利用水稻原生质体快速分析miRNA靶标RNA

Efficient transient expression to analyze miRNA targets in rice protoplasts

摘要:

与转基因方法相比，基因瞬时表达系统在基因表达研究上具有快速便捷的特点。为检验水稻miRNA与靶标基因之间的调控关系，将MIRNA基因与GFP /靶标序列融合基因 (或GFP /靶标突变序列融合基因) 构建在同一瞬时表达载体上，并转化水稻原生质体，通过观察含有GFP /靶标序列融合基因和GFP /靶标突变序列融合基因的载体之间的荧光强度差异，以及通过qRT-PCR方法检测靶标和非靶标mRNA水平差异来验证miRNA对靶标基因的调控。用osaMIR156和osaMIR397及其靶标序列对实验设计方法进行验证，荧光显微观察和qRT-PCR检测证明，osamiR156和osamiR397能降低相应靶标序列GFP融合基因的转录物水平和GFP荧光水平。此种水稻原生质体瞬时表达方法用于在体内进行大规模miRNA靶标基因检测。由于其他近缘单子叶植物很可能与水稻有近似的小RNA加工系统，因此对于其他单子叶植物miRNA功能研究也将有很好的应用前景。

Abstract:

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.