GmNAC2通过调控异黄酮的生物合成响应大豆虫害胁迫的机制研究

In today's agricultural production, various insect pests significantly affect the yield and quality of crops. Although the application of chemical pesticides can reduce the pests, this management technique inevitably causes serious pollution to the ecological environment. The use of host plant resistance to pest would contribute to integrated pest management in crops. Cultivating resistant germplasms may be a sustainable and economical approach for crops production. Soybean-specific secondary metabolites of isoflavones have been shown to play an important role in the insect resistance defense response of soybeans. However, the molecular mechanism of isoflavones in response to insect stress-induced synthesis is still unclear. In the early stage of the project, a transcription factor gene GmNAC2 that can both regulate isoflavone biosynthesis and response to soybean leaf-eating pest Spodoptera litura was identified, indicating that biosynthesis of isoflavones regulated by GmNAC2 is associated with soybean insect resistance defense response. Based on this, the project intends to obtain the GmNAC2 gene over-expression and knockout soybean material through the Agrobacterium-mediated stable cotyledon nodes genetic transformation system and then clarify the relationship between GmNAC2 regulation of isoflavone accumulation and insect resistance in soybean leaves. Meanwhile, combined with ChIP-Seq, dual luciferase reporter assay, electrophoretic mobility shift assay, yeast one-hybrid and co-expression to study the molecular network and mechanism of GmNAC2 gene in response to insect stress and regulation of isoflavone synthesis. The implementation of the project can provide a useful supplement to the molecular mechanism of the secondary metabolites acting on the defense response of herbivores.

当今的农业生产中，虫害严重制约着农作物的产量和品质。虽然化学农药的施用可在一定程度上减轻虫害，但不可避免地对生态环境造成严重污染。发掘利用作物自身的优异抗虫基因，培育持久高效的抗虫品种是农业科学家的当务之急。目前已证实大豆特有的异黄酮类次生代谢物在大豆抗虫防御反应过程中发挥重要作用。本项目前期鉴定到一个既可以调控异黄酮合成又响应大豆食叶性害虫斜纹夜蛾诱导的转录因子GmNAC2，暗示GmNAC2调控的异黄酮合成与大豆抗虫防御反应可能存在联系。本项目拟在此基础上通过大豆稳定遗传转化体系获得GmNAC2基因超量表达及敲除的大豆材料，明确GmNAC2调控大豆叶片异黄酮的积累与抗虫性的关系。结合染色质免疫沉淀测序、双荧光素酶检测、凝胶阻滞、酵母单杂交和共表达分析，研究GmNAC2基因响应虫害胁迫从而调控异黄酮合成的分子机制。该项目的实施可为解析次生代谢物作用于虫害胁迫防御反应的机制提供有益补充。

在影响大豆产量和品质的诸多因素中，各种虫害造成的损失尤为突出。虽然采用化学防治和耕作栽培措施等手段可以在一定程度上减轻虫害造成的产量损失，但无法从根本上解决虫害问题。充分发掘大豆自身的抗虫基因，减少化学杀虫剂的使用，培育具有持久高效抗虫的新品种，已成为大豆育种的迫切课题。 大豆特有的异黄酮类次生代谢物在大豆抗虫防御反应过程中发挥重要作用。本项目前期利用酵母单杂交的方法筛选到一个既调控异黄酮合酶基因GmIFS2又响应大豆食叶性害虫斜纹夜蛾诱导的转录因子基因GmNAC2。本项目以GmNAC2基因为研究对象，通过转基因技术获得了GmNAC2过表达株系和gmnac2突变体株系，采用双向选择和强迫喂养的方法对转基因材料进行了抗虫性的鉴定，结果显示GmNAC2基因的过表达降低了大豆对斜纹夜蛾的排趋性和抗生性，gmnac2突变体植株的抗虫性虽然有所升高，但是差异没有达到显著水平，推测可能是GmNAC2与其同源基因存在功能冗余。结合转录组测序（RNA-Seq）和DNA亲和纯化高通量测序（DAP-Seq）分析，共检测到1243个GmNAC2直接结合并调控的靶标基因，KEGG富集分析显示这些下游靶标基因显著富集于植物激素信号传导、植物-病原体互作、苯丙烷和异黄酮等防御代谢物合成途径，其中异黄酮合成途径的基因在GmNAC2过表达转基因株系中均下调表达。双荧光素酶报告系统分析验证了GmNAC2对异黄酮合成途径基因的表达主要起到抑制作用。利用反向酵母单杂交技术筛选获得了35个GmNAC2的结合基序。最后，广泛靶向代谢组学分析结果显示过表达GmNAC2可以抑制异黄酮等多种防御代谢物的积累。以上研究结果表明，GmNAC2负调控大豆对斜纹夜蛾的抗性是由该转录因子抑制异黄酮合成途径基因的表达从而降低异黄酮类化合物的积累导致的，通过同时敲除GmNAC2及其同源基因的方式有望获得大豆抗虫新品种，本研究揭示了GmNAC2通过调控异黄酮合成从而参与大豆虫害防御的分子机制，为抗虫基因工程育种提供了新基因。