抗病毒蛋白Y3与TMV CP的体内互作研究

发布时间：2018-03-29 22:25

本文选题：毛头鬼伞　切入点：烟草花叶病毒　出处：《南昌大学》2017年硕士论文

【摘要】：烟草花叶病毒(Tobacco mosaic virus,TMV)是一种RNA病毒,具有广泛的寄主,不仅能感染烟草等茄科植物,使它们引起花叶病,还能感染310种以上其他植物,因此,研究者都在迫切寻找烟草花叶病毒病的防治方法。相对于一些化学药剂所带来的环境污染和易对作物产生药害等问题,探索天然产物用于烟草花叶病毒的防治,显然是一条可持续发展途径。近十多年来,不断有研究工作者发现植物或真菌提取物对TMV具有抗性。Y3是一种能够降低TMV侵染率的天然活性蛋白质,它是从真菌毛头鬼伞(Coprinus comatus)中提取出的,有研究表明Y3蛋白对TMV的外壳蛋白(TMV CP)有体外钝化的作用,使病毒蛋白不能顺利合成,从而达到抑制烟草花叶病毒的作用。已有研究通过酵母双杂交技术证明了Y3蛋白能与TMV CP在体外有相互作用,为进一步探究Y3蛋白与TMV CP是否在烟草细胞内有相互作用且寻找互作位点,本研究采用双分子荧光互补技术,首先将y3基因、TMV CP基因分别与双分子荧光互补载体pSPYNE(R)173、pSPYCE(M)相连,成功获得了融合表达载体pSPYNE(R)173-y3和pSPYCE(M)-CP,然后将他们分别转化入农杆菌GV3101中,利用渗透注射法导入烟草叶片下表皮细胞中,其后用激光共聚焦扫描显微镜观察到烟草细胞内有荧光信号,结果表明Y3蛋白与TMV CP在烟草细胞内发生了相互作用。为进一步研究Y3蛋白与TMV CP的互作位点,利用蛋白质序列分析软件分析Y3蛋白的相关功能结构域,发现其第23到26位氨基酸是CK2磷酸化位点,从而利用PCR介导的定点突变技术,将与荧光载体pSPYNE(R)173相融合的Y3蛋白的第23位丝氨酸(TCA)突变为丙氨酸(GCA),再将获得的突变体与融合载体pSPYCE(M)-CP共同导入烟草叶片下表皮细胞中,利用激光共聚焦扫描显微镜观察到仍有荧光信号,证明此位点的突变没有影响到Y3蛋白与TMV CP的相互作用,由此可判断该位点可能不是这两个蛋白的相互作用位点。以上结果表明,Y3蛋白与TMV CP在烟草细胞内有相互作用,然而单独突变CK2磷酸化位点并不能阻止Y3蛋白与TMV CP的互作,这为进一步研究其作用位点,了解Y3蛋白抑制TMV的作用机制奠定良好的基础。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.41

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