Abstract
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.
Highlights
MicroRNAs, a class of small non-protein-coding RNAs, play essential roles in post-transcriptional regulation and many other biological processes in animals and plants [1, 2].PLOS ONE | DOI:10.1371/journal.pone.0171173 February 22, 2017Identification of miRNAs and their targets in Camellia sinensis
Following extraction of catechins from fully ground samples with 70% methanol in a water bath at 70 ̊C, catechin content was measured by High-performance liquid chromatography (HPLC) at a wavelength of 278 nm using a C18 column, with three biological repeats performed per sample [25]
To further understand the function of miRNAs in the regulation of catechin anabolism, we predicted potential miRNAs by subjecting miRNAs obtained from high-throughput sequencing to a BLAST search against catechin biosynthesis-related gene mRNAs from National Center for Biotechnology Information (NCBI) database and RNA sequencing (S6 Table)
Summary
MicroRNAs, a class of small non-protein-coding RNAs, play essential roles in post-transcriptional regulation and many other biological processes in animals and plants [1, 2]. MiRNA target genes can be predicted using bioinformatic methods, they are mainly identified by degradome sequencing or by RNA ligase-mediated rapid-amplification of 5’cDNA ends (5’-RLM-RACE). These two methods can be used to identify target genes of miRNA cleavage sites, which increase their utility for miRNA functional analysis. EGCG can restrict cancer cell growth, suppress androgen receptor functions and regulate cancer cell survival, angiogenesis and movement [10] Based on their molecular constituents, catechins can be classified as simple catechins and ester catechins [11]. We used high-throughput sequencing to identify conserved and novel miRNAs in tea and analyzed potential miRNA targets by 5’-RLM-RACE. The combination of 5’-RLM-RACE and expression analyses allowed us to discover new catechin biosynthetic regulators responsible for the cleavage of genes involved in the catechin biosynthetic pathway
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