水稻条纹病毒（RSV）编码的SP蛋白的结构与功能初探

发布时间：2018-01-04 20:17

  本文关键词：水稻条纹病毒（RSV）编码的SP蛋白的结构与功能初探　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 水稻条纹病毒 SP蛋白 纯化 互作

【摘要】：水稻是目前世界人口最重要的粮食作物之一,但是,水稻因为各种各样的病虫害导致的产量下降却屡见不鲜,其中包括严重影响我国水稻产量的一种单链RNA病毒——水稻条纹病毒(Rice stripe virus,RSV)。文献报道发现,水稻条纹病毒主要侵染水稻、烟草、甚至玉米等的叶片,使叶片出现枯色条纹状病变。关于水稻条纹病毒如何作用于宿主植物,包括,病毒蛋白如何通过宿主发挥其毒害作用的机制还不清楚。因此,开展关于水稻条纹病毒蛋白与宿主水稻蛋白之间的互作关系的研究具有十分重要的意义。研究发现,水稻条纹病毒的第四条RNA链正向控制编码的一种致病特异蛋白(disease-specific protein,SP)可能是参与病毒侵染植物,发挥其致病作用的一种关键蛋白。因此,本论文以该蛋白为研究重点,以期解析其结构;通过寻找该蛋白作用于宿主水稻的靶标蛋白,解析互作复合体的结构,从而分析其可能的致病机理。首先,论文采用大肠杆菌表达系统,体外表达SP蛋白,利用亲和纯化的方法,纯化获得该蛋白,并以期得到该蛋白的晶体,然后优化出最佳衍射数据的晶体,从而解析该蛋白的分子结构,分析其可能与宿主靶标蛋白互作的结构域或蛋白序列。其次,以新鲜的水稻叶片为材料,进行叶片全蛋白的提取,并将SP蛋白与MBP标签和GST标签融合表达。最后,利用pull-down技术,将纯化好的含有不同标签的SP蛋白与水稻叶片全蛋白进行孵育后,除去非特异性结合的杂蛋白,进行SDS-PAGE检测,银染显色,核磁鉴定互作蛋白的序列,并以期获得二者互作的晶体结构,分析互作的关键位点。结果显示,(1)体外表达获得了大量的纯度较高的SP蛋白;(2)戊二醛交联实验标明该蛋白可能存在通过自身互作形成三聚体;(3)报道称发现SP可与水稻叶绿体光合作用复合体Ⅱ中的PsbP蛋白互作,但根据等温滴定量热法(ITC)测定二者互作关系,未显示其可明显发生互作;(4)有报道标明,SP可能与某些短肽互作,但ITC结果未发现有明显的互作关系;(5)目前还未得到该蛋白的晶体衍射数据;(6)该蛋白与水稻叶片全蛋白的pull-down结果还未发现特异的互作蛋白。本论文采用以上方法获得的大量纯化较好的蛋白,为后面关于该蛋白功能的研究奠定了良好的基础,同时,也为进一步研究该病毒的其他蛋白的作用方式提供了新的思路。
[Abstract]:Rice is one of the most important food crops in the world at present. However, the decline of rice yield caused by various diseases and insect pests is common. It includes a single-stranded RNA virus, Rice stripe virus, which seriously affects rice yield in China. Rice stripe virus mainly infects the leaves of rice, tobacco, and even corn, causing the leaf to wither stripes. About how the rice stripe virus acts on host plants, including. The mechanism by which viral proteins play a toxic role through the host is unclear. It is of great significance to study the interaction between rice stripe virus protein and host rice protein. The 4th RNA strands of rice stripe virus may be involved in the infection of plants. It is a key protein to play its pathogenicity. Therefore, this paper focuses on this protein in order to analyze its structure. By looking for the target protein acting on host rice and analyzing the structure of the interaction complex, the possible pathogenic mechanism of the protein was analyzed. Firstly, the SP protein was expressed in vitro by the expression system of Escherichia coli. The protein was purified by affinity purification and the crystal of the protein was obtained. Then the crystal of the best diffraction data was optimized and the molecular structure of the protein was analyzed. The domain or protein sequence which may interact with host target protein was analyzed. Secondly, the whole protein was extracted from fresh rice leaves. The SP protein was fused with MBP tag and GST tag. Finally, the purified SP protein with different labels was incubated with the whole rice leaf protein by pull-down technique. The nonspecific binding proteins were removed for SDS-PAGE detection, silver staining, nuclear magnetic resonance (NMR) to identify the sequence of the interacting proteins, and to obtain the crystal structure of the interaction between the two proteins. The key sites of interaction were analyzed. The results showed that a large number of SP proteins with high purity were obtained. (2) the glutaraldehyde crosslinking test indicated that the protein might form trimer through self-interaction. It was reported that SP could interact with PsbP protein in rice chloroplast photosynthesis complex 鈪，

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