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A novel approach for the construction of plant amiRNA expression vectors

Paper ID Volume ID Publish Year Pages File Format Full-Text
23928 43483 2011 6 PDF Available
Title
A novel approach for the construction of plant amiRNA expression vectors
Abstract

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.

Keywords
Artificial microRNA; Seamless enzyme-free cloning; Mating-assisted genetically integrated cloning; Homologous recombination
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A novel approach for the construction of plant amiRNA expression vectors
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 151, Issue 1, 10 January 2011, Pages 9–14
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us