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A simple artificial microRNA vector based on ath-miR169d precursor from Arabidopsis

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Abstract

Artificial microRNA (amiRNA) is becoming a powerful tool for silencing genes in plants, and several amiRNA vectors have recently been developed based on the natural precursor structures of ath-miR159a, ath-miR164b, ath-miR172a, ath-miR319a and osa-miR528. In this study we generated a simple amiRNA vector (pAmiR169d) based on the structure of Arabidopsis miR169d precursor (pre-miR169d). Two unique restriction sites were created inside the stem region of pre-miR169d, which allows for the artificial miRNA sequences to be cloned as either ~80 bp synthetic oligonucleotides or PCR products. A β-glucuronidase:green florescent protein fusion gene (GUS-GFP) was efficiently silenced in transient assays using a pAmiR169d-derived construct targeting the GUS-GFP sequence. 5′-RACE showed that the target GUS-GFP transcript was cleaved precisely at the expected position across nucleotides 10 and 11 of the artificial miRNA. Thus, pAmiR169d allows for both easy construction of artificial miRNA constructs and efficient silencing of target genes in plants.

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Acknowledgments

This work was supported by the National Key Basic Research Program [Grant Numbers 2007CB109004]; and National High Technology Research and Development Program of China [Grant Number 2007AA10Z147].

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Authors and Affiliations

  1. Biotechnology Research Institute/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 100081, Beijing, China

    Chong Liu, Lan Zhang, Yanzhong Luo, Yun-Liu Fan & Lei Wang

  2. College of Agriculture/Key Laboratory of Oasis Ecology Agriculture of BINTUAN, Shihezi University, 832003, Shihezi, China

    Chong Liu & Jie Sun

  3. CSIRO Plant Industry, PO Box 1600, Canberra, ACT, 2601, Australia

    Ming-Bo Wang

Authors
  1. Chong Liu
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  2. Lan Zhang
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  3. Jie Sun
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  4. Yanzhong Luo
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  5. Ming-Bo Wang
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  6. Yun-Liu Fan
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  7. Lei Wang
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Correspondence to Lei Wang.

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Liu, C., Zhang, L., Sun, J. et al. A simple artificial microRNA vector based on ath-miR169d precursor from Arabidopsis. Mol Biol Rep 37, 903–909 (2010). https://doi.org/10.1007/s11033-009-9713-1

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  • DOI: https://doi.org/10.1007/s11033-009-9713-1

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