Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
BMC Plant Biol. 2012 Aug 15;12:146. doi: 10.1186/1471-2229-12-146.
Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.
To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.
We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.
小非编码 RNA（sRNA）在植物发育、生长以及对生物和非生物胁迫的响应中起着关键作用。至少有四类 sRNA 在植物中得到了很好的描述，包括重复相关的 siRNA（rasiRNA）、microRNA（miRNA）、反式作用 siRNA（tasiRNA）和天然反义转录物衍生的 siRNA。杉木（Cunninghamia lanceolata）是中国最重要的针叶常绿树种之一。迄今为止，尚未描述过来自杉木的 sRNA。
为了获得杉木中的 sRNA，我们使用 Illumina 高通量测序技术对来自种子、幼苗、叶片、茎和愈伤组织的 sRNA 文库进行了测序。获得了一套全面的 sRNA，包括保守和新的 miRNA、rasiRNA 和 tasiRNA。通过 BLASTN 和 MIREAP，我们总共鉴定出 115 个保守 miRNA，包括 40 个 miRNA 家族和一个具有前体序列的新 miRNA。通过 RT-PCR 检测了 16 个保守 miRNA 和 1 个新 miRNA 和 1 个 tasiRNA 的表达。利用实时 RT-PCR，我们揭示了四个保守 miRNA 和一个新 miRNA 在杉木中具有发育阶段特异性的表达模式。此外，209 个 unigenes 被预测为 30 个杉木 miRNA 家族的靶基因，其中五个靶基因通过 5' RACE 实验验证，包括一个花椰菜花叶病毒 35S 启动子结合蛋白基因、一个五肽重复蛋白基因、一个 BolA 样家族蛋白基因、AGO1 和一个未知功能的基因。我们还证明了 DCL3 依赖的 rasiRNA 生物发生途径，该途径在针叶树中被认为不存在，存在于杉木中。此外，阐明了 miR390-TAS3-ARF 调控途径。
我们通过高通量测序揭示了杉木中复杂的 sRNA 群体。这为杉木中 sRNA 的组成和功能提供了深入的了解，并为陆地植物 sRNA 进化提供了新的线索。
