本文關(guān)鍵詞：水稻條紋病毒（RSV）編碼的SP蛋白的結(jié)構(gòu)與功能初探　出處：《福建農(nóng)林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 水稻條紋病毒 SP蛋白 純化 互作

【摘要】：水稻是目前世界人口最重要的糧食作物之一,但是,水稻因?yàn)楦鞣N各樣的病蟲害導(dǎo)致的產(chǎn)量下降卻屢見不鮮,其中包括嚴(yán)重影響我國水稻產(chǎn)量的一種單鏈RNA病毒——水稻條紋病毒(Rice stripe virus,RSV)。文獻(xiàn)報(bào)道發(fā)現(xiàn),水稻條紋病毒主要侵染水稻、煙草、甚至玉米等的葉片,使葉片出現(xiàn)枯色條紋狀病變。關(guān)于水稻條紋病毒如何作用于宿主植物,包括,病毒蛋白如何通過宿主發(fā)揮其毒害作用的機(jī)制還不清楚。因此,開展關(guān)于水稻條紋病毒蛋白與宿主水稻蛋白之間的互作關(guān)系的研究具有十分重要的意義。研究發(fā)現(xiàn),水稻條紋病毒的第四條RNA鏈正向控制編碼的一種致病特異蛋白(disease-specific protein,SP)可能是參與病毒侵染植物,發(fā)揮其致病作用的一種關(guān)鍵蛋白。因此,本論文以該蛋白為研究重點(diǎn),以期解析其結(jié)構(gòu);通過尋找該蛋白作用于宿主水稻的靶標(biāo)蛋白,解析互作復(fù)合體的結(jié)構(gòu),從而分析其可能的致病機(jī)理。首先,論文采用大腸桿菌表達(dá)系統(tǒng),體外表達(dá)SP蛋白,利用親和純化的方法,純化獲得該蛋白,并以期得到該蛋白的晶體,然后優(yōu)化出最佳衍射數(shù)據(jù)的晶體,從而解析該蛋白的分子結(jié)構(gòu),分析其可能與宿主靶標(biāo)蛋白互作的結(jié)構(gòu)域或蛋白序列。其次,以新鮮的水稻葉片為材料,進(jìn)行葉片全蛋白的提取,并將SP蛋白與MBP標(biāo)簽和GST標(biāo)簽融合表達(dá)。最后,利用pull-down技術(shù),將純化好的含有不同標(biāo)簽的SP蛋白與水稻葉片全蛋白進(jìn)行孵育后,除去非特異性結(jié)合的雜蛋白,進(jìn)行SDS-PAGE檢測,銀染顯色,核磁鑒定互作蛋白的序列,并以期獲得二者互作的晶體結(jié)構(gòu),分析互作的關(guān)鍵位點(diǎn)。結(jié)果顯示,(1)體外表達(dá)獲得了大量的純度較高的SP蛋白;(2)戊二醛交聯(lián)實(shí)驗(yàn)標(biāo)明該蛋白可能存在通過自身互作形成三聚體;(3)報(bào)道稱發(fā)現(xiàn)SP可與水稻葉綠體光合作用復(fù)合體Ⅱ中的PsbP蛋白互作,但根據(jù)等溫滴定量熱法(ITC)測定二者互作關(guān)系,未顯示其可明顯發(fā)生互作;(4)有報(bào)道標(biāo)明,SP可能與某些短肽互作,但I(xiàn)TC結(jié)果未發(fā)現(xiàn)有明顯的互作關(guān)系;(5)目前還未得到該蛋白的晶體衍射數(shù)據(jù);(6)該蛋白與水稻葉片全蛋白的pull-down結(jié)果還未發(fā)現(xiàn)特異的互作蛋白。本論文采用以上方法獲得的大量純化較好的蛋白,為后面關(guān)于該蛋白功能的研究奠定了良好的基礎(chǔ),同時(shí),也為進(jìn)一步研究該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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