Identification and differential expression of cold-stress-responsive microRNAs in cold-tolerant and -susceptible Hemerocallis fulva varieties

Exploring microRNA (miRNA) expression patterns in Hemerocallis under low-temperature stress helps understanding the mechanisms of miRNA-mediated stress responses. Here, eight separate libraries were generated from the leaves and roots of two different varieties, cold-tolerant ‘Jinyan 7’ and cold-sensitive ‘Lucretius 4’, which were exposed to 20°C and −4°C for 8 h. In total, 333 cold-responsive miRNAs, including 85 putative novel and 248 conserved miRNAs, were identified during different temperature treatments. Under cold-stress conditions, two miRNAs were down-regulated and one was up-regulated in the roots, and no differentially expressed miRNAs were detected in the leaves in ‘Lucretius 4’. In ‘Jinyan 7’, there were two novel and one conserved differentially expressed miRNAs in leaves, whereas three conserved and two novel miRNAs were differentially expressed in roots. The target genes were mainly involved in metabolic processes and cellular components. The differential expression levels of 10 miRNAs were validated using quantitative RT–PCR. Some candidate miRNAs (e.g. ath-miR165a, ath-miR166a, ath-miR395a and novel-miR67) may be involved in plant response-related processes to low-temperature.探究低温胁迫下萱草（Hemerocallis）体内微小核糖核酸（microRNA，miRNA）的表达模式，有助于阐明miRNA介导的植物应激响应机制。本研究分别从耐寒品种‘金燕7号’与冷敏感品种‘Lucretius 4’这两个不同品种的叶片与根系中构建了8个独立文库，供试植株分别置于20°C与−4°C环境下处理8小时。在不同温度处理条件下，共鉴定得到333个低温响应miRNA，其中包含85个推定新型miRNA与248个保守miRNA。在低温胁迫条件下，‘Lucretius 4’的根系中存在2个下调表达、1个上调表达的miRNA，但其叶片中未检测到差异表达miRNA；‘金燕7号’的叶片中鉴定到2个新型与1个保守的差异表达miRNA，根系中则存在3个保守与2个新型的差异表达miRNA。该类miRNA的靶基因主要参与代谢过程与细胞组分相关功能。通过定量逆转录聚合酶链反应（quantitative RT–PCR）验证了10个miRNA的差异表达水平。ath-miR165a、ath-miR166a、ath-miR395a以及novel-miR67等候选miRNA，可能参与植物应对低温的相关响应过程。