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New Phytol. 2010 Jul;187(1):240-50. doi: 10.1111/j.1469-8137.2010.03253.x. Epub 2010 Apr 12.

One-step, zero-background ligation-independent cloning intron-containing hairpin RNA constructs for RNAi in plants.

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1
Protein Science Laboratory of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Abstract

*The hairpin-based RNA interference (RNAi) technique plays an important role in exploring gene function in plants. Although there are several methods for making hairpin RNA (hpRNA) constructs, these methods usually need multiple relatively laborious, time-consuming or high-cost cloning steps. Here we describe a one-step, zero-background ligation-independent cloning (OZ-LIC) method for making intron-containing hpRNA (ihpRNA) constructs by our vector pRNAi-LIC. *To generate the ihpRNA constructs with zero-background, this method only requires treating two PCR products of target gene flanked with different LIC sequences and SmaI-linearized pRNAi-LIC vector by T4 DNA polymerase respectively, and then transforming these treated DNA mixture into Escherichia coli. *The ihpRNA constructs generated with our OZ-LIC RNAi vector can efficiently induce not only transient silencing of the exogenous marker genes and the endogenous resistance-related Nicotiana benthamiana SGT1 gene, but also stable transgenic suppression of Arabidopsis SGT1b gene. *Our new OZ-LIC method and RNAi vector will represent a powerful tool for gene knockdown in plants and may facilitate high-throughput determination of plant gene function.

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