转录因子MYC2调控玉米抗虫代谢物丁布合成和抗虫性的分子机理研究

Benzoxazinoids (BXs) are a class of indole-derived plant chemical defenses comprising compounds with a 2-hydrooxy-2H-1,4-benzoxazin-3(4H)-one skeleton and their derivatives. These phytochemicals display antifeedant and insecticidal activities in maize, the important cereal crop. Although their effects against insect herbivores are well-known, how their biosynthesis is regulated remains unclear. Jasmonate signaling plays important roles in plant-herbivore interaction, in which MYC2 is the essential transcript factor controlling the expression of most jasmonate-responses genes. BXs are highly induced in maize after herbivore attack and jasmonic acid treatment. Furthermore, our preliminary work show that over-expression of MYC2 in maize protoplasts increased the concentrations of several BXs. Thus, we hypothesize that MYC2 regulates the concentrations of BXs by transcriptionally activating BX biosynthesis genes, and thus increasing the resistance to herbivores. To test this, we will silence the two MYC2 homologs, ZmMYC2-1and ZmMYC2-2, by stably-transforming the maize inbred A188 line with an invert-repeat construct. In the silenced plants, the resistance against Mythimna separatai and Ostrinia furnacalis, two main insect herbivores in maize, as well as the BX concentrations will be determined. Given that MYC2-binding motifs exist in the promoters of several BX biosynthesis genes, MYC2 likely binds directly to the promoters of these BX biosynthesis genes and activates their transcription. We will use multiple DNA-protein interaction assays to test this speculation. Furthermore, yeast two-hybrid screening will be employed to identify the proteins interacting with ZmMYC2 to explore other possible regulatory mechanisms of BX biosynthesis. This study will expand our knowledge on the mechanism underlying the regulation of BX production and provide a basis for breeding new insect-resistant maize lines.

丁布是玉米中重要的抗虫次生代谢物，调控其合成的分子机理还很不清楚。茉莉酸信号途径在植物抵御昆虫取食中发挥重要的功能，而转录因子MYC2是调控茉莉酸下游大多数基因表达的关键转录因子。前期的研究表明，昆虫取食及外源喷施茉莉酸均能使玉米中的丁布含量增加，由此我们推测，玉米MYC2蛋白通过调控丁布的合成提高植物的抗虫性。我们前期在玉米原生质体中的工作发现，过表达MYC2蛋白导致多种丁布类物质的含量显著上升10-30%，并且MYC2蛋白结合在多个丁布合成基因的启动子区并上调其表达。本项目中，我们将通过在玉米自交系A188中同时沉默两个玉米MYC2同源基因并测定沉默植物对玉米重要害虫粘虫和玉米螟的抗性及其中的丁布含量，以证实我们的推测。同时，我们还将通过核酸-蛋白及蛋白-蛋白互作分析解析玉米MYC2蛋白调控丁布合成的分子机理。该研究将为丁布的利用及玉米害虫防治奠定理论基础并提供新的思路。

茉莉酸信号途径中的核心转录因子MYC2在玉米抵御咀嚼式昆虫取食的过程中如何发挥作用还很不清楚。本项目利用CRISPR-Cas9技术，敲除了玉米中的两个MYC2同源蛋白MYC2a和MYC2b，获得了这两个蛋白的单突变体及双突变体。项目组首先检测了以上突变体对玉米的主要害虫粘虫及草地贪夜蛾的抗性，发现myc2双突变体对这两种害虫的抗性大大降低。进而，项目组比较了突变体及野生型植物被昆虫取食后，重要抗虫物质丁布类化合物的含量及其合成基因的表达情况，发现在MYC2突变体中昆虫取食诱导的多种丁布类化合物的含量大大降低，已知的14个丁布合成基因均受到MYC2的调控，特别是负责合成对咀嚼式昆虫强毒性的HDMBOA的BX10、BX11，其昆虫取食诱导的表达完全依赖MYC2。进一步的染色质免疫共沉淀等实验表明，MYC2通过与BX10及BX11的启动子结合，直接调控其转录。此外，在myc2双突变体中，具有吸引昆虫天敌作用的萜烯类挥发物（E）-α-佛手柑和（E）-β-法呢烯不能被检测到，其合成基因TPS10被昆虫取食处理后也不能被诱导表达。转录组分析表明共有1118个基因受昆虫取食诱导表达的基因被MYC2调控，MYC2调控的基因主要参与色氨酸合成与代谢、分支酸合成与代谢、防御与机械损伤响应、茉莉酸合成代谢及信号通路、吲哚烷基胺合成代谢等生物学过程。受MYC2调控的基因中有7.0%属于转录因子，其中bHLH115在玉米原生质体中过表达后多种丁布类化合物的含量上升。以上结果表明玉米MYC2a及其功能互补蛋白MYC2b在玉米抵御咀嚼式昆虫如粘虫及草地贪夜蛾的过程中，通过与一些下游转录因子形成转录调控分级网络，调控重要的抗虫次生代谢物丁布类物质及萜烯类挥发物的合成，发挥至关重要的作用。这些结果深化了人们对丁布类物质及萜烯类挥发物合成调控的理解，为玉米害虫的防控奠定了重要的理论基础。