Gene chans encoding anthocyanin synthase and application thereof
By cloning and expressing the anthocyanin synthase (ChANS) gene of Prunus cerasifera and constructing a recombinant expression vector, the problem of weak research on the molecular mechanism of anthocyanin synthesis in Prunus cerasifera was solved, and the anthocyanin content and color of Prunus cerasifera fruit were effectively regulated, promoting the efficient synthesis and enrichment of anthocyanins.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING UNIV OF CHINESE MEDICINE
- Filing Date
- 2026-05-22
- Publication Date
- 2026-06-23
AI Technical Summary
Research on the molecular mechanism of anthocyanin synthesis in Prunus cerasifera is weak, and the ANS gene has not yet been cloned and its function analyzed, which seriously restricts the improvement of Prunus cerasifera color and the breeding of high anthocyanin varieties.
The anthocyanin synthase (ChANS) gene of Prunus cerasifera was cloned and expressed, and a recombinant expression vector was constructed. Through genetic engineering technology, the anthocyanin content and color formation of the fruit were regulated, and colorless anthocyanins were catalyzed to be converted into colored anthocyanins.
This study effectively regulates the anthocyanin content and color formation in Prunus cerasifera fruit, promotes the efficient synthesis and enrichment of anthocyanins, and provides genetic resources and technical support for the improvement of Prunus cerasifera germplasm resources and the optimization of fruit color.
Smart Images
Figure CN122256384A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a gene ChANS encoding anthocyanin synthase and its applications. Background Technology
[0002] Anthocyanins are key secondary metabolites that determine the color of fruits and flowers, possessing strong antioxidant, anti-inflammatory, eye-protecting, and cardiovascular-protective health benefits. They are key targets for breeding functional fruits and ornamental plants. Anthocyanin synthase (ANS) is a key rate-limiting enzyme downstream of the anthocyanin synthesis pathway, responsible for catalyzing the conversion of colorless anthocyanins into colored anthocyanins, directly determining the final production and accumulation of anthocyanins. The expression level and enzyme activity of the ANS gene directly affect plant color phenotype, anthocyanin composition, and content, making it a key functional gene in molecular breeding.
[0003] Prunus cerasifera, a dwarf shrub native to China and belonging to the genus Prunus of the Rosaceae family, produces fruit rich in anthocyanins, calcium, and vitamins, making it a wild fruit tree and functional food ingredient with significant development potential. Currently, research on the molecular mechanisms of anthocyanin synthesis in Prunus cerasifera is weak; its ANS gene has not yet been cloned and its function analyzed, severely hindering research on color improvement, breeding of high-anthocyanin varieties, and the color regulation mechanisms of Rosaceae fruit trees. Summary of the Invention
[0004] The purpose of this invention is to provide an anthocyanin synthase (ChANS) related to anthocyanin synthesis in *Prunus armeniaca*, the encoding gene ChANS, and its applications.
[0005] Therefore, the present invention provides a gene ChANS encoding anthocyanin synthase, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0006] The present invention also provides an expression protein of the above-mentioned gene ChANS, the amino acid sequence of which is shown in SEQ ID NO.2.
[0007] The present invention also provides primers for cloning the gene ChANS, the sequences of which are as follows:
[0008] ChANS-F: ACCATTGCTTCATCAATCCACC;
[0009] ChANS-R:ACTATGCCGCAAATGGCTTC.
[0010] The present invention also provides an anthocyanin synthase encoded by the gene ChANS.
[0011] The present invention also provides a recombinant expression vector containing the ChANS gene.
[0012] The present invention also provides a genetically engineered bacterium containing the recombinant expression vector.
[0013] This invention also provides the application of the gene ChANS in regulating anthocyanin content and color formation in fruits.
[0014] The present invention also provides the application of the gene ChANS, the expressed protein, the anthocyanin synthase, the recombinant expression vector, or the genetically engineered bacteria in the in vitro synthesis of anthocyanins.
[0015] The present invention also provides a method for in vitro synthesis of anthocyanins, comprising the following steps: using colorless anthocyanins as a substrate, and catalyzing the production of anthocyanins with anthocyanin synthase; the amino acid sequence of the anthocyanin synthase is shown in SEQ ID NO. 2.
[0016] Specifically, the aforementioned colorless anthocyanins include colorless cyanidin.
[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0018] The ChANS gene encoding anthocyanin synthase provided in this invention was cloned and prepared from the *Prunus armeniaca* plant. *Prunus armeniaca* ChANS is a key functional enzyme in the anthocyanin biosynthesis pathway, effectively regulating the anthocyanin content and color formation process of the fruit. Based on the ChANS gene provided in this invention, the content of anthocyanins, a functional component, in *Prunus armeniaca* fruit can be directionally increased through genetic engineering techniques, promoting the efficient synthesis and enrichment of anthocyanins. This invention provides important genetic resources and technical support for the preparation of functional compounds containing anthocyanins in *Prunus armeniaca*, the improvement of *Prunus armeniaca* germplasm resources, and the optimization and upgrading of fruit color quality, possessing high scientific research value and application prospects.
[0019] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description
[0020] Figure 1 This is an agarose gel electrophoresis image of the anthocyanin synthase gene ChANS from Prunus armeniaca; where M: marker; 1: BL21(DE3) / E1; 2-4: BL21(DE3) / E1-ChANS.
[0021] Figure 2 These are the results of SDS-polyacrylamide gel electrophoresis of ChANS proteins.
[0022] Figure 3 This is a chromatogram of the colorless cyanidin product catalyzed by the ChANS protein.
[0023] Figure 4This is the mass spectrum of the colorless cyanidin product catalyzed by the ChANS protein. Detailed Implementation
[0024] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.
[0025] The following specific embodiments illustrate the effects of the gene ChANS encoding anthocyanin synthase of the present invention on its application.
[0026] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.
[0027] The TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit, pEASY®-Blunt Zero Cloning Kit, and pEASY®-Blunt E1 Expression Kit were purchased from Beijing TransGen Biotech Co., Ltd.; the GoledenView nucleic acid dye was purchased from Beijing Biomed Co., Ltd.; the Gel Extraction Kit was purchased from Omega Bio-Tek Inc. (USA); 2×Phanta Flash Master Mix (Dye Plus) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the T vector pEASY-Blunt Zero Cloning Kit, E. coli Transetta (DE3), and restriction endonucleases were purchased from Beijing TransGen Biotech Co., Ltd.; the His-tagged protein purification kit (reduction-resistant chelate type) was purchased from Beyotime Biotechnology Co., Ltd.; primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; PBS was purchased from Beijing Lamborghide Biotechnology Co., Ltd.; and other reagents were imported or domestically produced analytical grade reagents. Unless otherwise specified, all reagents were commercially available.
[0028] Example 1: Cloning of the ChANS gene, anthocyanin synthase, from Prunus armeniaca.
[0029] The full-length gene sequence fragment was obtained by annotating and screening the transcriptome data of *Prunus armeniaca*. Using *Prunus armeniaca* cDNA as a template, specific primers ChANS-F and ChANS-R were designed for PCR amplification.
[0030] ChANS-F: ACCATTGCTTCATCAATCCACC (SEQ ID NO.3);
[0031] ChANS-R: ACTATGCCGCAAATGGCTTC (SEQ ID NO. 4).
[0032] Amplification system: 15 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of each primer, 1 μL of template, and the remainder is made up to 30 μL with sterile double-distilled water.
[0033] Reaction conditions: 98℃ pre-denaturation for 30s, 98℃ denaturation for 10s, 57℃ annealing for 5s, 72℃ extension for 9s, 35 cycles followed by 72℃ extension for 1min, and storage at 4℃.
[0034] The PCR product was ligated into the pEASY®-Blunt Zero Cloning vector to obtain the pEASY-ANS vector; the vector was then transformed into trans T1 competent cells, and the sequence of the PCR product was sequenced.
[0035] The PCR product contains a gene whose nucleotide sequence is shown in SEQ ID NO.1. The gene is named ChANS, and the protein encoded by the gene is named ChANS. The amino acid sequence of the protein is shown in SEQ ID NO.2.
[0036] SEQ ID NO.1:
[0037]
[0038] SEQ ID NO.2:
[0039] MVSSDSVNSRVETLASSGIATIPKEYIRPKEELINIGDIFEQEKSTDGPQVPTIDLKEIDSENEKVREKCREELKKAAVDWGVMHLVNHGISDELMDRVRKAGKAFFDLPIEQKEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFHLIYPEDKRDLSIWPQTPADYIEATAEYAKE LRALATKVLRVLSLGLGLEEGRLEKEVGGLEELLLQMKINYYPVCPQPELALGVEAHTDVSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIVMHIGDTIEILSNGKYKSILHRGMVNKEKVRISWAAFCEPPKEKIILKPLPETVSETEPPIFPPRTFAEHIQHKLFRKSQEALLNK.
[0040] Example 2: Function of ChANS, anthocyanin synthase from Prunus armeniaca
[0041] 1. Construction of prokaryotic expression vectors
[0042] Using the pEASY-ChANS vector prepared in Example 1 as a template, PCR amplification was performed using ChANS-F and ChANS-R primers. The amplified fragments were detected by 1% agarose gel electrophoresis and then recovered from the gel. The recovered target fragment was ligated with the expression vector pEASY®-Blunt E1 at 25°C for 30 min to obtain the recombinant plasmid E1-ChANS.
[0043] The recombinant plasmid E1-ChANS was transformed into Escherichia coli Trans1-T1 competent cells, single clones were selected for sequencing, and plasmid E1-ChANS was extracted from positive clones by expanding the bacterial culture.
[0044] BL21(DE3) competent cells were transformed with recombinant plasmid E1-ChANS, and positive strains were screened (those amplified by ChANS-F and ChANS-R to obtain 1074bp were positive), and named BL21(DE3) / E1-ChANS.
[0045] The empty vector pEASY®-Blunt E1 was transformed into BL21(DE3) competent cells to obtain the control recombinant strain BL21(DE3) / E1. The gel electrophoresis results are as follows: Figure 1 As shown.
[0046] 2. Induction and purification of ChANS protein
[0047] Seed culture of positive strain BL21(DE3) / E1-ANS was added to LB medium containing Amp resistance at a ratio of 1:100. The culture was shaken at 37℃ and 200r / min until A600 = 0.6~0.8. IPTG was added to a final concentration of 0.2mM and induced overnight at 16℃.
[0048] Centrifuge the bacterial culture to remove the supernatant and obtain bacterial cells. Resuspend the cells in 6 mL of PBS and sonicate for 40 min in an ultrasonic homogenizer. Perform the operation on ice to avoid generating air bubbles. Centrifuge the sonicated lysate at 4 °C for 15 min to obtain the supernatant.
[0049] The Ni-NTA affinity chromatography column was treated with 0.1 mol / L EDTA solution to elute the original nickel ions on the column. The column was then washed with 5 column volumes of double-distilled water, followed by regeneration with 0.2 mol / L nickel sulfate solution. The column was equilibrated with 5 column volumes of Ni-NTA Binding Buffer. The supernatant sample was slowly injected into the treated Ni-NTA affinity chromatography column to ensure sufficient binding of the His-tagged target protein to the nickel ions on the column. The flow-through was collected for later use. After complete sample loading, the column was washed with approximately 5 column volumes of Ni-NTA Binding Buffer, followed by washing with Ni-NTA Washing Buffer until the baseline stabilized (detection wavelength 280 nm) to remove non-specifically bound proteins. Gradient elution was performed using Ni-NTA Elution Buffer. Pump A was set to Ni-NTA Binding Buffer, and pump B to Ni-NTA Elution Buffer at a flow rate of 5 mL / min. The concentration of pump B was linearly increased from 0% to 50% in a 100 mL feed volume, and the elution peak fraction was collected. A small amount of the eluted sample was used to prepare an SDS-PAGE electrophoresis sample, which was boiled in a metal bath at 100℃ for 1 min, followed by SDS-PAGE electrophoresis (220V constant voltage, 45 min). After Coomassie brilliant blue staining and destaining, the protein purification status was determined based on the electrophoresis results. The elution fraction containing the target protein was selected, dialyzed to remove imidazole, and purified recombinant Prunus armeniaca ANS protein was obtained.
[0050] SDS-PAGE gel electrophoresis results are as follows: Figure 2 As shown, a distinct specific protein expression band appears in BL21(DE3) / E1-ChANS at a molecular weight of approximately 43 kDa, consistent with the theoretical value.
[0051] 3. Verification of in vitro enzyme function expressed in prokaryotes
[0052] Colorless cyanidin (Shanghai Yuanye Biotechnology Co., Ltd., B23203) was used as the substrate for in vitro enzyme function verification. The reaction system consisted of 20 μL substrate (1 mmol / L), 10 μL of the target protein ChANS solution obtained in step 2 above, and 70 μL PBS. The mixture was incubated at 35°C for 1 h. The reaction was terminated by adding one volume of methanol to obtain the reaction product.
[0053] Replace the substrate with an equal volume of PBS solution as a blank control sample.
[0054] The reaction products were detected by HPLC under the following conditions: mobile phase A was water (containing 1% formic acid) and mobile phase B was acetonitrile (containing 1% formic acid). Gradient elution was employed under the following conditions: 0.02–2.0 min, 92.0%–88.0% A; 2.0–5.0 min, 88.0%–82.0% A; 5.0–8.0 min, 82.0%–80.0% A; 8.0–10.0 min, 80.0%–75.0% A; 10.0–13.0 min, 75.0%–70.0% A; 13.0–16.0 min, 70.0%–55.0% A; 16.0–18.0 min, 55.0%–92.0% A; 18.0–22.0 min, 92.0%–92.0% A; volumetric flow rate: 0.8 mL / min; detection wavelength: 520 nm; injection volume: 10 μL; column temperature: 35 °C.
[0055] The results are as follows Figure 3 As shown, the retention time of cyanidin standard (Shanghai Yuanye Biotechnology Co., Ltd., V98626) in HPLC is 8.2 min. The product obtained after the reaction of the target protein ChANS solution has a characteristic peak at a retention time of 8.2 min, while no corresponding characteristic peak was detected in the blank control sample. This indicates that the target protein ChANS is an anthocyanin synthase that can catalyze the conversion of colorless cyanidin to cyanidin.
[0056] The eluent with the characteristic peak at a retention time of 8.2 min was qualitatively detected using LC-MS.
[0057] Chromatographic conditions: Waters ACQUITY UPLCT3 column (2.1 mm × 100 mm, 1.7 μm, Waters, USA); mobile phase A: ultrapure water (with 0.1% acid added); mobile phase B: acetonitrile (with 0.1% formic acid added). Elution gradient: 0.00–9.00 min, 5%–95% B; 9.00–10.00 min, 95% B; 10.00–11.10 min, 95%–5% B; equilibrate to 5% for 14 min; flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL; flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 1 μL.
[0058] Mass spectrometry conditions: Electrospray ionization (ESI) temperature 500°C; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, with collision-induced ionization parameters set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. DP and CE for each MRM ion pair were optimized through further declustering voltage (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.
[0059] The results are as follows Figure 4 As shown, the sample exhibits a characteristic peak of cyanidin (primary ion [m+H]) at 4.42 min. + (m / z 287.06, secondary fragment ion: m / z 287.06), identified by MS analysis as cyanidin, thus further confirming that ChANS has the activity of catalyzing the synthesis of cyanidin from colorless cyanidin.
[0060] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.
Claims
1. A gene ChANS encoding anthocyanin synthase, characterized in that: The nucleotide sequence of the gene ChANS is shown in SEQ ID NO.
1.
2. The expression protein of the gene ChANS according to claim 1, characterized in that: The amino acid sequence of the expressed protein is shown in SEQ ID NO.
2.
3. Primers for cloning the ChANS gene as described in claim 1, characterized in that, The primer sequences are: ChANS-F: ACCATTGCTTCATCAATCCACC; ChANS-R: ACTATGCCGCAAATGGCTTC.
4. An anthocyanin synthase encoded by the gene ChANS as described in claim 1.
5. A recombinant expression vector containing the ChANS gene of claim 1.
6. A genetically engineered bacterium containing the recombinant expression vector of claim 5.
7. The application of the gene ChANS as described in claim 1 in regulating anthocyanin content and color formation in fruits.
8. The application of the gene ChANS as described in claim 1, the expressed protein as described in claim 2, the anthocyanin synthase as described in claim 4, the recombinant expression vector as described in claim 5, or the genetically engineered bacteria as described in claim 6 in the in vitro synthesis of anthocyanins.
9. A method for synthesizing anthocyanins in vitro, characterized in that, Includes the following steps: Anthocyanins are produced by using colorless anthocyanins as a substrate and anthocyanin synthase as a catalyst; the amino acid sequence of the anthocyanin synthase is shown in SEQ ID NO.
2.
10. The method for synthesizing anthocyanins in vitro as described in claim 9, characterized in that: The colorless anthocyanins include colorless cyanidin.