Yeh H H, Tian T, Medina V, Falk B W
Department of Plant Pathology, 1 Shields Avenue, University of California, Davis, California 95616, USA.
Virology. 2001 Oct 10;289(1):54-62. doi: 10.1006/viro.2001.1110.
Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1.
在原生质体中比较了生菜传染性黄化病毒（LIYV）RNA 2缺陷RNA（D RNA）的复制能力以及从重组D RNA构建体中表达绿色荧光蛋白（GFP）的能力。最初比较了四种不同基因组成的LIYV D RNA，但只有两种（LIYV D RNA M5和M18）能高水平复制。这两种都至少包含两个完整的开放阅读框（ORF），其中一个是位于3'端编码P26的ORF。使用与LIYV RNA 2的3'区域对应的探针进行的Northern杂交分析，检测到了感染LIYV RNA 1和2的原生质体或仅接种RNA 1加LIYV D RNA M5或M18的原生质体中的P26亚基因组RNA，这表明这些LIYV D RNA可作为生成P26亚基因组RNA的模板。将GFP编码区作为框内插入片段插入LIYV M5和M18 D RNA的P26编码区，得到M5gfp和M18gfp。当用M5gfp和M18gfp的转录本接种原生质体时，只有在与LIYV RNA 1共接种时才能看到明亮的荧光。荧光原生质体的百分比因实验而异，但高达5.8%。时间进程分析表明，在接种后48小时之前未检测到荧光，这与LIYV RNA 2和RNA 2 D RNA积累的时间相关，但与LIYV RNA 1的积累时间无关。
