The Key Lab of National Tobacco Cultivation, College of Life Sciences, Henan Agricultural University, Zhengzhou, China.
PLoS One. 2011;6(12):e28565. doi: 10.1371/journal.pone.0028565. Epub 2011 Dec 14.
Topping is an important cultivating measure for flue-cured tobacco, and many genes had been found to be differentially expressed in response to topping. But it is still unclear how these genes are regulated. MiRNAs play a critical role in post-transcriptional gene regulation, so we sequenced two sRNA libraries from tobacco roots before and after topping, with a view to exploring transcriptional differences in miRNAs.
METHODOLOGY/PRINCIPAL FINDINGS: Two sRNA libraries were generated from tobacco roots before and after topping. Solexa high-throughput sequencing of tobacco small RNAs revealed a total of 12,104,207 and 11,292,018 reads representing 3,633,398 and 3,084,102 distinct sequences before and after topping. The expressions of 136 conserved miRNAs (belonging to 32 families) and 126 new miRNAs (belonging to 77 families) were determined. There were three major conserved miRNAs families (nta-miR156, nta-miR172 and nta-miR171) and two major new miRNAs families (nta-miRn2 and nta-miRn26). All of these identified miRNAs can be folded into characteristic miRNA stem-loop secondary hairpin structures, and qRT-PCR was adopted to validate and measure the expression of miRNAs. Putative targets were identified for 133 out of 136 conserved miRNAs and 126 new miRNAs. Of these miRNAs whose targets had been identified, the miRNAs which change markedly (>2 folds) belong to 53 families and their targets have different biological functions including development, response to stress, response to hormone, N metabolism, C metabolism, signal transduction, nucleic acid metabolism and other metabolism. Some interesting targets for miRNAs had been determined.
CONCLUSIONS/SIGNIFICANCE: The differential expression profiles of miRNAs were shown in flue-cured tobacco roots before and after topping, which can be expected to regulate transcripts distinctly involved in response to topping. Further identification of these differentially expressed miRNAs and their targets would allow better understanding of the regulatory mechanisms for flue-cured tobacco response to topping.
打顶是烤烟的一项重要栽培措施，许多基因在打顶后表现出差异表达。但这些基因如何调控仍不清楚。miRNA 在转录后基因调控中起着关键作用，因此我们从前茬打顶后和后茬打顶后的烟草根部分别构建了两个 sRNA 文库，旨在探索 miRNA 的转录差异。
方法/主要发现：从前茬打顶后和后茬打顶后的烟草根部分别构建了两个 sRNA 文库。通过对烟草小 RNA 的 Solexa 高通量测序，共获得了 12104207 和 11292018 个reads，分别代表前茬打顶和后茬打顶的 3633398 和 3084102 个独特序列。测定了 136 个保守 miRNA（属于 32 个家族）和 126 个新 miRNA（属于 77 个家族）的表达。有三个主要的保守 miRNA 家族（nta-miR156、nta-miR172 和 nta-miR171）和两个主要的新 miRNA 家族（nta-miRn2 和 nta-miRn26）。所有这些鉴定出的 miRNA 都可以折叠成特征性的 miRNA 茎环二级发夹结构，并采用 qRT-PCR 进行验证和测定 miRNA 的表达。136 个保守 miRNA 和 126 个新 miRNA 中的 133 个都鉴定出了假定的靶标。在这些鉴定出靶标的 miRNA 中，变化明显（>2 倍）的 miRNA 属于 53 个家族，它们的靶标具有不同的生物学功能，包括发育、应激响应、激素响应、氮代谢、碳代谢、信号转导、核酸代谢和其他代谢。确定了一些 miRNA 的有趣靶标。
结论/意义：展示了打顶前后烤烟根中 miRNA 的差异表达谱，有望对调控与打顶反应明显相关的转录物产生显著影响。进一步鉴定这些差异表达的 miRNA 及其靶标，将有助于更好地理解烤烟对打顶反应的调控机制。
