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高通量测序和生物信息学分析鉴定杨桃 Eugenia uniflora 中的 microRNAs。

Identification of microRNAs from Eugenia uniflora by high-throughput sequencing and bioinformatics analysis.

机构信息

PPGGBM, Departamento de Genética, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, Rio Grande do Sul, Brazil.

出版信息

PLoS One. 2012;7(11):e49811. doi: 10.1371/journal.pone.0049811. Epub 2012 Nov 15.

Abstract

BACKGROUND

microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species.

RESULTS

Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology.

CONCLUSIONS

This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

摘要

背景

miRNAs 或 microRNAs 是小的非编码调控 RNA,通过靶向 mRNAs 进行降解或抑制蛋白质翻译,在转录后水平上对基因表达发挥重要功能。Eugenia uniflora 是一种原产于热带美洲的植物,具有药理学和生态学重要性,以前没有关于其基因表达和调控的研究。迄今为止,桃金娘科物种中尚未报道过 miRNAs。

结果

构建了小 RNA 和 RNA-seq 文库,以鉴定 Eugenia uniflora 中的 miRNAs 和 pre-miRNAs。使用 Solexa 技术对文库进行高通量测序,并用生物信息学工具分析获得的数据。从 14,489,131 个小 RNA 清洁读取中,我们获得了 1,852,722 个成熟 miRNA 序列,代表了在其他植物物种中已鉴定出的 45 个保守家族。使用 RNA-seq 组装的 contigs 进一步分析,预测了 25 个已知和 17 个新的 pre-miRNAs 的二级结构。使用 RT-PCR 测定还验证了 27 个鉴定的 miRNAs 的表达。根据序列同源性,预测了在鉴定的 pre-miRNAs 中最丰富的成熟 miRNAs 的潜在靶标。

结论

这项研究是首次在没有基因组序列资源的桃金娘科物种中大规模鉴定 miRNAs 及其潜在靶标。我们的研究提供了更多关于植物 miRNA 调控网络进化保守性的信息,并突出了物种特异性的 miRNAs。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eb16/3499529/5b8b9502c0cd/pone.0049811.g001.jpg

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