本文選題：毛頭鬼傘　切入點：煙草花葉病毒　出處：《南昌大學》2017年碩士論文

【摘要】：煙草花葉病毒(Tobacco mosaic virus,TMV)是一種RNA病毒,具有廣泛的寄主,不僅能感染煙草等茄科植物,使它們引起花葉病,還能感染310種以上其他植物,因此,研究者都在迫切尋找煙草花葉病毒病的防治方法。相對于一些化學藥劑所帶來的環(huán)境污染和易對作物產(chǎn)生藥害等問題,探索天然產(chǎn)物用于煙草花葉病毒的防治,顯然是一條可持續(xù)發(fā)展途徑。近十多年來,不斷有研究工作者發(fā)現(xiàn)植物或真菌提取物對TMV具有抗性。Y3是一種能夠降低TMV侵染率的天然活性蛋白質(zhì),它是從真菌毛頭鬼傘(Coprinus comatus)中提取出的,有研究表明Y3蛋白對TMV的外殼蛋白(TMV CP)有體外鈍化的作用,使病毒蛋白不能順利合成,從而達到抑制煙草花葉病毒的作用。已有研究通過酵母雙雜交技術證明了Y3蛋白能與TMV CP在體外有相互作用,為進一步探究Y3蛋白與TMV CP是否在煙草細胞內(nèi)有相互作用且尋找互作位點,本研究采用雙分子熒光互補技術,首先將y3基因、TMV CP基因分別與雙分子熒光互補載體pSPYNE(R)173、pSPYCE(M)相連,成功獲得了融合表達載體pSPYNE(R)173-y3和pSPYCE(M)-CP,然后將他們分別轉化入農(nóng)桿菌GV3101中,利用滲透注射法導入煙草葉片下表皮細胞中,其后用激光共聚焦掃描顯微鏡觀察到煙草細胞內(nèi)有熒光信號,結果表明Y3蛋白與TMV CP在煙草細胞內(nèi)發(fā)生了相互作用。為進一步研究Y3蛋白與TMV CP的互作位點,利用蛋白質(zhì)序列分析軟件分析Y3蛋白的相關功能結構域,發(fā)現(xiàn)其第23到26位氨基酸是CK2磷酸化位點,從而利用PCR介導的定點突變技術,將與熒光載體pSPYNE(R)173相融合的Y3蛋白的第23位絲氨酸(TCA)突變?yōu)楸彼?GCA),再將獲得的突變體與融合載體pSPYCE(M)-CP共同導入煙草葉片下表皮細胞中,利用激光共聚焦掃描顯微鏡觀察到仍有熒光信號,證明此位點的突變沒有影響到Y3蛋白與TMV CP的相互作用,由此可判斷該位點可能不是這兩個蛋白的相互作用位點。以上結果表明,Y3蛋白與TMV CP在煙草細胞內(nèi)有相互作用,然而單獨突變CK2磷酸化位點并不能阻止Y3蛋白與TMV CP的互作,這為進一步研究其作用位點,了解Y3蛋白抑制TMV的作用機制奠定良好的基礎。
[Abstract]:Tobacco mosaic virus (TMV) is a RNA virus with a wide range of hosts. It not only infects tobacco and other plants in the family Solanaceae, causing them to cause mosaic disease, but also affects more than 310 other plants. Researchers are urgently looking for ways to prevent and cure tobacco mosaic virus disease. In contrast to the environmental pollution caused by some chemical agents and the problems of easily harming crops, researchers are exploring natural products for the prevention and treatment of tobacco mosaic virus. Over the past decade, researchers have found that plant or fungal extracts are resistant to TMV. Y3 is a naturally active protein that can reduce the infection rate of TMV. It was extracted from the fungus Coprinus comatus. Some studies have shown that Y3 protein can passivate the TMV coat protein in vitro, so that the virus protein can not be synthesized smoothly. It has been proved by yeast two-hybrid technique that Y3 protein can interact with TMV CP in vitro. In order to further investigate whether Y3 protein and TMV CP interact in tobacco cells and find interaction sites, the y3 gene TMV CP gene was first linked to the bimolecular fluorescent complementary vector pSPYNERNER173pSPYCEM by using bimolecular fluorescence complementary technique. The fusion expression vectors pSPYNE(R)173-y3 and pSPYCECE-CPwere successfully obtained, and they were transformed into Agrobacterium tumefaciens GV3101 respectively, and then introduced into tobacco leaf epidermal cells by osmotic injection. The fluorescent signals in tobacco cells were observed by confocal laser scanning microscope. The results showed that Y3 protein interacted with TMV CP in tobacco cells. In order to further study the interaction sites between Y3 protein and TMV CP, protein sequence analysis software was used to analyze the functional domains of Y3 protein. It was found that its 23 to 26 amino acids were CK2 phosphorylation sites, thus using PCR mediated site-directed mutagenesis, The 23 th position serine TCA of Y3 protein fused with fluorescent vector pSPYNE(R)173 was mutated to alanine GCA. The obtained mutant and fusion vector pSPYCE(M)-CP were introduced into tobacco leaf epidermal cells. The fluorescence signal was observed by laser confocal scanning microscope, which showed that the mutation at this site did not affect the interaction between Y3 protein and TMV CP. These results suggest that Y3 protein and TMV CP interact with each other in tobacco cells, but the single CK2 phosphorylation site can not prevent the interaction between Y3 protein and TMV CP. This will lay a good foundation for further study of its action site and understanding of the mechanism of Y3 protein inhibiting TMV.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S432.41

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