Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.
- Oregon State University, Corvallis
- West Virginia University
- Oregon State University, Virginia Tech. University
- University of Florida
- ORNL
- Oregon State University
We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- DE-AC05-00OR22725
- OSTI ID:
- 931978
- Journal Information:
- Plant Biotechnology Journal, Vol. 5, Issue 5
- Country of Publication:
- United States
- Language:
- English
Similar Records
GENETIC MODIFICATION OF GIBBERELLIC ACID SIGNALING TO PROMOTE CARBON SEQUESTRATION IN TREE ROOTS AND STEMS
The histone H3K9 methylation and RNAi pathways regulate normalnucleolar and repeated DNA organization by inhibiting formation ofextrachromosomal DNAs