Abstract
MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica.
Highlights
Lonicera japonica Thunb. (FLJ), commonly known as Japanese Honeysuckle or “Ren Dong,” has been widely used in traditional medicine in East Asian countries for thousands of years (Jiang et al, 2009)
For the evaluation of potential endogenous normalizers in L. japonica, 16 candidate reference miRNAs were selected from small RNA libraries, which were chosen in preference according to the following criteria: the mean variation (RPKM value) in miRNA expression levels was 0.05)
Those precursors of selected miRNAs were compared with miRBase miRNA sequences2 to search for homologs of microRNA sequences (Table 2)
Summary
Lonicera japonica Thunb. (FLJ), commonly known as Japanese Honeysuckle or “Ren Dong,” has been widely used in traditional medicine in East Asian countries for thousands of years (Jiang et al, 2009). The content of active compounds differs significantly among the varieties and production areas of L. japonica, leading to the different pharmacological activities and medicinal qualities (Yuan et al, 2012; Shi et al, 2016; Wu et al, 2016). Chinensis (Watts; rFLJ), a Chinese endemic variety, has different active compound contents from those of FLJ (Yuan et al, 2012; Supplementary Figure S1) Both varieties of L. japonica are widely planted in different production areas of China, which can obviously affect the content of the bioactive components (Yuan et al, 2012; Li et al, 2013; Tan et al, 2016). It is necessary to focus on elucidating the molecular regulatory mechanisms of secondary metabolism in flower buds, stems, and leaves from both varieties of L. japonica in various production areas
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