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马铃薯病毒及类病毒病害严重影响了中国马铃薯的生产。为了培育抗病毒及类病毒的马铃薯新材料,本研究采用人工miRNA(artificial microRNA,amiRNA)策略,以马铃薯X病毒(potato virus X,PVX)与马铃薯Y病毒(potato virus Y,PVY)编码沉默抑制子P25蛋白和HC-pro蛋白的基因为靶标,利用5’RACE技术对P25基因、HC-Pro基因潜在的剪切热点进行预测。再采用overlapping PCR技术,以拟南芥mi R159a前体为骨架,设计特异性引物,构建分别靶向P25和HC-Pro基因的amiRNA表达载体。同时构建靶向马铃薯内源基因Virp1的amiRNA表达载体,将构建完成的三个amiRNA表达载体串联获得amiRNAs三联体,分别转入根癌农杆菌菌株EHA105,并用农杆菌介导法转化马铃薯品种克新13。经PCR鉴定,结果表明,目的基因片段已成功转入马铃薯中。
Potato virus and virus-like diseases have seriously affected the production of potatoes in China. In order to cultivate a new potato material with anti-virus and virus-like activity, we used artificial miRNA (artificial microRNA, amiRNA) strategy to encode silencing inhibition of potato virus X (PVX) and potato virus Y (PVY) P25 gene and HC-pro protein gene as targets, the use of 5’RACE technology P25 gene, HC-Pro gene potential of the hot-spot prediction. Using overlap PCR technique, the Arabidopsis thaliana mi R159a precursor was used as the backbone, and specific primers were designed to construct amiRNA expression vector targeting P25 and HC-Pro respectively. At the same time, the amiRNA expression vector targeting Virp1 of potato was constructed, and the constructed three amiRNA expression vectors were tandemly connected to get the triplet of amiRNAs and transformed into Agrobacterium tumefaciens strain EHA105 respectively. The Agrobacterium- 13. The results of PCR showed that the target gene fragment was successfully transferred into potato.