Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors

Autor:	Stephen P. DiFazio, Zamin K. Yang, Renee S. Arias, John M. Davis, Elizabeth Etherington, Amy M. Brunner, Gerald A. Tuskan, Steven H. Strauss, Sergei A. Filichkin, Udaya C. Kalluri
Rok vydání:	2007
Předmět:	Inverted repeat Genetic Vectors Mutant Arabidopsis MADS Domain Proteins Flowers Plant Science Gene Expression Regulation Plant RNA interference Cluster Analysis Gene silencing Vector (molecular biology) Gene Oligonucleotide Array Sequence Analysis Repetitive Sequences Nucleic Acid Genetics biology Arabidopsis Proteins Reverse Transcriptase Polymerase Chain Reaction Gene Expression Regulation Developmental Nuclear Proteins biology.organism_classification DNA-Binding Proteins RNA Interference DNA microarray Agronomy and Crop Science Biotechnology
Zdroj:	Plant Biotechnology Journal. 5:615-626
ISSN:	1467-7652 1467-7644
DOI:	10.1111/j.1467-7652.2007.00267.x
Popis:	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 cloningmore » throughput, number of events and degree of suppression required.« less
Databáze:	OpenAIRE
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