Identification of Taxus microRNAs and their targets with high-throughput sequencing and degradome analysis
Identification of Taxus microRNAs and their targets with high-throughput sequencing and degradome analysis
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DOI：
10.1111/j.1399-3054.2012.01668.x
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发表时间：
2012-12-01
影响因子：
6.4
通讯作者：
Liu, Ming
中科院分区：
文献类型：
--
作者：
Hao, Da-Cheng;Yang, Ling;Liu, Ming
植物microRNA（miRNAs）通过在转录后水平负调控基因表达，对发育、生长和代谢等生物学过程产生影响。然而，这些小分子在药用裸子植物红豆杉中的作用仍然难以捉摸。为了阐明miRNAs在红豆杉中的作用，我们使用深度测序方法分析了南方红豆杉叶片的小RNA和降解产物序列标签。对于miRNA，测序文库产生了1490万个短序列，产生了1310万个干净的读数。该文库主要含有21个核苷酸长度的小RNA，其次是19-nt和20-nt的小RNA。大约29%的小RNA与T. mairei转录组。通过序列比对，我们鉴定了871个成熟的miRNA，15个miRNA* 和869个代表已知植物miRNA家族的miRNA前体。有547个独特的小RNA匹配的miRNA前体。我们从未注释的小RNA中预测了37个候选的新miRNA，这些小RNA可以映射到参考转录组。首次通过实时逆转录聚合酶链反应定量了所选候选基因的表达。新的miRNA m0034原来来自紫杉醇生物合成基因紫杉二烯合酶的内含子序列。预测了9个新的miRNAs的21个潜在靶点。此外，通过高通量降解组测序方法鉴定了已知miRNA家族的56个靶标和新候选miRNA家族的15个靶标。发现紫杉醇生物合成基因紫杉烷13 a羟化酶和紫杉烷2a-O-苯甲酰转移酶分别是miR 164和miR 171的切割靶点。这项研究代表了裸子植物miRNAs和degradome的第一个基于转录组的分析。
Plant microRNAs (miRNAs) have an impact in the regulation of several biological processes such as development, growth and metabolism by negatively controlling gene expression at the post-transcriptional level. However, the role of these small molecules in the medicinal gymnosperm species Taxus remained elusive. To elucidate the role of miRNAs in Taxus we used a deep sequencing approach to analyze the small RNA and degradome sequence tags of Taxus mairei leaves. For miRNAs, the sequencing library generated 14.9 million short sequences, resulting in 13.1 million clean reads. The library contains predominantly small RNAs with 21 nucleotide length, followed by 19-nt and 20-nt small RNAs. Around 29% of total small RNAs are matched to the T. mairei transcriptome. By sequence alignment, we identified 871 mature miRNAs, 15 miRNA* and 869 miRNA precursors representing known plant miRNA families. There are 547 unique small RNA matching the miRNA precursors. We predict 37 candidate novel miRNAs from the unannotated small RNAs that could be mapped to the reference transcriptome. The expression of the selected candidates was for the first time quantified by real-time reverse transcription polymerase chain reaction. The novel miRNA m0034 turns out to be from the intron sequence of the paclitaxel biosynthetic gene taxadiene synthase. The 21 potential targets of nine novel miRNAs are also predicted. Additionally, 56 targets for known miRNA families and 15 targets for novel candidate miRNA families were identified by high-throughput degradome-sequencing approach. It is found that two paclitaxel biosynthetic genes, taxane 13a hydroxylase and taxane 2a-O-benzoyltransferase, are the cleavage targets of miR164 and miR171, respectively. This study represents the first transcriptome-based analysis of miRNAs and degradome in gymnosperms.