Application of grape VvNAC83 gene in regulating fruit firmness

Abstract

The present application belongs to the technical field of plant genetic engineering, and particularly relates to a VvNAC83 gene derived from grape, and application of the gene in regulating fruit firmness of grape and tomato, and further includes a recombinant carrier containing the gene, a recombinant genetically engineered bacterium, transgenic plant tissue and related products. The present application first clones and identifies the fruit firmness regulation function of the grape VvNAC83 gene, and through overexpression of the gene, the fruit firmness of grape and tomato can be significantly improved, and silencing of the gene can significantly reduce the fruit firmness of grape, which confirms that the gene is a positive regulation factor of fruit firmness. The gene can provide a precise target for molecular breeding, and lays an important foundation for creating new germplasm of grape and solanaceous crops with high firmness and storage and transportation resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving a VvNAC83 gene derived from grapes, and the application of this gene in regulating the firmness of grape and tomato fruits. It also includes recombinant vectors containing this gene, recombinant genetically engineered bacteria, transgenic plant tissues and related products. Background Technology

[0002] Fruit firmness, as a core quality trait, not only affects taste and flavor but is also closely related to post-harvest storage, transportation losses, and processing suitability—high-firm fruits are more resistant to storage and transportation, have a longer shelf life, and can significantly reduce industry costs and increase commercial value. However, fruit firmness is a typical quantitative trait, easily affected by environmental conditions and cultivation techniques. Conventional hybridization breeding has a long cycle and low selection efficiency, making it difficult to meet the market's urgent demand for specialized high-firmity varieties.

[0003] The NAC family of transcription factors is a class of important transcriptional regulators unique to plants, widely involved in various biological processes such as growth and development, stress response, and fruit ripening. In the field of fruit texture regulation, NAC family members form diverse regulatory mechanisms by targeting cell wall metabolism-related genes (such as PG, PL, and XTH): some NAC proteins (such as PavNAC56 in sweet cherry and FvRIF in strawberry) accelerate fruit softening through transcriptional activation; others (such as MaNAC154 in banana and SNAC9 in tomato) delay the softening process by inhibiting the expression of related genes or through protein interactions.

[0004] However, there are significant gaps in existing technologies: the specific functional genes and molecular mechanisms by which NAC transcription factors regulate fruit firmness in grapes have not yet been identified, and there is a lack of key targets that can be directly used for molecular breeding.

[0005] Therefore, identifying key NAC transcription factors in grapes that regulate fruit firmness and clarifying their functions is of great significance for improving the regulatory network of fruit ripening and softening and promoting crop quality improvement. Summary of the Invention

[0006] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the purpose of this invention is to provide the application of the VvNAC83 gene in regulating fruit firmness, laying an important foundation for creating new germplasm of grapes and solanaceous crops with high firmness and good storage and transportation resistance.

[0007] (II) Technical Solution In a first aspect, the present invention provides the application of the grape VvNAC83 gene in regulating fruit firmness, wherein the fruit includes grapes and tomatoes.

[0008] Optionally, the regulation is to improve fruit firmness, specifically including: introducing an overexpression vector of the VvNAC83 gene into grapes or tomatoes to achieve overexpression of the VvNAC83 gene.

[0009] Secondly, the present invention provides a product for regulating fruit firmness, wherein the product is a gene overexpression vector, and the gene overexpression vector, after being introduced into the host, can achieve overexpression of the VvNAC83 gene.

[0010] Optionally, the gene overexpression vector comprises any of the following nucleotide sequences: a) The nucleotide sequence shown in SEQ ID NO.1; b) Due to the degeneracy of the genetic codon, a degenerate sequence that is different from the sequence shown in a) but encodes the same amino acid sequence; c) The nucleotide sequence is ≥90% identical to that shown in a) or b), and the encoded protein has a nucleotide sequence with the same or similar function as the VvNAC83 protein.

[0011] Optionally, the gene overexpression vector comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0012] Thirdly, the present invention provides a method for regulating fruit firmness, which includes: regulating the expression of the VvNAC83 gene in grapes or tomatoes to regulate fruit firmness.

[0013] Optionally, the regulation is to improve fruit firmness, specifically including: constructing the gene overexpression vector and introducing it into grapes or tomatoes to increase the expression level of the VvNAC83 gene.

[0014] Preferably, the overexpression vector is pCAMBIA1301-VvNAC83-GFP, which is constructed by inserting the VvNAC83 gene into the modified pCAMBIA1301 vector through double digestion with SpeI and BstEII.

[0015] Fourthly, the present invention provides a recombinant vector containing the VvNAC83 gene; the nucleotide sequence of the VvNAC83 gene is selected from any of the following nucleotide sequences: a) the nucleotide sequence shown in SEQ ID NO.1; b) a degenerate sequence that is different from the sequence shown in a) but encodes the same amino acid sequence due to the degeneracy of the genetic codon; c) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in a) or b) and encodes a protein that has the same or similar function as the VvNAC83 protein, or a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO.2.

[0016] The recombinant vector for the VvNAC83 gene is pCAMBIA1301-VvNAC83-GFP, which is constructed by inserting the VvNAC83 gene (complete CDS coding sequence) into the modified pCAMBIA1301 vector through double digestion with SpeI and BstEII.

[0017] As a control experiment, this embodiment of the invention also constructed an interference vector, TRV2-EGFP-VvNAC83, which was constructed by inserting a VvNAC83 gene fragment (a specific gene fragment without a complete ORF) into the TRV2-GFP vector through double digestion with XhoI and EcoRI. By inserting the VvNAC83 gene fragment as a "silencing template," the plant itself is guided to degrade endogenous target gene mRNA, thereby achieving the purpose of inhibiting target gene expression.

[0018] Fifthly, the present invention provides a recombinant genetically engineered bacterium or transgenic plant tissue, which is obtained by transformation of the recombinant vector; the transgenic plant tissue is grape or tomato tissue.

[0019] Preferably, the recombinant genetically engineered bacteria is Escherichia coli DH5α or Agrobacterium GV3101 containing a recombinant vector; the transgenic plant tissue is grape fruit tissue or tomato plant tissue containing the VvNAC83 gene.

[0020] In a sixth aspect, the present invention provides the application of the above-mentioned gene overexpression vector or the above-mentioned recombinant vector in regulating fruit firmness, wherein the fruit includes grapes and tomatoes.

[0021] Preferably, the application is to increase the firmness of grape or tomato fruits by overexpressing the VvNAC83 gene, or to decrease the firmness of grape fruits by silencing the VvNAC83 gene.

[0022] (III) Beneficial Effects This invention is the first to clone and identify the fruit firmness regulation function of the grape VvNAC83 gene. Overexpression of this gene can significantly increase the fruit firmness of grapes and tomatoes, while silencing the gene can significantly reduce the fruit firmness of grapes, confirming that it is a positive regulator of fruit firmness.

[0023] The technical advantages of this invention are: 1. It provides a gene derived from grapes that can regulate fruit firmness, which can play a role in regulating fruit firmness in both grapes and tomatoes; 2. It clarifies the sequence characteristics and function of the VvNAC83 gene, providing a precise target for molecular breeding; 3. It constructs an efficient recombinant vector and transformation system, which can quickly achieve targeted improvement of crop fruit firmness.

[0024] By applying the technology of this invention, new grape and tomato germplasm with high hardness and good storage and transportation resistance can be created through genetic engineering, shortening the breeding cycle and reducing post-harvest losses. This has important practical application value for promoting the quality and efficiency of the fruit and vegetable industries. Attached Figure Description

[0025] Figure 1 The image shows the gene cloning and E. coli colony PCR results during the construction of the VvNAC83 gene overexpression vector. The marker in the figure is 2000.

[0026] Figure 2 The image shows the gene cloning and E. coli colony PCR results during the construction of the VvNAC83 gene VIGS vector. The marker in the figure is 2000.

[0027] Figure 3 Images of grape berries transformed with VvNAC83 gene overexpression vector, VIGS interference vector, and empty vector.

[0028] Figure 4 Comparison of the relative expression levels of the VvNAC83 gene and fruit firmness in grape fruits transformed with VvNAC83 gene overexpression vector, VIGS interference vector and empty vector.

[0029] Figure 5 Images of tomatoes transformed with the VvNAC83 gene overexpression vector and wild-type tomatoes.

[0030] Figure 6 Comparison of the relative expression levels of the VvNAC83 gene and fruit firmness in tomatoes transformed with the VvNAC83 gene overexpression vector and wild-type tomatoes. Detailed Implementation

[0031] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1 In this embodiment, the CDS sequence of the grape VvNAC83 gene was cloned using the 'Muscat' grape variety as the experimental material. The specific steps are as follows: Sample collection and processing: Grapes of 'Muscat' variety were collected 7-10 days before color change and rapidly frozen in liquid nitrogen for subsequent total RNA extraction.

[0033] Total RNA extraction from grape berries: Total RNA was extracted using the HiPure HP Plant RNA MiniKit (Catalog No. R4165-02, Magen). The procedure was as follows: (1) Use liquid nitrogen to pre-cool the sample grinder, grind the frozen fruit sample into powder, weigh 150 mg of powder into a 2 mL centrifuge tube, and immediately add 700 μL of PAL reagent containing 2-mercaptoethanol; (2) Vigorously vortex the sample to disperse it, and place it in a 65℃ water bath for 20 minutes; (3) Add 800 μL of chloroform to the lysis solution, vortex at high speed for 10 s to mix thoroughly, and centrifuge at 13000 g for 5 min at room temperature; (4) Transfer 600-700 μL of supernatant to a new centrifuge tube, add 1.5 times the volume of Buffer GXP2, vortex for 10s to mix, transfer the mixture to the adsorption column in multiple batches, centrifuge at 12000g for 30-40s each time, discard the filtrate in the collection tube, until all the mixture has been loaded. (5) Add 300 μL of Buffer RW1 to the adsorption column, centrifuge at 12000g for 1 min, and discard the filtrate; (6) To remove DNA contamination from nucleic acids, add 110 μL of DNAase I reaction solution to the center of the RNA column membrane, let it stand at room temperature for 30 min, then add 500 μL of Buffer RW1, let it stand at room temperature for 3 min, centrifuge at 12000g for 30 s and discard the filtrate. (7) Add 500 μL Buffer RW2 to the adsorption column, centrifuge at 12000g for 30s and discard the filtrate. Repeat this operation once. After discarding the filtrate for the second time, put the adsorption column back into the empty collection tube and centrifuge at 12000g for 2min. (8) After the empty separation is completed, transfer the adsorption column to a new centrifuge tube, open the cap and let it stand at room temperature for 2-3 minutes, add 30-100 μL of RNase Free Water to the center of the column membrane, close the cap and let it stand at room temperature for 2 minutes, then centrifuge at 12000g for 2 minutes to elute the RNA. (9) Discard the adsorption column, determine the concentration of eluted RNA, label it and freeze it at -80℃ for later use.

[0034] cDNA first-strand synthesis: cDNA was synthesized by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (catalog number R312-02, Vazyme) with 1 μg of extracted total RNA as a template. The procedure was as follows: (1) Using the total RNA extracted from grape fruits as a template, prepare an RNA mixture: 1 μg of total RNA, and add RNase-free water to make up to 8 μL; (2) Heat the RNA mixture at 65°C for 5 min, then quickly transfer it to ice and cool for 2 min; add 2 μL of gDNA wiper Mix to the above mixture, gently pipette to mix, and heat at 42°C for 2 min. (3) Add 1 μL of Random hexamers, 2 μL of 10×RT Mix, 2 μL of Enzyme Mix and 5 μL of RNase-free water to the above 10 μL mixture in sequence, and gently pipette to mix. Complete the reverse transcription according to the PCR reaction program of incubating at 25℃ for 5 min, reacting at 37℃ for 45 min, and heating at 85℃ for 5 s to inactivate the reverse transcriptase. After aliquoting the synthesized cDNA, store it in a -80℃ freezer.

[0035] VvNAC83 gene CDS sequence cloning: (1) Based on the CDS sequence of the grape VvNAC83 gene in the EnsemblePlants database, specific amplification primers were designed. The forward primer was SEQ ID NO.3 and the reverse primer was SEQ ID NO.4. (2) PCR amplification was performed using KOD high-fidelity enzyme. The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. (3) After the PCR reaction of high-fidelity DNA polymerase is completed, the amplification product is subjected to 1% agarose gel (w / v) electrophoresis. The size of the target gene band is determined according to DNA marker DL-2000. The target fragment is recovered from the gel according to the instructions of the gel recovery kit HiPure GelPureDNA Mini Kit (catalog number D211102, Magen). (4) The recovered target fragment was sequenced and identified to obtain the VvNAC83 gene CDS sequence as shown in SEQ ID NO.1. The sequence is 630 bp in length and encodes the amino acid sequence as shown in SEQ ID NO.2.

[0036] Table 1: PCR amplification system Table 2: PCR amplification system Example 2 This example demonstrates the construction of VvNAC83 overexpression and interference vectors, as well as a transient transformation experiment on grape fruits. The experimental method is as follows: I. Construction of overexpression vector pCAMBIA1301-VvNAC83-GFP and preparation of engineered bacteria 1. Using the pLB Zero Background Rapid Cloning Kit (catalog number VT206, Tiangen), the PCR product of the VvNAC83 gene recovered in Example 1 was ligated into the intermediate vector pLB Simple Vector.

[0037] 2. Design specific primers: the forward primer is SEQ ID NO.5, and the reverse primer is SEQ ID NO.6. Use the pLB Simple Vector containing the target gene as a template for PCR amplification to facilitate the amplification of the target gene from the pLB Simple Vector using specific primers. The amplification products are detected by agarose gel electrophoresis (see [link to product description]). Figure 1 A is the gene clone diagram during the construction of the VvNAC83 gene overexpression vector (Marker in the diagram is 2000). After that, the target fragment is recovered by gel extraction.

[0038] 3. The modified overexpression vector pCAMBIA1301 was double-digested with SpeI and BstEII restriction endonucleases. The digested vector was then detected by agarose gel electrophoresis and the linearized vector fragments were recovered by gel electrophoresis.

[0039] 4. The VvNAC83 gene DNA fragment recovered from the gel was ligated into the linearized pCAMBIA1301 vector to obtain the recombinant overexpression vector pCAMBIA1301-VvNAC83-GFP.

[0040] 5. Transform the recombinant overexpression vector into E. coli DH5α competent cells. The procedure is as follows: (1) Thaw 50 μL of Escherichia coli DH5α competent cells on ice, and gently tap the tube wall to resuspend the cells; add 10 μL of the above ligation product, gently tap a few times to mix, and incubate on ice for 30 min.

[0041] (2) Heat shock in a 42℃ water bath for 50s, then quickly transfer to ice to cool for 2min.

[0042] (3) Add 700 μL of LB liquid culture medium to the centrifuge tube and incubate at 37°C and 200 rpm for 60 min.

[0043] (4) Take 400 μL of bacterial solution and spread it evenly on LB solid medium containing kanamycin (kana, final concentration 50 mg / L). After the bacterial solution is completely absorbed by the medium, incubate it upside down at 37°C overnight.

[0044] Positive clones were identified using colony PCR (see [link]). Figure 1Image B is a PCR diagram of E. coli colonies during the construction of the VvNAC83 gene overexpression vector (Marker is 2000). The detection primers are the same as the primers for amplifying the target gene (SEQ ID NO.5-6).

[0045] 6. Transform Agrobacterium GV3101 competent cells with the recombinant plasmid that has been verified by sequencing. The operation steps are as follows: (1) Thaw 50 μL of Agrobacterium GV3101 competent cells on ice, add 5 μL of recombinant plasmid with correct sequencing, mix gently and incubate on ice for 5 min.

[0046] (2) Quick freeze in liquid nitrogen for 5 min, then incubate in a water bath at 37°C for 5 min, and then in an ice bath for 5 min.

[0047] (3) Add 700 μL of non-resistant LB liquid medium and incubate at 28°C and 200 rpm for 2 h.

[0048] (4) Spread 400 μL of bacterial culture onto LB solid medium containing kanamycin and rifampin (kana+rif, both with a final concentration of 50 mg / L) and incubate at 28°C upside down until a single colony grows.

[0049] (5) Select 6-8 single colonies for each gene for colony PCR verification. The detection primers are 35S-F (forward primer is SEQ ID NO.7) and Reverse (reverse primer is SEQ ID NO.8). Screen positive Agrobacterium clones.

[0050] II. Construction of VIGS interference vector TRV2-EGFP-VvNAC83 and preparation of engineered bacteria 1. Using a pLB Simple Vector linked with the VvNAC83 gene as a template, specific primers were designed: the forward primer (SEQ ID NO. 9) and the reverse primer (SEQ ID NO. 10). PCR amplification was performed, and the amplification products were detected by agarose gel electrophoresis (see [link to relevant documentation]). Figure 2 A is the gene cloning diagram of the VvNAC83 gene in the VIGS vector construction (Marker in the diagram is 2000), followed by gel recovery of the target fragment.

[0051] 2. The VIGS vector TRV2-EGFP was double-digested with XhoI and EcoRI restriction endonucleases. After the digestion products were detected by agarose gel electrophoresis, the corresponding bands of the large fragments of the vector were extracted and recovered by gel electrophoresis.

[0052] 3. The VvNAC83 gene DNA fragment recovered from the gel was ligated into the linearized TRV2-EGFP vector to obtain the recombinant interference vector TRV2-EGFP-VvNAC83.

[0053] 4. Following the steps 5-6 in section "I", transform the recombinant interference vector into *E. coli* DH5α competent cells, and identify positive clones by colony PCR (see...). Figure 2 B is the E. coli colony PCR diagram constructed using the VvNAC83 gene VIGS vector (Marker is 2000). The detection primers are TRV2-seq-F (forward primer SEQ ID NO.11) and TRV2-seq-R (reverse primer SEQ ID NO.12). The correctly sequenced recombinant plasmid was transformed into Agrobacterium GV3101 competent cells. The colony PCR verification primers were TRV2-seq-F (forward primer SEQ ID NO.11) and pTRV2-seq-R (reverse primer SEQ ID NO.12). Positive Agrobacterium clones were screened.

[0054] In the subsequent experiments, after Agrobacterium carrying the interference vector TRV2-EGFP-VvNAC83 infected grape fruits, the TRV virus replicated within plant cells, and the VvNAC83 gene fragment it carried transcribed into corresponding RNA. This exogenous RNA triggered the plant's post-transcriptional gene silencing (PTGS) defense mechanism. Plant cells recognized the exogenous VvNAC83 RNA and cleaved it into small interfering RNAs (siRNAs). These siRNAs bound to the RNA-induced silencing complex (RISC) within the plant, specifically recognizing and binding to endogenous VvNAC83 mRNA. The RISC then cleaved the endogenous VvNAC83 mRNA, preventing its translation into protein, ultimately silencing / downregulating the expression of the endogenous VvNAC83 gene. By inserting a VvNAC83 gene fragment as a silencing template, the plant itself was guided to degrade endogenous target gene mRNA, thereby inhibiting the expression of the target gene.

[0055] III. Agrobacterium-mediated transient transformation of grape berries 1. Agrobacterium overexpression vector infects grape berries (1) Select one of the activated Agrobacterium positive clones containing the target gene and inoculate it into 1 mL of LB liquid medium containing kana + rif (final concentration of 50 mg / L) and culture overnight at 28°C and 200 rpm.

[0056] (2) Take 0.5 mL of the overnight culture and transfer it to 5 mL of LB liquid medium containing kana + rif (final concentration of 50 mg / L). Culture at 28°C and 200 rpm on a shaker until the OD600 value of the culture is 0.6.

[0057] (3) Transfer the above 5 mL bacterial culture to 100 mL of LB liquid medium containing kana + rif (final concentration of 50 mg / L) and culture at 28 °C and 200 rpm on a shaker until the bacterial culture OD600 value is 0.8-1.0.

[0058] (4) Centrifuge the cultured bacterial solution at 5000 rpm for 10 min, discard the supernatant, add 80-100 mL of resuspension (containing MS basic salt + 200 μmol / L acetylsalicylic acid) to suspend the Agrobacterium cells, adjust the OD600 value of the bacterial solution to 0.8, and let it stand at room temperature for 3 h.

[0059] (5) Before use, vortex or pipette to resuspend the bacterial cells. Use a 1mL sterile syringe to draw 1mL of the resuspended solution and slowly inject it into the pulp from about 0.5cm from the stem end or stigma end of the 'Muscat' grape. To compensate for the large amount of leakage of the bacterial solution during the injection process, each fruit should be injected three times to ensure that at least 0.1mL of bacterial solution is retained in the pulp.

[0060] (6) Using Agrobacterium tumefaciens culture with empty vector as a control, grape fruits injected with Agrobacterium tumefaciens were cultured under suitable conditions (25±2℃, in the dark) and samples were taken after 5-7 days.

[0061] (7) Genomic DNA was extracted from the fruit using the Plant Genomic DNA Extraction Kit (CTAB, catalog number ZP309, Zoman). The transformation effect was detected by PCR. The PCR primers were 35S-F (forward primer was SEQ ID NO.7) and Reverse (reverse primer was SEQ ID NO.8). The results showed that the VvNAC83 gene was successfully integrated into the grape fruit genome, indicating that the transient transformation of the grape fruit was successful.

[0062] 2. Agrobacterium tumefaciens infecting fruit with VIGS interference vector. (1) Pick the two activated Agrobacterium positive clones containing the target gene and the Agrobacterium positive clones containing the TRV1 helper vector, and inoculate them into two tubes of 1 mL LB liquid medium containing kana+rif (final concentration of 50 mg / L) and culture overnight at 28°C and 200 rpm.

[0063] (2)-(3) The operation steps are the same as those described above. Agrobacterium overexpression vectors are used to infect grape fruit parts (2) and (3). The two Agrobacterium species are cultured by small shaking and large shaking respectively until the OD600 value of the bacterial solution is 0.8-1.0.

[0064] (4) Centrifuge the two cultured bacterial solutions separately, discard the supernatant, add resuspension solution to suspend the bacterial cells and adjust the OD600 value to 0.8 (the operation steps are the same as in the above-mentioned Agrobacterium tumefaciens infection of grape fruit part with overexpression vector (4)). Then mix TRV2-target gene bacterial solution and TRV1 auxiliary bacterial solution at a volume ratio of 1:1 and let stand at room temperature for 3 hours.

[0065] (5) The operation steps are the same as those described above. Agrobacterium overexpression vector is used to infect grape fruit (5). The mixed bacterial solution is injected into the grape pulp using a syringe. Each fruit is injected three times.

[0066] (6) Grape berries infused with a mixture of empty TRV1 and empty TRV2 vectors were used as controls. Subsequent fruit culture conditions were the same as those described above for the Agrobacterium tumefaciens overexpressing the vectors in grape berries (6).

[0067] (7) The operation steps are the same as those for Agrobacterium overexpression vector infection of grape fruits (7), except that the detection primers are changed to TRV2-seq-F (forward primer SEQ ID NO.11) and pTRV2-seq-R (reverse primer SEQ ID NO.12). The detection results show that the target gene fragment was successfully integrated into the grape fruit genome.

[0068] Example 3 This embodiment uses Agrobacterium-mediated genetic transformation to transfer the VvNAC83 gene into tomato, obtaining stably inherited transgenic tomato plants (MicroTom). The specific steps are as follows: Preparation of sterile tomato seedlings: Select plump, mature tomato seeds, disinfect them with 75% alcohol for 1 min, disinfect them with sodium hypochlorite solution for 10-20 min, and rinse them 4 times with sterile water; inoculate the disinfected seeds onto 1 / 2 MS solid medium, incubate them in the dark at 25℃ for 3 days, and then transfer them to a light incubator for 1 week of light culture to obtain sterile tomato seedlings.

[0069] Preparation of Agrobacterium tumefaciens bacterial suspension: Agrobacterium tumefaciens containing the recombinant overexpression vector of the VvNAC83 gene was streaked on LB solid medium containing kana + rif (final concentration of 50 mg / L) and cultured at 28°C for 2 days; single colonies were picked and inoculated into LB liquid medium containing the same antibiotics and cultured at 28°C and 200 rpm with shaking to prepare Agrobacterium tumefaciens bacterial suspension with an OD600 value of 0.6, which was used for infection of tomato explants.

[0070] Agrobacterium-mediated genetic transformation and plant regeneration of tomatoes: (1) Take newly unfolded cotyledons of 6-7 day old sterile tomato seedlings, cut off both ends, cut the middle section of the cotyledons into explants of appropriate size, and pre-culture them in a pre-medium for 24 hours; (2) Infect the pre-cultured explants in the above Agrobacterium solution for 8-10 minutes, gently shaking them during the process; after infection, remove the explants, blot the surface bacterial solution with sterile filter paper, and transfer them to a co-medium for dark culture at 28°C for 2-3 days; (3) After co-culture, transfer the explants sequentially to callus induction medium and bud differentiation medium containing screening antibiotics for screening and induction, and change the medium every 2 weeks; (4) When the resistant buds grow to about 2 cm, cut off the resistant buds and transfer them to a rooting medium to induce rooting, and obtain complete transgenic tomato plants; use untransformed wild-type (WT) tomato plants as controls.

[0071] Obtaining homozygous transgenic tomato lines: The seeds produced by the T0 generation transgenic tomato plants are harvested, sown, and screened to obtain the T1 generation plants. The T2 generation of stable transgenic tomato plants is obtained through further propagation and screening.

[0072] Transgenic identification: After the T2 generation plants produced fruit, genomic DNA was extracted from the fruit of the T2 generation transgenic tomato plants. The genomic DNA was extracted using a plant genomic DNA extraction kit and detected by PCR according to step "7" in Example 2 to verify the integration of the VvNAC83 gene. The primers used for PCR detection were primer 35S-F (forward primer SEQ ID NO. 7) and reverse primer (reverse primer SEQ ID NO. 8). The detection results showed that the target gene was successfully integrated into the tomato plant genome.

[0073] Example 4 This example demonstrates the detection of gene expression levels in VvNAC83 transgenic plants and a comparison of fruit firmness between transgenic plants and the control group.

[0074] I. Detection of VvNAC83 gene expression level in transgenic plants 1. Total RNA extraction and cDNA synthesis: Grape fruits that were transiently transformed in Example 2 and T2 generation transgenic tomato fruits in Example 3 were taken respectively, and total RNA was extracted and reverse transcribed to synthesize cDNA according to the methods in steps 2-3 of Example 1.

[0075] 2. Real-time quantitative PCR detection: The relative expression level of the VvNAC83 gene was detected using the HiScript II One Step RT-PCR Kit (catalog number P612-01, Vazyme). (1) Primer design for real-time PCR: The forward primer for the VvNAC83 gene is VvNAC83-F (SEQ ID NO.13), and the reverse primer is VvNAC83-R (SEQ ID NO.14); the grape Actin gene is used as an internal control, and the primers are Actin-F (SEQ ID NO.15) and Actin-R (SEQ ID NO.16); the tomato Slactin gene is used as an internal control, and the primers are Slactin-F (SEQ ID NO.17) and Slactin-R (SEQ ID NO.18).

[0076] (2) The reaction system was prepared according to the instructions of the real-time PCR kit, and 3 biological replicates and 3 technical replicates were set up. The amplification was performed on the real-time PCR instrument. The relative expression level of the VvNAC83 gene was calculated using the 2^(-ΔΔCt) method.

[0077] II. Measurement of the firmness of transgenic fruits The firmness of transgenic grapes and tomatoes was objectively and quantitatively determined using the multifaceted texture analysis (TPA) method. This method simulates human chewing motions, effectively avoiding subjective interference and achieving objective quantitative determination of fruit firmness. The instrument settings were as follows: grape fruit deformation 25%, interval between two compressions 5 s, and test and return speeds both 2 mm / s. Tomato fruits were measured using the same parameters; at least three biological replicates were performed for each sample, and fruit firmness values ​​were recorded.

[0078] See the results of genetically modified grape fruit testing. Figures 3-4 As shown in the diagram. OE or OE-VvNAC83 represents the VvNAC83 gene overexpression group, VIGS represents the VvNAC83 gene virus-induced silencing group, EV-VIGS represents the empty vector control of the VIGS silencing group (co-transformed into TRVI and blank TRV2 vectors), and EV-OE represents the empty vector control of the OE overexpression group (transformed into the blank pCAMBIA1301 vector). Figure 4 In A and B, EV represents the overexpression empty vector control group; in C and D, EV represents the VIGS silencing group empty vector control group. The phenotype of transgenic grape fruit is as follows: Figure 3 As shown. Gene expression levels are as follows. Figure 4 As shown in the results, compared with the empty vector control (EV), the expression level of the VvNAC83 gene was significantly upregulated in grape berries overexpressing the VvNAC83 gene, and the fruit firmness was also significantly improved. Figure 4 In grape berries treated with VIGS silencing, the expression level of the VvNAC83 gene was significantly downregulated, and the fruit firmness was also significantly reduced. Figure 4 (C, D).

[0079] See the results of genetically modified tomato fruit testing. Figures 5-6 As shown. WT represents wild type, and #6, #12, and #13 are the experimental numbers for transgenic tomatoes. The experimental results show that, compared with wild-type (WT) tomato fruits, the expression level of the VvNAC83 gene was significantly upregulated in the T2 generation tomato fruits overexpressing the VvNAC83 gene, and the fruit firmness was also significantly improved. Figure 6 This result is consistent with the results of the grape overexpression experiment.

[0080] The above results indicate that the VvNAC83 gene positively regulates the fruit firmness of grapes and tomatoes. Overexpression of this gene can significantly increase the fruit firmness of grapes and tomatoes, while silencing this gene will lead to a significant decrease in the fruit firmness of grapes.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the grape VvNAC83 gene in regulating fruit firmness.

2. The application according to claim 1, characterized in that, The regulation aims to improve fruit firmness, specifically by introducing an overexpression vector of the VvNAC83 gene into grapes or tomatoes to achieve overexpression of the VvNAC83 gene.

3. A product for regulating fruit firmness, characterized in that, The product is a gene overexpression vector, which, after being introduced into the host, enables the overexpression of the VvNAC83 gene.

4. The product according to claim 3, characterized in that, The gene overexpression vector contains any of the following nucleotide sequences: a) The nucleotide sequence shown in SEQ ID NO.1; b) Due to the degeneracy of the genetic codon, a degenerate sequence that is different from the sequence shown in a) but encodes the same amino acid sequence; c) The nucleotide sequence is ≥90% identical to that shown in a) or b), and the encoded protein has a nucleotide sequence with the same or similar function as the VvNAC83 protein.

5. The product according to claim 3, characterized in that, The gene overexpression vector contains a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

6. A method for regulating fruit firmness, characterized in that, include: Regulating the expression of the VvNAC83 gene in grapes or tomatoes can regulate fruit firmness.

7. The method according to claim 6, characterized in that, The regulation is to improve fruit firmness, specifically including: constructing the gene overexpression vector as described in any one of claims 3-5, introducing it into grapes or tomatoes, and increasing the expression level of the VvNAC83 gene.

8. A recombinant vector, characterized in that, Contains the VvNAC83 gene; the nucleotide sequence of the VvNAC83 gene is selected from any of the sequences described in claim 4 (a), (b), and (c), or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

9. A recombinant genetically engineered bacterium or transgenic plant tissue, characterized in that, It is obtained by transformation of the recombinant vector according to claim 8; the transgenic plant tissue is grape or tomato tissue.

10. The application of the gene overexpression vector according to any one of claims 3-5 or the recombinant vector according to claim 8 in regulating fruit firmness, wherein the fruit includes grapes and tomatoes.

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