Study Of H₂O₂-Responsive MiRNA169b Promoter Activity In Rice Seedlings

As a double-edged molecule sword, hydrogen peroxide （H₂O₂） plays a dual role in plants as the toxic product leading to oxidative destruction of cells and as a signaling molecule in signal transduction. microRNA （miRNA） is widespread in plants with 20²4 nt in length, and regulates many plant biological processes including growth, development, and responses to various biotic and abiotic stresses. Therefore, it is believed that miRNAs play a vital role in plant response to H₂O₂. In this study, we used H₂O₂-treated rice seedlings as experimental materials, applied high-throughput deep sequencing approach to analyze the small RNA libraries, and finally identified H₂O₂-responsive miRNAs in rice seedlings, whose expression level differentiation indicate they have important functions in rice response to H₂O₂.This study for the first time extensively compares both miRNA sequencing numbers of two small RNA libraries from rice seedlings either under H₂O₂ stress or non-inducing conditions to identify 7 probable H₂O₂-responsive miRNA families significant differentiation, one of which is osa-miR169.osa-miR169 family contains multiple members. however, we primarily focus on osa-miR169b, whose sequencing number （QH₂O₂） under H₂O₂-treated conditions is the highest, 1794,and in control, 1163.This indicates that osa-miR169b is H₂O₂-responsive miRNA, whose expression level is up-regulated in rice seedlings when treated with H₂O₂.To validate this result, we performed Northern blotting with miR169b in rice seedlings. The sequencing result indicates that its expressing level is induced by H₂O₂, which is consistent with Northern blotting experiment when treated with H₂O₂ （especially 0.6 and 3.0 mM H₂O₂ respectively）. They both fit very well. Hence, it confirms that miR169b is the right miRNA response to and induced by H₂O₂.Then we located miRNA169b in genome and selected the upstream 1500bp sequence of pre-miRNA169b start site as cis-element analytical targets, whose probable motifs were predicted by software. Using the engineered fusion plasmid containing promoter of miR169b and a reporter gene LUC, i.e. PmiR169b-LUC to transform tobacco and perform transient expression assay, we can find and identify the critical cis-element response to H₂O₂. Our study will contribute to discover the critical element response to oxidative stress, uncover the regulation and molecular mechanism of miRNA gene at transcriptional level.