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摘要：番茄斑萎病毒（Tomato spotted wilt virus, TSWV）寄主范围广，感染TSWV的植物多为系统性侵染，植株矮化，叶片皱缩且不对称生长，叶片上可见坏死环斑或不规则的坏死区，严重时整个叶片甚至整个植株坏死呈萎蔫状。前期研究发现瞬时表达TSWV编码的核衣壳蛋白（N）可导致寄主产生类似病毒侵染的系统性坏死症状。为深入探究TSWV导致植物产生系统性坏死症状的机制，本实验以TSWV N及其导致细胞死亡的关键区域N-N∆46C∆82为诱饵，利用酵母双杂交的方法筛选本氏烟（Nicotiana benthamiana）cDNA文库以获得与二者互作的蛋白。经过多次筛选，TSWV N暂未筛到阳性克隆；TSWV N-N∆46C∆82共筛到3个阳性克隆，测序分析后均为Nicotiana tabacum F-box protein FBW2-like，推测N-N∆46C∆82可能与寄主F-box类蛋白互作从而干扰其调节功能进而导致坏死症状。37270
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毕业论文关键词：番茄斑萎病毒；核衣壳蛋白；酵母双杂交；蛋白与蛋白相互作用
Screening of the Host Factors Interacting with Nucleocapsid Protein N of Tomato spotted wilt virus
Abstract: Tomato spotted wilt virus (TSWV) has a wide host range and always causes systemic infection. The infected plants are dwarf with shrunk and asymmetric leaves. In addition, there are always necrotic spots and irregular necrotic areas in the leaves of infected plants. . The entire leaf or even the whole plant is wilted and dead sometimes, when the disease is serious. The previous study showed that transient expression of the nucleocapsid protein (N) of TSWV resulted in systemic necrosis in Nicotiana benthamiana plants as similar to the plants infected with the virus. In this study, we used yeast two-hybrid method to screen the cDNA library of Nicotiana benthamiana to obtain interacting proteins with TSWV N and N-N∆46C∆82 (the key region for N caused cell death). After several screening, no positive interactors of TSWV were found. Three positive clones of TSWV N-NΔ46CΔ82 were found and all of them were found to be Nicotiana tabacum F-box protein FBW2-like. It is possible that TSWV N-NΔ46CΔ82 interacts with the host F-box proteins to interfere with its regulatory function and thus lead to the necrosis symptom.
Key words: Tomato spotted wilt virus; nucleocapsid protein; yeast two-hybird; protein-protein interaction
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法…2
1.1 材料 2
1.1.1 菌株、质粒、目的片段、文库2
1.1.2 培养基2
1.1.3 生化及分子生物学试剂…2
1.2 方法 2
1.2.1 质粒的提取3
1.2.2 目的片段的回收3
1.2.3 目的片段和载体酶切与回收…3
1.2.4 目的片段和载体的连接和转化4
1.2.5 菌落PCR并提取阳性菌株的质粒…4
1.2.6 PCR验证、扩增、测序分析…4
1.2.7 酵母感受态细胞的制备…4
1.2.8 诱饵质粒转化到酵母细胞5
1.2.9 文库杂交筛选阳性克隆…5
1.2.10 猎物蛋白质粒提取…5
1.2.11 文库质粒和诱饵质粒共转化到酵母细胞验证…5
2 结果与分析…5
2.1 重组诱饵蛋白载体…6
2.2 载体及诱饵蛋白自激活性检验6
2.3 文库筛选结果6
2.4 酵母共转化验证…8
3 讨论9
3.1 酵母双杂交系统…9
3.2 共转化验证中对照的设立…9
3.3 文库筛选结果分析9
3.4 存在的问题及解决措施9
3.4.1 TSWV N可形成多聚体结构9
3.4.2 培养基选择压力10
3.4.3 实验方案中需注意的问题10
3.5 后续研究计划…10