Application of slan2-like gene in regulating anthocyanin content in tomato

Abstract

The application discloses application of a SlAN2-like gene in regulating content of delphinidin-3-glucoside in tomatoes, a nucleotide sequence of the SlAN2-like gene is shown as SEQ ID NO:1, and overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in the tomatoes. Compared with the prior art, the application finds that overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in the tomatoes, and only one anthocyanin of delphinidin-3-glucoside is produced. Therefore, the SlAN2-like gene can be used to produce a tomato material containing only delphinidin-3-glucoside monomers, because anthocyanins in most plants are in the form of a mixture, and extraction and purification work is needed to obtain monomer anthocyanin substances. The created tomato material can be used to reduce technical barriers and costs of extracting delphinidin-3-glucoside.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, specifically involving the application of the SlAN2-like gene in regulating the content of delphinidin-3-glucoside in tomatoes. Background Technology

[0002] Anthocyanins are a class of natural water-soluble pigments, but because anthocyanin molecules are extremely unstable under natural conditions, they easily combine with glycosides and are stored in vacuoles as anthocyanins. In plants, anthocyanins often exist in mixed forms; for example, blueberries contain cyanidin-3-glucoside, cyanidin-3-arabinoside, delphinidin-3-galactoside, paeoniflorin-3-galactoside, and paeoniflorin-3-arabinoside. The forms in which anthocyanins exist make them difficult to purify and separate.

[0003] Delphinidin-3-glucoside is a secondary metabolite of anthocyanins found in plants. Recent studies have confirmed that delphinidin-3-glucoside possesses antioxidant, anti-inflammatory, anti-obesity, breast cancer prevention, liver cancer prevention, and cardiovascular disease prevention activities. Although delphinidin-3-glucoside has been proven to promote health and prevent disease, the isolation and extraction of high-purity delphinidin-3-glucoside faces technical difficulties and high costs. Currently, few studies have proposed specific purification methods, and due to the challenges in its isolation and purification, the standard product of delphinidin-3-glucoside is expensive in the market.

[0004] Tomatoes are an important fruit-type vegetable crop. Commonly cultivated tomatoes on the market do not contain anthocyanins. However, by introducing loci controlling anthocyanin synthesis from wild tomato relatives into cultivated tomatoes, tomatoes containing natural anthocyanins, such as Indigo Rose (InR), have been obtained. Research has found that the InR tomato genome contains two loci related to anthocyanin synthesis: Aft (Anthocyanin fruit) and atv (atroviolacium). Four R2R3-MYB candidate genes were identified at the Aft locus: SlAN2 (SlMYB75), SlANT1 (Slanthocyanin 1), SlANT1-like (SlMYB28), and SlAN2-like (SlMYB114). The atv locus encodes an anthocyanin synthesis repressor, SlMYBATV, which belongs to the R3-MYB transcription factor family. In InR fruit, mutations at this locus cause premature termination of SlMYBATV translation, resulting in a truncated protein with no biological function. Previous studies have shown that SlANT1 and SlAN2 are positive regulators of anthocyanin synthesis in tomatoes. Functional comparative analysis of four candidate genes at the Aft locus revealed that SlAN2-like genes exhibited the strongest ability to transcribe and activate genes related to anthocyanin biosynthesis. Therefore, it is inferred that SlAN2-like genes play a crucial role in promoting anthocyanin accumulation in tomatoes. However, in cultivated tomatoes (such as Ailsa Craig), intron polymorphism leads to alternative splicing of SlAN2-like genes, resulting in premature termination of protein translation and the generation of truncated proteins lacking the R3 domain containing the bHLH binding site. This prevents the formation of the MBW complex to induce transcription of downstream LBGs such as ANS, ultimately hindering the anthocyanin synthesis pathway. Therefore, there are currently no studies on the function of SlAN2-like genes in fresh-eating tomato varieties.

[0005] Chinese patent document CN109136233A discloses a method for increasing the anthocyanin content of tomatoes by overexpressing the SlBBX20 transcription factor. However, the anthocyanin content in the tomato materials obtained by this method only accumulates significantly in the leaves, while the anthocyanin content in the fruit remains unchanged.

[0006] Chinese patent document CN103966235B discloses a recombinant expression vector for the eggplant anthocyanin synthesis-related gene SmMYB1 and its application in cultivating purple tomatoes. This method increases the anthocyanin content and turns tomato fruits purple by overexpressing the exogenous gene SmMYB1. However, this tomato material contains multiple anthocyanins, and only focuses on the total anthocyanin content, making it difficult to purify and separate anthocyanin monomers. Summary of the Invention

[0007] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides the application of the SlAN2-like gene in regulating the content of delphinidin-3-glucoside in tomatoes.

[0008] Technical solution: To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the application of the SlAN2-like gene (Solyc10g086290) in regulating the content of delphinidin-3-glucoside in tomatoes. The nucleotide sequence of the SlAN2-like gene is shown in SEQ ID NO:1. Overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes.

[0010] As a specific implementation scheme, the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0011] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0012] Secondly, the present invention provides a protein expressed by the SlAN2-like gene, and the application of a vector or host cell containing the SlAN2-like gene in regulating the content of delphinidin-3-glucoside in tomatoes. The nucleotide sequence of the SlAN2-like gene is shown in SEQ ID NO:1. Overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes.

[0013] As a specific implementation scheme, the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0014] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0015] Thirdly, the present invention provides the application of SlAN2-like gene expression promoter in the preparation of products that regulate the content of delphinidin-3-glucoside in tomatoes, wherein the nucleotide sequence of the SlAN2-like gene is shown in SEQ ID NO:1, and the SlAN2-like gene expression promoter can increase the content of delphinidin-3-glucoside in tomatoes.

[0016] As a specific implementation scheme, the SlAN2-like gene expression promoter can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0017] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0018] Fourthly, the present invention provides the application of the SlAN2-like gene or SlAN2-like gene expression promoter in tomato breeding. The application obtains tomato varieties containing high purity delphinidin-3-glucoside by overexpressing the SlAN2-like gene with a nucleotide sequence as shown in SEQ ID NO:1.

[0019] As a specific implementation scheme, the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0020] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0021] Fifthly, the present invention provides a method for creating tomatoes containing high-purity delphinidin-3-glucoside, wherein the method involves overexpressing the SlAN2-like gene with a nucleotide sequence as shown in SEQ ID NO:1, and the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes.

[0022] As a specific implementation scheme, the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0023] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0024] In a sixth aspect, the present invention provides a method for extracting and separating high-purity delphinidin-3-glucoside, the method comprising extracting high-purity delphinidin-3-glucoside from tomatoes, wherein the tomatoes are transgenic tomatoes obtained by overexpressing the SlAN2-like gene with a nucleotide sequence as shown in SEQ ID NO:1.

[0025] As a specific implementation scheme, the overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside.

[0026] As a specific implementation, the tomato is a fresh tomato; preferably, the tomato is selected from the fresh tomato variety Ailsa Craig.

[0027] Beneficial Effects: Compared with existing technologies, this invention discovers that overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes, and produces only one anthocyanin, delphinidin-3-glucoside. Therefore, this SlAN2-like gene can be used to produce a tomato material containing only delphinidin-3-glucoside monomers. Since anthocyanins in most plants are in mixed forms, obtaining monomeric anthocyanins requires extraction and purification. The newly created tomato material can reduce the technical barriers and costs associated with extracting delphinidin-3-glucoside. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the overexpression vector pYLTAC380GW-AN2like.

[0029] Figure 2 The results are for PCR amplification products.

[0030] Figure 3 This is a visual representation of a tomato fruit at the ripening stage.

[0031] Figure 4 The qualitative and quantitative analysis of anthocyanins in T0 generation tomatoes transgenic with the SlAN2-like gene is presented. A is a high-performance liquid chromatogram of SlAN2-like tomato fruits overexpressing AilsaCraig tomato; B is a graph showing the content of delphinidin-3-glucoside in wild-type, AN2L#6, and AN2L#14 fruits at fresh weight. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0033] The reagents and formulations used in the following examples are as follows:

[0034] 1. Relevant reagents

[0035] Phanta Max Super-Fidelity DNA Polymerase (Catalog No.: P505-d1), 2×Taq Master Mix for PCR (Catalog No.: P112-01), DNA Recovery and Purification Kit The following kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.: Gel DNA Extraction Mini Kit (Catalog No.: DC301), HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Catalog No.: R312-01), and In-Fusion Recombination Kit ClonExpress II OneStep Cloning Kit (Catalog No.: C112-01). A plasmid extraction kit was also included. The Plasmid MiniPrep Kit (catalog number: EM101-02) was purchased from Beijing TransGen Biotech Co., Ltd., and the total RNA extraction kit was from EZN. The Plant RNA kit (catalog number: R6827) was purchased from OMEGA Biotechnology Co., Ltd.; the Plant Genomic DNA Extraction Kit (catalog number: DP305) was purchased from Tiangen Biotech (Beijing) Co., Ltd. Restriction endonucleases Nb.BtsⅠ, I-SceⅠ, PI-SceⅠ, and HiFi Taq DNA ligase were all purchased from NEB Corporation (USA).

[0036] Antibiotics such as kanamycin (Kan), rifampin (Rif), gentamicin (Gen), ampicillin (Amp), and chloramphenicol (Cm) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Biochemical reagents such as Na2EDTA·2H2O, Tris base, yeast extract, tryptone, sodium chloride, and sodium hydroxide were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Agarose was purchased from Beijing Qingke Biotechnology Co., Ltd. GelStain nucleic acid dye was purchased from Beijing TransGen Biotechnology Co., Ltd. Anhydrous ethanol was purchased from Shanghai Maclean Biotechnology Co., Ltd. DNA Marker and 10× DNA loading buffer were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0037] 2. Culture medium formulation

[0038] (1) MS (Murashige Skoog) basic medium (TO): Weigh 1.776 g MS salt and 12 g sucrose, dissolve in 800 mL deionized water, adjust pH to 5.80-5.82 with NaOH, and add 5.6 g agar. Autoclave at 121℃ for 20 min. Cool to about 55-65℃, pour into sterile glass culture bottles (about 80 mL / bottle) in a laminar flow hood, and let cool and solidify before use;

[0039] (2) Pre-culture medium (T1): Weigh 3.544 g MS salt and 24 g sucrose, dissolve them in 800 mL deionized water, adjust the pH to 5.80-5.82 with NaOH, and add 5.6 g agar. Autoclave at 121℃ for 20 min, cool to about 55-65℃, add 800 μL (working concentration 1 mg / L) and 800 μL (working concentration 0.1 mg / L) of 6-BA in a laminar flow hood, mix well, and pour into disposable culture dishes (about 20-25 mL / dish). Let cool and solidify before use.

[0040] (3) Differentiation-selective medium (T21): Weigh 3.544 g MS salt and 24 g sucrose, dissolve them in 800 mL deionized water, adjust the pH to 5.80-5.82 with NaOH, and add 5.6 g agar. Autoclave at 121℃ for 20 min, cool to about 55-65℃, add 160 μL hygromycin (working concentration 10 mg / L), 800 μL Ti (working concentration 200 mg / L), 800 μL IAA (working concentration 0.1 mg / L), and 800 μL ZT (working concentration 1 mg / L) in a clean bench, mix well, pour into disposable culture dishes (about 20-25 mL / dish), cool and solidify for later use;

[0041] (4) Bud elongation selective medium (T22): Weigh 3.544 g MS salt and 24 g sucrose, dissolve them in 800 mL of deionized water, adjust the pH to 5.80-5.82 with NaOH, and add 5.6 g agar. Autoclave at 121℃ for 20 min, cool to about 55-65℃, add 160 μL hygromycin (working concentration 10 mg / L), 800 μL Ti (working concentration 200 mg / L), 800 μL GA (working concentration 1 mg / L), and 800 μL ZT (working concentration 0.5 mg / L) in a clean bench, mix well, pour into sterile glass culture bottles (about 80 mL / bottle), cool and solidify for later use;

[0042] (5) Rooting medium (T3): Weigh 1.766 g MS salt and 24 g sucrose, dissolve them in 800 mL deionized water, adjust the pH to 5.80–5.82 with NaOH, and add 5.6 g agar. Autoclave at 121 °C for 20 min, cool to about 55–65 °C, add 80 μL hygromycin (working concentration 5 mg / L), 600 μL Ti (working concentration 150 mg / L), and 800 μL IBA (working concentration 2 mg / L) in a clean bench, mix well, pour into sterile glass culture flasks (about 80 mL / flask), and let cool and solidify before use.

[0043] Example 1: Obtaining the SlAN2-like gene

[0044] The steps to obtain the SlAN2-like gene against the Ailsa Craig tomato background are as follows:

[0045] 1. Using OMEGA's EZNA Total RNA was extracted from the pericarp of Ailsa Craig during the color-breaking stage using the Plant RNA Kit, and then reverse transcription was performed using the reverse transcription kit from Nanjing Weizan Biotechnology Co., Ltd. to obtain Ailsa Craig cDNA.

[0046] 2. After completing step 1, using Ailsa Craig's cDNA as a template, PCR amplification was performed on AN2like-Exon1 using primers AN2L-F1 and AN2L-R1, yielding a PCR amplification product of approximately 124 bp. PCR amplification was performed on AN2like-Exon2 using primers AN2L-F2 and AN2L-R2, yielding a PCR amplification product of approximately 130 bp. PCR amplification was performed on AN2like-Exon3 using primers AN2L-F3 and AN2L-R3, yielding a PCR amplification product of approximately 544 bp. The results are as follows. Figure 2 As shown.

[0047] AN2L-F1:5'-TGACCAGAATTCATGAATATTGCCA-3'

[0048] AN2L-R1:5'-GACATCTATTTAGACCAGCTCTAAAAG-3'

[0049] AN2L-F2: 5'-CTTTTAGAGCTGGTCTAAATAGATGTCG-3'

[0050] AN2L-R2:5'-ATAAGTGACCCATCTATTGCCTAGAA-3'

[0051] AN2L-F3:5'-TCTAGGCAATAGATGGTCACTTATTG-3'

[0052] AN2L-R3: 5'-TTTGAACGATCGGGGGATCCACTGGC-3'.

[0053] Reaction system (50 μL): Phanta Max from Nanjing Weizan Biotechnology Co., Ltd.

[0054] Construction system of Super-Fidelity DNA Polymerase. The system consisted of 25 μL 2×Phanta Flash Master buffer, 1 μL dNTP Mix, 2 μL forward primer (10 μM AN2L-F1, AN2L-F2, or AN2L-F3), 2 μL reverse primer (10 μM AN2L-R1, AN2L-R2, or AN2L-R3), 1 μL Ailsa Craig cDNA (containing 50-60 ng cDNA), 1 μL Phanta Flash Master DNA polymerase, and 18 μL double-distilled water. Reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 60 s, for a total of 34 cycles; 72℃ extension for 5 min.

[0055] 3. After completing step 2, the AN2like-Exon1, AN2like-Exon2, and AN2like-Exon3 PCR amplification products obtained in step 2 were recovered using the PCR product purification kit of Nanjing Weizan Biotechnology Co., Ltd., and the recovered fragments were obtained respectively.

[0056] 4. After completing step 3, the AN2like-Exon1, AN2like-Exon2, and AN2like-Exon2 were recombined using the In-Fusion Recombinant Recombinant Kit (ClonExpress II One Step Cloning Kit) from Nanjing Weizan Biotechnology Co., Ltd. via recombinant PCR.

[0057] AN2like-Exon3 was used for ligation. Homologous arms were designed for primers AN2L-R1 and AN2L-F2, and AN2like-Exon3 was used for ligation.

[0058] AN2like-Exon1 and AN2like-Exon2 were ligated. Homologous arms were designed for AN2L-R2 and AN2L-F3. The ligation products of AN2like-Exon1 and AN2like-Exon2 were used to ligate AN2like-Exon3, resulting in a ligation product of approximately 798 bp. The ligation product was recovered using a PCR product purification kit.

[0059] Reaction system (20 μL): The system was constructed using the ClonExpress II One Step Cloning Kit from Nanjing Weizan Biotechnology Co., Ltd. The system contained 0.02 μg of the fragment to be ligated (number of homologous arms × bp), 4 μL of 5×CE II buffer, 2 μL of Exnase II, and double-distilled water to a final volume of 20 μL. Reaction conditions: 37℃ for 30 min, followed by an ice bath for 5 min, and then storage at -20℃ for later use.

[0060] 5. Using the ClonExpress II One Step Cloning Kit, the recovered fragment from step four was ligated with the vector PUC57 to obtain the recombinant plasmid PUC57-AN2like.

[0061] 6. The recombinant plasmid PUC57-AN2like was sequenced by Beijing Qingke Biotechnology Co., Ltd. Sequencing results showed that the recombinant plasmid contained the nucleotide sequence of AN2like. The nucleotide sequence of the SlAN2-like gene under the Ailsa Craig tomato background is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0062] Example 2: Construction of the overexpression vector pTAC380GW+AN2like and preparation of recombinant Agrobacterium

[0063] I. Construction of the overexpression vector pTAC380GW+AN2like

[0064] 1. Using the PUC57-AN2like constructed in Example 1 as a template, PCR amplification of AN2like was performed using primers AN2-E8-F (5'-CGAATTCGGCAATGAATATTGCCAAGACATTGG-3') with an E8 promoter homologous arm and AN2-Nos-R (5'-CAGTTGATATTGACCTATGGAATCTATTTAATTAGCCCGATCGTTCAAACAT-3') with a Tnos terminator homologous arm. The reaction system and conditions were as shown in step 2 of Example 1. The amplified product was recovered using a PCR product purification kit.

[0065] 2. Using pEF 1-E8-4 plasmid as a template, PCR amplification of promoter E8 was performed using primers E8-F and E8-R as follows. The reaction system and conditions are as shown in step 2 of Example 1. The amplified product was then recovered using a PCR product purification kit.

[0066] E8-F: 5'-GAATTCGGATCTTCCAGAGATTGGAATTCGAGCTCGGTACC-3'

[0067] E8-R: 5'-GCAATATTCATTGCCGAATTCGGGGATCCG-3'

[0068] 3. Using pCAMBIA1300 plasmid as a template, the terminator Tnos was amplified by PCR using the following primers Tnos-F and Tnos-R, yielding a PCR product of approximately 251 bp. The amplified product was then recovered using a PCR product purification kit.

[0069] Tnos-F: 5'-CCAAATGTTTTGAACGATCGGGCTAATTAAATAGATT-3'

[0070] Tnos-R:5'-CGCGGTGTCATCTATGTTACT-3'

[0071] 4. Using the ClonExpress II One Step Cloning Kit, the recovered products from steps 1, 2, and 3 were ligated. The reaction system and conditions are as shown in step 4 of Example 1. The ligation product was then recovered using a PCR product purification kit to obtain the PE8-AN2like-Tnos expression cassette.

[0072] 5. The recovered product obtained in step 4 is ligated into the pCE-Zero vector to obtain the pCE-Zero-AN2like plasmid.

[0073] 6. The recombinant plasmid pCE-Zero-AN2like was sequenced by Beijing Qingke Biotechnology Co., Ltd. Sequencing results showed that the recombinant plasmid contained the nucleotide sequence of PE8-AN2like-Tnos. The nucleotide sequence of the PE8-AN2like-Tnos expression cassette is shown in SEQ ID NO:3.

[0074] 7. The pYLTAC380GW-UniE vector was linearized using the nicked endonuclease Nb.BtsⅠ.

[0075] Reaction system (50 μL): Nb. BtsⅠ nicked endonuclease construction system (NEB, USA). 10 μL of 10×rCutsmartBuffer, 10 U of Nb. BtsⅠ, 1 μg of pYL380GW-UniE plasmid, and finally double-distilled water to a final volume of 50 μL. Reaction conditions: 37℃ for 1 h, followed by 79℃ for 30 min.

[0076] 8. Using pCE-Zero-AN2like plasmid as a template, the AN2like gene expression cassette sequence was amplified by PCR using the specific primers from steps 2 and 3. This resulted in Nb.BtsⅠ restriction sites at both the 5' and 3' ends of the expression cassette sequence, as well as a 15nt adapter sequence that complements the 15nt end of the pYLTAC380GW-UNiE empty vector digested in step 7. The amplified product was recovered using a PCR product purification kit. 1 μg of the recovered product was digested according to the reaction in step 7, reacting at 37°C for 1 h, followed by reaction at 79°C for 30 min.

[0077] 9. The vector backbone from step 7 and the insertion expression cassette fragment from step 8 are ligated at a molar ratio of 1:3. The amount of the pYLTAC380GW-UNiE linearized vector is 100 ng, and the amount of the insertion expression cassette fragment is calculated based on the fragment length and molar ratio. The linearized vector and expression cassette fragment are added to 10 μL of 1×Taq DNA ligase reaction buffer, mixed, and incubated at 70°C for 1 min, then at 60°C for 1 min in a PCR instrument. 0.5 μL of HiFi Taq DNA ligase is added to the reaction solution. Reaction conditions: 46°C for 5 min, 55°C for 2 min, 60°C for 2 min, 65°C for 2 min, for a total of 18 cycles. The pYLTAC380GW-AN2like vector is obtained. A schematic diagram of the structure of the overexpression vector pYLTAC380GW-AN2like is shown below. Figure 1 .

[0078] II. Preparation of Reconstituted Agricultural Stalks

[0079] 1. Take 50 μL of Agrobacterium GV3101 electroporation competent cells, thaw them on ice, add 1 μL of pYLTAC380GW-AN2like plasmid, quickly transfer it to the middle groove of the electroporation cuvette, immediately insert the electroporation cuvette into the middle of the patch in the electroporator well, press the pulse button and immediately remove it, add 1 mL of room temperature SOC medium (without antibiotics), mix thoroughly by aspiration, and then aspirate the bacterial solution into a new centrifuge tube.

[0080] 2. Place the bacterial solution from step 1 in a shaker at 28°C for 1 hour to allow it to recover.

[0081] 3. Take 100 μL of bacterial culture from step 3 and spread it on LB solid medium (containing antibiotics Gen 50 μg / mL, Rif 50 μg / mL, and Kan 50 μg / mL), and incubate overnight at 28°C.

[0082] 4. Select a single colony from step 3 for PCR verification. Reaction system (20 μL): Select a single colony, add 10 μL of 2×Taq Master Mix, 1 μL of forward primer, 1 μL of reverse primer, and finally add double-distilled water to 20 μL. Reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 25 cycles; 72℃ extension for 5 min.

[0083] 5. After PCR amplification, 1.0% agarose gel electrophoresis was used to check whether the fragment size was consistent with the expected size.

[0084] 6. The selected single colonies were sequenced by Beijing Qingke Biotechnology Co., Ltd. The sequencing results were consistent with the expected sequence. A positive recombinant Agrobacterium was obtained and named GV3101 / pYLTAC380GW-AN2like.

[0085] Example 3: Obtaining T0 generation SlAN2-like gene-transformed tomatoes

[0086] I. Preparation of explants

[0087] 1. Place an appropriate amount of Micro-Tom seeds in a sterile petri dish, add 30 mL of 4% sodium hypochlorite, and shake the petri dish continuously and evenly for 8 minutes to sterilize the seed surface.

[0088] 2. Discard the sodium hypochlorite solution in the petri dish, add sterile water and wash, repeating 8 times for 1 minute each time.

[0089] 3. After cleaning, absorb the remaining water from the petri dish, and then use tweezers to evenly distribute the sterilized seeds on the T0 medium, about 30-40 seeds per bottle.

[0090] 4. Place the T0 culture bottle after sowing in a dark room and culture it in the dark for 4-5 days. When the seed germination length is 1-2cm, take out the culture bottle and place it under light (light intensity of about 2000LX, 16h / d) for about 2 days.

[0091] 5. Pour about 30mL of sterile water into a sterile petri dish. Use tweezers to remove the tomato seedling and place its roots into the petri dish containing sterile water. Use scissors to cut off the tips of the two cotyledons. Then cut the remaining cotyledons into squares of about 0.5cm × 0.5cm. Spread the leaves out evenly on T1 medium containing sterile filter paper with the back side facing up.

[0092] 6. Cover the culture dish with the lid, seal it with sealing film, and place it in a dark room at 23°C for 2 days to obtain explants.

[0093] II. Preparation of Agrobacterium tumefaciens infusion solution

[0094] 1. Streak the Agrobacterium strain GV3101 / pYLTAC380GW-AN2like on LB solid medium (containing antibiotics Gen 50μg / mL, Rif 50μg / mL, and Kan 50μg / mL) and incubate at 28℃ for two days until the single colony is large enough to be removed.

[0095] 2. Pick a single colony and place it in 3 mL of LB liquid medium (containing antibiotics Gen 50 μg / mL, Rif 50 μg / mL, and Kan 50 μg / mL), and incubate at 28℃ with constant temperature shaking at 220 rpm for 14-18 h.

[0096] 3. Transfer 200-300 μL of bacterial culture to 10 mL of LB liquid medium (containing antibiotics Gen 50 μg / mL, Rif 50 μg / mL, and Kan 50 μg / mL), and incubate at 28℃ with shaking at 220 rpm for about 6 hours until the OD600 value is 0.5-0.6.

[0097] 4. Centrifuge the bacterial solution from step 3 at 4℃ and 5000×g for 10 min, discard the supernatant, resuspend the bacterial cells in sterile water, and adjust the OD600 of the bacterial solution to 0.1-0.2 to obtain the Agrobacterium infection solution.

[0098] III. Obtaining T0 generation tomatoes with SlAN2-like gene transfer

[0099] 1. Transfer the small square tomato leaves from T1 medium to a sterile Petri dish, add the diluted infection solution from step 4 above, and gently shake the Petri dish for 5 minutes to ensure that the tomato explants are fully in contact with the bacterial solution.

[0100] 2. Discard the infection solution, absorb the remaining infection solution from the culture dish, place the explants face up in a T1 culture dish lined with sterile filter paper, and incubate in a dark room at 23°C for two days.

[0101] 3. Using tweezers, transfer the explants cultured in the dark for two days from the pre-medium (T1) to the differentiation selection medium (T21), seal the culture dish with sealing film, and incubate at 23℃ (16h light / d).

[0102] 4. After one week of culture under light, transfer the explants to a new T21 medium. Change the T21 medium every two weeks until the explants differentiate into shoots.

[0103] 5. Using tweezers, transfer the explants that have differentiated into shoots (with obvious growth points and good growth) from T21 medium to shoot elongation medium (T22). Place 2-3 explants in each culture bottle and incubate at 23℃ (16h light / d). Replace the explants with fresh T22 medium every half month until the top cap is reached.

[0104] 6. Use tweezers to remove the explant from the T22 culture flask and place it in a sterile culture dish. Use scissors to cut the bud that has differentiated into a growth point from the connection between the explant and the base of the stem. Gently insert it into the rooting medium (T3) with tweezers and incubate at 23℃ (16h light / d).

[0105] 7. After about one month of induction in T3 medium, the seedlings will grow into young roots. When the young roots are relatively thick (and root hairs grow from the lateral roots), they can be transplanted into soil for cultivation.

[0106] 8. Carefully remove the robust seedlings from the T3 medium and transplant them into pre-moistened soil. Immediately cover the newly transplanted tomato seedlings with a plastic film. After a noticeable water mist has accumulated inside the plastic film, poke holes in the film. Once the transplanted plants are growing well, remove the plastic film to obtain T0 generation SlAN2-like gene-transformed tomatoes.

[0107] 9. Fifteen T0 generation tomatoes transgenic with the SlAN2-like gene were named AN2L#1 to AN2L#15.

[0108] IV. Identification of DNA Levels in T0 Generation Tomatoes Transformed with the SlAN2-like Gene

[0109] (I) DNA extraction from transgenic tomato plants

[0110] 1. Take one leaf from each transgenic plant and wild-type (WT) plant and place it in a 2.0 mL centrifuge tube. Add small steel balls and grind them into powder using a grinder.

[0111] 2. Add 600 μL of CTAB (containing 2% β-mercaptoethanol), vortex for 1 min, mix thoroughly, and then incubate in a 65°C water bath for 30 min. During the water bath, invert the centrifuge tube 2-3 times to ensure complete lysis of the sample.

[0112] 3. Add 700 μL of DNA extraction buffer (phenol: chloroform: isopropanol = 125:24:1) to each tube and vortex until fully emulsified.

[0113] 4. Centrifuge at 13400×g for 10 min, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, add 0.7 times the volume of isopropanol to the aspirated supernatant, mix by inversion, and let stand at -20℃ for 2 h to precipitate.

[0114] 5. Centrifuge at 13400×g for 10 minutes. A white DNA precipitate will be visible at the bottom of the tube. Discard the supernatant.

[0115] 6. Add 1 mL of 70% ethanol, invert several times to rinse the DNA precipitate, centrifuge at 13400×g for 10 min, and discard the supernatant. Repeat this step once.

[0116] 7. Aspirate any remaining liquid, open the centrifuge tube cap, and air dry in a fume hood for 10 minutes to remove residual ethanol. Add 30-50 μL of TE buffer and incubate at 65°C for 30-60 minutes to dissolve the DNA. This yields the genomic DNA of the tomato plant.

[0117] (II) Hygromycin Identification in Transgenic Plants

[0118] 1. Using the genomic DNA of the tomato plant to be tested as a template, PCR amplification was performed using primer pair HPT-F: 5'-TTCGAGATAATATTAGAAGTAGG-3' and primer HPT-R: 5'-TCTTGTGCTTCCGTTATTAT-3' to obtain the PCR amplification product. If the PCR amplification product contains a DNA fragment of 760 bp, the corresponding T0 generation SlAN2-like gene-transgenic tomato is a T0 generation positive SlAN2-like gene-transgenic tomato; if the PCR amplification product does not contain a DNA fragment of 760 bp, the corresponding T0 generation SlAN2-like gene-transgenic tomato is a negative SlAN2-like gene-transgenic tomato.

[0119] 2. Reaction system (20 μL): composed of 10 μL of 2×Taq Master Mix, 1 μL of primer HPT-F (concentration of 10 μM), 1 μL of primer HPT-R (concentration of 10 μM), 1 μL of genomic DNA from the tomato plant to be tested (containing 50-60 ng DNA) and 7 μL of double-distilled water; reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 60 s, for a total of 28 cycles; 72℃ extension for 5 min.

[0120] 3. Using the genomic DNA of Micro-Tom plants as a template, the PCR amplification products obtained did not contain a 760bp DNA fragment; however, using the genomic DNA of AN2L#2, AN2L#3, AN2L#4, AN2L#6, AN2L#7, AN2L#8, AN2L#9, AN2L#10, AN2L#11, AN2L#12, AN2L#13, AN2L#14, and AN2L#15 plants as templates, the PCR amplification products obtained contained a 760bp DNA fragment. Therefore, AN2L#2, AN2L#3, AN2L#4, AN2L#6, AN2L#7, AN2L#8, AN2L#9, AN2L#10, AN2L#11, AN2L#12, AN2L#13, AN2L#14, and AN2L#15 are all positive T0 generation SlAN2-like gene transgenic tomatoes. A total of 15 tissue culture plants of the T0 generation were transplanted, and 14 of them were positive for hygromycin identification.

[0121] V. Phenotypic Identification of T0 Generation Tomatoes Transformed with SlAN2-like Gene

[0122] The tomatoes under test were observed to have reached the red-ripe stage, and the color of the fruit was then monitored. Some experimental results are shown below. Figure 3 The results showed that the fruits of Micro-Tom were red, while the fruits of AN2L#6 and AN2L#14 had varying degrees of purplish-black spots at the base. Furthermore, the freeze-dried powder of AN2L#6 and AN2L#14 fruits was darker in color compared to the wild-type fruits.

[0123] VI. Identification of anthocyanins in T0 generation tomatoes transgenic with the SlAN2-like gene

[0124] 1. Anthocyanins were detected using HPLC-QQQ-MS / MS. A Poroshell 120EC-C18 column (150 × 2.1 mm, 2.7 μm) was used for separation of anthocyanin compounds. Mobile phase A was 0.1% formic acid, and mobile phase B was an acetonitrile / methanol (50:50, v / v) solution containing 0.1% formic acid. The mobile phase flow rate was 0.4 mL / min. The mobile phase B program was as follows: 0–28 min, 10%–46%; 28–29 min, 46%–10%, and hold for 5 min. Mass spectra of the target compounds were acquired using ESI source positive ion mode and MRM mode. The ion source conditions are as follows: gas temperature, 350℃; gas flow rate, 12L / h; nebulizer, 35psi; sheath gas temperature, 350℃; sheath gas flow rate, 12L / min; capillary positive electrode voltage, 4000V; negative electrode voltage, 4000V.

[0125] 2. Some experimental results are shown below. Figure 4 The results showed that P1 in the high-performance liquid chromatography (HPLC) chromatogram of AN2-like tomato fruits overexpressing Ailsa Craig tomato background was delphinidin-3-glucoside, indicating that only one anthocyanin was present in these tomato fruits. Figure 4 A). Micro-Tom fruits contained no anthocyanins, while AN2L#6 and AN2L#14 fruits produced anthocyanins, and only one high-purity anthocyanin, delphinidin-3-glucoside, was produced. The content of delphinidin-3-glucoside was relatively higher in AN2L#14 fruits (7.83 mg / kg FW), compared to 1.25 mg / kg FW in AN2L#6 fruits. This substance was not detected in WT fruits. Figure 4 B). The above results indicate that overexpression of the SlAN2-like gene can significantly increase the total anthocyanin content of tomatoes and yield tomato material containing high-purity delphinidin-3-glucoside. This tomato material can be used for the separation and purification of delphinidin-3-glucoside, enabling simple, efficient, and low-cost monomeric extraction of delphinidin-3-glucoside.

[0126] The above embodiments are the best implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of the SlAN2-like gene in regulating the content of delphinidin-3-glucoside in tomatoes, characterized in that, The nucleotide sequence of the SlAN2-like gene is shown in SEQ ID NO:

1. Overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside. The tomatoes are selected from the fresh tomato variety Micro-Tom.

2. The application of proteins expressed by the SlAN2-like gene, and vectors or host cells containing the SlAN2-like gene, in regulating the content of delphinidin-3-glucoside in tomatoes, characterized in that... The nucleotide sequence of the SlAN2-like gene is shown in SEQ ID NO:

1. Overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside. The tomatoes are selected from the fresh tomato variety Micro-Tom.

3. A method for creating tomatoes containing high-purity delphinidin-3-glucoside, characterized in that, The method involves overexpressing the SlAN2-like gene with a nucleotide sequence as shown in SEQ ID NO:

1. Overexpression of the SlAN2-like gene can increase the content of delphinidin-3-glucoside in tomatoes and produce only one anthocyanin, delphinidin-3-glucoside. The tomatoes are selected from the fresh tomato variety Micro-Tom.

4. A method for extracting and separating high-purity delphinidin-3-glucoside, characterized in that, The extraction and separation method includes extracting high-purity delphinidin-3-glucoside from tomatoes, wherein the tomatoes are transgenic tomatoes obtained by overexpressing the SlAN2-like gene with a nucleotide sequence as shown in SEQ ID NO:1, and the tomatoes are selected from the fresh tomato variety Micro-Tom.

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