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A Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors

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Abstract

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a ‘vector modification fragment’. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose ‘Highly Efficient gene Silencing Compatible vector’ (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.

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Acknowledgments

We thank CAMBIA (Canberra, Australia) for supplying the binary vector pCAMBIA1300. We also thank Professor Mike Adams for his help in English modification of the manuscript. This work was supported by the Program of New Varieties of Genetically Modified Organism Cultivation of China [2009ZX08009-043B, 2009ZX08001-006B]; ‘863’ Program, [2008AA02Z125]; the Zhejiang Provincial Foundation for Natural Science [Z307451]; and special grant from the Zhejiang Provincial Department of Science and Technology [2007C12039].

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Authors and Affiliations

  1. Institute of Virology and Biotechnology (MOA and Zhejiang Provincial Key Laboratory for Plant Virology), Zhejiang Academy of Agricultural Sciences, Shiqiao Road 198#, Hangzhou, 310021, Zhejiang, People’s Republic of China

    Xuming Wang, Yong Yang, Chulang Yu, Jie Zhou, Ye Cheng, Chengqi Yan & Jianping Chen

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  1. Xuming Wang
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  2. Yong Yang
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  3. Chulang Yu
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  7. Jianping Chen
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Correspondence to Jianping Chen.

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Vectors mentioned in this work can be requested from Jianping Chen.

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Wang, X., Yang, Y., Yu, C. et al. A Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors. Mol Biotechnol 46, 211–218 (2010). https://doi.org/10.1007/s12033-010-9291-4

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