CN113005130A - Tanshinone IIA synthase gene and application thereof - Google Patents

Abstract

The invention discloses a gene of tanshinone IIA synthase in salvia miltiorrhiza, a coding protein thereof and a method for producing or improving the content of tanshinone IIA. The gene is obtained by cloning from various plants such as salvia miltiorrhiza bunge and the like for the first time, and the coded protein can catalyze cryptotanshinone to synthesize tanshinone IIA and can catalyze iso-cryptotanshinone to synthesize iso-tanshinone IIA. Therefore, the invention has application value in the aspect of producing effective active substances, provides a new way for producing tanshinone IIA in a large scale and lays a solid foundation for industrial production of other related tanshinone compounds.

Description

Tanshinone IIA synthase gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, particularly relates to the field of medicinal plant genetic engineering, and more particularly relates to a gene participating in a synthesis pathway of a tanshinone compound and application thereof.

Background

Salvia miltiorrhiza (Salvia miliiorhiza Bunge) belongs to Labiatae (Lamiaceae), is a traditional medicinal plant, is used as a medicine by root and rhizome, and is widely used for treating cardiovascular and cerebrovascular diseases and related diseases. The medicinal components of the salvia miltiorrhiza mainly comprise two types: one is fat-soluble diterpene tanshinone compound, and the other is water-soluble polyphenol acid compound. The diterpene tanshinone compound has strong biological activity and high content in Salvia miltiorrhiza Bunge. In recent years, new progress is continuously obtained on biosynthesis and regulation research of tanshinone, and the tanshinone has wide development potential in development and utilization.

The research on the biosynthesis pathway of tanshinone has important guiding significance for genetic breeding and the development of synthetic biology. In addition to CPS and KSL catalyzing GGPP to produce sub-tanshinone diene, various Cytochrome P450 enzymes (Cytochrome P450, CYP450) have also been identified, including CYP76AH1, CYP76AH3, CYP76AK1, etc., which catalyze sub-tanshinone dienes to produce ferruginol and its hydroxylated tanshinone intermediates (Guo J, Ma X, Cai Y, Ma Y, Zhan Z, ZHou YJ, Liu W, Guan M, Yang J, Cui G, Kang L, Yang L, Shen Y, Tang J, Lin H, Ma X, Jin B, Liu Z, Peters RJ, ZHao ZK, Huang L (2016) Cytochrome P450 probiotic strains to a biochemical synthetic pah, Yang P450 proteins, Yang J, Yang H, Yang X, Shu Z, Yang J, Yang H, Yang X, J, Yang Z, Peter RJ, Zhao Zhang L K, Huang J, Yang J, chen X, ZHao ZK, Huang L (2013) CYP76AH1 catalysis turn of milliradianes in biochemical and energetic heterologous products of cancer in yeases, proceedings of the National Academy of Sciences of the United States of America 110(29):12108-12113.doi: 10.1073/pnas.1218061110). Three CYP71D family proteins CYP71D375, CYP71D373, and CYP71D411 have recently been shown to participate in the biosynthetic pathway of tanshinone, among which CYP71D375 catalyzes the synthesis of cryptotanshinone (Ma Y, Cui G, Chen T, Ma X, Wang R, Jin B, Yang J, Kang L, Tang J, Lai C, Wang Y, ZHao Y, Shen Y, Zeng W, Peters RJ, Qi X, Guo J, Huang L (2021) Expansion with the CYP71 communication 71D subfamily driving the heterocyclic synthesis of tanshins synthesis in Salvia millicirculation 12 (685 1), doi:10.1038/s 4120959-1).

Among diterpene compounds of salvia miltiorrhiza, tanshinone IIA is one of the most important active ingredients. Tanshinone IIA sodium sulfonate has been widely used as a medicine and has activity on various diseases such as cardiovascular and cerebrovascular diseases, liver injury and the like, and recent studies show that Tanshinone IIA can relieve atherosclerosis by reducing the activity of outer membrane mast Cells (Meim X-D, Cao Y-F, Che Y-Y, Li J, Shang Z-P, Zhao W-J, Qiao Y-J, Zhang J-Y (2019) Danshenn: a phytochemical and pharmacological overview. Chinese Journal of national instruments 17(1) 59-80.doi:10.1016/s1875-5364(19) 30010-X; Park YK, Obiang-Obououou BW, Lee J, Lee TY MA, Hwang KS, Lee KB, Choi JS, Jang-BC (2017) International-Edou BW, Lee J, Lee TY MA, H-III, Na-H-III-V-III-V-III-V, however, the catalytic enzymes which catalyze the synthesis of tanshinone IIA have not been known so far.

Disclosure of Invention

The invention discloses a tanshinone IIA synthase gene and a coding protein thereof for the first time, and particularly relates to a tanshinone IIA synthase gene cloned from various plants such as salvia miltiorrhiza bunge and the like, the synthesis path of tanshinone IIA is researched and analyzed, and the important function of the gene in the accumulation process of tanshinone IIA is predicted. The present invention relates to the gene of the salvia miltiorrhiza TiAS in vivo and in vitro verification of the functions of plants such AS salvia miltiorrhiza and microorganisms.

The invention provides a gene TIIAS of tanshinone IIA synthase, which has one of the following nucleotide sequences:

1) the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 and SEQ ID No. 15;

2) the sequence obtained by adding, deleting and substituting one or more nucleotides in SEQ ID No.1, the encoded protein still has the activity of tanshinone IIA synthase.

3) The nucleotide sequence consistency and SEQ ID No.1 are more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% of homologous genes, and the coded protein still has the activity of tanshinone IIA synthase; preferably, it is derived from Salvia miltiorrhiza, Salvia przewalskii, Salvia splendens, Salvia pratense, Salvia rubra, Salvia przewalskii, Salvia praecox and Salvia flammans. More preferably, the tanshinone IIA synthase gene is a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 and SEQ ID No. 15.

Furthermore, the present invention provides a protein encoded by the gene TIIAS. Specifically, the protein has the amino acid sequence consistency of more than 80%, more than 85% or more than 90%, more than 95% or more than 99% of homologous protein with SEQ ID No.2, and still has the activity of tanshinone IIA synthase. Preferably, it is derived from Salvia miltiorrhiza, Salvia przewalskii, Salvia splendens, Salvia pratense, Salvia przewalskii, Salvia pratensis and Salvia flammans, more preferably the amino acid sequence is shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No. 16.

The invention also provides a recombinant vector containing the gene TIIAS, preferably an expression vector, more particularly a bacterial, fungal or plant expression vector, and further an escherichia coli and yeast expression vector.

Further provided are host cells, more particularly bacterial, fungal or plant cells, and further E.coli, yeast cells, containing said gene TIIAS or said recombinant vector.

The invention especially provides application of the gene TIIAS or the protein in producing or preparing tanshinone IIA or iso-tanshinone IIA.

Furthermore, the present invention provides a method for producing tanshinone IIA by catalyzing cryptotanshinone through fermentation or isotanshinon through a microorganism, wherein the microorganism is introduced and expresses the gene TIIAS; culturing microorganism containing TIIAS expressing the gene, such AS Escherichia coli and yeast, and extracting tanshinone IIA or isotanshinone IIA from thallus, wherein tanshinone or cryptotanshinone is added into the culture medium.

In addition, the invention also provides a method for improving the content of plant tanshinone IIA and/or isotanshinone IIA, which is characterized in that the gene TIIAS is introduced into a target plant to obtain a transgenic plant over-expressing the gene TIIAS; more specifically, the constructed agrobacterium or agrobacterium tumefaciens strain containing the gene TIIAS is used for transforming the salvia miltiorrhiza to obtain a transgenic salvia miltiorrhiza plant or hairy root over expressing the gene TIIAS, wherein the content of tanshinone IIA and/or iso-tanshinone IIA is improved.

The invention also provides a method for producing or preparing tanshinone IIA or iso-tanshinone IIA in vitro by using the protein, wherein the protein is adopted to catalyze cryptotanshinone to generate tanshinone IIA or catalyze iso-cryptotanshinone to generate iso-tanshinone IIA, preferably, the reaction system contains alpha-ketoglutaric acid and further contains Fe²⁺And/or ascorbic acid; preferably, the protein is obtained by extracting after fermentation of genetically engineered bacteria. In one embodiment, 200nM MES (pH6.5), 1mM alpha-ketoglutarate, 1mM ascorbic acid, 250. mu.M Fe²⁺1mM ATP and 50 μ g TIIAS protein, and the concentration of the substrate cryptotanshinone or iso-cryptotanshinone is 50 μ M. The reaction conditions are 20 ℃, 180rpm, the reaction is carried out for 30min by shaking, and then the ethyl acetate with twice volume of the reaction system is taken out to terminate the reaction. Further comprises a step of purifying tanshinone IIA or iso-cryptotanshinone.

The invention discloses a tanshinone IIA synthase gene and a coding protein thereof for the first time, and verifies that the tanshinone IIA synthase gene has the tanshinone IIA synthase gene in vivo and in vitro, thereby providing a new way for large-scale production of tanshinone IIA and laying a solid foundation for industrial production of other related tanshinone compounds.

Drawings

FIG. 1 shows that tanshinone IIA is generated by catalyzing cryptotanshinone with tanshinone IIA synthase (TIIIAS) in Escherichia coli prokaryotic system.

FIG. 2 shows that tanshinone IIA is produced by catalyzing isocytotanshinone with tanshinone IIA synthase (TIIAS) in Escherichia coli prokaryotic system.

FIG. 3 shows that TIIAS protein can catalyze CT to generate TIIAS in an in vitro enzyme activity reaction system.

FIG. 4 shows the content of tanshinone TIIA in transgenic Salvia miltiorrhiza hairy roots by TIIAS overexpression. 1 is cryptotanshinone, and 2 is tanshinone TIIA.

FIG. 5 shows that the accumulation of cryptotanshinone and the inhibition of tanshinone TIIA content in the transformed Salvia miltiorrhiza Bunge hairy root by TIIAS-RNAi. 1 is cryptotanshinone, and 2 is tanshinone TIIA.

FIG. 6 shows that CTs are catalyzed by SwowTIIIAS, SmeitTIIIAS, StriTIIIAS, SpriTIIIAS, SprzTIIIAS, SsigTIIIAS and SubTIIAS proteins to produce tanshinone IIA (TIIA).

Detailed Description

The invention is further described below by means of specific embodiments in order to facilitate a better understanding of the invention, without however constituting a limitation thereto. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The various kits and enzymes used in the examples described below were purchased from Tiangen Biochemical technology Ltd and Thermo Fisher.

Example 1 cloning of Salvia miltiorrhiza TiAS Gene and construction of corresponding overexpression, RNAi expression vector and E.coli expression vector

(1) TIIAS gene primer design and synthesis

Firstly, deep sequencing is carried out on transcriptomes of roots and leaves of salvia miltiorrhiza, then, a gene library highly expressed in the roots is determined according to screening of differentially expressed genes in the roots and the leaves, a candidate protein gene sequence is determined in the gene library highly expressed in the roots through homologous comparison by taking an alpha-ketoglutarate dependent oxidase (2-ODD) protein sequence reported in arabidopsis thaliana as a standard, and the function of the candidate protein is determined through in-vitro enzyme activity identification.

The overexpression and RNAi vector is constructed by adopting a gateway method, and the prokaryotic expression vector is constructed by adopting an enzyme digestion connection method. Primer premier5.0(PP5) is used for designing a primer, a 395bp section is selected from a TIIAS full-length gene for designing an RNAi vector according to the recommendation of PP5 software, and a Gateway recombination site and a restriction enzyme cutting site (underlined) are respectively added to the TIIAS fragment and primers at two ends of the full length during primer design.

TⅡASox-F：GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCACATCCAGCTTGAAAAATT

TⅡASox-R：GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAGACATTGTCTTCATCTTGCAAT

TⅡAS-RNAi-F：GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAGATGCGGAGGCGAAGAAGCAGTT

TⅡAS-RNAi-R：GGGGACCACTTTGTACAAGAAAGCTGGGTTTTGGAGAAGAATAGTTAGGAAACAA

TⅡAS-BamHI-F：cgggatccATGGCCACATCCAGCTTGAAAAATT

TⅡAS-NotI-R：ataagaatgcggccgcTTAGACATTGTCTTCATCTTGCAAT

The primer is synthesized by Shanghai.

(2) Cloning of the TIIAS Gene

The salvia miltiorrhiza root is taken as a material, total RNA of the salvia miltiorrhiza root is extracted according to the instruction of an RNA extraction kit, and the reverse transcription kit is used for carrying out reverse transcription to obtain cDNA of the salvia miltiorrhiza root.

Taking cDNA as a template, and respectively taking TIIASOx-F and TIIASOx-R; TIIAS-RNAi-F, TIIAS-RNAi-R and TIIAS-BamHI-F, TIIAS-NotI-R are used AS primers to carry out amplification reaction, and TIIAS fragment and full-length gene (the nucleotide sequence is shown AS SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown AS SEQ ID NO. 2) are obtained.

And (3) PCR reaction system: 1 μ L of cDNA, 2.5 μ L of upstream primer, 2.5 μ L of downstream primer, 10 μ L of phusion HF buffer, 1 μ L of dNTP mix, 0.5 μ L of phusion enzyme, and 32.5 μ L of ddH2O 32.5.

The PCR was programmed as follows: 1) heating at 98 deg.C for 3 min; 2) 10s at 98 ℃; 3) 20s at 55 ℃; 4)72 ℃ for 1 min; 5)72 ℃ for 5 min. Cycles from 2) to 4), cycle number 40.

After the PCR amplification is finished, agarose gel electrophoresis detection is carried out on the product, and 1183bp, 453bp and 1146bp gene fragments and the full length are obtained respectively.

(3) Construction of pDenar 207-TIIAbox and pDenar 207-TIIAS-RNAi vectors

The PCR products of 1183BP and 453BP in (2) were subjected to BP reaction after recovering specific bands using a Tiangen cutting gel recovery kit, and determining the concentration of the recovered products.

BP reaction: PCR product 100-200ng (3. mu.L), pDOnar 207100 ng (1. mu.L), 1 XTE 0.5. mu.L, BP clone enzyme 0.5. mu.L; the BP reaction was terminated by incubation at 25 ℃ for 5h, addition of 0.5. mu.L proteinase K, and incubation at 37 ℃ for 10 min.

Coli DH10B was transformed with the above reaction product, and the resulting bacterial suspension was spread on LB solid medium containing 20mg/L gentamicin and cultured overnight in an incubator at 37 ℃. The next day, single clones were picked for colony PCR validation, and positive clones were sent to the bio-corporation for sequencing. Plasmids are extracted according to the sequencing result to obtain plasmids pDOnar 207-TIIASOx and pDOnar 207-TIIAAS-RNAi.

(4) Construction of overexpression and RNAi expression vectors for TIIAS genes

pK7WG2R was used AS an overexpression vector for TIIAS gene, pK7GWIWG2R was used AS an RNAi expression vector, and the LR reaction was carried out after the concentration of the plasmid obtained by BP reaction in (3) was measured.

LR reaction: BP reaction plasmids pDOnar 207-TIIASOx and pDOnar 207-TIIAAS-RNAi 100-200ng (3. mu.L), pK7WG2R and pK7GWIWG2R 100ng (1. mu.L), 1 XTE 0.5. mu.L, LR clone enzyme 0.5. mu.L; the LR reaction was stopped by incubation at 25 ℃ for 5h, 0.5. mu.L proteinase K added, and incubation at 37 ℃ for 10 min. Coli DH10B was transformed with the above reaction product, and the resulting bacterial suspension was spread on LB solid medium containing 50mg/L spectinomycin (Spe) and cultured overnight in an incubator at 37 ℃. The next day, single clones were picked for colony PCR validation.

The plasmid is extracted from the obtained positive clone to obtain an over-expression vector pK7WG 2R-TIIAS containing the full length of the target gene and a plant RNAi vector pK7GWIWG 2R-TIIAS containing the gene fragment.

(5) Prokaryotic expression vector of TIIAS gene

The PCR product of 1146bp in (2) was used in a native excision gel recovery kit to recover a specific band, the concentration of the recovered product was measured, and then an enzyme digestion reaction was carried out, with restriction enzyme and T4 DNA ligase each purchased from Thermo.

And (3) enzyme digestion reaction: the reaction was a 20. mu.L system. PCR product 10 u L or pET32a plasmid DNA 5 u L, 10x Fastdigest buffer 2 u L; incubation is carried out for 10min at 37 ℃, the enzyme digestion product is recovered by using a tiangen gel cutting recovery kit, the concentration of the recovered product is determined, and then the ligation reaction is carried out.

And (3) connection reaction: the reaction was a 20. mu.L system. mu.L of the digested PCR product and 3. mu.L of the digested pET32a plasmid DNA were added with 2. mu.L of 10x T4 DNA Ligase buffer, 0.25. mu.L of Ligase, and reacted at 16 ℃ for 30 min.

Coli DH10B was transformed with the above reaction product, and the resulting bacterial suspension was spread on LB solid medium containing 100mg/L ampicillin and cultured overnight in an incubator at 37 ℃. The next day, single clones were picked for colony PCR validation, and positive clones were sent to the bio-corporation for sequencing. And extracting the plasmid according to the sequencing result to obtain a plasmid pET32 a-TIIAS.

Example 2 verification of function of protein of TanTIIAS gene in catalyzing cryptotanshinone in prokaryotic system

(1) Protein for expressing salvia miltiorrhiza TiAS gene in prokaryotic escherichia coli

The prokaryotic expression vector pET32 a-TIIAS is transferred into an escherichia coli Rosetta2(DE3) strain by a chemical conversion method to obtain an escherichia coli strain containing a target gene, meanwhile, empty plasmids containing pET32a are converted to be used AS negative control, the escherichia coli strain is grown overnight at 37 ℃ on LB solid culture medium containing 100mg/L of ampicillin, and positive clones are selected for subsequent experiments.

Positive monoclonals were then picked and grown overnight at 37 ℃ in 2mL LB liquid medium containing 100mg/L ampicillin. Then inoculating into 50mL LB liquid culture medium containing 100mg/L ampicillin, culturing OD600 to 0.5 at 37 deg.C, adding IPTG with final concentration of 0.5mM for induction, simultaneously adding cryptotanshinone with 50 μ M as substrate, inducing target protein expression and enzymatic reaction at 16 deg.C and 180 rpm. After 24h of culture, the cells were collected.

(2) After the collection of the cells, the cells were dissolved in 1mL of methanol and disrupted by sonication for 2 hours to extract the metabolites. Centrifuging at 12000rpm for 5min, collecting supernatant, and filtering with 0.22 μm organic filter membrane to obtain sample for UPLC detection.

UPLC detection conditions: agilent 1260 definition II Prime LC System, ACQUITY UPLC HSS C₁₈(2.1X 100mm,1.8 μm) column, detection wavelength 270 nm. Mobile phase conditions: the flow rate is 0.5mL/min, the mobile phase is 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B), the column temperature is 40 ℃, and gradient elution is carried out: 0-8min, 50% -80% B; 8-8.5min, 80% -100% B; 8.5-11min, 100% B; 11-11.5min, 100% -50% B; 11.5-14.5min, 50% B.

The results show that: in an escherichia coli prokaryotic system, the result is shown in fig. 1, no-load of pET32a is taken as a control, Cryptotanshinone (CT) is taken as a substrate, and a new product cannot be generated; in the catalytic system of tanshinone IIA synthase, CT is catalyzed to produce new tanshinone IIA (TIIA). The results indicate that tanshinone IIA synthase (TIIAS) is capable of catalyzing CT to produce TIIAS.

Example 3 verification of function of protein of TanTIIAS gene in catalyzing Isocryptotanshinone in prokaryotic system

(1) The protein of the Salvia miltiorrhiza TIIAS gene is expressed in prokaryotic Escherichia coli (the specific method is the same AS that in example 2).

Unlike example 2, the enzymatic reaction was carried out by adding 50. mu.M of Isocytotanshinone (iCT) as a substrate while inducing the protein.

(2) After collection of the cells, the extraction and UPLC detection methods were the same as in example 2.

The results show that: in the E.coli prokaryotic system, the results are shown in FIG. 2, the substrate Isocryptotanshinone (iCT) has no new product generated in the reaction system of no-load pET32, and the new product Isotanshinone TIIA (iTIIA) is generated under the catalysis of tanshinone IIA synthase (TIIAS). Thus indicating that TIIAS can catalyze the generation of the isotanshinone IIA by the isocytotanshinone.

Example 4: in-vitro enzyme activity reaction system, the function of catalyzing cryptotanshinone by the protein of the TIIAS gene is verified

(1) The protein of the Salvia miltiorrhiza TiAS gene was expressed in prokaryotic Escherichia coli Rosetta2(DE3) by reference to example 2, which is different from example 2 in that it was inoculated into 50mL of LB liquid medium containing 100mg/L ampicillin, and OD was cultured at 37 ℃₆₀₀When the concentration is 0.5, IPTG with a final concentration of 0.5mM is added for induction, the expression of the target protein is induced at 16 ℃ and 180rpm without adding any substrate, and after 24 hours of culture, the cells are collected for protein purification.

(2) To the cells, 5mL of Binding buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH7.4) was added to resuspend the cells, and the cell pellet was mixed well. After high pressure crushing, the mixture is placed in a centrifuge with precooling at 4 ℃ and centrifuged at 12000rpm for 10 min. The supernatant was taken into a support column containing 1mL of Ni-NAT nickel beads, and the supernatant was filtered at 4 ℃ and 15mL of Wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH7.4) was added to Wash the nickel column. When all wash buffers are to be filtered, 2mL of Elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH7.4) is added to the nickel column and the protein of interest is eluted from the nickel beads. The purified protein was concentrated by desalting by centrifugation at 4000rpm of a 4 ℃ precooled centrifuge using a 10K Millipore ultrafiltration tube for 30min, and the buffer in the tube was replaced with an aqueous MES solution (1M, pH6.5) to obtain a purified protein concentrate. Based on a protein concentration standard curve of bovine serum albumin, the concentration of the bovine serum albumin is detected by a Bradford method, and the final protein concentration is obtained by conversion according to an absorption value of an ultraviolet spectrophotometer.

(3) The in vitro enzyme activity reaction system is as follows: 200nM MES (pH6.5), 1mM alpha-ketoglutarate, 1mM ascorbic acid, 250. mu.M Fe²⁺1mM ATP and 50. mu.g TIIAS protein, and the substrate cryptotanshinone concentration is 50. mu.M. The reaction conditions are 20 ℃, 180rpm, the reaction is carried out for 30min by shaking, and then the ethyl acetate with twice volume of the reaction system is taken out to terminate the reaction.

(4) Violently shaking the centrifuge tube to fully mix the organic phase and the water phase, placing the mixture in a centrifuge at 12000rpm for 1min, transferring the upper organic phase into a new tube, vacuum drying until all liquid is volatilized, only keeping tanshinone solid at the bottom of the centrifuge tube, adding 200 mu L of methanol into the tube for redissolving, centrifuging, filtering through a 0.22 mu m filter, and detecting.

(5) The conditions and methods for UPLC detection were the same as those of example 2.

The results show that: the target protein TIIAS can be successfully obtained through a prokaryotic expression system (figure 3A). TiIAS can catalyze Cryptotanshinone (CT) to generate tanshinone IIA (TIIIA) in-vitro enzyme activity reaction, the reaction depends on alpha-ketoglutaric acid (2OG), and the reaction does not depend on Fe²⁺And ascorbic Acid (ASC), but both promote enzymatic activity to increase tiia production (fig. 3B). The result shows that TIIAS protein can catalyze CT to generate TIIAA in an in-vitro enzyme activity reaction system.

Example 5 overexpression of TIIAS validation Gene protein function in Salvia miltiorrhiza hairy root

The TIIAS overexpression vector pK7WG 2R-TIIAS was transferred into Agrobacterium tumefaciens strain C58C1 by chemical transformation, while an empty plasmid containing CaMV 35S promoter was transferred into the strain AS a negative control.

Leaves of wild tissue culture salvia miltiorrhiza are used as explant materials of transgenosis. Infecting a salvia miltiorrhiza leaf explant for 20min by using transgenic agrobacterium, cleaning for 3 times by using sterile water, then sucking water by using filter paper, placing the salvia miltiorrhiza leaf explant in a 6,7-V solid culture medium, co-culturing for 3d in a dark place, cleaning the infected explant by using sterile water for 3 times, then sucking water by using filter paper, placing the explant in a sterilization culture medium containing carbenicillin (400mg/L), and culturing until hairy roots grow. After 3 weeks of culture, the hairy roots are transferred to a 6,7-V liquid culture medium and cultured for 2 months at a rotating speed of 40rpm under the condition of keeping out of the sun.

The hairy root material is freeze-dried overnight and then ground into powder, and 0.05g of the powder is precisely weighed and ultrasonically crushed in 1mL of methanol for 2h to extract the metabolite. Centrifuging at 12000rpm for 5min, collecting supernatant, and filtering with 0.22 μm organic filter membrane to obtain sample for UPLC detection.

The UPLC assay conditions were the same as in example 2.

The results show that the content of tanshinone IIA (compound 2) in the salvia miltiorrhiza hairy roots transformed by the over-expression TIIAS is obviously improved (figure 4), and the content of the tanshinone IIA in the over-expression strain can reach 2.8 times of that of the control. Therefore, the TIIAS in the salvia miltiorrhiza participates in the biosynthesis of the tanshinone TIIA, and the increase of the expression quantity of the TIIAS can promote the biosynthesis of the tanshinone TIIA.

Example 6 verification of protein function of TIIAS Gene in Salvia miltiorrhiza hairy root RNAi

The RNA interference vector pK7GWIWG 2R-TIIAS was transferred into Agrobacterium tumefaciens strain C58C1 by chemical transformation, and an empty plasmid containing CaMV 35S promoter was transferred into the strain AS a negative control.

The method for transforming the roots with RNAi vector, culturing the roots and extracting the compound are the same as in example 5.

UPLC detection conditions: agilent 1260 definition II Prime LC System, ACQUITY UPLC HSS C₁₈(2.1X 100mm,1.8 μm) column, detection wavelength 270 nm. Mobile phase conditions: the flow rate is 0.5mL/min, the mobile phase is 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B), the column temperature is 40 ℃, and gradient elution is carried out: 0-8min, 50% -58% B; 8-20min, 58% -70% B; 20-20.5min, 70% -80% B; 20.5-24.5min, 80% -100% B; 24.5-25min, 100% -50% B; 25-28min, 50% B.

The results show that in the transformation of salvia miltiorrhiza hairy roots by TiIAS-RNAi (figure 5), the content of tanshinone IIA (compound 2) is obviously inhibited, the content of the TIIA in a control strain is 0.66mg/g (dry weight content), and the content of the TIIA in an interference strain is only 0.09mg/g (dry weight content); while the accumulation of cryptotanshinone (compound 1) as a substrate was increased to 1.82mg/g (dry weight content) in the control group, while the amount in the RNAi strain was increased to 8.46mg/g (dry weight content). This further suggests that tiias is involved in the biosynthesis of tanshinone tiia in salvia miltiorrhiza. Therefore, increasing the expression level of tiias can promote the biosynthesis of tanshinone tiia, which confirms the experimental results of example 5.

Example 7: the function of the TiIAS homologous protein was verified in 7 plants of the genus Salvia (Salvia spp.).

(1) Based on the transcriptome of south salvia miltiorrhiza (s.bowleyana), salvia sclarea (s.meiriensis), salvia pratensis (s.trijuga), salvia pratensis (s.prinitis), salvia przewalski, salvia digitalis (s.digitaloides) and sedum lineare (s.substolonifera), homologous proteins of tiias in these species were determined by blast, named respectively SbowT ii AS, SmeiT ii AS, StriT ii AS, SpriT ii AS, SprzT ii AS, SdigT ii AS and SsubT ii AS, with the nucleotide and amino acid sequences respectively: SEQ ID NO.3 and SEQ ID NO. 4; SEQ ID NO.5 and SEQ ID NO. 6; SEQ ID NO.7 and SEQ ID NO. 8; SEQ ID NO.9 and SEQ ID NO. 10; SEQ ID NO.11 and SEQ ID NO. 12; SEQ ID NO.13 and SEQ ID NO. 14; SEQ ID NO.15 and SEQ ID NO. 16. Construction of prokaryotic expression vectors plasmids pET32 a-SkowT II AS, pET32a-SmeiT II AS, pET32a-StriT II AS, pET32a-SpriT II AS, pET32a-SprzT II AS, pET32 a-SdugT II AS and pET32 a-SubT II AS were obtained, respectively, with reference to the restriction ligation method described in example 1. The primers used were as follows:

SbowTⅡAS-BamHI-F：cgggatccATGCAGGAAGACGATCGCGTGAAGG

SbowTⅡAS-NotI-R：ataagaatgcggccgcTTAGACATTGTCTTCATCTTGCAAT

SmeiTⅡAS-BamHI-F：cgggatccATGGCCACATCCAGCTTGAAAAATT

SmeiTⅡAS-NotI-R：ataagaatgcggccgcTTAGACATTGTCTTCATCTTGCAAT

StriTⅡAS-BamHI-F：cgggatccATGGCGGCAGACGATCGCGTGGAGG

StriTⅡAS-NotI-R：ataagaatgcggccgcTTAGACATTGTCTTCATCTTGCAAC

SpriTⅡAS-BamHI-F：cgggatccATGCAGGAAGACGATCGCGTGAAGG

SpriTⅡAS-NotI-R：ataagaatgcggccgcTCACAAAGTGTCAGCACAAAGAGAT

SprzTⅡAS-BamHI-F：cgggatccATGGAGGAAGACGATCGCATGGAGG

SprzTⅡAS-NotI-R：ataagaatgcggccgcTCACAAAGTGTCAGCACAAAGAGAT

SdigTⅡAS-BamHI-F：cgggatccATGGAGGAAGACGATCGCATGAAGG

SdigTⅡAS-NotI-R：ataagaatgcggccgcTCACAAAGTGTCAGCACAAAGAGAT

SsubTⅡAS-BamHI-F：cgggatccATGCATATTTCAACCATTTTTCTGC

SsubTⅡAS-NotI-R：ataagaatgcggccgcCTAAGTAATAATTAACCTGAACTCA

(2) the function of the SwowTIIAS, SmeitIIAS, StriTIIAS, SpriTIIAS, SprzTIIAS, SsigTIIAS and SubTIIAS proteins was verified in prokaryotic systems in the same manner AS in example 2.

The results show that: in an escherichia coli prokaryotic system, the result is shown in fig. 6, and a new product cannot be generated by taking pET32a no-load as a control and Cryptotanshinone (CT) as a substrate; in systems catalyzed by SwotIIAS, SmeitIIAS, StriTIIIAS, SpriTIIIAS, SprzTIIIAS, SsigtIIAS and SubTIIIAS proteins, CT is catalyzed to produce tanshinone IIA (TIIA). It is demonstrated that in species related to Salvia miltiorrhiza, such AS Salvia miltiorrhiza, Salvia meikochia, Salvia praeparata, Salvia sanguinea, Salvia calomel, Salvia praecox, and Salvia flammans, the homologous proteins of tanshinone IIA synthase (TIIIAS) can catalyze CT to produce TIIIA.

Sequence listing

<110> Shanghai mountain plant garden

<120> tanshinone IIA synthase gene and application

<160> 36

<170> PatentIn version 3.5

<210> 1

<211> 1122

<212> DNA

<213> Salvia miltiorrhiza

<400> 1

atggccacat ccagcttgaa aaattgctcg caagaaaacg aggccgatcg cgtgcacgag 60

ctgaacgctt tcgaggccac aaaagccggc gtgaaggggc tcaccgactc cggcgtccag 120

aaggttccga gaatgttcat caggccagcc gacgagctcg tcgaggagcg caaccggagc 180

cgctccccgc tgcaagctcc ggtgatagac ctcggccgga tcggggaggg cgaggggcgg 240

gagaaggccg tgagcgaggt gagatgggcg tcgaaggagc tcgggatctt ccagatcgtg 300

aaccacgggg tggccgtgga ggtcatggac gcgatgatcg acggcgtgag gaagtttcac 360

gagcaagatg cggaggcgaa gaagcagttc cacacgcgcg acgccatgcg caaggtgatg 420

tacgcgagca acgtcgatct gtacaagtcg cgcgccgcga attggaggga cacgttctcg 480

gtcgcgctca tgggttcgga ccgcgttgag ccggaagagt tgccggagat ttgcagagac 540

tcaacaatca agtatcttga tgaagtcacg aatctcgcac acactctatt tgagctgctc 600

tcggaagctc ttgggctcga acaaggttgc ctaggagcct tgaaatgtgg ccgaggacgc 660

acgttcgtcg gccagtacta ccccgcgtgc cccgagccgg agctcacgat gggcatgacc 720

aaccacaccg atccttgttt cctaactatt cttctccaag atcaaatcgg aggcctccaa 780

gctctgcaca atagtcagta cataaatgtg gagcctctgc ctgctagctt cgtcgtcaac 840

attggagata tgttgcagat tgtgacaaat gatgagttca taagcccaat tcatagagtc 900

cacgcaaatc gggccgggcc aagaatctcg gttgcgggct ttttcaccgg tgatgctatt 960

tcagggacaa tatatggccc gatcaaagag ttggtatcag agaacaatcg ggctcgatac 1020

aaagagttca cagtgggaga gtacatgtcc aagtttttag agcggccaat tgataaatct 1080

ggtcttgatg aatggagatt gcaagatgaa gacaatgtct aa 1122

<210> 2

<211> 373

<212> PRT

<213> Salvia miltiorrhiza

<400> 2

MATSSLKNCS QENEADRVHE LNAFEATKAG VKGLTDSGVQ KVPRMFIRPA DELVEERNRS 60

RSPLQAPVID LGRIGEGEGR EKAVSEVRWA SKELGIFQIV NHGVAVEVMD AMIDGVRKFH 120

EQDAEAKKQF HTRDAMRKVM YASNVDLYKS RAANWRDTFS VALMGSDRVE PEELPEICRD 180

STIKYLDEVT NLAHTLFELL SEALGLEQGC LGALKCGRGR TFVGQYYPAC PEPELTMGMT 240

NHTDPCFLTI LLQDQIGGLQ ALHNSQYINV EPLPASFVVN IGDMLQIVTN DEFISPIHRV 300

HANRAGPRIS VAGFFTGDAI SGTIYGPIKE LVSENNRARY KEFTVGEYMS KFLERPIDKS 360

GLDEWRLQDE DNV 373

<210> 3

<211> 1092

<212> DNA

<213> Salvia miltiorrhiza bge

<400> 3

atgcaggaag acgatcgcgt gaaggagctc caggctttcg aggccacaaa agccggcgtg 60

aaagggctaa tcgacgccgg catccagaag gttccgagaa tgttcatcag gccagccgat 120

gagctcgtgg aggagctcaa ctgcgcgcgc tcggctctgc aagttccggt gatagacctc 180

ggccggatcg gagaaggcga tcatcgcgag aaggttgtcg gcgaggtgag gagggcgtcg 240

aaggagctcg ggatcttcca gatcgtgaat cacggtgtgg ctgtggaggt tatggatgcg 300

atgctcgacg gcgtgaggaa gtttcacgag ctagatgcgg aggtgaagaa gcagttccac 360

tcgcgcgagc ctatgcagaa ggtcaagtat gcgagcaacg tcgatctgta ccggtcgcgc 420

gccgcgaatt ggagggattc gttgacgatc tcgctcatgg gctccgatcg tcttccgccg 480

gaggagttgc cggagatttg cagggactca acaatcaagt atcttgatga agtcacgagc 540

ctcggagaaa ctctatttga gctactctcc gaagctcttg gcctcaaacg aggttatcta 600

ggagccctga aatgtggcca aggacgcact ttcattggcc actactaccc tccgtgccct 660

gagccggagc tcacgatggg cacggccact cacaccgacc cttccttcct aacaattctt 720

ctacaagatc aaatcggagg cctccaagct ttgcacaata atcagtacat aaatgtggag 780

cctctacctg gtagcttggt tgtcaacatt ggagatatgt tgcagattgt gacaaatgat 840

gagttcataa gcccggatca tagagttgta gccaatcggg ccgggccaag aatctcggtt 900

gcgggcttgt tcacgggcga tgctctttct gggacgatat acggcccgat caaagagctg 960

atatcggaaa acaatcgcgc aagatacaaa gagttgacag tgagagacta catttcaaag 1020

ttttttgcgc ggccaattga taaatctggt cttgatgagt tcaagttatt gcaagatgaa 1080

gacaatgtct aa 1092

<210> 4

<211> 363

<212> PRT

<213> Salvia miltiorrhiza bge

<400> 4

MQEDDRVKEL QAFEATKAGV KGLIDAGIQK VPRMFIRPAD ELVEELNCAR SALQVPVIDL 60

GRIGEGDHRE KVVGEVRRAS KELGIFQIVN HGVAVEVMDA MLDGVRKFHE LDAEVKKQFH 120

SREPMQKVKY ASNVDLYRSR AANWRDSLTI SLMGSDRLPP EELPEICRDS TIKYLDEVTS 180

LGETLFELLS EALGLKRGYL GALKCGQGRT FIGHYYPPCP EPELTMGTAT HTDPSFLTIL 240

LQDQIGGLQA LHNNQYINVE PLPGSLVVNI GDMLQIVTND EFISPDHRVV ANRAGPRISV 300

AGLFTGDALS GTIYGPIKEL ISENNRARYK ELTVRDYISK FFARPIDKSG LDEFKLLQDE 360

DNV 363

<210> 5

<211> 1122

<212> DNA

<213> Salvia officinalis

<400> 5

atggccacat ccagcttgaa aaattgctcg caagaaaacg aggccgatcg cgtgcacgag 60

ctgaacgctt tcgaggccac aaaagccggc gtgaaggggc tcaccgactc cggcgtccag 120

aaggttccga gaatgttcat caggccagcc gacgagctcg tcgaggagct caaccggagc 180

cgctccccgc tgcaagttcc ggtgatagac ctcggccgga tcggggaggg cgaggggcgg 240

gagaaggtgg tgagcgaggt gagatgggcg tcgaaggagc tcgggatctt ccagatcgtg 300

aaccacgggg tggccgtgga ggtcatggac gggatgatcg acggcgtgag gaggtttcac 360

gagcaagatg cggaggcgaa gaagcagttc cacacgcgcg acgccatgcg caaggtgatg 420

tacgcgagca acgtcgatct gtacaagtcg cgcgcggcga attggaggga cacgttctcg 480

gtcgcgctca tgggttccga cagcgttgag ccggaagagt tgccggagat ttgcagagac 540

tcaacaatca agtatcttga tgaagtcacg aatctcgcaa acactctatt tgagctgctc 600

tcggaagctc ttgggctcga aaaaggttgc ctaggagcct tgaaatgtgg ccgaggacgc 660

acgttcgtcg gccagtacta ccccgcgtgc cccgagccgg agctcacgat gggcatgacc 720

aaccacaccg atccttgttt cctaactatt cttctccaag atcaaatcgg aggcctccaa 780

gctctgcaca ataatcagta cataaatgtg gagcctctgc ctgctagctt cgtcgtcaac 840

attggagata tgttgcagat tgtgacaaat gatgagttca taagcccaat tcatagagtc 900

cacgcaaatc gggccgggcc aagaatctcg attgcgggct ttttcaccgg tgatgctatt 960

tcagggacaa tatacggccc gatcaaagag ttggtatcag agaacaatcg ggctcgatac 1020

aaagagttca cagtgggaga gtacatgtcc aagtttttag agcggccaat tgataaatct 1080

ggtcttgatg aatggagatt gcaagatgaa gacaatgtct aa 1122

<210> 6

<211> 373

<212> PRT

<213> Salvia officinalis

<400> 6

MATSSLKNCS QENEADRVHE LNAFEATKAG VKGLTDSGVQ KVPRMFIRPA DELVEELNRS 60

RSPLQVPVID LGRIGEGEGR EKVVSEVRWA SKELGIFQIV NHGVAVEVMD GMIDGVRRFH 120

EQDAEAKKQF HTRDAMRKVM YASNVDLYKS RAANWRDTFS VALMGSDSVE PEELPEICRD 180

STIKYLDEVT NLANTLFELL SEALGLEKGC LGALKCGRGR TFVGQYYPAC PEPELTMGMT 240

NHTDPCFLTI LLQDQIGGLQ ALHNNQYINV EPLPASFVVN IGDMLQIVTN DEFISPIHRV 300

HANRAGPRIS IAGFFTGDAI SGTIYGPIKE LVSENNRARY KEFTVGEYMS KFLERPIDKS 360

GLDEWRLQDE DNV 373

<210> 7

<211> 1092

<212> DNA

<213> clover sage

<400> 7

atggcggcag acgatcgcgt ggaggagctc aaggctttcg aggcgacgaa agccggcgtg 60

aaggggctaa tcgacgccgg catccagaag gttccgagaa tgttcatcag gccggccgat 120

gagctcgtgg aggagctcaa ctgcgcgcgc tcggctctgc aagttccggt gatcgacctc 180

ggccggatcg gagaaggcgg cgatgcgcgc gagaaggtcg tcggcgaggt gagggcggcg 240

tcgaaggagc tagggatctt ccagatcgtg aatcacggcg tggctgcgga ggtcatggac 300

gcgatgctcg acggcgtgag gaggtttcac gagctagatg cggaggtgaa gaagctgttc 360

cacccgcgcg aggcgatgca gaaggtcgag tacgcgagca acgtcgatct gtaccggtcg 420

cgcgccgcga attggaggga ttcgttgaag atctcgctcg cgggctccga tcgcattcag 480

ccggaggagt tgccggagat ttgcagggac tcaacaatca agtatcttga tgaagtcacg 540

agcctcgcag acactctatt tgagctgctc tccgaagctc ttgggctcga aaaaggttgc 600

ctaggagcct tgaaatgtgg ccgaggacgc acgtttgtcg gccagtacta ccccgtgtgc 660

cccgagccgg agctcacaat gggcatgagc aaccacaccg acccttgttt cctaactatt 720

cttctccaag atgaaatcgg aggcctccaa gctttgcaca ataatcaata cataaacgtg 780

gagcctctgc ccggtagctt ggttgtcaac attggagata tgttgcagat tgtgacaaat 840

gatgagttca taagcccgat tcatagagtt catgccaatc gggccgggac aagaatctcg 900

gttgcgggct ttttcaccgg tgatgctatt tcagggacaa tatacggccc gatcaaagag 960

ttgatatcaa agaacaatcg ggctcgatac aaagagttca cagtgggaga ctacatgacc 1020

aagtttttag agaggccaat tgaaaattct ggtcttgatg atttcaggtt gcaagatgaa 1080

gacaatgtct aa 1092

<210> 8

<211> 363

<212> DNA

<213> clover sage

<400> 8

MAADDRVEEL KAFEATKAGV KGLIDAGIQK VPRMFIRPAD ELVEELNCAR SALQVPVIDL 60

GRIGEGGDAR EKVVGEVRAA SKELGIFQIV NHGVAAEVMD AMLDGVRRFH ELDAEVKKLF 120

HPREAMQKVE YASNVDLYRS RAANWRDSLK ISLAGSDRIQ PEELPEICRD STIKYLDEVT 180

SLADTLFELL SEALGLEKGC LGALKCGRGR TFVGQYYPVC PEPELTMGMS NHTDPCFLTI 240

LLQDEIGGLQ ALHNNQYINV EPLPGSLVVN IGDMLQIVTN DEFISPIHRV HANRAGTRIS 300

VAGFFTGDAI SGTIYGPIKE LISKNNRARY KEFTVGDYMT KFLERPIENS GLDDFRLQDE 360

DNV 363

<210> 9

<211> 1122

<212> DNA

<213> Salvia miltiorrhiza

<400> 9

atgcaggaag acgatcgcgt gaaggagctc caggctttcg aggccacaaa agccggcgtg 60

aaagggctaa tcgacgccgg catccagaag gttccgagaa tgttcatcag gccagccgat 120

gagctcgtgg aggagctcaa ctgcgcgcgc tcctctctgc aagttccggt gatagacctc 180

ggccggatcg gagaaggcga tcatcgcgag aaggttgtcg gagaggtgag aacggcgtcg 240

aaggatctcg ggatcttcca gatcgtgaat cacggtgtgg ctgtggaggt tatggatgcg 300

atgctcgacg gcgtgaggaa gtttcacgag ctagatgcgg aggtgaagaa gcagttccac 360

tcgcgcgagt ctatgcagaa ggtcaagtat gcgagcaacg tcgatctgta ccggtcgcgc 420

gccgcgaatt ggagggattc gttgacgatc tcgctcatgg gctccgatcg tcttccgccg 480

gaggagttgc cggagatttg cagggactca acaatcaagt atcttgatga agtcacgagc 540

ctcggagaaa ctctatttga gctactctcc gaagctcttg gcctcaaacg aggttaccta 600

ggagccctga aatgtggcca agcacgcact ttcattggcc actactaccc tccgtgccct 660

gagccggagc tcacgatggg cacggccact cacaccgacc cttctttcct aacaattctt 720

ctacaagatc aaatcggagg cctccaagct ttgcataata atcaatacat aaatgtggag 780

cctctgcctg gtagcttggt tgtcaacatt ggagatatgt tgcagattgt gacaaatgat 840

gagttcataa gcccggatca tagagttgta gccaatcggg ccgggccaag aatctcggtt 900

gcgggctttt tcacgggcgg tgctctttct gggacgatat acggcccgat caaagagctg 960

atatcggaaa acaatcgcgc aagatacaaa gagttcacag tgagagacta catttcaaag 1020

ttttttgcgc ggccaattga taaatctggt cttgatgagt tcaggttatt acaagatgaa 1080

gacaatgtcc aaattttatc tctttgtgct gacactttgt ga 1122

<210> 10

<211> 373

<212> PRT

<213> Salvia miltiorrhiza

<400> 10

MQEDDRVKEL QAFEATKAGV KGLIDAGIQK VPRMFIRPAD ELVEELNCAR SSLQVPVIDL 60

GRIGEGDHRE KVVGEVRTAS KDLGIFQIVN HGVAVEVMDA MLDGVRKFHE LDAEVKKQFH 120

SRESMQKVKY ASNVDLYRSR AANWRDSLTI SLMGSDRLPP EELPEICRDS TIKYLDEVTS 180

LGETLFELLS EALGLKRGYL GALKCGQART FIGHYYPPCP EPELTMGTAT HTDPSFLTIL 240

LQDQIGGLQA LHNNQYINVE PLPGSLVVNI GDMLQIVTND EFISPDHRVV ANRAGPRISV 300

AGFFTGGALS GTIYGPIKEL ISENNRARYK EFTVRDYISK FFARPIDKSG LDEFRLLQDE 360

DNVQILSLCA DTL 373

<210> 11

<211> 1128

<212> DNA

<213> Salvia przewalskii

<400> 11

atggaggaag acgatcgcat ggaggagctg aaggctttcg aggccacaaa agccggcgtg 60

aaagggctaa tcgacgccgg catccagaag gttccgagaa tgttcatcag gccagccgat 120

gagctcgtgg aggagctcaa ctgcggccgc tcggctctgc aagttccggt gatcgacctc 180

ggccggatcg gagaattagg agatcatcat cgcgagaagg ttgtcggcga ggtgaggagg 240

gcgtcgaagg agctcgggat cttccagatc gtgaatcacg gcgtggctgt ggaagttatg 300

gatgggatgc ttgatggcgt gaggaagttt cacgagcaag atgcggaggt gaagaagcag 360

ttccactcgc gcgagcctat gcagaaggtc aagtatgcca gcaacgtcga tctgtaccgg 420

tcgcgcgccg cgaattggag ggattcgctg acgatctcgc tcgcgggttc cgatcgcctt 480

cagccggagg agttgccgga gatttgcagg gactcgacaa tcaagtatct tgatcaagtc 540

acgagcctcg gagaaactct atttgagcta ctctccgaag ctcttgacct caaacgaggt 600

tacctaggag ccctgaaatg tggccaagga cgcactttca ttggccacta ctaccctccg 660

tgccctgagc cggagctcac gatgggcacg gccagtcaca ccgacccttc tttcataaca 720

attcttctac aagatcaaat cggaggcctc caagctttgc acaataatca atacataaat 780

gtggaccctc tgcctggtag cttggttgtc aacattggag atatgttgca gattgtgaca 840

aatgatgagt tcataagccc ggatcataga gttgtagcca atcgggccgg gccaagaatc 900

tcggttgcgg gcttactctt tggcgatact ctttctggga cgatatacgg cccgatcaaa 960

gagctgatat cggaaaacaa tcgcgcaaga tacaaagagt tcacagtgag agactacatt 1020

tcaaagtttt ttgcgcggcc aattgataaa tctggtcttg atgagttcaa gttattacaa 1080

gatgaagaca atgtccaaat tttatctctt tgtgctgaca ctttgtga 1128

<210> 12

<211> 375

<212> PRT

<213> Salvia przewalskii

<400> 12

MEEDDRMEEL KAFEATKAGV KGLIDAGIQK VPRMFIRPAD ELVEELNCGR SALQVPVIDL 60

GRIGELGDHH REKVVGEVRR ASKELGIFQI VNHGVAVEVM DGMLDGVRKF HEQDAEVKKQ 120

FHSREPMQKV KYASNVDLYR SRAANWRDSL TISLAGSDRL QPEELPEICR DSTIKYLDQV 180

TSLGETLFEL LSEALDLKRG YLGALKCGQG RTFIGHYYPP CPEPELTMGT ASHTDPSFIT 240

ILLQDQIGGL QALHNNQYIN VDPLPGSLVV NIGDMLQIVT NDEFISPDHR VVANRAGPRI 300

SVAGLLFGDT LSGTIYGPIK ELISENNRAR YKEFTVRDYI SKFFARPIDK SGLDEFKLLQ 360

DEDNVQILSL CADTL 375

<210> 13

<211> 1131

<212> DNA

<213> Salvia officinalis with rehmannia

<400> 13

atggaggaag acgatcgcat gaaggagctc aaggctttcg aggccacaaa agccggcgtg 60

aaaggtctaa tcgacgccgg catccagaag attccgagaa tgttcatcag gccagccgat 120

gagctcgtgg aggagctcaa ctgcggccgc tcggctctgc aagttccggt gatcgacctc 180

ggccggatcg gagaattagg agatcatcat cgcgagaagg ttgtcggtga ggtgaggagg 240

gcgtcgaagg agctcgggat cttccagatc gtgaatcacg gtgtggctgt ggaagttatg 300

gatgggatgc ttgacggcgt gaggaagttt cacgagcaag atgcggaggt gaagaagcag 360

ttccactcgc gcgagactat gcagaaggtc aagtatgcca gcagcaacgt cgatctgaac 420

cggtcgcgcg ccgcgaattg gagggattcg ttgaagatct cgctcgcggg ttccgatcgt 480

cttcagccgg aggagttgcc ggagatttgc agggactcaa caatcaagta tcttgatcaa 540

gtcacgagcc tcggagaaac tctatttgag ctactctccg aagctcttgg cctcaaacga 600

ggttacctag gagccctgaa atgtggccaa ggacgcactt tcatcggcca ctactaccct 660

ccgtgccctg agccggagct cacgatgggc acggccagtc acaccgaccc ttctttccta 720

acaattcttc tacaagatca aatcggaggc ctccaagctt tgcacaataa tcaatacata 780

aatgtggacc ctctgcctgg tagcttggtt gtcaacattg gagatatgtt gcagattgtg 840

acaaatgatg agttcataag cccggatcat agagttgtag ccaatcgggc cgggccaaga 900

atctcggttg cgggcttctt cactgacgat atcctttctg ggacgatata cggcccgatc 960

aaagagctga tatcggaaaa caatcgcgca agatacaaag agttcacagt gagagactac 1020

atttcaaagt tttttgcgcg gccaattgat aaatctggtc ttgatgagtt caggttatta 1080

caagatgaag acaatgtcca aattttatct ctttgtgctg acactttgtg a 1131

<210> 14

<211> 376

<212> PRT

<213> Salvia officinalis with rehmannia

<400> 14

MEEDDRMKEL KAFEATKAGV KGLIDAGIQK IPRMFIRPAD ELVEELNCGR SALQVPVIDL 60

GRIGELGDHH REKVVGEVRR ASKELGIFQI VNHGVAVEVM DGMLDGVRKF HEQDAEVKKQ 120

FHSRETMQKV KYASSNVDLN RSRAANWRDS LKISLAGSDR LQPEELPEIC RDSTIKYLDQ 180

VTSLGETLFE LLSEALGLKR GYLGALKCGQ GRTFIGHYYP PCPEPELTMG TASHTDPSFL 240

TILLQDQIGG LQALHNNQYI NVDPLPGSLV VNIGDMLQIV TNDEFISPDH RVVANRAGPR 300

ISVAGFFTDD ILSGTIYGPI KELISENNRA RYKEFTVRDY ISKFFARPID KSGLDEFRLL 360

QDEDNVQILS LCADTL 376

<210> 15

<211> 1149

<212> DNA

<213> Foguang grass

<400> 15

atgcatattt caaccatttt tctgctcaac aaatcagaac aaaaacttcc cccaaaagtt 60

aagaaaatgg cggaagacga ccgagtggag gagctcaagg ctttcgaggc gacgaaagcc 120

ggcgtgaagg ggctaatcga cgccggcatc cagaaggttc cgaggatgtt catcaggcca 180

gccgatgagc tcgtggagga gctcaactgc gcgcgctcgg ctctgcaagt tccggtgatc 240

gacctcggcc ggatcggaga gggcggcgat gcgcgcgagg aggtcgtcgg cgaggtgagg 300

gcggcgtcga aggagctcgg gatcttccag atcgtgaatc acggcgtggc cgcggaggtc 360

atgaacgcga tgctcgacgg cgtgaggagg tttcacgagc tagatgcgga ggtgaagaag 420

cggttccact cgcgcgaggc gatgcagaag gtcaagtatg cgagcaacgt tgatctgtac 480

cggtcgcgcg ccgcgaattg gagggattcg ttgacgatct cgctcgcggg cgccgatcgt 540

cttccgccgg aggagttgcc ggagatttgc agggactcaa caatcaagta tattgatcaa 600

gtcacgagcc tcggagaaac tctatttgag ctactctccg aagctcttgg cctcaaacga 660

ggttacctag gagccctgaa atgtggccaa cggcgcactt tcatcggcca ctactaccct 720

ccgtgccctg agccggagct cacgatgggc acggccagtc acaccgacct ttctttcata 780

acaattcttc tacaagatca aattggaggc ctccaagctt tgcacaataa tcagtacata 840

aacgtggagc ctctgcccgg tagcttggtt gtcaacattg gagatatgtt gcagattatg 900

acaaatgatg agttcataag cccggatcat agagttgtag ccaatcgggc cgggccaaga 960

atctcggttg cgggcttctt cagtggcgat ggtttttctg ggacaatata cggcccgatt 1020

aaagagctga tatcggaaga caatcgcgcg agatacaaag agttcacagt gagggactac 1080

atttcaaagt tttttgcgcg gccgattgat aaatccggtc ttgatgagtt caggttaatt 1140

attacttag 1149

<210> 16

<211> 382

<212> DNA

<213> Foguang grass

<400> 16

MHISTIFLLN KSEQKLPPKV KKMAEDDRVE ELKAFEATKA GVKGLIDAGI QKVPRMFIRP 60

ADELVEELNC ARSALQVPVI DLGRIGEGGD AREEVVGEVR AASKELGIFQ IVNHGVAAEV 120

MNAMLDGVRR FHELDAEVKK RFHSREAMQK VKYASNVDLY RSRAANWRDS LTISLAGADR 180

LPPEELPEIC RDSTIKYIDQ VTSLGETLFE LLSEALGLKR GYLGALKCGQ RRTFIGHYYP 240

PCPEPELTMG TASHTDLSFI TILLQDQIGG LQALHNNQYI NVEPLPGSLV VNIGDMLQIM 300

TNDEFISPDH RVVANRAGPR ISVAGFFSGD GFSGTIYGPI KELISEDNRA RYKEFTVRDY 360

ISKFFARPID KSGLDEFRLI IT 382

<210> 17

<211> 56

<212> DNA

<213> Artificial sequence

<400> 17

ggggacaagt ttgtacaaaa aagcaggctt catggccaca tccagcttga aaaatt 56

<210> 18

<211> 55

<212> DNA

<213> Artificial sequence

<400> 18

ggggaccact ttgtacaaga aagctgggtt ttagacattg tcttcatctt gcaat 55

<210> 19

<211> 56

<212> DNA

<213> Artificial sequence

<400> 19

ggggacaagt ttgtacaaaa aagcaggctt caagatgcgg aggcgaagaa gcagtt 56

<210> 20

<211> 55

<212> DNA

<213> Artificial sequence

<400> 20

ggggaccact ttgtacaaga aagctgggtt ttggagaaga atagttagga aacaa 55

<210> 21

<211> 33

<212> DNA

<213> Artificial sequence

<400> 21

cgggatccat ggccacatcc agcttgaaaa att 33

<210> 22

<211> 41

<212> DNA

<213> Artificial sequence

<400> 22

ataagaatgc ggccgcttag acattgtctt catcttgcaa t 41

<210> 23

<211> 33

<212> DNA

<213> Artificial sequence

<400> 23

cgggatccat gcaggaagac gatcgcgtga agg 33

<210> 24

<211> 41

<212> DNA

<213> Artificial sequence

<400> 24

ataagaatgc ggccgcttag acattgtctt catcttgcaa t 41

<210> 25

<211> 33

<212> DNA

<213> Artificial sequence

<400> 25

cgggatccat ggccacatcc agcttgaaaa att 33

<210> 26

<211> 41

<212> DNA

<213> Artificial sequence

<400> 26

ataagaatgc ggccgcttag acattgtctt catcttgcaa t 41

<210> 27

<211> 33

<212> DNA

<213> Artificial sequence

<400> 27

cgggatccat ggcggcagac gatcgcgtgg agg 33

<210> 28

<211> 41

<212> DNA

<213> Artificial sequence

<400> 28

ataagaatgc ggccgcttag acattgtctt catcttgcaa c 41

<210> 29

<211> 33

<212> DNA

<213> Artificial sequence

<400> 29

cgggatccat gcaggaagac gatcgcgtga agg 33

<210> 30

<211> 41

<212> DNA

<213> Artificial sequence

<400> 30

ataagaatgc ggccgctcac aaagtgtcag cacaaagaga t 41

<210> 31

<211> 33

<212> DNA

<213> Artificial sequence

<400> 31

cgggatccat ggaggaagac gatcgcatgg agg 33

<210> 32

<211> 41

<212> DNA

<213> Artificial sequence

<400> 32

ataagaatgc ggccgctcac aaagtgtcag cacaaagaga t 41

<210> 33

<211> 33

<212> DNA

<213> Artificial sequence

<400> 33

cgggatccat ggaggaagac gatcgcatga agg 33

<210> 34

<211> 41

<212> DNA

<213> Artificial sequence

<400> 34

ataagaatgc ggccgctcac aaagtgtcag cacaaagaga t 41

<210> 35

<211> 33

<212> DNA

<213> Artificial sequence

<400> 35

cgggatccat gcatatttca accatttttc tgc 33

<210> 36

<211> 41

<212> DNA

<213> Artificial sequence

<400> 36

ataagaatgc ggccgcctaa gtaataatta acctgaactc a 41

Claims (10)

1. The gene TIIAS of tanshinone IIA synthase has one of the following nucleotide sequences:

1) the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 and SEQ ID No. 15;

2) the sequence obtained by adding, deleting and substituting one or more nucleotides in SEQ ID No.1, the coded protein still has the activity of tanshinone IIA synthase;

3) the nucleotide sequence consistency and SEQ ID No.1 are more than 80%, more than 85%, more than 90%, more than 95% or more than 99% of homologous genes, and the coded protein still has the activity of tanshinone IIA synthase; preferably, it is derived from Salvia miltiorrhiza, Salvia przewalskii, Salvia splendens, Salvia pratense, Salvia rubra, Salvia przewalskii, Salvia praecox and Salvia flammans.

2. The gene tiias according to claim 1, wherein: the tanshinone IIA synthase gene is a nucleotide sequence shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 and SEQ ID No. 15.

3. A protein encoded by gene tiias of claim 1 or 2.

4. The protein of claim 3, wherein: the amino acid sequence is homologous protein which is more than 80%, more than 85%, more than 90%, more than 95% or more than 99% identical to SEQ ID No.2, and still has the activity of tanshinone IIA synthase, preferably, the protein is derived from Salvia miltiorrhiza Bunge, Salvia przewalskii Bunge, Salvia pratense, Salvia rubra Bunge, Salvia przewalskii Bunge, Salvia praecox and Salvia flammans, and more preferably, the amino acid sequence is shown as SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14 or SEQ ID No. 16.

5. A recombinant vector, preferably an expression vector, more particularly a bacterial, fungal or plant expression vector, further e.coli, yeast expression vector, comprising the gene tiias according to claim 1 or 2.

6. A host cell, more particularly a bacterial, fungal or plant cell, further E.coli, yeast cell, comprising the gene TIIAS AS defined in claim 1 or 2 or the recombinant vector AS defined in claim 5.

7. Use of a gene tiias AS defined in claim 1 or 2 or a protein AS defined in claim 3 or 4 for producing or preparing tanshinone iia or isotanshinone iia.

8. A process for producing tanshinone IIA by catalyzing cryptotanshinone by fermentation or isotanshinone by using a microorganism, which comprises introducing and expressing the gene TIIAS AS defined in claim 1 or 2 into the microorganism; culturing microorganism containing TIIAS expressing the gene, such AS Escherichia coli and yeast, and extracting tanshinone IIA or isotanshinone IIA from thallus, wherein tanshinone or cryptotanshinone is added into the culture medium.

9. A method for increasing the content of tanshinone iia and/or isotanshinone iia in plants, characterized in that the gene tiias according to claim 1 or 2 is introduced into a target plant to obtain a transgenic plant overexpressing the gene tiias; more specifically, the constructed agrobacterium or agrobacterium tumefaciens strain containing the gene TIIAS is used for transforming the salvia miltiorrhiza to obtain a transgenic salvia miltiorrhiza plant or hairy root over expressing the gene TIIAS, wherein the content of tanshinone IIA and/or iso-tanshinone IIA is improved.

10. A process for in vitro production or preparation of tanshinone IIA or isotanshinone IIA using the protein of claim 3 or 4, wherein the protein of claim 3 or 4 is used to catalyze cryptotanshinone to produce tanshinone IIA or isotanshinone to produce isotanshinone IIA, preferably the reaction system contains α -ketoglutaric acid and further contains Fe²⁺And/or ascorbic acid; preferably the protein of claim 3 or 4 is obtained by extracting after fermentation of genetically engineered bacteria; further preferably, the reaction system is 200nM MES (pH6.5), 1mM alpha-ketoglutarate, 1mM ascorbic acid, 250. mu.M Fe^{2 +}1mM ATP and 50 μ g TIIAS protein, and the concentration of the substrate cryptotanshinone or iso-cryptotanshinone is 50 μ M; the reaction conditions are 20 ℃, 180rpm, the mixture is taken out after shaking reaction for 30min, and ethyl acetate with twice volume of the reaction system is added to terminate the reaction; further comprises a step of purifying tanshinone IIA or iso-cryptotanshinone.

Images