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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mi RNA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q RT-PCR方法检测靶标和非靶标m RNA水平差异来验证mi RNA对靶标基因的调控。用osa MIR156和osa MIR397及其靶标序列对实验设计方法进行验证,荧光显微观察和q RT-PCR检测证明,osami R156和osami R397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mi RNA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mi RNA功能研究也将有很好的应用前景。
Compared with the transgenic method, gene transient expression system has the characteristics of quick and easy gene expression research. In order to test the regulatory relationship between miRNA and target genes in rice, the MIRNA gene was fused with the GFP / target sequence fusion gene (or GFP / target sequence fusion gene) on the same transient expression vector and transformed into rice protoplasts. Differences in fluorescence intensity between vectors containing GFP / target sequence fusion genes and GFP / target mutation sequence fusion genes and verification of miRNA target gene regulation by q RT-PCR method detection of differences in target and non-target m RNA levels. The experimental design methods were validated with osa MIR156 and osa MIR397 and their target sequences. Fluorescent microscopy and q RT-PCR showed that osami R156 and osami R397 could reduce the transcript level and GFP fluorescence level of GFP fusion gene . This transient method of rice protoplast expression is used to detect large-scale miRNA target genes in vivo. As other closely related monocotyledons are likely to have similar small RNA processing systems to rice, the study on the mi RNA function of other monocotyledons will also have good application prospects.