赤霉病菌中RNA沉默机制及其生物学功能的研究与利用

RNA-silencing pathway serves as an important regulator for viral defense, transposon silencing, heterochromatin formation and gene expression in almost all eukaryotes including fungi, plants and animals. Mechanisms of RNA silencing in fungi have been mainly explored in Neurospora crassa, but information on RNA-silencing in other fungi is very limited. Our previous studies have shown that the phenomena of RNA silencing is very clear in Fusarium graminearum, the mainly causal agent of what head blight. Genome-wide search showed that F. graminearum contains nine homologous components of RNA-silencing pathways. Thus, biological functions of each component will be investigated in current study. Since the application of RNA silencing to engineer transgenic plants resistant to various insects and pathogens has led to valuable outcomes, in this study, several dsRNA silencing vectors based on several key genes involved in mitosis in F. graminearum cells will be constructed and transferred in wheat cells, then transgenic plants with highly resistant to F. graminearum will be screened and identified in this study.

RNA沉默现象普遍存在于真菌、植物和动物等真核生物中，在抵御外源遗传因子、异染色质的形成、基因表达调控等方面发挥重要作用。目前人们对真菌RNA沉默机制的研究主要集中在粗糙脉孢霉，对其它真菌中RNA沉默机制研究很少。我们前期研究发现：小麦赤霉病菌中存在明显的RNA沉默现象，因此，本项目通过比较基因组学分析小麦赤霉病菌中RNA沉默途径，系统研究RNA沉默途径上各个元件的生物学功能及其相互关系。在此基础上，针对赤霉病菌细胞有丝分裂的多个关键基因，构建高效双链RNA（dsRNA）的沉默载体、并转入小麦中，进而筛选高抗赤霉病的小麦转基因材料。

禾谷镰刀菌含有9个参与RNAi途径的关键元件。利用同源重组技术构建了这些基因的敲除突变体，发现FgAgo1和FgDicer2在沉默过程中起关键作用，其中FgDicer2还参与禾谷镰刀菌中小RNA合成。在本研究测试条件下，未发现RNAi途径关键基因参与致病、生长、发育等过程。在禾谷镰刀菌中成功构建了dsRNA介导的沉默方法及沉默效果评价体系，并用于基因的功能分析。在解析500多个禾谷镰刀菌的基因功能基础上，筛选出能用于寄主诱导的基因沉默（HIGS）培育抗禾谷镰刀菌的靶基因FgSTE12、FgSGE1和FgPP1。利用基因枪法获得靶向禾谷镰刀菌FgSge1-Ste12-PP1的HIGS转基因小麦。接种试验表明，转基因小麦株系T1代的部分株系对禾谷镰刀菌的抗性水平显著提高。本项目的研究成果为后续利用HIGS技术培育抗赤霉病小麦品种建立了比较完善的技术体系。