A novel approach for the construction of plant amiRNA expression vectors

文献类型： 外文期刊

第一作者： Yan, Hong

作者： Yan, Hong;Deng, Xuan;Ma, Lixin;Yan, Hong;Zhao, Bin;Cao, Yinglong;Huang, Junyan

作者机构：

关键词： Artificial microRNA;Seamless enzyme-free cloning;Mating-assisted genetically integrated cloning;Homologous recombination

期刊名称：JOURNAL OF BIOTECHNOLOGY （ 影响因子：3.307； 五年影响因子：3.778 ）

ISSN：

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收录情况： SCI

摘要： Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.

分类号： Q81

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