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          Institute: MPI für Entwicklungsbiologie     Collection: Abteilung 6 - Molecular Biology (D. Weigel)     Display Documents



ID: 402898.0, MPI für Entwicklungsbiologie / Abteilung 6 - Molecular Biology (D. Weigel)
Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts
Authors:Khraiwesh, B.; Ossowski, S.; Weigel, D.; Reski, R.; Frank, W.
Date of Publication (YYYY-MM-DD):2008-10
Journal Abbrev.:Plant Physiol
Volume:148
Issue / Number:2
Start Page:684
End Page:693
Audience:Not Specified
Abstract / Description:MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, which include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome necessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexible inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.
Free Keywords:Base Sequence; Bryopsida/*genetics/metabolism; Gene Expression Regulation, Plant; *Gene Silencing; Gene Targeting; Genes, Plant; MicroRNAs/genetics/*metabolism; Molecular Sequence Data; Plants, Genetically Modified/genetics/metabolism; RNA, Messenger/genetics/metabolism; RNA, Plant/genetics/metabolism
External Publication Status:published
Document Type:Article
Affiliations:MPI für Entwicklungsbiologie/Abteilung 6 - Molekulare Biologie (Detlef Weigel)/
External Affiliations:%G eng
Identifiers:ISSN:0032-0889 (Print) %R pp.108.128025 [pii] 10.1104/pp.108.128025
URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=...
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