Molecular Biology Reports

, 37:903 | Cite as

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

  • Chong Liu
  • Lan Zhang
  • Jie Sun
  • Yanzhong Luo
  • Ming-Bo Wang
  • Yun-Liu Fan
  • Lei Wang


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.


miRNA Hairpin RNA Artificial microRNA Gene silencing 



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].


  1. 1.
    Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280CrossRefPubMedGoogle Scholar
  2. 2.
    Long D, Lee R, Williams P et al (2005) Principles of microRNA-target recognition. PLoS Biol 3:e85CrossRefGoogle Scholar
  3. 3.
    Llave C, Kasschau KD et al (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619CrossRefPubMedGoogle Scholar
  4. 4.
    Hervé V, Vazquez F, Crété P et al (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197CrossRefGoogle Scholar
  5. 5.
    Niu Q-W, Lin S-S, Reyes J-L et al (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428CrossRefPubMedGoogle Scholar
  6. 6.
    Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed microRNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333CrossRefPubMedGoogle Scholar
  7. 7.
    Parizotto E-A, Dunoyer P, Rahm N et al (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18:2237–2242CrossRefPubMedGoogle Scholar
  8. 8.
    Warthmann N, Chen H, Ossowski S et al (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS ONE 3:e1829CrossRefPubMedGoogle Scholar
  9. 9.
    Schwab R, Ossowski S, Riester M et al (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133CrossRefPubMedGoogle Scholar
  10. 10.
    Schwab R, Palatnik JF, Riester M et al (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527CrossRefPubMedGoogle Scholar
  11. 11.
    Wang L, Wang M-B, Tu J-X, Helliwell C-A et al (2007) Cloning and characterization of microRNAs from Brassica napus. FEBS Lett 581:3848–3856CrossRefPubMedGoogle Scholar
  12. 12.
    Wang L, Zhao J, Fan Y-L (2002) Cloning and function analysis of ABP9 protein which specifically binds toABRE2 motif of maize Cat1 gene. Chin Sci Bull 47(22):1871–1875CrossRefGoogle Scholar
  13. 13.
    Wang L, Luo Y-Z, Zhang L et al (2008) Rolling circle amplification-mediated hairpin RNA (RMHR) library construction in plants. Nucleic Acids Res 22:e149CrossRefGoogle Scholar
  14. 14.
    Ossowski S, Schwab R, Weigel D (2007) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant cell 53:674–690Google Scholar
  15. 15.
    Ding Y, Chan C-Y, Charles E, Lawrence (2004) Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res 32:w135–w141CrossRefPubMedGoogle Scholar
  16. 16.
    Long D, Lee R, Williams P et al (2007) Potent effect of target structure on microRNA function. Nat Struct Mol Biol 4:1038–1226Google Scholar
  17. 17.
    Kertesz M, Iovino N, Unnerstall U et al (2007) The role of site accessibility in microRNA target recognition. Nat Genet 39:1278–1285CrossRefPubMedGoogle Scholar
  18. 18.
    Wang MB, Helliwell CA, Wu LM et al (2008) Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA 14(5):903–913CrossRefPubMedGoogle Scholar
  19. 19.
    Khraiwesh B, Ossowski S, Weigel D et al (2008) Specific gene silencing by artificial microRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148:684–693CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Chong Liu
    • 1
    • 2
  • Lan Zhang
    • 1
  • Jie Sun
    • 2
  • Yanzhong Luo
    • 1
  • Ming-Bo Wang
    • 3
  • Yun-Liu Fan
    • 1
  • Lei Wang
    • 1
  1. 1.Biotechnology Research Institute/The National Key Facility for Crop Gene Resources and Genetic ImprovementChinese Academy of Agricultural SciencesBeijingChina
  2. 2.College of Agriculture/Key Laboratory of Oasis Ecology Agriculture of BINTUANShihezi UniversityShiheziChina
  3. 3.CSIRO Plant IndustryCanberraAustralia

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