Application of XTH gene in promoting grape fruit firmness formation by brassinosteroids

Abstract

The application provides application of an XTH gene in promotion of grape fruit hardness formation by brassinosteroids, finds that brassinosteroids (BR) can mediate synthesis of hemicellulose in grape fruits and thus significantly improve the hardness of the grape fruits by treating the grape fruits with the brassinosteroids at a veraison stage, finds two target genes VvXTH25 and VvXTH31 capable of regulating the hardness of the grape fruits through whole genome correlation analysis and transcriptome analysis, and determines the key genes and mechanism for regulating the hardness of the grape fruits, which regulates the hardness of the grape fruits by regulating the content of hemicellulose, has the action of providing the basis and means for regulating the texture of the grape fruits and improving the storage and transportation capacity, and provides a brand-new way for truly regulating the hardness of the grape fruits.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fruit cultivation, and particularly relates to application of XTH gene in promotion of grape fruit hardness formation by brassinosteroids. BACKGROUND

[0002] Grape (Vitis vinifera L.) is popular due to its sweet taste and rich nutrition. However, different grape varieties have different fruit hardness. Soft flesh grape has a limited shelf life at room temperature, and is easily damaged during postharvest transportation and storage, which seriously reduces its market value. In addition, soft flesh grape is limited in the sales market due to its poor storage and transportation, and is mainly used for domestic sales. The fruit flesh of hard flesh grape is dense and has less water, and the hard flesh grape fruit has a long brush, and the fruit flesh and the brush are closely combined, so that the fruit is not prone to falling and cracking. Therefore, increasing the hardness of grape fruit is conducive to promoting the expansion and upgrading of the grape industry. Therefore, if the hardness of grape fruit during growth and development can be independently regulated, the development of the grape industry will be greatly promoted.

[0003] At present, factors affecting grape fruit hardness are various, including internal factors and external factors. Among them, the structure of fruit cells and the special ion components contained therein affect the change of hardness, such as the content of cell wall structure, calcium ions and potassium ions. Exogenous hormones and external conditions have a certain influence on the hardness of grape fruit. There are many studies on improving postharvest fruit hardness by using hormones, including abscisic acid (ABA), auxin (IAA), gibberellin (GA), etc., which regulate fruit ripening by coordinating with various target genes in multiple biological processes. Brassinolide (BR) is a kind of hormone with sterol structure in plant body, and its core function is to promote cell elongation and division, improve photosynthesis, promote vessel differentiation, affect pollen development and fertility, and delay aging. As a plant growth regulator, brassinolide has the characteristics of high efficiency, non-toxicity and broad spectrum. The concentration is much lower than that of gibberellin, growth hormone, abscisic acid, cytokinin and ethylene, which are the five major plant growth regulators, and is only one thousandth of them, and is listed as the sixth major hormone of plants.

[0004] However, in the prior art, the influence of gibberellin on the fruit hardness of kiwi fruit, cherry, grape and other fruits has been mainly studied, and BR can improve the fruit setting rate and yield of grape, golden small jujube and apple. However, there is no research report on the influence of BR treatment on the maturity and hardness of grape fruit, and the specific regulation mechanism is not clear. SUMMARY

[0005] To solve the above problems, the application provides application of XTH gene in promotion of grape fruit hardness formation by brassinosteroids, finds that it can mediate synthesis of hemicellulose in grape fruit and thus significantly improve the hardness of grape fruit by using BR treatment at the grape color changing period, thus explores the mechanism, finds two target genes VvXTH25 and VvXTH31 capable of regulating grape fruit hardness through whole genome correlation analysis and transcriptomics analysis, and uses the overexpression vectors of the two target genes to treat grape fruits in the growth period, so that the hemicellulose content in grape fruits can be obviously improved and the fruit hardness can be improved, thereby providing a brand new way for realizing regulation of grape fruit hardness.

[0006] The application finds that BR can mediate synthesis of hemicellulose and thus improve the hardness of grape fruit, in order to study the mechanism, uses BR treatment on grape fruits at the color changing period, combines whole genome correlation analysis and transcriptomics, screens candidate genes for regulating grape fruit hardness by BR, and analyzes the functions of related genes through subcellular localization and stable overexpression of grape fruits, so as to provide a reference for elucidating the molecular mechanism of grape fruit hardness formation.

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

[0008] In order to prolong the preservation and storage period of grape fruits and improve the hardness of grape fruits, various means including spraying of exogenous hormones, temperature control, humidity control and the like are adopted in the existing market, wherein GA, cytokinin (CTK) and the like are mainly used in the exogenous hormone treatment. As a kind of sterol hormone capable of regulating growth and development, brassinosteroids (BR) has been proved to play a regulating function in promoting growth, division and differentiation of plant cells in the process of plant growth and development, and no research on regulation of fruit hardness has been conducted. Therefore, the application adopts BR spraying treatment on grape fruits, explores the relationship between BR and grape fruit hardness, and clarifies the regulation mechanism mediated by BR, thereby providing a new technical means for regulation of grape fruit hardness. Therefore, in some embodiments, different concentrations of BR are used to treat 'Yin Hong' grape, and the results show that the BR treatment has no obvious effect on the quality of grape fruits, but significantly improves the size, hardness and hemicellulose content of grape fruits, indicating that the method of spraying BR can significantly improve the hardness of grape fruits.

[0009] In some embodiments, the present application further targets the shape of grape fruit hemicellulose content, performs whole genome association analysis, and screens 552 candidate genes related to hemicellulose synthesis, finds genes related to cell wall polysaccharide metabolism through KEGG and GO enrichment analysis, and finds 8 cell wall metabolism related genes that may regulate grape fruit hemicellulose content. At the same time, the transcriptome analysis of the fruit treated by BR exogenous hormone is carried out, and 25 xyloglucan endotransglycosylase / hydrolases are screened out, so it can be speculated that it may play an important role in the depolymerization of grape fruit hemicellulose. The intersection of the results of whole genome association analysis and transcriptome analysis is found through FPKM value and expression amount analysis, and finally the key target genes VvXTH25 and VvXTH31 that may regulate grape pulp hardness and pericarp hardness are found.

[0010] Further, the overexpression vectors of VvXTH25 and VvXTH31 are constructed for treating grape fruits, and it is found that the VvXTH25 and VvXTH31 genes regulate the hardness of grape fruits significantly, and the hemicellulose content also proves similar conclusion, which proves that the hemicellulose content can be increased by increasing the expression of VvXTH25 and VvXTH31, so as to increase the hardness of grape fruits.

[0011] On the other hand, the present application provides a method for regulating the hardness of grape fruits by regulating the expression of VvXTH25 and / or VvXTH31 genes, so as to regulate the hardness of grape fruits; the nucleotide sequence of the VvXTH25 gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the VvXTH31 gene is shown as SEQ ID NO. 4.

[0012] In some embodiments, in order to explore the relationship between the change of the expression amount of VvXTH25 and VvXTH31 genes and the hardness of grape fruits, gene engineering technology means is adopted for operation. By constructing the overexpression vector containing VvXTH25 and VvXTH31 genes, the inflorescence dipping method is used to introduce them into grape fruit tissues, and then a series of physiological index detection is carried out on the treated fruits. The results show that the hemicellulose content of grape fruits overexpressing VvXTH25 and VvXTH31 genes is significantly increased, and the hardness and longitudinal diameter of grape fruits are significantly increased. This finding provides a key theoretical basis for the subsequent development of preparations and methods for improving the quality of grape fruits, and it is expected to improve the cell wall structure of grape fruits by artificially regulating the expression of the gene, to improve the hardness and storage and transportation capacity of the fruits, and to have a positive role in promoting the development of grape industry.

[0013] Further, by regulating the expression of VvXTH25 and / or VvXTH31 genes, the synthesis of hemicellulose is regulated, so as to regulate the hardness of grape fruits.

[0014] Further, by constructing a VvXTH25 and / or VvXTH31 gene overexpression vector, introducing it into grape fruits, and thereby improving the expression of the VvXTH25 and / or VvXTH31 gene.

[0015] In another aspect, the present application provides a method for promoting the synthesis of hemicellulose in grape fruits, thereby improving the hardness, wherein the grape fruits are treated with a reagent containing BR during the growth of the grape fruits.

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

[0017] In some modes, the fruits include grape.

[0018] In some modes, the VvXTH25 and VvXTH31 genes mainly regulate the hardness of grape fruits by regulating the synthesis of hemicellulose.

[0019] In another aspect, the present application provides a use of a gene for preparing a reagent for regulating the content of hemicellulose in grape fruits, thereby regulating the hardness of the grape fruits, wherein the gene includes a VvXTH25 and / or VvXTH31 gene, the sequence of the VvXTH25 gene is as shown in Seq ID NO. 3, and the sequence of the VvXTH31 gene is as shown in Seq ID NO. 4.

[0020] In another aspect, the present application provides a use of BR for preparing a reagent for promoting the synthesis of hemicellulose in grape fruits, thereby improving the hardness of the grape fruits.

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

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

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

[0024] 1. It is found that BR treatment of grape during growth and development can mediate the synthesis of hemicellulose in grape fruit, thereby significantly improving the hardness of grape fruit;

[0025] 2. Screening of candidate genes for BR regulation of grape fruit hardness, two target genes VvXTH25 and VvXTH31 are found, and it is found that BR can mediate the synthesis of hemicellulose in grape fruit by promoting the expression of VvXTH25 and VvXTH31, thereby improving the hardness of grape fruit;

[0026] 3. It is found that the construction of overexpression vector of target gene VvXTH25 or VvXTH31 can improve the hardness of grape fruit during growth and development, and the hardness of grape fruit is improved by mediating the synthesis of hemicellulose in grape fruit;

[0027] 4. The application innovatively finds that BR treatment can regulate grape fruit size and hardness, and the key genes for fruit hardness are screened, and it is verified that VvXTH25 and VvXTH31 positively regulate fruit hardness by mediating the synthesis of hemicellulose, which provides a theoretical basis for the reasonable regulation of grape fruit hardness and provides a scientific basis for improving the storage and transportation capacity of fresh grape by artificial means. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the mature grape fruit after different concentrations of BR spraying in example 1;

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

[0030] Figure 3 It is the titratable acid content of mature grape fruit after different concentrations of BR spraying in example 1;

[0031] Figure 4 It is the hardness of mature grape fruit after different concentrations of BR spraying in example 1;

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

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

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

[0035] Figure 8Figure 2 Manhattan plot and QQ-plot of genome-wide association study (GWAS) for hemicellulose content using Manhattan technique in Example 2, wherein A is Manhattan plot of mixed linear BLINK model, and B is QQ-plot;

[0036] Figure 9 Figure 3 Volcano plot of transcriptome data in Example 2;

[0037] Figure 10 Figure 4 GO enrichment analysis results of transcriptome data in Example 2;

[0038] Figure 11 Figure 5 KEGG analysis results of transcriptome data in Example 2;

[0039] Figure 12 Figure 6 25 xyloglucan endotransglycosylase / hydrolase genes screened from transcriptome in Example 2

[0040] Figure 13 Figure 7 Subcellular localization results in Example 3;

[0041] Figure 14 Figure 8 Tissue and spatiotemporal expression pattern analysis in Example 3;

[0042] Figure 15 Figure 9 Vector map of 35S::VvXTH25 overexpression vector in Example 4;

[0043] Figure 16 Figure 10 Vector map of 35S::VvXTH31 overexpression vector in Example 4;

[0044] Figure 17 Figure 11 Photographs of stable expression of VvXTH25 and VvXTH31 in grape fruits in Example 4;

[0045] Figure 18 Figure 12 Diagram of fruit transverse and longitudinal diameter and fruit firmness after stable expression of VvXTH25 and VvXTH31 in grape fruits in Example 4;

[0046] Figure 19 Figure 13 Diagram of qRT-PCR analysis of VvXTH25 and VvXTH31 expression levels after stable expression of VvXTH25 and VvXTH31 in grape fruits in Example 4;

[0047] Figure 20 Figure 14 Diagram of hemicellulose content after stable expression of VvXTH25 and VvXTH31 in grape fruits in Example 4;

[0048] Figure 21 Figure 15 Diagram of hemicellulose content after stable expression of VvXTH25 and VvXTH31 in grape fruits in Example 4;

[0049] Figure 22Grape fruit after injection of water (CK), empty vector (35S::FLAG), overexpression vector 35S::VvXTH31 and 35S::VvXTH31 in Example 4;

[0050] Figure 23 Grape fruit gene expression level in Example 4;

[0051] Figure 24 Grape fruit hardness in Example 4;

[0052] Figure 25 Grape fruit hemicellulose content in Example 4. DETAILED DESCRIPTION

[0053] The application will be further described in conjunction with the accompanying drawings and examples. It is to be noted that the following examples are intended to facilitate the understanding of the application, and do not limit the application in any way.

[0054] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0055] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0056] Example 1: Effect of BR treatment on grape fruit

[0057] In this example, ‘Yin Hong’ grape fruits were selected as test materials in a grape test base, and fruit surface spraying method was used for treatment at the turning color stage (about 70 days after flowering). Five concentration gradients of 0, 0.2, 0.4, 0.6, and 0.8 mg / L were set, with 3 biological repeats. A small sprayer was used to uniformly spray the fruit surface until dripping water. All treatment groups were grown under the same cultivation conditions, and the fruits were collected at the mature stage (about 100 days after flowering). The related properties of grape fruits in each group were recorded, and the quality indicators of fruit hardness, soluble solids content (TSS), titratable acid (TA), and hemicellulose content were detected, respectively.

[0058] 1. Related properties of grape fruits

[0059] In order to evaluate the effect of different concentrations of BR on the quality of ‘Yin Hong’ grape fruits, grape berry phenotype was determined, and the results are shown in Table 1 and Figure 1 .

[0060] Table 1. Related property record table of grape fruits treated by spraying different concentrations of BR

[0061]

[0062] From Figure 1As shown in Table 1, the exogenous hormone treatment of different concentrations has a significant effect on the quality regulation of grape fruit. The size and weight of the 'Yin Hong' grape fruit treated by BR are significantly increased.

[0063] 2. Soluble solids (TSS)

[0064] The portable refractometer was used to determine the soluble solids (TSS), and each treatment was set with 3 replicates, and the average value was taken. The detection results are shown in Figure 2 .

[0065] From Figure 2 it can be seen that the soluble solids content of each treatment group is higher than that of CK (blank control), but it is not significantly related to the concentration gradient. The content is the largest (17.90 birx) under the treatment of 0.6 mg / L, but the difference between each group is not large.

[0066] 3. Titration acid

[0067] The titration acid was determined by sodium hydroxide titration method, and each treatment was set with 3 replicates, and the average value was taken. The detection results are shown in Figure 3 .

[0068] According to Figure 3 , the titratable acid content of each group of grape fruit shows a downward trend, and the reduction degree of titratable acid is higher when 0.8 mg / L BR reaches the peak treatment, but it does not reach a significant level.

[0069] In addition, the sensory evaluation of each group of fruits was carried out in this embodiment, and it was found that there was no obvious difference in the sweet and sour taste of grape fruit after different concentration BR treatment. It can be seen that BR treatment does not affect the quality of grape fruit.

[0070] 4. Hardness

[0071] The flesh and peel hardness were determined by GY-4 hardness meter (Aidberg, Yueqing), and each treatment was set with 3 replicates, and the average value was taken. The detection results are shown in Figure 4 .

[0072] The hardness of grape fruit is mainly determined by the peel hardness and the flesh hardness. The BR treatment of different concentrations can significantly improve the hardness of the fruit. Among them, 0.2 mg / L has the most significant effect on the peel hardness, which is increased by 14.9% compared with CK (blank control), and the peel hardness is 6.8 N. The effect of 0.6 mg / L on the flesh hardness is the most significant, which is increased by 52.4% compared with CK (blank control).

[0073] 5. Hemicellulose content

[0074] Hemicellulose content was determined by the 3,5-dinitrosalicylic acid method. The specific detection method of hemicellulose content is as follows: the hemicellulose is hydrolyzed into reducing sugars under certain conditions, and the reducing sugars are oxidized into sugar acids and other products under alkaline conditions and heating. DNS is reduced to 3-amino-5-nitrosalicylic acid with a characteristic absorption peak at 540 nm. The content of hemicellulose can be quantitatively detected by the change of absorbance value. Each treatment was set in triplicate, and the results were averaged. The detection results are shown in Table 1. Figure 5 .

[0075] According to Figure 5 , different treatments have different effects on the hemicellulose content of the peel and pulp, and all reach a significant level. In the peel group, the change is most significant after 0.2 mg / L BR treatment. The hemicellulose content of grape pulp increases significantly compared with the control, and the hemicellulose content reaches the maximum after 0.6 mg / L BR treatment.

[0076] 6. Paraffin section

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

[0078] According to Figure 6 and Figure 7 , compared with CK (blank control), the cell wall is significantly thickened after BR treatment, the cell morphology is more compact, the intercellular space is reduced, and the cell morphology is 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 genome-wide association analysis was performed based on the BLINK model. The SNP sites with -log10(P)>5 were taken as significant sites. The Q-Q plot shows that the observed value in the lower left is consistent with the expected value, and the observed value in the upper right is upward, indicating that the BLINK model is suitable for this association study. Figure 8

[0082] In the 100 kb interval of SNP sites, 552 candidate genes related to hemicellulose synthesis were screened. Through KEGG and GO enrichment analysis, genes related to cell wall polysaccharide metabolism were found, and 8 cell wall metabolism-related genes that may regulate the hemicellulose content of grape fruits were found, 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, the candidate genes related to BR were identified by transcriptome analysis in this study. Figure 9 Based on the volcano plot of the transcriptome data, it was found that Figure 10 there were more up-regulated and down-regulated genes in the transcriptome. GO enrichment analysis Figure 11 showed that, compared with CK, DEGs were enriched in molecular function, cell component and biological process after BR treatment. KEGG enrichment analysis Figure 12 found that the differential genes (DEGs) were enriched in the biosynthesis of secondary metabolites, plant hormone signal transduction, starch and sucrose metabolism, galactose metabolism and other cell wall metabolism processes. Among them, 25 xyloglucan endotransglycosylase / hydrolases were screened out. It was speculated that they might play an important role in the depolymerization of hemicellulose in grape berry.

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

[0088] The intersection of the results of genome-wide association analysis and transcriptome analysis was found by FPKM value and expression analysis, and finally the target genes VIT_11s0052g01180 (VvXTH25) (Seq ID NO. 3) and VIT_11s0052g01300 (VvXTH31) (Seq ID NO. 4) that may regulate the firmness of grape pulp and peel were found, as shown in Figure 13 .

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

[0090] 1. Subcellular localization

[0091] The subcellular localization of VvXTH25 and VvXTH31 proteins was predicted by Cell PLOC software, and the prediction results showed that the proteins were located in the nucleus of grape berry cells. To verify this prediction result, the position of gene expression was determined by green fluorescent protein GFP tag signal Figure 14 , which was consistent with the prediction.

[0092] 2. Analysis of tissue, spatiotemporal expression patterns

[0093] The expression of VvXTH25 and VvXTH31 in different developmental stages and different tissues of grape was verified, respectively, and the results are as follows Figure 15The left panel shows the expression of VvXTH25 and VvXTH31 in different tissues, and the right panel shows the expression of VvXTH25 and VvXTH31 in different periods of fruit development. The results show that VvXTH25 and VvXTH31 are specifically expressed in different periods and different tissues.

[0094] Example 4: Verification of key functional genes

[0095] 1. Construction of overexpression vector

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

[0097] The 35S::VvXTH25 overexpression vector was modified from the pBWA(V)HS vector, and 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 method, and the primers were as follows:

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

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

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

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

[0102] VvXTH31-F: AACACGGGGGACTTTGCAACatgtttgggaagattgatatgcagctgaagc (Seq ID NO. 7)

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

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

[0105] 2、Stable overexpression of VvXTH25 and VvXTH31 on grape berry

[0106] At 5 days after flowering, Agrobacterium tumefaciens containing 35S::VvXTH25 overexpression vector and 35S::VvXTH31 overexpression vector were soaked into inflorescences of ‘Yin Hong’ grape to perform stable expression of grape berry. 35S::VvXTH25 overexpression grape and 35S::VvXTH31 overexpression grape were constructed, and compared with empty vector group (35S::FLAG) and blank control group (CK). At the mature stage, fruits were collected (Fig. 1) to detect gene expression, hemicellulose content, size and hardness of grape berry in each group. Figure 18 qRT-PCR was used to analyze the expression level of each group (Fig. 2). The results showed that the relative expression level of VvXTH31 in the 35S::VvXTH31 group was the highest in the grape berry peel, which was 4.4 times that of the control group, and the relative expression level of VvXTH25 in the 35S::VvXTH25 group was 3.2 times that of the control group. The relative expression level of VvXTH31 in the 35S::VvXTH31 group was the highest in the grape berry pulp, which was 4.1 times that of the control group, and the relative expression level of VvXTH25 in the 35S::VvXTH25 group was 3.3 times that of the control group. There was no significant difference in the relative expression level between the empty vector group and the control group.

[0107] Figure 19 The hemicellulose content of the pulp and peel of the overexpression group of grape berry was measured (Fig. 3). The results showed that overexpression of VvXTH25 and VvXTH31 could increase the hemicellulose content, which was significantly higher than that of the control group and the empty vector group. The hemicellulose content of the peel and pulp of the 35S::VvXTH31 group was significantly higher than that of the control group and the empty vector group, which was 104 mg / g and 13.8 mg / g, respectively. The hemicellulose content of the peel and pulp of the 35S::VvXTH25 group was also significantly higher than that of the control group and the empty vector group, which was 101 mg / g and 9.8 mg / g, respectively. It was speculated that VvXTH25 and VvXTH31 mainly regulated fruit hardness by regulating hemicellulose content.

[0108] The fruit size and fruit hardness of VvXTH25 and VvXTH31 in each group of grape were measured (Fig. 4). Compared with the empty vector group (35S::FLAG) and the control group (CK), the hardness of grape fruit and the longitudinal diameter of grape fruit showed significant differences after overexpression of VvXTH25 and VvXTH31 (Fig. 5). Figure 20

[0109] Figure 21 ​​​). The flesh firmness (5.0 N) and peel firmness (7.2 N) of the 35S::VvXTH31 group were significantly higher than those of the control group and the empty vector group, and the flesh firmness (5.2 N) and peel firmness (5.9 N) of the 35S::VvXTH25 group were also higher than those of the control group and the empty vector group. Among them, the peel firmness of the 35S::VvXTH31 group increased most significantly. In addition, compared with the empty vector group (35S::FLAG) and the control group (CK), the longitudinal diameter of the grape increased significantly (1.1 times and 1.3 times, respectively) after overexpression of VvXTH25 and VvXTH31, and the transverse diameter did not change significantly. Therefore, it is speculated that VvXTH25 and VvXTH31 may mainly affect the fruit firmness by regulating the peel firmness and affect the grape fruit size by regulating the longitudinal diameter, and the effect of the VvXTH31 overexpression vector on increasing the peel firmness and fruit size is more obvious than that of the VvXTH25 overexpression vector.

[0110] 3. Effect of transient overexpression on grape fruit

[0111] Before the fruit turning stage, Agrobacterium tumefaciens liquid containing 35S::VvXTH25 and 35S::VvXTH31 overexpression vectors (1 mL each) was injected into ‘Yin Hong’ grape fruits for transient overexpression treatment, and 35S::VvXTH25 and 35S::VXTH31 transient overexpression groups were constructed, with the empty vector (35S::FLAG) and wild type (CK) grape as controls. Figure 22 ). Samples were collected after 7 days, and the expression levels of related genes in the grape flesh and peel, fruit firmness, and hemicellulose content were detected.

[0112] The qRT-PCR analysis of the VvXTH25 and VvXTH31 expression levels of each group of grapes was performed, and the results are shown in Figure 23 . The relative expression level of VvXTH25 in the peel of the 35S::VvXTH25 group of grapes was the highest, which was 5 times that of the control group, and the relative expression amount in the flesh was 4.6 times that of the control. The relative expression level of VvXTH31 in the flesh of the 35S::VvXTH31 group of grapes was the highest, which was 5 times that of the control, and the relative expression amount in the peel was 3.5 times that of the control. There was no significant difference in the relative expression level in the peel between the empty vector group and the control group.

[0113] The hemicellulose content and firmness of the grape fruit flesh and peel were measured by sampling near the pinhole, and the results are shown in Figure 24 and 25As shown. It can be seen that overexpression of VvXTH25 and VvXTH31 genes can effectively improve the fruit hardness and hemicellulose content of grape in the growth and development stage. In the 35S::VvXTH31 overexpression group, the grape skin hardness (20.4 N) and pulp hardness (14.7 N), and the skin hemicellulose content (91 mg / g) and pulp hemicellulose content (15.6 mg / g) are significantly higher than those of the control group and the empty group, and the changes of fruit hardness and hemicellulose content are particularly significant. Similarly, the skin hardness (18.6 N), pulp hardness (15.2 N), skin hemicellulose content (80 mg / g) and pulp hemicellulose content (12 mg / g) of the 35S::VvXTH25 overexpression group are also significantly higher than those of the control group and the empty group. The above results show that VvXTH25 and VvXTH31 may mainly affect the overall hardness of the fruit by regulating the skin hardness, and the effect of VvXTH31 overexpression vector in improving the skin hardness is better than that of VvXTH25.

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

[0115] This example also uses tomato as a model species, and Agrobacterium tumefaciens liquid containing 35S::VvXTH25 and 35S::VvXTH31 overexpression vectors is used to infect tomato cotyledons to further verify the regulation of VvXTH25 and VvXTH31 genes on fruit hardness. When the cotyledons develop into complete plants, positive verification is carried out by DNA, and the mature stage tomato fruits are measured, and the results are shown in Table 1. Overexpression of VvXTH25 and VvXTH31 genes can indeed effectively improve the hardness of tomato fruits during growth and development. The pulp and skin hardness and hemicellulose content of 35S::VvXTH25 and 35S::VvXTH31 groups are significantly greater than those of the control group and the empty group, and the change of skin hardness is more obvious; the pulp and skin hardness and hemicellulose content of 35S::VvXTH31 group change more than those of 35S::VvXTH25 group. The above results further prove that VvXTH25 and VvXTH31 may mainly affect the overall hardness of the fruit by regulating the skin hardness, and the effect of VvXTH31 overexpression vector in improving the skin hardness is better than that of VvXTH25.

[0116] Table 3 Fruit hardness and hemicellulose content of tomato fruit after stable overexpression

[0117]

[0118] The application of the present application is not limited to the above. It can be extended according to 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 application. Therefore, the protection scope of the present application should be defined by the scope of the claims.

Claims

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

2.

2. A method for modulating the firmness of grape berries, characterized in that, by modulating VvXTH25 and / or VvXTH31 expression of a gene, thereby modulating grape berry firmness; said VvXTH25 nucleotide sequence of said gene is set forth in SEQ ID NO. 3, said VvXTH31 nucleotide sequence of said gene is set forth in SEQ ID NO.

4.

3. The method of claim 2, wherein, By modulating VvXTH25 and / or VvXTH31 expression of genes that regulate hemicellulose synthesis, thereby modulating berry firmness.

4. The method of claim 3, wherein, By constructing VvXTH25 and / or VvXTH31 overexpression vectors of the genes, introducing them into grape fruits, and thereby increasing VvXTH25 and / or VvXTH31 expression of the genes.

5. Use of a gene overexpression vector for the preparation of an agent for promoting the synthesis of hemicellulose in a fruit, thereby increasing the firmness of the fruit, characterized in that, The gene overexpression vector comprises a nucleotide sequence as shown in Seq ID NO. 1 and / or Seq ID NO. 2, and the fruit comprises grape.

6. Use of a gene for the preparation of an agent modulating the content of hemicellulose in the grape berry, and thus modulating the firmness of the grape berry, characterized in that, The genes include VvXTH25 and / or VvXTH31 genes, the VvXTH25 The sequence of the gene is shown as Seq ID NO. 3, the VvXTH31 The sequence of the gene is shown as Seq ID NO. 4.

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