Transcriptome analysis of leaves,roots and flowers of Panax notoginseng on ginsenoside and alkaloid biosynthesis and amelioration of vascular insufficiency conditions by saponins from the flower buds

摘要

OBJECTIVE To investigate the transcriptomic details on the biosynthetic pathways in different parts of the Panax notoginseng and the pharmacological activity of the saponins extracted from the flower(FS)on vascular insufficiency conditions.METHODS RNA sequencing of three different Panax notoginseng tissues was performed using next generation DNA sequencing and differential gene expression was validated by real-time PCR.In order to determine pro-angiogenic and therapeutic effects of FS on myocardial infraction(MI),FS was examined on the endothelial cell migration assay,vascular insufficiency model in zebrafish and MI model in rats.RESULTS After assembling the high quality sequencing reads into 107 340 unigenes,biochemical pathways were predicted and 9 908 unigenes were assigned to 135 KEGG pathways.Among them,270 unigenes were identified to be involved in triterpene saponin biosynthesis as well as 350 and 342unigenes were predicted to encode cytochrome P450 sand glycosyltransferases,respectively.One unigene was annotated as CYP716A53v2,probably participates in the formation of protopanaxatriol from protopanaxadiol and the differential expression of this gene was confirmed by real-time PCR.In addition,the pharmacological evaluation demonstrate that FS significantly promoted vascular endothelial growth factor(VEGF)induced the migration of human umbilical vein endothelial cells(HUVECs)and partially restored defective intersegmental vessels in a chemically induced vascular insufficiency model of zebrafish larva.Moreover,the two week posttreatment of the rat MI model with FS(25-50mg·kg-1·d-1)induced approximately 3-fold upregulation of VEGF mRNA expression,with a concomitant increase in blood vessel density in the peri-infarct area of the heart by 50.7%,compared to 41.4%in the MI group.Furthermore,TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment.CONCLUSION We have established a global transcriptome dataset for Panax notoginseng and provided additional genetic information for further genome-wide research and analyses.Candidate genes involved in ginsenoside biosynthesis,including putative cytochrome P450 sand glycosyltransferases were obtained.The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P.notoginseng.And the significant pro-angiogenic effect of FS in multiple experimental models renders the purified saponin preparation as potential preventive and therapeutic agent for cardiovascular diseases yet to be developed.