Exploring The Biosynthetic Pathway Of Phenolic Acids And Funcitonal Study Of The Involved Genes In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge (Dan-shen in Chinese), is one of the most importanttraditional Chinese herbal medicine. It was used for promoting circulation and improvingblood flow for longtime. Modern medicinal research suggested that the phenolic acidsincluding rosmarinic acid (RA) and Salvianolic acid B (SAB) are the important materialbasis of pharmacological activities of S. miltiorrhiza. So it aroused scientists'interest in thelast decades. By now, few of the research focused on the biosynthesis of phenolic acids inS. miltiorrhiza, the biosynthesis pathway of SAB was still unknown. Although thebiosynthesis pathway of the other important phenolic acids RA was reported, the researchabout this pathway was not fully enough. The important enzyme SmRAS andSmCYP98A14on the downstream of RA biosynthesis pathway were not cloned, and therewas no research about these two enzymes until now. So the study which focus on the abovequestions is important for developing the S. miltiorrhiza resources.In our study,13C tracer and precursor feeding experiment were used to reveal thebiosynthesis pathway of phenolic acids especially SAB. The dynamic observation of thephenolic acids metabolic flux described the whole metabolic process from L-phenylalanineto SAB. We found the differences of RA biosynthesis pathway in Salvia miltiorrhiza fromthat reported before. We also cloned3important enzymes SmRAS, SmCYP98A14andSmCPR involved in the downstream of RA biosynthesis pathway and studied on theirfunction. the results are as follows:On the first time, we revealed the biosynthesis process from RA to SAB. Two RAchange to SAE by condensation, and SAE then change to SAB by cyclization, this is thefirst time study on the biosynthesis of SAB. The results can provide valuable informationfor phenolic acids biosynthesis research in S. miltiorrhiza.We observed the metabolic process of phenolic acids by13C tracer, the result helps tore-understand the biosynthesis process of RA. Our research revealed that the Caffeoyl-CoAand the4-hydroxyphenyllactate esterified to Caffeoyl-4-hydroxyphenyllactate thenhydroxylated in position3' of the aromatic rings to RA. This process is different from theprevious report that4-coumaroyl-CoA and the4-hydroxyphenyllactate esterified to4-coumaroyl-4'-hydroxyphenyllactate then hydroxylated in positions3and3' of thearomatic rings to RA. This result helps us to reanalysis the biosynthesis process of RAbiosynthesis. We sketch a whole phenolic acids biosynthesis process from L-phenylalanine andL-tyrosine to RA and then to SAB, this work enlarge our knowledge about the biosynthesispathway of phenolic acids in S. miltiorrhiza, it can help us to understand the process ofphenolic acids biosynthesis clearly.Three genes on phenolic acids biosynthesis pathway were cloned, designated asSmRAS(Genbank accession No. ADA60182), SmCYP98A14(Genbank accession No.HQ316179), SmCPR(Genbank accession No. FR693803). The SmRAS cDNA has a totallength of1489bp with an open reading frame (ORF) of1284bp, and is predicted toencode a protein of428amino acid residues. The SmCYP98A14cDNA has a total length of1714bp with an open reading frame (ORF) of1527bp, and is predicted to encode aprotein of508amino acid residues. The SmCPR cDNA has a total length of2771bp withan open reading frame (ORF) of2118bp, and is predicted to encode a protein of705amino acid residues.The expression profile of the three genes in specific tissues and under induction withmethyl ester jasmonic acid(MeJA) and Abscisic acid (ABA) were also observed, theresult show that the three genes are expressed in all tissues, the highest expression ofSmRAS is in the Stem SmCYP98A14in root, SmCPR in leaves. These three gene were alsoinduced up-regulated by MeJA andABA.RNA-interference(RNAi) transgenic hairy roots confirms the function of SmRAS andSmCYP98A14in phenolic acids biosynthesis. The accumulation of RA and SAB weredramatically suppressed by RNAi SmRAS and SmCYP98A14, both RA and SAB contentdeclined to20%of CK. All the results prove the importance of SmRAS and SmCYP98A14on phenolic acids biosynthesis.The method used in our research can provide valuable information for otherbiosynthesis pathway exploration. In conclusion, our research explored the phenolic acidsbiosynthesis pathway in Salvia miltiorrhiza, cloned and functional studied on the keyenzymes SmRAS SmCYP98A14and SmCPR, this research expands the understanding ofphenolic acids biosynthesis, and promoted the research about the metabolic engineering ofphenolic acids in S. miltiorrhiza.