、参与间接防御进行分析。首先利用启动子-报告基因系统，确定调控ZmEREB58基因表达的顺式作用元件，再利用酵母单杂交方法获得调控ZmEREB58的上游调控基因，确定ZmEREB58和上游调控基因在JA信号途径中的功能。通过对玉米内源抗虫机制的研究，期望探索一条利用植物内源和外源相结合防治害虫新途径。

玉米受到害虫为害, 叶片会释放一系列萜类化合物吸引害虫的天敌，启动间接防御。玉米萜类合成酶基因TPS10可以催化产生法尼烯(E)-β-farnesene和香柑油烯(E)-α-bergamotene，而ZmEREB58基因是调控TPS10基因表达的转录因子。本项目通过对ZmEREB58启动子进行缺失分析，其功能区段包括两部分：-323至-270区段和-270至-183。将两段功能区段作为诱饵序列分别与拟南芥转录因子文库进行筛选，其中-270至-183区段筛选获得76个候选转录因子，MYB转录因子有22个，对这些候选基因正在鉴定中。对ZmEREB58基因的表达模式进行了分析，ZmEREB58在玉米的叶片中特异表达，受茉莉酸和乙烯诱导表达，在茉莉酸诱导途径阻断的MYC2A突变体中，EREB58不能表达，说明JA调控途径是ZmEREB58表达所必需的。为了进一步明确EREB58基因的功能，构建了EREB58基因过表达载体和CRISPR-Cas9基因敲除载体，获得了转基因玉米，EREB58基因的过表达和基因敲除之后，对玉米生长发育未见影响，将EREB58-OX(OVEREXPRESSION)、EREB58-KO(KNOCK OUT)及B73玉米进行了转录组分析，受EREB58基因影响，有39个转录因子表达量增高2倍以上，其中14个基因受JA调控上调表达，11个基因受JA调控下调表达； 有19个转录因子受EREB58影响表达下降，其中3个基因受JA调控上调表达，8个基因受JA调控下调表达，在受影响的转录因子中，WRKY、MYB和bHLH转录因子是受影响最大的转录因子类型，EREB58与WRKY、MYB和bHLH转录因子间的调控关系将继续研究。以上研究表明，EREB58是通过JA途径参与害虫防御，与多种转录因子级联调控萜类合成酶的表达量，继而影响次生代谢物的产生，调控着玉米的间接防御。