MAPK级联调控大豆异黄酮类植保素合成的机制及其抗病应用

Soybean, a major source of protein and oil in our diet, is an important crop in our agriculture production. Because of lower yield, a large portion of our annual soybean consumption relies on import. One possible approach to increase soybean production in our country is to reduce the loss due to disease caused by various pathogens. Phytoalexins, secondary metabolites induced after plant sensing of invading pathogens, play essential roles in plant disease resistance because of their anti-microbial activity. Daidzein and genistein, two isoflavones, are major soybean phytoalexins involved in broad-spectrum disease resistance. Our previous research demonstrated that mitogen-activated protein kinase (MAPK) cascade is the major signaling pathway in the regulation of phytoalexin biosynthesis in Arabidopsis and tobacco. In this project, we are going to study the regulatory function of MAPK cascade in the induction of isoflavone phytoalexins in soybean. Specific goals include, 1) to generate transgenic soybean with the constitutively active GmMKK4 and GmMKK5 under the control of steroid-inducible promoter to determine whether the activation of downstream GmMPK3/GmMPK6 is sufficient to induce soybean isoflavone phytoalexins; 2) to utilize the pathogen-responsive phenylalanine ammonia-lyase (PAL) gene promoter-driven GmMKK4 and GmMKK5 to elevate the induction of phytoalexin biosynthesis to achieve broad disease resistance in the transgenic soybean; and 3) to identify the transcription factor(s) downstream of GmMPK3/GmMPK6 cascade in inducing the expression of soybean phytoalexin biosynthetic genes. This project will provide a new venue for the engineering of soybean plants with enhanced disease resistance, therefore increase yield. In addition, because of the nutraceutical values of daidzein and genistein, enhancing their synthesis by manipulating MAPK activity also holds the possibility of enhancing the nutritional values of soybean crop.

大豆是我国重要粮油作物，在国民经济中占有重要的地位。提高大豆的抗病性，对发展我国大豆生产至关重要。大豆黄酮及染料木黄酮这两种异黄酮物质是大豆最重要的植保素,与抗病性密切相关。前期在拟南芥和烟草中研究发现，植保素类次生代谢物是受丝裂原活化蛋白激酶 (MAPK) 级联信号调控。本课题拟对大豆的MAPK级联反应在异黄酮植保素合成中的作用进行研究。本研究将：1）利用诱导型启动子，在转基因大豆中表达组成型活化态GmMKK4/MKK5蛋白, 检测内源性底物GmMPK3/GmMPK6和类黄酮的合成量,明确MAPK级联信号是否及如何参与大豆植保素合成的过程。2）用大豆病菌侵害诱导表达的PAL启动子，在转基因大豆中表达活化态GmMKK4/MKK5蛋白，检测转基因大豆类黄酮的累积量、及其对大豆病菌的抗性。3）分离参与调控类黄酮合成基因的转录因子。本研究结果对培育具有广谱抗病性的转基因大豆提供新思路和新种质。

本项目旨在探究大豆的 MAPK 级联信号在异黄酮类植保素合成中的调控作用，明确 MAPK 级联信号是否及如何参与大豆抗病过程中植保素的合成。本研究运用农杆菌介导的大豆子叶节遗传转化法，成功构建了条件诱导型的MAPK功能获得型转基因大豆株系GmMKK4DD。DEX诱导GmMKK4DD表达后下游GmMPK3/GmMPK6级联信号被激活。利用该研究体系所获得的研究结果证明，大豆中GmMKK4-GmMPK3/GmMPK6级联信号调控了大豆异黄酮类物质的动态合成。利用高分辨液相色谱-质谱联用技术，我们发现激活该MAPK级联信号通路之后，苯丙素合成途径被激活，代谢特异流向大豆异黄酮类物质合成。刺芒柄花素和鹰嘴豆芽素A合成被显著诱导，大豆苷元和染料木苷元分别被促进转化为2’-羟基-大豆苷元和2’-羟基-染料木苷元。进一步分析结果表明，2’-羟基-大豆苷元继续被诱导衍生为大豆抗毒素及香豆雌酚，两者在DEX处理过的GmMKK4DD大豆叶片内大量累积。诱导GmMKK4DD表达后，上述途径中涉及的关键催化酶的编码基因的表达量明显上调。使用大豆疫霉菌中的病原菌效应子NLP1（Nep1-Like Protein 1）处理野生型大豆叶片，GmMPK3/GmMPK6信号通路被激活，同时大豆异黄酮合成途径发生类似GmMKK4DD转基因植株中的动态代谢变化。表明GmMKK4-GmMPK3/GmMPK6级联信号很可能是通过调节大豆异黄酮及其衍生物的代谢而调控了大豆的抗病过程。我们的研究结果为培育具有广谱抗病性的转基因大豆提供新思路。