Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan,
Transgenic Res. 2013 Dec;22(6):1191-205. doi: 10.1007/s11248-013-9721-8. Epub 2013 Jun 8.
Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.
黄瓜花叶病毒是一种重要的植物病原体，具有广泛的宿主范围，包括许多植物物种。本研究通过转录后基因沉默展示了生产对黄瓜花叶病毒 O 株系和 Y 株系具有完全抗性的转基因马铃薯品系。使用了两种构建体，一种是 pEKH2IN2CMVai，它包含黄瓜花叶病毒 O 株系（CMV-O）RNA2 衍生的 1138bp 片段的缺陷复制酶基因的反向重复，另一种是基于 TRV 的 VIGS 载体（pTRV2CMVai），它包含相同的复制酶基因片段，但没有反向重复。这些构建体用于生产易感基因型马铃薯品种“Danshaku”的转基因马铃薯品系。源自 pEKH2IN2CMVai 的转基因品系在病毒挑战前后积累小干扰 RNA（siRNA），而源自 pTRV2CMVai 的转基因品系在病毒挑战后显示 siRNA 表达。当转基因品系受到 CMV-O 或 CMV-Y 挑战时，四条品系对两种株系均表现出完全（100%）抗性，而其他品系具有高抗性水平。在高抗性植物中，CMV-O 的感染性低于 CMV-Y。源自 pEKH2IN2CMVai 的植物和源自 pTRV2CMVai 的植物在抗性方面没有显著差异。抗性表型中存在 CMV 特异性 siRNA 表明抗性是通过 RNA 沉默获得的。
