05月03 【成功案例】百邁客云 小RNA測序分析平臺 助力 賴忠雄老師 發表《小RNA和降解組聯合測序揭示microRNA調節茶葉中的兒茶素生物合成》
MicroRNA是內源性非編碼小RNA，在植物中起著關鍵的調節作用。茶是一種風靡全球的非酒精飲料，具有豐富的促進健康的兒茶素。在本研究中，通過高通量測序鑒定出了調節644個靶基因的69個保守的和47個新的小RNA。小RNA預測的靶基因主要與植物的生長、信號轉導、形態發生和防御有關。為了進一步鑒定小RNA的靶基因，本研究同時開展了降解組測序與RLM-RACE。使用降解組測序，26個基因主要涉及轉錄因子，抗性蛋白和信號轉導蛋白質合成被鑒定為潛在的miRNA靶標基因，隨后驗證了其中5個基因。qRT-PCR結果顯示：novel-miR1, novel-miR2,csn-miR160a, csn-miR162a, csn-miR394 和 csn-miR396a與兒茶素含量負相關。6個miRNA（csn-miRNA167a，csn-miR2593e，csn-miR4380a，csn-miR3444b，csn-miR5251和csn-miR7777-5p.1）及其與兒茶素生物合成相關的靶基因的表達也通過qRT-PCR進行了分析；這些miRNA和兒茶素含量之間呈正相關和負相關，而在其目標基因和兒茶素含量之間呈正相關。這個結果表明這些miRNA可能通過下調它們來負調節兒茶素生物合成生物合成相關靶基因。綜上：本文的研究結果表明，miRNA是茶葉中關鍵的調節因子，5′-RLM-RACE和表達分析的結果揭示了miRNAs在兒茶素合成代謝中的重要作用。
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.