【作者】 张伟；

【导师】 谢从华； 田振东；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2015， 硕士

【摘要】 马铃薯是重要的粮食作物之一,致病疫霉Phytophthora infestans引起的晚疫病是马铃薯生产上威胁最大的病害之一。晚疫病效应子是病原菌在侵染过程中向植物体内释放的一类蛋白,它们通过与植物体内靶蛋白互作操控植物的抗病反应。本实验室与国外研究团队前期合作研究中筛选到一些重要晚疫病效应子,本氏烟草中瞬时表达这些效应子能够显著促进病原菌的侵染。本研究旨在通过农杆菌介导转基因技术获得这些效应子的马铃薯超量表达转基因材料,对效应子转基因材料的晚疫病抗病进行接种鉴定以及对flg22诱导的下游PTI maker基因进行分析,明确这些效应子在马铃薯中表达对马铃薯晚疫病抗性的影响。本研究主要结果如下:1.从晚疫病菌中克隆了7个效应子Pi17309、Pi09585、Pi13628、Pi04097、Pi04089、Pi04314、Pi02860基因,分别构建了效应子基因p RI101和p BI121植物表达载体;利用农杆菌介导法将这些效应子基因表达载体转入马铃薯受体材料鄂马铃薯3号(E3)中,通过PCR阳性检测和目标基因表达量分析,获得了其中6个效应子稳定表达的转基因株系:Pi17309(4个)、Pi09585(5个)、Pi04097(2个)、Pi04089(12个)、Pi04314(9个)和Pi02860(8个)。2.转基因株系晚疫病接种鉴定结果表明:Pi04089、Pi04314、Pi17309、Pi02860效应子转基因株系与对照E3相比晚疫病抗性明显下降;Pi04089、Pi17309、Pi02860部分转基因株系发病叶片孢子产量明显高于对照;而Pi09585、Pi04097转基因株系晚疫病抗性与对照E3相比无明显差异。3.为探究所得效应子转基因株系对PTI反应途径相关maker基因St WRKY7、St WRKY8、St Acre31的表达影响,分别在未处理前和flg22处理后0.5h进行表达量分析,结果表明:Pi04089和Pi04314转基因株系中未处理前maker基因的本底水平表达量明显低于对照E3,而flg22处理后的诱导表达量与对照相比并无明显差异;Pi02860转基因株系中未处理前maker基因的本底表达与对照E3无明显差异,但处理后的诱导表达量与对照相比明显被抑制;Pi09585、Pi04097转基因株系对flg22处理前后maker基因的表达均无抑制作用。4.对Pi17309转基因株系接种后60h St WRKY7、St WRKY8、St Acre31的表达分析结果表明:只有St WRKY7的表达与对照E3相比受到抑制,St WRKY8、St Acre31与对照E3无明显表达差异。本研究获得了6个晚疫病效应子基因稳定表达的马铃薯转基因株系,通过晚疫病接种和PTI相关maker基因分析初步明确了它们在马铃薯晚疫病抗性中功能,为更进一步研究这些效应子与植物靶蛋白的互作机理以及如何调控马铃薯免疫应答奠定了良好基础。更多还原

【Abstract】 Potato is the fourth crop in the world which plays an important role in food security,but the late blight caused by Phytophthora infestans has been the most destructive disease to potato crop.Plants develop two levels immune system,PTI and ETI.P.infestans can secretes an array of proteins by haustorium during its colonisation,known as “effectors”.Effectors can interact with host targets to suppress plant defense.In this research I focus on 7 P.infestans effectors that can suppress early flg22-triggered immunity and promote P.infestans colonisation in planta.We want to generate the effectors potato transgenic plants via Agrobacterium mediated stable transformation,and investigate their resistance function in potato by P.infestans inoculation and PTI maker genes expression analysis.The main results are as below:1.p RI101 and p BI121 vectors for overexpression of P.infestans effectors Pi17309,Pi09585,Pi13628,Pi04097,Pi04089,Pi04314 and Pi02860 were constructed,then 4,5,0,2,12,9 and 8 positive potato transgenic plants via Agrobacterium-mediated transformation were obtained,respectively.2.P.infestans inoculation results showed that Pi04089、Pi04314、Pi02860、Pi17309 transgenic plants developed larger lesions diameter and more sporangia harvest than E3 control,while Pi09585 and Pi04097 transgenic plants showed undetectable influence on potato late blight resistance.3.To further explore the effectors transgenic plants resistance function,three PTI maker genes St WRKY7、St WRKY8、St Acre31 expression were analysised before flg22 treatment and after 0.5h flg22 treatment: the maker genes were significantly suppressed in Pi040897 and Pi04314 transgenic lines compare to E3 while the expression of flg22-elevated maker genes expression were not affected.There was no difference between Pi02860 lines and E3 on the maker genes expression before flg22 treatment.In contrast,the flg22-elevated maker genes expression were significantly suppressed in Pi02860 transgenic lines.Suppression on the flg22-elevated marker genes expression were not observed in Pi04097 and Pi09585 transgenic plants.4.The maker genes analysis at 60 hours post inoculation showed only WRKY7 was suppressed on Pi17309 transgenic plants.In this research we obtain 6 effectors potato transgenic plants and confirm their preliminary function on potato late blight resistance,the results will perform a useful information for further research on the effectors interacting with their host target proteins and how their manipulate plants immune response.更多还原