Petsch, K. A., Chonglie, M., Scanlon, M. J., Jorgensen, R. A. (December 2010) Targeted forward mutagenesis by transitive RNAi. The Plant Journal, 61 (5). pp. 873-882.
Abstract
A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types. © 2010 Blackwell Publishing Ltd.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Arabidopsis thaliana Forward mutagenesis Laser capture microdissection Mesophyll cell RNAi |
| Subjects: | organism description > plant > Arabidopsis bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > cDNA |
| CSHL Authors: | |
| Communities: | CSHL Post Doctoral Fellows CSHL labs > Timmermans lab |
| Depositing User: | CSHL Librarian |
| Date: | 10 December 2010 |
| Date Deposited: | 05 Oct 2011 14:00 |
| Last Modified: | 03 May 2013 13:39 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/15504 |
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