Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA.
BMC Genomics. 2012 Jul 16;13:310. doi: 10.1186/1471-2164-13-310.
MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets.
In this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5' rapid amplification of cDNA ends (RLM-5'RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development.
The miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.
微小 RNA（miRNA）通过介导动植物基因沉默来调节靶基因的表达。利用计算预测、在转基因植物中转基因过表达的实验验证以及降解组或 PARE（RNA 末端平行分析）测序，已在拟南芥和水稻中广泛研究了 miRNA 靶标。然而，大豆（Glycine max）中的 miRNA 靶标大多仍未知。更具体地说，大豆发育种子中不同种子发育阶段的 miRNA 介导基因调控在很大程度上仍未被探索。为了剖析大豆发育种子中 miRNA 指导的基因调控，我们使用降解组测序进行了全转录组实验方法，直接检测切割的 miRNA 靶标。
在这项研究中，分别从小豆未成熟子叶的三个发育阶段和种皮的两个阶段制备降解组文库。从每个文库中测序和分析 10 到 4000 万个读数，鉴定了 53 个已知大豆 miRNA 的 183 个不同靶标。其中，一些仅在代表 25 个 miRNA 切割的子叶中发现，而另一些仅在反映 12 个 miRNA 切割的种皮中发现。16 个 miRNA 家族的大量靶标在两个组织中都被鉴定出来，而与阶段无关。有趣的是，我们在晚期种子成熟的干燥子叶中鉴定到的 miRNA 靶标比在未成熟种子中多。我们通过 RNA 连接酶介导的 5'快速扩增 cDNA 末端（RLM-5'RACE）验证了 4 个不同的生长素响应因子基因作为 gma-miR160 的靶标。基因本体论（GO）分析表明，miRNA 靶基因参与了种子发育过程中各种细胞过程。
通过对 miRNA 引导的信使 RNA 切割产生的富含片段进行高通量测序的综合实验证据，阐明了大豆种子发育的几个阶段的子叶和种皮中的 miRNA 靶标。近 50%的 miRNA 靶标是转录因子，它们参与的途径可能对建立或维持导致高质量大豆种子的发育程序很重要，而大豆种子是世界市场上蛋白质和油的主要来源之一。
