| Salvia miltiorrhiza is an important staple of traditional Chinese medicine, with thedeepening research on pharmacological effects, it shows that Salvia miltiorrhiza secondarymetabolites has a clear active pharmacological effect in the prevention and treatment ofcardiovascular so that makes the demand for medicines containing Salvia miltiorrhizacomponents rising. However, the traditional planting methods had been affected and limitedby the problems of regions, salinification and soilborne diseases, thus the supply of thetraditional planting method usually could not be ensured. Salvia miltiorrhiza hairy root systemis considered to be a potential alternative to traditional planting methods of producing Salviamiltiorrhiza secondary metabolites. However, the secondary metabolites content level is lowwhen under normal circumstances in hairy root.In our study, I have respectively take use ofendogenous and exogenous microbes to affect secondary metabolism of Salviamiltiorrhizahairy hairy roots, on the one hand in order to improve the secondary metabolitescontent of Salvia miltiorrhiza hairy roots, on the other hand by using metabolic engineeringstudies to provide the basis in order to improve the secondary metabolites content in hairyroot via the study of its mechanism and induction regularity. The main research contents ofthis thesis are roughly as follows:（1）The fermentation broth and mycelial pellets of Streptomyces pactum Act12hadsignificantly affected primary and secondary metabolites of Salvia miltiorrhiza hairy roots.Added the fermentation broth and Mycelial pellets of Streptomyces pactum Act12(SpFFMy),9days in the Gao1ST culture medium, together to the Salvia miltiorrhiza hairyroots cultured21days, the biomass of Salvia miltiorrhiza hairy roots changed little after theinduction with2%SpFFMy, while4%SpFFMy had a slightly inhibition to the growth ofSalvia miltiorrhiza hairy roots; the accumulation of total soluble sugar in hairy roots has beenpromoted by SpFFMy, the soluble sugar content in hairy root increased by23.2%comparedwith the control with the treatment of2%SpFFMy,but SpFFMy had a inhibition to theaccumulation of soluble protein in hairy root, the treatment of4%SpFFMy decreased thesoluble protein content by18.0%in hairy root compared with the control; the treatment of SpFFMy significantly promoted the accumulation of tanshinone in hairy roots, of whichcryptotanshinone content reachede33.6fold over control, with the treatment of2%and4%SpFFMy,the total tanshinone content reached respectively a maximum of9.2and12.6foldover control; the impact from SpFFMy to the water-soluble phenolic acids in hairy root is notconsistent, SpFFMy promoted the rosmarinic acid accumulation in hairy roots by blockingthe biosynthetic pathway from rosmarinic acid to salvianolic acid B; In addition, the treatmentof Act12also promoted the accumulation of danshensu in hairy roots and inhibited the contentof salvianolic acid A and cinnamic acid.（2）Joined SpFFMy in different periods of hairy root growth produced different effectsto Salvia miltiorrhiza hairy root growth and accumulation of secondary metabolites.Using3%SpFFMy to induce Salvia miltiorrhiza hairy roots at day0inhibitedsignificantly the biomass accumulation of hairy roots, inoculated3%SpFFMy at day7andday14, the hairy root biomass did not change significantly compared with the control. The3%SpFFMy at day21treatment promoted the growth of hairy roots, however, highconcentrations SpFFMy significantly inhibited the hairy root biomass; Three treatments asfollows;3%Act12at day0,3%SpFFMy and9%SpFFMy at day21, had the most obviouseffect of promoting total of tanshinone content, the total tanshinone content reached9.0,9.0and9.3fold over control at45days with these three treaments, however, since the treatmentof3%SpFFMy at day21promoted the accumulation of hairy root biomass, so the totaltanshinone production with the treatment of3%SpFFMy at day21was higher than withother treatments. The treatment of3%SpFFMy at day21promoted rosmarinic acid contentin hairy root, which made rosmarinic acid content increased by40.3%compared to thecontrol in hairy root, but the treatment in other time and concentrations had little effect onrosmarinic acid content in hairy root; but each treatment significantly inhibited theaccumulation of salvianolic acid B in in hairy roots.(3)The Cell Free Fermentation Filtrate (CFFF) of Streptomyces pactum Act12significantly affected primary and secondary metabolites of Salvia miltiorrhiza hairy roots.The inoculation of CFFF promoted the biomass in hairy root, after the treatment of3%CFFF,the growth of Salvia hairy root increased by15.8%, the accumulation of total solublesugars in hairy root also had been promoted by FAct12, while the accumulation of solubleproteins had been inhibited in hairy root; CFFF also had a significant promoting effect for thetanshinone synthetic in hairy root, of which the promoting effect to cryptotanshinone contentwas the most obvious,the treatments of3%and6%CFFF made cryptotanshinone contentincreased to a maximum of35.3and34.2fold over control in hairy root; Dihydrotanshinone Icontent reached a maximum of20.0and29.1fold over control, the treatments of3%and6% CFFF made the Total Ketone reached up to9.8and9.0fold over control, meanwhile, thehighest yield of tanshinone reached11.4and9.5fold over control; CFFF also promoted therosmarinic acid accumulation in in hairy roots by blocking biosynthetic pathway fromrosemary acid to salvianolic acid B, and significantly inhibited salvianolic acid B content,CFFF Treatment also increased Danshensu content in hairy roots, but had no obvious impacton salvianolic acid A, caffeic acid and cinnamic acid.(4) Treatments of SpFFMy and CFFF had significantly increased the expression of Keyenzyme gene involved in the biosynthesis pathway of tanshinone in hairy roots to improve thetanshinone content in Salvia miltiorrhiza hairy roots.The treatment of4%FAct had promoted the growth of Salvia miltiorrhiza hairy root,while the treatment of4%SpFFMy had significantly inhibited the biomass accumulation.Treatments of Act12and FAct12were both able to promote the soluble sugars accumulationin hairy root, but4%Act12treatment had a significant stronger inhibition of soluble proteincontent than the4%FAct12Treatment; treatments of4%Act12and FAct12could bothincrease gene expression of HMGR, DXS, DXR GGPPS so that tanshinone content hadsignificantly increased in hairy roots; the treatment of4%Act12had significantlyup-regulated the expression of GGPPS, and reached59.6fold over control at day7, on thesame day4%FAct12reached only10.2fold over control, throughout the harvest period, therelative expression of GGPPS with the treatment of4%Act12was always higher than thatwith the4%FAct12treatment, and the treatment of4%Act12had a stronger promotion effectto Tanshinone I and tanshinone IIA in hairy roots than that with the treatment of4%FAct12,so we speculate that accumulation of tanshinone I and tanshinone IIA in hairy root are morelikely to have a closer relationship with the expression of GGPPS.(5) Reactive oxygen species play an important role in the effects of SpFFMy to theSalvia miltiorrhiza hairy roots growth and the tanshinone accumulation.Our previous research indicated that the Streptomyces pactum Act12(Act12) had acertain promotional effect on tanshinone accumulation and upregulate the expression of genes3-hydroxy-3-methyglutary1-CoA reductase (HMGR) and1-deoxy-d-xylulose-5-phosphatereductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles ofreactive oxygen species in Streptomyces pactum Act12-induced tanshinone production inSalvia miltiorrhiza hairy roots. The generation of reactive oxygen species (ROS) in Salviamiltiorrhiza hairy roots was triggered by4%SpFFMy treatment. The relative expressions ofgenes HMGR and DXR in4%SpFFMy treatment were32.4and4.8-fold higher than those inthe control. And the total tanshinone in the hairy roots to become as higher as10.2times thatof the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in4%SpFFMy treatment, which induced thetotal tanshinone content was decreased by74.6%compared with the4%SpFFMy treatment.ROS mediated SpFFMy-induced tanshinone production. The SpFFMy via the ROS signalchannel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content inhairy roots was increased.(6) The Synergistic induction of CFFF and yeast extract (YE) could significantlyimprove the cryptotanshinone content in Salvia miltiorrhiza hairy root.The treatment of3%FAct12promoted the accumulation of Salvia miltiorrhiza hairy rootsbiomass, increased relatively by12.0%that of control, while using YE alone produced asignificant inhibition to the accumulation of Salvia miltiorrhiza hairy roots biomass,decreased19.6%that of control, while the treatment of CFFF+YE had significantly offsetinhibition of YE to the accumulation of hairy root biomass; the treatment of CFFF+YE hadsignificantly promoted the accumulation of cryptotanshinone whose content reached itsmaximum value of2.46mg·g-1DW at day11which was far higher than the maximum valueof1.52mg·g-1DW with the treatment of YE and the maximum value of.08mg·g-1DW withthe treatment of CFFF, but compared with the treatment of FAct12, the treatment of CFFF+YE had no significant difference on tanshinone I and tanshinone IIA content, theDihydrotanshinone I content at day11with the treatment CFFF+YE was higher that thetreatment of CFFF, however, the content remained the same at day14with the treatment ofFAct12.(7) The endophytic bacteria could significantly affect primary and secondary metabolitesof Salvia miltiorrhiza hairy roots.Our study found that except Novosphingobium resinovorum (B5) Salvia miltiorrhiza rootendophytic bacteria Pseudomonas brassicacearum sub sp. neoaurantiaca (B1), Rhizobiumradiobacter (B2), Pseudomonas thivervalensis (B3), Pseudomonas frederiksbergensis (B4)significantly improved the activity of key enzymes3-hydroxy-3-methyglutary1-CoAreductase (HMGR) and1-deoxy-D-xylulose-5-phosphate synthase (DXS) in the biosyntheticpathway of tanshinones. Specifically, HMGR activity with B1treatment increased2.1-foldthat of control, DXS activity with B2treatment increased5.0-fold that of control, whichcaused a significant increase in tanshinone content in the hairy roots. The dihydrotanshinone I(DT-I) and cryptotanshinone (CT) content under B1treatment increased19.2-fold and11.3-fold, respectively, and total tanshinone (TT) content increased3.7-fold that of control.The five endophytic bacteria B1, B2, B3, B4and B5all significantly decreased phenylalanineammonia-lyase (PAL) and tyrosine aminotransferase (TAT) activity in hairy roots, of which,B3treatment decreased PAL activity by46.2%and B2treatment decreased TAT activity by 44.7%compared with the control. Each of the five endophytic bacteria decomposedrosmarinic acid (RA) and salvianolic acid B (SAB), which caused a significant decrease inRA and SAB content in hairy roots, with B2treatment decreasing RA and SAB content by94.5%and89.0%, respectively, compared with the control. The five endophytic bacteria alsoinhibited the growth of S. miltiorrhiza hairy roots, of which, B2and B4treatment decreasedhairy root biomass by55.2%and51.3%, respectively, compared with the control, while hairyroots promoted the growth of B4and B5and inhibited the growth of B1and B3. |
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