Application of grape VvNAC17 gene in regulation and control of postharvest grape berry abscission and carrier

Abstract

The invention belongs to the technical field of fruits, vegetables and genes, and discloses application of a grape VvNAC17 gene in regulation and control of postharvest grape berry abscission and a carrier, the gene sequence of the VvNAC17 gene is as shown in SEQ ID NO.1, and the amino acid sequence of encoding protein of the VvNAC17 gene is as shown in SEQ ID NO.2. The VvNAC17 silencing technology disclosed by the invention is accurately regulated and controlled from a gene molecule level, the uncertainty influence of an external environment and storage and transportation conditions is reduced, the fruit grain falling rate is remarkably reduced, and the commodity and economic benefits during the storage and transportation period of grapes are improved. By constructing a VvNAC17-RNAi vector, the invention verifies the key positive regulation effect of the VvNAC17 gene on postharvest fruit falling for the first time. The gene silencing technology does not depend on complex storage and transportation conditions, potential influences of chemical agents on fruit quality and the environment are avoided, and an innovative solution is provided for the modern grape storage and transportation technology.

Description

Technical Field

[0001] The present invention belongs to the fields of fruits, vegetables and gene technology, and in particular relates to the application of grape VvNAC17 gene in regulating postharvest grape berry abscission and a vector. Background Art

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

[0003] In this context, as an important class of transcription factors in plants, NAC plays a key role in biological processes such as organ abscission, cell wall degradation, and hormone signal regulation. In recent years, significant progress has been made in the functional studies of NAC family genes in model plants (such as Arabidopsis thaliana) and food crops (such as rice and wheat). Some NAC genes have been confirmed to be closely related to leaf abscission, fruit ripening, and cell wall metabolism. However, compared with these model plants and food crops, the research on NAC transcription factors in grapes is still in its initial stage, especially the molecular mechanism related to postharvest fruit abscission is even more lacking. Among them, as a potential key gene in grapes, the function and regulatory mechanism of VvNAC17 have not been clarified, and systematic functional verification is lacking. In addition, there are bottlenecks in conducting gene function verification in grapes in the existing technologies, including problems such as low gene editing efficiency, complex vector construction, and significant influence of genetic background, which further hinder the progress of related research. These technical obstacles have led to the failure to achieve a postharvest berry abscission regulation strategy centered on VvNAC17.

[0004] Therefore, it is of great significance to clarify the specific mechanism of action of the VvNAC17 gene in postharvest grape berry abscission and develop corresponding molecular regulation technologies. By constructing an RNAi silencing vector of VvNAC17 and studying its function in the formation of the fruit stalk abscission zone and cell wall degradation, not only can the precise regulation of postharvest berry abscission be achieved at the molecular level, but also innovative solutions can be provided for modern grape storage and transportation. This method can effectively improve the marketability of grapes during storage and transportation, reduce fruit losses, and at the same time reduce the adverse effects of traditional external chemical regulation on the environment, providing theoretical support for modern grape storage and transportation technologies.

[0005] Through retrieval, the following patent publication documents related to this invention patent application were found:

[0006] Comparative Patent 1: Application of Grape Hydrogen Peroxide Oxidase Gene and Its Encoded Protein (CN113416737A)

[0007] This patent publication document discloses a method for improving the antioxidant properties and quality of grape fruits by regulating the expression of the VvHPCA1 gene to reduce the accumulation of reactive oxygen species (ROS). This technology has certain application value in solving the deterioration of fruit quality caused by oxidative stress. However, this patent publication document mainly focuses on the improvement of antioxidant properties and does not study the molecular mechanism of postharvest berry abscission from the perspective of the structure and cell decomposition of the fruit stalk abscission zone of berries, making it difficult to achieve precise control of the berry abscission phenomenon. The present invention takes the VvNAC17 gene as the core, focuses on the regulatory mechanism of the formation of the postharvest grape fruit stalk abscission zone, and intervenes in the cell wall degradation process of the fruit stalk abscission zone through RNAi technology to effectively reduce berry abscission, providing a new molecular regulation strategy for the regulation of postharvest grape berry abscission and making up for the deficiencies in the research of this patent in the field of berry abscission.

[0008] Comparative Patent 2: Grape NAC Transcription Factor Gene VaNAC08 and Its Application (CN113736793A)

[0009] This patent disclosure studied 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 conditions, its important role in stabilizing cell membranes and enhancing stress resistance was proposed. The NAC transcription factor family is diverse in plants, and different members have specific functions in growth and development, environmental stress responses, and organ abscission regulation. The VaNAC08 gene is mainly used to regulate stress resistance under salt stress conditions, while the functions of other genes in the NAC family in grapes remain to be further explored. This study further expanded the application of the NAC family in postharvest grape abscission regulation, enriched the functional research of NAC proteins, and provided new ideas for postharvest management techniques of grapes and other fruit plants.

[0010] Comparative Patent 3: Application of NAC17 Gene in Improving Salt Tolerance Traits of Populus davidiana var. japonica (CN115838739A)

[0011] This patent disclosure improved the traits of Populus davidiana var. japonica by overexpressing the NAC17 gene, mainly including enhancing salt tolerance traits and improving wood quality. This technology has important application value in enhancing the stress resistance of forest trees and improving the degree of lignification. However, this patent disclosure mainly focused on the functional research of the NAC17 gene in the overall traits of forest trees, and its expression pattern in other species and its specific functions in different biological processes have not been deeply explored. In particular, its potential role in regulating important traits such as fruit abscission in economic crops remains unclear.

[0012] In this invention, the VvNAC17 gene in grapes was silenced to systematically study its molecular function in the process of postharvest berry abscission. Focusing on the mitochondrial damage and cell wall degradation in the pedicel abscission zone, the regulatory mechanism of the VvNAC17 gene in postharvest grape management was analyzed. Through functional verification, this invention proposed a precision strategy centered on molecular regulation, significantly expanding the application scope of the NAC17 gene.

[0013] By comparison, this invention is essentially different from the above patent disclosures. Summary of the Invention

[0014] The purpose of this invention is to overcome the deficiencies in 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 problems is:

[0016] Application of a 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 its encoded protein is shown in SEQ ID NO.2.

[0017] Application of a silenced 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 its encoded protein is shown in SEQ ID NO.2.

[0018] Furthermore, the inhibition of grape cell wall degradation is to inhibit the degradation of the three main components of the cell wall in the abscission zone, namely protopectin, soluble pectin, and cellulose.

[0019] Application of a silenced grape VvNAC17 gene in inhibiting 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.

[0020] Application of a silenced 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.

[0021] A VvNAC17-RNAi vector containing a silenced grape VvNAC17 gene, 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.

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

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

[0024] Take 100 mg of the abscission zone sample of Thompson Seedless grapes frozen with liquid nitrogen, extract total RNA, and after measuring that the RNA concentration is qualified, reverse the extracted total RNA into cDNA, and screen for the grape VvNAC17 gene in NCBI;

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

[0026] 1) Gene amplification

[0027] Specific primers were designed according to the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R, and the sequences of these primers were SEQ ID NO.3 and SEQ ID NO.4. Using the cDNA of Thompson Seedless grape as a template for PCR amplification, the reaction system was as follows: 1 μL of cDNA template, 12.5 μL of 2×HieffDye, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and supplemented with ddH2O to 25 μL; the reaction program was: pre-denaturation at 98 °C for 30 s, denaturation at 98 °C for 10 s, annealing at 60 °C for 5 s, extension at 72 °C for 1 s, 30 cycles, and final extension at 72 °C for 2 min;

[0028] 2) Detection and recovery of PCR products

[0029] The specificity of the PCR products was detected by agarose gel electrophoresis. The voltage was set at 140 V, the current was 200 A, and the electrophoresis was carried out for 28 min; then 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 an ultraviolet gel cutter, and the recovered product was stored at -20 °C for later use;

[0030] 3) Ligation of the pNC-AEnTopo blunt-end cloning vector

[0031] The obtained PCR products were ligated as follows. Take 2 μL of the product and mix it with 1 μL of pNC-AEnTopo, and supplement with ddH2O to 5 μL. React at room temperature for 5 min, and then store at -20 °C for later use;

[0032] 4) Transformation of competent cells

[0033] For transformation, take 5 μL of the ligated product and add it to 50 μL of DH5α competent cells. Let it stand on ice for 5 min, place it in a 42 °C water bath for heat shock for 55 s, and then quickly place it on ice and let it stand for 2 min; add 700 μL of LB medium without antibiotics and shake it in a shaker at 37 °C for 2 h; then centrifuge at 5000 rpm for 3 min and coat it on an LB solid medium containing Amp antibiotic, and incubate it upside down in a 37 °C incubator for 20 h;

[0034] 5) Colony PCR and sequence detection

[0035] Pick single colonies for PCR: Perform according to the instructions of 2×HieffUltra-Rapid HotStart PCR Master Mix (With Dye). The reaction system is as follows: 2 μL of DNA template, 10 μL of 2×HieffMaster Mix, 1 μL of universal M13 forward primer at 10 μM, 1 μL of universal M13 reverse primer at 10 μM, and make up to 20 μL with ddH2O; The reaction program is: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 2 s, 30 cycles, and final extension at 72°C for 5 min; Detect the PCR products by agarose gel electrophoresis, and sequence the bacteria with bands.

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

[0037] Extract plasmids from the bacteria with correct sequencing, and then perform NC cloning. The reaction system is as follows: 80 ng each of VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid, 5 μL of Nimble Mix, and make up to 10 μL with ddH2O; After pipetting and mixing evenly, react at 50°C for 55 min; Transform 5 μL of the reaction product into DH5α competent cells, and the operation is as described in step 4), and change the plate antibiotic to Kana. After single colony PCR, sequence the bacterial solution with bands.

[0038] 7) Agrobacterium transformation

[0039] Extract plasmids from the bacteria with correct sequencing, and then perform transformation. The reaction conditions are: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37°C for 5 min, ice bath for 5 min; Then add 700 μL of LB medium without antibiotics and shake at 28°C for 2 h, centrifuge at 6000 rpm for 1 min, and coat the LB plates with Kana and Rfp; Place in a 28°C incubator for 2 - 3 days; Perform PCR on the grown single colonies, sequence the bacterial solution with bands, and correct sequencing indicates successful construction to obtain the VvNAC17-RNAi vector.

[0040] Furthermore, the method for preparing the abscission zone sample of Thompson Seedless grapes is as follows:

[0041] Take Xinjiang Thompson Seedless grapes. After picking, place them at room temperature for 2 h to release field heat; Immerse them in a 5% sodium hypochlorite aqueous solution for 10 min, then rinse 3 times with distilled water and air dry naturally; Use a blade to cut tissues with 2 mm on each of the upper and lower sides of the surface where the fruit stalk and fruit brush are connected as abscission zone samples.

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

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

[0044] The advantages and positive effects achieved by the present invention are as follows:

[0045] 1. Compared with traditional fruit abscission regulation methods, the VvNAC17 silencing technology of the present invention precisely regulates at the gene and molecular level, reducing the uncertainty effects of the external environment and storage and transportation conditions, significantly reducing the fruit drop rate, and enhancing the commerciality and economic benefits during grape storage and transportation. By constructing the VvNAC17-RNAi vector, the present invention first verified the key positive regulatory role of the VvNAC17 gene in postharvest fruit abscission. Compared with the traditional methods of using hormone regulators and optimizing the storage environment, the gene silencing technology does not depend on complex storage and transportation conditions and avoids the potential impacts of chemical agents on fruit quality and the environment. Experimental results show that after 8 days of storage, the fruit drop rate of the VvNAC17-RNAi group is significantly lower than that of the control group, with the specific drop rates being 5.83% and 16.47% respectively ( Figure 3 ). This technology is efficient and stable, providing an innovative solution for modern grape storage and transportation technology.

[0046] 2. The present invention reveals the molecular regulation mechanism of the postharvest pedicel abscission zone from the perspective of cell wall metabolism, providing new theories and technical supports for the research on postharvest grape fruit abscission. By detecting the content changes of the main components of the cell wall (protopectin, soluble pectin, and cellulose) ( Figure 4 ), the present invention clarified the inhibitory effect of VvNAC17 gene silencing on the degradation of the pedicel cell wall. The results show that the protopectin content of the VvNAC17-RNAi group is significantly higher than that of the control group, while the soluble pectin content decreases, and the degradation rate of cellulose also significantly slows down, indicating that silencing the VvNAC17 gene effectively inhibits cell wall degradation. Compared with the traditional research that failed to clarify the cell wall metabolism change rule, the present invention systematically explored the dynamic regulation of pedicel cell wall metabolism, providing a scientific basis for reducing fruit drop.

[0047] 3. By regulating the expression of genes related to cell wall metabolism, the present invention deeply analyzed the positive regulatory role of VvNAC17 in postharvest fruit abscission at the molecular level. The present invention detected the expression changes of genes related to cell wall metabolism (VvPG, VvPME, VvXTH, VvPL, VvCx, and Vvβ-GAL) ( Figure 5) The results showed that the expression levels of these genes were significantly inhibited in the VvNAC17-RNAi group. 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 inhibition of these genes effectively slowed down the cell wall degradation and significantly reduced the fruit drop rate, providing important data support for the molecular regulation of cell wall-related genes.

[0048] 4. From the perspective of the overall cell structure, the present invention optimized the stability of the fruit stalk abscission zone, further improving the cell integrity and fruit commerciality during grape storage and transportation. Through scanning electron microscopy observation, the surface of the abscission zone cells in the VvNAC17-RNAi group remained intact, and no significant lysis was observed, while the cell surface in the control group was severely lysed and the cell gap was significantly enlarged ( Figure 6 ). In addition, transmission electron microscopy observation further revealed the protective effect of the organelle structure ( Figure 7 ). The mitochondrial structure in the VvNAC17-RNAi group was intact, the outer membrane and inner membrane remained stable, and the matrix density was uniform, while the mitochondria in the control group showed phenomena such as outer membrane rupture and inner cristae disorder. The above results indicate that the silencing of the VvNAC17 gene not only reduces cell wall degradation but also significantly maintains the integrity of the abscission zone cells of the fruit stalk, providing new ideas for improving the scientificity and practicality of postharvest management of grapes.

[0049] 5. The present invention first constructed a postharvest fruit abscission regulation technology system with the VvNAC17 gene as the core, promoting the innovation of postharvest management technology for grapes. The present invention comprehensively revealed the mechanism of action of the VvNAC17 gene in the postharvest fruit stalk abscission zone by combining gene silencing technology, detection of cell metabolic components, and observation of organelle structure. Compared with the prior art, the present invention not only focuses on gene function but also integrates multi-dimensional research methods for cell wall metabolism and structural changes. The superiority of this technology in postharvest abscission regulation of grapes was verified by experiments, providing a basis for the development of precise molecular regulation tools.

[0050] 6. The present invention first constructed a molecular strategy for postharvest fruit abscission regulation with the grape VvNAC17 gene as the core.

[0051] By constructing an RNAi silencing vector of the VvNAC17 gene, the present invention systematically studied its molecular regulation mechanism in the formation of the fruit stalk abscission zone and cell wall degradation process in grapes. Based on the in-depth verification of gene function, the core role of the VvNAC17 gene in postharvest berry abscission was clarified. In view of the problem that the molecular mechanism of postharvest berry abscission has not been clarified, the present invention proposed a new method for precisely intervening in fruit abscission at the molecular level, providing a scientific basis for postharvest fruit management.

[0052] 7. The present invention expands the functions of NAC genes and for the first time reveals the application potential of the VvNAC17 gene in regulating postharvest abscission in grapes.

[0053] The present invention discovers and systematically verifies the function of the VvNAC17 gene in the formation of the abscission zone of postharvest grape pedicels and the process of fruit abscission, and clarifies its regulatory effects on cell wall degradation, mitochondrial structure and function, and hormone signaling. As an important member of the NAC family in grapes, the VvNAC17 gene has good functions and application prospects. The present invention further fills the gap in the research of grape NAC genes and lays a foundation for the realization of molecular regulatory strategies for postharvest abscission in grapes.

[0054] 8. Based on the overall regulatory network of the formation of the abscission zone of grape pedicels, the present invention develops a technical system for multi-dimensional regulation of postharvest fruit abscission.

[0055] The present invention not only focuses on the functional research of the VvNAC17 gene, but also further integrates various signal pathways and biological processes related to postharvest fruit abscission (such as hormone signaling, mitochondrial function, and cell wall degradation, etc.). By constructing a systematic molecular regulatory network for the formation of the abscission zone of grape pedicels, the present invention proposes a technical system for multi-dimensional comprehensive regulation of postharvest fruit abscission in combination with precise molecular tools. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a nucleic acid gel electrophoresis diagram for the construction of the VvNAC17-RNAi vector in the present invention, and the fragment size is 360 bp;

[0057] Figure 2 It is a diagram of the phenotypic changes of Thompson Seedless grapes in the present invention at 0, 2, 4, 6, and 8 days after treatment with EV-RNAi and VvNAC17-RNAi respectively;

[0058] Figure 3 It is a diagram of the shattering rate of Thompson Seedless grapes in the present invention after treatment;

[0059] Figure 4 It is a diagram of the contents of protopectin, soluble pectin, and cellulose, the components of the cell wall of Thompson Seedless grapes in the present invention;

[0060] Figure 5 It is a diagram of the expression levels of cell wall metabolic genes of Thompson Seedless grapes in the present invention;

[0061] Figure 6 It is a scanning electron microscope microscopic image of the abscission zone cells of Thompson Seedless grapes in the present invention;

[0062] Figure 7 It is a transmission electron microscope microscopic image of the abscission zone cells of Thompson Seedless grapes in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0064] For various experimental operations involved in the specific embodiments, they are all conventional techniques in the art. For parts not specifically annotated in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation.

[0065] Application of a grape VvNAC17 gene in regulating postharvest grape berry abscission, wherein the gene sequence of the VvNAC17 gene is as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is as shown in SEQ ID NO.2.

[0066] Application of a silenced grape VvNAC17 gene in inhibiting grape cell wall degradation, wherein the gene sequence of the VvNAC17 gene is as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is as shown in SEQ ID NO.2.

[0067] Preferably, the inhibition of grape cell wall degradation is to inhibit the degradation of the three main components of the cell wall in the abscission zone, namely protopectin, soluble pectin, and cellulose.

[0068] Application of a silenced grape VvNAC17 gene in inhibiting 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 as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is as shown in SEQ ID NO.2.

[0069] Application of a silenced 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 as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is as shown in SEQ ID NO.2.

[0070] A VvNAC17-RNAi vector containing a silenced VvNAC17 gene of the grape VvNAC17 gene, wherein the gene sequence of the VvNAC17 gene is as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is as shown in SEQ ID NO.2.

[0071] Preferably, the construction method of the vector comprises the following steps:

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

[0073] Take 100 mg of the abscission zone sample of Thompson Seedless grapes frozen with liquid nitrogen, extract total RNA, and after the RNA concentration is determined to be qualified, reverse the extracted total RNA into cDNA, and screen for the grape VvNAC17 gene in NCBI;

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

[0075] 1) Gene amplification

[0076] Design specific primers according to the CDS sequence of the VvNAC17 gene. The primers required for PCR amplification are VvNAC17-RNAi-F and VvNAC17-RNAi-R, and the sequences of these primers are SEQ ID NO.3 and SEQ ID NO.4. Use the cDNA of Thompson Seedless grapes as a template for PCR amplification. The reaction system is: 1 μL of cDNA template, 12.5 μL of 2×HieffDye, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and make up to 25 μL with ddH2O; The reaction program is: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 1 s, 30 cycles, and final extension at 72°C for 2 min;

[0077] 2) Detection and recovery of PCR products

[0078] Detect the specificity of the PCR products by agarose gel electrophoresis. Set the voltage to 140 V and the current to 200 A, and electrophorese for 28 min; Then place the gel in the gel imaging system to observe the target gene fragment, quickly cut the gel block containing the target gene fragment with a blade under the ultraviolet gel cutting instrument, and store the recovered product at -20°C for later use;

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

[0080] Ligate the obtained PCR products as follows. Take 2 μL of the product and mix it with 1 μL of pNC-AEnTopo, and make up to 5 μL with ddH2O. React at room temperature for 5 min, and then store at -20°C for later use;

[0081] 4) Transform competent cells

[0082] Perform transformation. Take 5 μL of the ligated product and add it to 50 μL of DH5α competent cells. Let it stand on ice for 5 min, place it in a 42°C water bath for heat shock for 55 s, and then quickly place it on ice and let it stand for 2 min; Add 700 μL of LB medium without antibiotics and shake it on a shaker at 37°C for 2 h; Then centrifuge at 5000 rpm for 3 min and coat it on an LB solid medium with Amp antibiotic, and incubate it upside down in a 37°C incubator for 20 h;

[0083] 5) Colony PCR and sequence detection

[0084] Pick single colonies for PCR: Perform according to the instructions of 2×HieffUltra-Rapid HotStart PCR Master Mix (With Dye). The reaction system is as follows: 2 μL of DNA template, 10 μL of 2×HieffMaster Mix, 1 μL of universal M13 forward primer at 10 μM, 1 μL of universal M13 reverse primer at 10 μM, and make up to 20 μL with ddH2O; The reaction program is: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 2 s, 30 cycles, and final extension at 72°C for 5 min; Detect the PCR products by agarose gel electrophoresis, and sequence the bacteria with bands.

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

[0086] Extract plasmids from the bacteria with correct sequencing, and then perform NC cloning. The reaction system is as follows: 80 ng each of VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid, 5 μL of Nimble Mix, and make up to 10 μL with ddH2O; After pipetting and mixing evenly, react at 50°C for 55 min; Transform 5 μL of the reaction product into DH5α competent cells, and the operation is as described in step 4), and change the plate coating antibiotic to Kana. After single colony PCR, sequence the bacterial liquid with bands.

[0087] 7) Agrobacterium transformation

[0088] Extract plasmids from the bacteria with correct sequencing, and then perform transformation. The reaction conditions are as follows: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37°C for 5 min, ice bath for 5 min; Then add 700 μL of LB medium without antibiotics and shake at 28°C for 2 h, centrifuge at 6000 rpm for 1 min, and coat on LB plates with Kana and Rfp; Place in an incubator at 28°C for 2 - 3 days; Perform PCR on the grown single colonies, and sequence the bacterial liquid with bands. Correct sequencing indicates successful construction, and the VvNAC17-RNAi vector is obtained.

[0089] Preferably, the method for preparing the abscission zone sample of Thompson Seedless grape is as follows:

[0090] Take Xinjiang Thompson Seedless grapes. After picking, place them at room temperature for 2 h to release field heat; Immerse them in a 5% sodium hypochlorite aqueous solution for 10 min, then rinse 3 times with distilled water and air dry naturally; Cut tissues with a blade, each 2 mm on the upper and lower sides including the surface where the fruit stalk and fruit brush are connected, and use them as abscission zone samples.

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

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

[0093] Specifically, the related preparation and detection are as follows:

[0094] I. Process steps and parameters

[0095] 1. Preparation of experimental materials

[0096] The experimental materials are Thompson Seedless grapes from Xinjiang. Twelve bunches were picked and transported to the laboratory within 2 hours, and left at room temperature for 2 hours to release field heat. After soaking in 5% sodium hypochlorite aqueous solution for 10 minutes, they were rinsed 3 times with distilled water and air-dried naturally. The whole bunch of grapes was used for the observation of apparent traits. Tissues about 2 mm on the upper and lower sides of the surface where the fruit stalk and fruit brush are connected were cut with a blade and used as abscission zone samples. After sampling, the abscission zone samples were immediately frozen with liquid nitrogen and stored in a -80°C refrigerator for subsequent experiments. Each experiment was repeated biologically three times and technically three times.

[0097] 2. Gene cloning steps

[0098] 2.1 Cloning of the VvNAC17 gene sequence

[0099] Take 100 mg of the abscission zone samples of Thompson Seedless grapes frozen with liquid nitrogen, extract total RNA using the Plant Total RNA IsolationKit Plus kit, and after determining that the RNA concentration is qualified. Use the Prime Script TM reverse transcription kit to reverse the extracted total RNA into cDNA. Screen the grape VvNAC17 gene in NCBI (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).

[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] Construction of 2.2VvNAC17-RNAi Vector

[0139] 2.2.1 Gene Amplification

[0140] Specific primers were designed according to the CDS sequence of the VvNAC17 gene (Table 1). The required primers for PCR amplification were VvNAC17-RNAi-F and VvNAC17-RNAi-R. Using the cDNA of Thompson Seedless grape as a template for PCR amplification, the reaction system was: 1 μL of cDNA template, 12.5 μL of 2×HieffDye, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), and supplemented with ddH2O to 25 μL. The reaction program was: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 1 s, 30 cycles, and final extension at 72°C 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 at 140 V, the current was 200 A, and the electrophoresis was carried out for 28 min. Subsequently, the gel was placed into 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 an ultraviolet gel cutter, and recovered using a DNA gel recovery kit (FastPure Gel DNA Extraction Mini Kit). The recovered product was stored at -20°C for later use.

[0143] 2.2.3 Ligation of pNC-AEnTopo Blunt-End Cloning Vector

[0144] The obtained PCR product was ligated according to the pNC-AEnTopo blunt-end cloning vector kit. Take 2 μL of the product and mix it with 1 μL of pNC-AEnTopo, and supplement with ddH2O to 5 μL. React at room temperature for 5 min, and then store at -20°C for later use.

[0145] 2.2.4 Transformation of Competent Cells

[0146] Transformation was carried out according to the Trelief5α Chemically Competent Cell instruction manual. Take 5 μL of the ligated product and add it to 50 μL of DH5α competent cells. Let it stand on ice for 5 min, then place it in a 42°C water bath for heat shock for 55 s and quickly place it on ice. Let it stand for 2 min. Add 700 μL of LB (antibiotic-free) medium and shake it in a shaker at 37°C for 2 h. Then centrifuge at 5000 rpm for 3 min and spread it on an LB solid medium with Amp antibiotic, and incubate it upside down in a 37°C incubator for 20 h.

[0147] 2.2.5 Colony PCR and sequence detection

[0148] Single colonies were picked for PCR. It was carried out according to the 2×HieffUltra-Rapid HotStart PCR Master Mix (With Dye) instruction manual. The reaction system was: 2 μL of DNA template, 10 μL of 2×HieffMaster Mix, 1 μL of forward primer (universal M13, 10 μM), 1 μL of reverse primer (universal M13, 10 μM), and made up to 20 μL with ddH2O. The reaction program was: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 2 s, 30 cycles, and final extension at 72°C for 5 min. The PCR products were detected by agarose gel electrophoresis, and the bacteria with bands were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequence detection.

[0149] 2.2.6 NC cloning and transformation of competent cells

[0150] Plasmids were extracted from the bacteria with correct sequencing, and then NC cloning was carried out. The reaction system was: 80 ng each of VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid, 5 μL of Nimble Mix, and made up to 10 μL with ddH2O. After pipetting and mixing evenly, react at 50°C for 55 min. Transform 5 μL of the reaction product into DH5α competent cells, and the operation is as described in 2.2.4, and the plate antibiotic is changed to Kana. After single colony PCR, the bacterial liquid with bands was sequenced.

[0151] 2.2.7 Agrobacterium transformation

[0152] Extract plasmids from the bacteria with correct sequencing, and then perform transformation according to the instructions of GV3101 (Vidi Biotechnology). The reaction conditions are as follows: ice bath for 5 min, liquid nitrogen for 5 min, water bath at 37 °C for 5 min, and ice bath for 5 min. Then add 700 μL of LB (antibiotic-free) medium and shake in a shaker at 28 °C for 2 h, centrifuge at 6000 rpm for 1 min, and spread on LB plates containing Kana and Rfp. Place in an incubator at 28 °C for 2 - 3 days. Perform PCR on the grown single colonies, and sequence the bacterial liquid with bands. Correct sequencing indicates successful construction, obtaining the VvNAC17-RNAi vector. At the same time, use the empty vector without homologous recombination as a control.

[0153] 2.2.8 Agrobacterium-mediated transformation of abscission zone cells of Thompson Seedless grapes

[0154] Activate the constructed VvNAC17-RNAi vector and the empty Agrobacterium. Add the Agrobacterium liquid containing pNC-Cam2304-RNAi (empty vector) and pNC-Cam2304-VvNAC17 recombinant plasmid to LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin respectively, and culture with shaking at 28 °C until the OD 600 reaches 1.2. Subsequently, centrifuge at 5000 rpm for 10 min at 28 °C to collect the bacterial cells, and discard the supernatant. Resuspend the bacterial cells with an infection solution containing 10 mM MgCl2, 10 mM MES, and 200 μM acetosyringone (As) at pH 5.6, and adjust the OD 600 to 1.0. Collect Thompson Seedless grape fruits, puncture several small holes at the fruit stalk with a sterile syringe, immerse the whole fruit in the bacterial suspension, and perform vacuum infiltration at a pressure of 0.75 MPa for 30 min. Place the infected grape fruits in MES liquid medium for 2 d, and then culture in an incubator at 26 °C. Collect samples 8 days after infection.

[0155] 3. Functional verification steps

[0156] 3.1 Determination of fruit phenotypic changes and abscission rate

[0157] For each experimental group, select 3 fixed bunches of grapes during storage to measure phenotypic changes and the shattering rate. Process the captured images with ImageJ. Use the ratio of the mass of abscised fruits to the total mass of grapes during storage as the abscission rate, and the calculation formula is as follows:

[0158]

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

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

[0161] Table 1 Primer sequences used for the construction of 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 Forward Primer (5’-3’) Reverse 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 contents of the main components of the abscission zone cell wall

[0166] The contents of three main cell wall components (including protopectin, soluble pectin, and cellulose) were mainly evaluated. The determination of protopectin and soluble pectin contents was performed using the carbazole method. In a fresh grape abscission zone sample (1.0 g), 10 mL of ethanol with a volume concentration of 95% was added. After mixing, it was placed in a boiling water bath for 30 min and centrifuged at 8,000×g for 10 min. The supernatant was discarded, and 4 mL of distilled water was added to the precipitate. It was heated in a water bath at 60 °C for 30 min. The solution was filtered and the residue was washed. The filtrate was collected and transferred to a 25 mL volumetric flask and made up to the mark with distilled water. This was the soluble pectin determination solution. The residue was collected and 5 mL of 0.5 mL L -1 sulfuric acid was added. After filtering after a 1 h boiling water bath, the residue was washed again and the washing solutions were combined. After cooling, the washing solution was transferred to a 25 mL volumetric flask and made up to the mark. This was the protopectin determination solution. 1 mL of the protopectin determination solution and the soluble pectin determination solution were respectively taken and 6 mL of concentrated sulfuric acid was added. After mixing, it was placed in a boiling water bath for 20 min. After cooling to room temperature, 0.2 mL of 1.5 g L -1 carbazole reagent was added. After mixing and placing in the dark for 2 h, the absorbance value of the reaction solution was measured at a wavelength of 530 nm. A standard curve was made using pectin as the standard, and the content was expressed as g kg -1 FW.

[0167] Add 1.0 mL of 80% ethanol by volume to a fresh grape abscission zone sample (0.3 g). After mixing, heat in a water bath at 90 °C for 20 min. After cooling, centrifuge at 25 °C and 6,000×g for 10 min. Discard the supernatant, and sequentially add 1.5 mL of 80% ethanol by volume and acetone to the precipitate. After mixing, centrifuge again (at 25 °C and 6,000×g for 10 min). Discard the supernatant, add 1.0 mL of dimethyl sulfoxide (DMSO), mix well, and let stand for 15 h. Then centrifuge the mixture at 25 °C and 6,000×g for 10 min. Discard the supernatant and dry the precipitate, which is the cell wall material (CWM). Place the CWM in an oven at 40 °C and dry to a constant weight. Sequentially add 0.5 mL of distilled water and 0.75 mL of concentrated sulfuric acid to the dried CWM and mix well. After 30 min, centrifuge the mixture at 4 °C and 8,000×g for 10 min. Dilute the supernatant 20-fold with distilled water and measure the absorbance at 630 nm. The cellulose content is expressed as g kg -1 。

[0168] 3.4 Scanning electron microscopy detection of the abscission zone surface

[0169] Observe the surface morphology of the abscission zone using a scanning electron microscope (SEM). Cut the tissue containing the abscission zone (including the fruit stalk and part of the fruit brush) with a blade and completely immerse it in a 2.5% glutaraldehyde solution by volume. Let stand at 4 °C for 12 h. After thoroughly rinsing the sample three times with 0.1 M phosphate buffer (pH = 7.0), fix it with 1% (w / v, mass concentration) osmium tetroxide solution for 2 h. After removing the osmium tetroxide waste liquid, rinse it again three times thoroughly with 0.1 M phosphate buffer (pH = 7.0). Then perform dehydration treatment with ethanol solutions of gradient concentrations (30%, 50%, 70%, 80%, 90%, 95%, and 100%, volume percentages), 15 min each time, and then place the sample in fresh 100% ethanol by volume. Further dehydrate the sample in a critical point dryer. Coat the dehydrated sample with a gold-palladium alloy in an ion sputtering instrument, and then observe with a scanning electron microscope.

[0170] 3.5 Transmission electron microscopy detection of the abscission zone cell structure

[0171] The ultrastructure of abscission zone cells was observed by transmission electron microscopy (TEM). Tissue blocks (about 3 mm×1 mm×1 mm) were manually cut with a blade at the junction of the fruit stalk and the fruit brush, and the samples were fixed with 2.5% glutaraldehyde solution and 1% (w / v) osmium tetroxide solution in the same steps as for SEM fixation. Then the samples were dehydrated with ethanol solutions of gradient concentrations (30%, 50%, 70% and 80%, v / v) for 15 min each time, and then dehydrated with acetone solutions of gradient concentrations (90%, 95% and 100%, v / v) for 20 min each time. After dehydration, the samples were immersed in a mixture of acetone and resin (1:1, v / v) for 1 h, immersed in a mixture of acetone and resin (1:3, v / v) for 3 h, and then immersed in pure resin embedding agent at room temperature overnight. The embedded samples were heated at 70 °C overnight, and the thin sections (70 nm) cut with an ultramicrotome were stained with lead citrate solution and 50% (v / v) ethanol saturated solution of uranyl acetate for 10 min each, and then observed with a transmission electron microscope.

[0172] II. Experimental data part:

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

[0174] Total RNA was extracted from the abscission zone tissue of Thompson Seedless grape stalks in the present invention. After synthesizing cDNA, specific primers were designed according to the VvNAC17-CDS sequence, and the VvNAC17 gene fragment was amplified by PCR technology. After the PCR products were detected by agarose gel electrophoresis, it was observed that the size of the amplified band was consistent with the expectation (~360 bp), further verifying the success of PCR amplification ( Figure 1 ). Subsequently, the amplified target fragment was inserted into the pNC-Cam2304-RNAi vector to construct the VvNAC17-RNAi recombinant plasmid, and the constructed vector was successfully transformed into competent Agrobacterium tumefaciens GV3101 by the Agrobacterium-mediated method. At the same time, Agrobacterium tumefaciens was transformed with the pNC-Cam2304-RNAi vector to construct an empty vector (EV-RNAi) control group. Through colony PCR and sequencing verification, the sequence of the recombinant plasmid was correct and completely consistent with the design. The clear display of the electrophoresis band and the correct insertion of the target fragment together indicated that the VvNAC17-RNAi vector had been successfully constructed, laying a foundation for subsequent research.

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

[0176] 1. Fruit phenotypic changes and abscission rate

[0177] Pattern diagram of fruit phenotypic changes during storage( Figure 2 ), significant differences between the EV-RNAi and VvNAC17-RNAi groups can be visually observed. The berries in the EV-RNAi group gradually dropped off as the storage time increased, with the most severe drop on the 8th day, and the dropping rate reached 16.47%; while the berries in the VvNAC17-RNAi group remained intact, with only a small amount of dropping, and the dropping rate was only 5.83% (p<0.01)( Figure 3 ). Statistical results showed that the dropping rate of the VvNAC17-RNAi group was significantly lower than that of the EV-RNAi group. The results indicated that the silencing of VvNAC17 could significantly reduce berry dropping. This result effectively verified the key regulatory role of the VvNAC17 gene in the abscission of postharvest grape berries.

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

[0179] By detecting the contents of the three main components (protopectin, soluble pectin, and cellulose) of the abscission zone cell wall( Figure 4 ), the regulatory effect of the VvNAC17 gene on cell wall degradation was further verified. The results showed that the protopectin content in the VvNAC17-RNAi group was 16.40% higher than that in the EV-RNAi group on the 8th day (p<0.01), indicating that the degradation of cell wall pectin was inhibited. At the same time, the soluble pectin content in the VvNAC17-RNAi group decreased by 27.63% compared with the EV-RNAi group. In addition, the degradation rate of cellulose in the VvNAC17-RNAi group was significantly slowed down, and the cellulose content on the 8th day was 14.24% higher than that in the EV-RNAi group (p<0.01). These results indicated that the silencing of the VvNAC17 gene effectively inhibited the degradation process of the abscission zone cell wall.

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

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

[0182] Example 3: Morphological and Structural Changes in the Pedicel Abscission Zone

[0183] 1. Scanning Electron Microscopy Observation of the Surface Morphology of the Abscission Zone

[0184] The effects on the surface and overall cell structure of the abscission zone were observed by scanning electron microscopy ( Figure 6 ). The results showed that in the EV-RNAi group, significant lysis occurred on the surface of abscission zone cells, and obvious decomposition was shown in the cell wall and surrounding tissues, and the overall cell structure lost its integrity. In addition, an enlarged cell gap and loose cell connection structure were observed at high magnification, suggesting the activation of the cell wall degradation and cell destruction processes. While in the VvNAC17-RNAi group, the cell surface was relatively smooth, the lysis phenomenon was significantly reduced, the overall structure remained intact, and the cell connections were tight, indicating that the silencing of VvNAC17 effectively prevented the structural damage of abscission zone cells and inhibited the processes of cell wall degradation and abscission layer formation. This result further demonstrated the role of VvNAC17 from a morphological perspective.

[0185] 2. Transmission Electron Microscopy Observation of the Ultrastructure of Abscission Zone Cells

[0186] Transmission electron microscopy observation revealed the changes in the ultrastructure of abscission zone cells, further demonstrating the details of cell wall degradation ( Figure 7)。In the EV-RNAi group, the cell wall was significantly thinned, organelles showed rupture phenomena, accompanied by the overflow of cytoplasm, resulting in serious damage to the integrity of the cell structure. The mitochondrial structure of the abscission zone cells in the EV-RNAi group was severely damaged, manifested as the rupture of the outer membrane, the breakage and disorder of the inner cristae, the sparseness of the matrix, and the swelling of mitochondria, suggesting that mitochondrial dysfunction may lead to cell metabolic disorders and trigger cell death. In the VvNAC17-RNAi group, the cell wall thickness was maintained, the cytoplasm was evenly distributed, and the cell structure remained intact. The outer and inner membranes of mitochondria were intact, the inner cristae were clear and neatly arranged, the matrix density was uniform, and the morphology remained stable. The above results indicate that the silencing of VvNAC17 effectively prevents the decomposition of the cell wall and the destruction of the cell structure at the cellular level. From the perspective of ultrastructure, the study demonstrates the protective effect of the silencing of the VvNAC17 gene on abscission zone cells.

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

Claims

1. Use of the grape VvNAC17 gene in regulating postharvest grape berry abscission. The gene sequence of the VvNAC17 gene is shown as SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown as SEQ ID NO.

2.

2. Use of silencing the grape VvNAC17 gene in inhibiting grape cell wall degradation. The gene sequence of the VvNAC17 gene is shown as SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown as SEQ ID NO.

2.

3. The application according to claim 2, wherein: The inhibition of grape cell wall degradation is to inhibit the degradation of the three main components of the cell wall in the abscission zone, namely protopectin, soluble pectin, and cellulose.

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

2.

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

2.

6. A VvNAC17 - RNAi vector for silencing the VvNAC17 gene containing the grape VvNAC17 gene. The gene sequence of the VvNAC17 gene is shown as SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown as SEQ ID NO.

2.

7. The VvNAC17-RNAi vector according to claim 6, characterized in that: The construction method of the vector includes the following steps: (1) Cloning of the VvNAC17 gene sequence Take 100 mg of the abscission zone sample of Thompson Seedless grapes frozen with liquid nitrogen, extract total RNA. After the RNA concentration is qualified, reverse the extracted total RNA into cDNA, and screen the grape VvNAC17 gene in NCBI; (2) Construction of the VvNAC17 - RNAi vector 1) Gene amplification Design specific primers according to the CDS sequence of the VvNAC17 gene. The required primers for PCR amplification are VvNAC17 - RNAi - F and VvNAC17 - RNAi - R, and the sequences of the primers are SEQ ID NO.3 and SEQ ID NO.

4. Use the cDNA of Thompson Seedless grapes as a template for PCR amplification. The reaction system is: 1 μL of cDNA template, 12.5 μL of 2×HieffDye, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and make up to 25 μL with ddH2O; The reaction program is: pre - denaturation at 98℃ for 30 s, denaturation at 98℃ for 10 s, annealing at 60℃ for 5 s, extension at 72℃ for 1 s, 30 cycles, and final extension at 72℃ for 2 min; 2) Detection and recovery of the PCR product The specificity of the PCR products was detected by agarose gel electrophoresis at a voltage of 140 V, a current of 200 A for 28 min. Subsequently, the gel was placed into 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 an ultraviolet gel cutter, and the recovered product was stored at -20 °C for later use. 3) Ligate the blunt-end cloning vector pNC-AEnTopo Ligate the obtained PCR products as follows. Take 2 μL of the product and mix it with 1 μL of pNC-AEnTopo, and make up to 5 μL with ddH2O. React at room temperature for 5 min, then store at -20 °C for later use. 4) Transform competent cells For transformation, take 5 μL of the ligated product and add it to 50 μL of DH5α competent cells. Incubate on ice for 5 min, then place in a 42 °C water bath for heat shock for 55 s and quickly transfer to ice. Incubate on ice for 2 min. Add 700 μL of LB medium without antibiotics and shake at 37 °C for 2 h. Then centrifuge at 5000 rpm for 3 min and spread on an LB solid medium containing Amp antibiotic. Incubate upside down in a 37 °C incubator for 20 h. 5) Colony PCR and sequence detection Pick single colonies for PCR: According to the instructions of 2×HieffUltra-Rapid HotStart PCR Master Mix (With Dye), the reaction system is: 2 μL of DNA template, 10 μL of 2×HieffMaster Mix, 1 μL of universal M13 forward primer at 10 μM, 1 μL of universal M13 reverse primer at 10 μM, and make up to 20 μL with ddH2O. The reaction program is: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 10 s, annealing at 60 °C for 20 s, extension at 72 °C for 2 s, 30 cycles, and final extension at 72 °C for 5 min. Detect the PCR products by agarose gel electrophoresis, and sequence the bacteria with bands. 6) NC cloning and transformation of competent cells Extract the plasmids from the bacteria with correct sequencing, and then perform NC cloning. The reaction system is: 80 ng each of VvNAC17 Topo plasmid and pNC-Cam2304-RNAi vector plasmid, 5 μL of Nimble Mix, and make up to 10 μL with ddH2O. Mix well by pipetting and react at 50 °C for 55 min. Transform 5 μL of the reaction product into DH5α competent cells, and the operation is as described in step 4). The plate antibiotic is changed to Kana. After single colony PCR, sequence the bacterial liquid with bands. 7) Agrobacterium transformation Extract the plasmids from the bacteria with correct sequencing, and then perform transformation. The reaction conditions are: ice bath for 5 min, liquid nitrogen for 5 min, 37 °C water bath for 5 min, ice bath for 5 min. Then add 700 μL of LB medium without antibiotics and shake at 28 °C for 2 h, centrifuge at 6000 rpm for 1 min, and spread on an LB plate containing Kana and Rfp. Place in a 28 °C incubator for 2 - 3 days. Perform PCR on the grown single colonies, and sequence the bacterial liquid with bands. Correct sequencing indicates successful construction, and the VvNAC17-RNAi vector is obtained.

8. The VvNAC17-RNAi vector according to claim 7, characterized in that: The method for preparing the abscission zone sample of Thompson Seedless grapes is as follows: Take Thompson Seedless grapes from Xinjiang. After picking, place them at room temperature for 2 h to release field heat. Immerse them in an aqueous solution of sodium hypochlorite with a mass concentration of 5% for 10 min, then rinse them 3 times with distilled water and air-dry naturally. Use a blade to cut tissues with a thickness of 2 mm on both the upper and lower sides of the surface where the fruit stalk and fruit brush are connected as the abscission zone sample.

9. The application of the VvNAC17-RNAi vector according to any one of claims 6 to 8 in regulating the abscission of postharvest grape berries.

10. The application of the VvNAC17-RNAi vector according to any one of claims 6 to 8 in inhibiting the degradation of grape cell walls.

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