Identification And Analysis Of The Genes Involved In The Biosynthesis Of Phenolic Acid In Salvia Miltiorrhiza And Dioscin In Dioscorea Nipponica

In the long-term interactions with their various and ever-changing biotic and abiotic stresses, terrestrial plants have developed to manufacture a remarkably diverse array of secondary metabolites which weren’t directly involved in the plant growth and development, and be known as "secondary metabolite", many of which possess biological activities related to human health. The producing organisms known collectively as officinal plant, the resource of more than 30% current human therapeutics, are also an important part of the modern medicine. Such as compound Danshen dripping pills whose main material is the officinal plant Salvia miltiorrhiza and Di’ao Xin Xue Kang Capsule mainly produced from Dioscorea nipponica possess excellent curative effect on the prevention and treatment of cardiovascular disease.S. miltiorrhiza, a well-known Chinese herb, has been widely used for treatment of inflammation and cardiovascular diseases. Hydrophilic phenolic acid derivatives are suggested to be the major active ingredients in conventional S. miltiorrhiza decoctions, particularly rosmarinic acid and its derivatives all possess attractive medicinal activities. The biosynthetic pathway for phenolic acids differs in this species from that of other investigated plants. However, the molecular basis for this is unknown, with systematic analysis of the genes involved not yet performed. As the first step towards unraveling this complex biosynthetic pathway in S. miltiorrhiza, the current genome assembly was searched for putatively involved genes. Twenty-nine genes were revealed,19 of which are described here for the first time. These include 15 genes predicted in the phenylpropanoid pathway; seven genes in the tyrosine-derived pathway; six genes encoding putative hydroxycinnamoyltransferases, and one CYP98A, namely CYP98A78. The promoter regions, gene structures and expression patterns of these genes were examined. Furthermore, conserved domains and phylogenetic relationships with homologous proteins in other species were revealed. Most of the key enzymes, including 4-coumarate:CoA ligase,4-hydroxyphenylpyruvate reductase and hydroxycinnamoyl-transferase, were found in multiple copies, each exhibiting different characteristics. Ten genes putatively involved in rosmarinic acid biosynthesis are also described. These findings provide a foundation for further analysis of this complex and diverse pathway, with potential to enhance the synthesis of water-soluble medicinal compounds in S. miltiorrhiza.D. nipponica is herbaceous, climbing, tropical monocot belonging to Dioscorea; its extracts have been reported to have anticancer and antiobesity activities with very high medicinal value. For habitat destruction and excessive excavation, its resources is sharply reduced, this species has been listed as a national second-class protected plant. The main medicinal component, dioscin exhibited many important activities, such as anti-neoplastic, anti-inflammatory, hypolipidemic, and hemolytic activities, in addition, this constituent is also the main material for synthesis of steroid hormone drugs, and has been honored as "medicinal gold". However, the biosynthetic pathway of dioscin, and the genetic information of this species are still poorly understood, systematic analysis of the genes involved in dioscin biosynthesis and high throughput sequencing analysis of this species are essential for next intensive investigation. The plant was identified by DNA barcoding technology including using chloroplast genome sequence as super barcode sequence, and then its genome was preliminarily assessed using flow cytometry and genome survey analysis, the genome size is about 500Mb, and its heterozygosity is 1.02%. The differential expression analysis results showed 482 genes were tissue-specific highly expressed in the roots, which mainly involved in stress response and transcriptional regulation predicted by Gene Ontology (GO) enrichment analysis. Eighteen genes related to cholesterol biosynthesis were identified, eight of them putatively involved in dioscin biosynthesis. One hundred and eighty-two P450 genes were annotated,10 genes were more likely to participate in dioscin biosynthesis. Forty-seven glycosyltransferase genes were found, nine genes highly expressed in roots, four of them could be selected as candidate genes correlated with dioscin biosynthesis for next investigation. Base on the data of significantly differentially expressed genes, the co-expression network was constructed,15 gene modules generated whose main functions were also predicted using GO enrichment analysis, and the genes putatively involved in dioscin biosynthesis were distributed in seven modules. KEGG analysis results showed many of the genes in the seven modules probably related to the regulation of dioscin biosynthesis or involved in its upstream pathway. Finally, the content of many intermediate products involved in this pathway were also analyzed, which were accordance with the gene analysis results. These results were helpful for molecular identification and genome research on Dioscorea plants, and for next intensive investigation of dioscin biosynthesis pathway.