Application and vector of grape VvNAC17 gene in regulating postharvest grape berry abscission

Abstract

This invention belongs to the fields of fruit and vegetable science and gene technology, and discloses the application and vector of the grape VvNAC17 gene in regulating postharvest grape berry abscission. The gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The VvNAC17 silencing technology of this invention provides precise regulation at the gene molecular level, reducing the uncertainties of external environment and storage and transportation conditions, significantly reducing berry abscission rate, and improving the marketability and economic benefits of grapes during storage and transportation. By constructing a VvNAC17-RNAi vector, this invention verifies for the first time the key positive regulatory role of the VvNAC17 gene in postharvest berry abscission. The gene silencing technology does not rely on complex storage and transportation conditions and avoids the potential impact of chemical agents on fruit quality and the environment, providing an innovative solution for modern grape storage and transportation technology.

Description

Technical Field

[0001] This invention belongs to the fields of fruit and vegetable and gene technology, and in particular to the application and vector of the grape VvNAC17 gene in regulating postharvest grape berry abscission. Background Technology

[0002] Grapes (Vitis vinifera L.) are an important economic crop widely cultivated globally, and their fruits are loved by consumers for their unique flavor and rich nutritional value. However, the problem of berry drop during postharvest handling and storage has long plagued all aspects of the industry chain. This problem seriously affects the marketability and sales value of grapes, directly reducing economic benefits. Currently, the problem of postharvest grape berry drop mainly manifests in the following aspects: First, the physiological and molecular mechanisms of postharvest berry drop are not yet clear. Fruit drop is mainly related to the formation of the abscission zone at the pedicel, and is regulated by plant hormone signals such as ethylene and abscisic acid. However, existing research on the molecular regulatory factors and mechanisms of action of berry drop during postharvest storage and transportation is not in-depth, making it difficult to provide a scientific basis for solving the problem. Second, existing postharvest management measures have obvious limitations. Traditional methods mainly include optimizing the storage environment (such as temperature and humidity control) and using hormone regulators (such as ethylene inhibitors) for external intervention. However, these methods are limited in practical application by high costs, inconsistent storage and transportation conditions, and the potential adverse effects of chemical regulators on fruit quality and the environment, making it difficult to meet the needs of the modern grape industry. In addition, the structural characteristics of grape stalks make them more susceptible to mechanical vibration and environmental stress during post-harvest storage and transportation. The degradation of stalk cell walls and the formation of abscission zones further accelerate berry drop. However, existing technologies lack in-depth research on the molecular regulation of stalk abscission zone formation, making it difficult to propose targeted solutions.

[0003] Against this backdrop, NACs, as an important class of transcription factors in plants, play a crucial role in biological processes such as organ abscission, cell wall degradation, and hormone signaling regulation. In recent years, significant progress has been made in functional studies of NAC family genes in model plants (such as Arabidopsis thaliana) and food crops (such as rice and wheat), with some NAC genes confirmed to be closely related to leaf abscission, fruit ripening, and cell wall metabolism. However, compared to these model plants and food crops, research on NAC transcription factors in grapes is still in its early stages, especially regarding the molecular mechanisms related to postharvest fruit abscission. Among these, VvNAC17, a potential key gene in grapes, has not yet had its function and regulatory mechanisms clearly defined, lacking systematic functional validation. Furthermore, current technologies for gene function validation in grapes face bottlenecks, including low gene editing efficiency, complex vector construction, and significant influence from genetic background, further hindering related research progress. These technical obstacles have prevented the realization of a postharvest berry abscission regulation strategy centered on VvNAC17.

[0004] Therefore, clarifying the specific mechanism of action of the VvNAC17 gene in postharvest grape berry abscission and developing corresponding molecular regulatory technologies is of great significance. By constructing an RNAi silencing vector for VvNAC17 and studying its function in pedicel abscission zone formation and cell wall degradation, we can not only achieve precise regulation of postharvest berry abscission at the molecular level, but also provide innovative solutions for modern grape storage and transportation. This method can effectively improve the marketability of grapes during storage and transportation, reduce fruit loss, and simultaneously mitigate the adverse environmental impacts of traditional external chemical regulation, providing theoretical support for modern grape storage and transportation technologies.

[0005] The search revealed the following patent publications related to this invention's patent application:

[0006] Comparison Patent 1: Application of Grape Peroxidase Gene and its Encoded Protein (CN113416737A)

[0007] The patent disclosure describes a method to reduce reactive oxygen species (ROS) accumulation by regulating VvHPCA1 gene expression, thereby improving the antioxidant properties and quality of grape berries. This technology has certain application value in addressing fruit quality deterioration caused by oxidative stress. However, the patent disclosure mainly focuses on improving antioxidant properties and does not study the molecular mechanisms of postharvest berry abscission from the perspective of berry stalk abscission zone structure and cell degradation, making it difficult to achieve precise control over berry abscission. This invention focuses on the VvNAC17 gene as the core, targeting the regulatory mechanism of postharvest grape stalk abscission zone formation. By intervening in the cell wall degradation process of the stalk abscission zone using RNAi technology, it effectively reduces berry abscission, providing a novel molecular regulatory strategy for the control of postharvest grape berry abscission and making up for the shortcomings of this patent in the field of berry abscission research.

[0008] Comparison Patent 2: Grape NAC transcription factor gene VaNAC08 and its application (CN113736793A)

[0009] This patent publication investigated the application of the VaNAC08 gene in improving the salt tolerance and stress resistance of grape cells. By regulating the physiological response of grapes under salt stress, it proposed its important role in stabilizing cell membranes and enhancing stress resistance. The NAC transcription factor family is diverse in plants, with different members having specific functions in growth and development, environmental stress response, and organ abscission regulation. The VaNAC08 gene is mainly used to regulate stress resistance under salt stress, while the functions of other NAC family genes in grapes remain to be further explored. This study further expands the application of the NAC family in regulating postharvest grape abscission, enriches the functional research of NAC proteins, and provides new ideas for postharvest management techniques for grapes and other fruit-bearing plants.

[0010] Comparison Patent 3: Application of NAC17 gene in improving salt tolerance trait of Populus tomentosa (CN115838739A)

[0011] This patent publication describes the improvement of traits in *Populus tomentosa* through overexpression of the NAC17 gene, primarily enhancing salt tolerance and wood quality. This technology has significant application value in strengthening the stress resistance of trees and improving lignification. However, the patent publication mainly focuses on the functional study of the NAC17 gene in overall tree traits; its expression patterns in other species and its specific functions in different biological processes have not been thoroughly explored, especially its potential role in regulating important traits such as fruit shedding in economic crops remains unclear.

[0012] This invention systematically studies the molecular function of the VvNAC17 gene in postharvest berry abscission by silencing the VvNAC17 gene in grapes. Focusing on mitochondrial damage and cell wall degradation in the pedicel abscission zone, the regulatory mechanism of the VvNAC17 gene in postharvest grape management was elucidated. Through functional validation, this invention proposes a precise strategy centered on molecular regulation, significantly expanding the application scope of the NAC17 gene.

[0013] By comparison, the present invention is fundamentally different from the aforementioned patent publications. Summary of the Invention

[0014] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of the grape VvNAC17 gene in regulating postharvest grape berry abscission.

[0015] The technical solution adopted by this invention to solve its technical problem is:

[0016] An application of the grape VvNAC17 gene in regulating postharvest grape berry abscission, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0017] An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0018] Furthermore, the inhibition of grape cell wall degradation refers to the inhibition of the degradation of the three main components of the cell wall in the isolated zone: protopectin, soluble pectin, and cellulose.

[0019] An application of silencing the grape VvNAC17 gene in suppressing the expression levels of grape cell wall metabolism-related genes VvPG, VvPME, VvXTH, VvPL, VvCx and Vvβ-GAL, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0020] An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation while maintaining the integrity of grape pedicel abscission zone cells, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0021] A VvNAC17-RNAi vector containing the grape VvNAC17 gene and silencing the VvNAC17 gene, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0022] Furthermore, the method for constructing the carrier includes the following steps:

[0023] (1) Cloning of the VvNAC17 gene sequence

[0024] Take 100 mg of seedless white grape isolated sample frozen in liquid nitrogen, extract total RNA, and after the RNA concentration is qualified, reverse the extracted total RNA into cDNA and screen the grape VvNAC17 gene in NCBI.

[0025] (2) Construction of VvNAC17-RNAi vector

[0026] 1) Gene amplification

[0027] Specific primers were designed based on the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R, with sequences of SEQ ID NO.3 and SEQ ID NO.4, respectively. PCR amplification was performed using cDNA from seedless white grapes as a template. The reaction system consisted of: 1 μL cDNA template, 12.5 μL 2×HieffDye, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 1 s, 30 cycles, and a final extension at 72℃ for 2 min.

[0028] 2) Detection and recovery of PCR products

[0029] The specificity of the PCR products was detected by agarose gel electrophoresis at a voltage of 140V and a current of 200A for 28 minutes. Subsequently, the gel was placed in a gel imaging system to observe the target gene fragment. The gel block containing the target gene fragment was quickly cut with a blade under a UV gel cutter. The recovered product was stored at -20℃ for later use.

[0030] 3) Ligate the pNC-AEnTopo blunt-ended cloning vector

[0031] The obtained PCR product was ligated according to the instructions. 2 μL of the product was mixed with 1 μL of pNC-AEnTopo and then added to 5 μL with ddH2O. The mixture was reacted at room temperature for 5 min and then stored at -20℃ for later use.

[0032] 4) Transformed competent cells

[0033] Transformation was performed, and 5 μL of the ligated product was added to 50 μL of LDH5α competent cells. The cells were placed on ice for 5 min, then heat-shocked in a 42°C water bath for 55 s, and then immediately placed on ice for 2 min. 700 μL of antibiotic-free LB medium was added and the cells were shaken at 37°C for 2 h. After centrifugation at 5000 rpm for 3 min, the cells were spread on LB solid medium containing Amp antibiotic and incubated upside down in a 37°C incubator for 20 h.

[0034] 5) Colony PCR and Sequence Detection

[0035] Single colonies were picked and subjected to PCR: The reaction was performed according to the instructions for the 2×Hieff Ultra-Rapid HotStart PCR Master Mix (With Dye). The reaction mixture consisted of: 2 μL DNA template, 10 μL 2×Hieff Master Mix, 1 μL universal M13, 10 μM forward primer, 1 μL universal M13, 10 μM reverse primer, and ddH2O to a final volume of 20 μL. The reaction program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 2 s, 30 cycles, and a final extension at 72℃ for 5 min. The PCR products were then analyzed by agarose gel electrophoresis, and the bacteria showing bands were sequenced.

[0036] 6) NC cloning and transformation of competent cells

[0037] Plasmids were extracted from the correctly sequenced bacteria, and then NC cloning was performed. The reaction system was as follows: VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid were both added at 80 ng, Nimble Mix 5 μL, and ddH2O was added to bring the total volume to 10 μL. After mixing by pipetting, the mixture was incubated at 50℃ for 55 min. 5 μL of the reaction product was transformed into DH5α competent cells, and the operation was as described in step 4). The antibiotic for plating was changed to Kana. After single colony PCR, the bacterial solutions with bands were sequenced.

[0038] 7) Agrobacterium-mediated transformation

[0039] Plasmids were extracted from the correctly sequenced bacteria and then transformed under the following conditions: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 28℃ for 2 h on a shaker, centrifuged at 6000 rpm for 1 min, and plated onto LB plates containing Kana and Rfp. The plates were then incubated at 28℃ for 2-3 days. Single colonies were subjected to PCR, and bacterial cultures showing bands were sequenced. Correct sequencing indicated successful construction, yielding the VvNAC17-RNAi vector.

[0040] Furthermore, the method for preparing the seedless white grape isolated sample is as follows:

[0041] Seedless white grapes from Xinjiang were harvested and left at room temperature for 2 hours to release field heat. They were then soaked in a 5% sodium hypochlorite aqueous solution for 10 minutes, rinsed three times with distilled water, and air-dried. Tissue samples, including the upper and lower sides of the fruit stem and fruit brush, were cut with a blade and used as samples from the isolated area.

[0042] The application of the VvNAC17-RNAi vector as described above in regulating postharvest grape berry abscission.

[0043] The application of the VvNAC17-RNAi vector in inhibiting grape cell wall degradation, as described above.

[0044] The advantages and positive effects of this invention are as follows:

[0045] 1. Compared with traditional methods for regulating fruit abscission, the VvNAC17 silencing technology of this invention precisely regulates fruit abscission at the gene molecular level, reducing the uncertainties of external environment and storage and transportation conditions, significantly reducing fruit abscission rate, and improving the marketability and economic benefits of grapes during storage and transportation. By constructing the VvNAC17-RNAi vector, this invention verifies for the first time the key positive regulatory role of the VvNAC17 gene in postharvest fruit abscission. Compared with traditional methods using hormone regulators and optimizing the storage environment, gene silencing technology does not rely on complex storage and transportation conditions and avoids the potential impact of chemical agents on fruit quality and the environment. Experimental results show that after 8 days of storage, the fruit abscission rate in the VvNAC17-RNAi group was significantly lower than that in the control group, specifically 5.83% and 16.47%, respectively. Figure 3 This technology is highly efficient and stable, providing an innovative solution for modern grape storage and transportation.

[0046] 2. This invention reveals the molecular regulatory mechanism of postharvest detachment in grapes from the perspective of cell wall metabolism, providing new theoretical and technical support for the study of grape postharvest detachment. This is achieved by detecting changes in the content of the main components of the cell wall (protopectin, soluble pectin, and cellulose). Figure 4 This invention clarifies the inhibitory effect of VvNAC17 gene silencing on fruit stalk cell wall degradation. Results showed that the protopectin content in the VvNAC17-RNAi group was significantly higher than that in the control group, while the soluble pectin content was reduced and the cellulose degradation rate was significantly slowed, indicating that silencing the VvNAC17 gene effectively inhibited cell wall degradation. Compared with traditional studies that failed to clarify the patterns of cell wall metabolic changes, this invention systematically explored the dynamic regulation of fruit stalk cell wall metabolism, providing a scientific basis for reducing fruit abscission.

[0047] 3. By regulating the expression of cell wall metabolism-related genes, this invention deeply elucidates the positive regulatory role of VvNAC17 on postharvest fruit abscission at the molecular level. This invention detected changes in the expression of cell wall metabolism-related genes (VvPG, VvPME, VvXTH, VvPL, VvCx, and Vvβ-GAL). Figure 5The results showed that the VvNAC17-RNAi group significantly suppressed the expression levels of these genes. Compared with the EV-RNAi group, the expression levels of VvPG and VvPME decreased by 41.23% and 83.08%, respectively, and the expression levels of other key genes such as VvCx and Vvβ-GAL also decreased significantly. The suppression of these genes effectively slowed down cell wall degradation and significantly reduced fruit abscission rate, providing important data support for the molecular regulation of cell wall-related genes.

[0048] 4. This invention optimizes the stability of the abscission zone from the perspective of overall cell structure, further improving cell integrity and fruit marketability during grape storage and transportation. Scanning electron microscopy revealed that the cell surface in the abscission zone of the VvNAC17-RNAi group remained intact with no significant lysis, while the control group showed severe cell surface lysis and significantly enlarged intercellular spaces. Figure 6 Furthermore, transmission electron microscopy revealed the protective role of organelle structures. Figure 7 In the VvNAC17-RNAi group, the mitochondrial structure remained intact, the outer and inner membranes were stable, and the matrix density was uniform, while the control group showed mitochondria with outer membrane rupture and disordered inner cristae. These results indicate that VvNAC17 gene silencing not only reduces cell wall degradation but also significantly maintains the integrity of cells in the pedicel abscission zone, providing a new approach to improving the scientific and practical aspects of postharvest management of grapes.

[0049] 5. This invention is the first to construct a postharvest fruit abscission regulation technology system centered on the VvNAC17 gene, promoting innovation in grape postharvest management technology. This invention combines gene silencing technology, cellular metabolic component detection, and organelle structure observation to comprehensively reveal the mechanism of action of the VvNAC17 gene in the postharvest fruit abscission zone. Compared with existing technologies, this invention not only focuses on gene function but also integrates multidimensional research methods on cell wall metabolism and structural changes. Experiments have verified the superiority of this technology in regulating grape postharvest abscission, providing a foundation for the development of precise molecular regulatory tools.

[0050] 6. This invention is the first to construct a molecular strategy for regulating postharvest fruit abscission, with the grape VvNAC17 gene as the core.

[0051] This invention constructs an RNAi silencing vector for the VvNAC17 gene to systematically study its molecular regulatory mechanism in the formation of the abscission zone and cell wall degradation in grapes. Based on in-depth verification of gene function, the core role of the VvNAC17 gene in postharvest berry abscission has been clarified. Addressing the issue that the molecular mechanism of postharvest berry abscission remains unclear, this invention proposes a novel method for precise intervention in fruit abscission at the molecular level, providing a scientific basis for postharvest fruit management.

[0052] 7. This invention expands the function of the NAC gene and reveals for the first time the application potential of the VvNAC17 gene in the regulation of grape postharvest abscission.

[0053] This invention discovered and systematically verified the function of the VvNAC17 gene in the formation of the abscission zone and fruit abscission process in grapes after harvest, clarifying its regulatory role in cell wall degradation, mitochondrial structure and function, and hormone signaling. As an important member of the NAC family in grapes, the VvNAC17 gene has promising functions and applications. This invention further fills the gap in grape NAC gene research and lays the foundation for realizing molecular regulatory strategies for grape postharvest abscission.

[0054] 8. This invention is based on the overall regulatory network formed by the grape stalk abscission zone, and develops a multi-dimensional technical system for regulating postharvest fruit drop.

[0055] This invention not only focuses on the functional study of the VvNAC17 gene, but also integrates multiple signaling pathways and biological processes related to postharvest fruit abscission (such as hormone signaling, mitochondrial function, and cell wall degradation). By constructing a systematic molecular regulatory network for grape pedicel abscission zone formation, this invention proposes a multi-dimensional comprehensive regulatory technology system for postharvest fruit abscission using precise molecular tools. Attached Figure Description

[0056] Figure 1 This is a gel electrophoresis image of the nucleic acid constructed by the VvNAC17-RNAi vector in this invention, with a fragment size of 360bp;

[0057] Figure 2 The phenotypic changes of seedless white grapes in this invention on days 0, 2, 4, 6, and 8 after treatment with EV-RNAi and VvNAC17-RNAi, respectively.

[0058] Figure 3 This is a graph showing the berry drop rate of seedless white grapes after treatment in this invention;

[0059] Figure 4 This is a diagram showing the content of protopectin, soluble pectin, and cellulose, the cell wall components of seedless white grapes in this invention.

[0060] Figure 5 This is a graph showing the expression levels of cell wall metabolism genes in seedless white grapes used in this invention.

[0061] Figure 6 These are scanning electron microscope images of the isolated cells of seedless white grapes in this invention.

[0062] Figure 7 This is a transmission electron microscope image of the isolated cells of seedless white grapes in this invention. Detailed Implementation

[0063] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0064] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0065] An application of the grape VvNAC17 gene in regulating postharvest grape berry abscission, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0066] An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0067] Preferably, the inhibition of grape cell wall degradation refers to the inhibition of the degradation of the three main components of the cell wall in the isolated zone: protopectin, soluble pectin, and cellulose.

[0068] An application of silencing the grape VvNAC17 gene in suppressing the expression levels of grape cell wall metabolism-related genes VvPG, VvPME, VvXTH, VvPL, VvCx and Vvβ-GAL, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0069] An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation while maintaining the integrity of grape pedicel abscission zone cells, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0070] A VvNAC17-RNAi vector containing the grape VvNAC17 gene and silencing the VvNAC17 gene, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0071] Preferably, the method for constructing the carrier includes the following steps:

[0072] (1) Cloning of the VvNAC17 gene sequence

[0073] Take 100 mg of seedless white grape isolated sample frozen in liquid nitrogen, extract total RNA, and after the RNA concentration is qualified, reverse the extracted total RNA into cDNA and screen the grape VvNAC17 gene in NCBI.

[0074] (2) Construction of VvNAC17-RNAi vector

[0075] 1) Gene amplification

[0076] Specific primers were designed based on the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R, with sequences of SEQ ID NO.3 and SEQ ID NO.4, respectively. PCR amplification was performed using cDNA from seedless white grapes as a template. The reaction system consisted of: 1 μL cDNA template, 12.5 μL 2×HieffDye, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 1 s, 30 cycles, and a final extension at 72℃ for 2 min.

[0077] 2) Detection and recovery of PCR products

[0078] The specificity of the PCR products was detected by agarose gel electrophoresis at a voltage of 140V and a current of 200A for 28 minutes. Subsequently, the gel was placed in a gel imaging system to observe the target gene fragment. The gel block containing the target gene fragment was quickly cut with a blade under a UV gel cutter. The recovered product was stored at -20℃ for later use.

[0079] 3) Ligate the pNC-AEnTopo blunt-ended cloning vector

[0080] The obtained PCR product was ligated according to the instructions. 2 μL of the product was mixed with 1 μL of pNC-AEnTopo and then added to 5 μL with ddH2O. The mixture was reacted at room temperature for 5 min and then stored at -20℃ for later use.

[0081] 4) Transformed competent cells

[0082] Transformation was performed, and 5 μL of the ligated product was added to 50 μL of LDH5α competent cells. The cells were placed on ice for 5 min, then heat-shocked in a 42°C water bath for 55 s, and then immediately placed on ice for 2 min. 700 μL of antibiotic-free LB medium was added and the cells were shaken at 37°C for 2 h. After centrifugation at 5000 rpm for 3 min, the cells were spread on LB solid medium containing Amp antibiotic and incubated upside down in a 37°C incubator for 20 h.

[0083] 5) Colony PCR and Sequence Detection

[0084] Single colonies were picked and subjected to PCR: The reaction was performed according to the instructions for the 2×Hieff Ultra-Rapid HotStart PCR Master Mix (With Dye). The reaction mixture consisted of: 2 μL DNA template, 10 μL 2×Hieff Master Mix, 1 μL universal M13, 10 μM forward primer, 1 μL universal M13, 10 μM reverse primer, and ddH2O to a final volume of 20 μL. The reaction program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 2 s, 30 cycles, and a final extension at 72℃ for 5 min. The PCR products were then analyzed by agarose gel electrophoresis, and the bacteria showing bands were sequenced.

[0085] 6) NC cloning and transformation of competent cells

[0086] Plasmids were extracted from the correctly sequenced bacteria, and then NC cloning was performed. The reaction system was as follows: VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid were both added at 80 ng, Nimble Mix 5 μL, and ddH2O was added to bring the total volume to 10 μL. After mixing by pipetting, the mixture was incubated at 50℃ for 55 min. 5 μL of the reaction product was transformed into DH5α competent cells, and the operation was as described in step 4). The antibiotic for plating was changed to Kana. After single colony PCR, the bacterial solutions with bands were sequenced.

[0087] 7) Agrobacterium-mediated transformation

[0088] Plasmids were extracted from the correctly sequenced bacteria and then transformed under the following conditions: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 28℃ for 2 h on a shaker, centrifuged at 6000 rpm for 1 min, and plated onto LB plates containing Kana and Rfp. The plates were then incubated at 28℃ for 2-3 days. Single colonies were subjected to PCR, and bacterial cultures showing bands were sequenced. Correct sequencing indicated successful construction, yielding the VvNAC17-RNAi vector.

[0089] Preferably, the method for preparing the seedless white grape isolated sample is as follows:

[0090] Seedless white grapes from Xinjiang were harvested and left at room temperature for 2 hours to release field heat. They were then soaked in a 5% sodium hypochlorite aqueous solution for 10 minutes, rinsed three times with distilled water, and air-dried. Tissue samples, including the upper and lower sides of the fruit stem and fruit brush, were cut with a blade and used as samples from the isolated area.

[0091] The application of the VvNAC17-RNAi vector as described above in regulating postharvest grape berry abscission.

[0092] The application of the VvNAC17-RNAi vector in inhibiting grape cell wall degradation, as described above.

[0093] Specifically, the relevant preparation and testing methods are as follows:

[0094] I. Process Steps and Parameters

[0095] 1. Preparation of experimental materials

[0096] The experimental material was seedless white grapes from Xinjiang. Twelve bunches were harvested and transported to the laboratory within 2 hours, then left at room temperature for 2 hours to release field heat. After soaking in a 5% sodium hypochlorite aqueous solution for 10 minutes, the grapes were rinsed three times with distilled water and air-dried. Whole bunches of grapes were used for observation of phenotypic characteristics. Tissue samples, approximately 2 mm in diameter, including the area where the stem and fruit brush connect, were cut with a blade to serve as isolates. After sampling, the isolates were immediately frozen in liquid nitrogen and stored at -80°C for subsequent experiments. Each experiment was performed in triplicate (biological replicates) and in triplicate (technical replicates).

[0097] 2. Gene cloning steps

[0098] 2.1 Cloning of the VvNAC17 gene sequence

[0099] 100 mg of seedless white grape isolate, frozen in liquid nitrogen, was used to extract total RNA using the Plant Total RNA Isolation Kit Plus. After confirming the RNA concentration met the acceptable levels, Prime Script was used. TM The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit. The grape VvNAC17 gene (the complete coding region gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2) was screened in NCBI.

[0100] SEQ ID NO.1:

[0101] ATGAAGGTGACAGTGGGTGATTCGTCGCCGTGCTTTGACGGAGACGAAAAGTCTGCGT

[0102] GGCCACCCGGGTTCCGATTCCATCCCACCGATGAAGAGCTGGTTCTGTATTATCTGAAGA

[0103] AGAAGATCTGCCGGCGACGGCTGAAGCTCGATATCATCGCCGAGGTCGATGTCTACAAG

[0104] TGGGACCCCGAGGATTTGCCTGGACTATCTAAATTGAAGACAGGAGATAGGCAATGGTT

[0105] CTTTTTTAGCCCCAGAGACAGGAAGTACCCTAATGGAGCTAGGTCTAATAGGGCAACCA

[0106] GGCATGGATACTGGAAAGCAACAGGAAAGGATCGAACTATTAGCTGTAATATTCGGTCA

[0107] GTTGGTGTGAAGAAGACCTTGGTTTTCTATAAAGGCCGTGCTCCAAGTAAAGAGCGCAC

[0108] AGACTGGGTGATGCATGAGTATACAATGGATGAAGAGGAGCTCAAGAGATGCCCGAATG

[0109] TGCAGGATTATTATGCACTTTATAAGGTCTTCAAGAAGAGTGGACCTGGTCCCAAAAATG

[0110] GTGAGCAATACGGGGCTCCATTTAAAGAAGAGGAATGGGCTGACGAAGATGACCTAGAT

[0111] GTTAGTAACTACTCTGTTGAAGAGACTCCTCCAGAGCAGTTGAATGGCGTTATTTCTGTC

[0112] AATAATTCCAAACCTAATGGGCAAGACTGTCAAGCAGATGCTTGGGATGACATCTGGAA

[0113] AGGACTTGCAGAAGCACCTCCAGTTGTTCCTCTGCGTGTTGATGATTATGTTAATCTACT

[0114] AGCTCAGGTTATTGGTGAAGAAGAAGCTCAAACTCCTTTGGTGGATTCATCACTCAATG

[0115] GAGCTTTCGTTGCTGATCCAATAAGCACAGTATTAACCCCTACCTCTCAGCAGTATGCTG

[0116] TGCCAGAGAACGTTGAGTTTACACAATCAGCCTCCTCTCAGTTGCAATTGCACGAGGCA

[0117] CCTGAGGTCACATCTGCTCCTAACATTAGTGAGCAGGAACGTGGATTAAGTGAGGAGGA

[0118] CTTTCTAGAAATGGATGATCTCCTTGGTCCAGAACCCATTCCTCAAAACTATGAAAAAAC

[0119] TGAGGAGAACTTGCAGTTTGAAGGCGATGGATTGAGCATACTTGACCTGTACCATGATG

[0120] CAGCCATGTTTCTTCGTGACATTGGCCCTATTGATCAAGGAACGGTTCCGCATCCATATTT

[0121] GAATACCATTGAGAATGAGATGGTGAACCAGTTGAATTACCAGCTGCAGCCCCATTCTGT

[0122] TGGTGCAGATCAGATTAGTGGTCAGCTGTGGACACTCGATCAAAGTGTCTGTACCTCAG

[0123] CAGAATCTATTCAGGGGATCATTGGGCAGCCAACCTCAGGTGTTGTATATGCCAGCAGTT

[0124] CTACAAATGTTCCCACCGAAGGAAATCAAAACATGAATGGCGAAGGGGGTAACGGTGC

[0125] AGGGAACCGATTCACTTCTGCTCTATGGTCCTTTGTGGAGTCAATACCTACCACACCTGC

[0126] ATCAGCTTCAGAAAATGCGTTGGTAAATCGGGCATTGGTGAGAATGTCTAGCTTTAGTAG

[0127] GATGAGAATGAATGCATTGAACACAAATGCAGGTAATGGAGGTGCAGCCACATGGAAG

[0128] GGAGGTATAAATAAGGGGGGATTCATCATTCTTTCAGTTATTGGAGCACTGATAGCTATATTCTGGGTCCTAATGCTAGGACCTGTGAAGATGTTAGGAAGATGCCTCCCCTCATGA.SEQ ID NO.2:

[0129] MKVTVGDSSPCFDGDEKSAWPPGFRFHPTDEELVLYYLKKKICRRRLKLDIIAEVDVYKWD

[0130] PEDLPGLSKLKTGDRQWFFFSPRDRKYPNGARSNRATRHGYWKATGKDRTISCNIRSVGVK

[0131] KTLVFYKGRAPSKERTDWVMHEYTMDEEELKRCPNVQDYYALYKVFKKSGPGPKNGEQY

[0132] GAPFKEEEWADEDDLDVSNYSVEETPPEQLNGVISVNNSKPNGQDCQADAWDDIWKGLAE

[0133] APPVVPLRVDDYVNLLAQVIGEEEAQTPLVDSSLNGAFVADPISTVLTPTSQQYAVPENVEFT

[0134] QSASSQLQLHEAPEVTSAPNISEQERGLSEEDFLEMDDLLGPEPIP

[0135] QNYEKTEENLQFEGDGLSILDLYHDAAMFLRDIGPIDQGTVPHPYLNTIENEMVNQLNYQL

[0136] QPHSVGADQISGQLWTLDQSVCTSAESIQGIIGQPTSGVVYASSSTNVPTEGNQNMNGEGG

[0137] NGAGNRFTSALWSFVESIPTTPASASENALVNRALVRMSSFSRMRMNALNTNAGNGGAATWKGGINKGGFIILSVIGALIAIFWVLMLGPVKMLGRCLPS.

[0138] 2.2 Construction of VvNAC17-RNAi vector

[0139] 2.2.1 Gene Amplification

[0140] Specific primers were designed based on the CDS sequence of the VvNAC17 gene (Table 1). The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R. PCR amplification was performed using cDNA from seedless white grapes as a template. The reaction mixture consisted of: 1 μL cDNA template, 12.5 μL 2×HieffDye, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and ddH2O to a final volume of 25 μL. The reaction program was: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 1 s, 30 cycles, followed by a final extension at 72℃ for 2 min.

[0141] 2.2.2 Detection and Recovery of PCR Products

[0142] The specificity of the PCR products was detected by agarose gel electrophoresis. The voltage was set to 140V, the current to 200A, and the electrophoresis time was 28 min. Subsequently, the gel was placed in a gel imaging system to observe the target gene fragment. The gel block containing the target gene fragment was rapidly cut with a blade under UV excimer and recovered using a DNA gel extraction kit (FastPure Gel DNA Extraction Mini Kit). The recovered product was stored at -20℃ for later use.

[0143] 2.2.3 Ligation of pNC-AEnTopo blunt-ended cloning vector

[0144] The obtained PCR product was ligated using the pNC-AEnTopo blunt-end cloning vector kit. 2 μL of the product was mixed with 1 μL of pNC-AEnTopo, and the volume was brought up to 5 μL with ddH2O. The mixture was incubated at room temperature for 5 min, and then stored at -20℃ for later use.

[0145] 2.2.4 Transformation of competent cells

[0146] Transformation was performed according to the Trelief5α Chemically Competent Cell instructions. 5 μL of the ligated product was added to 50 μL of LDH5α competent cells, incubated on ice for 5 min, then heat-shocked at 42°C for 55 s, and immediately placed on ice for 2 min. 700 μL of LB (antibiotic-free) medium was added, and the cells were incubated at 37°C with shaking for 2 h. After centrifugation at 5000 rpm for 3 min, the cells were spread onto LB solid medium containing Amp antibiotics and incubated upside down at 37°C for 20 h.

[0147] 2.2.5 Colony PCR and Sequence Detection

[0148] Single colonies were picked and subjected to PCR. The reaction was performed according to the instructions for the 2×Hieff Ultra-Rapid HotStart PCR Master Mix (With Dye). The reaction mixture consisted of: 2 μL DNA template, 10 μL 2×Hieff Master Mix, 1 μL forward primer (universal M13, 10 μM), 1 μL reverse primer (universal M13, 10 μM), and ddH2O to a final volume of 20 μL. The reaction program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 2 s, 30 cycles, followed by a final extension at 72℃ for 5 min. The PCR products were analyzed by agarose gel electrophoresis. Bacteria with visible bands were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing.

[0149] 2.2.6 NC Cloning and Transformation of Competent Cells

[0150] Plasmids were extracted from the correctly sequenced bacteria, followed by NC cloning. The reaction mixture consisted of 80 ng each of VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid, 5 μL of Nimble Mix, and ddH2O to a final volume of 10 μL. After mixing thoroughly by pipetting, the mixture was incubated at 50°C for 55 min. 5 μL of the reaction product was then transformed into DH5α competent cells, following the procedure described in 2.2.4, with the antibiotic changed to Kana for plating. Single-colony PCR was performed, and the banded bacterial cultures were sequenced.

[0151] 2.2.7 Agrobacterium-mediated transformation

[0152] Plasmids were extracted from the correctly sequenced bacteria, and then transformed according to the GV3101 (Weidi Biotechnology) instructions. The reaction conditions were: ice bath for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, ice bath for 5 min. Then, 700 μL of LB (antibiotic-free) medium was added, and the mixture was incubated at 28℃ for 2 h on a shaker, centrifuged at 6000 rpm for 1 min, and plated onto LB plates containing Kana and Rfp. The plates were then incubated at 28℃ for 2-3 days. Single colonies were subjected to PCR, and bacterial cultures showing bands were sequenced. Correct sequencing indicated successful construction, yielding the VvNAC17-RNAi vector. An empty vector without homologous recombination was used as a control.

[0153] 2.2.8 Agrobacterium-mediated transformation of seedless white grape isolated cells

[0154] The constructed VvNAC17-RNAi vector and the empty vector Agrobacterium were activated. Agrobacterium cultures containing pNC-Cam2304-RNAi (empty vector) and pNC-Cam2304-VvNAC17 recombinant plasmid were added to LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin, respectively, and cultured at 28°C with shaking until OD. 600 The OD value was 1.2. The cells were then collected by centrifugation at 5000 rpm for 10 min at 28°C, and the supernatant was discarded. The cells were resuspended in infection medium containing 10 mM MgCl2, 10 mM MES, and 200 μM acetylsyringone (As) at pH 5.6, and the OD value was adjusted. 600 The value was 1.0. Seedless white grape berries were collected, and several small holes were punctured at the stem end using a sterile syringe. The entire berry was then immersed in the bacterial suspension and vacuum-permeated at a pressure of 0.75 MPa for 30 minutes. The infected grape berries were then cultured in MES liquid medium for 2 days, followed by incubation at 26°C. Samples were collected 8 days after infection.

[0155] 3. Functional verification steps

[0156] 3.1 Fruit phenotypic changes and abscission rate determination

[0157] In each experimental group, three fixed bunches of grapes were selected during storage to measure phenotypic changes and berry drop rate. Images were processed using ImageJ. The berry drop rate was defined as the ratio of the weight of dropped grapes to the total weight of the grapes during storage, calculated using the following formula:

[0158]

[0159] 3.2 qRT-PCR detection of cell wall metabolism gene expression

[0160] RNA extraction and cDNA reverse transcription are performed as described in 2.1. Using... Premix Ex TaqTM The II kit was used to determine the quantification of mRNA expression. Then, 2... -ΔΔCT The relative quantification was calculated using this method. Primers used in the experiment are shown in Table 2.

[0161] Table 1 Primer sequences used for constructing the VvNAC17-RNAi vector

[0162] Primer name Primer sequence (5'-3') VvNAC17-RNAi-F AAGGCCGTGCTCCAAGTAAA VvNAC17-RNAi-R GCAGAGGAACAACTGGAGGT

[0163] Table 2 Primer sequences for real-time quantitative PCR (qRT-PCR)

[0164] Gene Upstream primer (5'-3') Downstream primer (5'-3') VvPG AGGAGGAAAGGCAGCCAAAG AGTGTGTGATCAGAGGATTCAG VvPME AGGTCCCATACACTGAGCCT TGCCACCACGATATTTGCCT VvXTH CCAACCTGTACAACCACGGA AAGTCCAGTTCATCGTGCGT VvPL TTGATGACTGCTGGCGATGT TGAGCTCCTGCTTCAGTGTG VvCx GGTTGCGTTGCAGAGCAGAT GAGAATGCCCTTGGTCAAAGC Vvβ-GAL CCTGATTGCAGAACCGTAGTCT GCACAGTGAGGGTTGGAGTT

[0165] 3.3 Detection of the content of major components of the cell wall in the isolated zone

[0166] The main assessment focused on the content of three major cell wall components: protopectin, soluble pectin, and cellulose. The protopectin and soluble pectin contents were determined using the carbazole method. 10 mL of 95% ethanol (v / v) was added to a 1.0 g sample of fresh grapes, mixed, and heated in a boiling water bath for 30 min. The mixture was then centrifuged at 8,000 × g for 10 min. The supernatant was discarded, and 4 mL of distilled water was added to the precipitate. The mixture was heated in a 60°C water bath for 30 min. The solution was filtered, and the residue was washed. The filtrate was transferred to a 25 mL volumetric flask and diluted to volume with distilled water; this was the soluble pectin assay solution. The residue was collected and 5 mL of 0.5 mL L-ethanol was added. -1 Sulfuric acid was added, boiled in a water bath for 1 hour, and then filtered. The residue was washed again, and the washings were combined. After cooling, the washings were transferred to a 25 mL volumetric flask and diluted to volume. This is the original pectin assay solution. 1 mL of the original pectin assay solution and the soluble pectin assay solution were added to 6 mL of concentrated sulfuric acid, mixed well, and boiled in a water bath for 20 minutes. After cooling to room temperature, 0.2 mL of 1.5 g L-sulfuric acid solution was added. -1 Carbazole reagent, after mixing and standing in the dark for 2 hours, was used to measure the absorbance of the reaction solution at a wavelength of 530 nm. A standard curve was prepared using pectin as a standard, and the content was expressed in g / kg. -1 FW.

[0167] Add 1.0 mL of 80% ethanol (v / v) to a fresh grape absorptio sample (0.3 g), mix well, and heat in a 90°C water bath for 20 min. After cooling, centrifuge at 6,000 × g for 10 min at 25°C. Discard the supernatant, add 1.5 mL of 80% ethanol and acetone to the precipitate, mix well, and centrifuge again (6,000 × g for 10 min at 25°C). Discard the supernatant, add 1.0 mL of dimethyl sulfoxide (DMSO), mix well, and let stand for 15 h. Then centrifuge the mixture at 6,000 × g for 10 min at 25°C. Discard the supernatant, and dry the precipitate, which is the cell wall material (CWM). Dry the CWM in a 40°C oven to constant weight. Add 0.5 mL of distilled water and 0.75 mL of concentrated sulfuric acid to the dried CWM and mix well. After 30 minutes, the mixture was centrifuged at 8,000 × g for 10 minutes at 4°C. The supernatant was diluted 20 times with distilled water, and the absorbance was measured at 630 nm. Cellulose content is expressed in g / kg. -1 .

[0168] 3.4 Scanning electron microscopy inspection of the surface of the off-zone

[0169] The surface morphology of the absorptive regions was observed using scanning electron microscopy (SEM). Tissue containing absorptive regions (including the fruit stalk and part of the fruit brush) was excised with a blade and completely immersed in a 2.5% glutaraldehyde solution (v / v) and incubated at 4°C for 12 h. The samples were then thoroughly rinsed three times with 0.1 M phosphate buffer (pH = 7.0) and fixed with 1% (w / v) osmium tetroxide solution for 2 h. After removing the osmium tetroxide waste solution, the samples were rinsed three more times with 0.1 M phosphate buffer (pH = 7.0). The samples were then dehydrated using a gradient concentration of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%, v / v) for 15 min each time, and then placed in fresh 100% ethanol. The samples were further dehydrated using a critical point desiccator. The dehydrated samples were then coated with a gold-palladium alloy using an ion sputtering apparatus and observed using a scanning electron microscope.

[0170] 3.5 Transmission electron microscopy examination of cell structures in the isolated region

[0171] The ultrastructure of the isolated cells was observed using transmission electron microscopy (TEM). A tissue block (approximately 3 mm × 1 mm × 1 mm) was manually excised from the junction of the fruit stalk and fruit brush. The sample was fixed with 2.5% glutaraldehyde solution and 1% (w / v) osmium tetroxide solution, following the same fixation procedure as for SEM. Dehydration was then performed using gradient concentrations of ethanol (30%, 50%, 70%, and 80%, v / v) for 15 min each time, followed by gradient concentrations of acetone (90%, 95%, and 100%, v / v) for 20 min each time. After dehydration, the sample was immersed in a mixture of acetone and resin (1:1 v / v) for 1 h, then in a mixture of acetone and resin (1:3 v / v) for 3 h, and finally immersed overnight in pure resin embedding medium at room temperature. The embedded sample was heated at 70°C overnight. The thin sections (70 nm) cut by an ultramicrotome were stained with lead citrate solution and 50% (volume percentage) ethanol saturated solution of uranium acetate for 10 min each, and then observed by transmission electron microscopy.

[0172] II. Experimental Data Section:

[0173] Example 1: Cloning of the VvNAC17 gene and construction of the RNAi vector

[0174] This invention extracts total RNA from the stalk-isolated region of seedless white grapes, synthesizes cDNA, and designs specific primers based on the VvNAC17-CDS sequence. The VvNAC17 gene fragment is amplified using PCR technology. After agarose gel electrophoresis, the amplified band size was observed to be consistent with expectations (~360 bp), further verifying the success of the PCR amplification. Figure 1 Subsequently, the amplified target fragment was inserted into the pNC-Cam2304-RNAi vector to construct the VvNAC17-RNAi recombinant plasmid. The constructed vector was then successfully transformed into GV3101 competent Agrobacterium using Agrobacterium-mediated transformation. Simultaneously, the pNC-Cam2304-RNAi vector was used to transform Agrobacterium to construct an empty vector (EV-RNAi) control group. Colony PCR and sequencing verification confirmed that the recombinant plasmid sequence was correct and completely consistent with the design. The clear display of electrophoretic bands and the correct insertion of the target fragment together indicate that the VvNAC17-RNAi vector has been successfully constructed, laying the foundation for subsequent research.

[0175] Example 2: Effects of VvNAC17 gene silencing on grape fruit abscission

[0176] 1. Fruit phenotypic changes and abscission rate

[0177] Through a diagram of fruit phenotypic changes during storage ( Figure 2 The significant differences between the EV-RNAi and VvNAC17-RNAi groups can be visually observed. In the EV-RNAi group, fruit granules gradually detached with increasing storage time, reaching the highest detachment rate of 16.47% on day 8; while in the VvNAC17-RNAi group, fruit granules remained intact with only a small amount of detachment, a detachment rate of only 5.83% (p<0.01). Figure 3 Statistical results showed that the abscission rate in the VvNAC17-RNAi group was significantly lower than that in the EV-RNAi group. This indicates that silencing VvNAC17 significantly reduces berry abscission. These results effectively validate the key regulatory role of the VvNAC17 gene in postharvest grape berry abscission.

[0178] 2. Changes in the main components of the cell wall in the exozone

[0179] By detecting the content of the three main components (protopectin, soluble pectin, and cellulose) of the cell wall in the isolated zone ( Figure 4 This study further validated the regulatory role of the VvNAC17 gene in cell wall degradation. Results showed that on day 8, the protopectin content in the VvNAC17-RNAi group was 16.40% higher than that in the EV-RNAi group (p<0.01), indicating that cell wall pectin degradation was inhibited. Simultaneously, the soluble pectin content in the VvNAC17-RNAi group was 27.63% lower than that in the EV-RNAi group. Furthermore, the cellulose degradation rate in the VvNAC17-RNAi group was significantly slowed, with the cellulose content on day 8 being 14.24% higher than that in the EV-RNAi group (p<0.01). These results indicate that VvNAC17 gene silencing effectively inhibits the degradation process of the cell wall in the isolated region.

[0180] 3. Expression of genes related to cell wall metabolism

[0181] To further explore the effects of VvNAC17-RNAi on the molecular mechanisms of fruit stalk abscission, the expression levels of cell wall metabolism-related genes (VvPG, VvPME, VvXTH, VvPL, VvCx, and Vvβ-GAL) were examined. Figure 5The results showed that on day 8, compared with the EV-RNAi group, the gene expression levels of VvPG, VvPME, VvXTH, VvPL, VvCx, and Vvβ-GAL in the VvNAC17-RNAi group decreased by 41.23%, 83.08%, 60.32%, 37.46%, 55.32%, and 55.61%, respectively (p<0.01). These results indicate that VvNAC17 is a positive regulator of key genes involved in cell wall degradation, and its silencing significantly reduced the expression of genes related to fruit stalk cell wall degradation. The changes in gene expression levels further validated the importance of the VvNAC17 gene in the regulation of cell wall metabolism, providing insights into the molecular mechanisms of postharvest grape berry abscission.

[0182] Example 3: Morphological and structural changes in the abscission zone of the fruit stalk

[0183] 1. Scanning electron microscopy observation of the surface morphology of the off-site area

[0184] The effects on the surface and overall cell structure of the detachment zone were observed using scanning electron microscopy. Figure 6 The results showed that in the EV-RNAi group, significant lysis was observed on the surface of cells in the abscess zone, with marked decomposition of the cell wall and surrounding tissues, resulting in a loss of overall cell structure integrity. Furthermore, at high magnification, increased intercellular spaces and looser cell junctions were observed, suggesting active cell wall degradation and cell destruction. In contrast, in the VvNAC17-RNAi group, the cell surface was smoother, lysis was significantly reduced, the overall structure remained intact, and intercellular junctions were tight, indicating that VvNAC17 silencing effectively prevented structural damage in the abscess zone and inhibited cell wall degradation and abscess formation. This result further demonstrates the role of VvNAC17 from a morphological perspective.

[0185] 2. Transmission electron microscopy observation of the ultrastructure of cells in the isolated region

[0186] Transmission electron microscopy revealed changes in the ultrastructure of cells in the isolated region, further demonstrating the details of cell wall degradation. Figure 7In the EV-RNAi group, the cell wall was significantly thinner, organelles ruptured, and cytoplasm leaked out, resulting in severe damage to cell structural integrity. The mitochondrial structure of the EV-RNAi group's exotropic cells was severely damaged, manifested as outer membrane rupture, cristae breakage and disorder, matrix thinning, and mitochondrial swelling, suggesting that mitochondrial dysfunction may lead to cellular metabolic disorders and trigger cell death. In contrast, in the VvNAC17-RNAi group, cell wall thickness was maintained, cytoplasm was uniformly distributed, and cell structure remained intact. The outer and inner mitochondrial membranes were intact, the cristae were clear and neatly arranged, the matrix density was uniform, and the morphology remained stable. These results indicate that VvNAC17 silencing effectively prevents cell wall degradation and cell structural damage at the cellular level. From an ultrastructural perspective, this study demonstrates the protective effect of VvNAC17 gene silencing on exotropic cells.

[0187] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. An application of silencing the grape VvNAC17 gene in reducing berry drop rate, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

2. An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

3. The application of silencing the grape VvNAC17 gene in suppressing the expression levels of grape cell wall metabolism-related genes VvPG, VvPME, VvXTH, VvPL, VvCx and Vvβ-GAL, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1 and the amino acid sequence of its encoded protein is shown in SEQ ID NO.

2.

4. An application of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation while maintaining the integrity of grape pedicel abscission zone cells, wherein the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.

2.

5. Application of VvNAC17-RNAi vector in reducing postharvest grape berry abscission; the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.

2.

6. Use according to claim 5, characterized in that: The method for constructing the carrier includes the following steps: (1) Cloning of the VvNAC17 gene sequence Take 100 mg of seedless white grape isolated sample frozen in liquid nitrogen, extract total RNA, and after the RNA concentration is qualified, reverse the extracted total RNA into cDNA and screen the grape VvNAC17 gene in NCBI. (2) Construction of VvNAC17-RNAi vector 1) Gene amplification Specific primers were designed based on the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R, with sequences of SEQ ID NO.3 and SEQ ID NO.4, respectively. PCR amplification was performed using cDNA from seedless white grapes as a template. The reaction system consisted of: 1 μL cDNA template, 12.5 μL 2×Hieff Dye, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 98 ℃ pre-denaturation for 30 s, 98 ℃ denaturation for 10 s, 60 ℃ annealing for 5 s, 72 ℃ extension for 1 s, 30 cycles, and a final extension at 72 ℃ for 2 min. 2) Detection and recovery of PCR products The specificity of the PCR products was detected by agarose gel electrophoresis at a voltage of 140V and a current of 200A for 28 min. The gel was then placed in a gel imaging system to observe the target gene fragment. The gel containing the target gene fragment was quickly cut with a blade under a UV gel cutter. The recovered product was stored at -20 ℃ for later use. 3) Ligate the pNC-AEnTopo blunt-ended cloning vector The obtained PCR product was ligated according to the instructions. 2 μL of the product was mixed with 1 μL of pNC-AEnTopo and then made up to 5 μL with ddH2O. The mixture was reacted at room temperature for 5 min and then stored at -20 ℃ for later use. 4) Transformed competent cells Transformation was performed, and 5 μL of the ligated product was added to 50 μL of DH5α competent cells. The cells were placed on ice for 5 min, then heat-shocked in a 42°C water bath for 55 s, and immediately placed on ice for 2 min. 700 μL of antibiotic-free LB medium was added and the cells were shaken at 37°C for 2 h. After centrifugation at 5000 rpm for 3 min, the cells were spread on LB solid medium containing Amp antibiotic and incubated upside down at 37°C for 20 h. 5) Colony PCR and Sequence Detection Single colonies were picked for PCR: The reaction was performed according to the 2×Hieff Ultra-Rapid HotStart PCR Master Mix (With Dye) instructions. The reaction mixture consisted of: 2 μL DNA template, 10 μL 2×Hieff Master Mix, 1 μL universal M13, 10 μM forward primer, 1 μL universal M13, 10 μM reverse primer, and ddH2O to a final volume of 20 μL. The reaction program was: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 10 s, 60 ℃ annealing for 20 s, 72 ℃ extension for 2 s, 30 cycles, and a final extension at 72 ℃ for 5 min. The PCR products were then analyzed by agarose gel electrophoresis, and the bacteria showing bands were sequenced. 6) NC cloning and transformation of competent cells Plasmids were extracted from the correctly sequenced bacteria, and then NC cloning was performed. The reaction system was as follows: VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid were both added at 80 ng, Nimble Mix 5 μL, and ddH2O was added to bring the total volume to 10 μL. After mixing by pipetting, the mixture was incubated at 50 ℃ for 55 min. 5 μL of the reaction product was transformed into DH5α competent cells, and the operation was as described in step 4). The antibiotic for plate coating was changed to Kana. After single colony PCR, the bacterial solutions with bands were sequenced. 7) Agrobacterium-mediated transformation Plasmids were extracted from the correctly sequenced bacteria and then transformed under the following conditions: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 28℃ for 2 h on a shaker, centrifuged at 6000 rpm for 1 min, and plated onto LB plates containing Kana and Rfp. The plates were then incubated at 28℃ for 2-3 days. Single colonies were subjected to PCR, and bacterial cultures showing bands were sequenced. Correct sequencing indicated successful construction, yielding the VvNAC17-RNAi vector.

7. Use according to claim 6, characterized in that: The method for preparing the seedless white grape isolated sample is as follows: Seedless white grapes from Xinjiang were harvested and left at room temperature for 2 hours to release field heat. They were then soaked in a 5% sodium hypochlorite aqueous solution for 10 minutes, rinsed three times with distilled water, and air-dried. Tissue samples, including the upper and lower sides of the fruit stem and fruit brush, were cut with a blade and used as samples from the isolated area.

8. Application of VvNAC17-RNAi vector in inhibiting grape cell wall degradation; the VvNAC17-RNAi vector contains the grape VvNAC17 gene, the gene sequence of the VvNAC17 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

9. Use according to claim 8, characterized in that: The method for constructing the carrier includes the following steps: (1) Cloning of the VvNAC17 gene sequence Take 100 mg of seedless white grape isolated sample frozen in liquid nitrogen, extract total RNA, and after the RNA concentration is qualified, reverse the extracted total RNA into cDNA and screen the grape VvNAC17 gene in NCBI. (2) Construction of VvNAC17-RNAi vector 1) Gene amplification Specific primers were designed based on the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R, with sequences of SEQ ID NO.3 and SEQ ID NO.4, respectively. PCR amplification was performed using cDNA from seedless white grapes as a template. The reaction system consisted of: 1 μL cDNA template, 12.5 μL 2×Hieff Dye, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 98 ℃ pre-denaturation for 30 s, 98 ℃ denaturation for 10 s, 60 ℃ annealing for 5 s, 72 ℃ extension for 1 s, 30 cycles, and a final extension at 72 ℃ for 2 min. 2) Detection and recovery of PCR products The specificity of the PCR products was detected by agarose gel electrophoresis at a voltage of 140V and a current of 200A for 28 min. The gel was then placed in a gel imaging system to observe the target gene fragment. The gel containing the target gene fragment was quickly cut with a blade under a UV gel cutter. The recovered product was stored at -20 ℃ for later use. 3) Ligate the pNC-AEnTopo blunt-ended cloning vector The obtained PCR product was ligated according to the instructions. 2 μL of the product was mixed with 1 μL of pNC-AEnTopo and then made up to 5 μL with ddH2O. The mixture was reacted at room temperature for 5 min and then stored at -20 ℃ for later use. 4) Transformed competent cells Transformation was performed, and 5 μL of the ligated product was added to 50 μL of DH5α competent cells. The cells were placed on ice for 5 min, then heat-shocked in a 42°C water bath for 55 s, and immediately placed on ice for 2 min. 700 μL of antibiotic-free LB medium was added and the cells were shaken at 37°C for 2 h. After centrifugation at 5000 rpm for 3 min, the cells were spread on LB solid medium containing Amp antibiotic and incubated upside down at 37°C for 20 h. 5) Colony PCR and Sequence Detection Single colonies were picked for PCR: The reaction was performed according to the 2×Hieff Ultra-Rapid HotStart PCR Master Mix (With Dye) instructions. The reaction mixture consisted of: 2 μL DNA template, 10 μL 2×Hieff Master Mix, 1 μL universal M13, 10 μM forward primer, 1 μL universal M13, 10 μM reverse primer, and ddH2O to a final volume of 20 μL. The reaction program was: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 10 s, 60 ℃ annealing for 20 s, 72 ℃ extension for 2 s, 30 cycles, and a final extension at 72 ℃ for 5 min. The PCR products were then analyzed by agarose gel electrophoresis, and the bacteria showing bands were sequenced. 6) NC cloning and transformation of competent cells Plasmids were extracted from the correctly sequenced bacteria, and then NC cloning was performed. The reaction system was as follows: VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid were both added at 80 ng, Nimble Mix 5 μL, and ddH2O was added to bring the total volume to 10 μL. After mixing by pipetting, the mixture was incubated at 50 ℃ for 55 min. 5 μL of the reaction product was transformed into DH5α competent cells, and the operation was as described in step 4). The antibiotic for plate coating was changed to Kana. After single colony PCR, the bacterial solutions with bands were sequenced. 7) Agrobacterium-mediated transformation Plasmids were extracted from the correctly sequenced bacteria and then transformed under the following conditions: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 28℃ for 2 h on a shaker, centrifuged at 6000 rpm for 1 min, and plated onto LB plates containing Kana and Rfp. The plates were then incubated at 28℃ for 2-3 days. Single colonies were subjected to PCR, and bacterial cultures showing bands were sequenced. Correct sequencing indicated successful construction, yielding the VvNAC17-RNAi vector.

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