Strawberry genes for regulating sugar content in strawberry fruits and uses thereof

Abstract

This invention relates to the field of molecular biology, and proposes a strawberry gene that regulates the sugar content of strawberry fruit and its application. The strawberry gene includes... FaSUS4g , FaSPS4b and FaSUT2d Any one of them; FaSUS4g The nucleotide sequence is shown in SEQ ID NO.1; FaSPS4b The nucleotide sequence is shown in SEQ ID NO.2; FaSUT2d The nucleotide sequence is shown in SEQ ID NO.3. The above technical solution solves the problem in related technologies that cannot provide precise gene targets and regulatory strategies for molecular breeding of strawberry fruit quality.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to strawberry genes that regulate the sugar content of strawberry fruits and their applications. Background Technology

[0002] Strawberries are perennial herbaceous fruit trees, prized for their vibrant color, rich aroma, and high nutritional value, making them a favorite among growers and consumers. The firmness, sugar-acid ratio, color, and aroma of strawberry fruits undergo significant changes during ripening. Sugar is the main carbon source in plants and a primary product of photosynthesis, providing energy for plant growth and development and playing a crucial role in the process.

[0003] The sugars accumulated in strawberries mainly include sucrose, glucose, and fructose. Sugar content is one of the important indicators for judging the internal quality of strawberries. Sugar content determines the sweetness of the fruit. Increasing sugar content can not only improve the sweetness of strawberries, but also help solve various metabolic processes in the fruit, such as the synthesis of key flavor compounds, pigments, amino acids, vitamins, and aromatic substances.

[0004] However, the understanding of regulatory genes and molecular mechanisms for sugar accumulation in strawberry fruits is still insufficient, making it difficult to achieve targeted and efficient regulation of sucrose, glucose, and fructose metabolic pathways, and thus unable to provide precise gene targets and regulatory strategies for molecular breeding of strawberry fruit quality. Summary of the Invention

[0005] This invention proposes a strawberry gene that regulates the sugar content of strawberry fruit and its application, in order to solve or alleviate at least one of the above-mentioned problems.

[0006] The technical solution of the present invention is as follows: This invention proposes a strawberry gene that regulates the sugar content of strawberry fruits, the strawberry gene comprising: FaSUS4g , FaSPS4b and FaSUT2d Any one of the genes; The FaSUS4g The nucleotide sequence is shown in SEQ ID NO.1; The FaSPS4b The nucleotide sequence is shown in SEQ ID NO.2; The FaSUT2d The nucleotide sequence is shown in SEQ ID NO.3.

[0007] The present invention also proposes an expression cassette containing the strawberry gene described above; When the strawberry gene is FaSUS4g At that time, the expression box is an interference. FaSUS4g The expression box; When the strawberry gene isFaSPS4b At that time, the expression box is overexpressed. FaSPS4b The expression box; When the strawberry gene is FaSUT2d At that time, the expression box is overexpressed. FaSUT2d The expression box.

[0008] The present invention also proposes a recombinant expression vector containing the strawberry gene or the expression cassette described above; When the strawberry gene is FaSUS4g At that time, the recombinant expression vector is FaSUS4g A silent carrier; When the strawberry gene is FaSPS4b At that time, the recombinant expression vector is FaSPS4b Overexpression vectors; When the strawberry gene is FaSUT2d At that time, the recombinant expression vector is FaSUT2d Overexpression vectors.

[0009] The present invention also proposes a recombinant microorganism containing the strawberry gene, the expression cassette, or the recombinant expression vector.

[0010] This invention also proposes the application of the strawberry gene in increasing the sugar content of strawberry fruits.

[0011] Preferably, when the strawberry gene is FaSUS4g At that time, reduce the FaSUS4g The expression level of the strawberry gene; when the strawberry gene is FaSPS4b At that time, improve the FaSPS4b The expression level of the strawberry gene; when the strawberry gene is FaSUT2d At that time, improve the FaSUT2d The amount of expression in.

[0012] The present invention also proposes a method for cultivating transgenic strawberries with high sugar content using the strawberry gene, wherein a recombinant expression vector of the strawberry gene is constructed, the recombinant expression vector is instantaneously transferred into strawberry fruits at the white ripe stage, the sugar content in the fruits is measured, and the gene function is verified. When the strawberry gene is FaSUS4g At that time, instantaneous transfer of the recombinant expression vector into white-ripe strawberry fruit can reduce the... FaSUS4g The amount of expression; When the strawberry gene is FaSPS4b At that time, instantaneous transfer of the recombinant expression vector into white-ripe strawberry fruits can improve the... FaSPS4b The amount of expression; When the strawberry gene is FaSUT2d At that time, instantaneous transfer of the recombinant expression vector into white-ripe strawberry fruits can improve the... FaSUT2d The amount of expression.

[0013] This invention also proposes the application of the strawberry gene in strawberry breeding, wherein the strawberry breeding is for selecting strawberries with high sugar content. When the strawberry gene is FaSUS4g At that time, the selection and breeding of the aforementioned FaSUS4g Strawberries with reduced expression levels; When the strawberry gene is FaSPS4b At that time, the selection and breeding of the aforementioned FaSPS4b Strawberries with increased expression levels; When the strawberry gene is FaSUT2d At that time, the selection and breeding of the aforementioned FaSUT2d Strawberries with increased expression levels.

[0014] The beneficial effects of this invention are as follows: This invention, through the measurement of physiological indicators, confirmed that 'Jimixiang' is a high-sugar strain, exhibiting high potential for widespread application. Therefore, further experiments were conducted using 'Jimixiang-JMX' (numbered 773-27) and its sister line 'CK' (numbered 773-4) as materials. Based on transcriptome sequencing and transient functional validation, it was found that... FaSUS4g , FaSPS4b and FaSUT2d To regulate key genes in strawberry fruit sugar accumulation and reduce FaSUS4g It will increase the sugar content of the fruit, while overexpression FaSPS4b , FaSUT2d These three factors can increase the sugar content of fruits and play a key role in sugar accumulation. FaSUS4g , FaSPS4b and FaSUT2d It can serve as a candidate target gene for improving strawberry fruit quality through molecular-assisted breeding in the future. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The graph shows the soluble solids content of 'JMX' and 'CK' fruits from different years and at different developmental stages in Example 1 of this invention. Figure: Scale bar: 1.0 cm. A, Morphological development of strawberry fruit; BD, Soluble solids content of upper, middle, and lower parts of 'JMX' and 'CK' during DG, IR, and FR stages in 2024; EG, Soluble solids content of upper, middle, and lower parts of 'JMX' and 'CK' during DG, IR, and FR stages in 2025; In BG, data are expressed as mean ± SD, **. P <0.01, t For the test, the small black dots in the bar chart represent the sample size, n=21-24.

[0017] Figure 2 This is a graph showing the total sugar, glucose, sucrose, and fructose content in the fruits of 'JMX' and 'CK' from different years and at different developmental stages in Example 2 of the present invention; In the figures: AD, total sugar, glucose, sucrose, and fructose content of 'JMX' and 'CK' during DG, IR, and FR periods in 2024; EH, total sugar, glucose, sucrose, and fructose content of 'JMX' and 'CK' during DG, IR, and FR periods in 2025; in AH, data are expressed as mean ± SD, **. P <0.01, t The test result is n=3.

[0018] Figure 3 This is a gene information analysis diagram of the SUS family in Arabidopsis thaliana, wild strawberry, and cultivated strawberry in Example 3 of the present invention; In the figure: A, constructing the phylogenetic tree of the SUS family in Arabidopsis thaliana, forest strawberry, and cultivated strawberry; branch length represents the number of replacements at each site; B, 6 FveSUSs Expression patterns in various tissues of *Strombocypripedium praecox*; TPM values ​​(per million transcripts) are shown in each box, with color indicating the Z-score obtained from the TPM values; the scale bar represents the TPM values ​​after log2 transformation. Arrows indicate two genes with high expression abundance.

[0019] Figure 4 This is a gene information analysis diagram of the SPS family in Arabidopsis thaliana, wild strawberry, and cultivated strawberry in Example 3 of the present invention; In the figure: A, constructing the phylogenetic tree of the SPS family in Arabidopsis thaliana, forest strawberry, and cultivated strawberry; branch length represents the number of replacements at each site. B, 10 sites. FveSPSs Expression patterns in various tissues of *Strombocypripedium praecox*; TPM values ​​(per million transcripts) are shown in each box, with color indicating the Z-score obtained from the TPM values; the scale bar represents the TPM values ​​after log2 transformation. Arrows indicate genes with high expression abundance.

[0020] Figure 5This is a gene information analysis diagram of the SUT family in Arabidopsis thaliana, wild strawberry, and cultivated strawberry in Example 3 of the present invention; In the figure: A, constructing the phylogenetic tree of the SUT family in Arabidopsis thaliana, forest strawberry, and cultivated strawberry; branch length represents the number of replacements at each site; B, 8 FveSUTs Expression patterns in various tissues of *Strombocypripedium praecox*; TPM values ​​(per million transcripts) are shown in each box, with color indicating the Z-score obtained from the TPM values; the scale bar represents the TPM values ​​after log2 transformation. Arrows indicate two genes with high expression abundance.

[0021] Figure 6 This is a transcriptome analysis diagram of the 'JMX' and 'CK' fruits during their developmental stages in Example 4 of this invention; Figure: A, PCA analysis of transcriptome data from 'JMX' and 'CK' fruits during the IR and FR periods (TPM values ​​of all expressed genes were transformed using log2), with sequencing performed on 'JMX' and 'CK' fruits during the IR and FR periods, including three biological replicates; B, a bar chart showing the number of differentially expressed genes (DEGs) in 'JMX' fruits during the IR and FR periods compared to 'CK' (fold change > 2). P <0.05; CD, KEGG analysis of upregulated and downregulated genes in IR period of 'JMX' compared with 'CK' ( P <0.05); X-axis represents enrichment factor; EF, KEGG analysis of upregulated and downregulated genes in 'JMX' during the FR period compared to 'CK' ( P <0.05); the X-axis represents the enrichment factor.

[0022] Figure 7 In Embodiment 5 of the present invention SUS , SPS and SUT Schematic diagram of the expression pattern of family genes in 'CK' and 'JMX' fruits; In the figure: AB, Venn diagrams show the genes co-upregulated and co-downregulated in 'JMX' during the IR and FR periods compared to 'CK'; CD, heatmaps show the genes co-upregulated and co-downregulated in 'JMX' compared to 'CK'. SUS , SPS and SUT Differential expression of family genes in 'JMX' fruit; TPM values ​​(per million transcripts) are shown in each box, with color indicating the Z-score obtained based on the TPM value; the scale bar represents the TPM value after log2 transformation; EG, detected by qRT-PCR. SUS , SPS and SUTExpression levels of differentially expressed genes in the family in 'CK'-IR, 'JMX'-IR, 'CK'-FR, and 'JMX'-FR, data are mean ± SD of three biological replicates. P Significant differences at the <0.05 level are indicated by different letters determined by the Tukey test.

[0023] Figure 8 In Embodiment 6 of the present invention FaSUS4g , FaSPS4b and FaSUT2d Functional verification result diagram; In the figure: A, transient overexpression in 'JMX' or 'CK' fruits, respectively. FaSUS4g , FaSPS4b and FaSUT2d Phenotypic results at 3 and 7 days post-surgery; BD, detected by RT-PCR. FaSUS4g , FaSPS4b and FaSUT2d Expression levels in transiently transformed fruits; E, transient overexpression FaSUS4g , FaSPS4b and FaSUT2d Soluble solids content in the post-fruit. FH, transient overexpression. FaSUS4g , FaSPS4b and FaSUT2d Subsequently, the glucose, sucrose, and fructose contents in the fruit were determined; the data for BD and FH are the mean ± SD of three biological replicates. P <0.01, t Test; the data in E are the mean ± SD. P Significant differences at the <0.05 level are represented by different letters determined by the Tukey test, n=10. Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0025] This invention confirmed that 'Jimixiang' is a high-sugar strain by measuring physiological indicators, demonstrating high potential for widespread application. Therefore, further experiments were conducted using 'Jimixiang-JMX' (numbered 773-27) and its sister strain 'CK' (numbered 773-4). Experimental results showed that at different developmental stages, the soluble solids, glucose, sucrose, and fructose content of 'Jimixiang' fruit were significantly higher than those of its sister strain 'CK', ​​indicating that 'Jimixiang' is a high-sugar strain. Phylogenetic analysis identified members of the SUS, SPS, and SUT families and elucidated the tissue expression patterns of their corresponding homologous genes in forest strawberries. Transcriptome sequencing revealed a large number of differentially expressed genes between the 'JMX' and 'CK' strains, enriched in sugar metabolism pathways, with 'JMX' containing 3... FaSUSs Expression levels were significantly lower in the IR and FR phases than in the 'CK' phase. FaSPSs and 8 FaSUTs The levels during the IR and FR periods were significantly higher than those of 'CK'; as the fruits of 'CK' and 'JMX' matured, the three... FaSUSs All five showed down-regulation. FaSPSs and 8 FaSUTs All upregulated expression; FaSUS4g yes FaSUSs The gene with the lowest expression level in the family during the 'JMX'-FR period. FaSPS4b and FaSUT2d They are FaSPSs and FaSUTs The gene with the highest expression level in the family during the 'JMX'-FR stage. Transient transformation validation showed that overexpression... FaSUS4g It will reduce the sugar content of the fruit, while overexpression FaSPS4b , FaSUT2d These three substances can increase sugar content and play a key role in sugar accumulation.

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will be described through embodiments.

[0027] A specific embodiment of the first aspect of the present invention provides a strawberry gene that regulates the sugar content of strawberry fruit. The strawberry gene includes... FaSUS4g , FaSPS4b and FaSUT2d Any one of them; FaSUS4g The nucleotide sequence is shown in SEQ ID NO.1; FaSPS4b The nucleotide sequence is shown in SEQ ID NO.2; FaSUT2d The nucleotide sequence is shown in SEQ ID NO.3.

[0028] A specific embodiment of the second aspect of the present invention provides a strawberry gene that regulates the sugar content of strawberry fruit, containing the strawberry gene provided in the specific embodiment of the first aspect; When the strawberry gene is FaSUS4g At that time, the expression box is a distraction. FaSUS4g The expression box; When the strawberry gene is FaSPS4b At that time, the expression box is overexpressed. FaSPS4b The expression box; When the strawberry gene is FaSUT2d At that time, the expression box is overexpressed. FaSUT2d The expression box.

[0029] A specific embodiment of the third aspect of the present invention provides a recombinant expression vector containing the strawberry gene provided in the specific embodiment of the first aspect or the expression cassette provided in the specific embodiment of the second aspect; When the strawberry gene is FaSUS4g At that time, the recombinant expression vector was FaSUS4g A silent carrier; When the strawberry gene is FaSPS4b At that time, the recombinant expression vector was FaSPS4b Overexpression vectors; When the strawberry gene is FaSUT2d At that time, the recombinant expression vector was FaSUT2d Overexpression vectors.

[0030] In one embodiment of the present invention, when the strawberry gene is FaSUS4g or FaSPS4b or FaSUT2d When any one of the following is true, a method for constructing a recombinant expression vector includes the following steps: using strawberry fruit cDNA as a template, amplifying and purifying the target gene with KOD enzyme; ligating the target fragment with a linearized vector to form a recombinant expression vector.

[0031] In this invention, the linearization carrier is utilized NdeI , EcoRI The 35SGFP-PRI101 vector was linearized by double enzyme digestion.

[0032] A specific embodiment of the fourth aspect of the present invention provides a recombinant microorganism containing the strawberry gene provided in the specific embodiment of the first aspect, or the expression cassette provided in the specific embodiment of the second aspect, or the recombinant expression vector provided in the specific embodiment of the third aspect.

[0033] In one embodiment of the present invention, when the strawberry gene is FaSUS4g or FaSPS4b or FaSUT2d At that time, a recombinant microorganism was obtained by transferring a recombinant expression vector into Agrobacterium GV3101 competent cells.

[0034] A specific embodiment of the fifth aspect of the present invention provides an application of using strawberry genes to increase the sugar content of strawberry fruits.

[0035] In one embodiment of the present invention, when the strawberry gene is FaSUS4g At that time, reduce FaSUS4g The expression level of the strawberry gene; when the strawberry gene is FaSPS4b At that time, improve FaSPS4b The expression level of the strawberry gene; when the strawberry gene is FaSUT2d At that time, improve FaSUT2d The amount of expression.

[0036] A specific embodiment of the sixth aspect of the present invention provides a method for increasing the sugar content of strawberry fruit by regulating the expression level of strawberry genes, specifically: When the strawberry gene is FaSUS4g At that time, reduce FaSUS4g The amount of expression; When the strawberry gene is FaSPS4b At that time, improve FaSPS4b The amount of expression; When the strawberry gene is FaSUT2d At that time, improve FaSUT2d The amount of expression.

[0037] A specific embodiment of the seventh aspect of the present invention provides a method for cultivating transgenic strawberries with high sugar content using strawberry genes, constructing a recombinant expression vector of strawberry genes, instantaneously transferring the recombinant expression vector into strawberry fruits at the white ripe stage, measuring the sugar content in the fruits, and verifying its function; When the strawberry gene is FaSUS4g At that time, transiently transferring the recombinant expression vector into strawberry fruits at the white ripe stage can reduce FaSUS4g The amount of expression; When the strawberry gene is FaSPS4b Transiently transferring the recombinant expression vector into strawberry fruits at the white-ripe stage can improve... FaSPS4b The amount of expression; When the strawberry gene is FaSUT2d Transiently transferring the recombinant expression vector into strawberry fruits at the white-ripe stage can improve... FaSUT2d The amount of expression.

[0038] The specific implementation of the eighth aspect of the present invention provides the application of strawberry genes in strawberry breeding to select strawberries with high sugar content; When the strawberry gene is FaSUS4g At that time, selection and breeding FaSUS4g Strawberries with reduced expression levels; When the strawberry gene is FaSPS4b At that time, selection and breeding FaSPS4b Strawberries with increased expression levels; When the strawberry gene isFaSUT2d At that time, selection and breeding FaSUT2d Strawberries with increased expression levels.

[0039] The present invention will now be described in detail with reference to preferred embodiments and comparative examples. The preferred embodiments of the invention described below can be modified in various ways, and therefore the scope of the invention should not be construed as limited to the preferred embodiments described in detail below. Preferred embodiments are provided to help those skilled in the art to more readily understand the invention.

[0040] Example 1 Sample Selection As strawberries ripen, their sugar content, acidity, firmness, color, and aroma undergo significant changes. Based on the morphological development of strawberry fruits, it can be divided into seven developmental stages: Small Green Stage (SG), Large Green Stage (BG), Degreening Stage (DG), White Ripe Stage (Wt), Early Red Stage (IR), Partial Red Stage (PR), and Full Red Stage (FR), which occur approximately 7, 14, 18, 21, 23, 25, and 28 days after pollination, respectively. Figure 1 A). Preliminary investigation and tasting revealed that the strawberry fruit numbered '773-27' had a higher sweetness, significantly superior to its sister line '773-4'. Therefore, strawberry lines numbered 773-27 (hereinafter referred to as 'JMX') and 773-4 (hereinafter referred to as 'CK') were selected for self-breeding at the Shijiazhuang Fruit Tree Research Institute of the Hebei Academy of Agricultural and Forestry Sciences (strawberry lines numbered 773-27 and 773-4 were obtained by crossbreeding 'Xiangye' as the female parent and '696-3' as the male parent; '696-3' was obtained by crossbreeding 'Dongxing' as the female parent and 'Hongyan' as the male parent). The materials from both strawberry lines were planted in a solar greenhouse and managed under conventional field conditions. In November and December of 2024 and 2025, the temperature in the solar greenhouse of the Shijiazhuang Fruit Tree Research Institute of the Hebei Academy of Agricultural and Forestry Sciences was 8-30 ℃, and the light duration was 8-9 h. Collect strawberry fruits at the degreening stage (DG), the initial red stage (IR), and the full red stage (FR). Cut them evenly into three parts: the upper (fruit tip), the middle, and the lower. Squeeze the juice out by hand and use a pipette to add 300 μL to a saccharimeter to determine the soluble solids content. The results showed that the soluble solids content of 'JMX' was significantly higher than that of 'CK' in both 2024 and 2025. Specifically, tests in 2024 showed that the soluble solids content of the upper, middle, and lower parts of 'JMX' fruit at the DG stage was 9.12%, 9.63%, and 10.97% higher than that of 'CK', ​​respectively. Figure 1 B); the soluble solids content during the IR period was 34.37%, 28.26%, and 25.10% higher than that of 'CK'. Figure 1C); the soluble solids content during the FR period was 44.04%, 41.09%, and 38.68% higher than that of 'CK'. Figure 1 D). Tests conducted in 2025 showed that the soluble solids content of the upper, middle, and lower parts of 'JMX' fruit during the DG period was 10.24%, 9.50%, and 11.65% higher, respectively, compared to 'CK'. Figure 1 E); the soluble solids content during the IR period was 38.85%, 29.35%, and 25.23% higher than that of 'CK'. Figure 1 F); the soluble solids content during the FR period was 43.82%, 42.01%, and 37.99% higher than that of 'CK'. Figure 1 G). The above data indicate that 'JMX' fruit has a high sweetness and this phenotype is stable, making it an ideal material for elucidating the molecular mechanism of sugar metabolism in cultivated strawberries.

[0041] Example 2: Determination and Analysis of Sugar Content Fruits of 'JMX' and 'CK' at DG, IR, and FR stages were collected, and the contents of total sugar, glucose, sucrose, and fructose were determined. The extraction and determination of sucrose, glucose, and fructose in strawberry fruit were performed according to previously reported methods (Peng Q, Cai Y, Lai E, et al., 2020. The sucrose transporter MdSUT4.1 participates in the regulation of fruit sugar accumulation in apple. BMC Plant Biology 20, 191. Xiao K, Fan J, Bi X, et al., 2024. A NAC transcription factor and a MADS-box protein antagonistically regulate sucrose accumulation in strawberry receptors. Plant Physiology 195, 2228–46.), and were detected by high performance liquid chromatography (HPLC). The determination procedure was as follows: 30 g of strawberry fruit sample ground with liquid nitrogen was weighed, an appropriate amount of ddH2O was added, and the mixture was incubated in a water bath at 100 ℃ for 30 min, followed by extraction using an ultrasonic-assisted method for 15 min. After the solution temperature cooled to room temperature, the volume was adjusted to 200 mL. Insoluble particles were removed using a 0.22 μm inorganic phase filter membrane and analyzed using an LC3000 semi-preparative high-performance liquid chromatography system. The mobile phase consisted of acetonitrile and water in a ratio of 75:25 (volume ratio; ammonia was used to adjust the pH), and the flow rate was fixed at 0.6 mL / min.

[0042] A standard curve was generated to quantitatively determine the content of the three sugars (sucrose, glucose, and fructose). The specific steps were as follows: First, the sugars were diluted with ultrapure water to 100 ng / mL and mixed at equimolar concentrations to obtain a stock solution. This solution was then sequentially diluted to obtain solutions with corresponding concentration gradients. Next, the diluted solutions were analyzed by high-performance liquid chromatography (HPLC) to determine the sugar content. Finally, a standard curve was generated using the sugar content as the x-axis and the peak area as the y-axis. Furthermore, the concentrations of the three sugars (sucrose, glucose, and fructose) in each sample were determined using the same method. Each analysis included multiple replicates, and the sugar content is expressed as mean ± standard deviation.

[0043] The results showed that the total sugar content of 'CK' and 'JMX' gradually increased with fruit ripening, and the total sugar content of 'JMX' was significantly higher than that of 'CK' at the DG, IR, and FR stages. Figure 2 (A and 2E) The contents of glucose, sucrose, and fructose in 'CK' and 'JMX' increase significantly with fruit ripening. Glucose is the most abundant sugar during the DG stage, while sucrose is the most abundant sugar during the IR and FR stages. Figure 2 BD and 2F-H). During the DG period, 'JMX' contained 3.39, 2.82, and 2.63 times more glucose, sucrose, and fructose than 'CK', ​​respectively. Figure 2 During the IR period, the glucose, sucrose, and fructose content in 'JMX' was 3.51, 4.68, and 3.47 times that of 'CK', ​​respectively. Figure 2 In C and 2G, the glucose, sucrose, and fructose content in 'JMX' during the FR period was 3.11, 4.36, and 3.12 times that of 'CK', ​​respectively. Figure 2 D and 2H).

[0044] The above results indicate that the increased content of glucose, sucrose, and fructose is the main reason for the high sweetness of 'JMX' fruit.

[0045] Example 3: Identification and analysis of SUS, SPS, and SUT family genes in cultivated and forest strawberries. Protein sequences were searched in databases of Arabidopsis thaliana, cultivated strawberry, and wild strawberry to construct phylogenetic trees and identify and analyze members of the SUS, SPS, and SUT families.

[0046] Methodology: By consulting relevant literature, we determined the number of homologous proteins in each of the SUS, SPS, and SUT families in Arabidopsis thaliana. The corresponding protein sequences were obtained from (https: / / www.arabidopsis.org / ), and the Blastalp command was used to find protein sequences with high homology in both wild and cultivated strawberries. These homologous protein sequences were aligned using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo) and manually corrected. After correction, the protein sequences were saved in FASTA format for later use. Phylogenetic analysis was performed using MEGA7 (http: / / www.megasoftware.net / ). The Neighbor-Joining (NJ) algorithm was used to calculate the similarity approximation number, and a phylogenetic tree of the SUS, SPS, and SUT family members was constructed.

[0047] Using published transcriptome data (Kang C, Darwish O, Geretz A, Shahan R, Alkharouf N, Liu Z, 2013. Genome-Scale Transcriptomic Insights into Early-Stage Fruit Development in Woodland Strawberry) Fragaria vesca . The Plant Cell25, 1960-78; Li Y, Pi M, Gao Q, Liu Z, Kang C, 2019. Updated annotation of the wild strawberry Fragaria vesca (V4 genome. Horticulture Research 6, 61.) This study analyzed the expression patterns of genes in the SUS, SPS, and SUT families in forest strawberry.

[0048] The tissues involved in the transcriptome data include: shoot apical meristem (SAM), floral meristem (FM), receptacle meristem (REM), anther, carpel, fleshy tissue adjacent to the fruit (Cortex), internal tissue of the stigma (Pith), Ruegen-red fruit, 15 / 22 days post-pollination (Rg15D / 22D), Yellow Wonder-white fruit, 15 / 22 days post-pollination (Yw15D / 22D), embryo, complete seed after removing the embryo (Ghost), leaf, ovule, pollen, seed, seedling, style, seed coat, root, and root tip (Root_P). The numbers after the tissues indicate different developmental stages.

[0049] The results showed that the SUS family includes 6, 6, and 19 genes in Arabidopsis thaliana, wild strawberry, and cultivated strawberry, respectively. Figure 3 A), FveSUS4a and FveSUS3 The expression level was high in various tissues of *Strombocybe spp.* Figure 3 B). During the fruit ripening process FveSUS4a Significantly downregulated expression (from 194.52 to 33.38). FveSUS3 The expression level remained almost unchanged (from 21.36 to 22.55). Figure 3 B). The SPS family includes 4, 10, and 12 genes in Arabidopsis thaliana, forest strawberry, and cultivated strawberry, respectively. Figure 4 A), FveSPS3d The highest expression level was observed in various tissues of *Strombocybe moniliforme*. Figure 4 B). During the fruit ripening process FveSPS3d Slightly upregulated expression (51.15 to 69.82). The SUT family includes 3, 8, and 24 genes in Arabidopsis thaliana, forest strawberry, and cultivated strawberry, respectively. Figure 5 A), FveSUT4 and FveSUT2g The expression level was high in various tissues of *Strombocybe spp.* Figure 5 B). During the fruit ripening process FveSUT4 Slightly down-adjusted expression (46.51 to 39.40). FveSUT2g The expression level remained almost unchanged (from 19.62 to 19.44). By constructing a phylogenetic tree, the member information of the SUS, SPS and SUT families in forest strawberry and cultivated strawberry was clarified, which facilitates subsequent analysis. FveSUSa , FveSUS3 , FveSPSd3 , FveSUT4 and FveSUT2g High expression in various tissues of *Strombocybe spp.* Figure 3 (B, 4B, and 5B) suggests that they may play a key role in the glucose metabolism pathway.

[0050] Example 4: Differential Gene Analysis Because 'CK' and 'JMX' had higher levels of soluble solids, total sugar, glucose, sucrose, and fructose during the IR and FR periods, and the differences were significant ( folds). Figure 1 and 2 Therefore, in 2025, 'CK' and 'JMX' fruits in the IR and FR stages were collected for transcriptome sequencing (RNA-seq), with each sample containing three biological replicates.

[0051] Methods: Total RNA was extracted using the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China; catno. R4151). 'CK' and 'JMX' samples each contained three biological replicates. RNA integrity (RIN value > 9) was assessed using an Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). Sequencing was performed at Shanghai Paisenno Biotechnology Co., Ltd. (China).

[0052] Data processing: Trimmomatic was used to remove 9 bp of low-quality bases from the 5' end of the transcriptome data, and then the Hisat2 program was used to align to the reference genome of *Strawberry 'Red Face'* (Bolger AM, Lohse M, Usadel B, 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-20; Kim D, Langmead B, Salzberg SL, 2015. HISAT: a fast splicedaligner with low memory requirements. Nature Methods 12, 357-60; Hu S, Zeng X, Liu Y, et al., 2024. Global characterization of somatic mutations and DNA methylation changes during vegetative propagation in strawberries. GenomeResearch 34, 1582-94.). Feature Counts was used to calculate the number of reads aligned to the gene CDS (Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014:30(7):923–930). Gene expression levels were calculated as transcripts per million reads (TPM) using TBtools (Chen C, Chen H, Zhang Y, et al., 2020. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Molecular Plant 13, 1194-202.). Differentially expressed genes (fold change > 2) were analyzed using the R package DESeq2. PExpression analysis of RNA <0.05 (Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014:15(12):550.). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using clusterProfile, and heatmaps were generated using MeV 4.9.0.

[0053] The results showed that Principal Component Analysis (PCA) indicated that the three biological replicates in the 'CK' and 'JMX' groups were tightly clustered, while the three biological replicates in the other two groups were more dispersed. Figure 6 A) indicates that the transcriptome data can be used to mine downstream genes. Analysis revealed that compared to 'CK', ​​4316 and 5511 genes were significantly upregulated in 'JMX' during the IR and FR periods, respectively, while 5701 and 6329 genes were significantly downregulated (fold change > 2). P (Value < 0.05) Figure 6 B). Enrichment analysis of these differentially regulated genes using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that genes upregulated during the IR phase were significantly enriched in starch and sucrose metabolic pathways. Figure 6 C), genes upregulated during the FR period were significantly enriched in starch and sucrose metabolism, glycolysis / glucose production, galactose metabolism, and fructose and mannose metabolism. Figure 6 E. Genes downregulated during the IR phase are enriched in other polysaccharide degradation pathways ( Figure 6 D), genes downregulated during the FR period are enriched in other polysaccharide degradation and glycosaminoglycan degradation pathways (D). Figure 6 F). The differential expression of the genes related to sugar metabolism mentioned above is likely the main reason for the high glucose, sucrose, and fructose content in 'JMX' fruit.

[0054] Example 5: Differential Expression Analysis Total RNA was extracted from 'JMX' tissue using an RNA extraction kit (Sangon Biotech, Shanghai, China, cat. NO. SK8631). RNA quality was assessed by 1% agarose gel electrophoresis, and RNA concentration was determined using Nanodrop 2000. 1 μg of total RNA was used for reverse transcription using a reverse transcription kit (TaKaRa, Shiga, Japan, cat. NO. RR047A), which included two reactions: step I (gDNA removal) and step II (reverse transcription). Reagents were added on ice.

[0055] The gDNA removal system was as follows: 5 x gDNA Eraser Buffer, 2 μL; gDNA Eraser, 1 μL; RNA, 1 μg; RNase-Free dH2O, to a final volume of 10 μL. The reverse transcription system was as follows: gDNA removal reaction solution, 10 μL; 5 x Primer Script Buffer 2, 4 μL; PrimerScript RT Enzyme Mix I, 4 μL; RTPrimer Mix, 4 μL; RNase-Free dH2O, 4 μL.

[0056] The reverse transcription program was as follows: 42 ℃, 2 min; 37 ℃, 15 min; 85 ℃, 5 sec. The reverse transcription product was diluted 5-fold for later use. Using the diluted cDNA as a template, the internal reference gene (Actin-) was amplified with Taq polymerase. FaCHP1 The cDNA quality was assessed to provide a template for subsequent real-time quantitative PCR. Reaction system: 2 x ES Taq Master Mix, 15 μL; positive primer, 2 μL; reverse primer, 2 μL; template, 1 μL; ddH2O, 10 μL. Reaction conditions: pre-denaturation 94 °C, 5 min; denaturation 94 °C, 30 sec; annealing 55 °C, 30 sec; extension 72 °C, 10 sec; final extension 72 °C, 5 min; cooling 4 °C, 5 min. 35 cycles from denaturation to extension were performed. The amplified fragment was examined using agarose gel electrophoresis to check the band size.

[0057] Real-time quantitative PCR: The reaction system was prepared on ice under low light conditions. SYBR Green fluorescent dye was purchased from Biorad. PCR amplification was performed on an ABI QuantStudio 7 Flex real-time quantitative PCR system. Fluorescent products were detected in the third step of each cycle. This experiment included 3 biological replicates and 3 technical replicates, using 2...-ΔΔCT Methods for analyzing experimental data, in order to FaCHP1 Standardization as an internal reference gene (Clancy MA, Rosli HG, Chamala S, Barbazuk WB, Civello PM, Folta KM, 2013. Validation of reference transcripts in strawberry). Fragaria spp (Molecular Genetics and Genomics 288, 671-81.), the primer sequences involved are shown in Table 1. The real-time quantitative reaction system is as follows: 2×SYBR Green PCRmaster mix, 5 μL; cDNA, 1 μL; positive or reverse primer, 0.25 μL; H2O not added to 10 μL, the reaction program is as follows: Stage I 50 ℃, 2 min; 95 ℃, 10 min; Stage II 95 ℃, 15 sec; 60 ℃, 20 sec; 72 ℃, 20 sec; 95 ℃, 15 sec; 50 cycles; Stage III 60 ℃, 1 min; 95 ℃, 15 sec.

[0058] Table 1 Primer sequences

[0059] Data Analysis and Results: Intersection of upregulated and downregulated genes in IR and FR showed that a total of 2860 genes were upregulated in both IR and FR periods. Figure 7 A), 3490 genes were downregulated in both IR and FR periods ( Figure 7 B). Since SUSs, SPSs, and SUTs are key rate-limiting enzymes and transporters in the glucose metabolism pathway and directly regulate glucose accumulation, the expression of these three gene families was first examined in genes that were co-upregulated and co-downregulated.

[0060] 3 in 'JMX' FaSUSs Expression levels were significantly lower in the IR and FR phases than in the 'CK' phase. FaSPSs and 8 FaSUTs The expression levels during IR and FR were significantly higher than those of 'CK'. Figure 7 CD). Subsequently, RT-PCR was used to detect the expression levels of these 16 genes during the IR and FR periods. The results showed that 3 genes in 'JMX'... FaSUSs Expression levels were significantly lower in IR and FR phases than in 'CK', ​​5 FaSPSs and 8 FaSUTs The levels during the IR and FR periods were significantly higher than those of 'CK'; as the fruits of 'CK' and 'JMX' matured, the three... FaSUSs All five showed down-regulation. FaSPSs and 8 FaSUTs All showed upregulation.

[0061] in, FaSUS4g yes FaSUSs The gene with the lowest expression level in the family during the 'JMX'-FR stage. FaSPS4b and FaSUT2d They are FaSPSs and FaSUTs The gene with the highest expression level in the family during the 'JMX'-FR period ( Figure 7 EG). The above results indicate that these 16 genes, especially FaSUS4g (The nucleotide sequence is shown in SEQ ID NO.1) FaSPS4b (The nucleotide sequence is shown in SEQ ID NO.2) and FaSUT2d (The nucleotide sequence is shown in SEQ ID NO.3) plays an important role in the sweetening process of 'JMX' fruit.

[0062] Example 6 FaSUS4g , FaSPS4b and FaSUT2d Verification of the regulation of sugar content in strawberry fruit Build FaSUS4g , FaSPS4b and FaSUT2d The overexpression vector, along with the empty vector, was transiently injected into 'JMX' or 'CK' white-ripe fruits. Figure 8 A).

[0063] Construction of overexpression vectors: based on FaSUS4g , FaSPS4b and FaSUT2d Primers were designed based on the gene sequence (with the stop codon removed), and the primer sequences are shown in Table 1.

[0064] (1) Using fruit cDNA as a template, the gene fragment was amplified with KOD enzyme. The reaction system was as follows: KOD plus, 1 uL; KOD plus buffer, 5 uL; positive primer, 5 uL; reverse primer, 5 uL; template, 50 ng; DMSO, 2.5 uL; dNTP, 5 uL; MgSO4, 3.5 uL; ddH2O to 50 uL. Reaction conditions: pre-denaturation 94 °C, 5 min; denaturation 94 °C, 35 s; annealing 60 °C, 35 s; extension 68 °C, 2 min; final extension 68 °C, 5 min; cooling 4 °C, 5 min. 35 cycles of denaturation to extension were performed. The amplified fragment was checked for band size by agarose gel electrophoresis, and the concentration was measured after purification for later use.

[0065] (2) The 35SGFP-PRI101 vector was subjected to [further treatment] at 37 °C. NdeI and EcoRI Enzyme digestion for 6 h; reaction system: 35SGFP-PRI101 vector plasmid, 4 μg; Nde I and EcoR I, 3 μL each; Cutsmart, 15 μL; Take 2 μL of the digested product and 1 μL of the undigested empty vector as controls, run gel electrophoresis to check if the digestion is complete. If the digestion is complete, purify the linearized vector and measure its concentration for later use.

[0066] (3) The amplified fragment and the linearized vector were ligated by homologous recombination reaction. The reaction system was as follows: 35SGFP-PRI101 linearized plasmid, 200 ng; target fragment, 200 ng; Exnase II, 2 μL; 5× CE II Buffer, 4 μL; water to 20 μL; reaction conditions were 37 ℃; reaction time was 1 h.

[0067] (4) The product after homologous recombination reaction was heat-shocked into colonic competent cells (DH5α). The specific process of converting colonic competent cells (DH5α) was as follows: 5 μL of the product after homologous recombination reaction was added to DH5α and placed in an ice bath for 30 min; heat-shocked at 42°C for 45 sec; placed on ice for 2 min; 200 μL of antibiotic-free liquid LB was added in a clean bench; cultured at 37°C with shaking for 1 h; and then spread onto solid LB medium containing antibiotics (LB+Km 50 mg / mL).

[0068] (5) After the single clones were grown, they were verified by PCR (reaction system: 2 x ES Taq Master Mix, 15 uL; positive primer, 2 uL; reverse primer, 2 uL; template, 1 uL; ddH2O, 10 uL. Reaction conditions: pre-denaturation 94 °C, 5 min; denaturation 94 °C, 30 sec; annealing 55 °C, 30 sec; extension 72 °C, 30 sec; final extension 72 °C, 5 min; cooling 4 °C, 5 min. 35 cycles from denaturation to extension were performed. The amplified fragments were checked for band size using agarose gel electrophoresis. Three single clones with correct band size were selected and first verified by enzyme digestion (using...). Nde I and EcoR I. The enzyme is digested (the specific process is as described above); then the single clone is sent to the company for sequencing to check whether the inserted sequence is correct.

[0069] (6) For correctly sequenced single clones, extract E. coli plasmids using Plasmid Mini Kit I (OMEGA). The specific procedure is as follows: Take 2 mL of bacterial culture, centrifuge at 1000 rpm for 1 min, and discard the supernatant; add 250 μL Solution I to resuspend; add 250 μL Solution II, shake gently, and let stand for 2 min; add 350 μL Solution III and immediately invert the tube to produce a white precipitate, centrifuge at 13000 rpm at room temperature for 10 min; carefully aspirate the supernatant into the adsorption column, centrifuge at 10000 rpm for 1 min, and discard the effluent; add 700 μL DNA Wash buffer, centrifuge at 10000 rpm for 1 min, and discard the effluent; repeat step 5; replace with a clean 1.5 mL centrifuge tube, add 50 μL ddH2O, let stand for 2 min, and centrifuge at 13000 rpm for 1 min; detect the concentration using agarose gel electrophoresis and NANODROP 2000. Store at -20 °C for later use.

[0070] (7) Transfer the positive plasmid into Agrobacterium (GV3101) as follows: Take 1 μL of plasmid, add it to GV3101, and incubate on ice for 30 min; place in liquid nitrogen for 5 min; incubate in a 37 °C water bath for 5 min; place on ice for 5 min; in a clean bench, add 200 μL of antibiotic-free liquid LB; incubate at 28 °C with shaking for 2 h; spread on solid LB medium containing antibiotics (LB+Km 50 mg / mL, Rif 50 mg / mL, Sm 50 mg / mL). After single colonies grow, perform PCR verification (the specific process is as described above). Mix the positive single colony culture with glycerol and store at -80 °C for later use.

[0071] (8) Transient transformation method for strawberry fruits: Select a single clone and place it into 6 mL of resistant liquid LB; culture overnight at 28 ℃ with shaking until the OD of the bacterial solution reaches 0.8~1.0; take 2 mL of bacterial solution into a centrifuge tube and centrifuge at 5000 rpm for 5 min; resuspend the bacterial solution in liquid MS containing 2% sucrose until the OD value of the bacterial solution reaches 0.8; select fruits at the white ripe stage, draw up the bacterial solution with a 1 mL syringe, and inject it into the fruit. 1 mL of bacterial solution can be injected into 3 strawberry fruits. Mark the fruits and observe the results about one week after injection. Inject each gene into more than 20 fruits.

[0072] Results: RT-PCR detection revealed... FaSUS4g , FaSPS4b and FaSUT2d The expression level was significantly upregulated ( Figure 8 (BD). Samples were collected after injection, and changes in soluble solids and sugar content were measured. Overexpression in 'JMX' was compared with the control. FaSUS4g This caused a significant decrease in the content of soluble solids, glucose, sucrose, and fructose in the fruit. Figure 8 A and 8E-F). Overexpressed in 'CK'. FaSPS4b and FaSUT2d This leads to a significant increase in the content of soluble solids, glucose, sucrose, and fructose in the fruit. Figure 8 A, 8E, and 8G-H). The above results indicate that... FaSUS4g , FaSPS4b and FaSUT2d It plays an important role in the sugar accumulation process of 'JMX' fruit.

[0073] Note: The statistical analysis sections in the above embodiments all use student data. t test(**, P Paired comparisons were performed for values ​​<0.01. For multiple sample comparisons, the Tukey test was used to determine the appropriate value. P Significant differences at the <0.05 level are indicated by different letters.

[0074] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strawberry gene that regulates the sugar content of strawberry fruit, characterized in that, The strawberry gene includes FaSUS4g , FaSPS4b and FaSUT2d Any one of them; The FaSUS4g The nucleotide sequence is shown in SEQ ID NO.1; The FaSPS4b The nucleotide sequence is shown in SEQ ID NO.2; The FaSUT2d The nucleotide sequence is shown in SEQ ID NO.

3.

2. An expression box, characterized in that, Contains the strawberry gene as described in claim 1; When the strawberry gene is FaSUS4g At that time, the expression box is an interference. FaSUS4g The expression box; When the strawberry gene is FaSPS4b At that time, the expression box is overexpressed. FaSPS4b The expression box; When the strawberry gene is FaSUT2d At that time, the expression box is overexpressed. FaSUT2d The expression box.

3. A recombinant expression vector, characterized in that, Contains the strawberry gene as described in claim 1 or the expression cassette as described in claim 2; When the strawberry gene is FaSUS4g At that time, the recombinant expression vector is FaSUS4g A silent carrier; When the strawberry gene is FaSPS4b At that time, the recombinant expression vector is FaSPS4b Overexpression vectors; When the strawberry gene is FaSUT2d At that time, the recombinant expression vector is FaSUT2d Overexpression vectors.

4. A recombinant microorganism, characterized in that, Contains the strawberry gene as described in claim 1, the expression cassette as described in claim 2, or the recombinant expression vector as described in claim 3.

5. The application of the strawberry gene as described in claim 1 in increasing the sugar content of strawberry fruit.

6. The application according to claim 5, characterized in that, When the strawberry gene is FaSUS4g At that time, reduce the FaSUS4g The expression level of the strawberry gene; when the strawberry gene is FaSPS4b At that time, improve the FaSPS4b The expression level of the strawberry gene; when the strawberry gene is FaSUT2d At that time, improve the FaSUT2d The amount of expression.

7. A method for increasing the sugar content of strawberry fruit using the strawberry gene described in claim 1, characterized in that, The expression level of the strawberry gene is regulated as follows: When the strawberry gene is FaSUS4g At that time, reduce the FaSUS4g The amount of expression; When the strawberry gene is FaSPS4b At that time, improve the FaSPS4b The amount of expression; When the strawberry gene is FaSUT2d At that time, improve the FaSUT2d The amount of expression.

8. A method for cultivating high-sugar transgenic strawberries using the strawberry gene described in claim 1, characterized in that, A recombinant expression vector for the strawberry gene was constructed, and the recombinant expression vector was transiently transferred into strawberry fruit. The sugar content in the fruit was measured to verify the gene function. When the strawberry gene is FaSUS4g At that time, instantaneous transfer of the recombinant expression vector into strawberry fruit can reduce the... FaSUS4g The amount of expression; When the strawberry gene is FaSPS4b At that time, instantaneous transfer of the recombinant expression vector into strawberry fruit can improve the... FaSPS4b The amount of expression; When the strawberry gene is FaSUT2d At that time, instantaneous transfer of the recombinant expression vector into strawberry fruit can improve the... FaSUT2d The amount of expression.

9. The method for cultivating high-sugar transgenic strawberries using strawberry genes according to claim 8, characterized in that, The instantaneous transfer operation is performed during the white ripening stage of the strawberry fruit.

10. The application of the strawberry gene as described in claim 1 in strawberry breeding, characterized in that, The selected strawberry varieties have high fruit sugar content; When the strawberry gene is FaSUS4g At that time, the selection and breeding of the aforementioned FaSUS4g Strawberries with reduced expression levels; When the strawberry gene is FaSPS4b At that time, the selection and breeding of the aforementioned FaSPS4b Strawberries with increased expression levels; When the strawberry gene is FaSUT2d At that time, the selection and breeding of the aforementioned FaSUT2d Strawberries with increased expression levels.

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