【作者】 苏晓峰；

【导师】 程红梅；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 棉花黄萎病（Verticillium Wilt of cotton）是一种土传、维管束系统性病害，被称为“棉花癌症”，给棉花的产量和棉纤维的质量都造成很大影响，其致病菌为大丽轮枝菌（Verticillium dahliae）。由于大丽轮枝菌极易产生新的生理小种，给研究带来相当大的难度和很多的不确定性，因此对大丽轮枝菌的研究一直以来都是热点。本文采用寄主诱导的基因沉默技术（Host-induced genesilencing），以高致病力的大丽轮枝菌株V991为实验材料，构建一系列针对大丽轮枝菌靶标基因的烟草脆裂病毒（tobacco rattle virus，TRV）干扰载体。通过农杆菌注射的方法转化本氏烟草（Nicotina benthamiana），并进行大丽轮枝菌接种，统计病情指数以及抗性反应级，并通过分子生物学手段检测真菌生物量以及靶标基因的转录水平，期望筛选到病原菌致病与生长发育的关键基因。本研究的主要结果如下：1、根据已公布的大丽轮枝菌数据库中的蛋白质序列，按照生物学功能进行分类，大致分为能量代谢相关、蛋白质代谢相关、DNA复制相关以及RNA转录相关等12类；从每组中挑选5-10条序列作为靶标序列，构建92个TRV干扰载体，并转化至农杆菌GV3101；2、建立寄主诱导的基因沉默（Host-induced gene silencing，HIGS）体系并筛选候选基因。首先优化了农杆菌注射浓度以及病原菌接菌最佳时间。将构建的92个TRV干扰载体通过农杆菌注射本氏烟草，然后接种病原菌后调查病情指数。与对照组相比，注射含有靶标基因片段农杆菌的植株，病情指数均有所降低，抗性反应型都有所提高。其中，病情指数显著降低与抗性反应级明显提高的基因共有20个，主要为糖代谢、能量代谢与蛋白质代谢相关的基因。3、对腺苷酸激酶（adenylate kinase，AK）、ADP，ATP载体蛋白（ADP,ATP carrier protein，AACP）和寡糖基转移酶STT3亚基（oligosaccharyl transferase STT3subunit，OST STT3）进行分子水平检测。通过生物量测定发现，与对照组相比，转入靶标基因的本氏烟草根、茎和叶中生物量明显减少，下降幅度均达到50%以上，这一结果与叶片中的台盼蓝染色结果相一致；通过对植物体内寄生的病原菌靶标基因的转录水平进行检测发现，靶标基因的转录水平都明显下降，对照组中靶标基因的表达量分别为干扰组的3.5倍（AK），3.7倍（AACP）和6.9倍（OSTSTT3）。4、构建AK、AACP和OSTSTT3稳定遗传的Gateway干扰载体，转化本氏烟草，目前已获得部分阳性植株。本实验利用HIGS技术在本氏烟草内进行大丽轮枝菌致病与生长发育关键基因的筛选，摸索一种更加直接、细致的观察植株与真菌互作、统计病情指数的新方法，并从分子生物学层面上进行验证方案的可行性，为农作物病虫害的防治提供了一定的实验数据和理论基础。更多还原

【Abstract】 Verticillium Wilt of cotton, named “cotton cancer”, is a kind of soil-borne, systematic vasculardisease, which produces the heavy influence of cotton production and cotton fiber qualify. Thepathogenic fungus was Verticillium dahliae. The V. dahliae was easy variation to generate newphysiological strains, which brought enormous difficulties and nondeterminacy. The research of V.dahliae has being a hot topic for a long time. The study was used host-induced gene silencing (HIGS)technology and the highly virulent strain V991as experimental materials. A series of tobacco rattlevirus (TRV) RNA interference (RNAi) vectors were constructed aiming to fungal targeting genes. TheRNAi vectors were transformed into the seedlings of Nicotina benthamianaby agroinfiltration. Then theV. dahliae spore was inoculated into the plants. In the end, the disease index and reactive type wasrecorded and statistics. Meanwhile, the fungal biomass and transcriptional level of targeting level wasdetected by molecular biological way. The ultimate goal is to get the key developmentalandpathogenicgenes.The main results of this research were listed as follows:1. The statistics of the published the peotein sequence of V. dahliae in database. According to thebiological function, the sequences were classified into12groups according the biological function,including energetic metabolism, protein metabolism, DNA replication, RNA transcription, and so on.The5-10sequences from different groups were selected and cloned as targeting genes from the V991transcriptome. The92TRV RNAi vectors were constructed and transformed into Agrobacteriumtumefaciens strain GV3101.2.The host-induced gene silencing (HIGS) platform was built to select the targeting genes. Firstly, theconcentration of injected Agrobacterium and the inoculated time of V. dahliaewere optimized.Afteragroinfiltration of92TRV RNAi vectors and fungal inoculation, the disease index was statistics.Compared to the control groups, the disease index reduced in the plants injected with the Agrobacteriumcontaining targeting genes. Meanwhile, the resistant active type was improved. Among them, the20genes were significant resistance to V. dahliae, mainly including glycometabolism, energy metabolism,protein metabolism, and so on.3.The AK (adenylate kinase), AACP (ADP,ATP carrier protein) and OST STT3(oligosaccharyltransferase STT3subunit) was further studied. Compared to the negative groups, the fungal biomass ofthe RNAi groups significantly decreased in root, stem and leaves, up to50%. These results wereconsistent to the trypan blue staining. Meanwhile, the transcriptional level of targeting genes wasremarkably higher in negative groups,3.5fold (AK),3.7fold (AACP) and6.9fold (OST STT3)respectively.4. To get the transgenic plants, the Gateway RNAi vectors were constructed and transformed into N.benthamiana. The research was focused on obtaining the key developmental and virulent genes in virtue of HIGS.A new way to observe the plant-pathogen interaction directly and record the disease index was exploredand detected by the molecular analysis. This study provided the experimental dataand theoretical base toprevent the agricultural pest and disease.更多还原