Abstract
Universal amiRNA vectors (pUAs) for constructing plant amiRNAs in Arabidopsis and rice have been developed. By using type IIg restriction enzyme, BaeI, a single amiRNA construct can be produced using only one PCR and one ligation reaction. Thus, only one pair of primers is required for each amiRNA vector and these can be designed to be compatible with existing or newly developed methods. Because the BaeI recognition sequence is completely digested, there is no modification to the miRNA backbone, therefore avoids the risk of sequence changes that may affect downstream analysis. Based on these vectors, specific amiRNA constructs were created and verified. With optimized parameters, 38–45 % colonies for each amiRNA construct contain insertions with the expected orientation, and approximately 80 % of these colonies have the correct sequences.
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Acknowledgments
This work was supported by the Program of New Varieties of Genetically Modified Organism Cultivation of China (2011ZX08009-003-001), National Natural Science Foundation of China for young scholar (30900263 30900098 31101208 31201482) and the Program for Zhejiang Leading Team of Science and Technology Innovation and the Program for Leading Team of Agricultural Research and Innovation of Ministry of Agriculture, China. We thank CAMBIA (Canberra, Australia) for supplying the binary vector pCAMBIA1300 and Professor M. J. Adams, Rothamsted Research, UK for his correction of the English manuscript.
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Zhou, J., Yu, F., Chen, B. et al. Universal vectors for constructing artificial microRNAs in plants. Biotechnol Lett 35, 1127–1133 (2013). https://doi.org/10.1007/s10529-013-1197-3
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DOI: https://doi.org/10.1007/s10529-013-1197-3