烟草NtTCTP基因抗马铃薯Y病毒功能及其分子机制初步研究

发布时间：2019-06-08 16:57

【摘要】：烟草是我国重要的经济作物,马铃薯Y病毒(potato virus Y,PVY)是烟草生产上的重要病害之一。近年来PVY在我国烟区爆发成灾并日益严重,成为威胁烟草生产的重要因素之一。目前生产上对于PVY的防治没有有效的方法,主要靠抗PVY烟草品种的选育。因此,挖掘烟草中的抗PVY的基因是抗病育种的关键。烟草NtTCTP基因是本实验室前期研究中发现的与PVY抗性密切相关的基因,具有重要的研究价值。为了更深入地研究NtTCTP基因的抗PVY功能,本研究通过在室内室外两个条件下对NtTCTP超表达株系和NtTCTP沉默株系进行PVY攻毒实验来分析烟草NtTCTP的抗PVY功能。结果显示,NtTCTP沉默后的烟草与对照相比,PVY发病速度和发病程度都显著降低,室内人工接毒后只有20%左右的发病率,在田间更是基本没有自然发病。NtTCTP超表达后不管在室内还是田间,烟草的发病速率和发病率都没有明显变化。以上结果表明,在烟草中沉默NtTCTP基因对PVY具有很强的抗性作用,该基因是烟草抗PVY的一个隐性基因。植物中eiF4E (真核起始因子4E)基因是目前唯一的抗PVY的基因,为了更好地了解烟草NtTCTP基因抗PVY的机理及与其他已知抗病基因的关系,本研究将烟草NtTCTP基因、eiF4E基因和PVY各蛋白基因都构建到酵母双杂交载体上进行酵母双杂交试验来验证烟草NtTCTP和eiF4E、PVY各蛋白之间的互作关系。结果显示,NtTCTP和eiF4E之间并不存在互作关系,说明NtTCTP对PVY的抗性并不是通过与eiF4E进行互作来实现的,可能存在一个新的抗PVY途径。烟草NtTCTP与PVY各个蛋白的互作实验显示,NtTCTP与PVY的CP(coat protein)存在互作关系。由于CP是病毒在细胞间扩散和在寄主体内长距离运输的关键蛋白,因此推测NtTCTP是CP行使功能所必需的寄主互作蛋白,它的存在对于PVY的运输是至关重要的。本研究表明,烟草NtTCTP对PVY表现出强抗性是由于与PVY的CP蛋白互作的关系,为进一步解析NtTCTP与PVY的互作机制奠定了基础。
[Abstract]:Tobacco is an important cash crop in China. Potato Y virus (potato virus Y, PVY) is one of the important diseases in tobacco production. In recent years, the outbreak of PVY in tobacco areas in China has become increasingly serious, which has become one of the important factors threatening tobacco production. At present, there is no effective method for the control of PVY in production, which mainly depends on the breeding of tobacco varieties resistant to PVY. Therefore, mining PVY resistant genes in tobacco is the key to disease resistance breeding. Tobacco NtTCTP gene is a gene closely related to PVY resistance found in our laboratory, which has important research value. In order to study the anti-PVY function of NtTCTP gene, the anti-PVY function of tobacco NtTCTP was analyzed by PVY challenge test on NtTCTP overexpressed lines and NtTCTP silent lines under indoor and outdoor conditions. The results showed that compared with the control, the incidence rate and degree of PVY in tobacco after NtTCTP silencing were significantly lower than those in the control, and the incidence of PVY was only about 20% after indoor manual exposure. There was no natural disease in the field. After the overexpression of NtTCTP, there was no significant change in the incidence rate and incidence of tobacco, both indoors and in the field. These results suggest that silencing NtTCTP gene has a strong resistance to PVY in tobacco, and this gene is a recessive gene resistant to PVY in tobacco. EiF4E (eukaryotic initiation factor 4e) gene is the only gene resistant to PVY in plants. In order to better understand the mechanism of PVY resistance of tobacco NtTCTP gene and its relationship with other known resistance genes, tobacco NtTCTP gene was used in this study. EiF4E gene and PVY protein gene were constructed on yeast two-hybrid vector to verify the interaction between tobacco NtTCTP and eiF4E,PVY proteins. The results showed that there was no interaction between NtTCTP and eiF4E, which indicated that the resistance of NtTCTP to PVY was not realized by interacting with eiF4E, and there might be a new anti-PVY pathway. The interaction between NtTCTP and PVY proteins in tobacco showed that there was an interaction between NtTCTP and CP (coat protein) of PVY. Because CP is the key protein for virus diffusion between cells and long distance transport in host, it is speculated that NtTCTP is a necessary host interaction protein for CP to function, and its existence is very important for PVY transport. The results showed that the strong resistance of tobacco NtTCTP to PVY was due to the interaction with CP protein of PVY, which laid a foundation for further analysis of the interaction mechanism between NtTCTP and PVY.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.72

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