Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis)

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RNA（microRNAs，miRNAs）是一类内源性非编码小RNA，在植物中发挥关键调控作用。茶作为全球广受欢迎的非酒精饮品，富含有益人体健康的儿茶素。本研究通过高通量测序技术，共鉴定出69个保守型miRNA与47个新型miRNA，这些miRNA总计靶向644个基因。经预测，这些miRNA的靶基因主要参与植物生长、信号转导、形态建成以及防御反应等生物学过程。为进一步鉴定茶树miRNA的靶基因，本研究采用了降解组测序（degradome sequencing）与RNA连接酶介导的5'cDNA末端快速扩增技术（RNA ligase-mediated rapid amplification of 5’cDNA ends, RLM-RACE）。通过降解组测序，本研究鉴定出26个潜在miRNA靶基因，这些基因主要参与转录因子、抗性蛋白及信号转导蛋白的合成过程，其中5个靶基因得到后续验证。实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）结果显示，novel-miR1、novel-miR2、csn-miR160a、csn-miR162a、csn-miR394与csn-miR396a的表达模式与儿茶素含量呈负相关。本研究还通过qRT-PCR分析了6个miRNA（csn-miRNA167a、csn-miR2593e、csn-miR4380a、csn-miR3444b、csn-miR5251及csn-miR7777-5p.1）及其参与儿茶素生物合成的靶基因的表达情况。针对该6个miRNA的分析结果显示，其与儿茶素含量间分别存在负相关或正相关关系，而其靶基因与儿茶素含量则呈正相关。该结果提示，这些miRNA可能通过下调其儿茶素生物合成相关靶基因的表达，负调控儿茶素的生物合成过程。综上，本研究结果证实miRNA是茶树中的关键调控因子，5'-RLM-RACE与表达分析结果进一步揭示了miRNA在儿茶素合成代谢中的重要作用。本研究成果可为后续解析miRNA在茶树儿茶素生物合成中的功能提供重要参考。