Novel Stratage Of Secondary Metabolic Engineering In Salvia Miltiorrhiza-Based On The Reform Of MeJA Biosynthetic Pathway

Plant secondary metabolites are not necessary in normal growth, development and reproduction processes, but often play an important role in plant defense against herbivory and other interspecies defenses. They are a wide variety of low-molecular weight compounds which often have pharmacal activities. Jasmonic acid methyl ester (MeJA) is important signaling molecules in plants, especially in defense reactions to environmental stresses, such as mechanical wounding or pathogen attack.On account of this theorem, we are going to exploit an novel stratagem of secondary metabolic engineering, over express the key enzyme genes in MeJA biosynthetic pathway, to result in the increase of endogenous MeJA content,then , to stimulate the biosynthesis and accumulation of multiple secondary metabolites.Allene oxide cycalse(AOC) and jasmonic acid carboxyl methyltransferase(JMT)are key enzymes in MeJA synthetic pathway. First, we cloned the AOC gene from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) and designated it as SmAOC (GenBank accession: HM156740). The SmAOC cDNA has a total length of 910 bp with an open reading frame (ORF) of 738 bp, and is predicted to encode a protein of 245 amino acid residues, sharing high degree of homology with AOCs from other plants. A 123bp intron was present in the genomic DNA. Southern-blot analysis revealed that SmAOC was a low copy gene. Real-time fluorescent semiquantitative PCR (RT-QPCR) analysis revealed that SmAOC was preferentially expressed in leaves compared with other tissues (roots and stems). When S. miltiorrhiza seedlings were induced by abiotic stimuli such as MeJA and low temperature (4℃) , SmAOC expression was markedly increased within 24h, but not obviously by ultraviolet irradiation (UV). The full-length of ORF was inserted into prokaryotic expression vector pET32a, which was then transferred into E. coli BL21 (DE3). The recombinant pET32a-SmAOC in bacteria induced by IPTG and the expression of fusion protein was revealed by SDS-PAGE. Overproduction of recombinant SmAOC resulted in an increased tolerance to salinity in transgenic E. coli.S. miltiorrhiza contain two major classes of chemicals, a class of lipid-soluble diterpene quinone pigments, generally known as tanshinones, and a class of water-soluble phenolic acids including salvianolic acids B, rosmarinic acid (RA).SmAOC from Salvia miltiorrhiz, HnAOC from Hyoscyamus niger and AtJMT from Arabidopsis thaliana were overexpressed in S. miltiorrhiz hairy roots which were Inducted by Agrobacterium rhizogenes C58C1. In the three transgenic affairs, higher contents of secondary metabolites were obtained in different degrees with the maximum of TanshinoneⅡA 16.6-fold and Salvianolic acid B 78% over control. The contents of other active compounds(rosmarinic acid,tanshinoneⅠ,cryptotanshinone and dihydrotanshinone) in transgentic hairy roots increased in different degrees, and so did rosmarinci acid biosynthetic compounds. Simultaneously, the RT-QPCR result illustrated that the expression of intermediates in MeJA,rosmarinci acid and terpenoid biosynthetic pathway were up-regulated accordingly.This study revealed that overexpression of MeJA biosynthetic pathway genes resulted in higher yield of secondary metabolites. Moreover, the higher yield was herediable and more stable and persistent than exogenous induction. Therefore, it indicated that genetic manipulation of MeJA biosynthetic pathway genes could be an alternative approach in metabolic engineering for the production of valuable secondary metabolites.