| Salvia miltiorrhiza is one of the most important Chinese herbal medicine,its secondary metabolites such as salvianolic acid and tanshinones are widely used in the prevention and treatment of hyperlipidemia cardio-cerebral vascular disease and acute ischemic stroke diseases.It has antioxidant,anti-tumor,anti-virus,anti-bacterial,antiinflammatory,lipid-lowering and other medicinal activity.With the gradual enhancement of public health consciousness and the increasing incidence of cardiovascular disease,the market and clinical demand for S.miltiorrhiza are increasing year by year.Due to the relatively low content of these compounds in plants and the difficulty of chemical synthesis,it is necessary and urgent to improve the quality of S.miltiorrhiza and increase the content of effective components based on genetic engineering strategies.Based on the establishment of Agrobacterium tumerfaciens mediated leaf disc transformation system with Basta herbicide resistance selection,and hairy root incucible transformation system with Agrobacterium tumefaciens habouring rooting inducing plasmid,we investigated that overexpression of transcription factor gene AtEDT1,SmSAMDC and inhibition of key biosynthetic genes(SmHPPD and SmCCR)in competitive pathways enhanced accumulation of salvianolic acids and tanshinones.The main contents of this paper are as follows:1.The A.tumerfaciens mediated transformation system with herbicide Basta as a selection marker was established in S.miltiorrhiza.To establish an efficient plant transformation system,the concentration of Basta in transgenic plant selection was optimized,and 0.6 mg/l phosphinothricin was the most suitable concentration.The transgenic positive lines were finally confirmed through herbicide Basta resistance selection,targeted gene PCR detection and GUS histochemical staining.2.To reduce Ri plasmid T-DNA redundancy,the hair root induced by rolA,rolB and rolC single gene and rolABC multi-gene in S.miltiorrhiza.The effect of the strategy was to maximize the application of hairy root system in the secondary metabolic engineering of S.miltiorrhiza.3.Based on the Basta resistant transformation system,Arabidopsis transcription factor AtEDT1(Arabidopsis Enhanced Drought Tolerance1)was overexpressed in S.miltiorrhiza.The results showed that the root length of transgenic plantlets was dramatically increased and the drought tolerance was significantly improved comparing with wild type plants.Further analysis showed that the overexpression of AtEDT1 significantly increased the accumulation of salvianolic acids(rosmarinic acid,lithospermic acid and salvianolic acid B)in transgenic roots,and dramatically upregulated the expression levels of salvianolic acid biosynthetic genes(SmTAT,SmHPPR,SmPAL,SmC4 H,Sm4CL,and SmRAS)correspondingly.On the other hand,overexpression of AtEDT1 had suppressive effect on the production of tanshinones(dihydrotanshinone,cryptotanshinone,tanshinone I and tanshinone IIA)and affected the expression levels of corresponding biosynthesis related key enzyme genes(SmHMGR,SmMDS and SmIPPI).It surggested that overexpression of AtEDT1 not only promoted the growth of the roots in transgenic lines,but also affected the synthesis and accumulation of secondary metabolites in roots.4.Based on the hairy root regeneration system,the efficient mi RNA-induced gene silencing(MIGS)was successfully confirmed by inhibition of GUS reporter gene expression.In order to increase the accumulation of salvianolic acid,two endogenous by passway genes(SmHPPD and SmCCR)included in salvianolic acid biosynthesis was choosen to inhibition.And the MIGS knockdown construction of a single gene(SmHPPD or SmCCR)or double genes(SmHPPD and SmCCR)were transferred into the hair root respectively.The results showed that the knockdown of key enzymes genes SmHPPD and SmCCR in salvianolic acid biosynthesis bypathway could significantly increase the content of rosmarinic acid in the hairy roots of S.miltiorrhiza.5.The tissue expression-specific expression pattern of SmSAMDC was analyzed,which was the key gene of polyamine metabolism in S.miltiorrhiza,then the overexpression vector was constructed based on the herbicide selection.Through Agrobacterium tumefaciens-mediated genetic transformation,the transgenic material of SmSAMDC was created,trying to control the secondary metabolites of S.miltiorrhiza by regulation of polyamine production.The identification of molecular level in transgenic lines was positive,but there were no obvious phenotypic differences between the transgenic lines and wild type.Given the phenomenon of transgenic silencing in the homologous transformation,the heterogeneous expression of SmSAMDC in tobacco was analyzed.The physiological and biochemical data indicated that transgenic plants showed a higher relative water content and activities of antioxidant enzyme,this provided the basic data for the effective application of SmSAMDC in the metabolic engineering of S.miltiorrhiza.In conclusion,based on the establishment of Agrobacterium tumefaciens mediated leaf disc transformation system with Basta herbicide resistance selection,and hairy root incucible transformation system with Agrobacterium tumefaciens habouring rooting inducing plasmid,the overexpression of the transcription factor AtEDT1 and inhibition expression of salvianolic acid synthesis genes SmHPPD and SmCCR in S.miltiorrhiza could influense the secondary metabolites regulation in S.miltiorrhiza.This research work provides a reliable altenative transformation system,secondary metabolites regulation target genes and case for the effective application of genetic engineering in S.miltiorrhiza. |
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