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Open AccessArticle

Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice

by Dandan Zhang 1,2,†, Nannan Zhang 1,3,†, Wenzhong Shen 1,* and Jian-Feng Li 1,*
State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
Guangdong Provincial Key Laboratory of Sugarcane Improvement and Biorefinery, Guangdong Bioengineering Institute, Guangzhou 510316, China
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(22), 5620; (registering DOI)
Received: 24 September 2019 / Revised: 2 November 2019 / Accepted: 8 November 2019 / Published: 10 November 2019
(This article belongs to the Special Issue Plant Genomics 2019)
Plant genome sequences are presently deciphered at a staggering speed, due to the rapid advancement of high-throughput sequencing technologies. However, functional genomics significantly lag behind due to technical obstacles related to functional redundancy and mutant lethality. Artificial microRNA (amiRNA) technology is a specific, reversible, and multiplex gene silencing tool that has been frequently used in generating constitutive or conditional mutants for gene functional interrogation. The routine approach to construct amiRNA precursors involves multiple polymerase chain reactions (PCRs) that can increase both time and labor expenses, as well as the chance to introduce sequence errors. Here, we report a simplified method to clone and express amiRNAs in Arabidopsis and rice based on the engineered Arabidopsis miR319a or rice miR528 precursor, which harbor restriction sites to facilitate one-step cloning of a single PCR product. Stem-loop reverse-transcriptase quantitative PCR (RT-qPCR) and functional assays validated that amiRNAs can be accurately processed from these modified precursors and work efficiently in plant protoplasts. In addition, Arabidopsis transgenic plants overexpressing the modified miR319a precursor or its derived amiRNA could exhibit strong gene silencing phenotypes, as expected. The simplified amiRNA cloning strategy will be broadly useful for functional genomic studies in Arabidopsis and rice, and maybe other dicotyledon and monocotyledon species as well. View Full-Text
Keywords: plant genome; artificial microRNA; gene silencing; Arabidopsis; rice plant genome; artificial microRNA; gene silencing; Arabidopsis; rice
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Zhang, D.; Zhang, N.; Shen, W.; Li, J.-F. Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice. Int. J. Mol. Sci. 2019, 20, 5620.

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