Kollàr A, Dalmay T, Burgyàn J
Agricultural Biotechnology Center, Hungary.
Virology. 1993 Mar;193(1):313-8. doi: 10.1006/viro.1993.1127.
Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.
兰花生环斑番茄病毒的缺陷干扰（DI）RNA被克隆到噬菌体T7 RNA聚合酶启动子下游。体外合成的RNA与亲本基因组RNA共同接种到本氏烟草植株上时具有生物活性，并可防止顶端坏死的发生。本氏烟草植株用相对于花椰菜花叶病毒35S启动子呈正向和反向的DI RNA序列进行转化。使用DNA提取物的PCR扩增和RNA提取物的Northern印迹分析验证了DI RNA序列在植物基因组中的整合。在未感染的转基因植物中检测到DI RNA相关转录本，但用亲本病毒接种仅在以正向表达DI RNA的转基因植物中诱导DI RNA的复制。DI RNA积累的转基因植物受到保护，不会发生顶端坏死和死亡。
