05月03 【成功案例】百迈客云 小RNA测序分析平台 助力 赖忠雄老师 发表《小RNA和降解组联合测序揭示microRNA调节茶叶中的儿茶素生物合成》
MicroRNA是内源性非编码小RNA，在植物中起着关键的调节作用。茶是一种风靡全球的非酒精饮料，具有丰富的促进健康的儿茶素。在本研究中，通过高通量测序鉴定出了调节644个靶基因的69个保守的和47个新的小RNA。小RNA预测的靶基因主要与植物的生长、信号转导、形态发生和防御有关。为了进一步鉴定小RNA的靶基因，本研究同时开展了降解组测序与RLM-RACE。使用降解组测序，26个基因主要涉及转录因子，抗性蛋白和信号转导蛋白质合成被鉴定为潜在的miRNA靶标基因，随后验证了其中5个基因。qRT-PCR结果显示：novel-miR1, novel-miR2,csn-miR160a, csn-miR162a, csn-miR394 和 csn-miR396a与儿茶素含量负相关。6个miRNA（csn-miRNA167a，csn-miR2593e，csn-miR4380a，csn-miR3444b，csn-miR5251和csn-miR7777-5p.1）及其与儿茶素生物合成相关的靶基因的表达也通过qRT-PCR进行了分析；这些miRNA和儿茶素含量之间呈正相关和负相关，而在其目标基因和儿茶素含量之间呈正相关。这个结果表明这些miRNA可能通过下调它们来负调节儿茶素生物合成生物合成相关靶基因。综上：本文的研究结果表明，miRNA是茶叶中关键的调节因子，5′-RLM-RACE和表达分析的结果揭示了miRNAs在儿茶素合成代谢中的重要作用。
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 corre- lations 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 cru- cial 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.