Method for synthesizing tanshinone precursor and protein combination used by same

Abstract

The invention discloses a method for synthesizing a tanshinone precursor and a protein combination used by the method. The protein combination comprises at least one of a SmKSL1 protein as shown in SEQ ID NO: 18, a SmCPS1 protein as shown in SEQ ID NO: 20, a SmCPR1 protein as shown in SEQ ID NO: 22, a SmCYP76AH1 protein as shown in SEQ ID NO: 24, a SmCYP76AH3 protein as shown in SEQ ID NO: 26 and a SmCYP76AK1 protein as shown in SEQ ID NO: 28. Experiments prove that tanshinone precursors such as hypotanshinone diene, ferruginol, 11-hydroxy ferruginol, 11-hydroxy Cryptotaxol, 11, 20-hydroxy ferruginol, 11, 20-hydroxy Cryptotaxol and the like can be synthesized by introducing the gene for coding the protein combination into Benyi tobacco. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for synthesizing a tanshinone precursor and a protein combination used therein. Background Art

[0002] Danshen was first recorded in Shennong's Herbal Classic. It is the dried root and rhizome of Salvia miltiorrhiza Bunge, a plant of the genus Salvia in the family Lamiaceae. It has the effects of promoting blood circulation and removing blood stasis, relieving pain, clearing the heart and relieving vexation, cooling blood and eliminating carbuncle, and is widely used to treat cardiovascular and cerebrovascular diseases. The main chemical components of Danshen include fat-soluble tanshinone and water-soluble salvianolic acid. Tanshinone belongs to the abietane-type diterpene compound, which is a secondary metabolite with important research and medicinal value. It has multiple pharmacological activities such as anti-tumor, anti-inflammatory, liver protection, anti-lung injury, antioxidant, antibacterial, and anti-fibrosis.

[0003] Diterpenoids are usually formed by four isoprenyl diphosphates under the action of trans-isoprenyl transferase (TPT) to form a linear primary metabolite geranylgeranyl pyrophosphate (GGPP), which is then catalyzed by diterpene synthase to form a diterpene skeleton. Figure 1 The diterpene precursor GGPP is cyclized under the action of terpene synthase to generate a carbon skeleton structure - tanshinone diene. Tanshinone diene is a precursor of abietane diterpene compounds such as triptolide, tanshinone, and carnosic acid. Subsequently, three CYP450 enzymes oxidatively modify the abietane diterpene precursor - tanshinone diene. CYP76AH1 catalyzes the C-12 hydroxylation of tanshinone diene to generate ferruginol; CYP76AH3 further catalyzes the C-11 hydroxylation and C-6 carbonylation of ferruginol to form 11-hydroxyferruginol and 11-hydroxycedar phenol, respectively; then CYP76AK1 catalyzes the C-20 hydroxylation of 11-hydroxyferruginol and 11-hydroxycedar phenol, respectively, to generate 11,20-hydroxyferruginol and 11,20-hydroxycedar phenol, respectively.

[0004] The chemical diversity of tanshinones is largely attributed to the catalytic activity from many enzyme families. Heterologous genes can be expressed in plant tissues by two methods: stable expression and transient expression. Transient expression in plants has the advantages of simplicity, rapidity, and short cycle, while stable expression efficiency, high conversion rate and high biosafety. The Agrobacterium tumefaciens-mediated transient expression system is a fast and effective method for analyzing gene function, and can be applied to transgenic complementation, promoter function analysis and protein expression. The model plant Nicotiana benthamiana is increasingly used in plant factories and can be used for bioreactors of heterologous biosynthetic pathways of natural products. Transient expression of Nicotiana benthamiana leaves using Agrobacterium-mediated transformation can be used to mix different genes together to study the catalytic function of multiple enzymes.

[0005] Compared with microbial hosts, metabolic engineering of Nicotiana benthamiana provides an optimal environment for mRNA processing and protein folding, sorting, splicing and localization in plants. The supply of precursors, primary metabolic intermediates and coenzyme factors required to support plant biosynthetic pathways are also very abundant in the plant chassis. CYP450 is an enzyme that catalyzes the formation of most diterpene skeletons. Tobacco can also express the reductase partner required by CYP450. Plant hosts have obvious advantages in the rapid reconstruction of long and complex metabolic pathways.

[0006] The combinatorial expression of genes encoding biosynthetic enzymes in Nicotiana benthamiana is a powerful platform for characterizing gene function and large-scale production of metabolites and recombinant proteins. Elizabeth S. Sattely et al. combinatorially verified the functional genes of the rice husk ketone biosynthesis pathway in rice in Nicotiana benthamiana and reconstructed the complete pathway of husk ketone in Nicotiana benthamiana (the rice husk ketone yield increased by more than 10 times compared with rice). Elizabeth S. Sattely et al. combinatorially verified the genes of the colchicine biosynthesis pathway in Gloriosa glorifolia and reconstructed the biosynthetic pathway of colchicine precursors starting from phenylalanine and tyrosine in Nicotiana benthamiana. Bastien Christ et al. used the Agrobacterium tumefaciens-mediated transient gene expression system to test the function of CYP450 genes in Nicotiana benthamiana, screened the key CYP450 genes for diosgenin synthesis, heterologously expressed the diosgenin biosynthesis pathway in tobacco, and heterologously produced diosgenin. Summary of the invention

[0007] The purpose of the present invention is to synthesize a tanshinone precursor, which is at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood.

[0008] The present invention firstly protects a protein combination, which may include SmKSL1 protein and SmCPS1 protein.

[0009] The protein combination may also include SmCPR1 protein and SmCYP76AH1 protein.

[0010] The protein combination may also include SmCYP76AH3 protein.

[0011] The protein combination may also include SmCYP76AK1 protein.

[0012] Any of the above protein combinations may specifically be K1), K2), K3) or K4).

[0013] K1) is composed of SmKSL1 protein and SmCPS1 protein.

[0014] K2) is composed of SmKSL1 protein, SmCPS1 protein, SmCPR1 protein and SmCYP76AH1 protein.

[0015] K3) is composed of SmKSL1 protein, SmCPS1 protein, SmCPR1 protein, SmCYP76AH1 protein and SmCYP76AH3 protein.

[0016] K4) is composed of SmKSL1 protein, SmCPS1 protein, SmCPR1 protein, SmCYP76AH1 protein, SmCYP76AH3 protein and SmCYP76AK1 protein.

[0017] Any of the above-mentioned SmKSL1 proteins may be any one of A1)-A4):

[0018] A1) the amino acid sequence is the protein shown in SEQ ID NO: 18;

[0019] A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A1);

[0020] A3) a protein having terpene synthase activity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in A1) or A2);

[0021] A4) A protein having an amino acid sequence homology of 80% or more to the protein shown in A1) or A2) and having terpenoid synthase activity.

[0022] In order to facilitate the purification of the protein in A1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 18. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0023] In the protein of A3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0024] The protein in A3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0025] The coding gene of the protein in A3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 17, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag at its 5′ end and / or 3′ end.

[0026] Any of the above-mentioned SmCPS1 proteins may be any one of B1)-B4):

[0027] B1) the amino acid sequence is the protein shown in SEQ ID NO: 20;

[0028] B2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B1);

[0029] B3) A protein having terpene synthase activity obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in B1) or B2);

[0030] B4) A protein having an amino acid sequence homology of 80% or more to the protein shown in B1) or B2) and having terpenoid synthase activity.

[0031] In order to facilitate the purification of the protein in B1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 20. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0032] In the protein of B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0033] The protein in B3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0034] The coding gene of the protein in B3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 19, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag to its 5′ end and / or 3′ end.

[0035] Any of the above-mentioned SmCPR1 proteins may be any one of C1)-C4):

[0036] C1) the amino acid sequence is the protein shown in SEQ ID NO:22;

[0037] C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in C1);

[0038] C3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in C1) or C2) and having CYP450 reductase activity;

[0039] C4) A protein having an amino acid sequence homology of 80% or more to the protein shown in C1) or C2) and having CYP450 reductase activity.

[0040] In order to facilitate the purification of the protein in C1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 22. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0041] In the protein in C3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0042] The protein in C3) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0043] The coding gene of the protein in C3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 21, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag to its 5′ end and / or 3′ end.

[0044] Any of the above-mentioned SmCYP76AH1 proteins may be any one of D1)-D4):

[0045] D1) the amino acid sequence is the protein shown in SEQ ID NO: 24;

[0046] D2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in D1);

[0047] D3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in D1) or D2) and having CYP450 enzyme activity;

[0048] D4) A protein having an amino acid sequence homology of 80% or more to the protein shown in D1) or D2) and having CYP450 enzyme activity.

[0049] In order to facilitate the purification of the protein in D1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 24. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0050] In the protein of D3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0051] The protein in D3) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0052] The coding gene of the protein in the above D3) can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 23, and / or performing missense mutation of one or several base pairs, and / or connecting a coding sequence with a tag at its 5′ end and / or 3′ end.

[0053] Any of the above-mentioned SmCYP76AH3 proteins may be any one of E1)-E4):

[0054] E1) the amino acid sequence is the protein shown in SEQ ID NO: 26;

[0055] E2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in E1);

[0056] E3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in E1) or E2) and having CYP450 enzyme activity;

[0057] E4) A protein having an amino acid sequence homology of 80% or more to the protein shown in E1) or E2) and having CYP450 enzyme activity.

[0058] In order to facilitate the purification of the protein in E1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 26. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0059] In the protein of the above E3), the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0060] The protein in E3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0061] The coding gene of the protein in the above E3) can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 25, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of a tag at its 5′ end and / or 3′ end.

[0062] Any of the above-mentioned SmCYP76AK1 proteins may be any one of F1)-F4):

[0063] F1) the amino acid sequence is the protein shown in SEQ ID NO: 28;

[0064] F2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in F1);

[0065] F3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in F1) or F2) and having CYP450 enzyme activity;

[0066] F4) A protein having an amino acid sequence homology of 80% or more to the protein shown in F1) or F2) and having CYP450 enzyme activity.

[0067] In order to facilitate the purification of the protein in F1), a tag may be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 28. The tag may be a Poly-Arg tag (RRRRR), a FLAG tag (DYKDDDDK), a Strep-tag II tag (WSHPQFEK) or a c-myc tag (EQKLISEEDL).

[0068] In the protein of F3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0069] The protein in F3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0070] The coding gene of the protein in F3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 27, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag at its 5′ end and / or 3′ end.

[0071] Nucleic acid molecules encoding any of the above-mentioned protein combinations also fall within the protection scope of the present invention.

[0072] The nucleic acid molecule encoding any of the above-mentioned SmKSL1 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 17 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 17.

[0073] The nucleic acid molecule encoding any of the above-mentioned SmCPS1 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 19 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 19.

[0074] The nucleic acid molecule encoding any of the above-mentioned SmCPR1 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 21 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 21.

[0075] The nucleic acid molecule encoding any of the above-mentioned SmCYP76AH1 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 23 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 23.

[0076] The nucleic acid molecule encoding any of the above-mentioned SmCYP76AH3 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 25 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 25.

[0077] The nucleic acid molecule encoding any of the above-mentioned SmCYP76AK1 proteins may be a DNA molecule whose coding region is shown in SEQ ID NO: 27 or a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 27.

[0078] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0079] A person skilled in the art can easily mutate the nucleotide sequence encoding any of the above-mentioned proteins (SmKSL1 protein, SmCPS1 protein, SmCPR1 protein, SmCYP76AH1 protein, SmCYP76AH3 protein or SmCYP76AK1 protein) of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher homology with the nucleotide sequence of any of the above-mentioned proteins isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode any of the above-mentioned proteins.

[0080] The term "homology" as used herein refers to sequence similarity to a natural amino acid sequence. "Homology" includes amino acid sequences having 80% or more, or 85% or more, or 90% or more, or 95% or more homology to the amino acid sequence of any of the above-described proteins of the present invention (SmKSL1 protein, SmCPS1 protein, SmCPR1 protein, SmCYP76AH1 protein, SmCYP76AH3 protein or SmCYP76AK1 protein). Homology can be evaluated by the naked eye or by computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0081] The present invention also protects an expression cassette, a recombinant vector, a recombinant cell or a recombinant organism containing any of the above-mentioned nucleic acid molecules.

[0082] The recombinant vector containing any of the above-mentioned nucleic acid molecules can be specifically the recombinant vector pC1300-TP2-P19-SmKSL1, the recombinant vector pC1300-TP2-P19-SmCPS1, the recombinant vector

[0083] pC1300-TP2-P19-SmCPR1, recombinant vector pC1300-TP2-P19-SmCYP76AH1, recombinant vector pC1300-TP2-P19-SmCYP76AH3, recombinant vector pC1300-TP2-P19-SmCYP76AK1, recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3 or recombinant vector

[0084] pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1.

[0085] Any of the above-mentioned recombinant organisms may be recombinant microorganisms. The recombinant microorganism containing any of the above-mentioned nucleic acid molecules may be obtained by transforming a recombinant vector containing any of the above-mentioned nucleic acid molecules into Agrobacterium tumefaciens to obtain a recombinant Agrobacterium. The Agrobacterium tumefaciens may be Agrobacterium tumefaciens GV3101.

[0086] The present invention also protects the use of any of the above-mentioned protein combinations, any of the above-mentioned nucleic acid molecules, or expression boxes, recombinant vectors, recombinant cells or recombinant organisms containing any of the above-mentioned nucleic acid molecules in synthesizing tanshinone precursors.

[0087] In the above application, the tanshinone precursor is at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0088] In the above application, the synthetic tanshinone precursor can use geranylgeranyl pyrophosphate as a substrate.

[0089] Specifically, the present invention protects the use of a protein combination consisting of any of the above-mentioned SmKSL1 proteins and any of the above-mentioned SmCPS1 proteins in the synthesis of at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0090] Specifically, the present invention protects the use of a protein combination consisting of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins and any of the above-mentioned SmCYP76AH1 proteins in the synthesis of at least one of ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0091] Specifically, the present invention protects the use of a protein combination consisting of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins, any of the above-mentioned SmCYP76AH1 proteins, and any of the above-mentioned SmCYP76AH3 proteins in the synthesis of at least one of 11-hydroxyferruginol, 11-hydroxycedarol, 11,20-hydroxyferruginol, and 11,20-hydroxycedarol. Preferably, any of the above-mentioned SmCYP76AH1 proteins and any of the above-mentioned SmCYP76AH3 proteins are co-expressed. That is, the coding gene of any of the above-mentioned SmCYP76AH1 proteins and the coding gene of any of the above-mentioned SmCYP76AH3 proteins are preferably co-expressed.

[0092] Specifically, the present invention protects the use of a protein combination consisting of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins, any of the above-mentioned SmCYP76AH1 proteins, any of the above-mentioned SmCYP76AH3 proteins and any of the above-mentioned SmCYP76AK1 proteins in the synthesis of 11,20-hydroxyferruginol and / or 11,20-hydroxycedarol. Preferably, any of the above-mentioned SmCYP76AH1 proteins, any of the above-mentioned SmCYP76AH3 proteins and any of the above-mentioned SmCYP76AK1 proteins are co-expressed. That is, the coding gene of any of the above-mentioned SmCYP76AH1 proteins, the coding gene of any of the above-mentioned SmCYP76AH3 proteins and the coding gene of any of the above-mentioned SmCYP76AK1 proteins are preferably co-expressed.

[0093] The present invention also protects a method for synthesizing a tanshinone precursor, which may include the following steps:

[0094] (1) increasing the activity and / or expression level of a protein in any of the above-mentioned protein combinations in a starting plant to obtain a transgenic plant; the starting plant can produce geranylgeranyl pyrophosphate;

[0095] (2) Isolating tanshinone precursor from the transgenic plant.

[0096] In the above method, the tanshinone precursor may be at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0097] In the above method, the plant may be tobacco, and the tobacco may specifically be Nicotiana benthamiana.

[0098] In the above, the protein combination composed of any of the above-mentioned SmKSL1 proteins and any of the above-mentioned SmCPS1 proteins can synthesize at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0099] In the above, the protein combination composed of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins and any of the above-mentioned SmCYP76AH1 proteins can synthesize at least one of ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

[0100] In the above, the protein combination consisting of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins, any of the above-mentioned SmCYP76AH1 proteins and any of the above-mentioned SmCYP76AH3 proteins can synthesize at least one of 11-hydroxyferruginol, 11-hydroxycedarol, 11,20-hydroxyferruginol and 11,20-hydroxycedarol. Preferably, any of the above-mentioned SmCYP76AH1 proteins and any of the above-mentioned SmCYP76AH3 proteins are co-expressed. That is, the coding gene of any of the above-mentioned SmCYP76AH1 proteins and the coding gene of any of the above-mentioned SmCYP76AH3 proteins are preferably co-expressed.

[0101] In the above, the protein combination consisting of any of the above-mentioned SmKSL1 proteins, any of the above-mentioned SmCPS1 proteins, any of the above-mentioned SmCPR1 proteins, any of the above-mentioned SmCYP76AH1 proteins, any of the above-mentioned SmCYP76AH3 proteins and any of the above-mentioned SmCYP76AK1 proteins can synthesize 11,20-hydroxyferruginol and / or 11,20-hydroxycedarol. Preferably, any of the above-mentioned SmCYP76AH1 proteins, any of the above-mentioned SmCYP76AH3 proteins and any of the above-mentioned SmCYP76AK1 proteins are co-expressed. That is, the coding gene of any of the above-mentioned SmCYP76AH1 proteins, the coding gene of any of the above-mentioned SmCYP76AH3 proteins and the coding gene of any of the above-mentioned SmCYP76AK1 proteins are preferably co-expressed.

[0102] Tanshinone is the main active ingredient in Danshen, which is highly oxidized in structure, has pharmacological effects such as anti-tumor, anti-liver and lung damage, anti-oxidation, antibacterial and anti-fibrosis, has high pharmacological activity, and is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinone is mainly biosynthesized in the root of Danshen, and the tanshinone biosynthetic pathway is analyzed. The heterologous synthesis of tanshinone in microorganisms or plants has opened up the possibility for the large-scale sustainable generation of tanshinone. The inventors of the present application cloned 5 functional genes (respectively SmKSL1, SmCPS1, SmCYP76AH1, SmCYP76AH3 and SmCYP76AK1, SmKSL1 and SmCPS1 all encode terpenoid synthase, SmCYP76AH1, SmCYP76AH3 and SmCYP76AK1 all encode CYP450 enzymes) and CYP450 reductase (SmCPR1 gene encoding) involved in the upstream pathway of tanshinone biosynthesis from Danshen root. The above genes were further gradually introduced into Nicotiana benthamiana leaves for transient co-expression, and Nicotiana benthamiana leaves were collected. The Nicotiana benthamiana leaf extract was detected and analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (UPLC-QTOF / MS); then the upstream pathway of tanshinone biosynthesis was reconstructed according to the test results. Six tanshinone precursor compounds, including subtanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedar phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedar phenol, were synthesized de novo in Nicotiana benthamiana into which the genes of the tanshinone biosynthesis pathway were introduced. The yields were detected, and the content of subtanshinone diene in freeze-dried tobacco could reach 59.8 mg / g, ferruginol content could reach 43.2 mg / g, 11-hydroxycedar phenol content could reach 29.8 mg / g, 11-hydroxyferruginol content could reach 33.4 mg / g, and 11,20-hydroxyferruginol content could reach 10.7 mg / g. The invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 It is the upstream pathway for the biosynthesis of tanshinone.

[0104] Figure 2 Schematic diagram of the TP2 expression cassette module structure of the pC1300-TP2-P19 vector.

[0105] Figure 3 GC-MS analysis of two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein) transiently co-expressed in tobacco.

[0106] Figure 4 This is a GC-MS analysis of two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), one CYP450 enzyme (i.e., SmCYP76AH1 protein) and one CYP450 reductase (i.e., SmCPR1 protein) transiently co-expressed in tobacco.

[0107] Figure 5 UPLC-QTOF / MS analysis of two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), two CYP450 enzymes (i.e., SmCYP76AH1 protein and SmCYP76AH3 protein) and one CYP450 reductase (i.e., SmCPR1 protein) transiently co-expressed in tobacco.

[0108] Figure 6 UPLC-QTOF / MS analysis of two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), three CYP450 enzymes (i.e., SmCYP76AH1 protein, SmCYP76AH3 protein and SmCYP76AK1 protein) and one CYP450 reductase (i.e., SmCPR1 protein) transiently co-expressed in tobacco.

[0109] Figure 7 GC-MS and UPLC-QTOF / MS analysis before and after optimization of tobacco transient expression system.

[0110] Figure 8 Reconstructed upstream pathway of tanshinone biosynthesis in Nicotiana benthamiana. DETAILED DESCRIPTION

[0111] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0112] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0113] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0114] The Salvia miltiorrhiza Bunge. plant was collected from the Henan Academy of Agricultural Sciences in September 2020. It is a cultivated biennial plant. After collection, it was washed with clean water, and the roots of the Salvia miltiorrhiza plant (i.e., the Salvia miltiorrhiza root sample) were quick-frozen with liquid nitrogen and then frozen at -80°C.

[0115] The cultivation conditions of Nicotiana benthamiana are as follows: temperature 25°C, relative humidity 70%, and alternating light and dark cultivation (16 h light / 8 h dark).

[0116] Escherichia coli DH5α competent cells were a product of Beijing Quanshijin Biotechnology Co., Ltd.

[0117] Agrobacterium tumefaciens GV3101 is a product of Beijing Qingke Technology Co., Ltd.

[0118] In the following examples, the GC-MS detection method is as follows:

[0119] Gas phase: Agilent HP-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm) was used; programmed temperature (initial temperature 50 ° C, maintained for 2 min; heated to 80 ° C at 7.0 ° C / min; heated to 300 ° C at 50.0 ° C / min, maintained for 10 min; injection port temperature was 280 ° C; carrier gas was high-purity helium (99.999%); carrier gas flow rate was 1.0 mL / min; split ratio was 50:1; solvent delay was 3 min;

[0120] Mass spectrometry: EI ion source; transfer line temperature of 300°C; ion source temperature of 300°C; electron energy of 70 eV; multiplier voltage of 1.2 kV; scanning range m / z: 50-500.

[0121] In the following examples, the UPLC-QTOF / MS detection method is as follows:

[0122] UPLC-QTOF / MS chromatographic conditions were as follows: chromatographic column was BEH column (2.1 mm×100 mm, 2.7 μm), mobile phase: phase A was 0.1% formic acid-water, phase B was 100% acetonitrile; liquid phase conditions were: 0-10 min: 80%-40% A, 10-15 min: 40%-2% A, 15-17 min: 2% A, 17-18 min: 2%-80% A, 18-20 min: 80% A. The injection volume was 3 μL, the column temperature was 40°C, and the mobile phase flow rate was 0.4 mL / min.

[0123] Mass spectrometry conditions: electrospray ionization (ESI) source, scanning and collecting MS data in positive and negative ion modes; scanning range: m / z: 50-800.

[0124] The names of the primers and their nucleotide sequences involved in the following examples are shown in Table 1.

[0125] Table 1

[0126]

[0127]

[0128] Example

[0129] 1. Experimental steps and methods

[0130] (I) Gene cloning and vector construction

[0131] 1. Take a sample of Salvia miltiorrhiza root stored at -80°C, grind it with liquid nitrogen using a ball mill, and then use an RNA rapid extraction kit (a product of Beijing Huayueyang Biotechnology Co., Ltd., product catalog number 0416-50) to extract RNA to obtain total RNA from the Salvia miltiorrhiza root.

[0132] 2. Use reverse transcriptase to reverse transcribe the total RNA of Salvia miltiorrhiza root to obtain cDNA of Salvia miltiorrhiza root.

[0133] 3. Using cDNA from Salvia miltiorrhiza root as template, high-fidelity enzyme ( KD Plus PCR SuperMix) and a primer pair consisting of primer KSL1-SpeⅠ-F and primer KSL1-SpeⅠ-R were used for PCR amplification, and then a PCR amplification product of about 1788 bp containing the SmKSL1 gene was recovered using a gel recovery kit (HiPure Gel Pure DNAMini Kit).

[0134] The nucleotide sequence of the SmKSL1 gene is shown in SEQ ID NO: 17. The SmKSL1 gene encodes the SmKSL1 protein. The amino acid sequence of the SmKSL1 protein is shown in SEQ ID NO: 18.

[0135] 4. Digest the pC1300-TP2-P19 vector with restriction endonuclease Spe I, and use a gel recovery kit to recover the approximately 13334 kb digested vector backbone. Use a seamless splicing enzyme ( The recombinant vector pC1300-TP2-P19-SmKSL1 was obtained by ligating with the Seamless Cloning and Assembly Kit and then transforming into Escherichia coli DH5α competent cells.

[0136] The pC1300-TP2-P19 vector is a recombinant plasmid obtained by replacing the DNA fragment between the restriction endonucleases EcoRI and SalI of the vector pCAMBIA1300 (a product of Abcam, with a product catalog number of ab275754) with the target DNA fragment shown in the nucleotide sequence of SEQ ID NO:29 from the 7th to 4408th position from the 5' end, and the other nucleotide sequences remain unchanged. The target DNA fragment includes P19 module (i.e., P19 expression cassette), T1 module (i.e., pCMV-MCS1-3×HA-tOCS expression cassette), T2 module (i.e., pSUPER-MCS2-3×FLAG-tNOS expression cassette) and T3 module (i.e., p35S-MCS3-3×MYC-tRBCS expression cassette) from the 5' end to the 3' end.

[0137] The P19 module includes the MAS promoter, P19 (RNA silencing suppressor 19KDa protein) gene and MAS terminator from the 3' end to the 5' end.

[0138] The pCMV-MCS-3×HA-tOCS expression cassette includes CMV promoter, multiple cloning site 1 (MCS1), HA tag and OCS terminator from the 5' end to the 3' end.

[0139] The pSUPER-MCS-3×FLAG-tNOS expression cassette includes SUPER promoter, multiple cloning site 2 (MCS2), FLAG tag and NOS terminator from the 3' end to the 5' end.

[0140] The p35S-MCS-3×MYC-tRBCS expression cassette includes 35S promoter, multiple cloning site 3 (MCS3), MYC tag and RBCS terminator from the 5' end to the 3' end.

[0141] The schematic diagram of the TP2 expression cassette module structure in the pC1300-TP2-P19 vector is shown in Figure 2 The nucleotide sequence of the TP2 Expression Module is shown in SEQ ID NO:29.

[0142] The recombinant vector pC1300-TP2-P19-SmKSL1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmKSL1 was a recombinant plasmid obtained by inserting the SmKSL1 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0143] 5. According to the above steps 3 and 4, the primer KSL1-SpeⅠ-F was replaced with CPS1-SpeⅠ-F, and the primer KSL1-SpeⅠ-R was replaced with CPS1-SpeⅠ-R. The other steps remained unchanged to obtain the recombinant vector pC1300-TP2-P19-SmCPS1. The recombinant vector pC1300-TP2-P19-SmCPS1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that pC1300-TP2-P19-SmCPS1 was a recombinant plasmid obtained by inserting the SmCPS1 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0144] The nucleotide sequence of the SmCPS1 gene is shown in SEQ ID NO: 19. The SmCPS1 gene encodes the SmCPS1 protein. The amino acid sequence of the SmCPS1 protein is shown in SEQ ID NO: 20.

[0145] The SmKSL1 protein is the kaurene synthase SmKSL1, and the SmCPS1 protein is the copalyl pyrophosphate synthase SmCPS1. Both the kaurene synthase SmKSL1 and the copalyl pyrophosphate synthase SmCPS1 are terpene synthases.

[0146] 6. According to the above steps 3 and 4, the primer KSL1-SpeⅠ-F was replaced with CPR1-SpeⅠ-F, and the primer KSL1-SpeⅠ-R was replaced with CPR1-SpeⅠ-R. The other steps remained unchanged to obtain the recombinant vector pC1300-TP2-P19-SmCPR1. The recombinant vector pC1300-TP2-P19-SmCPR1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmCPR1 was a recombinant plasmid obtained by inserting the SmCPR1 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0147] The nucleotide sequence of the SmCPR1 gene is shown in SEQ ID NO: 21. The SmCPR1 gene encodes the SmCPR1 protein, which is a CYP450 reductase. The amino acid sequence of the SmCPR1 protein is shown in SEQ ID NO: 22.

[0148] 7. According to the above steps 3 and 4, replace the primer KSL1-SpeⅠ-F with CYP76AH1-SpeⅠ-F, and replace the primer KSL1-SpeⅠ-R with CYP76AH1-SpeⅠ-R. The other steps remain unchanged to obtain the recombinant vector.

[0149] pC1300-TP2-P19-SmCYP76AH1. The recombinant vector pC1300-TP2-P19-SmCYP76AH1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmCYP76AH1 was a recombinant plasmid obtained by inserting the SmCYP76AH1 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0150] The nucleotide sequence of the SmCYP76AH1 gene is shown in SEQ ID NO:23.

[0151] The SmCYP76AH1 gene encodes the SmCYP76AH1 protein. The amino acid sequence of the SmCYP76AH1 protein is shown in SEQ ID NO:24.

[0152] 8. According to the above steps 3 and 4, replace the primer KSL1-SpeⅠ-F with CYP76AH3-SpeⅠ-F, and replace the primer KSL1-SpeⅠ-R with CYP76AH3-SpeⅠ-R. The other steps remain unchanged to obtain the recombinant vector.

[0153] pC1300-TP2-P19-SmCYP76AH3. The recombinant vector pC1300-TP2-P19-SmCYP76AH3 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmCYP76AH3 was a recombinant plasmid obtained by inserting the SmCYP76AH3 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0154] The nucleotide sequence of the SmCYP76AH3 gene is shown in SEQ ID NO:25.

[0155] The SmCYP76AH3 gene encodes the SmCYP76AH3 protein. The amino acid sequence of the SmCYP76AH3 protein is shown in SEQ ID NO:26.

[0156] 9. According to the above steps 3 and 4, replace the primer KSL1-SpeⅠ-F with CYP76AK1-SpeⅠ-F, and replace the primer KSL1-SpeⅠ-R with CYP76AK1-SpeⅠ-R. The other steps remain unchanged to obtain the recombinant vector

[0157] pC1300-TP2-P19-SmCYP76AK1.

[0158] The recombinant vector pC1300-TP2-P19-SmCYP76AK1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmCYP76AK1 was a recombinant plasmid obtained by inserting the SmCYP76AK1 gene into the restriction endonuclease Spe I of the pC1300-TP2-P19 vector.

[0159] The nucleotide sequence of the SmCYP76AK1 gene is shown in SEQ ID NO:27.

[0160] The SmCYP76AK1 gene encodes the SmCYP76AK1 protein. The amino acid sequence of the SmCYP76AK1 protein is shown in SEQ ID NO:28.

[0161] SmCYP76AH1 protein, SmCYP76AH3 protein and SmCYP76AK1 protein are all CYP450 enzymes.

[0162] 10. Construction of the recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3

[0163] (1) Using cDNA of Salvia miltiorrhiza root as template, PCR amplification was performed using high-fidelity enzyme and a primer pair consisting of primer CYP76AH1-StuⅠ-F and primer CYP76AH1-StuⅠ-R. A PCR amplification product 1 of about 1488 bp containing the SmCYP76AH1 gene was recovered using a gel recovery kit.

[0164] (2) Using the cDNA of Salvia miltiorrhiza root as a template, PCR amplification was performed using a high-fidelity enzyme and a primer pair consisting of primer CYP76AH3-SpeⅠ-F and primer CYP76AH3-SpeⅠ-R. A PCR amplification product 2 of approximately 1485 bp containing the SmCYP76AH3 gene was recovered using a gel recovery kit.

[0165] (3) The pC1300-TP2-P19 vector was digested with restriction endonuclease Stu I, and the approximately 13334 kb digested vector backbone 1 was recovered using a gel recovery kit. The digested vector backbone 1 and the PCR amplification product 1 recovered in step (1) were ligated using a seamless splicing enzyme, and then transformed into Escherichia coli DH5α competent cells to obtain an intermediate vector.

[0166] (4) The intermediate vector was digested with restriction endonuclease Spe I, and the digested vector skeleton 2 was recovered using a gel recovery kit. The digested vector skeleton 2 and the PCR amplification product 2 recovered in step (2) were connected using a seamless splicing enzyme, and then transformed into Escherichia coli DH5α competent cells to obtain the recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3.

[0167] The recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3 was a recombinant plasmid containing both CYP76AH1 and CYP76AH3 genes by inserting the SmCYP76AH1 gene into the pC1300-TP2-P19 vector with restriction endonuclease StuⅠ and inserting the SmCYP76AH3 gene into the restriction endonuclease SpeI.

[0168] 11. Construction of recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1

[0169] (1) Using cDNA of Salvia miltiorrhiza root as template, PCR amplification was performed using high-fidelity enzyme and a primer pair consisting of primer CYP76AH1-StuⅠ-F and primer CYP76AH1-StuⅠ-R. A PCR amplification product 1 of about 1488 bp containing the SmCYP76AH1 gene was recovered using a gel recovery kit.

[0170] (2) Using the cDNA of Salvia miltiorrhiza root as a template, PCR amplification was performed using a high-fidelity enzyme and a primer pair consisting of primer CYP76AH3-SpeⅠ-F and primer CYP76AH3-SpeⅠ-R. A PCR amplification product 2 of approximately 1485 bp containing the SmCYP76AH3 gene was recovered using a gel recovery kit.

[0171] (3) Using the cDNA of Salvia miltiorrhiza root as a template, PCR amplification was performed using a high-fidelity enzyme and a primer pair consisting of primer CYP76AK1-KpnⅠ-F and primer CYP76AK1-KpnⅠ-R. A PCR amplification product of approximately 1512 bp containing the SmCYP76AK1 gene was recovered using a gel recovery kit.

[0172] (4) The pC1300-TP2-P19 vector was digested with restriction endonuclease Stu I, and the approximately 13334 kb digested vector backbone 1 was recovered using a gel recovery kit. The digested vector backbone 1 and the PCR amplification product 1 recovered in step (1) were ligated using a seamless splicing enzyme, and then transformed into E. coli DH5α competent cells to obtain an intermediate vector 1.

[0173] (5) The intermediate vector 1 is digested with restriction endonuclease Spe I, and the digested vector skeleton 2 is recovered using a gel recovery kit. The digested vector skeleton 2 and the PCR amplification product 2 recovered in step (2) are connected using a seamless splicing enzyme, and then transformed into Escherichia coli DH5α competent cells to obtain the intermediate vector 2.

[0174] (6) The intermediate vector 2 was digested with restriction endonuclease KpnⅠ, and the digested vector skeleton 3 was recovered using a gel recovery kit. The digested vector skeleton 3 and the PCR amplification product 3 recovered in step (2) were connected using a seamless splicing enzyme, and then transformed into Escherichia coli DH5α competent cells to obtain the recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1.

[0175] The recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the recombinant vector

[0176] pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1 is obtained by inserting the SmCYP76AH1 gene into the restriction endonuclease StuⅠ of the pC1300-TP2-P19 vector, inserting the SmCYP76AH3 gene into the restriction endonuclease Spe I, and inserting the SmCYP76AK1 gene into the restriction endonuclease KpnⅠ, thereby obtaining a recombinant plasmid containing the CYP76AH1, CYP76AH3 and CYP76AK1 genes at the same time.

[0177] (II) Obtaining transient co-expression of Nicotiana benthamiana

[0178] 1. The plasmids to be tested (recombinant vector pC1300-TP2-P19-SmKSL1, recombinant vector pC1300-TP2-P19-SmCPS1, recombinant vector pC1300-TP2-P19-SmCPR1, recombinant vector pC1300-TP2-P19-SmCYP76AH1, recombinant vector pC1300-TP2-P19-SmCYP76AH3, recombinant vector pC1300-TP2-P19-SmCYP76AK1, recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3, recombinant vector

[0179] pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1 or pC1300-TP2-P19 vector) was transformed into Agrobacterium tumefaciens GV3101 competent cells using the freeze-thaw transformation method to obtain recombinant Agrobacterium.

[0180] 2. Inoculate a single colony of recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmKSL1 into 1 mL of YEB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture at 28°C and 220 rpm for 24 h to obtain culture solution 1. Then transfer culture solution 1 to 30 mL of YEB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin at a ratio of 1:100, and culture at 28°C and 200 rpm with shaking until an OD of 0.05 is obtained. 600nm The culture solution 2 was about 0.5. The culture solution 2 was centrifuged at room temperature and 5000 rpm for 10 minutes to collect the bacteria. Then, an equal volume of MMA impregnation solution (solute and concentration were 10 mM MgCl2, 10 mM MES and 200 μM acetosyringone, solvent was water, pH value was 5.6) was used to resuspend and obtain OD 600nm The resuspension was placed on a shaker at 28°C and 50 rpm for 1 to 2 hours to obtain the KSL1 infection solution.

[0181] According to the above steps, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmKSL1 was replaced with the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCPS1, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCPR1, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCYP76AH1, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCYP76AH3, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCYP76AK1, and the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCPS1. The recombinant Agrobacterium transformed with SmCYP76AH1-SmCYP76AH3, the recombinant Agrobacterium transformed with the recombinant vector pC1300-TP2-P19-SmCYP76AH1-SmCYP76AH3-SmCYP76AK1, and the recombinant Agrobacterium transformed with the pC1300-TP2-P19 vector, while the other steps remain unchanged, successively obtain CPS1 infection fluid, CPR1 infection fluid, CYP76AH1 infection fluid, CYP76AH3 infection fluid, CYP76AK1 infection fluid, CYP76AH1-CYP76AH3 infection fluid, CYP76AH1-CYP76AH3-CYP76AK1 infection fluid and pC1300-TP2-P19 infection fluid.

[0182] 3. Take healthy Nicotiana benthamiana plants that have grown to 4-5 weeks of age, select Nicotiana benthamiana leaves in their prime and inject the target infection solution for transient expression to obtain transiently expressed Nicotiana benthamiana. The specific steps are as follows: water the Nicotiana benthamiana plants one day before the injection, and inject directly during the day the next day; during the injection, use a 1mL syringe with the needle removed to gently poke the back of the Nicotiana benthamiana leaves, and draw 1ml of the target infection solution each time from the leaf wound and inject it into the Nicotiana benthamiana leaves until the leaves are completely infected. Each Nicotiana benthamiana plant is injected with 3 leaves, and each target infection solution is injected into 3 Nicotiana benthamiana plants, and cultured at room temperature for 4-5 days to obtain transiently co-expressed Nicotiana benthamiana.

[0183] The target infection solution is a single infection solution or a mixed infection solution.

[0184] The single infection solutions were KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, CYP76AH1 infection solution, CYP76AH3 infection solution, CYP76AK1 infection solution and pC1300-TP2-P19 infection solution.

[0185] The mixed infection solution is made by mixing equal volumes of each single infection solution.

[0186] The mixed infection solution 1 is a mixture of equal volumes of KSL1 infection solution and CPS1 infection solution.

[0187] The mixed infection solution 2 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution and CYP76AH1 infection solution.

[0188] The mixed infection solution 3 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, CYP76AH1 infection solution and CYP76AH3 infection solution.

[0189] The mixed infection solution 4 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, CYP76AH1 infection solution, CYP76AH3 infection solution and CYP76AK1 infection solution.

[0190] The mixed infection solution 5 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CYP76AH1 infection solution, CYP76AH3 infection solution and CYP76AK1 infection solution.

[0191] The mixed infection solution 6 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution and CYP76AH1-CYP76AH3 infection solution.

[0192] The mixed infection solution 7 is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, and CYP76AH1-CYP76AH3-CYP76AK1 infection solution.

[0193] (III) Preparation of standard curve

[0194] 1. Drawing of the standard curve of tanshinone diene

[0195] Ethyl acetate was used to dilute tanshinone diene to obtain a series of reference solutions with mass concentrations of 0.17, 0.34, 3.4, 6.8, 13.6, 27.2, and 54.4 mg / L, respectively, and GC-MS injection was used for detection. Regression processing was performed with the peak area as the ordinate and the mass concentration as the abscissa to draw a standard curve.

[0196] 2. Drawing of the standard curve of ferric rust alcohol

[0197] Ferrocarbol was diluted with ethyl acetate to obtain a series of reference solutions with mass concentrations of 0.18, 0.36, 3.6, 7.2, 14.3, 28.6, and 57.2 mg / L, respectively, and tested by GC-MS injection. Regression processing was performed with the peak area as the ordinate and the mass concentration as the abscissa to draw a standard curve.

[0198] 3. Drawing of the standard curve of 11-hydroxyferruginol

[0199] Methanol was used to dilute 11-hydroxyferruginol, and cedarwood was used as the internal standard to obtain a series of reference solutions with mass concentrations of 2.5, 5, 10, 12.5, 50 and 100 μM, respectively, and UPLC-QTOF / MS was used for sample injection and detection. Regression processing was performed with the peak area as the ordinate and the mass concentration as the abscissa to draw a standard curve.

[0200] 4. Drawing of the standard curve of 11-hydroxycedarol

[0201] Methanol was used to dilute 11-hydroxycedarwood phenol, and carnosic acid was used as the internal standard to obtain a series of reference solutions with mass concentrations of 2.5, 5, 10, 12.5, 25, and 50 μM, respectively. UPLC-QTOF / MS was used for sample injection and detection. Regression processing was performed with the peak area as the ordinate and the mass concentration as the abscissa to draw a standard curve.

[0202] 5. Drawing of the standard curve of 11,20-hydroxyferruginol

[0203] Methanol was used to dilute 11,20-hydroxyferruginol, and carnosic acid was used as the internal standard to obtain a series of reference solutions with mass concentrations of 10, 12.5, 25, 50, 100, and 200 μM, respectively, and UPLC-QTOF / MS was used for sample injection and detection. Regression processing was performed with the peak area as the ordinate and the mass concentration as the abscissa to draw a standard curve.

[0204] (IV) Detection of chemical components in leaves of transiently co-expressed Nicotiana benthamiana

[0205] 1. Cut the leaves of the transiently co-expressed Nicotiana benthamiana obtained in step (ii), freeze them with liquid nitrogen, and then freeze-dry them in a freeze dryer to obtain freeze-dried Nicotiana benthamiana leaves. Then, grind the freeze-dried Nicotiana benthamiana leaves into powder using a ball mill to obtain Nicotiana benthamiana leaf powder.

[0206] 2. Chemical component extraction and detection

[0207] The chemical components in Nicotiana benthamiana leaf powder were extracted using ethyl acetate extraction method. The details are as follows:

[0208] (1) Add 5 mL of chromatographically pure ethyl acetate to 200 mg of Nicotiana benthamiana leaf powder, vortex for 5 min, sonicate for 30 min, centrifuge at 5000 rpm for 5 min, collect the supernatant into a new EP tube, and blow the supernatant to dryness using a nitrogen blower;

[0209] (2) After completing step (1), add 5 mL of chromatographically pure ethyl acetate to the EP tube, vortex for 5 min, sonicate for 30 min, centrifuge at 5000 rpm for 5 min, collect the supernatant into a new EP tube, and blow the supernatant to dryness with nitrogen using a nitrogen blower;

[0210] (3) After completing step (2), add 500 μL of ethyl acetate or methanol to the EP tube for re-dissolution, centrifuge at 12000 rpm for 30 min, collect about 100 μL of supernatant into a liquid phase vial and dilute it 2 times with ethyl acetate or methanol, and then use GC-MS or UPLC-QTOF / MS for detection. GC-MS is used to detect tanshinone diene and ferruginol. UPLC-QTOF / MS is used to detect 11-hydroxyferruginol, 11-hydroxycedar phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedar phenol.

[0211] 3. Use the standard curve method to calculate the content of the target compound in Nicotiana benthamiana leaves. Specifically, substitute the peak area into the corresponding standard curve to obtain the corresponding mass concentration.

[0212] 2. Experimental Results and Conclusions

[0213] The MVA pathway exists in Nicotiana benthamiana and can produce sufficient GGPP compounds. In order to evaluate the applicability of Nicotiana benthamiana as a plant platform for de novo biosynthesis of tanshinone precursor compounds, Agrobacterium-mediated transient expression of tanshinone biosynthesis pathway genes was performed in Nicotiana benthamiana leaves.

[0214] (I) Expression of terpene synthase in Nicotiana benthamiana

[0215] The mixed infection solution 1, which was a mixture of equal volumes of KSL1 infection solution and CPS1 infection solution, was injected into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were freeze-dried and chemical components were extracted. The results were tested by GC-MS. Figure 3 As shown (KSL1+CPS1 are Nicotiana benthamiana leaves injected with mixed infection solution 1, the control group are Nicotiana benthamiana leaves injected with pC1300-TP2-P19 infection solution, and the standard is tanshinone diene).

[0216] The results showed that a new peak 1 with a molecular weight of 272.28 could be detected in the leaves of Nicotiana benthamiana injected with mixed infection solution 1 at a retention time of 11.08 min, which was consistent with the retention time and mass spectrometry fragmentation rules of the standard substance tanshinone diene; tanshinone diene, a rosinane-type diterpene skeleton compound, could be detected in the leaves of Nicotiana benthamiana injected with two terpene synthases, while tanshinone diene was not detected in the leaves of Nicotiana benthamiana injected with pC1300-TP2-P19 infection solution in the control group. To detect the content of tanshinone diene, a standard curve was established as y=112309x-104692 (R=0.9993); further calculations showed that the maximum content of tanshinone diene in Nicotiana benthamiana leaves was 59.8 mg / g.

[0217] The introduction of two terpene synthases from Salvia miltiorrhiza into Nicotiana benthamiana leaves can cyclize the GGPP produced by Nicotiana benthamiana itself to form the rosinane-type diterpene skeleton compound tanshinone diene, providing the possibility for the de novo synthesis of tanshinone precursors and tanshinone compounds as well as other rosinane-type diterpene compounds in Nicotiana benthamiana.

[0218] (II) Expression of CYP450 enzymes in Nicotiana benthamiana

[0219] 1. Use the mixed infection solution 2 made by mixing equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution and CYP76AH1 infection solution to inject into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were cut, freeze-dried and chemical components were extracted. GC-MS was used for detection. The results are as follows Figure 4 As shown (KSL1+CPS1+AH1+CPR1 are Nicotiana benthamiana leaves injected with mixed infection solution 2, the control group is Nicotiana benthamiana leaves injected with pC1300-TP2-P19 infection solution, and the standard is ferruvic alcohol).

[0220] The results showed that a new peak 2 with a molecular weight of 286.27 could be detected in the leaves of Nicotiana benthamiana injected with mixed infection solution 2 at a retention time of 11.68 min, which was consistent with the retention time and mass spectrometry fragmentation pattern of the standard substance ferruginol. No ferruginol was detected in the leaves of Nicotiana benthamiana injected with pC1300-TP2-P19 infection solution in the control group. To detect the yield of ferruginol, the ferruginol standard curve was established as y=48310556.22x+51887.60 (R=0.9998); further calculations showed that the maximum ferruginol content in freeze-dried Nicotiana benthamiana leaves was 43.2 mg / g.

[0221] Two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), one CYP450 enzyme (i.e., SmCYP76AH1 protein) and one CYP450 reductase (i.e., SmCPR1 protein) derived from Salvia miltiorrhiza were introduced into the leaves of Nicotiana benthamiana. The SmCYP76AH1 protein can further oxidatively modify the abietane-type diterpene skeleton compound tanshinone diene produced by the cyclization of Nicotiana benthamiana's own GGPP compound, and synthesize the oxidation product of tanshinone diene, ferruginol, in the leaves of Nicotiana benthamiana.

[0222] 2. The mixed infection solution 3, which is a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, CYP76AH1 infection solution and CYP76AH3 infection solution, was injected into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were cut, freeze-dried and chemical components were extracted. UPLC-QTOF / MS was used for detection. The UPLC-QTOF / MS negative ion detection results are shown in FIG. Figure 5(KSL1+CPS1+CPR1+AH1+AH3 are Nicotiana benthamiana leaves injected with mixed infection solution 3, the control group is Nicotiana benthamiana leaves injected with pC1300-TP2-P19 infection solution, the standard substance in A is 11-hydroxycedarwood phenol, and the standard substance in B is 11-hydroxyferruginol) as shown.

[0223] The results showed that a new peak 3 was detected at a retention time of 10.58 min in the leaves of Nicotiana benthamiana injected with mixed infection solution 3, with a molecular weight of 315.1992 ([MH] - ), the retention time and mass spectrometry fragmentation pattern of the standard 11-hydroxycedar phenol were consistent. No 11-hydroxycedar phenol was detected in the leaves of Nicotiana benthamiana injected with the pC1300-TP2-P19 infection solution in the control group; a new peak 4 was detected at a retention time of 14.08 min in the leaves of Nicotiana benthamiana injected with the mixed infection solution 3, with a molecular weight of 301.2171 ([MH] - ), the retention time and mass spectrometry fragmentation pattern were consistent with those of the standard 11-hydroxyferruginol. No 11-hydroxyferruginol was detected in the leaves of Nicotiana benthamiana injected with the pC1300-TP2-P19 infection solution in the control group.

[0224] Two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), two CYP450 enzymes (i.e., SmCYP76AH1 protein and SmCYP76AH3 protein) and one CYP450 reductase (i.e., SmCPR1 protein) derived from Salvia miltiorrhiza were introduced into the leaves of Nicotiana benthamiana. CYP76AH3 further catalyzed the C-11 hydroxylation and C-6 carbonylation of ferruginol to form 11-hydroxyferruginol and 11-hydroxycedarol, respectively.

[0225] 3. A mixed infection solution 4 consisting of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution, CYP76AH1 infection solution, CYP76AH3 infection solution and CYP76AK1 infection solution was injected into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were freeze-dried and chemical components were extracted. UPLC-QTOF / MS was used for detection. The UPLC-QTOF / MS negative ion detection results were as follows: Figure 6 (KSL1+CPS1+CPR1+AH1+AH3+AK1 are Nicotiana benthamiana leaves injected with mixed infection solution 4, the control group is Nicotiana benthamiana leaves injected with pC1300-TP2-P19 infection solution, the standard substance in A is 11,20-hydroxycedarwood phenol, and the standard substance in B is 11,20-hydroxyferruginol) as shown.

[0226] The results showed that a new peak 5 was detected at a retention time of 9.57 min in the leaves of Nicotiana benthamiana injected with mixed infection solution 4, with a molecular weight of 331.1942 ([MH]- ), the retention time and mass spectrometry fragmentation pattern of the standard 11,20-hydroxycedar phenol were consistent. No 11,20-hydroxycedar phenol was detected in the leaves of Nicotiana benthamiana injected with the pC1300-TP2-P19 infection solution in the control group; a new peak 6 was detected at a retention time of 12.99 min in the leaves of Nicotiana benthamiana injected with the mixed infection solution 4, with a molecular weight of 317.2127 ([MH] - ), which was consistent with the retention time and mass spectrometry fragmentation pattern of the standard 11,20-hydroxyferruginol. No 11,20-hydroxyferruginol was detected in the leaves of Nicotiana benthamiana injected with the pC1300-TP2-P19 infection solution in the control group.

[0227] Two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein), three CYP450 enzymes (i.e., SmCYP76AH1 protein, SmCYP76AH3 protein and SmCYP76AK1 protein) and one CYP450 reductase (i.e., SmCPR1 protein) derived from Salvia miltiorrhiza were introduced into the leaves of Nicotiana benthamiana. The CYP76AK1 protein catalyzed the C-20 hydroxylation of 11-hydroxyferruginol and 11-hydroxycedarol to produce 11,20-hydroxyferruginol and 11,20-hydroxycedarol, respectively.

[0228] (III) Optimization of the transient expression system of Nicotiana benthamiana

[0229] As the number of tanshinone pathway genes increases, the yield of the target product decreases. Two terpene synthases (i.e., SmKSL1 protein and SmCPS1 protein) and three CYP450 enzymes (i.e., SmCYP76AH1 protein, SmCYP76AH3 protein, and SmCYP76AK1 protein) from Salvia miltiorrhiza were transferred into Nicotiana benthamiana leaves. The leaves of Nicotiana benthamiana were infected with a mixed infection solution of equal volumes of KSL1 infection solution, CPS1 infection solution, CYP76AH1 infection solution, CYP76AH3 infection solution, and CYP76AK1 infection solution. After 5 days of infection, the chemical components were extracted and detected by GC-MS. The results are shown in Figure 5. Figure 7 As shown in the figure (before optimization, KSL1+CPS1+AH1+AH3+AK1 were leaves of Nicotiana benthamiana injected with mixed infection solution 5, peak 1 was tanshinone diene, and peak 2 was ferruginol), it was found that the intermediate products tanshinone diene and ferruginol accumulated in large quantities. It was speculated that with the increase of pathway genes, the pathway was too long, so that the CYP450 enzymes in the downstream pathway did not efficiently perform their catalytic functions. In order to increase the yield of the target product, multiple CYP450 genes were constructed on the same pC1300-TP2-P19 vector to ensure that the CYP450 enzymes could be effectively expressed.

[0230] 1. First, the two CYP450 genes CYP76AH1 and CYP76AH3 in the mixed infection solution 3 were constructed on the same pC1300-TP2-P19 vector to obtain the recombinant expression vector pC1300-TP2-P19-CYP76AH1-CYP76AH3, which was transformed into Agrobacterium tumefaciens GV3101 to obtain the CYP76AH1-CYP76AH3 infection solution. The mixed infection solution 6, which was a mixture of equal volumes of KSL1 infection solution, CPS1 infection solution, CPR1 infection solution and CYP76AH1-CYP76AH3 infection solution, was injected into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were collected, freeze-dried, and chemical components were extracted and detected using UPLC-QTOF / MS.

[0231] The results showed that high levels of 11-hydroxycedar phenol and 11-hydroxyferruginol could be detected in tobacco leaves. The yield was detected by the standard curve method: the standard curve of 11-hydroxycedar phenol was y=0.079x+0.006 (R=0.9969), and 5 batches of freeze-dried Nicotiana benthamiana leaves were detected, and the highest content of 11-hydroxycedar phenol in freeze-dried Nicotiana benthamiana leaves was calculated to be 29.8 mg / g; the standard curve of 11-hydroxyferruginol was y=0.0171x-0.0173 (R=0.9982), and 5 batches of freeze-dried Nicotiana benthamiana leaves were detected, and the highest content of 11-hydroxyferruginol in freeze-dried Nicotiana benthamiana leaves was calculated to be 33.4 mg / g.

[0232] 2. The three CYP450 genes CYP76AH1, CYP76AH3 and CYP76AK1 in the mixed infection solution 4 were constructed on the same pC1300-TP2-P19 vector to obtain a recombinant expression vector

[0233] pC1300-TP2-P19-CYP76AH1-CYP76AH3-CYP76AK1 was transformed into Agrobacterium tumefaciens GV3101 to obtain CYP76AH1-CYP76AH3-CYP76AK1 infection fluid. The mixed infection fluid 7, which was mixed with equal volumes of KSL1 infection fluid, CPS1 infection fluid, CPR1 infection fluid and CYP76AH1-CYP76AH3-CYP76AK1 infection fluid, was injected into Nicotiana benthamiana leaves for transient co-expression. After 5 days of co-expression, the Nicotiana benthamiana leaves were freeze-dried and chemical components were extracted, and then detected by GC-MS and UPLC-QTOF / MS.

[0234] Test results such as Figure 7(After optimization, KSL1+CPS1+CPR1+AH1-AH3-AK1 is a leaf of Nicotiana benthamiana injected with mixed infection solution 7, peak 3 is 11-hydroxycedar phenol, peak 4 is 11,20-hydroxycedar phenol, peak 5 is 11-hydroxyferruginol, and peak 6 is 11,20-hydroxyferruginol). The results show that in the optimized transient expression system of Nicotiana benthamiana, subtanshinone diene and ferruginol are basically completely converted, that is, the precursor compounds are basically converted into the final product. The yield of 11,20-hydroxyferruginol was detected, and the standard curve of 11,20-hydroxyferruginol was established as y=0.033x+0.002 (R=0.9956); 5 batches of freeze-dried Nicotiana benthamiana leaves were tested, and the highest content of 11,20-hydroxyferruginol in freeze-dried Nicotiana benthamiana leaves was calculated to be 10.7 mg / g.

[0235] It can be seen that the present application reconstructed the upstream pathway of tanshinone biosynthesis in Nicotiana benthamiana by gradually introducing genes of the upstream pathway of tanshinone biosynthesis into Nicotiana benthamiana leaves for transient co-expression (see Figure 8 ), six tanshinone precursors, including tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol, can be synthesized from scratch in Nicotiana benthamiana, the tanshinone biosynthetic pathway was analyzed, and the functional genes of the tanshinone biosynthetic pathway were identified to provide a Nicotiana benthamiana chassis platform.

[0236] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Protein combination, including SmKSL1 protein and SmCPS1 protein; The SmKSL1 protein is any one of A1)-A4): A1) the amino acid sequence is the protein shown in SEQ ID NO: 18; A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A1); A3) a protein having terpene synthase activity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in A1) or A2); A4) a protein having an amino acid sequence homology of 80% or more to the protein shown in A1) or A2) and having terpenoid synthase activity; The SmCPS1 protein is any one of B1)-B4): B1) the amino acid sequence is the protein shown in SEQ ID NO: 20; B2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in B1); B3) A protein having terpene synthase activity obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in B1) or B2); B4) A protein having an amino acid sequence homology of 80% or more to the protein shown in B1) or B2) and having terpenoid synthase activity.

2. The protein combination according to claim 1, characterized in that: The protein combination also includes SmCPR1 protein and SmCYP76AH1 protein; The SmCPR1 protein is any one of C1)-C4): C1) the amino acid sequence is the protein shown in SEQ ID NO:22; C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in C1); C3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in C1) or C2) and having CYP450 reductase activity; C4) a protein having an amino acid sequence homology of 80% or more to the protein shown in C1) or C2) and having CYP450 reductase activity; The SmCYP76AH1 protein is any one of D1)-D4): D1) the amino acid sequence is the protein shown in SEQ ID NO: 24; D2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in D1); D3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in D1) or D2) and having CYP450 enzyme activity; D4) A protein having an amino acid sequence homology of 80% or more to the protein shown in D1) or D2) and having CYP450 enzyme activity.

3. The protein combination according to claim 2, characterized in that: The protein combination also includes SmCYP76AH3 protein; The SmCYP76AH3 protein is any one of E1)-E4): E1) the amino acid sequence is the protein shown in SEQ ID NO: 26; E2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in E1); E3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in E1) or E2) and having CYP450 enzyme activity; E4) A protein having an amino acid sequence homology of 80% or more to the protein shown in E1) or E2) and having CYP450 enzyme activity.

4. The protein combination according to claim 3, characterized in that: The protein combination also includes SmCYP76AK1 protein; The SmCYP76AK1 protein is any one of F1)-F4): F1) the amino acid sequence is the protein shown in SEQ ID NO: 28; F2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in F1); F3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in F1) or F2) and having CYP450 enzyme activity; F4) A protein having an amino acid sequence homology of 80% or more to the protein shown in F1) or F2) and having CYP450 enzyme activity.

5. The protein combination according to any one of claims 1 to 4, characterized in that: The protein combination is K1), K2), K3) or K4): K1) consists of the SmKSL1 protein described in claim 1 and the SmCPS1 protein described in claim 1; K2) consisting of the SmKSL1 protein of claim 1, the SmCPS1 protein of claim 1, the SmCPR1 protein of claim 2, and the SmCYP76AH1 protein of claim 2; K3) consisting of the SmKSL1 protein of claim 1, the SmCPS1 protein of claim 1, the SmCPR1 protein of claim 2, the SmCYP76AH1 protein of claim 2, and the SmCYP76AH3 protein of claim 3; K4) consists of the SmKSL1 protein described in claim 1, the SmCPS1 protein described in claim 1, the SmCPR1 protein described in claim 2, the SmCYP76AH1 protein described in claim 2, the SmCYP76AH3 protein described in claim 3 and the SmCYP76AK1 protein described in claim 4.

6. A nucleic acid molecule encoding the protein combination according to any one of claims 1 to 5.

7. An expression cassette, a recombinant vector, a recombinant cell or a recombinant organism comprising the nucleic acid molecule of claim 6.

8. Use of the protein combination according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, or an expression cassette, a recombinant vector, a recombinant cell or a recombinant organism containing the nucleic acid molecule according to claim 6 in synthesizing a tanshinone precursor.

9. The use according to claim 8, characterized in that: The tanshinone precursor is at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

10. A method for synthesizing a tanshinone precursor, comprising the following steps: (1) increasing the activity and / or expression level of a protein in the protein combination according to any one of claims 1 to 5 in a starting plant to obtain a transgenic plant; the starting plant can produce geranylgeranyl pyrophosphate; (2) isolating tanshinone precursors from the transgenic plants; Preferably, the tanshinone precursor is at least one of tanshinone diene, ferruginol, 11-hydroxyferruginol, 11-hydroxycedarwood phenol, 11,20-hydroxyferruginol and 11,20-hydroxycedarwood phenol.

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