Bhardwaj Ankur R, Joshi Gopal, Pandey Ritu, Kukreja Bharti, Goel Shailendra, Jagannath Arun, Kumar Amar, Katiyar-Agarwal Surekha, Agarwal Manu
Department of Botany, University of Delhi, Delhi, India.
Department of Plant Molecular Biology, University of Delhi, South Campus, Delhi, India.
PLoS One. 2014 Mar 26;9(3):e92456. doi: 10.1371/journal.pone.0092456. eCollection 2014.
Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of B. juncea seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.
微小RNA（miRNA）参与多种生物学过程，包括对非生物胁迫的适应性反应。为了揭示可能调控非生物胁迫耐受性决定因素的小RNA，尤其是miRNA，我们对经受高温、高盐和干旱条件的芥菜型油菜幼苗进行了下一代测序。借助UEA sRNA工作台软件包，鉴定出了属于30个miRNA家族的51个保守miRNA。由于芥菜型油菜的基因组信息有限，我们以低覆盖率生成并组装了其基因组序列。以生成的序列和其他公开可用的芸苔属基因组/转录组资源作为映射参考，首次在芥菜型油菜中发现了126个新的（未在任何植物物种中报道过的）miRNA。进一步分析还分别揭示了保守miRNA和新miRNA存在32个和37个星序列。通过基于通用TaqMan的实时PCR，重新验证了所选保守miRNA和新miRNA在不同非生物胁迫条件下的表达。在白菜型油菜中预测了已鉴定的保守miRNA和新miRNA的假定靶标，以深入了解芥菜型油菜miRNA所表现出的功能作用。此外，实验验证了类SPL2、类ARF17和一个含NAC结构域的蛋白分别是miR156、miR160和miR164的靶标。对与已知和新miRNA靶标相关的基因本体的研究预测了它们参与各种生物学功能。
