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A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.

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CaMV:

cauliflower mosaic virus

DDCT method:

the comparative cycle threshold (CT) method

dsRNA:

double-stranded RNA

GUS:

β-glucuronidase

MCS:

multiply clone site

miRNAs:

microRNAs

mRNA:

messenger RNA

MS medium:

Murashige and Skoog medium

NOS:

nopaline synthase

PCR:

polymerase chain reaction

RNAi:

RNA interference

siRNAs:

small interfering RNAs

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-d-GlcUA

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1. Changbin Chen

   Present address: Department of Biology, Pennsylvania State University, 16802-5301, University Park, PA, USA

1. Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, 100093, Beijing, China

   Zhen Wang, Changbin Chen, Yunyuan Xu, Rongxi Jiang, Ye Han, Zhihong Xu & Kang Chong

1. Zhen Wang
2. Changbin Chen
3. Yunyuan Xu
4. Rongxi Jiang
5. Ye Han
6. Zhihong Xu
7. Kang Chong

These authors contributed equally to this work.

Wang, Z., Chen, C., Xu, Y. et al. A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Mol Biol Rep 22, 409–417 (2004). https://doi.org/10.1007/BF02772683

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• Published: 03 September 2012

• Issue Date: December 2004

• DOI: https://doi.org/10.1007/BF02772683

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