Sun Ping, Cheng Chunzhen, Lin Yuling, Zhu Qiufang, Lin Jinke, Lai Zhongxiong
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China.
Anxi College of Tea Science, Fujian Agriculture and Forestry University, Fuzhou, China.
PLoS One. 2017 Feb 22;12(2):e0171173. doi: 10.1371/journal.pone.0171173. eCollection 2017.
MicroRNAs are endogenous non-coding small RNAs playing crucial regulatory roles in plants. Tea, a globally popular non-alcoholic drink, is rich in health-enhancing catechins. In this study, 69 conserved and 47 novel miRNAs targeting 644 genes were identified by high-throughout sequencing. Predicted target genes of miRNAs were mainly involved in plant growth, signal transduction, morphogenesis and defense. To further identify targets of tea miRNAs, degradome sequencing and RNA ligase-mediated rapid amplification of 5'cDNA ends (RLM-RACE) were applied. Using degradome sequencing, 26 genes mainly involved in transcription factor, resistance protein and signal transduction protein synthesis were identified as potential miRNA targets, with 5 genes subsequently verified. Quantitative real-time PCR (qRT-PCR) revealed that the expression patterns of novel-miR1, novel-miR2, csn-miR160a, csn-miR162a, csn-miR394 and csn-miR396a were negatively correlated with catechin content. The expression of six miRNAs (csn-miRNA167a, csn-miR2593e, csn-miR4380a, csn-miR3444b, csn-miR5251 and csn-miR7777-5p.1) and their target genes involved in catechin biosynthesis were also analyzed by qRT-PCR. Negative and positive correlations were found between these miRNAs and catechin contents, while positive correlations were found between their target genes and catechin content. This result suggests that these miRNAs may negatively regulate catechin biosynthesis by down-regulating their biosynthesis-related target genes. Taken together, our results indicate that miRNAs are crucial regulators in tea, with the results of 5'-RLM-RACE and expression analyses revealing the important role of miRNAs in catechin anabolism. Our findings should facilitate future research to elucidate the function of miRNAs in catechin biosynthesis.
微小RNA是植物中发挥关键调控作用的内源性非编码小RNA。茶是一种全球流行的非酒精饮料，富含具有保健作用的儿茶素。在本研究中，通过高通量测序鉴定出69个保守的和47个新的靶向644个基因的微小RNA。微小RNA的预测靶基因主要参与植物生长、信号转导、形态发生和防御。为了进一步鉴定茶微小RNA的靶标，应用了降解组测序和RNA连接酶介导的5' cDNA末端快速扩增（RLM-RACE）。通过降解组测序，鉴定出26个主要参与转录因子、抗性蛋白和信号转导蛋白合成的基因作为潜在的微小RNA靶标，随后验证了其中5个基因。定量实时PCR（qRT-PCR）显示，新miR1、新miR2、csn-miR160a、csn-miR162a、csn-miR394和csn-miR396a的表达模式与儿茶素含量呈负相关。还通过qRT-PCR分析了6个微小RNA（csn-miRNA167a、csn-miR2593e、csn-miR4380a、csn-miR3444b、csn-miR5251和csn-miR7777-5p.1）及其参与儿茶素生物合成的靶基因。这些微小RNA与儿茶素含量之间存在负相关和正相关，而它们的靶基因与儿茶素含量之间存在正相关。该结果表明，这些微小RNA可能通过下调其生物合成相关靶基因来负调控儿茶素生物合成。综上所述，我们的结果表明微小RNA是茶叶中的关键调节因子，5'-RLM-RACE和表达分析结果揭示了微小RNA在儿茶素合成代谢中的重要作用。我们的发现应有助于未来阐明微小RNA在儿茶素生物合成中功能的研究。
