ABSTRACT
Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.
ABSTRACT
The development and metabolism of medicinal plant is affected by many factors, among which the effect from endophytic fungi has been noticed recently and has become one of hot fields. In order to explore the effect of endophytic fungi on gene expression in R. crenulata, RNA-sequencing was used to find genes involved in metabolic pathways, and the differential genes were verified by real-time fluorescent quantitative PCR. The method of 2-△△Ct was used to analyze the relative expression levels of genes in related metabolic pathways, which was used to verify the result of transcriptomics sequencing. The results showed that the endophytic fungus, P. fortinii, could up-regulate the gene expression in lipid metabolic pathway of R. crenulata. In signal transduction pathway, the genes were influenced at different level but the gene expressions were significantly increased under control of Notch signaling pathway, which was 34 times of that in control. The gene expressions of environmental adaption pathway were up-regulated in R. crenulata after inoculation of P. fortinii. This study could provide help for further understanding on mechanism of plant-fungus interaction, root cause of geoherbalism of medicinal plant and exploring bio-function of endophytic fungi.