Salvia Ggpp Synthase Gene Cloning And Expression Analysis

Salvia miltiorrhiza Bunge is a Chinese medicinal plant. Its roots (called Danshen in China) is a well-known traditional Chinese medicine (TCM), and widely used for treating coronary heart diseases, Cerebrovascular disease, hepatitis, hepatocirrhosis, chronic renal failure, dysmenorrheal and neuroasthenic insomnia. Danshen contains two major classes of chemicals, one class of water-soluble phenolic acids inculding salvianolic, rosmarinic acid, protocatechualdenyde, and the other class of lipid-soluble diterpene quinone pigments, generally known as tanshinones, including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone, which are the relatively abundant tanshinones.Tanshinone belongs to diterpene quinone pigments. In higher plants, geranylgeranyl diphosphate synthase (GGPPS) is one of the key enzymes of biosynthesis of diterpene, and controls the process of which the precursors—GGPP converts to diterpene quinone pigments. In our study, a GGPP synthases gene (named as SmGGPPS) from S. miltiorrhiza were cloned using degenerate primers and DNA Walking technology. The promotor elements in 5' flank sequence of SmGGPPS were analysized by bioinformatic methods. Then the SmGGPPS mRNA expression quantities in different organs or under different factors were analyzed by real-time quantitative PCR. At last, the changes of tanshinones accumulations in seedlings treated by different factors were detected by HPLC. This study was laying a foundation for elucidating the mechanism of the synthesis of the lipid-soluble diterpene quinone pigments of 5. miltiorrhiza and discussed the factors of tanshinones accumulation.The main results were as follows:(1) SmGGPPS from S. miltiorrhiza was cloned using degenerate primers and DNA Walking technology. The entire sequence was 2758 bp, which contained an intron and an ORF (1086 bp), which coded an amino acid sequence (361 aa). Homology analysis showed SmGGPPS and its amino acid sequence shared more than 64% identities with the GGPPS sequences reported in other species.(2) Amino acid sequence of SmGGPPS contained trans-isoprenyl diphosphate synthases domain, belonging to trans-isoprenyl diphosphate synthases family. The sequence had two aspartate-rich motifs (DDXX (XX) D) as the characteristic structure of GGPPS family. Bioinformatic analysis indicted that SmGGPPS was a chloroplast protein ,and coil was its mainly protein structure elements.(3) The promoter prediction showed SmGGPPS had core element (TATA box) and many cis-acting elements related to light, wound, heat, drought, GA, SA, ABA and pathogen. We presumed the SmGGPPS mRNA could be induced by these factors.(4) The real-time PCR results showed SmGGPPS mainly expressed in leaves, and could be induced by dark, heat, pathogen, wounding, NaCl solution, GA₃, ABA and SA.(5) 500 mM NaCl solution, heat, 50 mM GA₃ and 10 mM SA promoted the accumulations of tanshinones. Among these four elicitors, the effort of 10 mM SA was highest. Though 100 mM ABA and dark could induce the SmGGPPS expression, but they could not promote the accumulation of tanshinones.