拟南芥At5g01510和At5g49820基因人工microRNAs的构建与鉴定

以拟南芥内源MIR319a前体为骨架，构建沉默DUF647家族基因At5t01510和At5g49820表达的人工microRNAs，研究其对目的基因表达的抑制效果。利用WMD平台设计分别靶向At5g01510和At5g49820的amiRNAs序列，通过重叠PCR改造拟南芥MIR319a骨架序列，使其包含我们设计的特异amiRNAs序列。构建35S：：amiR-At5g0150和35S：：amiR-At5g49820融合基因，以农杆菌介导的花苞浸染法转化获得转基因拟南芥。RT-PCR分析表明，人工microRNAs能够显著抑制靶基因的表达，获得了抑制效果明显的转基因植株。本工作为进一步研究这两个基因的功能奠定了良好的基础。

Construction and Identification of Artificial MicroRNAs Targeting Arabidopsis At5gO1510 and At5949820 Genes

Using the endogenous Arabidopsis MIR319a precursor as a backbone, we constructed two artificial microRNAs （amiRNAs） to knock down the expressions of At5g01510 and At5g49820 genes. By using the WMD （Web MicroRNA Designer） platform, we designed two amiRNAs targeting the genes At5g01510 and At5g49820, respectively. Both amiRNAs were engineered into the MIR319a precursor using overlapping PCR to replace the endogenous miRNA sequences, and the amiRNAs backbones were fused to the constitutive promoter within the binary vector pCHF3. The amiR-At5g01510 and amiR-At5949820 expression constructs were introduced into Arabidopsis wild type （Col-0） by Agrobacterium-mediated floral dipping. RT-PCR analysis showed that the target genes were efficiently knocked down in transgenic lines harboring the overexpression constructs. This work has laid a foundation for further study of the functions of At5g01510 and At5949820 genes.