Tanshinone Compounds Of Biological Synthesis Cloning And Functional Studies

Danshen is the dried root of Salvia miltiorrhiza Bunge that belongs to the family of Labiatae.It is one of the most versatile Chinese herbal drugs that recorded firstly in "Shen Nong's herbal classic",and commonly used for the treatment of cardiovascular diseases such as angina pectoris,myocardial infarction and stroke. The content of danshen can be separated into lipid-soluble and water-soluble fractions.Its lipid-soluble fraction contains more than 30 diterpenoid tanshinones, the major active constituents include tanshinoneâ… ,â…¡A,B,cryptotanshinone, dihydrotanshinoneâ… ,methylenetanshinone,and isotanshinoneâ…¡A,etc.,and used for the treatment of cardiovascular diseases,infectious diseases,and cancer etc.,because of its properties of improving microcirculation,causing coronary vasodilatation, suppressing the formation of thromboxane,inhibiting platelet adhesion and aggregation,protecting against myocardial ischemia,and cytotoxicity etc..Tanshinones is a species of diterpenoid secondary metabolite in Salvia miltiorrhiza Bunge,and the research of tanshinones secondary metabolism, especially the downstream biosynthetic pathway of GGPP only commence with the work.The clones and functional identification is the base of the secondary metabolism,so the related genes from Salvia miltiorrhiza cloning and their functional identification carrying out are the key research of initiating biosynthesis of diterpenoid tanshinones.These researches will become the scientific evidence of tanshinones generating mechanism and Danshen quality,and provide feasible projects for enhance the contents of tanshinones or intermediate by biotechnology at another hand.In this thesis,the full length cDNA sequences were isolated from Salvia miltiorrhiza hairy root using RACE and PCR,and subcloned into prokaryotic expression vector,then expressed in Escherichia coli BL21 trxB(DE3) cells induced by IPTG.The soluble recombinant proteins were used for enzymatic assay, and the function of clones were identified by product structural analysis.Several detailed works focused on the full length cDNA cloning and functional analysis of genes involved in biosynthesis of diterpenoid tanshinones were carried out as followed:1 Molecular cloning and analysis of a novel copalyl diphosphate synthase full length cDNA from Salvia miltiorrhizaA novel full length copalyl diphosphate synthase gene was isolated from Salvia miltiorrhiza hairy root using RACE and PCR.The cDNA has an open reading frame of 2382 nucleotides,which encodes 793 amino acids with a calculated molecular weitht of 90362 Da and an estimated pI of 6.45.The deduced amino acid sequence resembles the sequences of other cloned copalyl diphosphate synthase(≤48% identity),but contain a DXDD motif in common to mediate protonation of a carbon-carbon double bond in an acid catalyzed reaction.A phylogenetic tree of plant diterpene cyclases indicates that SmCPS is the classâ…¡terpene synthase, resembling other copalyl diphosphate synthase in dicotyledons,and other than those in monocotyledons in the GA biosynthesis pathway.In addition,the level of expression of the SmCPS transcript in Salvia miltiorrhiza hairy root increased drastically in response to MJ treatment,whereas expression of its transcript was not induced by MJ.These results suggest that SmCPS are responsible for the biosynthesis oftanshinoneâ…¡A and cryptotanshinone.2 Molecular cloning and analysis of a novel kaunene synthase-like gene from Salvia miltiorrhizaA novel full length kaunene synthase-like gene was isolated from Salvia miltiorrhiza hairy root using RACE and PCR.The cDNA has an open reading frame of 2110 nucleotides,which encodes 595 amino acids with a calculated molecular weitht of 68326 Da and an estimated pI of 6.00.The deduced amino acid sequence resembles the sequences of other cloned kaunene synthase(≤38%identity),but contain a DDXXD metal binding motif in common that facilitates the metal ion assisted allylic diphosphate ionization reactions commonly associated with terpene synthases.A phylogenetic tree of plant terpene cyclases indicates that SmKSL is the classâ… terpene synthase,resembling the terpene cyclases in Nicotiana tabacum that did not characterized,and forming a main category with other diterpene cyclases in gymnosperm,whereas other diterpene cyclases monocotyledons and dicotyledons in the GA(gibberellic acid )biosynthesis pathway.The level of expression of the SmKSL transcript in Salvia miltiorrhiza hairy root increased drastically in response to MJ treatment,whereas expression of its transcript was not induced by MJ.These results suggest that SmKSL are responsible for the biosynthesis of tanshinoneâ…¡A and cryptotanshinone.3 Escherichia coli expression,purification and functional identification of Salvia miltiorrhiza copalyl diphosphate synthaseThe SmCPS full length open reading frame was subcloned into prokaryotic expression vector pET32a(+) in frame with HIS-tag resulting in pET32CPS,then was transformed into E coli BL21 trxB(DE3) resulting in recombinant strain E.coli [pET32CPS].The induction of E.coli[pET32CPS]in different temperatures, induction time,IPTG concentrations and A₆₀₀ values of E.coli were performed.The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis,and the ratio of the interest protein(108KDa) to total proteins reached to 35.6%.The recombinant SmCPS protein purified by Ni²⁺ affinity chromatography column,and was identified by Western blotting and LC-ESI-MS/MS.Enzymatic analysis was carried out in assay buffer with the purified recombinant SmCPS protein,and initiated by the addition of GGPP.The catalysates were dephosphorylated by calf intestinal phosphatase,and the dephosphorylated compounds were then extracted with hexanes for gas chromatography mass spectrometry(GC-MS).Comparison of the enzymatically formed compound to similarly dephosphorylated authentic samples of ent- and syn-CPP demonstrated that the enzyme produces ent-CPP.Therefore,we have functionally identified Salvia miltiorrhiza ent-CPP synthase(SmCPS).Furthermore, the purified recombinant SmCPS protein were used for rabbit immunization,and the polyclonal antibody against SmCPS was prepared by Western blotting analysis, which will be used for the further tagging research of SmCPS in Salvia miltiorrhiza.4 Escherichia coli expression and functional identification of Salvia miltiorrhiza kaunene synthase-likeThe SmKSL full length open reading frame was subcloned into prokaryotic expression vector pET32a(+) in frame with HIS-tag resulting in pET32KSL,then was expressed in E coli BE21 trxB(DE3) with IPTG inducing.SDS-PAGE result showed that a protein with an apparent molecular mass of 86KDa appeared, supporting that the novel kaurene synthase like gene clone could be normally translated into a complete trxA-fusion protein as expected,which was then identified by LC-ESI-MS/MS.Enzymatic assays with partially purified recombinant SmKSL protein was carried out with the catalysates of SmCPS as substrate.After the reaction,enzymatic turnover of GGPP was analyzed by gas chromatography mass spectrometry(GC-MS) of the resulting extracts.Comparison of the enzymatically formed compound to similarly authentic samples of kaur-16-ene,the similarity is 74%,but there are two even-electron ion including 134 and 148 m/z in compound MS,therefore the compound maybe a novel chemcial constitution other than kaur-16-ene.The functionally identified of Salvia miltiorrhiza kaurene synthase like still need the further structural analysis of enzymatically product.Biosynthesis of active ingredient in medicinal plant is conjunct catalytic outcome of many related key enzymes in plant secondary metabolism pathway.The cloning and functional identification of SmCPS and SmKSL will be the important first step of tanshinones biosynthesis,wherein the biololgical functional analysis of SmKSL show that tanshinones biosynthesis could have a original pathway in diterpenoid secondary metabolite other than GA biosynthesis pathway.