MicroRNA-guided regulation of heat stress response in wheat. MicroRNA-guided regulation of heat stress response in wheat

Sequencing of 24 small RNA libraries produced 55.2M reads while 404M reads were obtained from the corresponding 24 PARE libraries. From these, 202 miRNAs were ascertained, of which mature miRNA evidence was obtained for 104 and 36 were found to be differentially expressed after heat stress. The PARE analysis identified 589 transcripts targeted by 84 of the ascertained miRNAs. PARE sequencing validated the targets of the conserved members of miRNA156, miR166 and miR393 families as squamosa promoter-binding-like, homeobox leucine-zipper and transport inhibitor responsive proteins, respectively. Heat stress responsive miRNA targeted superoxide dismutases and an array of homeobox leucine-zipper proteins, F-box proteins and protein kinases. Query of miRNA targets to interactome databases revealed a predominant association of stress responses such as signalling, antioxidant activity and ubiquitination to superoxide dismutases, F-box proteins, pentatricopeptide repeat-containing proteins and mitochondrial transcription termination factor-like proteins. Overall design: A total of 144 Triticum aestivum cv Chinese Spring wheat plants were grown in a PG-40 growth cabinet under long-day conditions of 16 hours of light (300 μmol m−2 s−1) at 18°C and 8 hours of darkness at 16°C. At the boot stage, a set of 72 plants were exposed to a 37°C heat stress for 5 days while the other 72 plants remained under the control conditions mentioned above. The heat stressed plants were amply watered twice per day during the 5-day stress period to avoid a confounding effect with drought stress. Leaf tissue was collected from all heat-stressed and control individual plants immediately at the end of the stress period, i.e., time point zero day after treatment (0 DAT), and at one and four days after treatment (1 and 4 DAT). Equimolar amounts of RNA from the 18 samples representing a replicate, treatment and time point were pooled to create the 24 total RNA samples representing the four biological replicates, two treatments and three time points. These 24 total RNA samples were used to construct sRNA libraries.

对24个小RNA（small RNA, sRNA）文库进行高通量测序，共产出55.2百万条reads；对应24个PARE（Parallel Analysis of RNA Ends）文库则获得4.04亿条reads。从中共鉴定得到202个miRNA（microRNA），其中104个获得了成熟miRNA的表达证据，另有36个miRNA在热胁迫处理后呈现差异表达。
PARE分析共鉴定到84个已鉴定miRNA的589个靶标转录本。PARE测序验证了miRNA156、miR166及miR393保守家族的靶标，分别为Squamosa启动子结合蛋白样（squamosa promoter-binding-like）蛋白、同源域亮氨酸拉链（homeobox leucine-zipper）蛋白以及转运抑制因子响应蛋白。
响应热胁迫的miRNA靶标包括超氧化物歧化酶，以及一系列同源域亮氨酸拉链蛋白、F-box蛋白和蛋白激酶。对miRNA靶标进行互作组数据库检索后发现，信号转导、抗氧化活性及泛素化等应激响应通路，与超氧化物歧化酶、F-box蛋白、五肽重复序列（pentatricopeptide repeat）包含蛋白及线粒体转录终止因子样蛋白存在显著关联。
实验整体设计：共培育144株普通小麦（Triticum aestivum）品种中国春植株，在PG-40生长箱中采用长日照培养条件：光照时长16小时，光强300 μmol·m⁻²·s⁻¹，温度18℃；黑暗时长8小时，温度16℃。在孕穗期，将72株植株置于37℃下进行5天热胁迫处理，剩余72株保持上述对照培养条件。热胁迫处理期间，每日为胁迫组植株充分浇水两次，以排除干旱胁迫对实验结果的干扰。
分别在胁迫处理结束时（即处理后0天，0 DAT）、处理后1天（1 DAT）及处理后4天（4 DAT），从所有胁迫组及对照组植株中采集叶片组织。将每个重复、处理组及时间点对应的18份样本的RNA按等摩尔浓度混合，共构建得到24个总RNA样本，对应4个生物学重复、2个处理组及3个时间点。利用这24个总RNA样本构建sRNA文库。