PmiREN: a comprehensive encyclopedia of plant miRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that function as diverse endogenous gene regulators at the post-transcriptional level. In the past two decades, as research effort on miRNA identification, function and evolution has soared, so has the demand for miRNA databases. However, the current plant miRNA databases suffer from several typical drawbacks, including a lack of entries for many important species, uneven annotation standards across different species, abundant questionable entries, and limited annotation. To address these issues, we developed a knowledge-based database called Plant miRNA Encyclopedia (PmiREN, http://www.pmiren.com/), which was based on uniform processing of sequenced small RNA libraries using miRDeep-P2, followed by manual curation using newly updated plant miRNA identification criteria, and comprehensive annotation. PmiREN currently contains 16,422 high confidence novel miRNA loci in 88 plant species and 3,966 retrieved from miRBase. For every miRNA entry, information on precursor sequence, precursor secondary structure, expression pattern, clusters and synteny in the genome, potential targets supported by Parallel Analysis of RNA Ends (PARE) sequencing, and references is attached whenever possible. PmiREN is hierarchically accessible and has eight built-in search engines. We believe PmiREN is useful for plant miRNA cataloguing and data mining, therefore a resource for data-driven miRNA research in plants.

Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions.

Codonopsis pilosula is a well-known medicinal plant. Although its transcriptome sequence has been published, suitable reference genes have not been systematically identified for conducting expression analyses via quantitative real-time polymerase chain reaction (qRT-PCR). To screen appropriate genes for use with this species, we applied four different methods-GeNorm, NormFinder, BestKeeper, and RefFinder-to evaluate the stability of 13 candidates: CpiEF1Bb, CpiCACS, CpiF-Box, Cpiß-Tubulin, CpiGAPDH, CpiActin2, CpiAPT1, CpiActin7, CpiActin8, CpiRPL6, CpiHAF1, CpiTubulin6, and CpiUBQ12. Expression was examined by qRT-PCR for various tissue types, chemical treatments, and developmental stages. For all tested samples, CpiGAPDH proved to be the most stable. Comprehensive analysis indicated that the most stable internal reference genes were CpiF-Box and CpiCACS in different tissues and at different developmental stages, respectively. Under NaCl stress, CpiAPT1 was the best internal reference gene. For methyl jasmonate and abscisic acid treatments, CpiGAPDH and CpiF-Box, respectively, presented the highest degree of expression stability. Based on these findings, we chose CpiSPL9 as the target gene for validating the suitability of these selected reference genes. All of these results provide a foundation for accurate quantification of expression levels by genes of interest in C. pilosula.

Establishment of in vitro culture system for Codonopsis pilosula transgenic hairy roots.

The aim of the study was to establish a reliable system of transgenic hairy roots in Codonopsis pilosula through Agrobacterium-mediated genetic transformation. For this, we optimized several steps in the process of A. rhizogenes strain C58C1 mediated hairy root induction, including the most appropriate medium, explant type, time for infection and co-cultivation. We achieved an induction rate of up to 100% when the roots of C. pilosula seedlings were used as explants, infected with A. rhizogenes C58C1 harboring pCAMBIA1305 for 5 min, followed by induction on 1/2MS supplemented with 0.2 mg/L naphthylacetic acid and 200 mg/L cefotaxime sodium. The co-transformed hairy roots were confirmed by PCR amplification of hygromycin phosphotransferase II gene and histochemical GUS assay, and the efficiency of transformation was 70% and 68.3%, respectively, when no hygromycin selection pressure was exerted. To increase biomass production, we excised and self-propagated the transformed hairy roots, which produce saponins. Our successful establishment of an in vitro culture system of transgenic hairy root for this species lays the foundation not only for assessing gene expression and function but also for obtaining high levels of secondary metabolites through genetic engineering technology.