CsANS1, a gene encoding anthocyanin synthase, and its applications

Abstract

This invention discloses a gene CsANS1 encoding anthocyanin synthase and its applications, belonging to the field of biotechnology. The nucleotide sequence of the gene CsANS1 is shown in SEQ ID NO.1. The gene CsANS1 or related biological materials can be used to synthesize anthocyanins and quercetin in vitro. This invention verifies through recombinant engineered bacteria that the anthocyanin synthase CsANS1 not only has the activity of catalyzing the conversion of colorless anthocyanins to anthocyanins, but also has a strong catalytic activity of catalyzing the conversion of quercetin, effectively overcoming the limitations of existing technologies in the production of anthocyanins and quercetin. The bifunctional catalytic properties of the gene CsANS1 broaden the application prospects of ANS enzymes in metabolic engineering and help promote the development and utilization of natural active ingredients in the fields of food, medicine, and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a gene CsANS1 that encodes anthocyanin synthase and its applications. Background Technology

[0002] Anthocyanins and quercetin, as important representatives of flavonoids, have wide applications in the food, health product, and pharmaceutical fields. They not only give plants rich colors but also possess various biological activities such as antioxidant, anti-inflammatory, and anti-tumor effects. Currently, the main source of anthocyanins and quercetin still relies on plant extraction, but this method is limited by factors such as plant resources, season, and extraction efficiency, resulting in high costs and limited yields. While chemical synthesis can achieve large-scale production, it involves cumbersome steps, produces numerous byproducts, causes significant environmental pollution, and makes it difficult to guarantee the natural configuration and biological activity of the products. Therefore, developing an efficient, green, and sustainable biosynthetic method is of great significance.

[0003] In plants, anthocyanin biosynthesis depends on the flavonoid metabolic pathway, with anthocyanin synthase (ANS) being a key enzyme catalyzing the conversion of colorless anthocyanins into colored anthocyanins. The ANS gene has been cloned and functionally validated in various plants such as rice, Arabidopsis thaliana, and strawberry, and its expression is positively correlated with anthocyanin accumulation. However, whether the ANS gene in tea (Camellia sinensis) possesses multi-substrate catalytic capabilities, and whether it can simultaneously and efficiently synthesize anthocyanins and quercetin, remains unclear. Current research on the function of the tea ANS gene is insufficient, lacking systematic validation of its role in quercetin synthesis, which limits its application in metabolic engineering.

[0004] Furthermore, current research on the production of anthocyanins or quercetin using engineered microorganisms is mostly focused on single products, lacking efficient enzyme resources and supporting processes capable of simultaneously synthesizing multiple flavonoid compounds. Therefore, cloning and identifying the tea plant ANS gene with bifunctional catalytic activity, constructing an efficient expression system, and achieving the synergistic biosynthesis of anthocyanins and quercetin have significant application prospects for promoting the green manufacturing of flavonoid compounds. Summary of the Invention

[0005] The purpose of this invention is to provide a gene encoding anthocyanin synthase, CsANS1, and its applications to address the problems existing in the prior art. This invention verifies through engineered bacteria that the anthocyanin synthase CsANS1 not only possesses activity in catalyzing the conversion of colorless anthocyanins into anthocyanins, but also exhibits strong activity in catalyzing the conversion of quercetin, effectively overcoming the limitations of existing technologies in the production of anthocyanins and quercetin. The bifunctional catalytic properties of the CsANS1 gene broaden the application prospects of ANS enzymes in metabolic engineering, contributing to the development and utilization of natural active ingredients in the food, pharmaceutical, and cosmetic fields.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of a gene CsANS1 encoding anthocyanin synthase or related biological materials in the in vitro synthesis of anthocyanins and quercetin, wherein the nucleotide sequence of the gene CsANS1 is shown in SEQ ID NO.1.

[0008] Optionally, the relevant biological materials include the protein encoded by the gene CsANS1, a recombinant vector containing the gene CsANS1, and a recombinant microorganism containing the gene CsANS1.

[0009] Optionally, the protein encoded by the gene CsANS1 has the amino acid sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a method for in vitro synthesis of anthocyanins and quercetin, comprising the steps of using colorless anthocyanins as substrates and catalyzing the production of anthocyanins and quercetin by anthocyanin synthase under α-ketoglutarate conditions.

[0011] The amino acid sequence of the anthocyanin synthase is shown in SEQ ID NO.2.

[0012] Optionally, the anthocyanin synthase is induced to express by a recombinant bacterium containing the gene CsANS1; the nucleotide sequence of the gene CsANS1 is shown in SEQ ID NO.1.

[0013] The present invention also provides a gene CsANS1 encoding anthocyanin synthase, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0014] The present invention also provides an anthocyanin synthase encoded by the gene CsANS1.

[0015] The present invention also provides a recombinant expression vector containing the gene CsANS1.

[0016] The present invention also provides a recombinant microorganism containing the recombinant expression vector.

[0017] The present invention discloses the following technical effects:

[0018] This invention isolates a gene CsANS1 encoding anthocyanin synthase from fresh tea leaves. By constructing a recombinant engineered bacterium containing this gene, it is verified that the anthocyanin synthase CsANS1 expressed by the engineered bacterium not only has the activity of catalyzing the production of anthocyanins from colorless anthocyanins, but also has a strong activity of catalyzing the production of quercetin.

[0019] This invention, through the application of the CsANS1 gene and related biomaterials, effectively solves the problems of resource constraints, cumbersome procedures, significant environmental pollution, and difficulty in guaranteeing the natural conformation of products associated with the production of anthocyanins and quercetin, which rely on plant extraction or chemical synthesis. It overcomes the limitations of traditional methods and lays a solid foundation for the large-scale production of flavonoids. Simultaneously, the bifunctional catalytic properties of the CsANS1 gene broaden the application prospects of ANS enzymes in metabolic engineering, contributing to the development and utilization of natural active ingredients in the food, pharmaceutical, and cosmetic fields. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 SDS-PAGE protein electrophoresis analysis of recombinant engineered bacterial culture containing the CsANS1 gene; lane 1 shows the protein electrophoresis results of the bacterial culture before induction of expression; lane 2 shows the protein electrophoresis results of the bacterial culture after induction of expression; lane 3 shows the protein electrophoresis results of the supernatant after induction of expression and cell disruption; lane 4 shows the protein electrophoresis results of the supernatant after cell disruption and purification.

[0022] Figure 2 The images show the UPLC detection results of the enzyme-catalyzed reaction products. The broken lines from top to bottom represent the detection results of quercetin standard at 360 nm wavelength, the detection results of the sample under test at 360 nm wavelength, the detection results of cyanidin standard at 540 nm wavelength, and the detection results of the sample under test at 540 nm wavelength.

[0023] Figure 3 The image shows the UPLC-MS / MS detection results of the enzyme-catalyzed reaction products; where A is the characteristic peak at a specific mass-to-charge ratio (303 / 153); B is the characteristic peak at a specific mass-to-charge ratio (287 / 231); and C is the characteristic peak of the anthocyanin standard. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The anthocyanin synthase gene in this invention is the tea plant gene encoding α-ketoglutarate-dependent anthocyanin synthase, named CsANS1, and its nucleotide sequence is as follows:

[0030]

[0031] The amino acid sequence of the protein encoded by the CsANS1 gene is as follows:

[0032] MVATVAGIRVESLASSGLESIPKEYIRPQEELTSIGNVFEEEKKEEGPQVPTVDLKDLVAEDKEVRERCREALKKAATEWGVMHLVNHGIPDELMERVKAAGEGFFNQPVEEKEKYANDHDSGNIQGYGSKLANNASGQLEWEDYFFHLVFPEDKRDMSIWPKTPSDYIPATSEYAKQL RGLATKVLSALSLGLGLEEGRLEKEVGGMEELHLQMKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIIMHIGDTVEILSNRKYKSILHRGLVNKEKVRISWAVFCEPPKEKIILQPLPETVTEEEPPLFPPRTFAQHIQHKLFRKTQVLGGK (SEQ ID NO.2).

[0033] The present invention will be described in detail below with specific embodiments. The materials and reagents involved in the embodiments of the present invention are as follows:

[0034] 1. Biomaterials

[0035] Tea variety: Zijuan (Camellia sinensis (L.) O.Kuntze cv. Zijuan). Fresh leaves were quickly frozen with liquid nitrogen after collection and stored at -80℃ for later use.

[0036] Vector: pMAL-c5X prokaryotic expression vector;

[0037] Strains: Escherichia coli BL21(DE3)pLysS competent cells (purchased from Beijing TransGen Biotech Co., Ltd.), Escherichia coli DH5α competent cells (for vector cloning).

[0038] 2. Preparation of culture medium and reagents

[0039] LB liquid medium: Weigh 25g of broth, add 1L of deionized water and stir to dissolve, autoclave at 121℃ for 15min, cool and store at room temperature;

[0040] LB solid medium: Add 15g of agar powder to LB liquid medium, sterilize, pour into plates while hot, cool and solidify, and store at 4℃.

[0041] Ampicillin (Amp) + Stock solution (50mg / mL): Weigh 500mg of ampicillin powder, dissolve in 10mL of sterile water, filter through a 0.22μm filter membrane for sterilization, dispense into 1mL / tube, store at -20℃, and dilute at a ratio of 1:500 before use (final concentration 100μg / mL).

[0042] Isopropyl thio-β-D-galactopyranoside (IPTG) stock solution (1 mol / L): Weigh 2.383 g IPTG powder, dissolve in 10 mL of sterile water, filter sterilize through a 0.22 μm filter membrane, dispense into 1 mL / tube, store at -20℃, and dilute at a ratio of 1:1000 (final concentration 1 mmol / L) before use.

[0043] Protein purification buffer: Column loading buffer: Weigh 0.37g EDTA, 11.67g NaCl, 2.42g Tris, and 0.15g DTT, add 800mL of ultrapure water and stir to dissolve. Adjust the pH to 7.4 with dilute hydrochloric acid, bring the volume to 1L, sterilize, and store at 4℃.

[0044] Elution buffer: Take 1L of column loading buffer, add 3.60g of maltose and stir to dissolve, then sterilize and store at 4℃;

[0045] Tris-HCl buffer (100mM, pH 7.5): Weigh 1.1214g Tris powder, add 90mL of ultrapure water and stir to dissolve. Adjust the pH to 7.5 with 1mol / L HCl, bring the volume to 100mL, sterilize and store at 4℃.

[0046] UPLC mobile phase: Mobile phase A (containing 1% acetic acid aqueous solution): Pipette 10 mL of chromatographic grade acetic acid, add 990 mL of ultrapure water, mix well, and filter through a 0.22 μm filter membrane; Mobile phase B (100% acetonitrile): Use chromatographic grade acetonitrile directly.

[0047] Example

[0048] 1. Cloning of the CsANS1 gene in tea plants

[0049] 1.1 RNA extraction and cDNA synthesis

[0050] According to the EASYspin Plus kit instructions, 0.1g of frozen fresh leaves of Zijuan tea tree were ground into powder and added to the lysis buffer. Total RNA was extracted by centrifugation, washing, and elution. Using the PrimeScript RT reagent kit, cDNA was synthesized using the total RNA as a template by reverse transcription at 50℃ for 30 min and inactivation at 85℃ for 5 min. The cDNA product was stored at -20℃.

[0051] 1.2 PCR amplification of the CsANS1 gene

[0052] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., as follows:

[0053] Upstream primer: 5'-ATGGTGGCTACTGTGGCAGG-3', SEQ ID NO.3;

[0054] Downstream primer: 5'-TCATTTCCCTCCAAGCACCT-3', SEQ ID NO.4.

[0055] PCR reaction system (50μL): 2×Taq Master Mix 25μL, upstream primer (10μmol / L) 2μL, downstream primer (10μmol / L) 2μL, cDNA template 1μL and sterile water 20μL.

[0056] PCR reaction program: 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 62℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles, 72℃ extension for 10 min; incubate at 4℃.

[0057] PCR product processing: The amplification products were detected by 1% agarose gel electrophoresis, and the target fragment (approximately 1000 bp in size, corresponding to the nucleotide sequence shown in SEQ ID NO.1) was recovered using a gel recovery kit. The recovered products were stored at -20℃.

[0058] 1.3 Vector Ligation and Transformation Verification

[0059] The recovered PCR product was ligated with the pEASY-T&B CTB501 vector according to the kit instructions (incubation at 25°C for 30 min), and the ligation product was transformed into DH5α competent cells.

[0060] Add 10 μL of ligation product to 100 μL of DH5α competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 2 min; add 900 μL of antibiotic-free LB liquid medium and incubate at 37℃ with shaking at 200 rpm for 1 h; spread 100 μL of the bacterial culture onto a medium containing 100 μg / mL Ampicillin. + LB agar plates were incubated upside down at 37°C for 12-16 hours; single colonies were picked for colony PCR verification, and positive colonies were inoculated with a solution containing 100 μg / mL Ampicillin. + The plasmid was cultured overnight at 37°C and 200 rpm in LB liquid medium. The plasmid was extracted and sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. The sequence was confirmed to be consistent with SEQ ID NO.1, and a T vector containing the CsANS1 gene was obtained (named pEASY-T-CsANS1).

[0061] 2. Prokaryotic expression of CsANS1 gene and purification of recombinant protein

[0062] 2.1 Construction of recombinant expression plasmid pMAL-c5X-CsANS1

[0063] Homologous arm amplification: Using the T vector (pEASY-T-CsANS1) containing the CsANS1 gene as a template, the CsANS1 gene fragment containing homologous arms was amplified using the primer pairs shown in SEQ ID NO. 5-6, and the product was recovered. The primer pairs are as follows:

[0064] Upstream primer: 5'-GCGATATCGTCGACGGATCCATGGTGGCTACTGTGGCAGG-3', SEQ ID NO.5;

[0065] Downstream primer: 5'-TAATTACCTGCAGGGAATTCTCATTTCCCTCCAAGCACCT-3', SEQ ID NO.6.

[0066] Vector linearization: pMAL-c5X vector was double-digested with BamHⅠ and EcoRⅠ (reacted at 37℃ for 2 h), and the linearized vector was recovered by 1% agarose gel electrophoresis.

[0067] Seamless cloning: Following the instructions of the seamless cloning kit, the CsANS1 fragment containing the homologous arm was mixed with the linearized pMAL-c5X vector (fragment:vector = 3:1), and reacted at 50℃ for 15 min to obtain the recombinant plasmid pMAL-c5X-CsANS1.

[0068] 2.2 Construction and Screening of Recombinant Engineered Bacteria

[0069] pMAL-c5X-CsANS1 was transformed into E. coli BL21(DE3)pLysS competent cells:

[0070] Add 10 μL of recombinant plasmid to 100 μL of BL21(DE3)pLysS competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s, incubate on ice for 2 min, add 900 μL of antibiotic-free LB liquid medium, and incubate at 37℃ and 200 rpm for 1 h; spread 100 μL of the bacterial culture onto a medium containing 100 μg / mL Ampicillin. + LB solid plates were incubated upside down at 37°C for 12-16 hours; single colonies were picked for colony PCR verification, and positive colonies were recombinant engineered bacteria containing the CsANS1 gene (named BL21-pMAL-CsANS1).

[0071] 2.3 Induction and purification of recombinant proteins

[0072] Induction of expression: Inoculate positive engineered bacteria into 100 mL of solution containing 100 μg / mL Amp + LB liquid medium, cultured at 37°C with shaking at 200 rpm until OD 600 ≈0.6, add IPTG to a final concentration of 1 mmol / L, and incubate overnight at 37℃ and 200 r / min (12 h).

[0073] Bacterial cell disruption: Centrifuge at 5000 r / min for 10 min to collect bacterial cells, resuspend in 10 mL of column buffer, freeze at -20℃ overnight, and thaw in an ice bath; disrupt bacterial cells using an ultrasonic disruptor (15% power, 3 s operation, 5 s interval) for 10 min, centrifuge at 12000 r / min and 4℃ for 20 min, and collect the supernatant.

[0074] Protein purification: Load the supernatant onto a pre-equilibrated (washed with 3 column volumes of loading buffer) amylose affinity column, and proceed sequentially as follows:

[0075] (1) Wash with 5 column volumes of buffer to remove contaminating proteins;

[0076] (2) Elute with elution buffer for 3 column volumes and collect the eluent (containing recombinant protein, fused with 42.5 kDa maltose-binding protein MBP, with a total molecular weight of approximately 82.6 kDa).

[0077] Protein detection: The purification effect was detected by SDS-PAGE (12% separating gel, 5% stacking gel). After Coomassie brilliant blue staining, a single band appearing at 70-95 kDa indicates the purified CsANS1 recombinant protein.

[0078] The results are as follows Figure 1 As shown, Figure 1 As can be seen, after transformation of the pMal-c5X-CsANS1 recombinant plasmid into the host bacterium BL21(DE3), the recombinant protein was expressed after induction (lane 2) compared to before induction (lane 1), and the size of the recombinant protein band was consistent with the prediction. After adding the 42.5kDa maltose-binding protein (MBP) recombinant tag, the protein size was 82.6kDa. Therefore, there was a clear recombinant protein band in the 70-95kDa range in the SDS-PAGE protein electrophoresis analysis. After ultrasonic disruption and centrifugation of the bacterial cells after induction, soluble recombinant protein was found in the supernatant (lane 3), indicating that the induced recombinant protein was expressed in the supernatant and could be used for further purification and analysis. After purification of the supernatant protein by a straight-chain starch resin column, a relatively pure recombinant protein was obtained (lane 4), and the purified protein could be used for further enzymatic analysis.

[0079] 3. Enzymatic activity detection of CsANS1 recombinant protein

[0080] 3.1 Construction of the enzyme-catalyzed reaction system

[0081] For enzyme activity assay, prepare a 50 μL reaction system with the following concentrations: 20 mM pH 7 potassium phosphate buffer, 1 mM α-ketoglutarate, 0.4 mM ferrous(II) ammonium sulfate, 4 mM ascorbic acid, 100 mM sodium chloride, 10 mM maltose, 5 mM DTT, with 200 μM colorless anthocyanins as the reaction substrate, and 5-10 μg of purified recombinant protein.

[0082] Set up a control group: except that α-ketoglutarate was not added, the other components were the same as those in the reaction group.

[0083] Both the reaction group and the control group were incubated in a 30℃ water bath for 1 h. The reaction was terminated by adding 50 μL of pure methanol and 1 μL of 1 mol / L HCl. The mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was used for product detection.

[0084] 3.2 Product identification (UPLC and UPLC-MS / MS)

[0085] (1) UPLC detection conditions

[0086] Chromatograph: ACQUITY Arc system (Waters);

[0087] Chromatographic column: Waters XBridge, C18 column (3.5μm, 4.6mm×150mm);

[0088] Column temperature: 40℃;

[0089] Flow rate: 0.5 mL / min;

[0090] Injection volume: 10 μL;

[0091] Detection wavelengths: 360nm (quercetin), 540nm (anthocyanins);

[0092] Mobile phase A is a solution containing 1% (v / v) acetic acid; mobile phase B is a 100% acetonitrile solution.

[0093] The gradient program was set as follows: 0-2 min: 5%B; 2-8 min: 5-10%B; 8-18 min: 10-18%B; 18-20 min: 18%-25%B; 20-25 min: 25-95%B; 25-27 min: 95-5%B; 27-32 min: 5%B.

[0094] The enzyme reaction products were analyzed using UPLC and quercetin standards, and the results are as follows: Figure 2As shown in the figure. Compared with the quercetin standard shown in the first line graph from top to bottom, a distinct quercetin characteristic peak appeared at a specific retention time (26.8 min) at a wavelength of 360 nm, proving that the CsANS1 recombinant protein can catalyze the substrate to generate quercetin; compared with the anthocyanin standard shown in the third line graph from top to bottom, an anthocyanin characteristic peak appeared at a specific retention time (20.2 min) at a wavelength of 540 nm, proving that the CsANS1 recombinant protein can catalyze the generation of anthocyanins.

[0095] (2) UPLC-MS / MS detection conditions

[0096] Chromatograph: Nexera LC-40 (Shimadzu);

[0097] Mass spectrometer: QTRAP 6500+ (AiboCaisi);

[0098] Chromatographic column: Waters XBridge, C18 column (3.5μm, 4.6mm×150mm);

[0099] Column temperature: 40℃;

[0100] Flow rate: 0.4 mL / min;

[0101] Injection volume: 10 μL;

[0102] Mobile phase A is an aqueous solution containing 0.1% (v / v) formic acid; mobile phase B is a methanol solution containing 0.1% (v / v) formic acid.

[0103] The UPLC gradient program was set to 0-1 min, 5%B; 1-2 min, 5-10%B; 2-13 min, 10-50%B; 13-14 min, 50-95%B; and 14-15 min, 95%B.

[0104] In the MS qualitative identification of the compounds, an ESI electrospray ionization source in positive ion mode was used; the capillary voltage was 5 kV, the ion source temperature was 350 °C, the nebulizing gas (nitrogen) flow rate was 10 L / min, the mass-to-charge ratio range for compound detection was set to m / z 100-2000, and the collision voltage was 30 V. The results are as follows: Figure 3 As shown.

[0105] Depend on Figure 3 It can be seen that under multi-stage reaction monitoring (MRM), a distinct quercetin characteristic peak appears at a specific mass-to-charge ratio (303 / 153) in the reaction system, indicating that the CsANS1 recombinant protein can catalyze the formation of quercetin from the substrate. Figure 3 A); and Figure 3Compared to the anthocyanin standard shown in Figure C, under the conditions of a parent ion of 287 and a daughter ion of 231, the characteristic peak of cyanidin appeared at the corresponding retention time (approximately 4.5 min), proving that the CsANS1 recombinant protein can also simultaneously catalyze the production of anthocyanins. Figure 3 B).

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a gene CsANS1 encoding anthocyanin synthase or its related biological material in the simultaneous synthesis of anthocyanin and quercetin in vitro, characterized in that, The nucleotide sequence of the gene CsANS1 is shown as SEQ ID NO.

1. The related biological material is the protein encoded by the gene CsANS1, a recombinant vector containing the gene CsANS1, and a recombinant microorganism containing the gene CsANS1.

2. Use according to claim 1, characterized in that, The protein encoded by the gene CsANS1 has an amino acid sequence shown as SEQ ID NO.

2.

3. A method for the simultaneous synthesis of cyanidin and quercetin in vitro, characterized in that, The step of producing anthocyanin and quercetin by using anthocyanin as a substrate, using alpha-ketoglutaric acid as a condition, and using anthocyanin synthase as a catalyst. The amino acid sequence of the anthocyanin synthase is shown as SEQ ID NO.

2.

4. The method of claim 3, wherein, The anthocyanin synthase is induced and expressed by a recombinant bacterium containing the gene CsANS1, and the nucleotide sequence of the gene CsANS1 is shown as SEQ ID NO. 1.

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