Abstract
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.
Highlights
With the advent of whole-genome sequencing technologies, plant genomic data are expanding at an explosive rate
We provided evidence that Artificial microRNA (amiRNA) produced in this way can be effective in protoplasts or transgenic plants as those produced using the traditional approach
These data indicate that the same strategy could be applied to pre-miR528 engineering, which leads to production of functional amiRNAs in rice, but with a simple cloning procedure
Summary
With the advent of whole-genome sequencing technologies, plant genomic data are expanding at an explosive rate. Artificial microRNA (amiRNA) technology has already been successfully developed to silence target gene expression by producing artificially designed miRNAs using the naturally existing miRNA precursor as a backbone [16,17]. AmiRNA-expressing plasmids are constructed according to the method described by Schwab et al [17], as follows: The miRNA and miRNA* of pre-miR319a are replaced by amiRNA/amiRNA* sequences through site-directed mutagenesis using overlapping polymerase chain reactions (PCRs). This method is time-consuming and cost ineffective because it involves four PCRs using three pairs of primers. We provided evidence that amiRNAs produced in this way can be effective in protoplasts or transgenic plants as those produced using the traditional approach
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