Improving transgenic RNAi in plants

Double-stranded RNA (dsRNA)-induced gene silencing, or RNA interference (RNAi), is a conserved gene control mechanism in eukaryotes that has been exploited extensively as a tool for gene knockdown. In plants, RNAi has been achieved mainly through the expression of transgenes encoding long hairpin-shaped RNA (hpRNA), which has proven to be a powerful tool in gene function studies and crop improvement. A typical hpRNA transgene construct is comprised of a perfect inverted repeat with a sense and antisense sequence of the target gene, separated by a spacer sequence. A number of studies from us and others have indicated that hpRNA transgenes are subject to self-induced transcriptional silencing (self-silencing), compromising the efficiency and long-term stability of RNAi against target genes. We assumed that self-silencing of hpRNA transgenes is due to the perfect invertedrepeat DNA structure and small RNA-directed DNA methylation. My thesis was aimed at developing and testing new hpRNA transgene designs that could minimize transcriptional self-silencing and give stable and effective target gene silencing...