关键词: 水稻, miRNA, 荧光素酶, 靶基因验证, 瞬时表达系统

Abstract: The aim of this study is to develop an accurate,rapid and convenient assay for experimental validation of target rice miRNA genes in transient expression systems and to lay a foundation for study the biological function of rice miRNAs. Many studies have confirmed that osa-miR169o may cleave the mRNA of OsNF-YA4（LOC_Os03g48970.1）. Here,the precursor gene miR169o（MIR169o）was co-expressed respectively with LUC empty vector,LUC-48970,and LUC-48970m3 fusion vector in transient expression systems in rice protoplast and tobacco. The LUC activity of each reaction was measured by the CCD imager and Luminometer. Combined with the miR169o levels detected by stem-loop qRT-PCR at the given time points,an optimized investigative system to validate targets of miRNAs was developed. In the rice protoplast system,LUC activity and miR169o expression level were increased gradually after transformation;the LUC activity attached the peak at 24 h. Meanwhile,the level of miR169o increased about 10-fold. Thus,the optimal detection time in rice protoplast was between 24 h and 36 h after transformation. In Agrobacterium-mediated transient expression in tobacco,LUC activity peaked at 72 h and the level of miR169o increased to 20 folds at 48h after co-infiltration. The optimal detection time was between 48 h and 72 h after co-infiltration in tobacco. In sum,this system may provide an easy,fast,and almost in vivo method to validate the candidate target gene of rice miRNAs.

Key words: rice, miRNA, LUC, validation of target gene, transient expression system