VvNAC53, application and method for regulating content of terpenoid compounds in grape

Abstract

This invention relates to the field of genetic engineering technology, specifically to the grape transcription factor VvNAC53, its application, and a method for regulating the content of terpenoids in grapes. The nucleotide sequence encoding the grape transcription factor VvNAC53 is shown in SEQ ID NO.1. VvNAC53 can be used to regulate the content of terpenoids in grapes. The method involves transferring the grape transcription factor VvNAC53 into grapes and overexpressing it, thereby reducing the content of terpenoids in the grape fruit and enabling the targeted cultivation of grape germplasm with different aroma profiles.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the grape transcription factor VvNAC53, its application, and methods for regulating the content of terpenoid compounds in grapes. Background Technology

[0002] Grapes (Vitis vinifera L.) are an important economic crop widely cultivated worldwide. Their fruit is not only rich in nutrients but also possesses a unique flavor and aroma, making them popular for fresh consumption, winemaking, or drying. The aromatic compounds in grapes are mainly distributed in the skin and pulp. The skin is a crucial source of aroma compounds, especially volatile compounds such as terpenes, aldehydes, and esters, which play a key role in the aroma characteristics of grapes. The pulp also contains a certain amount of aroma compounds, but their concentration is usually lower than that of the skin. Therefore, in the winemaking process, skin maceration is often performed before or after fermentation, allowing the grape juice to come into close contact with the skin. This promotes the dissolution of volatile compounds from the grape skin, such as terpenes for floral and fruity aromas and aldehydes for spicy aromas, into the wine, giving it a rich and complex aroma.

[0003] Terpenes are an important component of grape aroma, with monoterpenes contributing primarily to floral and fruity aromas, directly influencing the aroma quality of grapes and the flavor characteristics of wine. Based on terpenoid content, grape varieties can be classified into three categories: non-aromatic (below 1 mg / L), non-Muscat aromatic (1-4 mg / L), and Muscat (above 6 mg / L). Muscat grapes have the highest terpenoid content and a rich aroma, but excessive terpenes can make the aroma too strong, masking other aroma components and resulting in a singular and unharmonious aroma. Different grape varieties also exhibit significant differences in terpenoid composition. For example, the main free monoterpenoids in Muscat grapes are linalool, trans-β-ocimene, β-cis-ocimene, geraniol, and β-myrcene, while the main free monoterpenoids in strawberry grapes are trans-β-ocimene, γ-terpinene, and β-citronellol. Limonene and β-myrcene are the main free monoterpenoids in neutral grapes. Furthermore, geraniol, linalool, and terpineol were detected in white grape varieties in Spanish non-aromatic grape tests, but not in red grape varieties. The composition and accumulation of terpenoids in fruit are mainly influenced by their genotype, transcription factor regulation, and climatic factors.

[0004] Transcription factors are a class of protein molecules that specifically recognize and bind to particular gene sequences, regulating the expression levels of target genes under specific spatiotemporal conditions, thereby influencing the synthesis and accumulation of plant secondary metabolites. As core components of the plant secondary metabolism regulatory network, transcription factors precisely regulate the synthesis of secondary metabolites by activating the transcription of key functional genes in metabolic pathways. This regulatory mechanism plays a crucial role in plant secondary metabolism, providing important theoretical basis and technical support for metabolic engineering and synthetic biology.

[0005] Currently, the reported transcription factors regulating terpene synthesis in plants mainly originate from the ARF, MYB, bHLH, MYC, WRKY, AP2 / ERF, and bZIP families, and all of them are transcription factors that regulate terpene synthase metabolism. For example, overexpression of CrWRKY1 in periwinkle can downregulate the expression levels of ORCA2 / 3, CrMYC2, and ZCTs, thereby regulating monoterpene synthesis. In Masson pine, PmWRKY33 regulates terpene synthesis by affecting the expression of NtDXS1 and NtDXS2. In peony, overexpression or silencing of PsMYB7 and PsMYB18 can regulate the expression of PsDXS, PsGPPS, PsTPS5, PsTPS9, and PsDXR genes, thereby regulating the terpene content in peony. In grape berries, studies have also shown that Agrobacterium-mediated transformation leading to overexpression of VvbZIP61 in grape callus significantly increased the accumulation of several monoterpenes, including hesperidin, linalool, geraniol, geraniol, β-myrcene, and D-limonene. The WRKY family transcription factor VviWRKY40 can regulate the expression of VviGT14, thereby affecting the accumulation of glycoside-bound monoterpenes in 'Little White Rose' grapes.

[0006] However, the specific regulatory mechanisms of the NAC family in grape terpene metabolism remain unclear. Identifying and regulating key transcription factors that regulate terpene synthesis in grapes is crucial for understanding the molecular mechanisms of grape aroma formation. Furthermore, how to utilize these transcription factors for targeted breeding to improve grape aroma quality and wine flavor requires further exploration. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for regulating the content of grape transcription factor VvNAC53, its application, and terpenoid compounds.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] The present invention provides a grape transcription factor VvNAC53, wherein the coding region nucleotide sequence of the grape transcription factor VvNAC53 is shown in SEQ ID NO.1.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, the amino acid sequence of the grape transcription factor VvNAC53 is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant vector comprising the coding region nucleotide sequence as described above for encoding the grape transcription factor VvNAC53.

[0013] Furthermore, primer pairs are used for construction, the primer pairs including an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0014] Furthermore, the backbone plasmid of the recombinant vector is pCAMBIA1300.

[0015] The present invention also provides an engineered bacterium containing the recombinant vector as described above.

[0016] The present invention also provides an application of the grape transcription factor VvNAC53 as described above, which can be used to regulate the content of terpenoids in grapes.

[0017] This invention also provides a method for regulating the content of terpenoid compounds in grapes, using the grape transcription factor VvNAC53 as described above.

[0018] Furthermore, the regulation method includes the following steps: transferring the grape transcription factor VvNAC53 into grapes and overexpressing it to reduce the content of terpenoids in grape fruits.

[0019] Furthermore, the terpenoids include free linalool, p-cymene, nerol, and bound trans-rose oxide.

[0020] The beneficial effects of this invention are as follows:

[0021] The grape transcription factor VvNAC53 can be used to regulate the content of terpenoids in grapes. By overexpressing the VvNAC53 gene in grapes, grape plants with lower terpenoid content compared to wild types can be obtained, effectively reducing the content of some terpenoid compounds in grape skins. This allows for the targeted breeding of grape germplasm with different aroma types and accelerates the breeding process. Attached Figure Description

[0022] Figure 1This is an agarose gel electrophoresis image of the grape transcription factor VvNAC53 of the present invention;

[0023] Figure 2 The relative expression levels of the VvNAC53 gene in grape skins transiently transformed with VvNAC53 and in grape skins without any transformation treatment are compared in Experiment 1, which is the grape transcription factor VvNAC53 of the present invention.

[0024] Figure 3 The figure shows a comparison of terpene content in grape skins transiently transformed with VvNAC53 and grape skins without any transformation treatment, based on the grape transcription factor VvNAC53 of this invention. Figure 3 In this context, 'a' represents free linalool. Figure 3 b represents free p-cymene. Figure 3 c represents free nerol. Figure 3 In the figure, d represents the bound state of trans-oxidized rose, and * indicates that P < 0.05. Detailed Implementation

[0025] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] The grape transcription factor VvNAC53 of the present invention has a coding region nucleotide sequence as shown in SEQ ID NO.1. This grape transcription factor VvNAC53 can be used to regulate the content of terpenoids in grapes.

[0027] The grape transcription factor VvNAC53 of this invention belongs to the NAC class of transcription factors. It participates in the regulation of terpene metabolism in grapes. By overexpressing the VvNAC53 gene in grapes, the content of some terpene compounds in grape skins can be effectively reduced, thereby enabling the targeted breeding of grape germplasm with different aroma types.

[0028] Specifically, the gene encoding VvNAC53 has a DNA length of 6123 bp and a cDNA length of 1680 bp, encoding a total of 559 amino acids. The amino acid sequence is shown in SEQ ID NO.2.

[0029] The present invention relates to a method for regulating the content of terpenoids in grapes by transferring the grape transcription factor VvNAC53 into grapes and overexpressing it, thereby reducing the content of terpenoids in grape fruits.

[0030] Preferably, the terpenoids include free linalool, p-cymene, nerol, and bound trans-rose oxide.

[0031] Specifically, a recombinant vector containing the VvNAC53 nucleotide sequence can be constructed, and then the recombinant vector can be transferred into engineered bacteria. The engineered bacteria can then be used to infect cells of grape fruits or peels to overexpress the grape transcription factor VvNAC53, thereby reducing the content of terpenoids.

[0032] In one embodiment of the present invention, the specific steps are as follows:

[0033] (1) The DNA of grape transcription factor VvNAC53 was obtained by cloning. The specific experimental procedure is as follows:

[0034] The RNA from grape skins (extracted using an RNA extraction kit) was reverse-engineered into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The specific systems for each step are shown in Tables 1-3.

[0035] Table 1. Step-1 Reverse Transcription System and Reaction Conditions

[0036]

[0037] Table 2. Second step reverse transcription system and reaction conditions

[0038]

[0039] Table 3. Reverse transcription system and reaction conditions for step 3

[0040]

[0041]

[0042] Specific primers were designed using SnapGene, and the full-length cDNA of VvNAC53 was amplified by PCR. The PCR system is shown in Table 4, the primer sequences are shown in Table 5, and the PCR reaction extent is shown in Table 6.

[0043] Table 4. PCR amplification reaction system and procedure

[0044] Components volume cDNA template 1.0μL upstream primer 0.5μL Downstream primer 0.5μL 2×Phanta Flash Master Mix(Dye Plus) 5μL <![CDATA[ddH2O]]> 3μL system 10μL

[0045] Table 5 Primer sequences

[0046]

[0047] Table 6 PCR reaction procedures

[0048]

[0049] The target fragment was detected by 1% agarose gel electrophoresis, and three parallel experiments were performed. In one embodiment of the present invention, the electrophoresis results are as follows: Figure 1 As shown in the image. Comparison of the markers reveals that the DNA length of all three bands is approximately 1680 bp, which corresponds to the VvNAC53 target band, indicating successful cloning. After electrophoresis, the target fragment was recovered using the Hipure Gel Pure DNA Mini Kit agarose gel DNA recovery kit.

[0050] (2) Construction of recombinant vector (overexpression vector) pCAMBIA1300-VvNAC53.

[0051] The plasmid of the overexpression vector pCAMBIA1300 was extracted, and pCAMBIA1300 was double-digested with restriction enzymes SacI and BamHI. After digestion, the pCAMBIA1300 vector was purified to obtain a linearized vector.

[0052] The gel recovery products were ligated to the linearization vector pCAMBIA1300 using the One Step Cloning Kit C112. The ligation system is shown in Table 7.

[0053] Table 7. Connection Reaction System and Reaction Conditions

[0054]

[0055] The ligation system was transformed into DH5α competent cells. The cloned competent cells were thawed on ice. 10 μL of the recombinant product was added to 50 μL of competent cells, and the mixture was gently tapped to mix. The cells were then placed on ice for 30 min, heat-shocked at 42°C for 45 sec, and incubated on ice for another 5 min. Under aseptic conditions, 800 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 220 rpm for 1 h on a shaker. The cells were centrifuged at 4000 × g for 5 min, and 500 μL of supernatant was discarded. The cells were resuspended in the remaining medium, and 100 μL was spread onto a solid medium containing kanamycin. The cells were incubated upside down at 37°C for 16 h until colonies reached approximately 2 mm in diameter. The colonies were then identified using the system described in Table 8 via PCR. Positive clones were selected for sequencing, and a portion of the bacterial culture was mixed with glycerol at a 1:1 ratio and stored at -80°C.

[0056] Table 8 Bacterial PCR System

[0057]

[0058] Dip a single colony into a pipette tip, pipette twice to dissolve it in 10 μL of LB broth, and then use 1 μL as a template. Extract the plasmid from the correctly sequenced positive clone to obtain the recombinant plasmid pCAMBIA1300-VvNAC53.

[0059] (3) Construction of recombinant bacteria.

[0060] Agrobacterium transformation was performed using the freeze-thaw method. Competent Agrobacterium GV3101 cells were thawed on ice. After thawing, 10 μL of recombinant plasmid was added to 100 μL of competent cells, and the mixture was gently pipetted to mix. The cells were then sequentially placed on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, incubated in a 37°C water bath for 5 min, and then incubated on ice for 5 min. In a clean bench, 900 μL of antibiotic-free liquid LB medium was added, mixed thoroughly, and incubated at 28°C with shaking at 200 rpm for 3 h. The cells were harvested by centrifugation at 4000×g for one minute. Approximately 100 μL of the supernatant was collected, gently resuspended, and spread onto LB + Rif (50 μg / mL) + Kan (50 μg / mL) solid medium. After drying, the medium was inverted and incubated in the dark at 28°C for 3 days.

[0061] Select a single colony of GV3101 and inoculate it into 4 mL of LB broth + Rif (50 μg / mL) + Kan (50 μg / mL). Incubate at 28°C and 200 rpm for 16 h. Perform PCR detection on positive clones. Inoculate 150 μL of the bacterial culture from the positive clone into 4 mL of the above-mentioned double-antibiotic LB broth and incubate at 28°C and 200 rpm for 5-8 h until OD (Organic Dioxide) is reached. 600 The concentration is 0.6-0.8. Take 1 mL of the bacterial culture, add an equal volume of sterilized glycerol, quick-freeze in liquid nitrogen, and store at -80℃.

[0062] (4) Obtain VvNAC53 instantaneous conversion of grape fruits.

[0063] Add 100-200 μL of the Agrobacterium tumefaciens bacterial suspension containing the target gene and the GV3101 empty vector bacterial suspension to 4-5 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium, respectively, and incubate at 28°C for 16 h with shaking. Transfer the activated bacterial suspension to 40 mL of LB (Kan + Rif) medium for subculture. Incubate the second-generation culture at 28°C and 220 rpm for 6-7 h until OD (Organic Demand) is reached. 600 To achieve an OD of 1-1.2, centrifuge the second-generation culture at 4000×g for 5 min and resuspend it 1:1 in Agrobacterium infection buffer. Centrifuge three times to discard the supernatant and wash the bacterial cells. Resuspend the second-generation culture pellet again in Agrobacterium infection buffer to an OD of 1-1.2. 600 The pH should be 0.9, and the solution should be allowed to stand at room temperature in the dark for 3 hours. The specific preparation of the Agrobacterium infection buffer is shown in Table 9, and the pH needs to be adjusted to 5.2 using KOH.

[0064] Each grape berry was punctured with 30 holes using a syringe and immersed in the infection solution. A vacuum was applied for 15 minutes, during which the berries were continuously agitated to ensure the infection solution penetrated the berries. The berries were then removed and cultured naturally for 3 days and 3 nights. Samples of the resulting grapes were then frozen in liquid nitrogen and stored at -80°C. The preparation of the infection solution is shown in Table 9. The pH was adjusted to 5 using KOH before use.

[0065] Table 9. Infection solution ratio

[0066] Components Added amount MES 2.13g <![CDATA[MgCl2·6H2O]]> 2.03g Add acetylsuccione (As) before use. 200μM sterile water 1L

[0067] Through the specific steps described above, the VvNAC53 gene can be successfully transferred into grapes and overexpressed. This treatment yields grapevines with lower terpene content compared to wild-type grapes, which plays a positive role in the targeted breeding of new varieties with different aroma profiles and in accelerating the breeding process.

[0068] The transcription factor of this invention that regulates the synthesis of grape terpenoids is of great significance for elucidating the molecular mechanism of grape fruit aroma formation and for improving the aroma quality of grape fruit using genetic engineering techniques.

[0069] The present invention will be specifically described below through examples. Unless otherwise specified, the experimental materials, instruments and reagents used in the following examples were obtained through conventional means.

[0070] Example 1: Relative Gene Expression Analysis of VvNAC53 Transiently Transformed Grape Skin

[0071] The experimental procedure in this embodiment is as follows:

[0072] RNA (extracted using an RNA extraction kit) was reverse-engineered into cDNA using the HiScript IIQ RT SuperMix for qPCR + gDNA wiper (R223) kit for qRT-PCR. The specific steps are as follows:

[0073] (1) Genomic DNA removal: Add 500 ng of RNA to an RNase-Free tube, add 4 μL of 4×g DNA wiperMix, then add RNase-ddH2O to a total liquid volume of 16 μL. Gently mix with a pipette and place in a PCR instrument. React at 42℃ for 2 min.

[0074] (2) Prepare the first strand cDNA synthesis reaction solution: Add 4 μL of 5×HiScript II qRTSuperMix II to the above system, place it in a PCR instrument, react at 55℃ for 15 min, react at 85℃ for 5 s, terminate the reaction, and store the product at -20℃ for later use.

[0075] In this embodiment, Ubiquitin was used as an internal reference gene for quantitative real-time PCR. Reagents were added according to the reaction system in Table 10, and the reaction was performed in triplicate. The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, 39 cycles; melting curve analysis was performed from 65℃ to 95℃. Primer sequences are shown in Table 11.

[0076] Table 10. Real-time PCR reaction system

[0077]

[0078]

[0079] Table 11 Primer sequences

[0080]

[0081] The results of quantitative real-time PCR are as follows Figure 2 As shown in the figure, "OE-VvNAC53" indicates that the VvNAC53 of this invention instantaneously transforms grape skins, and "CK" indicates grape skins without any transformation treatment. Figure 2 It can be seen that the expression level of VvNAC53 increased by 1.76 times in the overexpressing plants.

[0082] Experimental Example 2: Extraction and Detection of Terpenoids from Grape Peels Transformed by VvNAC53

[0083] In this embodiment, the contents of terpenoids were extracted from grape skins transiently transformed with VvNAC53 (OE-VvNAC53) and grape skins without any transformation treatment (CK) and compared to verify the regulatory effect of VvNAC53 overexpression on the content of terpenoids.

[0084] The extraction process of terpenoids in this embodiment is as follows:

[0085] Immediately after peeling the grapes, they were frozen with liquid nitrogen, ground into powder using a grinder pre-cooled with liquid nitrogen, and stored at -80℃. 1g of powder was added to 5mL of citric acid / phosphate buffer (0.2mol / L, pH=5.0), soaked at 4℃ for 16h, centrifuged at 5000×g for 15min at 4℃, and the supernatant was used for subsequent extraction and detection of aroma substances.

[0086] Solid phase extraction (SPE) was used to extract bound terpenoids from the pericarp. The extraction process and conditions were as follows: A Cleanert PEP-SP (150 mg / 6 mL) extraction column was activated with 10 mL of anhydrous methanol and 10 mL of purified water to remove impurities from the extraction column; then 2 mL of the supernatant was added, and the column was eluted sequentially with 5 mL of purified water and 5 mL of dichloromethane to remove pigments, low molecular weight sugars, acids and other polar compounds, as well as free nonpolar aroma compounds; finally, 20 mL of anhydrous methanol was used to elute the glycoside-bound aroma substances into a test tube. The flow rate was maintained at 2 mL / min throughout the elution process to obtain a methanol solution of glycoside aroma substances. The collected methanol solution containing glycosidic aroma compounds was evaporated to dryness using a rotary evaporator at 30℃, and then reconstituted with 10 mL of citric acid / phosphate buffer solution (0.2 mol / L, pH = 5.0). After reconstitution, 100 μL of glycosidase AR2000 (100 g / L) was added, and the solution was incubated at 37℃ for 16 h. After enzymatic hydrolysis, the pH of the hydrolysate was adjusted to 3.0 with citric acid. The hydrolysate was used for the detection of glycosidic aroma compounds.

[0087] Free and bound terpenoids in grape skins were detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS). Specific conditions were as follows: an Agilent 6890 gas chromatograph equipped with an Agilent 5975 mass spectrometer was used for GC-MS; the capillary column used was a polar HP-INNOWAX column (60m × 0.25mm × 0.25μm, J&W Scientific, Folsom, CA, USA).

[0088] Add 5 mL of fruit peel extract or enzymatic hydrolysate and 1.0 g of sodium chloride to a 20 mL gas chromatograph sample vial, along with 10 μL of internal standard (4-methyl-2-pentanol, 1.0 g / L). Seal the vial with a cap fitted with a polytetrafluoroethylene septum. HS-SPME pretreatment was performed using a CTC CombiPAL multi-functional autosampler (CTC Analytic, Zwingen, Switzerland). After equilibration at 40 °C for 30 min, the activated polydimethylsiloxane / carbon sieve / divinylbenzene (DVB / CAR / PDMS) 50 / 30 μm extraction head (Supelco, Bellefonte, PA, USA) was inserted into the headspace of the sample vial, and the sample was extracted with shaking at 500 rpm for 30 min. After extraction, the extraction head was inserted into the GC inlet at 250 °C, and splitless injection thermal desorption was performed for 8 min. The carrier gas was helium (purity > 99.999%) at a flow rate of 1 mL / min. Temperature program: 50℃ held for 1 min; then increased to 220℃ at a rate of 3℃ / min and held for 5 min. The mass spectrometry interface temperature was 250℃, the mass spectrometry ion source was an electron ionization (EI) source with an ion energy of 70 eV, and the mass scan range was 29-350 u.

[0089] Qualitative analysis was performed based on the retention index and mass spectrometry information of the compound under the same chromatographic conditions. For aroma compounds without standards, semi-qualitative analysis was performed using the retention index of the compound under similar chromatographic conditions reported in the literature and the comparison results with the NIST05 standard spectral library (NIST Chemistry WebBook, http: / / webbook.nist.gov / chemistry / ). For aroma compounds for which the retention index under similar chromatographic conditions was not reported in the literature, semi-qualitative analysis was performed based on the comparison results with the NIST05 standard spectral library.

[0090] The quantification of aroma substances is analyzed using the corresponding standard curve: for aroma substances with available standards, the standard curve of the corresponding standard is used for direct quantification; for aroma substances without available standards, the standard curve of a standard with a similar chemical structure is used for semi-quantification. The content of terpenoids is expressed as ng / g.

[0091] The results of terpene component detection in grape skins of OE-VvNAC53 and CK groups are as follows: Figure 3 As shown.

[0092] Depend on Figure 3It was found that the contents of free linalool, p-cymene, nerol, and bound trans-oxidized rose were significantly reduced in grape skins overexpressing the transcription factor VvNAC53. Among them, the content of free linalool was reduced by 1.5 times compared with the control group CK, indicating that VvNAC53 participates in the regulation of terpene metabolism in grapes and can significantly reduce the contents of the above-mentioned terpene components.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A grape transcription factor VvNAC53 The application is characterized by, The application method is to use the grape transcription factor VvNAC53 Transformed into grapes and overexpressed, it reduces the content of terpenoids in grape berries; used to encode the grape transcription factor. VvNAC53 The coding region nucleotide sequence is shown in SEQ ID NO.1; the terpene substance is free linalool, p - At least one of umbelliferone, nerol, and bound trans-oxidized rose.

2. A method for regulating the content of terpenoid compounds in grapes, characterized in that, The grape transcription factor encoded by the nucleotide sequence as shown in SEQ ID NO. 1 VvNAC53 is introduced into grape and overexpressed, reducing the content of terpenoids in grape fruits, said terpenoids being at least one of: p - linalool, geraniol, at least one of trans-oxiderosa, in bound state.

3. A method for regulating the content of terpenoid compounds in grapes, characterized in that, The amino acid sequence of the grape transcription factor is shown in SEQ ID NO.

2. VvNAC53 Regulation is performed; the regulation method is to regulate the grape transcription factor. VvNAC53 The substance was transferred into grapes and overexpressed, reducing the content of terpenoids in the grape berries; the terpenoids were free linalool, p - At least one of umbelliferone, nerol, and bound trans-oxidized rose.

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