Application of VvENO2 gene in regulating fruit development of plants

Abstract

This invention discloses VvENO2 The application of genes in regulating plant fruit development belongs to the field of plant genetic engineering technology. Previous research in this invention found that hydrogen peroxide (H2O2) treatment significantly promotes grape fruit ripening. Transcriptome analysis of H2O2-treated grapes revealed… VvENO2 Gene expression levels decreased. To further clarify... VvENO2 The role of the gene in plant fruit development was investigated through subcellular localization experiments and promoter activity analysis, demonstrating its nucleus location and that its promoter can activate the GUS reporter protein. Furthermore, treatment with ABA and H2O2 enhanced its activity. In addition, transgenic Arabidopsis and transgenic grape were constructed, and phenotypic observations confirmed... VvENO2 Genes negatively regulate the ripening of plant fruits. This invention provides a new regulatory site for breeding early-maturing plant varieties.

Description

Technical Field

[0001] This invention relates to VvENO2 The application of genes in regulating plant fruit development falls under the field of plant genetic engineering technology. Background Technology

[0002] Grapes belong to the genus *Vitis* of the family Vitaceae. Vitis Grapes (L.) are deciduous vines and one of the earliest cultivated and most widely distributed fruit trees in the world. Globally, the grape industry ranks fifth among agricultural products. The International Organisation of Vine and Wine (OIV) reported that the global grape-growing area in 2023 was approximately 7.3 million hectares. With the development of my country's fruit industry, grapes have become one of the fastest-growing fruit tree species, ranking alongside bananas, citrus fruits, apples, pears, and peaches as one of my country's six major fruits. my country has also become the world's second-largest grape-growing country, with table grapes accounting for 84.1% of its grape cultivation area.

[0003] Grape fruit development is a crucial trait in the growth and development of grapevines, primarily divided into three stages: fruit set, growth, and ripening. The fruit set stage is the first phase of fruit development, lasting approximately 4 to 7 weeks. During this stage, cell division is active, the number of cells increases rapidly, and subsequently, cell volume expands rapidly. The growth stage is the pit hardening stage or seed development stage. During this stage, fruit enlargement slows down, and growth ceases. Early-maturing varieties may have a growth period of only a few days, while late-maturing varieties may have a growth period of up to 4 weeks. After the growth period, the skin begins to fade, entering the color-changing stage. The ripening stage is the period from the start of the color-changing stage to fruit maturity. During ripening, the fruit begins to soften, colored varieties begin to color, sugar content increases dramatically, acid content begins to decrease, and flavor compounds gradually form. In the later stages of ripening, the pectin in the cell walls of juicy varieties degrades, filling the fruit with juice. Ripe fruit is considered ready when it reaches its desired color and flavor. Normal grape varieties have a relatively long cycle from bud break to fruit ripening, and the ripening period of grapes is closely related to economic benefits. Therefore, the development of early-ripening varieties can not only effectively meet the diverse needs of consumers but also further enrich my country's grape germplasm resource bank. With the continuous development of science and technology, research on grape fruit ripening and quality improvement using molecular biology and bioengineering methods has become a hot topic. Therefore, understanding and clarifying the molecular regulatory mechanisms of grape fruit ripening is of great significance for further promoting the breeding of early-ripening varieties.

[0004] Enolase (ENO, 2-phospho-D-glycerate hydrolyase), also known as 2-phospho-D-glycerate hydrolase, catalyzes the conversion of 2-phospho-D-glycerate (2-PGA) to phospho-enolpyruvate (PEP). PEP is a high-energy phosphate molecule that plays a crucial role in organisms. PEP not only provides a large amount of energy but is also a precursor in fatty acid secondary metabolism pathways. Enolases are not only ubiquitous in organisms but are also among the most abundant cytoplasmic proteins expressed in many organisms. Therefore, enolases play an important role in cellular energy metabolism. In recent years, significant progress has been made in the study of plant ENO2, revealing that in addition to its involvement in glycolysis, it also plays a vital role in plant growth and reproductive development, and possesses multiple functions such as responding to abiotic stresses and participating in transcriptional regulation. However, ENO2 The role of genes in grape fruit development and ripening is not well understood. Summary of the Invention

[0005] The purpose of this invention is to provide VvENO2 The application of genes in regulating plant fruit development provides a gene locus that can effectively regulate plant fruit development.

[0006] To achieve the above objectives, in this invention VvENO2 The technical approach used in the application of genes to regulate plant fruit development is as follows:

[0007] VvENO2 The application of genes in regulating plant fruit development, the aforementioned VvENO2 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1.

[0008] The beneficial effects of the above technical solution are as follows: This invention VvENO2 The application of genes in regulating plant fruit development is a pioneering invention. The inventors previously discovered that hydrogen peroxide (H2O2) treatment significantly promotes grape fruit ripening. Transcriptome analysis of H2O2-treated grapes revealed… VvENO2 The gene expression level was reduced, suggesting that it may be involved in the development of grape fruit.

[0009] To further clarify VvENO2 The function of the gene in grape fruit development was investigated, and its subcellular localization assay revealed that it is located in the cell nucleus. This was further validated by GUS staining. VvENO2The promoter activity of the gene was investigated, and the results showed that the promoter of this gene can activate the GUS reporter protein, and treatment with ABA (abscisic acid) and H2O2 (hydrogen peroxide) can enhance its activity. These results suggest that this gene may work synergistically with other genes to regulate fruit development.

[0010] To further clarify VvENO2 The influence of genes on fruit development was constructed. VvENO2 Gene overexpression vectors were transformed into Arabidopsis thaliana, and the results showed that overexpression... VvENO2 The Arabidopsis thaliana gene delayed fruit ripening. Next, a gene for transferring the gene into grapes was constructed. VvENO2 Gene overexpression vectors were used and transiently transformed into grapes. Results showed that... VvENO2 Gene silencing significantly promoted the color change of grape berries, while VvENO2 Gene overexpression does not have this effect. Furthermore, a key indicator of grape ripeness is the change in grape skin color. Therefore, the above results fully demonstrate... VvENO2 Genes participate in regulating the development process of grape fruits, playing a negative regulatory role in fruit ripening. This invention uses the important model plant Arabidopsis thaliana and the common economic crop grape as research subjects to clarify... VvENO2 Genes participate in regulating the development of plant fruits and can negatively regulate the ripening of plant fruits. This provides a new regulatory network for the regulation of fruit development and ripening, enriches the molecular regulatory network of fruit development, and lays the foundation for the use of molecular biology and bioengineering methods to improve plant varieties.

[0011] As a further improvement, the regulation of plant fruit development is to regulate the ripening of plant fruits.

[0012] As a further improvement, the regulation of plant fruit ripening includes regulating the coloring of plant fruits and / or the ripening of pods.

[0013] As a further improvement, overexpression VvENO2 Genes that inhibit the ripening of plant fruits; inhibit VvENO2 Gene expression promotes the ripening of plant fruits.

[0014] As a further improvement, the overexpression VvENO2 Genes for construction VvENO2 Gene overexpression vectors, transformed into plants, have the effect of VvENO2 The gene is overexpressed.

[0015] As a further improvement, the suppression VvENO2 Gene expression is the basis for construction VvENO2 RNAi vectors of genes, transformed into plants, have the effect of VvENO2 Gene expression is suppressed.

[0016] As a further improvement, the plant is a dicotyledonous plant.

[0017] As a further improvement, the dicotyledonous plant is grape or Arabidopsis thaliana. Attached Figure Description

[0018] Figure 1 In Embodiment 1 of the present invention VvENO2 Gene cloning and sequence analysis (in the figure, A represents...) VvENO2 PCR amplification results of the genes: B shows the phylogenetic relationship between VvENO2 and other ENOs; C shows the predicted conserved domains of VvENO2; and D shows the multiple sequence alignment and domain prediction of ENO2, namely: maize ENO2 (NP_001388142.1), poplar ENO2 (AXY97901.1), sunflower ENO (AMR99129.1), Arabidopsis ENO (AEC09265.1), and soybean ENO (KAH1258723.1).

[0019] Figure 2 This refers to the subcellular localization of VvENO2 in Example 2 of the present invention;

[0020] Figure 3 The image shows GUS histochemical staining after treatment with plant hormones and oxidants in Example 3 of this invention (in the image, GUS is the negative control (0390-GUS empty vector), and CAMV35S::GUS is the positive control (0390-35S-GUS vector)).

[0021] Figure 4 For the transfer in Embodiment 4 of the present invention VvENO2 Identification of transgenic Arabidopsis thaliana (in the figure, A shows the growth of transgenic Arabidopsis thaliana on Kan medium, B shows the PCR identification results of transgenic Arabidopsis thaliana (M: DL2000 molecular marker; H2O: water as template; WT: empty vector as template; #1-#3: 3 transgenic lines), C shows the phenotypic diagram of transgenic Arabidopsis thaliana plants).

[0022] Figure 5 For the identification of transgenic 'Kyoho' grapes in Example 5 of this invention (in the figure, A is the phenotypic observation of 'Kyoho' grapes after transgenication, and B is the phenotypic observation of 'Kyoho' grapes after transgenication). VvENO2 Gene expression analysis, * represents P<0.05, **** represents P<0.001). Detailed Implementation

[0023] Existing technologies for developing early-maturing varieties of economic crops such as grapes not only effectively address diverse consumer demands but also further enrich the germplasm resource bank for these crops. With the continuous development of science and technology, molecular biology and bioengineering have become effective means of plant variety improvement. However, fruit development is a complex biological process involving intricate regulatory networks, and the regulatory mechanisms and molecular regulatory networks for fruit ripening remain unclear. Based on this, this invention provides… VvENO2 The application of genes in regulating plant fruit development.

[0024] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0025] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0026] Plant materials:

[0027] In this embodiment of the invention, 'Kyoho' grapes from the vineyard of the Henan University of Science and Technology experimental base were used as materials. Transcriptome sequencing was performed on grapes treated with H2O2 and those treated as controls, and grape RNA and DNA were extracted from the samples. Naturally developed grapes were used as materials for transgenic experiments. Nicotiana benthamiana was used as material for subcellular localization and promoter activity analysis.

[0028] The primers used in the following embodiments of the present invention are shown in Table 1.

[0029] Table 1 Primer Sequences

[0030]

[0031] This invention VvENO2 Specific examples of the application of genes in regulating plant fruit development:

[0032] Previous research in this invention found that hydrogen peroxide (H2O2) treatment can significantly promote grape ripening. Transcriptome analysis of grapes treated with H2O2 revealed that... VvENO2 Gene expression levels were reduced. Further analysis using subcellular localization, promoter activity analysis, Arabidopsis transformation, and grape fruit transformation confirmed this. VvENO2 Genes play a negative regulatory role in plant fruit development. The specific implementation steps are as follows:

[0033] Example 1 VvENO2 Gene cloning and sequence analysis

[0034] In this embodiment, total RNA was extracted from 'Kyoho' grape berries using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) (Tiangen Biotech (Beijing) Co., Ltd.). The total RNA was reverse transcribed into cDNA using the Hi Fi Script cDNA Synthesis Kit (Beijing Kangwei Biotechnology Co., Ltd.). DNA was extracted from the 'Kyoho' grape berries using the FastPure Plant DNAIsolation Mini Kit-BOX2 (DC104-01) (Nanjing Novizan Biotechnology Co., Ltd.). Specific procedures were performed according to the kit's instruction manual.

[0035] Obtained through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). VvENO2 The CDS sequence of the (Vitvi08g01520) gene was obtained. Primers were designed using Premier 5 software (SEQ ID NO. 2-3 in Table 1). Using cDNA as a template, high-fidelity enzyme was used for cloning. VvENO2 The full-length coding region of the gene was analyzed. The reaction system consisted of 25 μL of high-fidelity enzyme, 19 μL of water, 2 μL each of forward and reverse primers (VvENO2-F / R), and 2 μL of cDNA template. The PCR amplification program was as follows: denaturation for 10 s (98℃), annealing for 5 s (61℃), extension for 10 s (72℃), and 34 cycles followed by a further extension for 10 min (72℃). The PCR amplification products were analyzed by 1% agarose gel electrophoresis to determine the band size, and the target band was recovered using a gel extraction kit.

[0036] Using the Expasy-Translate tool (https: / / web.expasy.org / translate) online website, the translation tool can be translated into English. VvENO2 The CDS sequence was translated into a protein sequence, and homology analysis was performed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The sequences were then analyzed using MEGA 7.0 software for homology alignment, amino acid sequence analysis, and phylogenetic tree analysis.

[0037] In this embodiment, cDNA from mature 'Kyoho' grapes was used as a template for gene cloning, resulting in a specific band of 1335 bp. Figure 1 (As shown in A), grapes VvENO2The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1. A phylogenetic tree of the VvENO2 protein was constructed using MEGA 7.0 software. In the VvENO2 cluster ( Figure 1 As shown in B), grapes ( Vitis vinifera L.) and sunflower ( Helianthus annuus (L) clustered together, showing a close homology. Homologous sequences of grape VvENO2 were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Comparison of the amino acid sequences of VvENO2 with ENO2 proteins from other species using software revealed that ENO2 contains two relatively conserved domains ( Figure 1 (shown in CD).

[0038] Example 2 Subcellular localization of VvENO2

[0039] In this embodiment, Primers were designed using Primer Premier 5.0 software to clone the full-length gene. To ensure that transcription did not terminate prematurely, the stop codon was removed from the reverse primer. The vector used in this embodiment was 101LYFP, with Kan antibiotic resistance, and EcoRI and BamHI were used for digestion. The vector was constructed using homologous recombination, with cDNA from 'Kyoho' grape berries as a template. PCR amplification was performed using primers with homologous arms. The primers used (SEQ ID NO. 6~7 in Table 1) are listed. The PCR system was as follows: 25 μL high-fidelity enzyme, 19 μL water, 2 μL each of forward and reverse primers (YFP-VvENO2-F / R), and 2 μL cDNA template. Denaturation was performed for 10 s (98℃), annealing for 5 s (61℃), extension for 10 s (72℃), and after 34 cycles, a further extension for 10 min (72℃) was performed. The PCR products were detected by agarose gel electrophoresis (1.0%), and the target gene fragment was recovered using a gel extraction kit. Simultaneously, the 101LYFP vector was digested with EcoRI and BamHI restriction endonucleases. The digestion products were then recovered via gel electrophoresis. Next, Beyotime's seamless cloning ligase was used to ligate the target gene. VvENO2 The gel recovery product was ligated with the gel recovery product of the linearized vector and transformed into Escherichia coli strain Trans5α. Positive single clones were selected, and bacterial PCR was performed. After enzyme digestion identification, the samples were sent to a biotechnology company for sequencing. The YFP-VvENO2 plasmid was extracted and transformed into Agrobacterium strain GV3101 for later use.

[0040] Agrobacterium suspension containing YFP-VvENO2 was shaken, with 100 μL of the suspension added to 50 mL of liquid LB, and shaken at 28°C until the OD600 was 0.6-0.8. Simultaneously, Agrobacterium suspensions without nuclear markers, P19, and 101LYFP were shaken at 28°C until the OD600 was 0.6-0.8. The cells were collected by centrifugation at 5000 rpm for 10 min and resuspended using a resuspension buffer (100 mL resuspension buffer: 5 mL of 200 Mm MgCl2 solution + 5 mL of 200 Mm EMS solution + 75 μL of 200 Mm AS solution + H2O) until the OD600 was 0.6-0.8. A mixture of bacterial suspension carrying the target gene, nuclear marker bacterial suspension, and P19 in a ratio of 4:3:3 was prepared to form an injection solution. Similarly, a mixture of 101YFP empty vector, nuclear marker bacterial suspension, and P19 in a ratio of 4:3:3 was prepared. These solutions were injected into the underside of tobacco leaves until the entire leaf was soaked. At least three leaves were used in each experiment. The leaves were incubated in the dark for 12 hours, followed by 2 days of light incubation. On the third day, tissue samples from the injected tobacco leaves were collected, spread on glass slides, and fluorescence signals were observed using a laser scanning-ning confocal microscopy (LSCM / CLSM).

[0041] Through the VvENO2 Sequence analysis of the gene-encoded protein revealed signals indicating nuclear localization, suggesting... VvENO2 The gene may be located in the cell nucleus. To verify our hypothesis, the gene with the stop codon removed was ligated into the empty YFP vector, and subcellular localization of VvENO2 was performed in tobacco leaves. The results showed that the fluorescent localization signal of the empty YFP vector was distributed throughout the entire cell, and mCherry was located in the cell nucleus. This was used as a nuclear marker control (e.g., ...). Figure 2 (As shown). Connection VvENO2 The recombinant vector of the gene showed overlap between the green fluorescent protein signal and the mCheey signal, indicating that VvENO2 is localized in the cell nucleus, which is consistent with the prediction. VvENO2 The location of genes.

[0042] Example 3 VvENO2 Gene promoter activity analysis

[0043] This embodiment uses a DNA extraction kit to extract template DNA from a mixture of roots, stems, and leaves of the 'Kyoho' grape variety. The DNA was obtained from the EnsemblPlants database. VvENO2The promoter sequence 2000 bp upstream of the gene start codon was used to design promoter primers containing the ABRE cis-acting element using Primerpremier 5.0 software, and a 500 bp promoter sequence was cloned. The vector used in this example was pCambia0390-GUS, with Kan antibiotic resistance. The vector was constructed using homologous recombination after digestion with EcoRI and HindIII. PCR amplification was performed using primers with homologous arms. The primers used (SEQ ID NO. 4-5 in Table 1) are listed. Using a homologous recombination kit, the target gene and the digested pCambia0390-GUS vector were ligated. The ligation product was transformed into *E. coli* strain DH5α, positive clones were screened, and culture PCR was performed. After enzyme digestion identification, the clones were sent to a biotechnology company for sequencing. Extraction VvENO2 The plasmid containing the gene promoter was transformed into Agrobacterium strain GV3101 for later use. Agrobacterium-mediated transient transformation of tobacco was performed using Pro::GUS as a negative control and 35S::GUS as a positive control.

[0044] Cloning was predicted using the PlantCARE database (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). VvENO2 The promoter sequence contains cis-acting elements. Instantaneous conversion of tobacco leaves was performed using a vacuum infiltration method. The vacuum pump pressure was 0.085 MPa, and the treatment time was 30 minutes. The infected area appeared water-soaked. This process was repeated until the entire leaf was successfully infected. The infiltrated leaf tissue was then removed, and excess bacterial solution was removed from the leaf surface using sterile absorbent paper. The leaf was placed with its abaxial surface facing upwards in a tray, and the petiole was wrapped with sterile, water-moistened absorbent cotton and covered with plastic wrap. To understand... VvENO2 To determine whether promoter activity is induced by hormones and oxidants, leaves were sprayed several times with 100 μmol / L abscisic acid (ABA) and 300 mmol / L H2O2, respectively, and cultured for 24 h. The cultured tobacco leaves were then placed in Petri dishes, and the prepared GUS staining solution was added. The dishes were incubated overnight at 37°C, followed by destaining with 70% ethanol for 5–6 h, with the ethanol being replaced several times. The leaves were then incubated with 90% ethanol at 37°C until complete destaining. The staining was observed and photographed.

[0045] Based on the staining results, the 35S GUS vector positive control stained more deeply, containing... VvENO2 The promoter leaves showed a lighter blue color, while the negative control using the empty GUS vector showed no staining effect. This was to investigate the effects of hormones and oxidants on... VvENO2 The effect of promoter activity was investigated by treating transiently transformed tobacco leaves with ABA and H2O2, respectively, and observing GUS staining. GUS histochemical staining results showed...VvENO2 Promoter activity is influenced by hormones and oxidants. Both ABA and H₂O₂ can enhance it. VvENO2 The promoter activity is significantly enhanced (e.g., Figure 3 (As shown).

[0046] Example 4 VvENO2 Negative regulation of fruit ripening in Arabidopsis thaliana by genes

[0047] In this embodiment, primers were designed using Primer Premier 5.0 software to clone the full-length gene. The vector used in this embodiment was pCAMBIA2300, with Kan antibiotic resistance, and BamHI digestion was performed. The vector was constructed using homologous recombination, using cDNA from 'Kyoho' grape berries as a template. PCR amplification was performed using primers with homologous arms. The primers used (SEQ ID NO. 8-9 in Table 1) are listed. The purified PCR product was ligated into the pCAMBIA2300 vector using a seamless cloning ligase provided by Beyotime Biotechnology Co., Ltd. The ligated product was transformed into Trans5α competent E. coli cells, and positive clones were screened on agarose plates supplemented with Kan antibiotic. Subsequently, the correctness of the inserted sequence in the positive clones was verified by bacterial culture PCR. The successfully transformed E. coli culture was amplified and used for plasmid extraction. This plasmid, along with the E. coli culture, was sent to Zhengzhou Sangon Biotech Co., Ltd. for sequencing analysis. The correctness of the vector construction was verified by comparing the sequencing results with the expected gene sequence.

[0048] Disinfection of Arabidopsis thaliana: First, disinfect with 75% alcohol for 30 seconds, invert and shake to remove the alcohol. Wash 2-3 times with sterile water, then disinfect again with 1% sodium hypochlorite solution for 6-7 minutes, wash 4-5 times with sterile water, plant on MS solid medium, air dry to remove excess moisture, vernalize in a 4°C refrigerator for 24 hours, then place in a culture room for about two weeks. When the second true leaf emerges, transplant into sterilized nutrient soil with added vermiculite.

[0049] Arabidopsis management: Water regularly. When Arabidopsis grows to the bolting stage, the stem tissue should be cut off. Allow it to grow for a period of time until the entire Arabidopsis plant bolts. Cut off the seed pods, leaving the flower buds. When the Arabidopsis grows normally and has a large number of inflorescences, carry out stable transformation with Agrobacterium.

[0050] The specific steps for transgenic Arabidopsis thaliana are as follows:

[0051] (1) Prepare the buffer (1L) formula: MS powder + 50g sucrose + 10μL 6-BA + 50μL surfactant (silwetL-77) (concentration is 1mg / mL).

[0052] (2) Streak the preserved 2300-ENO2 Agrobacterium on LB (Rif+Kan) solid medium and incubate in the dark for 2-3 days. Pick a single colony from the above plate and inoculate it into 4 mL of liquid LB (Rif+Kan) medium. Incubate overnight (18 h) at 220 rpm / min in the dark at 28°C. Then, take about 1 mL of the small-shake culture and shake it vigorously in 100 mL of liquid LB (Rif+Kan) medium overnight.

[0053] (3) Pour the shaken bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 6000 rpm and 28℃ for 8 min, collect the bacterial cells, discard the supernatant culture medium, and centrifuge multiple times.

[0054] (4) Add 2 mL of buffer solution to the centrifuge tube, resuspend the bacterial culture by pipetting with a 1 mL pipette, and add buffer solution until the OD600 value of the bacterial culture is about 0.8. Then add surfactant to the bacterial culture, stir well with a glass rod until foam appears.

[0055] (5) Arabidopsis flower soaking. Place the flower buds of Arabidopsis in a centrifuge tube containing Agrobacterium tumefaciens permeate for 10 min. After infection, gently dab off excess bacterial solution on absorbent paper, invert the tube, and incubate in the dark for 12 h before normal culture. After the peak flowering period of Arabidopsis, the plants can be tied to bamboo skewers for easy seed harvesting.

[0056] (6) After the Arabidopsis seeds mature and are harvested, the transgenic Arabidopsis seeds will be verified.

[0057] When transgenic Arabidopsis seeds were planted on a selection medium supplemented with Kan (50 mg / L⁻¹), most of the Arabidopsis died, while a small portion grew normally. Figure 4 (As shown in Figure A). Seeds from T3 generation transgenic Arabidopsis plants were collected from individual plants and replanted. Three transgenic Arabidopsis plants were selected for identification, with the wild type as a control. Leaves were collected from each plant for DNA extraction, which served as templates. Using OE-VvENO2-F forward primers and a sequence from the pCAMBIA2300 vector as a reverse primer, PCR amplification was performed. The results showed that all transgenic lines had bands in the correct positions. Figure 4 (As shown in Figure B). The results indicate that, VvENO2 The gene was successfully transferred into Arabidopsis thaliana plants. Phenotypic observation was conducted on the three lines with the highest expression levels (OE-VvENO2#1-3). In the early growth stage, overexpression was observed. VvENO2 After the gene, Arabidopsis thaliana has fewer branches, and its pods mature later than the wild type. Figure 4 (As shown in C). This result indicates VvENO2 Genes have an inhibitory effect on plant growth.

[0058] Example 5VvENO2 Negative regulation of grape fruit ripening by genes

[0059] This embodiment first constructs... VvENO2 Gene overexpression and RNAi vector were used, followed by transformation into grape berries. Real-time quantitative PCR was then used to analyze the transforming grape berries. VvENO2 The expression level of the gene was detected, and the role of the gene in grape fruit ripening was analyzed. The specific procedures are as follows:

[0060] 1. Construction and transient conversion of overexpression and silencing vectors

[0061] Using BamHI and XbaI enzyme digestion to VvENO2 The CDS subcloned into the pIR vector was amplified by PCR using primers with homologous arms. The primers used (SEQ ID NO. 10-11 in Table 1) are listed, and the constructed vector was named pIR-OE-VvENO2. Similarly, the CDS was digested with BamHI and XbaI enzymes to... VvENO2 The reverse subcloning was performed into the pIR vector, and PCR amplification was carried out using primers with homologous arms. The primers used (SEQ ID NO. 12~13 in Table 1) are listed, and the constructed vector was named pIR-RNAi-VvENO2. These constructs were extracted from proliferating *E. coli* cells under carbenicillin selection. Positive bacterial cultures identified by the biotechnology company were used for plasmid extraction and purification using a plasmid large-scale extraction and purification kit (Vigrass, Beijing, China), and then stored at -20°C.

[0062] Following the methods of Voinnet (see: Voinnet O, Rivas S, Mestre P et al. Retracted: an enhanced transient expression system in plants based on suppression of genesilencing by the p19 protein of tomato bushy stunt virus. Plant J, 2003, 33, 949-956.) and Jia (see: Jia H, Zuo Q, Sadeghnezhad E, et al. HDAC19 recruits ERF4 to the MYB5a promoter and diminishes anthocyanin accumulation during grape ripening. Plant J, 2023, 113(1): 127-144.), a hypodermal injection needle was used to puncture the stem of 'Kyoho' grape bunches approximately 30 days after flowering. A capillary tube was inserted into the formed hole, and plasmid DNA (50 μL, approximately 200 ng) extracted from the bacterial culture was pipetted into the tube until it was completely absorbed by the grape bunches. The injection was performed three times, with an interval of 7 days.

[0063] 2. Real-time quantitative PCR detection

[0064] Designed using Primer Blast on the NCBI website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). VvENO2 The primers for quantitative PCR of the gene (SEQ ID NO. 14~15 in Table 1) were used to detect the gene using the Cham QTM Universal SYBR qPCR Master Mix fluorescent dye from Novizan and a Quantagne q225 quantitative PCR instrument from Beijing Coolbot Technology Co., Ltd. The qPCR program was as follows: Amplification was performed in a 10 μL reaction mixture consisting of 5 μL 2× SYBR Green PCR Master Mix, 0.3 μL primers, and 200 ng cDNA at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 58°C for 30 seconds, and 95°C for 15 seconds. Each sample was repeated three times. Action was used as an internal reference gene in all experiments, based on 2... （-ΔΔCT） The algorithm normalizes the expression levels of the detected genes.

[0065] 3. Results

[0066] To further explore VvENO2 To investigate the regulatory role of genes in grape ripening, this example constructed an overexpression vector (pIR-VvENO2-OE) and a silencing vector (pIR-VvENO2-RNAi), and overexpressed and silenced these genes in grape culture. VvENO2 Genes were used, with the pIR empty vector serving as a control. Different bunches of grapes on the same vine were injected with bacterial suspensions of pIR-VvENO2-OE, pIR-ENO2-RNAi, and pIR empty vector, respectively. The injections were performed three times, on fruits 32 days (June 12), 39 days (June 19), and 46 days (June 26) after flowering. The growth status of the fruits was observed one week after each injection.

[0067] Phenotypic observation of grape bunches at three stages of viral vector infection revealed that, compared with fruits infected with the pIR empty vector, overexpression of [virus name missing] significantly increased the viral load. VvENO2 After gene administration, a delayed ripening process in grapes was observed, and silencing of the gene resulted in this effect. VvENO2 After gene modification, the ripening process of grapes is accelerated. Figure 5 (As shown in Figure A). This indicates that VvENO2 Genes play a negative regulatory role in grape ripening. Total RNA was extracted from grapes on the third day after treatment, and cDNA was obtained by reverse transcription, followed by identification by quantitative real-time PCR. Data analysis was performed using GraphPad software. The quantitative real-time results showed that the transient expression system was successfully established in grape berries. Figure 5 (As shown in B).

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The VvENO2 The coding region nucleotide sequence of the gene is shown in SEQ ID NO.1, which inhibits... VvENO2 Gene expression promotes the ripening of plant fruits, and the plants mentioned are grapes and Arabidopsis thaliana.

2. As described in claim 1 VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The promotion of fruit ripening includes promoting fruit coloring.

3. As described in claim 1 VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The promotion of plant fruit ripening includes promoting plant pod ripening.

4. As described in claim 1 VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The promotion of plant fruit ripening includes promoting fruit coloring and pod ripening.

5. The method according to claim 1 VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The inhibition VvENO2 Gene expression is the basis for construction VvENO2 RNAi vectors of genes, transformed into plants, have the effect of VvENO2 Gene expression is suppressed.

6. The method according to any one of claims 1 to 5 VvENO2 The application of genes in promoting fruit ripening in plants is characterized by: The grape variety mentioned is "Kyoho".

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