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Development and validation of a method for the detection of altered resistance in transgenic plants against herbivore-pathogen-complexes

机译:开发和验证检测转基因植物对草食动物病原体复合体抗性变化的方法

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摘要

Enhanced or reduced uptake of viruses by vectors and changes in resistance level can be sensitive indicators for metabolic changes in transgenic plants caused by the new trait and not observed by conventional methods. In addition, the plant transformation process itself can lead to such changes. To be able to investigate this hypothesis in cereals we decided to use two highly important insect-transmitted viruses infecting them - _Barley yellow dwarf virus_ (BYDV) and _Wheat dwarf virus_ (WDV). Corresponding molecular tools for their quantification in plants as well as virus vectors had to be developed. Both viruses cause similar symptoms: dwarfing, stunting, leaf discoloration leading to yield losses or death of plant. BYDV (_Luteoviridae_) is one of the most important cereal-infecting viruses worldwide causing substantial yield losses. In Germany, the strain PAV is widespread. It is transmitted by the aphids _Rhopalosiphum padi_ and _Sitobion avenae_ in a persistent manner. WDV (_Geminiviridae_) is found in Germany since the early 1990s and has gained importance over the last years. It is transmitted by the leafhopper _Psammotettix alienus_ in a persistent manner. Real-time PCR is the state of the art method for specific detection of viruses even in minor quantities. It allows exact quantification of the virus content of plants and vectors and is hence suited to monitor changes of it. We developed qPCR assays for WDV and RT-qPCR assays for BYDV-PAV based on TaqMan probe technology as well as the DNA-binding dye SybrGreen. The qPCR assays proved to be more sensitive than DAS-ELISA. For example, WDV is detected even at dilutions of 1:10^8^, whereas the corresponding threshold for ELISA is about 1:10^4^. As nucleic acid extraction procedures proved to be time consuming and expensive, detection methods are adopted now for immunocapture procedures. These methods will be applied to the analysis of several transgenic wheat lines.
机译:载体对病毒吸收的增加或减少以及抗性水平的变化可能是转基因植物新特性引起的代谢变化的敏感指标,而常规方法并未观察到。另外,植物转化过程本身也会导致这种变化。为了能够研究谷物中的这一假设,我们决定使用两种非常重要的昆虫传播病毒来感染它们-大麦黄矮病毒(BYDV)和小麦矮矮病毒(WDV)。必须开发用于在植物中定量的相应分子工具以及病毒载体。两种病毒都有类似的症状:矮化,发育迟缓,叶片变色导致产量下降或植物死亡。 BYDV(_Luteoviridae_)是世界范围内最重要的谷物感染病毒之一,导致大量产量损失。在德国,PAV株很普遍。它由蚜虫_Rhopalosiphum padi_和_Sitobion avenae_持续传播。 WDV(_Geminiviridae_)自1990年代初开始在德国发现,并在最近几年中变得越来越重要。它由叶蝉_Psammotettix alienus_持续传播。实时PCR是即使少量也可以特异性检测病毒的最新方法。它可以精确定量植物和载体的病毒含量,因此适合监控其变化。我们开发了基于TaqMan探针技术以及DNA结合染料SybrGreen的WDV的qPCR测定法和BYDV-PAV的RT-qPCR测定法。事实证明,qPCR分析比DAS-ELISA更灵敏。例如,即使在1:10 ^ 8 ^的稀释度下也检测到了WDV,而ELISA的相应阈值约为1:10 ^ 4 ^。由于核酸提取程序被证明是耗时且昂贵的,因此现在将检测方法用于免疫捕获程序。这些方法将用于几种转基因小麦品系的分析。

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