Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress / 中国中药杂志

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

SCAR Molecular Identification of Polygonatum filipe / 中国药学杂志

OBJECTIVE: To establish a rapid molecular identification method for Polygonatum filipe species. METHODS: Polymorphism analysis on DNA of P. filipe and P. cyrtonema was performed by using inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers. Differential ISSR and SRAP bands between the two species were sequenced and species-specific sequence characterized amplified region (SCAR) primers were designed for the identification of P. filipe and P. cyrtonema. RESULTS: Under respective optimal annealing temperature, three pairs of SCAR primers can specifically amplify three fragments of 150, 354 and 518 bp only from P. filipe, respectively, not from P. cyrtonema. The SCAR-PCR test was simple and convinent to operate, and reproducible. The molecular identification technology based on SCAR markers was further validated by testing 8 samples of Polygonatum tubes sold in market. CONCLUSION: SCAR molecular technology developed in this study can be used for the assistant identification of P. filipe species.