通过高通量测序鉴定三七根中新型和保守的微小RNA

Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.

作者信息

Wei Rongchang, Qiu Deyou, Wilson Iain W, Zhao Huan, Lu Shanfa, Miao Jianhua, Feng Shixin, Bai Longhua, Wu Qinghua, Tu Dongping, Ma Xiaojun, Tang Qi

机构信息

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China.

Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China.

出版信息

BMC Genomics. 2015 Oct 22;16:835. doi: 10.1186/s12864-015-2010-6.

DOI:10.1186/s12864-015-2010-6

PMID:26490136

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4618736/

Abstract

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

摘要

背景

微小RNA（miRNA）是一类小的非编码RNA，是基因表达的重要调节因子，在植物发育及其对环境的响应中发挥主要作用。三七根提取物含有三萜皂苷作为其主要生物活性成分，并具有药用特性。为了研究三七中新型和保守的miRNA，构建了来自1年生、2年生和3年生根的三个小RNA文库，这些根中的皂苷水平各不相同，并进行了高通量测序。

方法

分别从1年生、2年生和3年生的三七根中提取并纯化RNA，构建三个小文库并进行新一代测序。

结果

对这三个文库进行测序，从三七根中获得了67,217,124条干净 reads。共鉴定出316个保守miRNA（属于67个miRNA家族和1个未分类家族）和52个新型miRNA。MIR156和MIR166是最大的miRNA家族，而miR156i和miR156g显示出最高的miRNA种类丰度。使用直系同源簇、基因本体论和京都基因与基因组百科全书对潜在的miRNA靶基因进行了预测和注释。比较根样本之间的这些miRNA，发现2年生和1年生根之间有33个差异表达（8个上调，25个下调），3年生和1年生根之间有27个差异表达（7个上调，20个下调），3年生和2年生根之间有29个差异表达（8个上调，21个下调）。选择两个差异显著的miRNA和四个预测靶向参与萜类骨架生物合成途径基因的miRNA，并通过定量逆转录PCR进行验证。此外，还分析了这六个miRNA在不同发育阶段三七根、茎和叶中的表达模式。