A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor |
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| Authors: | Yan Fei Lu Yuwen Wu Gentu Peng Jiejun Zheng Hongying Lin Lin Chen Jianping |
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| Institution: | State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China. |
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| Abstract: | Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently. |
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