抑制赤霉菌RNAi分子的筛选及抗小麦赤霉病功能研究

Wheat Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease in China, causing huge losses of yield and producing various mycotoxins highly toxic to humans and domostic animals. Breeding wheat for Fusarium head blight resistance is a worldwide hard nut to crack, and the main bottle neck for that is the dearth of resistance germplasm. In this study, key genes involved in virulence and development of F. graminearum will be selected as RNAi-targeting genes. DNA fragments from the selected genes will be constructed in fungal RNAi vectors and transformed into F. graminearum. Fungal transformants expressing RNAi molecules and displaying impaired growth and development compared with non-transgenic wild type strain will be selected. These RNAi molecules with inhibition activity against F. graminearum will be constructed in wheat expression vectors under regulation by constitutive promoter or tissue-specific or pathogen-inducible promoters. Wheat varieties with high transformation efficiencies will be transformed with resistant RNAi and combinations of different RNAi molecules derived from different genes or pathways of F. graminearum via Agrobacterium-mediated method. Inoculation of seedlings and spikes of the transgenic wheat plants with F. graminearum will be performed and their resistance to initial infection, fungal spreading and mycotoxins will be evaluated. Fungal colonization and growth on transgenic wheat will be visualized by confocal microscopy and small RNAs within the fungi coming from host cells will be analyzed during in vitro culture and different passages; correlation between disease resistance and levels of transgene expression and target gene transcripts will be analyzed. Combined data will be used for dissection of resistance mechanism. Completion of this project would provide excellent resistant genes for breeding wheat varieties against FHB; this may generate a breakthrough of novel resistance germplasms and their application for FHB breeding in wheat.

小麦赤霉病是我国小麦生产上的重要病害，不仅严重降低产量，还产生危害人、畜健康的毒素。小麦抗赤霉病育种为世界性难题，其主要瓶颈是抗病资源匮乏。本研究从赤霉菌致病发育关键基因中，选择RNAi靶基因，构建RNAi分子表达载体，首先在赤霉菌中表达，筛选抑制赤霉菌RNAi分子，分析靶基因表达及抑菌特点；然后将具有抑菌功能的RNAi分子，构建到小麦组成型和组织特异及病菌诱导型启动子下，经农杆菌介导转化高转化率模式小麦品种，接种鉴定表达RNAi分子小麦的赤霉病抗性；分析抗病植株上的赤霉菌生长发育特征、赤霉菌中小RNA传递规律，以及RNAi分子和靶基因表达量与小麦赤霉病抗性和籽粒毒素残留量的相关性；研究不同RNAi分子抗侵入、抗扩展和抗毒素的综合能力及其抗病机理。本项目的完成，将为小麦抗赤霉病新品种培育提供优良抗病新基因，可望在小麦抗赤霉病新资源开发和应用上取得突破性进展。

本研究从赤霉菌致病发育关键基因中，选择了与细胞壁合成相关的重要基因Chs7，构建了7个RNAi分子表达载体，首先在赤霉菌中表达，筛选到了抑制赤霉菌的RNAi分子4个；挑选2个抑制赤霉菌生长表型明显的Chs7-3和Chs7-4串联构建双片段干扰载体，发现Chs7-3和7-4串联后抑菌效果高于单片段干扰载体；将Chs7-4构建细菌表达载体，大量表达纯化dsRNA处理小麦品种X76后接种，植株病小穗率降低73%；将Chs7-3和Chs7-4串联以茎环形式构建了2种植物表达载体，分别构建到小麦组成型和组织特异及病菌诱导型启动子ubiquitin和lem2下，经农杆菌介导转化小麦品种Y158，转ubiquitin-Chs7-3-Chs7-4的小麦植株在T3代稳定遗传，接种鉴定获得2个赤霉病抗性增加的株系；转Lem2-Chs7-3-Chs7-4的小麦植株在T5代稳定遗传，接种鉴定获得3个赤霉病抗性增加的株系，且这3个株系T6代种子的毒素含量分别比野生型降低92%，87%，53%。本项目的完成具有重要意义，可为小麦抗赤霉病新品种培育提供优良抗病新基因和种质资源。