【摘要】：煙草是我國重要的經濟作物,馬鈴薯Y病毒(potato virus Y,PVY)是煙草生產上的重要病害之一。近年來PVY在我國煙區(qū)爆發(fā)成災并日益嚴重,成為威脅煙草生產的重要因素之一。目前生產上對于PVY的防治沒有有效的方法,主要靠抗PVY煙草品種的選育。因此,挖掘煙草中的抗PVY的基因是抗病育種的關鍵。煙草NtTCTP基因是本實驗室前期研究中發(fā)現的與PVY抗性密切相關的基因,具有重要的研究價值。為了更深入地研究NtTCTP基因的抗PVY功能,本研究通過在室內室外兩個條件下對NtTCTP超表達株系和NtTCTP沉默株系進行PVY攻毒實驗來分析煙草NtTCTP的抗PVY功能。結果顯示,NtTCTP沉默后的煙草與對照相比,PVY發(fā)病速度和發(fā)病程度都顯著降低,室內人工接毒后只有20%左右的發(fā)病率,在田間更是基本沒有自然發(fā)病。NtTCTP超表達后不管在室內還是田間,煙草的發(fā)病速率和發(fā)病率都沒有明顯變化。以上結果表明,在煙草中沉默NtTCTP基因對PVY具有很強的抗性作用,該基因是煙草抗PVY的一個隱性基因。植物中eiF4E (真核起始因子4E)基因是目前唯一的抗PVY的基因,為了更好地了解煙草NtTCTP基因抗PVY的機理及與其他已知抗病基因的關系,本研究將煙草NtTCTP基因、eiF4E基因和PVY各蛋白基因都構建到酵母雙雜交載體上進行酵母雙雜交試驗來驗證煙草NtTCTP和eiF4E、PVY各蛋白之間的互作關系。結果顯示,NtTCTP和eiF4E之間并不存在互作關系,說明NtTCTP對PVY的抗性并不是通過與eiF4E進行互作來實現的,可能存在一個新的抗PVY途徑。煙草NtTCTP與PVY各個蛋白的互作實驗顯示,NtTCTP與PVY的CP(coat protein)存在互作關系。由于CP是病毒在細胞間擴散和在寄主體內長距離運輸的關鍵蛋白,因此推測NtTCTP是CP行使功能所必需的寄主互作蛋白,它的存在對于PVY的運輸是至關重要的。本研究表明,煙草NtTCTP對PVY表現出強抗性是由于與PVY的CP蛋白互作的關系,為進一步解析NtTCTP與PVY的互作機制奠定了基礎。
[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.
【學位授予單位】：浙江農林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.72

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