感病基因StDND1调节马铃薯抗晚疫病的分子机制研究

DND1 (defense no death), as susceptibility gene, is involved in a variety of pathogen infection processes in plants. Silencing of this gene in potato increases resistance (or decreased susceptibility) to Phytophthora infestans, but the reason behind it is not clear. ..In this study, biochemical methods and microscopy studies will be used to analyze the surface changes of RNAi::StDND1 leaves. The aim is to investigate after inoculation of wild type plants, RNAi::StDND1 silenced plants, the development of P. infestans spores on the plant surface and their molecular changes during the infection process. Also, the interaction proteins of StDND1 with P. infestans and the StDND1 related susceptibility genes will be identified and compared by yeast two-hybrid, co-immunoprecipitation, sequencing and bio-informatics. By combining these data with the RNA-seq data, we will try to explain how StDND1 silencing contributes to a very early disturbance of the interaction with spores of late blight. ..In addition, tetra-allelic loss of function mutation of StDND1 and its related genes will be induced by CRISPR/Cas9 technology in potato, selected and compared with StDND1 silenced potato. That result would be of advantage in research and development of agricultural biotechnology in China. This is probably more directly leading to acceptance of such tetra-allelic mutants which can be induced in existing potato varieties. The new susceptibility genes which identified in this stuy will enrich the breeding source for improved resistance.

DND1作为感病基因参与了多种病原菌侵染植物的过程。前期我们研究发现，沉默马铃薯中的DND1直系同源基因，可以使马铃薯提高对晚疫病的抗性，但对于其抗病机理却尚不清楚。本研究在前期工作的基础上分析沉默StDND1的马铃薯植株叶片表面生理变化，了解马铃薯晚疫病病原菌（致病疫霉）在植株表面的生存状况；采用酵母双杂、免疫共沉淀、测序技术和生物信息学分析确定该基因的互作蛋白，结合该基因沉默后的马铃薯相关材料的转录组数据，挖掘与StDND1相关的晚疫病感病候选基因并进行功能验证，全面解析由其介导的抗病调控途径。同时利用新一代的基因编辑技术CRISPR/Cas9，构建StDND1及其相关候选基因的CRSIPR/Cas9马铃薯植株。依据获得的实验结果，挖掘新的感病基因，丰富我国抗病育种的种质资源。

DND1作为感病基因参与了多种病原菌侵染植物的过程。前期我们研究发现，沉默马铃薯中的DND1直系同源基因，可以使马铃薯提高对晚疫病的抗性，但对于其抗病机理却尚不清楚。为明确StDND1基因参与致病疫霉侵染植物的阶段，明确感病基因StDND1参与致病疫霉侵染植物的分子机制，在项目的支持下，我们利用带有GUS和GFP标记的致病疫霉的生理小种分别侵染沉默StDND1的马铃薯植株，观察致病疫霉孢子在沉默StDND1的马铃薯植株上的生长情况，检测了病程相关基因的表达。此外，我们还以栽培种马铃薯Desiree叶片接种致病疫霉后的样品构建了酵母双杂交文库，筛选了与StDND1互作的蛋白。目前，本项目已经明确马铃薯的StDND1基因沉默后，可影响致病疫霉的孢子发育和吸附导致了其没有完成进一步的侵染；通过分析沉默StDND1的马铃薯植株接种晚疫病后植物防御及细胞坏死相关基因的表达，证明沉默StDND1后植物的防御能力有所提高，本研究成果在马铃薯抗晚疫病育种中具有一定的价值，丰富了马铃薯的抗晚疫病资源，为进一步在抗病育种中利用该感病基因提供了理论依据。