Abstract
RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.
Highlights
We constructed several vectors capable of expressing long hp-dsRNAs suitable for suppressing the expression of GFP, DsRed2, and KAN in plant cells
Transformants were allowed to self-pollinate and seeds from individual plants were pooled, surface sterilized, and allowed to germinate on one-halfstrength Murashige and Skoog medium (0.8% [w/v] agar and 3% [w/v] Suc) supplemented with the appropriate combinations of KAN and basta
About 310 bp of the DsRed2 open reading frame (ORF) were cloned as a PCR-amplified fragment from pSAT6-DsRed2-C1 (Tzfira et al, 2005) using the primers 5#-CATGCCATGGGGATCCCCTGGGTCACGGTCGC and 5#-CCGCTCGAGAACGTCATCACCGAGTTCATGC
Summary
We constructed several vectors capable of expressing long hp-dsRNAs suitable for suppressing the expression of GFP, DsRed, and KAN in plant cells. About 500 bp of the GFP open reading frame (ORF) were cloned as a PCR-amplified fragment from pSAT6-EGFP-C1 using the primers 5#-CATGCCATGGGGATCCAAGTTCAGCGTGTCCGGCG and 5#-CCGCTCGAGGGGCCGTCGCCGATGGGGG This fragment was digested by either NcoI-XhoI or XhoI alone, and the fragments were successively cloned, first into the NcoI-XhoI sites of MCS-I and into the SmaI-SalI sites of MCS-II of pSAT6.sup.RNAi, producing pSAT6.sup.GFPi. A similar strategy was employed using the plasmid pSAT5.nosP.RNAi to produce pSAT5. About 310 bp of the nptII ORF were cloned as a PCR-amplified fragment from pSAT4-nptII (Tzfira et al, 2005) using the primers 5#-CATGCCATGGGGATCCAAGCGGGAAGGGACTGGC and 5#-CCGCTCGAGCGACGAGATCATCGCCGTC This fragment was digested by either BamHI-XhoI or NcoI-XhoI alone, and the fragments were successively cloned, first into the BamHI-SalI sites of MCS-II and into the NcoI-XhoI sites of MCS-I of pSAT4.35SP.RNAi, producing pSAT4.35S.KAN
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