Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

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.

Effect of multiple factors on seeds germination of Paris polyphylla var. yunnanensis and polygerm varieties / 中草药

Objective: To explore the impacts of temperature, endogenous hormone, and pollination methods on the seeds germination and emergence of Paris polyphylla var. yunnanensis and polygerm varieties, and establish the technical system to promote the germination of P. polyphylla var. yunnanensis seeds and provide scientific guidance for the cultivation of Paris L., To ease the bottleneck problem of current serious shortage of high quality source of seeding material and commercial herbal products of Paris L., and highest market price. Methods: Using wet sand cultured P. polyphylla var. yunnanensis seeds under different conditions and determined the rate of germination and the mean germination time, the cultivated seeds were planted in the field to investigate the rate of seedling emergence and the average time of emergence. Results: The higher germination rate and emergence rate were obtained at the temperature of 4-18℃; Gibberellin (GA3) treatment could promote the seed germination, and reduce the mean germination time, especially in 100 and 200 mg/L; The germination rate and emergence rate of artificial pollination were significantly higher than natural pollination (P < 0.05); There is no difference between P. polyphylla var. yunnanensis and polygerm varieties seeds. Conclusion: The peeled seeds are treated by 100 mg/L gibberellin, and cultured to sprout by wet sand at the temperature of 4-18℃, then sown in the field. It can shorten the seed emergence period one year and up to more than 80% of emergence rate.

Determination of seven steroidal saponins in Paridis Rhizoma and polygerm varieties from different regions in Yunnan province by UPLC and establishment of fingerprint / 中草药

Objective: To establish a quantitative analysis method of seven steroidal saponins in Paridis Rhizoma and its polygerm varieties from different regions in Yunnan province, to simultaneously determine the contents of seven steroidal saponins, to establish the fingerprint for the polygerm varieties in Gaoligong Mountain by UPLC-ELSD, and to evaluate the qualities. Methods: It was detected with an Acquity UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column, and gradually diluted with acetonitrile-water at a flow rate of 0.2 mL/min, drift tube temperature of 55℃, nitrogen pressure 275.8 kPa, and gain 500 by UPLC-ELSD. Results: The 23 samples could be separated and analyzed of seven steroids within 23 min. All the indexes of the methodological investigation met the requirements. The results showed that the contents of saponins from 19 samples met the requirement in Chinese Pharmacopoeia 2015. The average content of four saponins was 1.42% and among them the content from the sample in Gaoligong Mounta was up to 1.660%. The similarity was lower than 0.9 in the samples of Gaoligong Mountain, and there were eight common peaks in the fingerprints, which were identified four characteristic peaks. The type and content of seven saponins in the samples could not be clearly classified by PCA analysis and system cluster analysis. Conclusion: The method is convenient, accurate, and suitable for the quantitative analysis of Paridis Rhizoma and polygerm varieties. There is no obvious difference between Paridis Rhizoma and polygerm varieties in chemical components and contents. Paridis Rhizoma (polygerm varieties) in Gaoligong Mountain is a variety worth popularization and in-depth researches.