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

Date Published: February 22, 2017

Publisher: Public Library of Science

Author(s): Ping Sun, Chunzhen Cheng, Yuling Lin, Qiufang Zhu, Jinke Lin, Zhongxiong Lai, Zhi Min Yang.


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.

Partial Text

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]. Based on the base-pair complementary mechanism, miRNAs recognize and combine the target genes to regulate their expression mainly in two ways: direct cleavage of target mRNAs and inhibition of target gene transcription [3]. Studies have shown that miRNAs are involved in a variety of plant growth and development processes, such as root, leaf and flower morphogenesis [4], signal transduction, hormone responses [5, 6] and nutrient metabolism [7]. Plant miRNAs are mainly identified by cloning, bioinformatic prediction, high-throughput sequencing and Northern blotting. The most popular of these techniques is high-throughput sequencing because of its affordability and easier and faster access to large numbers of miRNAs, especially those in low abundance [8]. Because miRNAs in plants usually act by cleaving their target genes or inhibiting their expression, target gene determination is crucial for miRNA functional analysis. Although 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 also be used to identify target genes of miRNA cleavage sites, which increase their utility for miRNA functional analysis.




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