The Key Laboratory of Forest Genetics and Gene Engineering of the Ministry of Education, Nanjing Forestry University, Nanjing, China.
PLoS One. 2012;7(8):e43451. doi: 10.1371/journal.pone.0043451. Epub 2012 Aug 29.
To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).
METHODOLOGY/PRINCIPAL FINDINGS: A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.
Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.
迄今为止，已有多项研究表明 microRNAs（miRNAs）在调控植物发育中发挥着重要作用，但 miRNA 对体细胞胚胎发育的调控作用还知之甚少，尤其是在硬木植物体细胞胚胎发生的早期阶段。本研究采用 Solexa 测序和 miRNA 微流控芯片技术，发现杂交鹅掌楸（Liriodendron tulipifera×L. chinense）体细胞胚胎发生过程中保守和种特异性 miRNAs。
方法/主要发现：从小RNA 文库中提取小 RNA 后，通过 Solexa 测序获得了代表 7421623 个独特序列的 17214153 个reads。通过深度测序和生物信息学分析相结合的方法，我们从 33 个保守 miRNA 家族和 273 个种特异性候选 miRNA 中发现了 83 个与已知 miRNA 完全匹配的序列。miRNA 微阵列结果表明，许多保守和种特异性 miRNAs 在杂交鹅掌楸胚胎中表达。此外，微阵列还检测到另外 149 个潜在 miRNA，属于 29 个保守家族，这些 miRNA在深度测序分析中未被发现。通过对拟南芥 GenBank 序列进行 BLAST 搜索，并对结果进行基因本体论分析，预测了这些 miRNA 靶基因的生物过程和分子功能。
本研究采用 Solexa 测序和微阵列杂交技术，从 61 个 miRNA 家族和 273 个种特异性 miRNA 中发现了 232 个候选保守 miRNA 和 273 个候选种特异性 miRNA。在这些预测的 miRNA 中，有 64 个保守 miRNA 和 177 个种特异性 miRNA 通过测序和微阵列杂交都被检测到。研究结果表明，miRNAs 在该经济重要物种的体细胞胚胎发生的各个阶段都具有广泛的特征和重要作用。
