Application of grape VvPMEI8 gene in delaying fruit softening

Abstract

The invention belongs to the technical field of gene engineering, and discloses an application of a grape VvPMEI8 gene in delaying fruit softening, and the CDS sequence of the grape VvPMEI8 gene is as shown in SEQ ID NO. 1. The molecular mechanism of grape fruit softening is clarified from the molecular level, a direction is provided for breeding storage and transportation-resistant grape varieties, the shelf life of table grapes is prolonged, loss is reduced for farmers, and healthy development of the grape industry is promoted.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a grape VvPMEI8 Application of genes in delaying fruit softening. Background Technology

[0002] Grape( Vitis vinifera Grapes (L.) are one of the most widely cultivated fruit trees in my country, with the cultivation area and yield of table grapes ranking first in the world for many years. During the storage and transportation of table grapes, soft-fleshed grape varieties are particularly susceptible to spoilage and damage due to their relatively soft fruit, resulting in significant post-harvest losses. Reports indicate that annual post-harvest losses for grapes are approximately 15%–20%. Fruit firmness is a key quality trait determining storage and transport resistance and shelf life. Therefore, to meet market demands, exploring the molecular mechanisms by which grape genes regulate fruit softening is of great significance for breeding storage-resistant varieties and extending the shelf life of grapes.

[0003] Pectin methylesterase inhibitor (PMEI) inhibits the demethylation of pectin by PME by binding to the active site of PME, thus jointly regulating the mode and extent of pectin demethylation. This regulation further affects the physicochemical properties of pectin, ultimately influencing plant growth and development. In cotton, GhPME3 It can effectively inhibit GhPME2 / GhPME31 The activity of [the substance] enhances the resistance of cotton to Verticillium wilt; in Arabidopsis thaliana, AtPMEI18 Direct inhibition AtPME31 The activity of [the substance] is reduced, pectin demethylation is decreased, stomatal size is reduced, and the dynamic response of stomatal tissue to environmental changes is enhanced; in tartary buckwheat, FtPMEI13 By inhibiting FtPME44 / 61 The activity of [the substance] influences the degree of pectin methylation, thereby regulating stomatal closure; in grapes, VvPMEI1 PME activity is inhibited in the early stages of flower and fruit development. However, genes related to PMEI regulation of grape fruit softening have been rarely reported. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a grape... VvPMEI8 The application of genes in delaying fruit softening provides direction for breeding grape varieties that are resistant to storage and transportation, which is conducive to extending the shelf life of fresh grapes, reducing losses for growers, and promoting the healthy development of the grape industry.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a grape VvPMEI8 The application of genes in delaying fruit softening, in grapes VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, the fruit is a grape or tomato fruit.

[0007] Secondly, the present invention provides a grape VvPMEI8 The gene encodes a protein, the sequence of which is shown in SEQ ID NO.2.

[0008] Thirdly, the present invention provides a product containing grapes VvPMEI8 Plant expression vectors for genes, specifically grapes VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0009] Fourthly, the present invention provides a product containing grapes VvPMEI8 Recombinant bacteria of the gene, the grape VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1. Agrobacterium is preferred.

[0010] Fifthly, the present invention provides a method for delaying fruit softening, comprising the following steps: [The method involves] placing a mixture of grapes... VvPMEI8 The gene recombination vector is transferred into the fruit, making the grape... VvPMEI8 Stable overexpression of the gene, the grape VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0011] Furthermore, the fruit is a grape or tomato fruit.

[0012] Sixthly, the present invention provides a method for delaying fruit softening, comprising the following steps: overexpressing grape... VvPME19 Transient overexpression of the gene in transgenic grapes VvPMEI8 Genes, the grape VvPMEI8 Genes suppress grape VvPME19 The enzyme activity increases fruit firmness and pectin content, thereby delaying fruit softening. The grapes... VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1, and the grape... VvPME19 The CDS sequence of the gene is shown in SEQ ID NO.3.

[0013] Furthermore, the fruit is a grape or tomato fruit.

[0014] Seventhly, the present invention provides a grape VvPMEI8 Gene-linked grapes VvPME19 The application of genes in the breeding of plant varieties that are resistant to storage and transportation, wherein the plant is grape or tomato.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovers and verifies the grape VvPMEI8 Genes can delay fruit softening, and grapes VvPMEI8- VvPME19 Gene interactions can also delay fruit softening. Elucidating the molecular mechanism of grape fruit softening at the molecular level provides direction for breeding grape varieties that are resistant to storage and transportation, which is conducive to extending the shelf life of fresh grapes, reducing losses for growers, and promoting the healthy development of the grape industry. Attached Figure Description

[0016] Figure 1 A graph showing the predicted interactions between VvPMEIs and VvPMEs proteins.

[0017] Figure 2 Electrophoresis diagram of the recombinant overexpression vectors of grape VvPMEI8 and VvPME19.

[0018] Figure 3 For overexpression VvPME19 Phenotypic identification and related indicators in 'Giant Rose' grapes, including: A) phenotypic changes over 3–21 days; B) transient overexpression. VvPME19 C) Expression level of the target gene in grape berries after fertilization; C) Transient overexpression VvPME19 Changes in grape berry firmness; D) Transient overexpression VvPME19 Changes in protopectin content in grape berries; E) transient overexpression VvPME19 Changes in PME enzyme activity in grape berries.

[0019] Figure 4 For grapes VvPME19 The role of genes in the identification of positive transgenic tomato plants and related indicators, including: A) phenotypic identification of transgenic and wild-type fruits; B) VvPME19 C) Relative expression levels at different developmental stages of fruit; D) Changes in firmness at different developmental stages of transgenic and wild-type fruit; E) Changes in protopectin content at different developmental stages of transgenic and wild-type fruit; MG: Green-ripe stage; BR: Color-changing stage; B3: 3 days after color change; B7: 7 days after color change; B15: 15 days after color change.

[0020] Figure 5 For overexpression VvPMEI8 Phenotypic identification and related indicators in 'Giant Rose' grapes, including: A) phenotypic changes over 3–21 days; B) transient overexpression. VvPMEI8 C) Expression level of the target gene in grape berries after fertilization; C) Transient overexpression VvPMEI8 Changes in grape berry firmness; D) Transient overexpression VvPMEI8 Changes in protopectin content in grape berries; E) transient overexpression VvPMEI8 Changes in PME activity in grape berries.

[0021] Figure 6 For grapes VvPMEI8 Identification of positive transgenic tomato plants and related indicators, including: A) overexpression of genes. VvPMEI8 Phenotypic identification of transgenic and wild-type fruits at different stages; B) VvPME19 C) Relative expression levels at different developmental stages of fruit; D) Changes in firmness at different developmental stages of transgenic and wild-type fruit; E) Changes in protopectin content at different developmental stages of transgenic and wild-type fruit; MG: Green-ripe stage; BR: Color-changing stage; B3: 3 days after color change; B7: 7 days after color change; B15: 15 days after color change.

[0022] Figure 7 To validate the luciferase complementation (LCI) assay VvPMEI8 Protein and VvPME19 Protein-protein interactions.

[0023] Figure 8 To verify via bimolecular fluorescence complementation (BIFC) assay VvPMEI8 Protein and VvPME19 Protein-protein interactions.

[0024] Figure 9 To verify the inhibition of VvPMEI8 protein in in vitro enzymatic experiments VvPME19 Protease activity.

[0025] Figure 10 In order to be in VvPME19 Transient overexpression in genetically modified tomato fruits VvPMEI8 verify VvPMEI8-VvPME19 The effect of interactions on delaying fruit softening, including: A) phenotypic identification of transgenic and wild-type fruits; B) VvPME19 C) Relative expression levels at different developmental stages of fruit; D) Changes in PME enzyme activity at different developmental stages of transgenic and wild-type fruit; E) Changes in firmness at different developmental stages of transgenic and wild-type fruit; BR: color-changing period; B3: 3 days after color change; B7: 7 days after color change; B15: 15 days after color change.

[0026] Figure 11 To verify the overexpression using a low-methyl esterified pectin-specific antibody (LM19) VvPMEI8 , VvPME19 and VvPMEI8-VvPME19The interaction modulates the effect of pectin demethylation in tomato fruit during fruit softening, including: A) Immunolocalization of hypomethylated pectin. The LM19 probe can recognize hypomethylated pectin, while fluorescent whitening agent 28 (FB28) can recognize plant cell walls. Scale bars represent 0.050 mm. B) Fluorescence intensity of LM19 immunolabeling during tomato fruit softening. The graph shows the relative fluorescence signal of the LM19 antibody. Different letters indicate significant differences between groups (one-way ANOVA combined with Tukey's test). p <0.05). Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the technical means used in the examples are all conventional techniques well known to those skilled in the art, such as tissue culture, molecular biology, biochemistry, and bioinformatics. "Gene overexpression" refers to the insertion of the DNA sequence of a target gene into the circular DNA molecule of an "expression vector," causing the expression level of a certain gene in tissues to be significantly higher than its normal physiological level.

[0028] The 'Giant Rose' grape variety used in this invention was grown in the Science and Education Park of Henan Agricultural University. The transient transformation experiment used 'Giant Rose' grapes of similar maturity from vines with uniform growth 7 weeks after flowering. Vitislabrusca L.× Vitisvinifera L.) were used as experimental materials. The tomato used for transgenic testing was 'Micro Tom', and the seeds were purchased from Nanjing Fengshuo Horticulture Co., Ltd. The tobacco used in the transient infection experiment was 'Ben's tobacco, and the seeds were obtained from long-term laboratory storage, but could also be purchased commercially.

[0029] Example 1: Screening for genes that delay fruit softening Fruits from the soft-fleshed grape variety 'Hanxiangmi' at different developmental stages (E-L32 swelling stage, E-L34 softening stage, and E-L38 ripening stage) were selected and measured using a texture analyzer (TA-XT2i, PlusStable Micro Systems, UK) with a P100 (100mm diameter) probe. The measurement parameters were as follows: pre-test speed 5.00 mm / s, test speed 1 mm / s, post-test speed 25 mm / s, grape pulp compression deformation rate set at 30%, and the two compression pause times and trigger forces were 2 s and 5 g, respectively. Three grape bunches of uniform maturity were selected, and 10 grapes from each bunch were measured. The average result was taken. Ten grapes from each grape bunch were immediately frozen in liquid nitrogen. Total RNA was extracted from the grape samples at three key softening stages using the column-based plant polysaccharide and polyphenol total RNA extraction kit (RH1401) from Beijing Prosea Biotechnology Co., Ltd. The specific procedures were performed according to the kit's instructions. After extraction, RNA quality was assessed by agarose gel electrophoresis to confirm the absence of contamination and degradation. The A260 / A280 ratio of total RNA was measured to determine RNA purity. RNA integrity (RIN value ≥7 and normal 28S / 18S ratio) was checked using Agilent Bioanalyzer to accurately determine RNA concentration. RNA reverse transcription was performed using the HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (R323-01, Novizan, Nanjing). cDNA library construction and sequencing were commissioned to Shenzhen BGI Genomics Co., Ltd. Pectin methylesterase was analyzed. PMEs The expression levels of family genes were screened to identify genes that were highly expressed throughout the fruit softening process. VvPME19 Filtered through the STRING website to find related VvPME19 Pectin methylesterase inhibitors of protein-protein interactions VvPMEI8 protein( Figure 1 ).

[0030] Example 2: Gene Cloning With cultivar PN40024 ( V . vinifera Using the telomere-to-telomere (T2T) assembled genome as a reference genome, and SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 as primers, and 'Hanxiangmi' cDNA as a template, amplification was performed. VvPMEI8, VvPME19 Genes. The sequences of SEQ ID NO.5 to SEQ ID NO.8 are shown below: VvPMEI8-F: 5'-ATGGGTTCTACTGGTTTGTTGTT-3' (SEQ ID NO. 5); VvPMEI8-R: 5'-CCAATCAAGAAGATCAATAATTCC-3' (SEQ ID NO. 6).

[0031] VvPME19-F: 5'-ATGGCTCTTTACTCAGGACAAG-3' (SEQ ID NO. 7); VvPME19-R: 5'-AACTGCTAATCCTGCTGAATATTT-3' (SEQ ID NO. 8).

[0032] Using cDNA from 'Pinot Noir' grapes as templates (Pinot Noir reference genome URL: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_030704535.1 / , version number: ASM3070453v1), PCR amplification was performed. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2.

[0033] Table 1 PCR reaction system Table 2 PCR reaction procedure Sequencing results showed that the cDNA obtained using 'Pinot Noir' cDNA as a template and primers SEQ ID NO.5 and SEQ ID NO.6 was obtained. VvPMEI8 The coding region (CDS) sequence is 549 bp (as shown in SEQ ID NO.1), and the protein it encodes consists of 182 amino acids (as shown in SEQ ID NO.2). VvPMEI8 Gene locus: 100262664, reference sequence: NC_081808.1, 24514880~24515959 bp, length 1079 bp.

[0034] Sequencing results showed that the cDNA obtained using 'Pinot Noir' cDNA as a template and primers SEQ ID NO.7 and SEQ ID NO.8 was... VvPME19 The coding region (CDS) sequence is 1596 bp (as shown in SEQ ID NO.3), and the protein it encodes consists of 531 amino acids (as shown in SEQ ID NO.4). VvPME19Gene locus: 100233113, reference sequence: NC_081816.1, 24226412~24228517 bp, length 2105 bp.

[0035] Example 3 Acquisition VvPMEI8 Gene overexpression grapes 1. Construction VvPMEI8 Gene overexpression vector The vector pSAK277 (laboratory-preserved vector) was linearized and digested using restriction endonuclease (NEB). The specific digestion system is shown in Table 3 below. Digestion was performed at 37 °C for 30 min.

[0036] Table 3 PCR enzyme digestion system The above PCR product ( VvPMEI8 and VvPME19 The coding region (CDS) and the linearized product of the pSAK277 vector were detected by agarose gel electrophoresis. Figure 2 The amplified products were purified using a microcolumn DNA concentration gel extraction kit (Zhuangmeng, Beijing). Further purification was performed using the ClonExpress Ultra One Step Cloning Kit V2 (C116, Novizan, Nanjing) on ​​ice according to the homologous recombination reaction system shown in Table 4, followed by ligation at 50 °C for 30 min. VvPMEI8 and VvPME19 The genes were ligated into the pSAK277 vector to construct the recombinant vectors pSAK277-VvPMEI8 and pSAK277-VvPME19.

[0037] Table 4 Homologous recombination reaction system 2. Transformation of Agrobacterium with overexpression vector The recombinant vectors pSAK277-VvPMEI8 and pSAK277-VvPME19 were transformed into Agrobacterium tumefaciens GV3101 competent cells (Zhuangmeng, Beijing). The specific procedures were as follows: 3 μL of the successfully constructed fusion plasmid was added to the competent cells. The cells were incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ℃ water bath for 5 min, and on ice for 5 min. 900 μL of LB broth was added to a clean bench, and the cells were amplified in a shaker at 28 ℃ for 2–3 h. After centrifugation at 5000 rpm for 2 min, the supernatant was discarded, and the remaining 100 μL of liquid was mixed and spread onto LB agar (50 mg / L spectinomycin + 25 mg / L rifampin). The cells were incubated at 28 ℃ for 24 h. The next day, uniformly sized single colonies were selected for colony PCR detection using universal primers for the pSAK277 vector. Positive clones were selected and stored at -80 ℃ with 30% glycerol at a 1:1 ratio for later use.

[0038] The overexpression vector suspensions of Agrobacterium tumefaciens GV3101 (containing pSAK277-VvPMEI8, pSAK277-VvPME19, and pSAK277 suspensions) stored at -80 ℃ were streaked onto LB solid medium (50 mg / L spectinomycin + 25 mg / L rifampin) and incubated at 28 ℃ for 48 h for activation. Single colonies containing the overexpression vector were picked and incubated in 5 mL of liquid LB medium (50 mg / L spectinomycin + 25 mg / L rifampin) at 28 ℃ with shaking for 12–16 h. Using 1 μL of the bacterial suspension as a template, colony PCR was performed using universal primers for the pSAK277 vector for verification. Positive bacterial suspensions were transferred to 50 mL of LB liquid medium (containing the same antibiotics) and cultured at 28 ℃ with shaking for 8–12 h until OD. 600 ≈0.6. Collect bacterial cells by centrifugation at 5000 rpm for 5 min, wash 2-3 times with resuspending buffer (10 mM MES pH 5.6 + 10 mM MgCl2 + 150 μM AS), and finally resuspend to OD. 600 =0.8~1.0, incubate statically at 28 ℃ for 2-3 h.

[0039] 3. Obtain VvPMEI8 , VvPME19 Genetically modified grapes and tomatoes Select 'Giant Rose' grapes that are uniformly ripe, 7-8 weeks after flowering. Vitislabrusca L. × VitisviniferaL.) grape bunches were momentarily injected with the above-mentioned resuspended solution to verify the effect of overexpression of the target gene on fruit softening. Genetic transformation was performed using the momentary injection method, specifically as follows: One side of each grape bunch was injected with an Agrobacterium suspension containing an empty vector as a control group, while the other side was injected with Agrobacterium suspensions containing recombinant vectors of the target gene as experimental groups. A total of five grape bunches were injected; one bunch was used for observing phenotypic changes on the tree, and samples were taken from the other four bunches at 3, 7, 14, and 21 days post-injection. For each sample, 10 injected fruits were randomly selected from the top, middle, and bottom of the bunch, placed in an icebox, and quickly transported back to the laboratory.

[0040] First, the firmness of the grape samples was determined using a texture analyzer. After cryogenic grinding in liquid nitrogen, total RNA was extracted from grape fruits at 0, 3, 7, 14, and 21 days of age using a polysaccharide and polyphenol plant RNA extraction kit (Beijing Proha Biotechnology Co., Ltd.). The specific procedures were strictly followed according to the kit instructions. The RT III All-in-One Mix with dsDNase reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.) was used to synthesize the first strand of cDNA using 1 μg of total RNA as a template. The cDNA was diluted 5-fold with sterile, enzyme-free ddH2O, aliquoted, and stored at -20 ℃ for later use. Using the quantitative primers listed in SEQ ID NO. 9–12, qRT-PCR analysis was performed on an ABI Pirism 7500 FAST real-time fluorescence quantitative PCR instrument to detect the relative expression level of the target gene. The sequences of SEQ ID NO. 9–SEQ ID NO. 12 are shown below: qVvPMEI8-F: 5'-ATTGGTCGGCTCTGCTAATG-3' (SEQ ID NO.9); qVvPMEI8-R: 5'-GTTCCGGTGGATAAGGCAAT-3' (SEQ ID NO. 10); qVvPME19-F: 5'-TCCTGGTGAAGAAGGGCTTA-3' (SEQ ID NO. 11); qVvPME19-R: 5'-TTCTGGCCTAATGGCATCAC-3' (SEQ ID NO. 12).

[0041] Transient overexpression VvPME19 The phenotype of the 'Giant Rose' grape was observed. Figure 3 A). As can be seen from the figure, transient overexpression... VvPME19 The grapes in the experimental group began to change color 7 days after injection, earlier than the control group. Quantitative real-time PCR detection of the target gene revealed that the experimental group... VvPME19 The expression level was significantly increased ( Figure 3 B), indicating VvPME19 It was successfully overexpressed in grapes. Relevant physiological data were measured, and the fruit firmness and protopectin content of the experimental group were significantly lower than those of the control group; simultaneously, the pectin methyl esterase (PME) activity of the experimental group was also significantly reduced. In conclusion, transient overexpression... VvPME19 It can accelerate the softening of the fruit. Figure 3 C, 3D, and 3E).

[0042] To further verify reliability, VvPME19 Stable transformation of tomato plants yielded the transgenic line OE. VvPME19- 1. OE VvPME19- 2. OE VvPME19- 3. Observe the phenotypic changes of tomato fruits during the softening process. Figure 4 A). First, the seeds were pretreated by soaking them in 55 ℃ warm water for 30 min, followed by disinfection with 75% ethanol for 30 s and 4% sodium hypochlorite solution for 15 min, and finally rinsed three times with sterile water to thoroughly remove the disinfectant. The disinfected seeds were placed in tissue culture bottles containing 1 / 2 MS medium and incubated in the dark at 25 ℃ until germination. After germination, the culture bottles were transferred to a tissue culture room with a photoperiod of 16 h light / 8 h dark for further culture. After 7 days of culture, when the seedlings began to grow true leaves, Agrobacterium infection experiments on tomato cotyledons were conducted using the leaf disc method. For transgenic tomato fruits, the expression level of the target gene was first detected by quantitative real-time PCR. The results showed: VvPME19 Gene expression levels peak during the BR phase. Figure 4 B). Compared to the wild type, the transgenic tomato fruit firmness was significantly reduced; at the BR stage, the transgenic lines (OE) showed significantly lower firmness. VvPME19- 1. OE VvPME19- 2. OE VvPME19- 3) The fruit firmness decreased by 36%, 45% and 28% respectively. Figure 4 C). In addition, VvPME19 Overexpression of PME enzymes leads to increased PME enzyme activity and decreased protopectin content. Figure 4 D and 4E). Speculative overexpression. VvPME19 It may promote fruit softening by increasing PME enzyme activity.

[0043] instantaneous transformation VvPMEI8 The phenotype of grape berries infused with the recombinant vector was observed. Figure 5 It can be seen that in the control group, the grapes began to change color after 3 days, compared to the instantaneous transformation. VvPMEI8 The grapes in the experimental group ripened earlier. Figure 5 A). Compared with the control group, the experimental group's grapes showed better results after injection. VvPMEI8 The expression level of [something] was significantly increased, indicating that... VvPMEI8 It was overexpressed in all grape berries of the experimental group. Figure 5 B). Measuring relevant physiological data revealed transient transformations. VvPMEI8 It significantly increased grape berry firmness and protopectin content, and significantly reduced pectin methyl esterase (PME) activity, thus delaying berry softening. Figure 5 C~5E). At the same time, VvPMEI8 Stable transformation of tomato fruits yielded the transgenic line OE. VvPMEI8- 1. OE VvPMEI8- 2. OE VvPMEI8- 3. Observe the phenotypic changes of tomato fruits during the softening process. Figure 6 A). The results showed that, compared with the control, VvPMEI8 The expression levels were relatively high in the BR and B3 stages. Figure 6 B). Measurements of relevant physiological indicators revealed that the firmness and protopectin content of tomato fruits during ripening were significantly higher than the control. Figure 6 C and 6D). Especially in the OE-VvPMEI8-2 strain, PME enzyme activity decreased sharply by 81% in the B3 stage (C and 6D). Figure 6 E).

[0044] 4. Verification VvPMEI8 Protein and VvPME19 Protein-protein interactions Each VvPMEI8 and VvPME19 The coding regions (SEQ ID NO.1 and SEQ ID NO.3, respectively) were inserted into the pCAMBIA1300-cLuc and pCAMBIA1300-nLuc vectors. Using specific primers (SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8), and the reaction systems listed in Tables 1-4 above, pCAMBIA1300- was constructed via homologous recombination. VvPMEI8 -cLuc and pCAMBIA1300- VvPME19 -nLuc fusion vector.

[0045] The pCAMBIA1300- constructed above are respectively VvPMEI8 -cLuc、pCAMBIA1300- VvPME19The vectors p-nLuc, pCAMBIA1300-cLuc, and pCAMBIA1300-nLuc were transformed into Agrobacterium tumefaciens GV3101 (pSoup) strain (ZC1406, Zhuangmeng, Beijing). Positive bacterial suspensions were selected and incubated at 28 ℃ with shaking at 220 rpm for 8–12 h. The bacterial cells were collected by centrifugation and resuspended in resuspending buffer (10 mM MES, 10 mM MgCl2, and 100 μM AS) to OD200. 600 =0.8-1.0. The fusion vectors were mixed at a 1:1 (volume ratio) and incubated in a 28 ℃ incubator in the dark for 3 h. The mixed resuspension was slowly injected into the tobacco leaves from the underside, and then cultured in the dark for 12 h, followed by 12 h under light. Forty-eight h after transfection, imaging was performed using a LASER900 multifunctional plant imaging system (BIO-0I, Japan).

[0046] To further verify the in vivo interactions, 'Ben's tobacco seeds were sown in nutrient pots containing a substrate (peat moss:vermiculite:perlite = 3:2:1), covered with plastic wrap for warmth and moisture retention, and placed in a culture chamber at 24℃ for 16 h light / 8 h darkness. After the tobacco seeds germinated two cotyledons (approximately 10 days), they were transplanted into 7 cm diameter nutrient pots and continued to be placed in a culture chamber at 24℃ for 16 h light / 8 h darkness. After approximately 4 weeks, tobacco plants with uniform growth were used for transient injection. The luciferase signal in the tobacco was observed. The results showed that only plants co-transfected with pCAMBIA1300- VvPMEI8 -cLuc and pCAMBIA1300- VvPME19 A strong luciferase signal was observed in the sample containing the -nLuc vector, indicating that VvPMEI8 It can be found in tobacco leaves and VvPME19 interaction( Figure 7 ).

[0047] Will VvPMEI8 and VvPME19 The coding regions were inserted into the vectors pNC-BiFC-Ecn and pNC-BiFC-Enn via homologous recombination, respectively. Specific primers were the sequences shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. Using the Nimble Cloning (NC001, NC biotech, Hainan) kit, and following the reaction system in Table 5, ligation was performed at 50℃ for 30–60 min to construct the corresponding vectors. VvPMEI8 -Ecn and VvPME19 -Enn fusion carrier.

[0048] Table 5 PCR reaction system for BIFC The above-mentioned Ecn, Enn, and VvPMEI8 -Ecn and VvPME19 -Enn fusion carrier is injected with tobacco using the above method, and injected with... VvPMEI8 -Ecn and Enn, VvPME19 Tobacco leaves treated with Enn and Ecn bacterial suspensions served as controls. Fluorescence signals in tobacco leaves were observed 2 days later using a high-resolution laser confocal microscope (Nikon, Japan). Results showed that only co-injected... VvPMEI8 -Ecn and VvPME19 A strong GFP fluorescence signal was detected in the nuclei of lower epidermal cells of tobacco leaves from Enn. Picture 8 This result confirms... VvPMEI8 and VvPME19 Direct protein-protein interactions exist in plant cell membranes.

[0049] Will VvPMEI8 and VvPME19 The coding region (CDS) was cloned into the vector PET-30a via homologous recombination. The PET-30a-VvPMEI8 and PET-30a-VvPME19 fusion vectors were constructed using the systems described in Tables 1-4, with specific primers as shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, SEQ ID NO.8, respectively. Purification was induced using a tagged protein purification kit (P2247S, Beyotime, China). VvPMEI8 and VvPME19 Protein. PME activity was determined using a pectin methyl esterase activity assay kit (JW-7-230319, Shanghai Jiwei Biotechnology Co., Ltd.) for in vitro validation. VvPMEI8 protein pairs VvPME19 Inhibitory effect of protein. The results showed that... VvPMEI8 Inhibition can be achieved through direct interaction VvPME19 enzyme activity ( Picture 9 ).

[0050] To further verify VvPMEI8 and VvPME19 The effect of interaction on fruit softening, in overexpression VvPME19 Transient overexpression of [a substance] in the fruit of transgenic tomato lines VvPMEI8, And observe the phenotypic changes of tomato fruits during the softening process. Picture 10 A). The results show that: VvPME19 - OE Transient overexpression in fruit VvPMEI8 It can significantly delay the softening process of the fruit. Picture 10 B). Simultaneously, PME enzyme activity was significantly inhibited, especially in the B3 stage where PME enzyme activity was inhibited by 76% (B). Picture 10 C). Compared to VvPME19-OE Plant, instantaneous expression VvPMEI8 This significantly improved fruit firmness. In stage B3, fruit firmness was significantly higher with overexpression of the inhibitor compared to... VvPME19-OE The plant is 1.2 times taller ( Picture 10 D). Furthermore, the protopectin content increased significantly ( Picture 10 E).

[0051] Furthermore, low-methyl esterified pectin-specific antibody (LM19) was used on wild-type (WT) tomatoes during the BR stage of fruit processing. OE VvPME19 , OE VvPMEI8 and OE VvPMEI8 + OE VvPME19 Immunochemical analysis of the fruit ( Picture 11 The results showed that overexpression VvPME19 The LM19-labeled pectin exhibited significantly enhanced fluorescence intensity, indicating a significant increase in the degree of pectin demethylation. Overexpression VvPMEI8 This leads to a decrease in the fluorescence intensity of LM19-labeled pectin. VvPME19-OE Transient expression in tomato fruit VvPMEI8 It will reduce the fluorescence intensity of LM19-labeled pectin (but will still be higher). VvPMEI8-OE These results indicate that VvPME19- OE The high level of demethylated pectin in tomato fruit may be due to VvPMEI8 The offset.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A type of grape VvPMEI8 The application of genes in delaying fruit softening is characterized by, The grapes VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The fruit is either a grape or a tomato.

3. A type of grape VvPMEI8 Gene-encoded protein, characterized by, The protein sequence is shown in SEQ ID NO.

2.

4. A type of product containing grapes VvPMEI8 Plant gene expression vectors, characterized in that, The grapes VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.

1.

5. A type of grape-containing VvPMEI8 Recombinant bacteria of genes, characterized in that, The grapes VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.

1.

6. A method for delaying fruit softening, characterized in that, Includes the following steps: Will contain grapes VvPMEI8 The gene recombination vector is transferred into the fruit, making the grape... VvPMEI8 Stable overexpression of the gene, the grape VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.

1.

7. The method according to claim 6, characterized in that, The fruit is either a grape or a tomato.

8. A method for delaying fruit softening, characterized in that, Includes the following steps: Overexpression of grapes VvPME19 Transient overexpression of genes in transgenic grapes VvPMEI8 Genes, the grape VvPMEI8 Genes suppress grape VvPME19 The enzyme activity increases fruit firmness and pectin content, thereby delaying fruit softening. The grapes... VvPMEI8 The CDS sequence of the gene is shown in SEQ ID NO.1, and the grape... VvPME19 The CDS sequence of the gene is shown in SEQ ID NO.

3.

9. The method according to claim 8, characterized in that, The fruit is either a grape or a tomato.

10. A type of grape VvPMEI8 The application of genes in breeding plant varieties resistant to storage and transportation is characterized by, The plant in question is either a grape or a tomato.

Images