Grape transcription factor VvNAC53, application and method for regulating content of terpenoids in grapes

Abstract

The invention relates to the technical field of gene engineering, in particular to a grape transcription factor VvNAC53, application of the grape transcription factor VvNAC53 and a method for regulating and controlling the content of terpenoids in grapes. The nucleotide sequence for coding the grape transcription factor VvNAC53 is as shown in SEQ ID NO. 1; the content of terpenoids in grapes can be regulated and controlled by adopting VvNAC53; the regulation and control method comprises the following steps: transferring a grape transcription factor VvNAC53 into grapes and performing overexpression to reduce the content of terpenoids in grape fruits so as to directionally cultivate grape germplasm with different aroma types.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular, to a grape transcription factor VvNAC53, its application, and a method for regulating the content of terpenoids in grapes. Background Art

[0002] Grapes (Vitis vinifera L.) are important economic crops widely planted worldwide. Their fruits are not only rich in nutrients but also have unique flavors and aromas. They can be used for fresh consumption, winemaking, or drying, and are deeply loved by consumers. The aroma substances in grape fruits are mainly distributed in the pericarp and pulp. The pericarp is an important source of aroma substances, especially volatile compounds such as terpenes, aldehydes, esters, etc. These substances play a key role in the aroma characteristics of grapes. The pulp also contains a certain amount of aroma substances, but their concentration is usually lower than that of the pericarp. Therefore, during the winemaking process, skin maceration is often carried out before and after fermentation to allow the grape juice to come into close contact with the pericarp, promoting the dissolution of volatile compounds in the grape skin, such as floral and fruity terpenes and spicy aldehydes, into the wine, giving the wine a rich and layered aroma.

[0003] Terpenoids are an important component of the aroma of grape fruits. Among them, monoterpenes mainly contribute to floral and fruity aromas, directly affecting the aroma quality of grape fruits and the flavor characteristics of wines. According to the content of terpenoids, grape varieties can be divided into non-aromatic types (below 1 mg / L), non-muscat aromatic types (1 - 4 mg / L), and muscat types (above 6 mg / L). Muscat grapes have the highest content of terpenoids and a strong aroma, but their excessive terpenoids may make the aroma too strong, masking other aroma components and resulting in a single and uncoordinated aroma. There are also significant differences in the terpenoid composition among different grape varieties. For example, the main free monoterpenoid compounds in muscat grapes are linalool, trans-β-ocimene, cis-β-ocimene, geraniol, and β-myrcene, while the main free monoterpenoid compounds in strawberry-type grapes are trans-β-ocimene, γ-terpinene, and β-citronellol. Limonene and β-myrcene are the main free monoterpenoid compounds in neutral-type grapes. In addition, in the detection of terpenoids in non-aromatic grapes in Spain, geraniol, linalool, and terpineol were detected in white grape varieties, but not in red grape varieties. The composition and accumulation of terpenoids in fruits are mainly affected by their own genotypes, transcription factor regulation, and climate factors.

[0004] Transcription factors are a class of protein molecules that can specifically recognize and bind to specific gene sequences, regulate the expression level of target genes under specific spatio-temporal conditions, and thus affect the synthesis and accumulation of plant secondary metabolites. As the core components in the plant secondary metabolism regulation network, transcription factors achieve precise regulation of the synthesis of secondary metabolites by activating the transcription of key functional genes in the metabolic pathway. This regulation mechanism plays an important role in the process of 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 come from families such as ARF, MYB, bHLH, MYC, WRKY, AP2 / ERF, bZIP, etc., and they are all transcription factors regulating the metabolism of terpene synthases. For example, overexpression of CrWRKY1 in Catharanthus roseus can down-regulate the expression levels of ORCA2 / 3, CrMYC2, and ZCTs, thereby regulating the synthesis of monoterpenes. In Pinus massoniana, PmWRKY33 regulates the synthesis of terpenoids by affecting the expression of NtDXS1 and NtDXS2. Overexpression or silencing of PsMYB7 and PsMYB18 in Paeonia suffruticosa can regulate the expression of genes such as PsDXS, PsGPPS, PsTPS5, PsTPS9, and PsDXR, thus regulating the content of terpenoids in Paeonia suffruticosa. In grape berries, there is also a study that overexpression of VvbZIP61 in grape callus by Agrobacterium-mediated transformation significantly increased the accumulation of several monoterpenes in the callus, including syringol, linalool, geranial, geraniol, β-myrcene, and D-limonene. The WRKY family transcription factor VviWRKY40 can regulate the expression of VviGT14, thus affecting the accumulation of glycoside-bound monoterpenes in 'Xiaobai Rose'.

[0006] However, the specific regulation mechanism of the NAC family in the terpene metabolism of grape berries is still unclear. Exploring and identifying key transcription factors regulating the synthesis of terpenoids in grape berries is of great significance for analyzing the molecular mechanism of grape berry aroma formation. In addition, how to use these transcription factors for directional breeding to improve the aroma quality of grape berries and the flavor of wine still needs further exploration. Summary of the Invention

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

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

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

[0010] Based on the above technical solution, the present invention can also be 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, which comprises the coding region nucleotide sequence for encoding the grape transcription factor VvNAC53 as described above.

[0013] Furthermore, a primer pair is used for construction, wherein the primer pair includes 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, wherein the engineered bacterium contains the above-mentioned recombinant vector.

[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 terpenoid substances in grapes.

[0017] The present invention also provides a method for regulating the content of terpenoid compounds in grapes, which uses the grape transcription factor VvNAC53 as mentioned above for regulation.

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

[0019] Furthermore, the terpenoid substances include free linalool, p-cymene, nerol, and bound trans-oxidized rose.

[0020] The beneficial effects of the present invention are:

[0021] The grape transcription factor VvNAC53 can be used to regulate the content of terpenes in grapes. By overexpressing the VvNAC53 gene in grapes, grape plants with lower terpenes than the wild type can be obtained, effectively reducing the content of some terpenoid compounds in the grape skin, thereby cultivating grape germplasm with different aromas and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Agarose gel electrophoresis pattern of grape transcription factor VvNAC53 of the present invention;

[0023] Figure 2 For grape transcription factor VvNAC53 of the present invention, in the grape pericarp with transient transformation of VvNAC53 and the grape pericarp without any transformation treatment in Experimental Example 1, the comparative diagram of the relative expression levels of the VvNAC53 gene;

[0024] Figure 3 For grape transcription factor VvNAC53 of the present invention, in Experimental Example 2, the comparative diagram of the terpene content in the grape pericarp with transient transformation of VvNAC53 and the grape pericarp without any transformation treatment, Figure 3 where a is free linalool, Figure 3 where b is free p - cymene, Figure 3 where c is free nerol, Figure 3 where d is bound trans - rose oxide, * indicates P < 0.05. Detailed implementation manners

[0025] The principles and features of the present invention will be described below in conjunction with 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] For grape transcription factor VvNAC53 of the present invention, the nucleotide sequence of its coding region is shown as SEQ ID NO.1. This grape transcription factor VvNAC53 can be used to regulate the content of terpenoids in grapes.

[0027] Grape transcription factor VvNAC53 of the present invention belongs to the NAC - type transcription factor. It participates in the regulation of terpenoid metabolism in grapes. By overexpressing the VvNAC53 gene in grapes, the content of some terpenoid compounds in the grape pericarp can be effectively reduced, thereby enabling the directional cultivation of grape germplasms with different fragrance types.

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

[0029] The method for regulating the content of terpenoid compounds in grapes of the present invention is to transfer and overexpress grape transcription factor VvNAC53 into grapes to reduce 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 engineering bacteria, and the engineering bacteria can be used to infect the cells of grape fruits or peels, so that the grape transcription factor VvNAC53 is overexpressed, thereby reducing the content of terpenoids.

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

[0033] (1) Obtain the DNA of the grape transcription factor VvNAC53 by cloning. The specific experimental process is as follows:

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

[0035] Table 1 The first-step reverse transcription system and reaction conditions

[0036]

[0037] Table 2 The second-step reverse transcription system and reaction conditions

[0038]

[0039] Table 3 The third-step reverse transcription system and reaction conditions

[0040]

[0041]

[0042] Use SnapGene to design specific primers, and use PCR to amplify the full-length cDNA of VvNAC53. The PCR system is shown in Table 4, the primer sequences are shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0043] Table 4 The PCR amplification reaction system and reaction program

[0044] Component Volume cDNA template 1.0 μL Forward primer 0.5 μL Reverse primer 0.5 μL 2×Phanta Flash Master Mix (Dye Plus) 5 μL <![CDATA[ddH2O]]> 3 μL System 10 μL

[0045] Table 5 Primer sequences

[0046]

[0047] Table 6 The PCR reaction procedure

[0048]

[0049] The target fragment was detected by 1% agarose gel electrophoresis, and three parallel experiments were conducted. In a certain embodiment of the present invention, the electrophoresis results are as Figure 1 shown. By comparing with the Marker, it can be seen that the DNA lengths of the three bands are all about 1680 bp, which are the target bands of VvNAC53, and the cloning was successful. After the electrophoresis detection was completed, the Hipure Gel Pure DNA Mini Kit agarose gel DNA recovery kit was used to recover the target fragment from the gel.

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

[0051] The plasmid of the overexpression vector pCAMBIA1300 was extracted, and the pCAMBIA1300 was double digested with the restriction enzymes SacI and BamHI. After digestion, the linearized pCAMBIA1300 vector was obtained by purification.

[0052] The One Step Cloning Kit C112 was used to ligate the gel recovery product with the linearized vector pCAMBIA1300. The ligation system is shown in Table 7.

[0053] Table 7 Ligation reaction system and reaction conditions

[0054]

[0055] The ligation system was transformed into DH5α competent cells. The cloning competent cells were thawed on ice. 10 μL of the recombinant product was added to 50 μL of the competent cells. After gently flicking the tube wall to mix, it was placed on ice for 30 min, heat shocked at 42 °C for 45 sec, then placed on ice for incubation for 5 min. Under sterile conditions, 800 μL of antibiotic-free LB medium was added, and it was placed in a shaker at 37 °C with a rotation speed of 220 r / min for 1 h. Centrifuged at 4000×g for 5 min, 500 μL of the supernatant was discarded, the cell pellet was resuspended with the remaining medium, and 100 μL was aspirated and spread on a solid medium containing kanamycin. Incubated upside down in a 37 °C incubator for 16 h. When the colonies grew to about 2 mm in diameter, the system in Table 8 was used for colony PCR identification. The positive clone bacterial solution was selected for sequencing, and part of the bacterial solution was mixed with glycerol in a 1:1 ratio and stored at -80 °C.

[0056] Table 8 Colony PCR system

[0057]

[0058] Dip the tip into a single colony, pipette it up and down twice to dissolve it in 10 μL of LB, and then aspirate 1 μL as a template. Extract the plasmid of the positive clone bacteria with correct sequencing, and finally obtain the recombinant plasmid pCAMBIA1300-VvNAC53.

[0059] (3) Construction of recombinant bacteria.

[0060] Agrobacterium transformation was carried out by the freeze-thaw method. Thaw the Agrobacterium competent GV3101 on ice. After thawing, add 10 μL of the recombinant plasmid to 100 μL of the competent cells, pipette gently to mix, and place it on ice for 5 min, quick-freeze in liquid nitrogen for 5 min, incubate in a water bath at 37 °C for 5 min, and then incubate on ice for 5 min. In a laminar flow hood, add 900 μL of antibiotic-free liquid LB medium, mix well, and incubate at 28 °C with shaking at 200 r / min for 3 h. Centrifuge at 4000×g for 1 min to collect the bacteria, leave about 100 μL of the supernatant, pipette gently to resuspend the bacterial pellet, and spread it on an LB + Rif (50 μg / mL) + Kan (50 μg / mL) solid medium. After air-drying, invert it and incubate in the dark at 28 °C for 3 d.

[0061] Pick a single colony of GV3101 into 4 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium, shake the bacteria at 28 °C and 200 r / min for 16 h, and perform PCR to detect positive clones. Aspirate 150 μL of the bacterial liquid of the positive clone and inoculate it into 4 mL of the above-mentioned double-antibiotic LB liquid medium, and culture it at 28 °C and 200 r / min for 5 - 8 h until the OD 600 reaches 0.6 - 0.8. Take 1 mL of this bacterial liquid, add an equal volume of sterilized glycerol, quick-freeze in liquid nitrogen, and store it at -80 °C.

[0062] (4) Obtain transient transformation of grape fruits with VvNAC53.

[0063] Add 100 - 200 μL of the above-mentioned Agrobacterium bacterial liquid containing the target gene and the Agrobacterium GV3101 empty vector bacterial liquid into 4 - 5 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium respectively, and shake the bacteria at 28 °C for 16 h. Pour all the activated bacterial liquid into 40 mL of LB (Kan + Rif) medium for subculture. The second-generation culture was cultured at 28 °C and 220 rpm for 6 - 7 h until the OD 600 reaches 1 - 1.2. Centrifuge the second-generation culture at 4000×g for 5 min and resuspend it 1:1 in the Agrobacterium infection buffer. Centrifuge and discard the supernatant 3 times to wash the bacterial cells. Resuspend the precipitate of the second-generation culture in the Agrobacterium infection buffer again until the OD 600 reaches 0.9, and let it stand in the dark at room temperature for 3 h. The specific composition of the Agrobacterium infection buffer is shown in Table 9, and its pH needs to be adjusted to 5.2 with KOH.

[0064] Use a syringe to puncture each grape fruit 30 times, soak it in the infection solution, evacuate for 15 min, and continuously shake during this period to promote the entry of the infection solution into the fruit interior. Take out the fruit, culture it for 3 days and 3 nights under natural rhythm, sample the obtained grape fruit, freeze it in liquid nitrogen, and then store it in a -80 °C refrigerator. The composition ratio of the infection solution is shown in Table 9, and the pH value is adjusted to 5 with KOH when in use.

[0065] Table 9 Composition ratio of the infection solution

[0066] Component Dosage MES 2.13g <![CDATA[MgCl2·6H2O]]> 2.03g +Add acetosyringone As before use 200 μM Sterile water 1L

[0067] Through the above specific steps, the VvNAC53 gene can be successfully transferred into grapes and overexpressed. After the above treatment, grape plants with lower terpene content compared to wild type can be obtained, which has a positive effect on the directional cultivation of new varieties with different fragrance types and accelerating the breeding process.

[0068] The transcription factor for regulating the synthesis of grape terpenes in the present invention is of great significance for analyzing the molecular mechanism of grape fruit aroma formation and improving the aroma quality of grape fruits by means of genetic engineering.

[0069] The present invention is specifically described below through examples. Unless otherwise specified, the experimental materials, experimental instruments and reagents used in the following examples are all obtained through conventional channels.

[0070] Example 1 Relative expression analysis of the gene of VvNAC53 transiently transformed into grape peel

[0071] The experimental process of this example is as follows:

[0072] Use the HiScript II Q RT SuperMix for qPCR + gDNA wiper (R223) kit to reverse RNA (extracted using an RNA extraction kit) into cDNA 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×gDNA wiperMix, then add RNase-ddH2O to a total liquid volume of 16 μL. Gently pipette and mix well, then place it in a PCR instrument and react at 42 °C for 2 min.

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

[0075] In this example, Ubiquitin was used as the internal reference gene for fluorescence quantitative PCR. After adding reagents according to the reaction system in Table 10, the reaction was carried out, and three parallel replicates were set. The qRT-PCR reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, for 39 cycles; melting curve analysis was performed from 65°C to 95°C. The primer sequences are shown in Table 11.

[0076] Table 10 Fluorescence quantitative PCR reaction system

[0077]

[0078]

[0079] Table 11 Primer sequences

[0080]

[0081] The results of fluorescence quantitative PCR are as Figure 2 shown. In the figure, "OE-VvNAC53" represents the transient transformation of VvNAC53 in grape peel in the present invention, and "CK" represents the grape peel without any transformation treatment. It can be Figure 2 seen that in the overexpressing plants, the expression level of VvNAC53 increased by 1.76 times.

[0082] Experimental Example 2 Extraction and detection of terpenoid components in VvNAC53 transiently transformed grape peel

[0083] In this example, the contents of terpenoid substances in VvNAC53 transiently transformed grape peel (OE-VvNAC53) and grape peel without any transformation treatment (CK) were extracted and compared respectively to verify the regulatory effect of VvNAC53 overexpression on the content of terpenoid substances.

[0084] The process of extracting terpenoid substances in this example is as follows:

[0085] After the grape peel was taken, it was immediately frozen with liquid nitrogen, put into a powder grinder pre-cooled with liquid nitrogen for powdering, and stored at -80°C. 1 g of powder was added with 5 mL of citric acid / phosphate buffer (0.2 mol / L, pH = 5.0), soaked at 4°C for 16 h, centrifuged at 4°C and 5000×g for 15 min, and the supernatant was taken for the subsequent extraction and detection of aroma substances.

[0086] The bound terpenoid compounds in the pericarp were extracted by solid phase extraction (SPE). The extraction process and conditions were as follows: The Cleanert PEP-SP (150 mg / 6 mL) extraction column was activated with 10 mL of anhydrous methanol and 10 mL of pure water respectively to remove impurities in the extraction column. Then, 2 mL of the above supernatant was added, and it was eluted successively with 5 mL of pure water and 5 mL of dichloromethane to remove polar compounds such as pigments, small molecular weight sugars, acids, and free non-polar 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-bound aroma substances. The methanol solution containing glycoside-bound aroma substances collected was evaporated to dryness on a rotary evaporator at 30 °C, and then re-dissolved with 10 mL of citric acid / phosphate buffer solution (0.2 mol / L, pH = 5.0). After re-dissolution, 100 μL of glycosidase AR2000 (100 g / L) was added and enzymatically hydrolyzed in a constant temperature incubator at 37 °C for 16 h. After the enzymatic hydrolysis was completed, the pH of the enzymatic hydrolysate was adjusted to 3.0 with citric acid. The enzymatic hydrolysate was used for the detection of glycoside-bound aroma.

[0087] The free and bound terpenoid compounds in grape pericarp were detected by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS). The specific conditions were as follows: The GC-MS instrument used was an Agilent 6890 gas chromatograph equipped with an Agilent 5975 mass spectrometer. The capillary chromatographic column used was a polar chromatographic column HP-INNOWAX (60 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA, USA).

[0088] Add 5 mL of fruit peel leaching solution or enzymatic hydrolysis solution and 1.0 g of sodium chloride into a 20 mL headspace vial, and add 10 μL of internal standard (4-methyl-2-pentanol, 1.0 g / L). Seal it with a cap equipped with a polytetrafluoroethylene septum. The HS-SPME pretreatment is completed by a CTC CombiPAL multi-functional autosampler (CTC Analytic, Zwingen, Switzerland). After equilibration at 40 °C for 30 min, insert the activated polydimethylsiloxane / carboxen / divinylbenzene (DVB / CAR / PDMS) 50 / 30 μm extraction head (Supelco, Bellefonte, PA, USA) into the headspace of the sample vial, and oscillate and extract the sample at 500 r / min for 30 min. After extraction, insert the extraction head into the GC injection port at 250 °C and perform splitless injection and thermal desorption for 8 min. The carrier gas is helium (purity > 99.999%), and the flow rate is 1 mL / min. Programmed temperature rise: hold at 50 °C for 1 min; increase to 220 °C at a rate of 3 °C / min and hold for 5 min. The mass spectrometry interface temperature is 250 °C, the mass spectrometry ion source is an electron ionization (EI) source, the ion energy is 70 eV, and the mass scanning range is 29 - 350 u.

[0089] Qualitative analysis is carried out based on the retention index and mass spectrometry information of the compound under the same chromatographic conditions. For aroma substances without standard samples, semi-qualitative analysis is carried out by using the retention index of the compound under similar chromatographic conditions reported in the literature and the comparison results of the NIST05 standard spectral library (NIST Chemistry WebBook. http: / / webbook.nist.gov / chemistry / ); for aroma substances whose retention indices under similar chromatographic conditions have not been reported in the literature, semi-qualitative analysis is carried out according to the comparison results of the NIST05 standard spectral library.

[0090] Quantification of aroma substances is analyzed by the corresponding standard curve: for aroma substances with standard samples, they are directly quantified using the standard curve of the corresponding standard samples; for aroma substances without standard samples, semi-quantification is carried out using the standard curve of standard samples with similar chemical structures. The content of terpenoids is expressed as ng / g.

[0091] The detection results of terpenoid components in the grape peels of the OE-VvNAC53 and CK groups are as Figure 3 shown.

[0092] From Figure 3It can be seen that in the grape pericarp with overexpression of the transcription factor VvNAC53, the contents of free linalool, p-cymene, nerol and conjugated trans-rose oxide are all significantly reduced. Among them, the content of free linalool is reduced by 1.5 times compared with the control group CK, indicating that VvNAC53 is involved in the regulation of terpene metabolism in grapes and can significantly reduce the contents of the above-mentioned terpene components.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A grape transcription factor VvNAC53, characterized in that, The nucleotide sequence encoding the grape transcription factor VvNAC53 is shown in SEQ ID NO.

1.

2. The grape transcription factor VvNAC53 according to claim 1, characterized in that, The amino acid sequence of the grape transcription factor VvNAC53 is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector includes the nucleotide sequence encoding the coding region of the grape transcription factor VvNAC53 as described in claim 1.

4. A recombinant vector according to claim 3, characterized in that, It is constructed using a primer pair, and the primer pair includes an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.

4.

5. The recombinant vector according to claim 3, characterized in that, The backbone plasmid of the recombinant vector is pCAMBIA1300.

6. An engineered bacterium, characterized in that, The engineered bacteria contain the recombinant vector as described in any one of claims 3-5.

7. Use of the grape transcription factor VvNAC53 as described in claim 1 or 2, characterized in that, It can be used to regulate the content of terpenoids in grapes.

8. A method for regulating the content of terpenoid compounds in grapes, characterized in that, Regulation is carried out using the grape transcription factor VvNAC53 as described in claim 1 or 2.

9. A method for regulating the content of terpenoid compounds in grapes according to claim 8, characterized in that, 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.

10. A method for regulating the content of terpenoid compounds in grapes according to claim 9, characterized in that, The terpenoids include free linalool, p-cymene, nerol, and bound trans-rose oxide.

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