Application of an amino acid sequence and a nucleotide sequence in the synthesis of anisodine

Abstract

The present invention discloses an application of an amino acid sequence and a nucleotide sequence in synthesizing anisodine, and relates to the field of biotechnology. The amino acid sequence is shown in SEQ ID NO.2, or the sequence shown in SEQ ID NO.2 is substituted, mutated or deleted by several amino acid residues and encodes an amino acid sequence with the same function, which is the amino acid sequence of anisodine synthase, and the enzyme is used to catalyze the synthesis of anisodine. The nucleotide sequence is shown in SEQ ID NO.1, or the sequence shown in SEQ ID NO.1 is substituted, mutated or deleted by several nucleotides and encodes a nucleotide sequence with the same function, which is used to encode the anisodine synthase. The present invention first discovered an amino acid sequence for synthesizing anisodine synthase, verified the function of anisodine synthase in synthesizing anisodine by tobacco transient expression experiments, and further verified its function by prokaryotic expression in vitro enzyme activity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of an amino acid sequence and a nucleotide sequence in synthesizing anisodine. Background Art

[0002] Anisodine is one of the medicinal tropane alkaloids. Its chemical structure is similar to that of atropine and scopolamine. It is a neurotropic anticholinergic drug. It is clinically used to treat cerebral vascular occlusion, vascular headache, retinal vasospasm, central retinopathy, Parkinson's disease, motion sickness, organophosphorus pesticide poisoning, and has good effects in traditional Chinese medicine anesthesia. Because of its high medicinal value and small side effects on the human body, the demand for anisodine as a basic drug with multifunctional uses is still growing rapidly. At present, anisodine is still completely extracted from the root of the Solanaceae medicinal plant Anisodus tanguticus (Maxim.) Pascher. Anisodendron tanguticum is the main commercially cultivated medicinal plant for the production of anisodine, which is included and specified in the "Chinese Pharmacopoeia". Because the growth of Anisodendron tanguticum is restricted by the environment and the content of anisodine in the plant is low, it is difficult to obtain, resulting in tight supply and demand and high prices, which seriously hinders the development and application of related drugs.

[0003] Anisodine is an atropine-type anticholinergic neurological drug. From the perspective of chemical configuration, its structure is an ester formed by the condensation of scopolamine and hydroxyhyoscyamine. α-Ketoglutarate / Fe(II)-dependent dioxygenases (2-ODDs, also known as 2-OGDs) are a class of mononuclear iron atom proteins that do not contain heme and are widely distributed in animals, plants, and microorganisms in nature. 2-ODD and cytochrome P450 oxygenases (CYPs) are the two most abundant oxygenases in nature that catalyze reactions, but 2-ODD only uses two or three ligands to chelate Fe(II). Therefore, compared with CYP450 (ferrous ions are chelated by four porphyrins), 2-ODD has higher flexibility, and the reactions it catalyzes are more diverse and complex. It plays an important role in the regulation of plant and animal growth and development, transcriptional regulation, nucleotide modification or nucleic acid repair, fatty acid metabolism, and the biosynthesis of some secondary metabolites with medicinal value. Studies have found that 2-ODD is involved in the biosynthesis of scopolamine, and hyoscyamine 6β-hydroxylase (H6H) catalyzes the epoxidation of scopolamine at the 6 and 7 positions to form scopolamine. However, so far, the enzyme / gene that catalyzes the synthesis of anisodine has not been reported. The gene encoding anisodine synthase AS is necessary for the production of anisodine by biosynthesis. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention provides an application of an amino acid sequence and a nucleotide sequence in the synthesis of anisodine.

[0005] One of the purposes is to provide an application of an amino acid sequence in the synthesis of anisodine, wherein the amino acid sequence is as shown in SEQ ID NO.2, or the sequence shown in SEQ ID NO.2 is substituted, mutated or deleted with several amino acid residues and encodes an amino acid sequence with the same function, which is the amino acid sequence of anisodine synthase, and the enzyme is used to catalyze the synthesis of anisodine.

[0006] The second purpose is to provide an application of a nucleotide sequence in the synthesis of anisodine, wherein the nucleotide sequence is as shown in SEQ ID NO.1, or the sequence shown in SEQ ID NO.1 is substituted, mutated or deleted with several nucleotides and encodes a nucleotide sequence with the same function, which is used to encode the anisodine synthase.

[0007] The beneficial effects of the present invention are as follows: the present invention has discovered for the first time an amino acid sequence that can be used to synthesize anisodine synthase, the content of anisodine is highest in the root, and it is speculated that the gene is highly expressed in the root. The function of the anisodine synthase in synthesizing anisodine has been verified by a tobacco transient expression experiment, and its function has been further verified by prokaryotic expression in vitro enzyme activity, which is of great significance for the production of anisodine by biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the expression profile analysis diagram of anisodine in various tissues of Anisodine;

[0009] Figure 2 This is the Western blot analysis of AS recombinant protein;

[0010] Figure 3 The in vitro enzyme activity LC-MS ion current diagram of the embodiment;

[0011] Figure 4 A is a graph showing the change of target product with reaction time, B is a graph showing the test of the optimum reaction temperature, and C is a graph showing the test of the optimum reaction pH.

[0012] Figure 5 The figure shows the content of anisodine in the hairy roots of the control and the AS-expression-interfered plants.

[0013] Figure 6 The graph shows the content of anisodine in the hairy roots of control and overexpressed AS.

[0014] Figure 7 The LC-MS extracted ion chromatogram of anisodine in transient expression of AS in tobacco;

[0015] Figure 8 This is the LC-MS secondary mass spectrum of anisodine in transiently expressed AS in tobacco. DETAILED DESCRIPTION

[0016] Example 1 Cloning and functional identification of anisodine synthase AS

[0017] (1) Genomic analysis to screen for potential candidate genes for α-ketoglutarate / Fe(II)-dependent dioxygenases

[0018] 2-ODDs are widely distributed in nature, in animals, plants and microorganisms, and participate in the regulation of plant and animal growth and development, transcriptional regulation, nucleotide modification or nucleic acid repair, fatty acid metabolism and the biosynthesis of some secondary metabolites with medicinal value. The corresponding Hidden Markov Model (HMM) accession number in the Pfam database is PF03171. Using the PF03171 model, 175 family member genes were identified in the genome of Anisodora. The above family member genes were clustered by tissue expression heat map analysis, and a 2-ODDs family gene highly expressed in the root was obtained. It has a high correlation with the expression of enzyme genes in the tropane alkaloid synthesis pathway, and it is speculated that it may be related to the synthesis of anisodine, and it is named anisodine synthase AS.

[0019] From the cluster analysis of the expression heat map of Anisodactyla tissue, another result was obtained, such as Figure 1 As shown, anisodine was highly expressed in the taproot.

[0020] (2) Cloning of anisodine synthase AS

[0021] Take an appropriate amount of fibrous root tissue of Anisodus scoparia, grind it in liquid nitrogen, add it to a 1.5 mL Eppendorf (EP) centrifuge tube containing lysis solution, shake it thoroughly, and extract RNA using the plant total RNA extraction kit DP419 of Tiangen Biochemical Technology (Beijing) Co., Ltd.; determine the RNA concentration on a spectrophotometer; use the extracted total RNA as a template to synthesize cDNA according to the instructions of the FastKing cDNA first-strand synthesis kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.; design a pair of gene-specific primers to amplify the AS gene from the total cDNA of Anisodus scoparia, and the primer sequences are as follows:

[0022] AS-F: 5'-atggccgaaactctcacagttttca-3'

[0023] AS-R: 5'-tcatatcattgctgcttctattggtcttctcc-3'.

[0024] Using the cDNA of Scoparia scoparia as a template, PCR amplification was performed using primers specific to the gene coding region, and the size of the PCR product was confirmed by agarose electrophoresis. The target band was recovered and sequenced, and the cDNA sequence of AS was successfully obtained. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0025] (3) Construction of prokaryotic vector and protein purification

[0026] The gene sequence is shown in SEQ ID NO: 1. A pair of primers with BamHI and HindIII restriction sites were used to amplify the gene sequence and inserted into plasmid pET28a to obtain pET28a-AS. The primers are as follows:

[0027] pET28a-AS-F: 5'-aatgggtcgcggatccatggccgaaactctcacagtttt-3'

[0028] pET28a-AS-R: 5'-gtgcggccgcaagctttatcattgctgcttctattggt-3';

[0029] Then, the plasmid pET28a-AS was transformed into competent Escherichia coli BL21 (DE3) to obtain the prokaryotic expression strain BL21-pET28a-AS; it was inoculated into LB liquid culture medium and cultured at 37°C with shaking until OD600 was 0.5; IPTG with a final concentration of 0.15 mM was added and cultured at 16°C with shaking for 16 h to induce the expression of the target protein; and HisPur-Ni-NTA resin (ThermoFisher Scientific) was used to purify the 6×His-tagged AS recombinant protein.

[0030] (4) Western blot detection of prokaryotic expressed proteins

[0031] The protein can be directly used for Western blot analysis. The specific steps of Western blot are as follows:

[0032] 1) Add 5× SDS loading buffer to the supernatant sample obtained in the previous step, and separate the sample by 12% SDS-PAGE;

[0033] 2) When the electrophoresis is about to end, cut a PVDF membrane of appropriate size, activate it with anhydrous methanol for 15 seconds, wash it twice with ultrapure water, and soak it in ultrapure water for 2-3 minutes. At the same time, prepare filter paper of similar size to the PVDF membrane;

[0034] 3) Wash the gel twice with ultrapure water and soak it in ultrapure water for 2 minutes;

[0035] 4) Immerse the gel, filter paper and PVDF membrane in transfer buffer;

[0036] 5) Transfer the protein to the PVDF membrane by wet transfer, and use constant current mode for transfer, with the current set to 200 mA and the time for 90 min;

[0037] 6) The PVDF membrane was washed once with ultrapure water and blocked with blocking solution (5% skim milk powder dissolved in TBST buffer) for 1 h;

[0038] 7) HA Tag Mouse Monoclonal antibody was diluted 3000 times with blocking solution and incubated on PVDF membrane for 1 hour, and then washed 3 times with TBST, each time for 5 minutes;

[0039] 8) Incubate with horseradish peroxidase-crosslinked goat anti-mouse IgG (diluted 2000 times in blocking buffer) secondary antibody for 1 hour, and wash with TBST buffer three times, 5 minutes each time;

[0040] 9) Slowly add an appropriate amount of ECL chemiluminescent substrate to the PVDF membrane, incubate at room temperature for 3-5 minutes, expose with X-ray film, and finally fix and develop with an automatic X-ray film processor (such as Figure 2 ).

[0041] (5) In vitro enzyme activity LC-MS detection

[0042] The enzyme activity reaction system (total volume 50μl) contained 20μg purified AS, 1mM α-ketoglutaric acid, 4mM ascorbic acid, 0.4mM ferrous ion and 50mM Tris-HCl (pH 7.6); heat-inactivated AS was used as a negative control; the reaction was allowed to proceed for 1h. An equal volume of methanol was added to the reaction system to terminate the reaction. After mixing, the mixture was centrifuged at 12000rpm. The supernatant was filtered and the product was analyzed by LC-MS (ThermalOrbitrap 120). The column temperature was set at 35°C, the flow rate was 0.3mL / min, the injection volume was 2μL, the mass spectrometer used an electrospray ionization source (ESI), and the ion mode was positive ion mode. The results of enzyme activity analysis showed that AS could catalyze scopolamine to produce the product anisodine (such as Figure 3 ), with a retention time of 3.08 min, which is consistent with that of the anisodine standard. No corresponding product was detected in the negative control.

[0043] (6) Optimal reaction conditions

[0044] The enzyme activity reaction system (total volume 50 μl) contained 20 μg purified AS, 1 mM α-ketoglutaric acid, 4 mM ascorbic acid, 0.4 mM ferrous ion and 50 mM Tris-HCl (pH 7.6). The reaction was performed for 5 min, 15 min, 30 min, 1 h, 2 h, 3 h and 6 h, respectively. The amount of the target product anisodine increased with time (e.g. Figure 4 A).

[0045] The enzyme activity reaction system (total volume 50 μl) contained 20 μg purified AS, 1 mM α-ketoglutarate, 4 mM ascorbic acid, 0.4 mM ferrous ion and 50 mM Tris-HCl (pH 7.6), and the reaction was carried out at 4 °C, 10 °C, 16 °C, 25 °C, 30 °C, 37 °C, 42 °C and 55 °C for 30 min, respectively, and the optimal reaction temperature was 16 °C (e.g. Figure 4 B).

[0046] The enzyme activity reaction system (total volume 50 μl) contained 20 μg purified AS, 1 mM α-ketoglutaric acid, 4 mM ascorbic acid, and 0.4 mM ferrous ion, which were reacted at 30 °C for 30 min in Na2HPO4-citrate buffer (pH 5-6), Na2HPO4-NaH2PO4 buffer (pH 6-7.5), Tris-HCL buffer (pH7.5-9), and Glycine-NaOH buffer (pH 9-10), respectively, to obtain the optimal pH of 7.5, and Tris-HCL buffer (e.g. Figure 4 C).

[0047] Example 2: AS interference with hairy root acquisition and molecular detection

[0048] In order to evaluate whether interfering with AS in plants affects the changes in the content of anisodine in Scots dasyphylla, this study constructed an interference vector and conducted plant metabolic engineering research using the hairy roots of Scots dasyphylla.

[0049] (1) Construction of interference vector

[0050] 1) Amplify the AS-specific 275 bp fragment using a high-fidelity polymerase, use XbaI and HindIII to digest the interference intermediate vector pHANNIBAL, purify the digestion product and PCR product using a kit, and recombinant the PCR fragment into pHANNIBAL using a seamless cloning method;

[0051] RNAi-AS-HindIII-F1:5'-cgaaatcgataagctttacaaataaaagtccaaccatcctctctc-3';

[0052] RNAi-AS-Xbal-R1:5'-aaagcaggactctagagttgtccggcaggggatg-3';

[0053] 2) The recombinant product was transformed into competent E. coli DH5α, positive clones were screened, and sequencing analysis was performed to obtain the intermediate vector pHANNIBAL-PPRi-F1 connected to an interference fragment;

[0054] 3) Use XhoI and KpnI to digest pHANNIBAL-PPRi-F1 and AS interference fragment PCR products, purify the digestion products with a kit, and then connect them with T4 DNA ligase;

[0055] RNAi-AS-XhoI-F2:5'-ggcctcgaggttgtccggcaggggatg-3';

[0056] RNAi-AS-KpnI-R2:5'-ggcggtacctacaaataaaagtccaaccatcctc-3';

[0057] 4) The ligation product was transformed into competent E. coli DH5α, positive clones were screened, and sequencing analysis was performed to obtain the intermediate vector pHANNIBAL-ASi-F1+F2 connecting the two interference fragments;

[0058] 5) The pBin19 vector was digested with endonucleases XbaI and SacI, and the intermediate vector pHANNIBAL-ASi-F1+F2 was digested with restriction endonucleases SpeI and SacI. The digestion products were purified using a kit, and the purified DNA molecules were ligated with T4 DNA ligase. The ligation products were transformed into competent Escherichia coli DH5α, positive clones were screened, and sequencing analysis was performed to obtain the plant expression binary vector pBin19-AS-RNAi for AS interference.

[0059] (2) AS interferes with hairy root acquisition

[0060] All operations were performed under sterile conditions and the cultures were placed in a dark room at 25°C. The specific methods are as follows:

[0061] 1) The AS interference vector was used to transform the hairy root Agrobacterium C58C1 to obtain the engineered strain C58C1-pBin19-AS-RNAi for hairy root transformation, and the C58C1 strain transformed with pBin19 was used as a plasmid control;

[0062] 2) The engineered bacteria were cultured in a liquid YEP culture medium containing the corresponding antibiotics in a shaking incubator at 28°C;

[0063] 3) Collect the cells at 4000 rpm at room temperature, resuspend them in transformation solution (MS liquid medium containing 100 μM acetosyringone), and place them at room temperature for later use;

[0064] 4) Take sterile leaves of Scots daphne seedlings and cut them into about 1 mm size. Use a blade to make a small wound on the leaves. Put the cut leaves into the resuspended bacterial solution and immerse them for 8-10 minutes. Use absorbent paper to absorb the bacterial solution on the surface of the leaves as much as possible.

[0065] 5) Place the leaves on MS solid medium containing 100 μM acetosyringone and co-cultivate for 48 h;

[0066] 6) After the co-cultivation, the leaves were transferred to MS solid screening medium containing 400 mg / L cephalothin and the medium was changed once a week until there was no Agrobacterium in the hairy roots;

[0067] 7) Completely sterilized hairy roots can be cultured on MS solid medium for a long time;

[0068] 8) Inoculate 5 hairy roots of about 5 cm in length into 100 mL of liquid MS medium. Culture 3-5 biological replicates for each hairy root strain and culture at 25°C on a shaker at 110 rpm;

[0069] 9) After 30 days of cultivation, the hairy roots were harvested and 100 mg of fresh material was used for genomic DNA and total RNA extraction. The remaining materials were all placed in tin foil bags, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0070] (3) AS interferes with LC-MS detection of anisodine content in hairy roots

[0071] After grinding the dried leaves, accurately weigh 20 mg, add 1 mL of 30% methanol and 1 / 1000 formic acid aqueous solution, place the sample on a horizontal shaker at 28°C for extraction for 3 hours, and use the supernatant for metabolite analysis after centrifugation. Take the supernatant and filter it, and analyze the product with LC-MS (ThermalOrbitrap 120). Use a Waters Symmetry C18 column (2.1×100mm, 3.5μm) to separate the samples, set the column temperature to 35°C, the flow rate to 0.3mL / min, the injection volume to 1μL, and the mass spectrometer detector to use an electrospray ionization source (ESI), and the ion mode to positive ion mode. In the hairy roots of AS-RNAi scopolamine, the content of anisodine was significantly lower than that of the control group (such as Figure 5 ).

[0072] Example 3. Acquisition of AS-overexpressing hairy roots and detection of anisodine content

[0073] (1) Construction of AS overexpression vector

[0074] The AS coding region was amplified using primers BglII-AS-F and BstEII-AS-R, and the vector p1305.1 was double-digested with BglII and BstEII. The PCR fragment was recombined into p1305.1 using the seamless cloning method. After sequencing, the plasmid was named p1305.1-AS.

[0075] BglII-AS-F:5'-tgaccatggtagatctatggccgaaactctcacagttttca-3'

[0076] BstEII-AS-R:5'-attcgagctggtcacctcatatcattgctgcttctattggtc-3'.

[0077] (2) Obtaining hairy roots by AS overexpression

[0078] All operations were performed under sterile conditions and the cultures were placed in a dark room at 25°C. The specific methods are as follows:

[0079] 1) The AS overexpression vector was used to transform the hairy root Agrobacterium C58C1 to obtain the engineered strain C58C1-p1305.1-AS for hairy root transformation, and the C58C1 strain transformed with p1305.1 was used as a plasmid control;

[0080] 2) The engineered bacteria were cultured in a liquid YEP culture medium containing the corresponding antibiotics in a shaking incubator at 28°C;

[0081] 3) Collect the cells at 4000 rpm at room temperature, resuspend them in transformation solution (MS liquid medium containing 100 μM acetosyringone), and place them at room temperature for later use;

[0082] 4) Take sterile leaves of Scots daphne seedlings and cut them into about 1 mm size. Use a blade to make a small wound on the leaves. Put the cut leaves into the resuspended bacterial solution and immerse them for 8-10 minutes. Use absorbent paper to absorb the bacterial solution on the surface of the leaves as much as possible.

[0083] 5) Place the leaves on MS solid medium containing 100 μM acetosyringone and co-cultivate for 48 h;

[0084] 6) After the co-cultivation, the leaves were transferred to MS solid screening medium containing 400 mg / L cephalothin and the medium was changed once a week until there was no Agrobacterium in the hairy roots;

[0085] 7) Completely sterilized hairy roots can be cultured on MS solid medium for a long time;

[0086] 8) Inoculate 5 hairy roots of about 5 cm in length into 100 mL of liquid MS medium. Culture 3-5 biological replicates for each hairy root strain and culture at 25°C on a shaker at 110 rpm;

[0087] 9) After 30 days of cultivation, the hairy roots were harvested and about 100 mg of fresh material was used for genomic DNA and total RNA extraction. The remaining materials were all placed in tin foil bags, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0088] (3) LC-MS detection of anisodine content in hairy roots with AS overexpression

[0089] After grinding the dried leaves, accurately weigh 20 mg, add 1 mL of 30% methanol and 1 / 1000 formic acid aqueous solution, place the sample on a horizontal shaker at 28°C for extraction for 3 hours, and use the supernatant for metabolite analysis after centrifugation. Take the supernatant and filter it, and analyze the product with LC-MS (ThermalOrbitrap 120). Use a Waters Symmetry C18 column (2.1×100mm, 3.5μm) to separate the samples, set the column temperature to 35°C, the flow rate to 0.3mL / min, the injection volume to 1μL, and the mass spectrometer uses an electrospray ionization source (ESI) in the positive ion mode. In AS-overexpressing hairy roots, the content of anisodine increased significantly compared with the control (such as Figure 6 ).

[0090] Example 4: Synthesis of anisodine in tobacco

[0091] In order to evaluate the function of AS in the biosynthesis of anisodine and its application in plant synthetic biology, the AS gene was transiently transformed into the leaves of Nicotiana benthamiana to analyze whether it has the ability to synthesize anisodine. According to the multiple cloning site of the tobacco transient expression vector PEAQ-HT, upstream and downstream primers with AgeI and XhoI restriction endonuclease cleavage sites were designed. The specific primers are as follows:

[0092] PEAQ-DO2-F: 5'-caaattcgcgaccggtatggccgaaactctcacagttttca-3';

[0093] PEAQ-DO2-R: 5'-agttaaaggcctcgagtcatatcattgctgcttctattggtcttctcc-3'.

[0094] After PCR amplification, the PEAQ-HT plasmid was double-digested and recovered and then connected. After successful sequencing, the recombinant plasmid PEAQ-DO2 was finally obtained. Agrobacterium GV3101 was transformed by freeze-thaw method to obtain the PEAQ-DO2 transient expression engineering strain (GV3101-PEAQ-DO2), and (GV3101-PEAQ) was used as an empty vector control. After the two engineering bacteria were expanded and cultured separately, the bacteria were resuspended in buffer (10mM MES, pH 5.7, 10mM MgCl2 and 150μM AS), OD600 was adjusted to 0.6, and placed at room temperature for 3h before being used for tobacco injection. Tobacco injected with only buffer was used as a negative control. After the tobacco was placed in a weak light environment for 48h, the substrates were injected respectively: hyoscyamine, anisodamine, and scopolamine. All were dissolved in water to a final concentration of 1 mM. After the substrate was injected, the tobacco leaves were harvested after continued cultivation for 24 hours and freeze-dried for subsequent analysis. After the dried leaves were ground and crushed, 20 mg was accurately weighed, 1 mL of 30% methanol and 1 / 1000 formic acid aqueous solution were added, and the sample was placed on a horizontal shaker at 28 ° C for extraction for 3 hours. After centrifugation, the supernatant was used for metabolite analysis. The supernatant was filtered and the product was analyzed by LC-MS (ThermalOrbitrap 120). The samples were separated using a Waters Symmetry C18 column (2.1×100mm, 3.5μm), the column temperature was set at 35 ° C, the flow rate was 0.3mL / min, the injection volume was 1μL, the mass spectrometer used an electrospray ionization source (ESI), and the ion mode was positive ion mode. LC-MS analyzed the corresponding metabolites in tobacco leaf cells, and the retention time of the anisodine standard was 3.08min. No anisodine was detected in tobacco leaves with no load as control; anisodine was also not detected in tobacco leaf cells with hyoscyamine and anisodamine as substrates; anisodine was detected in tobacco leaf cells with scopolamine as substrate, with a retention time of 3.04 min ( Figure 7 ), which is completely consistent with the retention time of anisodine standard, and the secondary mass spectrometry results are completely consistent with the standard ( Figure 8 ), indicating that expressing AS in tobacco leaves and supplementing the substrate scopolamine can synthesize anisodine.

Claims

1. An application of anisodine synthase in synthesizing anisodine, characterized in that: The amino acid sequence of the anisodine synthase is shown in SEQ ID NO.2, and the enzyme is used for catalyzing the synthesis of anisodine.

2. An application of anisodine synthase in synthesizing anisodine, characterized in that: The nucleotide sequence of the anisodine synthase is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: Used to synthesize anisodine in E. coli expression system.

4. The use according to claim 1 or 2, characterized in that: Used for metabolic engineering improvement of scopolamine or tobacco to synthesize anisodine.

5. The use according to claim 2, characterized in that Used to express anisodine synthase through tobacco transient expression technology to synthesize anisodine.

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