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.
Int J Mol Sci. 2019 Nov 10;20(22):5620. doi: 10.3390/ijms20225620.
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.
目前，由于高通量测序技术的快速发展，植物基因组序列的破译速度令人惊叹。然而，由于功能冗余和突变体致死性等技术障碍，功能基因组学的发展明显滞后。人工 microRNA (amiRNA) 技术是一种特异性、可逆性和多重基因沉默工具，常用于生成组成型或条件性突变体，以进行基因功能研究。构建 amiRNA 前体的常规方法涉及多个聚合酶链反应 (PCR)，这不仅增加了时间和劳动力成本，而且增加了引入序列错误的机会。在这里，我们报道了一种基于工程化拟南芥 miR319a 或水稻 miR528 前体的简化方法，用于在拟南芥和水稻中克隆和表达 amiRNA，该方法在前体中包含限制酶切位点，可方便地进行单个 PCR 产物的一步克隆。茎环反转录定量 PCR (RT-qPCR) 和功能分析验证了这些修饰前体可以准确地加工 amiRNA，并在植物原生质体中高效工作。此外，过表达修饰的 miR319a 前体或其衍生的 amiRNA 的拟南芥转基因植物表现出预期的强烈基因沉默表型。这种简化的 amiRNA 克隆策略将广泛用于拟南芥和水稻的功能基因组学研究，也可能适用于其他双子叶植物和单子叶植物。
