Two-step method for constructing Arabidopsis artificial microRNA vectors
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Xuming Wang1, Yong Yang, Jie Zhou
1State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, MOA Key Laboratory for Plant Protection and Biotechnology, Zhejiang Provincial Key Laboratory of Plant Virology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People's Republic of China.
Abstract:
Artificial microRNA (amiRNA) technology is used for gene silencing in Arabidopsis. We describe a method for constructing amiRNA vectors that requires only one PCR and one ligation reaction. Vectors produced by this method are the same as those from the method of Schwab et al. (Plant Cell 2006, 18:1121-1133). Transgenic plants created by this method can therefore be tested in the same way or compared with existing transgenic material without the risk of alteration to the amiRNA skeleton. With optimized parameters, 36-42 % colonies had the insertion in the expected orientation and 85-95 % of these had the correct sequence. Using this method, a transient gene knock-down analysis in Arabidopsis could be completed in 4-5 days.
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