Molecular Cloning And Characterization Of Phenylalanine Branch's Genes Involved In The Biosynthetic Pathways Of Rosmarinic Acid From Salvia Miltiorrhiza Bung

Rosmarinic acid, one of the important water-soluble components, is extracted from a wild Chinese traditional medicinal plant Salvia miltiorrhiza Bung. It has a lot of bioactivities such as antiinflammatory, antibacterial and antioxidant etc. People have tried to use a lot of methods to product useful secondary metabolite since 1970s'. Biosynthesis pathway is a new method to gain the useful components. Recently the research shows that there are two parallel branches involved in a few enzymes in the biosynthesis of rosmarinic acid. Caffeic acid and hydroxyphenyllactic acid, the productions of the two branches, are catalyzed into rosmarinic acid by rosmarinic acid synthase (RAS). Many genes of this pathway are cloned;however, few genes of this pathway are isolated from S. miltiorrhiza.In this paper, SmC4H and SmPAL genes are firstly isolated from young leaves of phenylalanine branch from 5. miltiorrhiza by rapid amplification of cDNA ends (RACE) technique. Characterization, functional prediction and construction of the two novel genes are studied. The analysis of the genes is as follows.1. SmC4H gene: Primers are designed based on sequences of other C4H genes. The full-length cDNA and its genomic DNA sequence are isolated. The comparison between cDNA and genomic DNA sequences reveals that SmC4H gene has three exons and two introns. The full-length cDNA of SmC4H is 1800 bp, containing an open reading frame (1512 bp) encoding a peptide of 504 amino acids. Sequence alignment shows that SmC4H protein shares 89.7% identities to C4H proteins from Agastache rugosa. The results of analysis show that the gene is predicted as C4H gene of S. miltiorrhiza. Expression analysis reveals that the signaling components and stimulus such as abscisic acid (ABA), methyl jasmonate (MeJA) and ultraviolet-B radiation (UV-B) up-regulate the SmC4H transcript levels over the control. Calcium chloride (CaCl₂) and hydrogen peroxide (H2O2), nevertheless, have no significant effect on SmC4H expression. The result of RT-PCR shows that the SmC4H gene expresses in root, stem and leaf. The expression level in root and stem much higherthan that in leaf.2. SmPAL gene: The full-length cDNA of SmPAL is 2354 bp, containing an open reading frame (2133 bp) encoding a peptide of 711 amino acids. The results of BLAST and the spatial structure prediction show that the SmPAL gene is the PAL gene of S. miltiorrhiza.3. The coding sequence of the SmC4H was constructed into the Pet28a and Pet32a (Novagen). The recombinant vectors were transformed into the expression hosts BL21.The SmC4H and SmPAL genes are very helpful to study the biosynthesis pathway of the rosmarinic acid of 5. miltiorrhiza. The research has significant effect to gain more independence intellectual property rights genes and through genetic engineering technique to raise the content of target secondary metabolite.