Bai Bin, Shi Bo, Hou Ning, Cao Yanli, Meng Yijun, Bian Hongwu, Zhu Muyuan, Han Ning
Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Institute of Genetics and Regenerative Biology, College of Life Sciences, Zhejiang University, Zhejiang, Hangzhou, 310058, China.
College of Life and Environmental Sciences, Hangzhou Normal University, Zhejiang, Hangzhou, 310036, China.
BMC Plant Biol. 2017 Sep 6;17(1):150. doi: 10.1186/s12870-017-1095-2.
Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. However, detailed spatial and temporal studies of miRNA-mediated regulation, which can reflect links between seed development and germination are still lacking.
In this study, we extended our investigation on miRNAs-involved gene regulation by a combined analysis of seed maturation and germination in barley. Through bioinformatics analysis of small RNA sequencing data, a total of 1324 known miRNA families and 448 novel miRNA candidates were identified. Of those, 16 known miRNAs with 40 target genes, and three novel miRNAs with four target genes were confirmed based on degradome sequencing data. Conserved miRNA families such as miR156, miR168, miR166, miR167, and miR894 were highly expressed in embryos of developing and germinating seeds. A barley-specific miRNA, miR5071, which was predicted to target an OsMLA10-like gene, accumulated at a high level, suggesting its involvement in defence response during these two developmental stages. Based on target prediction and Kyoto Encyclopedia of Genes and Genomes analysis of putative targets, nine highly expressed miRNAs were found to be related to phytohormone signalling and hormone cross-talk. Northern blot and qRT-PCR analysis showed that these miRNAs displayed differential expression patterns during seed development and germination, indicating their different roles in hormone signalling pathways. In addition, we showed that miR393 affected seed development through targeting two genes encoding the auxin receptors TIR1/AFBs in barley, as over-expression of miR393 led to an increased length-width ratio of seeds, whereas target mimic (MIM393)-mediated inhibition of its activity decreased the 1000-grain weight of seeds. Furthermore, the expression of auxin-responsive genes, abscisic acid- and gibberellic acid-related genes was altered in miR393 misexpression lines during germination and early seedling growth.
Our work indicates that miRNA-target pairs participate in gene expression regulation and hormone interaction in barley embryo and provides evidence that miR393-mediated auxin response regulation affects grain development and influences gibberellic acid and abscisic acid homeostasis during germination.
小RNA和降解组测序已在植物种子中鉴定出大量的miRNA-靶标对。然而，仍缺乏能够反映种子发育与萌发之间联系的miRNA介导调控的详细时空研究。
在本研究中，我们通过对大麦种子成熟和萌发的综合分析，扩展了对miRNA参与的基因调控的研究。通过对小RNA测序数据的生物信息学分析，共鉴定出1324个已知的miRNA家族和448个新的miRNA候选物。其中，基于降解组测序数据确认了16个已知的miRNA及其40个靶基因，以及3个新的miRNA及其4个靶基因。保守的miRNA家族，如miR156、miR168、miR166、miR167和miR894在发育中和萌发种子的胚中高表达。一个预测靶向类OsMLA10基因的大麦特异性miRNA miR5071高水平积累，表明其在这两个发育阶段参与防御反应。基于靶标预测和对假定靶标的京都基因与基因组百科全书分析，发现9个高表达的miRNA与植物激素信号传导和激素相互作用有关。Northern杂交和qRT-PCR分析表明，这些miRNA在种子发育和萌发过程中表现出差异表达模式，表明它们在激素信号通路中具有不同作用。此外，我们发现miR393通过靶向大麦中两个编码生长素受体TIR1/AFB的基因影响种子发育，因为miR393的过表达导致种子长宽比增加，而靶标模拟物（MIM393）介导的其活性抑制降低了种子的千粒重。此外，在萌发和幼苗早期生长过程中，miR393表达失调的株系中生长素响应基因、脱落酸和赤霉素相关基因的表达发生了改变。
我们的工作表明，miRNA-靶标对参与大麦胚中的基因表达调控和激素相互作用，并提供了证据表明miR393介导的生长素反应调控影响籽粒发育并在萌发过程中影响赤霉素和脱落酸稳态。
