【作者】 薛杨；

【导师】 安德荣； 李明福；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2006， 硕士

【摘要】 我国现有的经过准确鉴定的葡萄病毒和类病毒只有9种,而世界上现有的葡萄病毒和类病毒共有55种,对于我国来说,相当一部分的葡萄病毒病都属于检疫性病害。因此对葡萄病毒检测方法的研究,对我国葡萄产业的发展具有重要意义。目前对葡萄病毒病的检测方法主要有生物学方法、电子显微镜检测法、血清学检测法、分子生物学检测法,国内常用的检测方法是血清学检测法和分子生物学检测法。南芥菜花叶病毒(Arabis mosaic nepo virous,ArMV),是我国的二类检疫性植物病毒。对于ArMV的检测,我国目前主要是血清学方法。该方法检测植物病毒需要大量地制备高纯度、高特异性的抗血清。传统的抗体制备是以提纯的病毒粒子为抗原的,该方法难以获得大量的高纯度的抗原,制备出的抗血清在检测时会产生假阳性反应,影响结果的真实性。本研究用血清学方法对进口葡萄种苗进行检测,并建立ArMV的RT-PCR检测体系。同时,为了克服传统制备血清的局限性,运用分子生物学方法,克隆ArMV外壳蛋白的部分基因,构建外壳蛋白的原核表达载体,表达外壳蛋白,来制备特异性更高的抗血清。以RT-PCR为基础的多重RT-PCR可以在一个反应体系中同时提供常规的数次反应的扩增信息,与单重RT-PCR相比,多重RT-PCR更快速、更高效、更节约,能够一次性检测出两种以上的病毒,这种高效快速的优势使其在病毒的诊断中与无病毒苗快速繁育推广中具有重要意义。番茄环斑病毒(Tomato ringsopt virus/nepovirus,ToRSV)和烟草环斑病毒( Tobacco ringspot virus,TRSV)也是危害葡萄的两个重要病毒,分别是我国的一类和二类检疫有害生物。由于这两种病毒通常复合侵染,传统的血清学检测方法同时检测这两种病毒费时费力,而单一的RT-PCR同时检测两种病毒费用较高,本研究在单重RT-PCR体系的基础上,经过优化反应条件,建立多重PCR检测体系。为检测这两种病毒减少了工作量,节约了检测费用,提高了检测效率。通过以上研究,我们得到了以下结果:(1)用血清学方法从进境葡萄上检测到了ArMV;(2)成功的建立了ArMV的RT-PCR检测方法;(3)克隆ArMV外壳蛋白的部分基因,构建了外壳蛋白的原核表达载体,优化表达条件后,在大肠杆菌BL 21中成功的表达了外壳蛋白,以表达的蛋白为抗原免疫家兔,制备了异性强的抗血清,效价达到1/12800。(4)在成功建立葡萄两种检疫性病毒病ToRSV和TRSV的单重RT-PCR检测方法的更多还原

【Abstract】 In China, only 9 grape and virod species was exactly identified. However, the grape and virod species were 55 in total in the world. Therefore, some plant virus diseases are quarantine plant disease to China and it is important to study the method for detecting the plant virus, which may be very useful for the development of the Chinese grape industry. By now, methods used to detect ArMV are biological detection, serological detection , imaging the virus particles and molecular biology detection in China, among which serological detection and molecular biology detection are in common use.Arabis mosaic nepo virous(ArMV)has been defined as an important quarantine plant virus(on the A2 list). By now, methods used to detect ArMV are serological detection, which need to prepare quantity of antiserum with high pureness and specificity for detecting the plant virus by serological method. But it is difficult to obtain quantity of antigen with high pureness with the traditional method of purifying the virus particles from the infected plant. And the antiserum prepared by traditional method is not absolute specificity, which may cause false positive results in the process of detecting the plant virus. In this study, we detected ArMV in the in the grape seeding imported from the other country by serological detection, and established a RT-PCR method to detect ArMV. At the same time, in order to overcome the limitation of the preparation of antiserum by traditional method, we use molecular biology method to clone the partial coat protein gene of ArMV, construct expression vector, express the coat protein and prepare the antiserum with high specificity.Multiple RT-PCR, based on RT-PCR, could offer much more information in one reaction system than the common RT-PCR. Compared with single RT-PCR, multiple RT-PCR is more quick, more efficient and more economic in detecting two and above two species virus at the same time. So it is very useful in detecting viruses and breeding the grape seed without viruses.Tomato ringsopt virus/nepovirus(ToRSV)and Tobacco ringspot virus(TRSV), which seriously infect grape, are on the A1 and A2 list of quarantine plant virus. Because they always infect grape together, it took more time and cost more money to detect them at the更多还原