Molecular Cloning And Regulation Analysis Of Important Genes Involved In Tyrosine-branch Pathway Of Rosmarinic Acid Metabolism From Salvia Miltiorrhiza Bunge

Rosmarinic acid (RA), with lots of bioactivities such as antiinflammatory, antibacterial and antioxidant etc., widely exists in plants and more often can be found from Labiatae and Boraginaceae plants. Researches on RA metabolic pathways from plants have showed that there are two parallel branches which initiated individually from the aromatic amino acids phenylalanine and tyrosine. Caffeic acid and hydroxyphenyllactic acid, the products of the two branches, are catalyzed into RA by rosmarinic acid synthase (RAS).The dried root and rhizome of Salvia miltiorrhiza Bunge (Dan-shen in Chinese), a famous Traditional Chinese Medicine (TCM), has been widely used in China. The important water-soluble components extracted from Dan-shen including RA and lithospermic acid B have been the hot study points from 1990′s. However, there are few research reports on RA metabolic pathway involved in Salvia miltiorrhiza Bunge and till now, many related enzymes involved in this pathway have never been cloned from Salvia miltiorrhiza Bunge, which may result in some difficulties to further elucidate regulation mechanism of RA biosynthesis and to improve characters of its germplasm resource by gene engineering or other biological techniques.In this paper, tyrosine aminotransferase (SmTAT), 4-hydroxyphenylpylpyruvate reductase (SmHPPR) and 4-hydroxyphenylpyruvate dioxygenase (SmHPPD) genes were firstly isolated from Salvia miltiorrhiza Bunge by rapid amplification of cDNA ends (RACE) and genomic DAN walking techniques. Characterization, function prediction and regulation characters of these three novel genes were analyzed. 1. SmTAT gene: Gene Specific primers (GSP) of SmTAT were designed based on sequences blast of other tat genes. The full-length cDNA and its genomic DNA sequence were isolated. The full-length cDNA of SmTAT is 1603 bp long with an open reading frame (ORF) of 1233 bp encoding a polypeptide of 411 amino acid residues.The deduced amino acid sequence of the SmTAT gene shared high homology with other known TATs. Analysis of SmTAT genomic DNA reveals that it contains 6 exons and 5 introns. The analysis of SmTAT promoter region and terminator region was also presented. Semi-quantitative RT-PCR analysis reveals that the constitutive expression of SmTAT in stem is much higher than that in root or leaf. Further expression analysis reveals that the signaling components of defense/stress pathways, such as methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA) and ultraviolet-B radiation (UV-B), could up-regulate the SmTAT transcript levels compared with the control.2. SmHPPR gene: Hydroxyphenylpyruvate reductase (HPPR) catalyses the first specific biosynthetic step in the biosynthesis of RA from the aromatic amino acids phenylalanine and tyrosine. A novel HPPR gene was cloned from Salvia miltiorrhiza for the first time. The full-length cDNA of SmHPPR is 1066 bp with a 939 bp ORF encoding a polypeptide of 313 amino acid residues. The deduced protein of Smhppr includes a putative catalytic domain (aa 4-98) and a NAD(P)H-binding domain (aa 100-279) identified as typical domains for the family of D-isomerspecific 2-hydroxyacid dehydrogenases. Characterizations analysis shows SmHPPR shares high similarity with the proved hppr gene invoved in rosmarinic acid biosynthesis from Coleus blumei. Analysis of SmHPPR genomic DNA reveals that it contains 2 exons and 1 intron. The analysis of SmTAT promoter region and terminator region was also presented. Semi-quantitative RT-PCR reveals the expression pattern of Smhppr in different orgenons. The gene expression is the highest in stem, the second in root and the lowest in leaf. Further expression analysis reveals that MeJA, SA, GA3 and ABA treatments could up-regulate the SmHPPR transcription level and UV-B and H2O2 treatments could down-regulate its transcription level. All our findings suggest that SmHPPR, a metabolic pathway gene, might be influenced by JA, ABA, SA and ROS signaling pathways.3. SmHPPD gene: To share the same catalyzing substrate as HPPR, 4- hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4- hydroxyphenylpyruvate to 2, 5-dihydroxyphenylacetate (homogentisate) which as an intermediate is transformed to tocopherols and plastoquinones, conveying electrons to assure plant photosynthesis working effectively, in plastids of many plants. Based on sequences of other HPPD genes, one novel HPPD gene was cloned from Salvia miltiorrhiza for the first time. The full-length cDNA of SmHPPD is 1736 bp long with an ORF of 1443 bp encoding a polypeptide of 481 amino acid residues. According to BLAST of protein to protein, the deduced amino acid sequence of the SmHPPD gene shares high homology with other known HPPDs. Analysis of SmHPPD genomic DNA reveals that it contains 2 exons and 1 intron. The analysis of SmHPPD promoter region and terminator region is also found some regulatory elements of controlling gene expression. Semi-quantitative RT-PCR analysis reveals that the constitutive expression of SmHPPD in stem is much higher than that in root and leaf. Compared treatments of UV-B and dark to nature light, UV-B could down-regulate the SmHPPD transcript levels over the control. It implied that the expression of SmHPPD is affected by light.Based on clone and function analysis of the three metabolic pathway genes involved in tyrosine-branch will enable us to transfer these key biosynthetic genes into Salvia miltiorrhiza or other model plants to study RA biosynthesis pathway deeply and to study metabolic flux of the whole transgenic plant in the future. The research may also provide a possibility to elevate the content of valuable secondary metabolites and to improve characters of Salvia miltiorrhiza germplasm resource by genetic engineering techniques.