[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Illuminating the biosynthesis pathway genes involved in bioactive specific monoterpene glycosides in Paeonia veitchii Lynch by a combination of sequencing platforms.

BACKGROUND: Paeonia veitchii Lynch, a well-known herb from the Qinghai-Tibet Plateau south of the Himalayas, can synthesize specific monoterpene glycosides (PMGs) with multiple pharmacological activities, and its rhizome has become an indispensable ingredient in many clinical drugs. However, little is known about the molecular background of P. veitchii, especially the genes involved in the biosynthetic pathway of PMGs. RESULTS: A corrective full-length transcriptome with 30,827 unigenes was generated by combining next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRT) of six tissues (leaf, stem, petal, ovary, phloem and xylem). The enzymes terpene synthase (TPS), cytochrome P450 (CYP), UDP-glycosyltransferase (UGT), and BAHD acyltransferase, which participate in the biosynthesis of PMGs, were systematically characterized, and their functions related to PMG biosynthesis were analysed. With further insight into TPSs, CYPs, UGTs and BAHDs involved in PMG biosynthesis, the weighted gene coexpression network analysis (WGCNA) method was used to identify the relationships between these genes and PMGs. Finally, 8 TPSs, 22 CYPs, 7 UGTs, and 2 BAHD genes were obtained, and these putative genes were very likely to be involved in the biosynthesis of PMGs. In addition, the expression patterns of the putative genes and the accumulation of PMGs in tissues suggested that all tissues are capable of biosynthesizing PMGs and that aerial plant parts could also be used to extract PMGs. CONCLUSION: We generated a large-scale transcriptome database across the major tissues in P. veitchii, providing valuable support for further research investigating P. veitchii and understanding the genetic information of plants from the Qinghai-Tibet Plateau. TPSs, CYPs, UGTs and BAHDs further contribute to a better understanding of the biology and complexity of PMGs in P. veitchii. Our study will help reveal the mechanisms underlying the biosynthesis pathway of these specific monoterpene glycosides and aid in the comprehensive utilization of this multifunctional plant.