Cloning And Characterization Of DtC4H Of Rosmarinic Acid Biosynthesis From Dracocephalum Tanguticum Maxim

The Tibetan medicine Dracocephalum tanguticum Maxim.is a perennial herb of the genus Dracocephalum in labiaceae.It is distributed in dry river valleys,fields,grass beaches or pine forests of Tibet,Qinghai,Sichuan and Gansu.It is mainly used in the folk to treat hepatitis,gastritis,dizziness,yellow water sores,bleeding and other diseases.In recent years,studies have found that Cymbidium sinense has many pharmacological effects,such as antioxidant,anti hypoxia,antibacterial,anti-inflammatory,antiviral and so on.Rosemarinic acid Acid(RA)is a natural water-soluble phenolic acid compound,a secondary metabolite in plants,widely distributed in plants,mainly distributed in the family of Labiatae,gourd and lavender.As a natural antioxidant,rosmarinic acid itself has the functions of anti-oxidation,anti-inflammatory,antibacterial,antiviral,anti-tumor,anti depression et al.There are few reports on the key enzymes in the biosynthesis of rosemary acid in cyanobacteria glaucum.The rosemarinic acid biosynthesis pathway consists of two parallel branches,namely phenylalanine and tyrosine.C4 H gene is the key gene in the pathway of phenylpropane metabolism,and is one of the key enzymes involved in the synthesis of flavonoids,lignin and other secondary metabolites.C4 H The cloning and functional verification of the gene are of great significance to improve the content of rosmarinic acid in the blue and white blue orchid.In this study,the 2 004 bp electronic sequence of Dt C4 H gene was cloned by homology alignment.There were 206 bp 5’UTR,280 bp 3’UTR and 1 518 bp CDS in the 2 004 bp electronic sequence,which encoded 505 amino acids.The predicted molecular weight(MW)was about 57.9 k Da,the theoretical isoelectric point(p I)was 9.12,and Dt C4 H had membrane anchor region Typical conserved sites of C4 H gene,such as anchor,proline rich regions,threonine containing binding and heme binding,predicted that the protein encoded by this gene had signal peptide,which could guide protein transmembrane transport.The relative expression of Dt C4 H gene in root was the highest,followed by stem,and similar to other C4 H genes in plant tissues.Dt C4 H was expressed in Saccharomyces cerevisiae,the results of enzyme activity in vitro showed that Dt C4 H had cinnamate4-hydroxylase activity.Overexpression and interference expression of hairy roots were obtained by infecting the leaves of Dracocephalum tanguticum Maxim,The results showed that the content of rosmarinic acid in Dt C4 H overexpression hairy roots increased by 1.1-1.7 times,the gene expression increased by 1-8 times,the content of rosmarinic acid in interference expression hairy roots decreased by 24%-82%,and the gene expression decreased by 50%-90%,which indicated that Dt C4 H was involved in the synthesis of rosmarinic acid in Dracocephalum tanguticum Maxim.