Abstract
Artificial microRNA (amiRNA) is a powerful tool for silencing genes in many plant species. Here we provide an easy method to construct amiRNA vectors that reinvents the Golden Gate cloning approach and features a novel system called top speed amiRNA construction (TAC). This speedy approach accomplishes one restriction-ligation step in only 5 min, allowing easy and high-throughput vector construction. Three primers were annealed to be a specific adaptor, then digested and ligated on our novel vector pTAC. Importantly, this method allows the recombined amiRNA constructs to maintain the precursor of osa-miR528 with exception of the desired amiRNA/amiRNA* sequences. Using this method, our results showed the expected decrease of targeted genes in Nicotiana benthamiana and Oryza sativa.
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Acknowledgments
We really appreciate Professor Zhiyong Gao to provide us the N. benthamiana. This work was supported in part by the National Programme on Research and Development of Transgenic Plants (Grant No. 2011ZX08001-001), the National Natural Science Foundation of China for Innovative Research Team (Grant No. 30521004) and the Key Grant Project of Chinese Ministry of Education (Grant No. 313039).
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Yang Li and Yang Li have contributed equally to this work.
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Li, Y., Li, Y., Zhao, S. et al. A simple method for construction of artificial microRNA vector in plant. Biotechnol Lett 36, 2117–2123 (2014). https://doi.org/10.1007/s10529-014-1570-x
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DOI: https://doi.org/10.1007/s10529-014-1570-x