Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.
Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.
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DOI：
10.1186/s12864-015-2010-6
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发表时间：
2015-10-22
期刊：
影响因子：
4.4
通讯作者：
Tang Q
中科院分区：
文献类型：
--
作者：
Wei R;Qiu D;Wilson IW;Zhao H;Lu S;Miao J;Feng S;Bai L;Wu Q;Tu D;Ma X;Tang Q
MicroRNA（miRNAs）是一类小分子非编码RNA，是基因表达的重要调控因子，在植物发育和对环境的响应中起重要作用。三七根提取物含有三萜皂苷作为其主要生物活性成分，并显示出药用特性。为了研究三七中新的和保守的miRNAs，从1年，2年和3年的根中构建了三个小RNA文库，其中根皂苷水平不同，进行了高通量测序。三七根，纯化从1-，2-，和3岁的根，分别提取RNA。构建三个小文库并进行下一代测序。三个文库的测序产生了来自三七根的67，217，124个干净读段。共鉴定出316个保守的miRNAs（属于67个miRNAs家族和1个未分类家族）和52个新的miRNAs。MIR 156和MIR 166是最大的miRNA家族，而miR 156 i和miR 156 g显示了最高丰度的miRNA种类。使用Cluster of Orthopathic Groups、Gene Ontology和京都基因和基因组百科全书预测和注释潜在的miRNA靶基因。比较根样品之间的这些miRNA，发现33个在2年和1年的根之间差异表达（8个增加，25个减少），27个在3年和1年的根之间差异表达（7个增加，20个减少），29个在3年和2年的根之间差异表达（8个增加，21个减少）。选择两个显著差异表达的miRNAs和四个预测靶向参与萜类骨架生物合成途径的基因的miRNAs，并通过定量逆转录PCR进行验证。此外，还分析了这6种miRNAs在三七根、茎和叶不同发育阶段的表达模式。本研究鉴定了大量三七miRNAs及其靶基因、功能注释和基因表达模式。它提供了三七根发育周期的第一个已知的miRNA谱。本文的在线版本（doi：10.1186/s12864-015-2010-6）包含补充材料，可供授权用户使用。
MicroRNAs (miRNAs) are small, non-coding RNAs that are important regulators of gene expression, and play major roles in plant development and their response to the environment. Root extracts from Panax notoginseng contain triterpene saponins as their principal bioactive constituent, and demonstrate medicinal properties. To investigate the novel and conserved miRNAs in P. notoginseng, three small RNA libraries constructed from 1-, 2-, and 3-year-old roots in which root saponin levels vary underwent high-throughput sequencing. P. notoginseng roots, purified from 1-, 2-, and 3-year-old roots, were extracted for RNA, respectively. Three small libraries were constructed and subjected to next generation sequencing. Sequencing of the three libraries generated 67,217,124 clean reads from P. notoginseng roots. A total of 316 conserved miRNAs (belonging to 67 miRNA families and one unclassified family) and 52 novel miRNAs were identified. MIR156 and MIR166 were the largest miRNA families, while miR156i and miR156g showed the highest abundance of miRNA species. Potential miRNA target genes were predicted and annotated using Cluster of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Comparing these miRNAs between root samples revealed 33 that were differentially expressed between 2- and 1-year-old roots (8 increased, 25 decreased), 27 differentially expressed between 3- and 1-year-old roots (7 increased, 20 decreased), and 29 differentially expressed between 3- and 2-year-old roots (8 increased, 21 decreased). Two significantly differentially expressed miRNAs and four miRNAs predicted to target genes involved in the terpenoid backbone biosynthesis pathway were selected and validated by quantitative reverse transcription PCR. Furthermore, the expression patterns of these six miRNAs were analyzed in P. notoginseng roots, stems, and leaves at different developmental stages. This study identified a large number of P. notoginseng miRNAs and their target genes, functional annotations, and gene expression patterns. It provides the first known miRNA profiles of the P. notoginseng root development cycle. The online version of this article (doi:10.1186/s12864-015-2010-6) contains supplementary material, which is available to authorized users.