玉米植保素kauralexin二萜合酶基因相关转录因子鉴定及调控机制解析

Kauralexin is the diterpenoid phytolexin produced by maize in response to pathogen infection and exhibits high anti-fungal activity. However, the metabolism and regulatory mechanism of kauralexin have not been clarified, which limits the application of kauralexin in breeding and agriculture. Phytoalexin accumulation is primarily correlated with gene expression level of their key biosynthetic enzymes. Based on the identification and characterization of key diterpene synthases in kauralexin biosynthesis, we propose to elucidate the regulatory mechanism of these diterpene synthase genes in this project. Firstly, the cis-element of diterpene synthase genes will be identified. Secondly, transcription factor candidates will be screened and transiently expressed in maize protoplast in which the diterpene synthase gene expression will be analyzed under overexpression or RNAi of these transcription factors. In addition, the binding of transcription factors with the promoters and cis-elements of these diterpene synthase genes will be analyzed further through transient expression in maize protoplast and tobacco leaf, yeast one-hybrid and EMSA. Finally, the transcription factors will be transformed into maize for overexpression or RNAi stably. In the mean time, the Uniform Mu mutant will be searched and acquired from MaizeGDB for these transcription factors. Kauralexin accumulation and diterpene synthase gene expression will be analyzed in the transgenic maize plants and mutants to elucidate the regulatory mechanism of these transcription factors. This research will lay the foundation for clarification of kauralexin metabolism and regulatory mechanism, and provide the theoretic evidence for elucidation of differential accumulation of kauralexin in inbred lines and application in resistance breeding.

Kauralexin是玉米受到病害侵染时产生的二萜植保素，具有良好的抗病原真菌活性，但其代谢调控机制不清楚，限制了其在育种和农业生产上的应用。植保素积累与其生物合成关键酶基因的表达量密切相关，本项目基于前期kauralexin生物合成关键二萜合酶功能的鉴定研究，进一步提出解析其基因表达的调控机制。拟首先确定kauralexin二萜合酶基因启动子关键顺式元件，筛选相关转录因子并在玉米原生质体中进行瞬时过表达和RNAi，分析其对二萜合酶基因表达的调控作用，进一步通过共转化瞬时表达、酵母单杂交和EMSA等方法研究转录因子与二萜合酶基因启动子的结合作用，最后通过玉米过表达和RNAi及突变体分析，解析转录因子对二萜合酶基因表达和kauralexin生物合成的调控机制，为全面阐明kauralexin代谢调控机制奠定基础，也为解析kauralexin的种群差异性积累机理并应用于抗性育种提供理论支撑。

病原菌侵染导致玉米产量与品质下降，威胁粮食安全。植保素是植物受到病原真菌侵染时产生的小分子次生代谢物，具有良好的广谱抑菌活性。本项目通过共相关分析，从多个转录组数据中进行筛选，结合同源搜索，鉴定了多个调控玉米植保素kauralexins生物合成基因表达的转录因子，并对调控分子机制进行了深入分析。. 以玉米植保素kauralexins合成途径中的限速酶基因An2作为诱饵基因，利用多种胁迫下玉米转录组数据进行共相关分析，筛选到多个高度相关的转录因子。通过玉米叶肉原生质体瞬时过表达，确定了ZmWRKY79、ZmEREB92与ZmMYC2对玉米植保素生物合成基因的正调控作用，进一步分析了三个转录因子对植保素合成基因启动子的激活作用，通过渐变缺失与定点突变确定了启动子的关键顺式作用元件，明确了三个转录因子与植保素合成基因启动子的结合情况，阐明了三个转录因子调控玉米植保素生物合成的分子机制。. 通过MAPK级联抑制剂处理，发现玉米叶片对禾谷镰刀菌抗性降低，植保素合成相关基因的诱导受到抑制，表明玉米植保素生物合成受MAPK级联调控。分析发现ZmWRKY79、ZmEREB92与ZmMYC2在玉米体受到磷酸化修饰，且磷酸化影响三个转录因子对下游植保素合成基因的调节。此外还发现玉米自交系黄早四中植保素含量显著高于Mo17，且其生物合成基因的表达也更高。通过分析两个自交系中植保素生物合成基因启动子，鉴定了其序列差异和顺式作用元件变异，明确了这些变异对植保素生物合成基因启动子活性、基因表达以及植保素积累的影响。.本项目成功鉴定了多个调控玉米植保素生物合成的转录因子，并解析了其调控机制；对不同自交系中植保素含量差异的机理进行了探究，明确了磷酸化对植保素代谢的调控，为深入理解玉米植保素代谢调控机理和全面解析植保素差异性积累及种群抗性差异提供了基础，也为抗性育种及绿色防控提供理论支撑。