High-throughput sequencing of small RNAs in Nicotiana tabacum

Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Nicotiana tabacum tissues (including leaves, flowers and pods). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and pods of Nicotiana tabacum. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and flowers, and the Plant RNA Purification Reagent (Invitrogen) for pods, and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Barbara Baker for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.

小RNA（Small RNAs，长度21-24 nt）是基因表达的关键调控因子，可介导多种真核生物（涵盖几乎所有植物类群）中的转录及转录后基因沉默通路。微RNA（MicroRNAs，miRNAs）与小干扰RNA（short interfering RNAs，siRNAs）是两类主要的小RNA，二者均在植物发育、胁迫响应及病原体抗性中发挥已被证实的重要作用。本研究采用深度测序技术（边合成边测序，Sequencing-By-Synthesis，简称SBS），构建了烟草（Nicotiana tabacum）不同组织（叶片、花及荚果）的小RNA序列资源库。所得数据集的高测序深度使得我们能够详细解析该物种中小RNA的多项关键特征，包括长度分布、组织特异性调控以及不同器官间的序列保守性。本研究同时开发了数据库资源与专用网站（http://smallrna.udel.edu/），配套计算工具以供其他研究者识别参与特定调控通路的新型miRNAs或siRNAs、验证这些序列在其他植物物种中的保守程度，以及将小RNA定位到目标基因或研究中的玉米基因组更大区域上。小RNA文库来源于烟草的叶片、花及荚果。其中，叶片与花的总RNA采用TriReagent（Molecular Research Center公司）提取，荚果的总RNA则采用Plant RNA Purification Reagent（Invitrogen公司）提取；随后样本被送至Illumina公司（美国加利福尼亚州海沃德市，http://www.illumina.com），参照Lu等人2007年发表的方法并稍作修改，完成小RNA文库构建。小RNA文库通过Illumina的边合成边测序（SBS）技术完成测序。研究人员设计了PERL脚本以去除接头序列，并确定每一种独特小RNA的表达丰度。本研究感谢Barbara Baker提供植物材料，同时感谢Kan Nobuta与Gayathri Mahalingam在计算方法方面提供的协助。