Cloning Of SmJAZ3 Gene And JAZ Regulating Function Of Secondary Metabolism Of Salvia Miltiorrhiza

JAZ (Jasmonate-ZIM Domain) protein acts as the negative regulators of JA signa-ling pathway. JAZ family was first found in Arabidopsis thaliana, contains 12 members, and shares three conserved regions, named NT, ZIM and Jas domain, respectively. At present, JAZs were reported in rice, grapes, tobacco, soybean, corn and many other species, while the functions were various in different species.Salvia miltiorrhiza Bunge, a perennial herb, is a well-known traditional Chinese medicine. Its dry roots have been widely used for hundreds of years in the prevention and treatment of cardiovascular diseases. The active ingredients of S. miltiorrhiza are divided into two main groups:lipid-soluble tanshinones and water-soluble phenolic acids. Both are secondary metabolites, levels of which are not only controlled by plant genotype but also enhanced by various biotic and abiotic stresses. Previous studies shown that medicinal ingredients of S. miltiorrhizae were significantly induced under the treatment of JA and its derivatives. However, the molecular mechanism was still unclear. At present, JAZ protein is one of the key links in JA signal pathway. It participates in the regulation of secondary metabolism in Arabidopsis thaliana and the tobacco. We speculated that inhibiting JAZ activity could be a new strategy for inducing the active ingredients accumulation in S. miltiorrhiza. Therefore, cloning and functional studies of the JAZ gene is important to investigate the regulation mechanism of secondary metabolite synthesis in S. miltiorrhiza.Our main results and conclusions are as follows.1. Cloning a complete open reading frame of the SmJAZ gene, named SmJAZ3, through Blast analysis of S.miltiorrhiza transcriptome database Amino acid sequence analysis showed that JAZ3 contains the typical domain of JAZ proteins, Jas and conservative TIFY domain, and weak conservative N-terminal domain. Expression pattern analysis shown that SmJAZ3 expresses constitutively in roots, stems and leaves, with highest expression in roots, and its expression increases during the flowering phase. A total of 966 bp 5’ flanking sequences of SmJAZ3 was cloned by DNA walking, and the cis-elements were predicted by PlantCARE software. Consistent with promoter analysis, the expression of SmJAZ3 was induced by the biotic and abiotic stresses, including MeJA, mechanical injury, ABA, and SA.2. Comparative analysising of SmJAZ3 with the reported SmJAZ1 and SmJAZ8, including cis-elemnts of the promoters, the structures of the genes, the structures of the proteins, the expression patterns of the tissues and organs, and the functions of the genes. Results showed that the three genes have similar promoter elements, typical of JAZ protein structure domain, conserved gene structure, express in different tissues and organs, and were induced by a variety of biotic and abiotic stress. It is suggested that the three genes maybe redundant in function.3. Detection of six SmJAZ transgenic lines(co10/11/13/15 and it6/16). The contents of total phenolic and flavonoids in roots, stems and leaves in above transgenic lines and controls were compared. Results showed that the total contents of phenolics and flavonoids in transgenic plants were increased significantly. In the roots, total phenolic acid contents were 1.10-2.00 times than that of controls. In the stems and leaves, total phenolic acid contents were 1.36-2.13 times than that of controls. The total flavonoid contents were 1.35-1.50 times higher than that of the control in root, and 1.44-1.59 times in the stems and leaves.4. Determing the contents of rosmarinic acid (RA) and salvianolic acid B (SA) in the roots, stems and leaves of one-year-old transgenic plants (co13/15 and it6/16) by HPLC. Compared with the controls, contents of RA in roots, stems and leaves were decreased, while the SA and tanshinone IIA contents were various in different lines.5. Analysising the key genes expression in the SA, lignin anthocyanin, and isoprenoid biosynthesis (including tanshinone IIA) pathways in transgenic lines (co13/15 and it6/16) by real-time PCR. Results showed that the expression of key enzymes of secondary metabolic pathways in different transgenic lines were diverse. Six (PAL2,4CL1, TAT, HPPR, RAS and CYP98A14), four (HCT, CCR, COMT and CAD), two (F3’H and F3’5’H), and six (HMGR2, HMGR3, IP11, DXS1, CPS1 and GGPPS1) key enzyme genes in each pathway were increased at different degrees, indicating that JAZ gene is involved in regulation of the synthesis of secondary metabolites. Thus, we speculate that SmJAZ, act as a repressor protein, maybe involved in jasmonic acid signaling pathway directly, and the negative regulation of secondary metabolite biosynthesis indirectly.Our studies provide certain data for research of S.miltiorrhiza secondary metabolism, and pave the way for the improvement of the quality of S. miltiorrhiza by genetic engineering.