Ramesh S V, Mishra A K, Praveen S
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi-110012, India.
Oligonucleotides. 2007 Summer;17(2):251-7. doi: 10.1089/oli.2006.0063.
RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.
利用短干扰RNA（siRNA）进行的RNA干扰（RNAi）已被广泛用于抑制细胞内病毒靶标mRNA。基于我们之前通过病毒的反义复制酶基因（AC1）在体内稳定沉默番茄卷叶病毒的工作以及将AC4鉴定为一种小RNA调节因子（除了其在致病性中的作用），我们使用了四种不同的基于质粒载体的siRNA生成策略来沉默番茄卷叶病毒的病毒基因（AC1和AC4）。RNAi靶序列是根据AC1中的保守区域以及AC4基因的重叠序列从番茄卷叶病毒（ToLCV）的DNA A中选择的。为了对病毒产生高效且可预测的抗性，采用了不同的发夹RNA介导策略，如反义、自我互补反向重复、内含子剪接发夹RNA和小发夹RNA。在此我们展示，经过适当设计的siRNA不仅能防止RNAi抑制，还有助于培育性状稳定的转基因植株。这些策略表明，针对AC4和保守病毒序列的ToLCV复制酶驱动的RNAi为抑制番茄中广泛的ToLCV感染提供了一种有前景的方法。
