Characterization and expression analysis of conserved miRNAs (miR171 and miR482) and their targets in Pinus densa
Characterization and expression analysis of conserved miRNAs (miR171 and miR482) and their targets in Pinus densa
复制标题
浓松保守 miRNA（miR171 和 miR482）及其靶标的表征和表达分析
作者：
Zong-Bo Qiu
MicroRNAs(MiRNAs)是一种小的非编码RNA，通过切割或翻译抑制其靶标mRNAs在植物生长发育中发挥重要的调节作用。虽然在许多植物中已经预测和实验发现了越来越多的保守的miRNAs及其靶标，但这些miRNAs及其靶标还需要进一步的验证和表征。但到目前为止，关于在密松中保守的miRNAs及其靶基因的实验或计算鉴定知之甚少。在本研究中，采用实验验证的方法对密松中保守的miRNAs、miR171和miR482进行了鉴定。对miR171和miR482的保守性分析表明，这些miRNAs在其他植物中高度保守。此外，对miR171和miR482的前体进行了RT-PCR和测序验证。利用实时定量聚合酶链式反应(qRT-PCR)，发现miR171和miR482及其相应靶点在密松针叶、树干和树根中差异表达。此外，利用RNA连接酶介导的5‘端快速扩增基因末端快速扩增技术(RLM-RACE)，进一步验证了GRAS家族转录因子蛋白基因和NBS-LRR抗性蛋白基因分别是pDe-miR171和pde-miR482的靶基因。本研究所做的miR171和miR482在松树体内的实验验证和表达谱分析，以及目的基因的预测，可以加深我们对miR171和miR482在针叶树生长发育中的调控机制的理解。
MicroRNAs (miRNAs) are small non-coding RNAs that play significant regulatory roles in plant growth and development via cleavage or translational inhibition of their target mRNAs. Although a growing number of conserved miRNAs and their targets have been predicted and experimentally discovered in many plants, these miRNAs and their targets need to be further validated and characterized. But to date, little is known about the experimental or computational identification of conserved miRNAs and their target genes in Pinus densata. In the present study, the approach of experimental validation was adopted for characterization of conserved miRNAs, miR171 and miR482 from Pinus densata. Conservation analysis of miR171 and miR482 revealed that these miRNAs are highly conserved in other plant species. In addition, the precursors of miR171 and miR482 were validated by RT-PCR and sequencing. Using quantitative real-time PCR (qRT-PCR), miR171 and miR482 and their corresponding targets were found to be differentially expressed in needles, stems and roots of Pinus densata. Furthermore, two target genes, including a GRAS family transcription factor protein gene and one NBS-LRR resistance protein gene, were experimentally verified to be the targets of pde-miR171 and pde-miR482, respectively using RNA ligase-mediated 5’ rapid amplification of cDNA ends (RLM-RACE). The experimental validation and expression profiling as well target gene prediction of miR171 and miR482 in Pinus densata as done in this study can enhance our understanding of miR171 and miR482 regulatory mechanisms in the growth and development in conifers.