【作者】 谷宇；

【导师】 何农跃； 陈集双；

【作者基本信息】 东南大学 ， 生物医学工程， 2004， 硕士

【摘要】 马铃薯在全世界79%的国家广泛种植,其经济价值在世界作物中占第四位。目前在马铃薯作物上已发现的病毒、类病毒多达30余种,是影响马铃薯产量和质量的主要病害,引起的常年马铃薯产量损失达15%~20%。因此马铃薯病毒检测及其如何利用基因工程手段进行防治引起了各国的极大重视。开发高通量、快速、灵敏、规范化的检测体系是病毒检测发展的必然方向。基因芯片以其高通量、高信息量、且自动化程度高的特点成为检测病毒核酸的最佳方法之一。本研究通过RT-PCR和克隆、测序,在各地马铃薯样品上获得了三种病毒分离物(PVA、PVY、TMV)的3′-末端cDNA片段和类病毒PSTVd的两个分离物基因组全序列。并通过与不同地区来源的病毒分离物的相应序列的同源性比较进行了归组研究,分析结果表明:从CP基因核苷酸水平,可以将PVA分为3组、PVY分为4个株系、TMV分为三组;PSTVd的基因组序列较为保守,可以分两个亚组。对序列变异与地区来源之间关系的分析结果表明:PVA的基因变异与地区来源关系不大, PVY的各株系内的分离物表现出一定的地区适应性变异, TMV和PSTVd有通过基因组RNA的突变适应地域的趋势。本课题针对侵染马铃薯的十种主要病毒、类病毒(TMV、AlMV、CMV、PVA、PVX、PVY、PMTV、PVM、PLRV、PSTVd)检测芯片的制作和应用方面进行了深入的研究,并制备了相应的病毒检测芯片,取得了较为满意的结果。我们首先参照文献设计了能特异扩增靶序列的引物和特异性探针,设计制备了可以同时对上述病毒和类病毒进行检测的集成式芯片。并对从染病植物组织中提取出的RNA进行PCR扩增,同时标记荧光。为了使操作简化,我们探索了一步反应的RT标记技术,实现了对四种病毒(CMV、TMV、PVX、PVY)的四重RT标记。在芯片杂交过程中系统摸索并优化了杂交温度、杂交模板等对杂交结果的影响,制备的芯片可以同时检测7种病毒和类病毒,并且没有交叉杂交信号。本课题还进行了玻片固定总RNA的初步研究:探索了一种能在经过修饰的玻片上直接点上样品总RNA,并与特定病毒或类病毒的相应荧光标记的探针进行杂交检测的方法。与膜杂交相比,具有一次性检测量大,时间少,无辐射等优点。本研究以CMV为检测对象,对玻片固定RNA的方法、水合温度、荧光标记探针的长度等对杂交效果的影响进行了探索。将该方法应用于40个马铃薯样品的PSTVd的检测分析,结果表明其准确度超过膜杂交方法。更多还原

【Abstract】 Potato is a kind of popular and important economic plant that grows in most of countries of the world. The diseases caused by viruses or viroids bring about substantial losses in agricultural production. There are more than 30 viruses and viroids can infect potato. The proper diagnosis of causative viruses and viroids is the first requirement for the control of such diseases. So developing a high throughput, rapid, sensitive and specific method is necessary. Genechip, giving a large amount of sequence information directly and simultaneously, has become one of the best solutions to detect virus genes.In this study, using RT-PCR and cloning method, we got the 3( end sequences of genome of three kind of virus isolates (PVA, PVY and TMV), and all sequence of genome of two PSTVd isolates which were found in potato. The above sequence data obtained in our study were then compared with previously reported sequences of other isolates obtained from other parts of the world. The PVA isolates were then found belonging to three groups according to nucleic acid similarity of coat protein, and PVY isolates can be classified into four strains, and TMV isolates can be classified into three groups. PSTVd genome sequence is very conservative, the isolates belonging to two sub-groups. No genomic adoption of the genomic variation to geological origination was found when compared with genomic similarity among reports of PVA. In certain degree, similarity of CP gene of TMV and PSTVd and isolates of each PVY strain was found related with the geological origination.In this research, we fabricate an oligonucleotide microarray , which can simultaneously detect ten kinds of potato virus and viroid (TMV、AlMV、CMV、PVA、PVX、PVY、PMTV、PVM、PLRV、PSTVd).The virus-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the target regions that are highly conserved. These Cy5-labeled target cDNAs of different viruses were amplified by Reverse transcriptase PCR(RT-PCR) respectively, using specific primers. These fluorescent cDNAs were then hybridized to their specific oligonucleotide probe-coated glass slides. After optimizing the hybridization protocols, the fabricated gene-chip can detect seven kinds of virus and viroid at one time, with absence of cross-hybridization between the unrelated oligonucleotide probes and each virus produced a unique hybridization pattern. To simplify the operation, we try to label target by reverse transcriptase. Four kinds of virus (CMV, PVX, PVY and TMV) were detected by this multiplex RT labeling and <WP=6>hybridization method.We also first established a new method for the diagnosis of plant RNA viruses and viroid using dot blot hybridization to glass slide and using a fluorescently labeled probe by directly spotting large-scale extracted RNA samples onto a silanated glass slide and then hybridization with CY5-labeled probe of specific virus or viroid prepared by PCR. Factors affecting the result of hybridization were demonstrated by performing large numbers of conditional experiment in the course of detecting CMV. By the testing of 40 potato samples singly with PSTVd-specific probes, this method was demonstrated more accurate and reliable than nylon membrane hybridization with 32P-labelled probes.更多还原