Genome-wide analysis of microRNAs and their targets involving in leaf senescence of rice

Enhancing grain production of rice (Oryza sativa L.) is a top priority in ensuring food security for human being. One approach to increase yield is to delay leaf senescence and to extend the available time for photosynthesis. microRNAs (miRNAs) are key regulators for aging and cellular senescence in eukayotes. However, miRNAs and their roles in rice leaf senescence remain unexplored. Here, we report identification of miRNAs and their putative target genes by deep sequencing of six small RNA libraries, six RNA-seq libraries and two degradome libraries from the leaves of two super hybrid rice, Nei-2-You 6 (N2Y6, age-resistant rice) and Liang-You-Pei 9 (LYP9, age-sensitive rice). Totally 372 known miRNAs and 162 miRNA candidates were identified, and 1145 targets were identified. Compared with the expression of miRNAs in the leaves of LYP9, the numbers of miRNAs up-regulated and down-regulated in the leaves of N2Y6 were 47 and 30 at early stage of grain-filling, 21 and 17 at the middle stage, and 11 and 37 at the late stage, respectively. Six miRNA families, osa-miR159, osa-miR160 osa-miR164, osa-miR167, osa-miR172 and osa-miR1848, targeting the genes encoding APETALA2 (AP2), zinc finger proteins, salicylic acid-induced protein 19 (SIP19), Auxin response factors (ARF) and NAC transcription factors, respectively, were found to be involved in leaf senescence through phytohormone signaling pathways. These results provided valuable information for understanding the miRNA-mediated leaf senescence of rice, and offered an important foundation for rice breeding. Overall design: [miRNA] sample 1:The flag leaves at early stage of grain-filling of N2Y6 rice; sample 2: The flag leaves at middle stage of grain-filling of N2Y6 rice;sample 3:The flag leaves at late stage of grain-filling of N2Y6 rice; sample 4:The flag leaves at early stage of grain-filling of LYP9 rice; sample 5: The flag leaves at middle stage of grain-filling of LYP9 rice;sample 6:The flag leaves at late stage of grain-filling of LYP9 rice. [DGE]: samples 7-12 [degradome (targets)]: samples 13:The flag leaves at mixed stages of grain-filling of N2Y6 rice; sample 14:The flag leaves at mixed stages of grain-filling of LYP9 rice

提高水稻（Oryza sativa L.）籽粒产量是保障人类粮食安全的核心要务。提升水稻产量的有效途径之一是延缓叶片衰老，延长光合作用的有效持续时长。微小RNA（microRNAs, miRNAs）是真核生物衰老及细胞衰老的关键调控因子，但目前关于miRNAs及其在水稻叶片衰老中的调控作用仍未得到充分解析。
本研究以两份超级杂交水稻——耐衰老型品种内优6号（Nei-2-You 6, N2Y6）与早衰型品种两优培9（Liang-You-Pei 9, LYP9）的叶片为材料，构建6个小RNA文库、6个RNA测序文库及2个降解组文库，通过深度测序技术鉴定得到372个已知miRNAs、162个候选miRNAs以及1145个靶基因。
与两优培9叶片中的miRNA表达水平相比，内优6号叶片在灌浆早期、中期及晚期的上调miRNA数量分别为47、21和11个，下调miRNA数量则分别为30、17和37个。
研究发现，osa-miR159、osa-miR160、osa-miR164、osa-miR167、osa-miR172及osa-miR1848这6个miRNA家族，分别靶向编码APETALA2（AP2）蛋白、锌指蛋白、水杨酸诱导蛋白19（salicylic acid-induced protein 19, SIP19）、生长素响应因子（Auxin response factors, ARF）以及NAC转录因子的基因，它们通过植物激素信号通路参与调控水稻叶片衰老。
上述研究结果为解析miRNA介导的水稻叶片衰老调控机制提供了宝贵的科学依据，同时为水稻遗传育种工作奠定了重要基础。
整体实验设计如下：
[微小RNA测序（miRNA-seq）] 样本1：内优6号水稻灌浆早期旗叶；样本2：内优6号水稻灌浆中期旗叶；样本3：内优6号水稻灌浆晚期旗叶；样本4：两优培9水稻灌浆早期旗叶；样本5：两优培9水稻灌浆中期旗叶；样本6：两优培9水稻灌浆晚期旗叶。
[数字基因表达谱（DGE）] 样本7-12
[降解组测序（degradome）] 样本13：内优6号水稻混合灌浆时期旗叶；样本14：两优培9水稻混合灌浆时期旗叶