【作者】 艾涛波；

【导师】 郭兴启；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 近年来,人工合成的microRNA(amiRNA)的应用为植物抗病育种提供了新的途径。microRNA(miRNA)是一类由核基因编码的,转录后在细胞质中经一系列剪切而形成的小RNA,它能指导靶基因的剪切或翻译抑制来调控基因的表达。amiRNA技术是在内源基因沉默机制上发展起来的,利用该策略,设计特异的amiRNA就可以使靶基因发生高效的基因沉默。理论上讲,运用该策略也能用于培育抗病毒的转基因植株。本研究中,利用拟南芥的三个内源的miRNA前体(pre-miR159a,pre-miR167b和pre-miR171a)为骨架,设计特异的amiRNA,分别靶向马铃薯Y病毒(Potato virus Y, PVY)编码沉默抑制子的HC-Pro基因和马铃薯X病毒(Potato virus X, PVX)编码沉默抑制子的TGBp1/p25(p25)基因,并对所得的T1代转基因植株进行抗病性分析。主要实验结果如下:1)分别培育了三类针对PVY和PVX病毒的转基因植株,每类植株由不同的改造后的前体转化而来。Northern blot结果表明,三类转基因植株均能产生amiRNA,但效率不同,其中pre-miR159a植株的效率最高。2)病毒接种实验证明,三类转基因植株都具有一定的病毒抗性。ELISA和荧光定量PCR分析表明,植株的抗病性与amiRNA的表达量呈正相关。3)5′RLM-RACE实验证明,amiRNA能准确的指导靶序列的剪切。对接种后的转基因植株的Northern blot分析表明,病毒RNA的含量与amiRNA的表达水平呈负相关。4)不同株系的病毒接种实验表明,所得的amiRNA转基因植株对其所针对的病毒具有广谱的抗性,但其抗性水平受到靶序列的影响。5)表达pre-amiRNA二聚体的转基因植株能同时产生两类amiRNA,并能介导对PVX和PVY的双重抗性。6)对单抗植株的T2代进行抗性分析表明,混合接种时不发生协同效应,且其抗性具有可遗传性。更多还原

【Abstract】 Recently, the application of artificial microRNA (amiRNA) strategy has supplied a new way for generating virus-resistant transgenic plant. MicroRNAs (miRNAs) processed from nuclear-encoded transcripts control the expression of target transcripts by directing cleavage or translational inhibition. amiRNAs that exploit this endogenous gene silencing mechanism can be designed to target any gene of interest and provide a highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants. Thus, this strategy can be used to obtain virus-resistant transgenic plant in theory. Here, using Arabidopsis thaliana miR159a, miR167b, and miR171a precursors as backbones, we designed two types of amiRNAs targeting sequences encoding the silencing suppressor HC-Pro of Potato virus Y (PVY) and p25 of Potato virus X (PVX), respectively. Then, we investigated the virus-resistance of the T1 generation, and the main results are as follows:1) Three kinds of transgenic tobacco plants harboring different modified backbones which target to PVY or PVX were obtained. Northern blot assays revealed that all of them can generate amiRNAs, but with different efficiency. The plant with the pre-miR159a backbone was the most effective in generating amiRNAs.2) Through virus-challenging experiment, it was showed that all three kinds of transgenic plants could resist to their virus to some extent. ELISA assays and quantitative real-time RT-PCR have showed that the different resistance levels of the transgenic plants were correlated with the expression levels of amiRNAs.3) 5′RLM-RACE assay has showed that amiRNAs could mediated a precise cleavage of their target viral RNAs. Northern blot was carried out in the transgenic plants and the results indicated that the expression level of amiRNA was inversely correlated to the virus RNA accumulation.4) Inoculated the transgenic plants with different virus isolations of the one kind of virus. It was revealed that transgenic plants could present a wide range of resistance against their target viruses. However, the resistance was strongly influenced by the complementarity of the target sequence.5) Two different amiRNAs could be generated from transgenic plants with a dimeric amiRNA precursor, and these amiRNAs mediated double resistance against both PVX and PVY.6) The virus-challenging experiment of the T2 generation transgenic plants with single virus resistance verified that the resistance mediated by amiRNAs is hereditable, and when inoculated with mixed viruses, no synergistic effect was observed.更多还原