Application of XTH gene in promotion of grape fruit hardness formation by brassinolide

Abstract

The invention provides an application of an XTH gene in promotion of grape fruit hardness formation by brassinolide, brassinolide (BR) is adopted for treatment in a grape color changing period, it is found that the XTH gene can mediate synthesis of hemicellulose in grape fruits so as to significantly improve the hardness of the grape fruits, and through whole genome correlation analysis and transcriptomics analysis, the XTH gene can be used for promoting the hardness formation of the grape fruits. The two target genes VvXTH25 and VvXTH31 capable of regulating and controlling the hardness of the grape fruits are found, key genes and mechanisms for regulating and controlling the hardness of the grape fruits are defined, the hardness of the fruits is regulated and controlled by regulating and controlling the content of hemicelluloses, and the VvXTH25 and VvXTH31 have the effects of providing a basis and a means for regulating and controlling the texture of the grape fruits and improving the storage and transportation resistance; a brand-new way is provided for really regulating and controlling the hardness of the grape fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit cultivation, and particularly relates to the application of XTH genes in promoting the formation of grape fruit firmness through brassinolide. Background Art

[0002] Grapes (Vitis vinifera L.) are popular for their sweet taste and rich nutrition. However, grape varieties are diverse, and the firmness of each berry varies greatly. Soft-fleshed grapes have a limited shelf life at room temperature and are highly susceptible to mechanical damage during post-harvest transportation and storage, significantly reducing their market value. Furthermore, their poor storage and transportation resistance limits their market share, resulting in their primary use domestically. Firm-fleshed grapes, on the other hand, have denser flesh, less moisture, and are more durable in storage and transportation. Furthermore, firm-fleshed grapes have longer berries, which are tightly bound to the berries and less prone to fruit drop and cracking. Therefore, increasing grape berry firmness can promote the expansion and upgrading of the grape industry. Therefore, being able to independently regulate the firmness of growing grape berries will undoubtedly significantly boost the development of the grape industry.

[0003] Currently, grape berry firmness is influenced by multiple factors, both internal and external. Internal factors include the structure of the fruit's cells and the specific ions they contain, such as cell wall structure and calcium and potassium ion content. External factors, such as exogenous hormones and environmental conditions, also influence grape berry firmness. Numerous studies have explored the use of hormones to improve postharvest fruit firmness, including abscisic acid (ABA), auxin (IAA), and gibberellins (GA). These hormones regulate fruit ripening by coordinating with target genes in multiple biological processes. Brassinolide (BR) is a sterol-containing hormone in plants. Its core functions include promoting cell elongation and division, enhancing photosynthesis, promoting vessel differentiation, influencing pollen development and fertility, and delaying aging. As a plant growth regulator, brassinolide is highly effective, non-toxic, and broad-spectrum. Its use concentration is far lower than that of the five major plant growth regulators: gibberellins, growth hormones, abscisic acid, cytokinins, and ethylene, at only one thousandth of their concentration. It is therefore classified as the sixth major hormone in plants.

[0004] However, in the existing technology, the main research focuses on the effects of gibberellins on the fruit hardness of fruits such as kiwi, cherry, and grape, and BR can increase the fruit set rate and yield of fruits such as grapes, golden thread dates, and apples. However, there are currently no research reports on the effects of BR treatment on the ripening and hardness of grape fruits, and the specific regulatory mechanism is also unclear. Summary of the Invention

[0005] To address the above problems, the present invention provides the use of the XTH gene in promoting the formation of grape fruit firmness through brassinolide. By treating grapes with BR during the color change period, it was found that BR can mediate the synthesis of hemicellulose in grape fruits, thereby significantly improving the firmness of grape fruits. The mechanism was then explored. Through whole-genome association analysis and transcriptomics analysis, two target genes, VvXTH25 and VvXTH31, that can regulate grape fruit firmness were identified. Using overexpression vectors of the two target genes to treat growing grape fruits can significantly increase the hemicellulose content in the grape fruits and improve the fruit firmness, providing a new approach to regulating grape fruit firmness.

[0006] The present invention has found that BR can mediate hemicellulose synthesis and thus increase grape fruit firmness. To study its mechanism, BR was used to treat grape fruits at the color change period. Combined with whole-genome association analysis and transcriptomics, candidate genes that regulate grape fruit firmness through BR were screened. The functions of related genes were analyzed through subcellular localization and stable overexpression in grape fruits, in order to provide reference for clarifying the molecular mechanism of grape fruit firmness formation.

[0007] In one aspect, the present invention provides a product for regulating grape fruit firmness, comprising a gene overexpression vector having a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

[0008] To extend the shelf life and storage of grapes and improve their firmness, various methods are currently being used, including spraying exogenous hormones, controlling temperature and humidity, and more. Among these, GA and cytokinins (CTKs) are commonly used as exogenous hormones. Brassinosteroids (BRs), a steroid hormone that regulates growth and development, have been previously demonstrated to promote plant cell growth, division, and differentiation, but their role in regulating fruit firmness has been largely unstudied. Therefore, the present invention utilizes BR spraying to treat grapes, exploring the link between BR and fruit firmness and elucidating the BR-mediated regulatory mechanism, thereby providing a novel technical approach for regulating grape fruit firmness. In some embodiments, BR treatment of Yinhong grapes with varying concentrations of BR was performed. Results showed that BR treatment had no significant effect on grape quality, but significantly increased fruit size, firmness, and hemicellulose content, demonstrating that spraying BR can significantly improve grape fruit firmness.

[0009] In some embodiments, the present invention further conducted a genome-wide association analysis targeting grape fruit hemicellulose content, identifying 552 candidate genes related to hemicellulose synthesis. KEGG and GO enrichment analyses identified genes related to cell wall polysaccharide metabolism, with eight genes identified as potentially regulating grape fruit hemicellulose content. Furthermore, transcriptomic analysis of fruit treated with BR (exogenous hormone) identified 25 xyloglucan endoglycosyltransferases / hydrolases, suggesting their potential role in grape fruit hemicellulose depolymerization. The results of the genome-wide association analysis and transcriptomic analysis were intersected, and FPKM and expression level analyses revealed the key target genes VvXTH25 and VvXTH31, which may regulate grape flesh and skin firmness.

[0010] Overexpression vectors were then constructed for VvXTH25 and VvXTH31 to treat grape berries. It was found that the VvXTH25 and VvXTH31 genes had significant effects on regulating the firmness of grape berries. Their hemicellulose content also confirmed similar conclusions, proving that by increasing the expression of VvXTH25 and VvXTH31, the hemicellulose content can be increased, thereby increasing the firmness of grape berries.

[0011] In another aspect, the present invention provides a method for regulating grape berry firmness by regulating the expression of VvXTH25 and / or VvXTH31 genes, thereby regulating grape berry firmness; the nucleotide sequence of the VvXTH25 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the VvXTH31 gene is shown in SEQ ID NO.4.

[0012] In some embodiments, genetic engineering techniques were employed to investigate the relationship between changes in VvXTH25 and VvXTH31 gene expression and grape berry firmness. Overexpression vectors containing the VvXTH25 and VvXTH31 genes were constructed and introduced into grape berry tissue using the inflorescence dip method. The treated berries were then tested for a series of physiological indicators. Results revealed that grape berries overexpressing the VvXTH25 and VvXTH31 genes exhibited significantly increased hemicellulose content, firmness, and longitudinal diameter. This discovery provides a key theoretical basis for the subsequent development of formulations and methods for improving grape berry quality. By artificially manipulating the expression of these genes, it is expected that the cell wall structure of grape berries could be improved, enhancing firmness and storage and transportation resistance, which would have a positive impact on the development of the grape industry.

[0013] Furthermore, by regulating the expression of VvXTH25 and / or VvXTH31 genes, the synthesis of hemicellulose is regulated, thereby regulating the firmness of grape berries.

[0014] Furthermore, by constructing an overexpression vector of the VvXTH25 and / or VvXTH31 gene, the vector is introduced into grape berries, thereby increasing the expression of the VvXTH25 and / or VvXTH31 gene.

[0015] In another aspect, the present invention provides a method for promoting the synthesis of hemicellulose in grape berries to increase their hardness. During the growth of the grape berries, the grape berries are treated with a reagent containing BR.

[0016] In another aspect, the present invention provides a use of a gene overexpression vector for preparing a reagent for promoting hemicellulose synthesis in fruit and thereby increasing fruit hardness, wherein the gene overexpression vector has the nucleotide sequence shown in Seq ID NO.1 and / or Seq ID NO.2.

[0017] In some embodiments, the fruit comprises grapes.

[0018] In some embodiments, the VvXTH25 and VvXTH31 genes regulate grape berry firmness primarily by regulating the synthesis of hemicellulose.

[0019] In another aspect, the present invention provides a use of genes for preparing a reagent for regulating the hemicellulose content in grape fruit, thereby regulating the firmness of grape fruit. The genes include VvXTH25 and / or VvXTH31 genes. The sequence of the VvXTH25 gene is shown in Seq ID NO. 3, and the sequence of the VvXTH31 gene is shown in Seq ID NO. 4.

[0020] In another aspect, the present invention provides use of BR for preparing a reagent for promoting hemicellulose synthesis in grape berries, thereby increasing the firmness of the grape berries.

[0021] Furthermore, the BR promotes cellulose synthesis by promoting the expression of grape VvXTH25 and / or VvXTH31 genes. The sequence of the VvXTH25 gene is shown in Seq ID NO.3, and the sequence of the VvXTH31 gene is shown in Seq ID NO.4.

[0022] In another aspect, the present invention provides use of BR for preparing a reagent for promoting expression of VvXTH25 and / or VvXTH31 genes in grape berries, wherein the sequence of the VvXTH25 gene is shown in Seq ID NO. 3, and the sequence of the VvXTH31 gene is shown in Seq ID NO. 4.

[0023] The present invention has the following beneficial effects:

[0024] 1. It was found that BR treatment of grapes during growth and development can mediate the synthesis of hemicellulose in grape berries, thereby significantly improving the firmness of grape berries;

[0025] 2. Screening candidate genes for BR regulation of grape berry firmness, we identified two target genes, VvXTH25 and VvXTH31. We found that BR can mediate the synthesis of hemicellulose in grape berries by promoting the expression of VvXTH25 and VvXTH31, thereby improving the firmness of grape berries.

[0026] 3. We discovered that constructing an overexpression vector for the target gene VvXTH25 or VvXTH31 can increase the firmness of grape berries during growth and development, and that this can improve the firmness of grape berries by mediating the synthesis of hemicellulose in grape berries;

[0027] 4. The present invention innovatively discovered that BR treatment can regulate the size and firmness of grape berries, and screened for key genes that affect fruit firmness. Through stable transformation of grape berries, it was verified that VvXTH25 and VvXTH31 positively regulate fruit firmness by mediating the synthesis of hemicellulose. This provides a theoretical basis for the rational regulation of grape fruit firmness and a scientific basis for improving the storage and transportation tolerance of fresh grapes through artificial means. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are the ripe grape fruits after spraying with different concentrations of BR in Example 1;

[0029] Figure 2 is the soluble solid content of mature grape fruit after spraying with different concentrations of BR in Example 1;

[0030] Figure 3 is the titratable acid content of mature grape berries after spraying with different concentrations of BR in Example 1;

[0031] Figure 4 is the firmness of ripe grape fruit after spraying with different concentrations of BR in Example 1;

[0032] Figure 5 is the hemicellulose content of mature grape fruit after spraying with different concentrations of BR in Example 1;

[0033] Figure 6 is the cell length of mature grape fruit after spraying with different concentrations of BR in Example 1;

[0034] Figure 7 is the cell thickness of mature grape fruit after spraying with different concentrations of BR in Example 1;

[0035] Figure 8The results of the genome-wide association analysis of hemicellulose content using the Manhattan technique in Example 2, where A is the Manhattan plot of the mixed linear BLINK model and B is the QQ-plot;

[0036] Figure 9 The transcriptome analysis results in Example 2;

[0037] Figure 10 This is the volcano plot of the differences between groups in the transcriptome data analysis in Example 2;

[0038] Figure 11 The GO enrichment analysis results of the transcriptome data in Example 2;

[0039] Figure 12 The KEGG analysis results of the transcriptome data in Example 2;

[0040] Figure 13 25 xyloglucan endoglycosyltransferase / hydrolase genes screened from the transcriptome in Example 2

[0041] Figure 14 This is the subcellular localization result in Example 3;

[0042] Figure 15 For the tissue, spatiotemporal expression pattern analysis in Example 3;

[0043] Figure 16 This is the vector map of the 35S::VvXTH25 overexpression vector in Example 4;

[0044] Figure 17 This is the vector map of the 35S::VvXTH31 overexpression vector in Example 4;

[0045] Figure 18 This is a photo of the grape fruit showing stable expression in Example 4;

[0046] Figure 19 This is a schematic diagram of the horizontal and vertical diameters and firmness of grape fruits after stable expression in Example 4;

[0047] Figure 20 Schematic diagram of qRT-PCR analysis of the expression levels of VvXTH25 and VvXTH31 after stable expression in grape berries in Example 4;

[0048] Figure 21 This is a schematic diagram of the hemicellulose content in grape fruit after stable expression in Example 4;

[0049] Figure 22These are grape berries injected with clean water (CK), empty vector (35S::FLAG), overexpression vector 35S::VvXTH31, and 35S::VvXTH31 in Example 4;

[0050] Figure 23 is the gene expression level of transiently expressed grape fruit in Example 4;

[0051] Figure 24 This is a schematic diagram of the instantaneous expression of fruit firmness of grapes in Example 4;

[0052] Figure 25 This is a schematic diagram of the transient expression of hemicellulose content in grapes in Example 4. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] Example 1: Effects of BR treatment on grape berries

[0057] In this study, 'Yinhong' grape berries were selected at a grape experimental base and treated by spraying the fruit surface with the drug during the veraison period (approximately 70 days after anthesis). Five concentration gradients, 0, 0.2, 0.4, 0.6, and 0.8 mg / L, were set, with three biological replicates. A small sprayer was used to evenly spray the fruit surface until water dripped. All treatment groups were grown under identical cultivation conditions. Fruit was harvested at maturity (approximately 100 days after anthesis), and relevant traits were recorded for each group. Quality indicators such as firmness, total soluble solids (TSS), titratable acidity (TA), and hemicellulose content were also tested.

[0058] 1. Related characteristics of grape fruit

[0059] To evaluate the effects of different concentrations of BR on the berry quality of 'Yinhong' grape, the grape berry phenotypes were determined, and the results are shown in Tables 1 and Figure 1 .

[0060] Table 1 Record of related characteristics of grape berries after spraying with different concentrations of BR

[0061]

[0062] Depend on Figure 1As shown in Table 1, different concentrations of exogenous hormone treatments significantly regulated the quality of grape berries. The size and quality of the 'Yinhong' grape berries increased significantly after BR treatment.

[0063] 2. Total Soluble Solids (TSS)

[0064] The soluble solids (TSS) were determined using a portable refractometer. Three replicates were set for each treatment and the average value was taken. The test results are shown in Figure 2 .

[0065] from Figure 2 It can be seen that the soluble solids content of each treatment group was higher than that of CK (blank control), but had no significant correlation with the concentration gradient. The content was the highest (17.90birx) under the 0.6 mg / L treatment, but the difference between the groups was not large.

[0066] 3. Titratable acid

[0067] The titratable acid was determined by sodium hydroxide titration. Three replicates were set for each treatment and the average value was taken. Figure 3 .

[0068] according to Figure 3 It can be seen that the titratable acid content of grape berries in each group showed a downward trend. When the 0.8 mg / L BR treatment reached its peak, the reduction in titratable acid was greater, but it did not reach a significant level.

[0069] In addition, in this example, sensory evaluation was conducted on each group of fruits, and it was found that there was no significant difference in the sweet and sour taste of the grape fruits after treatment with different concentrations of BR. It can be seen that BR treatment does not affect the quality of the grape fruits.

[0070] 4. Hardness

[0071] The GY-4 hardness tester (Adberg, Yueqing) was used to measure the hardness of the flesh and peel respectively. Three replicates were set for each treatment and the average value was taken. The test results are shown in Figure 4 .

[0072] Grape fruit hardness is mainly determined by the hardness of the skin and the hardness of the flesh. Different concentrations of BR treatment can significantly improve the fruit hardness. Among them, the effect on the skin hardness is most significant when 0.2 mg / L is used, which is 14.9% higher than that of CK (blank control) and the skin hardness is 6.8N. The effect on the flesh hardness is most significant when 0.6 mg / L is used, which is 52.4% higher than that of CK (blank control).

[0073] 5. Hemicellulose content

[0074] The hemicellulose content was determined using the 3,5-dinitrosalicylic acid method. The specific method for detecting the hemicellulose content is as follows: utilizing the property of hemicellulose to be hydrolyzed into reducing sugars under certain conditions, the reducing sugars are then heated with 3,5-dinitrosalicylic acid (DNS) under alkaline conditions to be oxidized into sugar acids and other products. DNS is reduced to brown-red 3-amino-5-nitrosalicylic acid, which has a characteristic absorption peak at 540 nm. The hemicellulose content can be quantitatively detected by the change in absorbance. Each treatment was repeated 3 times, and the results were averaged. The test results are shown in the table. Figure 5 .

[0075] according to Figure 5 Different treatments had varying effects on the hemicellulose content of the peel and flesh, all reaching significant levels. In the peel group, the range was 0.2 mg / L > 0 mg / L > 0.4 mg / L > 0.6 mg / L > 0.8 mg / L, with the most significant change after the 0.2 mg / L BR treatment. Grape flesh hemicellulose content increased significantly compared to the control, reaching its maximum after the 0.6 mg / L BR treatment.

[0076] 6. Paraffin sections

[0077] Grape berries from different treatment groups were collected, fixed with FAA fixative, and stored at 4°C. Paraffin sections were prepared, and cell length and width were measured by observing the paraffin sections.

[0078] according to Figure 6 and Figure 7 Compared with CK (blank control), the cell wall was significantly thickened after BR treatment, the cell morphology was more compact, the intercellular space was reduced, and the cell morphology was elongated.

[0079] Example 2: Screening of key functional genes

[0080] 1. Genome-wide association analysis

[0081] The hemicellulose content of more than 120 grape varieties was used as the target trait, and a genome-wide association analysis was conducted based on the BLINK model. SNP sites with -log10(P)>5 were considered as significant sites. The QQ plot shows ( Figure 8 ), the observed values ​​in the lower left corner are consistent with the expected values, and the observed values ​​in the upper right corner are upward, indicating that the BLINK model is suitable for this association study.

[0082] A total of 552 candidate genes related to hemicellulose synthesis were screened within the 100 kb region of the SNP site. KEGG and GO enrichment analyses identified genes related to cell wall polysaccharide metabolism, and a total of eight cell wall metabolism-related genes were found that may regulate grape fruit hemicellulose content, as shown in Table 2.

[0083] Table 2 Cell wall metabolism-related genes

[0084]

[0085] 2. Transcriptome Data Analysis

[0086] To explore the regulatory mechanism of BR on grape berry firmness, this study used transcriptome analysis to identify related candidate genes ( Figure 9 ). Based on the difference volcano plot in transcriptome data ( Figure 10 ), there are more up-regulated and down-regulated genes in the transcriptome. GO enrichment analysis ( Figure 11 ) showed that after BR treatment, compared with CK, DEGs were enriched in categories such as molecular function, cellular components and biological processes. KEGG enrichment analysis ( Figure 12 ) found that differentially expressed genes (DEGs) were enriched in cell wall metabolism processes such as secondary metabolite biosynthesis, plant hormone signaling, starch and sucrose metabolism, and galactose metabolism. Among them, 25 xyloglucan endoglycosyltransferases / hydrolases were screened. These enzymes are speculated to play an important role in the depolymerization of hemicellulose in grape fruit.

[0087] 3. Combining transcriptome analysis and genome-wide association analysis

[0088] The results of genome-wide association analysis and transcriptome analysis were intersected, and through FPKM value and expression level analysis, the target genes VIT_11s0052g01180 (VvXTH25) (Seq ID NO.3) and VIT_11s0052g01300 (VvXTH31) (Seq ID NO.4) that may regulate grape flesh firmness and skin firmness were finally discovered. Figure 13 shown.

[0089] Example 3: Location and analysis of key functional genes

[0090] 1. Subcellular localization

[0091] Cell PLOC software was used to predict the subcellular localization of VvXTH25 and VvXTH31 proteins. The prediction results showed that the proteins were located in the nuclei of grape fruit cells. To verify this prediction, the green fluorescent protein (GFP) tag signal was used to determine the location of gene expression and emit fluorescent signals in the cell nucleus ( Figure 14 ), which is consistent with the prediction.

[0092] 2. Analysis of organizational and spatiotemporal expression patterns

[0093] The expression of VvXTH25 and VvXTH31 in different developmental stages and tissues of grapes was verified. Figure 15The left figure shows the expression of VvXTH25 and VvXTH31 in different tissues, and the right figure shows the expression of VvXTH25 and VvXTH31 at different stages of fruit development. The results showed that VvXTH25 and VvXTH31 were specifically expressed at different stages and in different tissues.

[0094] Example 4: Verification of key functional genes

[0095] 1. Construction of overexpression vector

[0096] In this example, a 35S::VvXTH25 overexpression vector and a 35S::VvXTH31 overexpression vector were constructed.

[0097] The 35S::VvXTH25 overexpression vector was modified from the pBWA(V)HS vector. The construction process was as follows: The VvXTH25 gene overexpression vector (Seq ID NO. 1) was constructed by homologous recombination and golden gate seamless cloning. The primers were:

[0098] VvXTH25-F: AACACGGGGGACTTTGCAACatggcttctccttctacttcaaatctttcacc (SeqID NO.5)

[0099] VvXTH25-R: TCCTCGCCCTTCACGATACAggacgtggtagtggcagtgc (Seq ID NO.6)

[0100] The vector map of 35S::VvXTH25 overexpression vector is shown in Figure 16 .

[0101] The 35S::VvXTH31 overexpression vector was modified from the pBWA(V)HS vector. The VvXTH31 overexpression vector (Seq ID NO. 2) was constructed by homologous recombination and golden gate seamless cloning. The primers were:

[0102] VvXTH31-F: AACACGGGGGACTTTGCAACatgtttgggaagattgatatgcagctgaagc (Seq IDNO.7)

[0103] VvXTH31-R: TCCTCGCCCTTCACGATACAagacatcgtggcggcagtgc (Seq ID NO.8)

[0104] The vector map of 35S::VvXTH31 overexpression vector is shown in Figure 17 .

[0105] 2. Effects of stable overexpression on grape berries

[0106] Five days after flowering, Agrobacterium tumefaciens containing the 35S::VXTH25 overexpression vector and the 35S::VvXTH31 overexpression vector were immersed in the inflorescence of 'Yinhong' grapes to infect them and achieve stable expression in the grapes. 35S::XTH25 overexpression grapes and 35S::VvXTH31 overexpression grapes were constructed and compared with the empty vector group (35S::FLAG) and the blank control group (CK). Fruits were collected at maturity ( Figure 18 ), and tested the gene expression, hemicellulose content, size, and hardness of each group of grape berries.

[0107] The expression levels of each group were analyzed by qRT-PCR ( Figure 19 ), the results showed that the relative expression level of VvXTH31 in the grape skin of the 35S::VvXTH31 group was the highest, 4.4 times that of the control group, and the relative expression level of VvXTH25 in the grape skin of the 35S::VvXTH25 group was 3.2 times that of the control; the relative expression level of VvXTH31 in the grape pulp of the 35S::VvXTH31 group was the highest, 4.1 times that of the control group, and the relative expression level of VvXTH25 in the grape pulp of the 35S::VvXTH25 group was 3.3 times that of the control. There was no significant difference in the relative expression levels between the empty vector group and the control group.

[0108] The hemicellulose content of the flesh and skin of the grape berries in the overexpression group was measured. Figure 20 . Overexpression of VvXTH25 and VvXTH31 can increase the hemicellulose content, which is significantly greater than that of the control group and the empty load group. Among them, the hemicellulose content of the peel and flesh of the grapes in the 35S::VvXTH31 group was significantly higher than that of the control group and the empty load group, which were 104 mg / g and 13.8 mg / g respectively. The hemicellulose content of the peel and flesh of the grapes in the 35S::VvXTH25 group was also significantly higher than that of the control group and the empty load group, which were 101 mg / g and 9.8 mg / g respectively. It is speculated that VvXTH25 and VvXTH31 mainly regulate fruit firmness by regulating hemicellulose content.

[0109] The fruit size and firmness of the grapes of each group were measured. Compared with the empty group (35S::FLAG) and the control group (CK), the fruit firmness and longitudinal diameter of the grapes after overexpressing VvXTH25 and VvXTH31 were significantly different ( Figure 21The 35S::VvXTH31 group showed significantly higher flesh firmness (5.0 N) and peel firmness (7.2 N) than the control and no-carrier groups. The 35S::VvXTH25 group also showed higher flesh firmness (5.2 N) and peel firmness (5.9 N) than the control and no-carrier groups. The 35S::VvXTH31 group showed the most significant improvement in peel firmness. Furthermore, compared with the no-carrier group (35S::FLAG) and the control group (CK), overexpression of VvXTH25 and VvXTH31 significantly increased the longitudinal diameter of the grapes (1.1-fold and 1.3-fold, respectively), while the transverse diameter remained unchanged. Therefore, it is speculated that VvXTH25 and VvXTH31 may affect fruit firmness primarily by regulating peel firmness and fruit size by regulating longitudinal diameter. Furthermore, the VvXTH31 overexpression vector has a more pronounced effect on peel firmness and fruit size than the VvXTH25 overexpression vector.

[0110] 3. Effects of transient overexpression on grape berries

[0111] Before the fruit veraison period, Agrobacterium tumefaciens culture fluids (1 mL each) containing 35S::VvXTH25 and 35S::VvXTH31 overexpression vectors were injected into 'Yinhong' grape berries for transient overexpression. 35S::VvXTH25 and 35S::VXTH31 transient overexpression groups were constructed, and empty vector (35S::FLAG) and wild-type (CK) grapes were used as controls ( Figure 22 After 7 days, samples were collected to detect the expression levels of related genes, fruit firmness, and hemicellulose content in the flesh and skin of each group of grapes.

[0112] The expression levels of VvXTH25 and VvXTH31 in each group of grapes were analyzed by qRT-PCR. Figure 23 As shown, the relative expression level of VvXTH25 in the grape skin of the 35S::VvXTH25 group was the highest, 5-fold higher than that of the control group; the relative expression level in the flesh was 4.6-fold higher than that of the control group. The relative expression level of VvXTH31 in the grape flesh of the 35S::VvXTH31 group was the highest, 5-fold higher than that of the control group; the relative expression level in the skin was 3.5-fold higher than that of the control group. There was no significant difference in the relative expression level in the skin between the empty vector group and the control group.

[0113] Samples were taken near the pinhole to measure the hemicellulose content and hardness of the grape pulp and skin. The results are as follows: Figure 24 and 25As shown in the figure, overexpression of the VvXTH25 and VvXTH31 genes effectively increased grape fruit firmness and hemicellulose content during the growth and development stages. In the 35S::VvXTH31 overexpression group, grape skin firmness (20.4 N) and flesh firmness (14.7 N), as well as skin hemicellulose content (91 mg / g) and flesh hemicellulose content (15.6 mg / g) were significantly higher than those in the control and no-load groups, with changes in fruit firmness and hemicellulose content being particularly significant. Similarly, the 35S::VvXTH25 overexpression group showed significantly higher skin firmness (18.6 N), flesh firmness (15.2 N), skin hemicellulose content (80 mg / g), and flesh hemicellulose content (12 mg / g) than those in the control and no-load groups. The above results indicate that VvXTH25 and VvXTH31 may affect the overall firmness of the fruit mainly by regulating the firmness of the peel, and the VvXTH31 overexpression vector is more effective than VvXTH25 in improving the firmness of the peel.

[0114] 4. Effects of stable overexpression on tomato fruit

[0115] This example also used tomato as a model species. Tomato cotyledons were infected with Agrobacterium tumefaciens containing overexpression vectors for 35S::VvXTH25 and 35S::VvXTH31, respectively, to further validate the regulatory effects of the VvXTH25 and VvXTH31 genes on fruit firmness. Once the cotyledons developed into full plants, DNA positive results were confirmed, and ripe tomato fruits were assayed. The results are shown in Table 1. Overexpression of the VvXTH25 and VvXTH31 genes effectively increased tomato fruit firmness during growth and development. The flesh and peel firmness and hemicellulose content of tomatoes in the 35S::VvXTH25 and 35S::VvXTH31 groups were significantly greater than those in the control and unexpressed groups, with even more pronounced changes in peel firmness. The changes in flesh and peel firmness and hemicellulose content of tomatoes in the 35S::VvXTH31 group were greater than those in the 35S::VvXTH25 group. The above results further proved that VvXTH25 and VvXTH31 may affect the overall firmness of the fruit mainly by regulating the firmness of the peel, and the VvXTH31 overexpression vector is more effective than VvXTH25 in improving the firmness of the peel.

[0116] Table 3 Fruit firmness and hemicellulose content in tomato fruit after stable overexpression

[0117]

[0118] The application of the present invention is not limited to this. It can be expanded based on its application scope in environmental protection. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims.

Claims

1. A product for regulating the firmness of grape fruit, characterized in that: It comprises a gene overexpression vector having a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.

2.

2. A method for regulating grape fruit hardness, characterized in that: By regulating the expression of VvXTH25 and / or VvXTH31 genes, the firmness of grape berries is regulated; the nucleotide sequence of the VvXTH25 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the VvXTH31 gene is shown in SEQ ID NO.

4.

3. The method according to claim 2, wherein By regulating the expression of VvXTH25 and / or VvXTH31 genes, the synthesis of hemicellulose is regulated, thereby regulating the firmness of grape berries.

4. The method according to claim 3, wherein By constructing an overexpression vector of the VvXTH25 and / or VvXTH31 gene and introducing it into grape berries, the expression of the VvXTH25 and / or VvXTH31 gene is increased.

5. A method for promoting the synthesis of hemicellulose in grape fruit to increase its hardness, characterized in that: During the growth of grape berries, they are treated with a reagent containing BR.

6. Use of a gene overexpression vector for preparing a reagent for promoting hemicellulose synthesis in fruit and thereby increasing fruit firmness, characterized in that: The gene overexpression vector has the nucleotide sequence shown in Seq ID NO.1 and / or Seq ID NO.2, and the fruit includes grapes.

7. A use of a gene for preparing a reagent for regulating the content of hemicellulose in grape fruit, thereby regulating the firmness of grape fruit, characterized in that: The genes include VvXTH25 and / or VvXTH31 genes. The sequence of the VvXTH25 gene is shown in Seq ID NO.3, and the sequence of the VvXTH31 gene is shown in Seq ID NO.

4.

8. Use of BR for preparing a reagent for promoting the synthesis of hemicellulose in grape berries and thus increasing the firmness of grape berries.

9. The use according to claim 8, characterized in that The BR promotes cellulose synthesis by promoting the expression of grape VvXTH25 and / or VvXTH31 genes. The sequence of the VvXTH25 gene is shown in Seq ID NO.3, and the sequence of the VvXTH31 gene is shown in Seq ID NO.

4.

10. Use of BR for preparing an agent for promoting the expression of VvXTH25 and / or VvXTH31 genes in grape berries, characterized in that: The sequence of the VvXTH25 gene is shown in Seq ID NO.3, and the sequence of the VvXTH31 gene is shown in Seq ID NO.4.

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