Identification of soybean seed developmental stage-specific and tissue-specific miRNA targets by degradome sequencing.
Identification of soybean seed developmental stage-specific and tissue-specific miRNA targets by degradome sequencing.
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DOI：
10.1186/1471-2164-13-310
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发表时间：
2012-07-16
期刊：
影响因子：
4.4
通讯作者：
Vodkin L
中科院分区：
文献类型：
--
作者：
Shamimuzzaman M;Vodkin L
在植物和动物中，微小RNA（miRNAs）通过介导基因沉默来调节靶基因的表达。通过计算预测、转基因植物中过表达的实验验证以及degradome或帕雷（RNA末端平行分析）测序，已经在拟南芥和水稻中对miRNA靶点进行了广泛研究。然而，在大豆（Glycine max）中，miRNA靶标大多仍是未知的。更具体地说，在大豆种子发育的不同阶段，miRNA介导的基因调控在很大程度上是未知的。为了剖析大豆发育种子中miRNA引导的基因调控，我们使用降解组测序进行了全转录组实验方法以直接检测切割的miRNA靶。在这项研究中，降解组文库分别从代表三个发育阶段的未成熟大豆子叶和两个阶段的种皮制备。对每个文库的1000万至4000万个读段进行测序和分析，鉴定出53种已知大豆miRNAs的183种不同靶标。其中，一些仅在子叶中发现，代表由25个miRNA切割，而另一些仅在种皮中发现，反映由12个miRNA切割。在两种组织中鉴定了16个miRNAs家族的大量靶点，而与阶段无关。有趣的是，我们在种子成熟后期的干燥子叶中比在未成熟种子中鉴定出更多的miRNA靶点。我们通过RNA连接酶介导的cDNA末端的5'快速扩增（RLM-5' RACE）验证了四种不同的生长素应答因子基因作为gma-miR 160的靶标。基因本体（GO）分析表明miRNA靶基因参与种子发育过程中的各种细胞过程。大豆种子发育的几个阶段的子叶和种皮中的miRNA靶点已经通过来自对由miRNA引导的信使RNA切割产生的富集片段的全面、高通量测序的实验证据阐明。近50%的miRNA靶点是转录因子，这些转录因子在设定或维持高质量大豆种子的发育程序中可能很重要，而大豆种子是世界市场上蛋白质和油的主要来源之一。
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.
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