Use of TRX-M4 gene related biological material in preparation of product for regulating soluble sugar content of fruit

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically involving TRX-M4 Application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits. Here, regulating the soluble sugar content of fruits refers to increasing or decreasing the content of soluble sugars in the fruit, including fructose, glucose, and sucrose. The present invention provides... TRX-M4 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention, through QTL mapping analysis, screened a key candidate gene related to the regulation of sugar content in apple fruit. TRX-M4 Overexpression in Apple TRX-M4 Genes can reduce fructose, glucose, and sucrose content, while silencing them... TRX-M4 TRX-M4 It can increase the content of fructose, glucose and sucrose, thereby promoting the accumulation of several major soluble sugars in genetically modified apples.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving TRX-M4 Application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits. Background Technology

[0002] Apples are one of the world's most important economic fruit trees. The content of soluble sugars such as fructose (Fru), glucose (Glc), and sucrose (Suc) in apples is a key indicator determining the flavor quality and commercial value of the fruit. Therefore, how to effectively regulate the soluble sugar content of apples has always been a research hotspot in the fields of fruit tree breeding and agricultural biotechnology.

[0003] Currently, in the field of plant genetic engineering, products or methods for regulating sugar content in apple fruits are mainly achieved by discovering and utilizing biomaterials related to genes involved in sugar metabolism or transport. Existing biomaterials related to these genes primarily focus on sugar transporter genes or key enzyme genes involved in sugar metabolism, such as the SWEET family, HT family, and REDL6 family genes; as well as sucrose invertase genes and sucrose synthase genes. Researchers typically use these known genes to construct overexpression or knockout vectors, thereby preparing corresponding transgenic plants to alter sugar accumulation in the fruit. For example, researchers have disclosed an apple fruit sugar transporter gene. MdERDL6 The application will MdERDL6 When overexpressed in tomatoes, the flowering and fruiting of the plants were significantly delayed, the main stem thickened, and the fructose content of the fruit increased by about 5 times. In addition, some transcription factor-related biological materials, such as CBF and NAC, have also been applied to the study of fruit sugar regulation.

[0004] However, the existing products and gene-related biomaterials for regulating the soluble sugar content of fruits still have a major technical problem in practical applications: they can only regulate the single sugar component, and can only regulate the multiple soluble sugar components in fruits. Summary of the Invention

[0005] To discover a new strategy for simultaneously and efficiently regulating the content of multiple soluble sugar components (especially fructose, glucose, and sucrose) in apple fruit, and to address the limitations of existing products and gene-related biomaterials that only regulate a single soluble sugar component, this invention provides... TRX-M4 Application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits. To achieve the above objective, the present invention adopts the following technical solution.

[0006] Given the scarcity of target genes capable of precisely and efficiently regulating multiple key soluble sugar components in apples (i.e., reducing sugar content through overexpression and increasing sugar content through silencing), this invention provides a novel key gene capable of simultaneously and efficiently regulating the content of multiple soluble sugar components (especially fructose, glucose, and sucrose) in apple fruit. TRX-M4 The application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits, the aforementioned TRX-M4 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] described TRX-M4 Gene-related biomaterials can improve TRX-M4 The substances and energy of gene expression are reduced. TRX-M4 Substances involved in gene expression.

[0008] The ability to improve TRX-M4 The gene-expressed substance is used to reduce the content of soluble sugars in fruits, which can reduce... TRX-M4 The gene-expressed substance is used to increase the content of soluble sugars in fruits.

[0009] The soluble sugars include fructose, glucose, and sucrose.

[0010] The TRX-M4 The gene is a thioredoxin gene, which can enhance... TRX-M4 The gene expression of substances reduces fructose, glucose, and sucrose content, or can reduce... TRX-M4 The gene-expressed substance increases the content of fructose, glucose, and sucrose, thereby promoting the accumulation of soluble sugars in transgenic apples, which can be used to prepare products that regulate the soluble sugar content of fruits. This invention utilizes apples... TRX-M4 The application of gene-related biomaterials can simultaneously increase and / or decrease the content of multiple soluble sugar components (such as fructose, glucose, and sucrose) in fruits, rather than regulating only a single sugar component. This solves the problem of existing technologies having a single regulatory function and being unable to synergistically improve the overall sugar metabolism and flavor quality of fruits, thus achieving more comprehensive and efficient genetic improvement of fruit sugar content.

[0011] Among them, the TRX-M4 The gene acts as a negative regulatory gene, used to increase the fructose, glucose, and sucrose content in apple fruits.

[0012] Furthermore, the fruit includes apples.

[0013] Furthermore, the energy can reduce TRX-M4 The substances expressed by genes include the above. TRX-M4 RNA interference vectors or CRISPR gene knockout vectors for genes.

[0014] Furthermore, the aforementioned TRX-M4 RNA interference vectors for genes contain TRX-M4 Recombinant expression vectors for gene interference sequences.

[0015] The TRX-M4 The CRISPR gene knockout vector contains a target gene. TRX-M4 CRISPR / Cas9 gene knockout vector containing the sgRNA sequence of the gene.

[0016] Furthermore, the aforementioned TRX-M4 RNA interference vectors for genes are used to... TRX-M4 The gene was constructed by cloning the CDS coding region (240bp) into the pK7GWIWG2D(Ⅱ) vector.

[0017] Furthermore, the aforementioned ability to improve TRX-M4 The substances involved in gene expression include TRX-M4 Gene overexpression vectors.

[0018] Furthermore, the aforementioned TRX-M4 Gene overexpression vectors are used to express the gene. TRX-M4 It was constructed by cloning the gene into a plant expression vector.

[0019] Furthermore, the aforementioned TRX-M4 Gene overexpression vectors are used to express the gene. TRX-M4 The gene was constructed by cloning the full-length CDS coding region into the pCAMBIA2300 vector.

[0020] Furthermore, the regulation of fruit soluble sugar content refers to simultaneously increasing or simultaneously decreasing the content of fructose, glucose and sucrose in the fruit.

[0021] This invention also provides a method for increasing the fructose, glucose, and sucrose content in apples, wherein the method involves reducing the fructose, glucose, and sucrose content in apples through genetic engineering. TRX-M4 Gene expression levels.

[0022] Furthermore, by using genetic engineering techniques to reduce the [specific content] in apples TRX-M4 The process of gene expression level is as follows: The TRX-M4 Introducing an RNA interference vector or CRISPR gene knockout vector into an apple reduces the... TRX-M4 Gene expression, thereby increasing the content of fructose, glucose and sucrose in apple fruits.

[0023] This invention also provides a method for reducing the fructose, glucose, and sucrose content in apples, wherein the method involves increasing the content of these substances in apples through genetic engineering. TRX-M4 Gene expression levels.

[0024] Furthermore, by using genetic engineering techniques to enhance the aforementioned properties of apples... TRX-M4 The process of gene expression level is as follows: The TRX-M4 Gene overexpression vectors were introduced into apples to enhance the expression of the gene. TRX-M4 Gene expression is reduced, thereby decreasing the content of fructose, glucose, and sucrose in apples.

[0025] Furthermore, the plant includes apples.

[0026] The present invention also provides a method for increasing the fructose, glucose and sucrose content in apple fruits, comprising increasing the fructose, glucose and sucrose content in apple plants. TRX-M4 Genes undergo RNA interference.

[0027] Furthermore, the aforementioned in apple plants TRX-M4 RNA interference with genes is the process of... TRX-M4 The RNA interference vector of the gene was transformed into apple plants, resulting in apple plants with increased fructose, glucose and sucrose content.

[0028] The present invention also provides a method for cultivating apple plants with high fructose, glucose and sucrose content, comprising the following steps: The apple plant described TRX-M4 RNA interference was performed on the genes to obtain transgenic apple plants.

[0029] Furthermore, the aforementioned in apple plants TRX-M4 RNA interference with genes is the process of... TRX-M4 An RNA interference vector was transferred into apple plants to obtain apple plants with increased soluble sugar content.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1. To discover a new strategy for simultaneously and efficiently regulating the content of multiple soluble sugar components (especially fructose, glucose, and sucrose) in apple fruit, this invention provides... TRX-M4 Application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits. Regulating the soluble sugar content of fruits refers to increasing or decreasing the content of soluble sugars in fruits, including fructose, glucose, and sucrose. This invention improves the content of soluble sugars in fruits by increasing... TRX-M4 The gene-expressed substance reduces fructose, glucose, and sucrose content, or by reducing... TRX-M4 The gene-expressed substance promotes the accumulation of soluble sugars in transgenic apple fruits, and can be used to prepare products that regulate the soluble sugar content of fruits. This invention utilizes apples... TRX-M4The application of gene-related biomaterials can simultaneously regulate (increase and / or decrease) the content of fructose, glucose, and sucrose in fruits, rather than regulating only a single sugar component. This solves the problem that existing products and gene-related biomaterials that regulate the content of soluble sugars in fruits only regulate multiple soluble sugar components in fruits and can only regulate a single sugar component. This achieves a more comprehensive and efficient genetic improvement of the soluble sugar content of fruits.

[0031] 2. This invention, through QTL mapping analysis, screened a key candidate gene related to the regulation of sugar content in apple fruit. TRX-M4 Overexpression in Apple TRX-M4 Genes can reduce fructose, glucose, and sucrose levels, while RNA interference... TRX-M4 The gene can increase the content of fructose, glucose, and sucrose, thereby promoting the accumulation of soluble sugars in transgenic apples. Simultaneously, four potential interacting proteins of TRX-M4 were screened using IP-MS, including the small subunit of ADP-glucose pyrophosphorylase (APS1), starch synthase 1 (SS1), disproportionating enzyme 1 (DPE1), and α-glucan phosphorylase 2 (PHS2). Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed the interaction between TRX-M4 and these four proteins. Biochemical experiments further verified that TRX-M4 can promote the enzyme activities of ADP-glucose pyrophosphorylase and starch synthase, thereby promoting starch synthesis in apples; while having no significant effect on the enzyme activities of disproportionating enzyme and α-glucan phosphorylase. The above data indicate that TRX-M4 promotes starch synthesis in apple fruit by interacting with adenosine diphosphate glucose pyrophosphorylase, starch synthase, superoxide dismutase, and α-glucan phosphorylase, and by enhancing the enzyme activities of adenosine diphosphate glucose pyrophosphorylase and starch synthase. This, in turn, inhibits the accumulation of fructose, glucose, and sucrose in apple fruit by altering carbon partitioning. In production practice, limited by the long juvenile stage of apple trees, this invention obtained heterologous overexpression and CRISPR gene knockout tomato fruits, and confirmed... TRX-M4 Its negative regulatory function on the content of fructose, glucose and sucrose in fruits confirms its practical application value. Attached Figure Description

[0032] Figure 1 Overexpression and RNA interference in this invention TRX-M4 Identification of transgenic apple lines; including: A represents overexpression and RNA interference. TRX-M4 Phenotypes of transgenic apple lines; Scale bar, 5cm; B represents overexpression and RNA interference. TRX-M4 In transgenic apple lines TRX-M4 Relative expression level; n=3, Mean±SD, * indicates P The difference was statistically significant at levels <0.05. C represents detection at the DNA level. TRX-M4 Overexpression lines. Lines #1, #2, and #5 were found to be transgenic in both DNA and CDS fragment lengths.

[0033] D represents detection at the DNA level. TRX-M4 RNA interference strains, namely strains #3, #4 and #14, showed amplified bands indicating transgene positivity.

[0034] Figure 2 In this invention TRX-M4 The contents of total reducing sugar (A), fructose (B), glucose (C), and sucrose (D) in transgenic apple leaves after overexpression and RNA interference; among which: A represents the total reducing sugar content; B represents the fructose content; C represents glucose content; D represents the sucrose content; n=3, Mean±SD, * indicates P There was a significant difference at the <0.05 level.

[0035] Figure 3 For the VIGS injection in this invention TRX-M4 Overexpression and silencing of genetically modified apple fruits TRX-M4 The relative expression levels of genes (A), fructose (B), glucose (C), and sucrose (D); among which: A is TRX-M4 The relative expression level of genes; B represents the fructose content; C represents glucose content; D represents the sucrose content; n=3, Mean±SD, * indicates P There was a significant difference at the <0.05 level.

[0036] Figure 4 In this invention TRX-M4 The redox state of transgenic apple seedlings; among which: A is TRX-M4 Total free sulfhydryl content in transgenic apple lines; B is TRX-M4The content of reduced glutathione (GSH) in transgenic apple lines; C is TRX-M4 Content of oxidized glutathione (GSSG) in transgenic apple lines; D is TRX-M4 Total glutathione content in transgenic apple strains; E is TRX-M4 GSH / GSSG ratio in transgenic apple lines; n=3, Mean±SD, * indicates P There was a significant difference at the <0.05 level.

[0037] Figure 5 This invention verifies the interaction relationship between TRX-M4 and APS1, SS1, DPE1, and PHS2; wherein: A represents yeast two-hybrid validation; in each row, multiple samples are, in order, APS1, SS1, DPE1, and PHS2 proteins fused with the AD domain; B and C are for bimolecular fluorescence complementarity verification; multiple samples in each row are sequentially compared with YFP. C Fusion empty vector control, APS1, SS1, DPE1, and PHS2 proteins; where: B is the protein with YFP N The fusion empty vector control; C is the control with YFP. N Fusion TRX-M4 protein; scale bar, 5 μm.

[0038] Figure 6 This invention relates to the effect of TRX-M4 on the enzyme activity of starch-metabolizing enzymes in plants; wherein: A is TRX-M4 Enzymatic activity of adenosine diphosphate glucose pyrophosphorylase (AGPase) in transgenic lines; B is TRX-M4 The enzyme activity of starch synthase (SS) in transgenic lines; C is TRX-M4 The enzyme activity of superoxide dismutase (DPE) in transgenic lines; D is TRX-M4 Enzymatic activity of α-glucan phosphorylase (PHS) in transgenic lines; n=3, Mean±SD, * indicates P There was a significant difference at the <0.05 level.

[0039] Figure 7 In this invention TRX-M4 Identification of heterologous overexpression transgenic tomato seedlings; including: A is TRX-M4 Phenotype of heterologous overexpression transgenic tomato seedlings; B is TRX-M4 DNA level identification of heterologous overexpression transgenic tomato seedlings; C is TRX-M4 RNA level identification in heterologous overexpression transgenic tomato seedlings; n=3, Mean±SD, * indicates P There was a significant difference at the <0.05 level.

[0040] Figure 8 CRISPR gene knockout apple in this invention TRX-M4 Identification of transgenic tomato seedlings with homologous genes; including: A is a CRISPR gene knockout apple. TRX-M4 Phenotype of transgenic tomato seedlings with homologous genes; B is a CRISPR gene knockout apple. TRX-M4 DNA level identification of homologous gene transgenic tomato seedlings; C represents CRISPR gene knockout in tomatoes and apples. TRX-M4 A schematic diagram of a dual-target vector design for homologous genes.

[0041] Figure 9 CRISPR gene knockout apples TRX-M4 Sequencing validation results of homologous transgenic tomato seedlings; among which: The multiple samples in each row are, in order, wild-type control (WT), CRISPR gene knockout line 1 (KO #1), CRISPR gene knockout line 2 (KO #2), and CRISPR gene knockout line 3 (KO #3); KO #1 and KO #3 are homozygous lines with two targets, while KO #2 is homozygous line with one target.

[0042] Figure 10 for TRX-M4 Heterologous overexpression and CRISPR gene knockout apples TRX-M4 Phenotypic characteristics of transgenic tomato fruits with homologous genes at five typical developmental stages; among which: The samples in each row are, in order: wild-type control (WT), overexpression line 1 (OE #1), overexpression line 2 (OE #2), overexpression line 3 (OE #3), CRISPR knockout line 1 (KO #1), CRISPR knockout line 2 (KO #2), and CRISPR knockout line 3 (KO #3). The five developmental stages are: young fruit stage (IMG), green ripe stage (MG), color change stage (B), orange ripe stage (O), and red ripe stage (R).

[0043] Figure 11 Heterologous overexpression in this invention TRX-M4 CRISPR gene knockout apples TRX-M4Determination of fructose, glucose, and sucrose content in transgenic tomato fruits of homologous genes; among which: A represents heterologous overexpression. TRX-M4 CRISPR gene knockout apples TRX-M4 Fructose content in homologous transgenic tomato fruits at five typical developmental stages; B represents heterologous overexpression. TRX-M4 CRISPR gene knockout apples TRX-M4 Glucose content in transgenic tomatoes with homologous genes at five typical developmental stages; C represents heterologous overexpression. TRX-M4 CRISPR gene knockout apples TRX-M4 Sucrose content in genetically modified tomatoes at five typical developmental stages; Mean±SD, * indicates P There was a significant difference at the <0.05 level. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1. Test materials The apple tissue culture seedlings ('Gala') explants were provided by the Horticultural Science Research Center of Northwest A&F University. These explants are standard materials for apple genetic transformation and have been widely used in apple gene function research. The apple tissue culture seedlings were subjected to a 16-hour light / 8-hour dark (140 μmol / m²) exposure. -2 s -1 Cultured at 24℃. TRX-M4 Transgenic apple and tomato seedlings with overexpression, RNA interference, or CRISPR gene knockout were acclimatized under natural light in a plastic greenhouse at the Jingyang Vegetable Experiment Station in Yangling District, Xianyang City, Shaanxi Province. Fully expanded leaves were harvested 60 days after transplanting and sampled for subsequent analysis. Tomato fruits were sampled at five developmental stages (IMG, MG, B, O, R: young fruit stage, green-ripe stage, color-changing stage, orange-ripe stage, and red-ripe stage, respectively). Apple fruits used for VIGS injection were freshly purchased from the local market.

[0046] 2. Test methods 2.1 Apple TRX-M4 Gene sequence cloning Obtained from the GDDH13 v1.1 apple genome database TRX-M4CDS sequence of the gene. Total RNA and genomic DNA were extracted from the pulp tissue of 'Qin Guan' using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (catalog number: DP441) and the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (catalog number: DP360) from Tiangen Biotech (Beijing) Co., Ltd. Total RNA was reverse transcribed into cDNA using the Takara Reverse Transcription Kit (catalog number: 6210A), and specific primers were designed based on the CDS sequence. TRX- M4- F and TRX-M4- R), using cDNA as a template, was used for PCR amplification to obtain TRX-M4 -CDS ( TRX-M4 Full-length CDS sequence of the gene TRX-M4 The CDS sequence of the gene was obtained by PCR amplification using DNA as a template. TRX-M4 -DNA ( TRX-M4 Full-length DNA sequence of the gene TRX-M4 (The DNA sequence of the gene).

[0047] The source of the 'Qin Guan' material is the Yangling Agricultural Comprehensive Experiment and Demonstration Station of Northwest A&F University.

[0048] Specific primers include TRX-M4- F and TRX-M4-R.

[0049] TRX-M4- The nucleotide sequence of F is shown in SEQ ID NO.1: 5'-ATGGCTACAGTCCTCGACTCCG-3'.

[0050] TRX-M4- The nucleotide sequence of R is shown in SEQ ID NO.2: 5'-CTACAAGAACTTCTCTATGCTGGTG-3'.

[0051] TRX-M4 The full-length coding sequence of the gene (CDS, from start codon ATG to stop codon TAG) is shown in SEQ ID NO. 3: .

[0052] 2.2 Genetic transformation of apple leaves Primers p2300-TRX-M4-F and p2300-TRX-M4-R were used to amplify the expression vector containing the adapter. TRX-M4 Full-length CDS sequence of the gene, including adapters TRX-M4 The full-length CDS sequence of the gene was inserted into the overexpression vector pCAMBIA2300 to obtain the p2300-TRX-M4 recombinant plasmid.

[0053] The nucleotide sequence of p2300-TRX-M4-F is shown in SEQ ID NO.4: 5'-ACGGGGGACGAGCTCGGTACCCATGGCTACAGTCCTCGACTCCG-3'.

[0054] The nucleotide sequence of p2300-TRX-M4-R is shown in SEQ ID NO.5: 5'-GGTGTCGACTCTAGAGGATCCCAAGAACTTCTCTATGCTGGTG-3'.

[0055] The overexpression vector pCAMBIA2300 (see authorized patent: CN119685345B) was preserved by the Apple Stress Resistance and Quality Improvement Innovation Team of the College of Horticulture, Northwest A&F University.

[0056] Primers PK7-TRX-M4-F and PK7-TRX-M4-R were used to amplify the adapter-containing RNA interference vector. TRX-M4 The gene CDS sequence (240 bp) will contain the adapter. TRX-M4 The gene CDS sequence (240 bp) was inserted into the RNA interference vector pK7GWIWG2D(Ⅱ) to obtain the PK7-TRX-M4 recombinant plasmid.

[0057] The nucleotide sequence of PK7-TRX-M4-F is shown in SEQ ID NO.6: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCGCAACATGGCAGTCACTTGTC-3'.

[0058] The nucleotide sequence of PK7-TRX-M4-R is shown in SEQ ID NO.7: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCGGCATCTTTCTTCTCGCCACC-3'.

[0059] The RNA interference vector pK7GWIWG2D(Ⅱ) (see published patent: CN117230092A) is preserved by the Apple Stress Resistance and Quality Improvement Innovation Team of the College of Horticulture, Northwest A&F University.

[0060] The constructed recombinant plasmids p2300-TRX-M4 and PK7-TRX-M4 were transformed into EHA105 Agrobacterium competent cells to obtain... TRX-M4 Agrobacterium overexpression and RNA interference.

[0061] The established experimental method was used to infect explants from apple tissue culture seedlings ('Gala'): leaves of 4-week-old apple tissue culture seedlings ('Gala') were wounded and then infected with... TRX-M4 Agrobacterium overexpression and RNA interference were used for infection (OD 0.8, 10 min), co-cultured in the dark for 2 days, and then screened under 16-hour light conditions to obtain... TRX-M4 Overexpression and RNA interference transgenic materials.

[0062] Performed by polymerase chain reaction (PCR) TRX-M4 DNA and RNA levels were validated in overexpression and RNA interference transgenic materials. DNA level validation for overexpression materials was performed using TRX-M4 Cross intron transgenic-identification primers F and R; DNA level validation for RNA interference materials was performed using PK7-TRX-M4-F and PK7-TRX-M4-R; RNA level validation for both overexpression and RNA interference materials was performed using qPCR-TRX-M4 primers F and R.

[0063] The nucleotide sequence of primer F for TRX-M4 cross intron transgenic identification is shown in SEQ ID NO.8: 5'-ATCAGCTCTCCGAACTCGC-3'.

[0064] The nucleotide sequence of primer R for TRX-M4 Cross intron transgenic-identification is shown in SEQ ID NO.9: 5'-ACGGCATCTTTCTTCTCGC-3'.

[0065] The nucleotide sequence of PK7-TRX-M4-F is shown in SEQ ID NO.6; the nucleotide sequence of PK7-TRX-M4-R is shown in SEQ ID NO.7.

[0066] The nucleotide sequence of qPCR-TRX-M4 primer F is shown in SEQ ID NO.10: 5'-TCAATTGCAACCCGATATG-3'.

[0067] The nucleotide sequence of primer R for qPCR-TRX-M4 is shown in SEQ ID NO.11: 5'-TCTATGCTGGTGGTCAATG-3'.

[0068] 2.3 Determination of total reducing sugar, fructose, glucose, and sucrose content in transgenic materials TRX-M4 Total reducing sugar in leaf tissues of apple seedlings with gene overexpression and RNA interference was detected using a reducing sugar content kit (catalog number: G0502F) from Suzhou Greens Biotechnology Co., Ltd.

[0069] The fructose, glucose, and sucrose contents were determined by gas chromatography-mass spectrometry (GC-MS): 100 mg of the mixture was ground in liquid nitrogen. TRX-M4 Leaf tissue samples from overexpression and RNA interference transgenic materials were extracted with 75% methanol ((v / v), containing 400 ppm ribitol, 95 °C, 30 min), phase-separated with chloroform, vacuum dried, derivatized with methoxamine and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and then analyzed for fructose, glucose, and sucrose content using a Shimadzu GCMS-2010 SE instrument (Shimadzu Corporation, Tokyo, Japan) equipped with a DB-5 MS capillary column (20 m × 18 mm × 0.18 µm) and a 5 m guard column (Agilent, California, USA).

[0070] 2.4 VIGS Instantaneous Injection into Apple Fruit The pTRV2-TRX-M4 primer F and pTRV2-TRX-M4 primer R were used to amplify the gene silencing vector containing the adapter. TRX-M4 The gene CDS sequence (240 bp) will contain the adapter. TRX-M4 The gene CDS sequence (240 bp) was inserted into the gene silencing vector pTRV2 to obtain the recombinant plasmid pTRV2-TRX-M4. The constructed recombinant plasmids p2300-TRX-M4 and pTRV2-TRX-M4 were transformed into GV3101 Agrobacterium competent cells to obtain... TRX-M4 Agrobacterium-mediated gene overexpression and gene silencing.

[0071] The resuspended Agrobacterium was directly injected into fresh apples ('Australian Green Apple') purchased from the Yangling Fruit and Vegetable Market in Xianyang City, Shaanxi Province using a 1 mL syringe. After one day of dark incubation followed by two days of light incubation, samples were collected. RNA was extracted and reverse transcribed. Relative expression levels were identified using qPCR-TRX-M4 primers F and R. Samples with correct identification were collected. TRX-M4 Apple fruit samples with gene overexpression and gene silencing were used for subsequent determination of relevant indicators.

[0072] The nucleotide sequence of primer F for pTRV2-TRX-M4 is shown in SEQ ID NO.12: 5'-CTAGTCTAGAGCAACATGGCAGTCACTTGTC-3'.

[0073] The nucleotide sequence of primer R for pTRV2-TRX-M4 is shown in SEQ ID NO.13: 5'-ACGCGGATCCGGCATCTTTCTTCTCGCCACC-3'.

[0074] The RNA interference vector pTRV2 (see published patent: CN121610525A) was preserved by the Apple Stress Resistance and Quality Improvement Innovation Team of the College of Horticulture, Northwest A&F University.

[0075] GV3101 Agrobacterium competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., product number AC1001.

[0076] The nucleotide sequence of qPCR-TRX-M4 primer F is shown in SEQ ID NO.10; the nucleotide sequence of qPCR-TRX-M4 primer R is shown in SEQ ID NO.11.

[0077] 2.5 Determination of Redox Characteristics of Transgenic Apple Seedlings TRX-M4 Free thiol groups in leaf tissues of apple seedlings with gene overexpression and RNA interference were detected using the DTNB kit (catalog number: S0138) from Shanghai Beyotime Biotechnology Co., Ltd. TRX-M4 Reduced glutathione (GSH) and oxidized glutathione (GSSG) in leaf tissues of apple seedlings with gene overexpression and RNA interference were detected using GSH and GSSG kits from Shanghai Tongwei Industrial Co., Ltd.

[0078] 2.6 Yeast Two-Hybrid The yeast two-hybrid pGBKT7 vector with adapters was amplified using BK-TRX-M4 primers F and R. TRX-M4 The CDS sequence of the gene will include the adapter. TRX-M4 The CDS sequence of the gene was inserted into the yeast two-hybrid vector pGBKT7 to obtain the BK-TRX-M4 recombinant plasmid; the adapter-containing plasmid for constructing the yeast two-hybrid vector pGBKT7 was amplified using AD-APS1 primers F and R, AD-SS1 primers F and R, AD-DPE1 primers F and R, and AD-PHS2 primers F and R. APS1 , SS1 , DPE1 and PHS2 The CDS sequence of the gene will include the adapter. APS1 , SS1 , DPE1 and PHS2The CDS sequence of the gene was inserted into the yeast two-hybrid vector pGADT7 to obtain recombinant plasmids AD-APS1, AD-SS1, AD-DPE1, and AD-PHS2. Yeast two-hybrid transformation was performed using Vidi Y2H competent cells (catalog number: YC1002S), and subsequent interaction verification was conducted according to the manufacturer's instructions.

[0079] The nucleotide sequence of primer F for BK-TRX-M4 is shown in SEQ ID NO.14: 5'-ATGGCCATGGAGGCCGAATTCATGGCTACAGTCCTCGACTCCG-3'.

[0080] The nucleotide sequence of primer R for BK-TRX-M4 is shown in SEQ ID NO.15: 5'-TGCGGCCGCTGCAGGTCGACCAAGAACTTCTCTATGCTGGTG-3'.

[0081] The nucleotide sequence of AD-APS1 primer F is shown in SEQ ID NO.16: 5'-CCATGGAGGCCAGTGAATTCATGGCGTCCTCTTCGATGTCAG-3'.

[0082] The nucleotide sequence of AD-APS1 primer R is shown in SEQ ID NO.17: 5'-CAGCTCGAGCTCGATGGATCCGATTACTGTTCCACTAGGAATC-3'.

[0083] The nucleotide sequence of AD-SS1 primer F is shown in SEQ ID NO.18: 5'-CCATGGAGGCCAGTGAATTCATGGGGTCTCTGAAATTCACTA-3'.

[0084] The nucleotide sequence of AD-SS1 primer R is shown in SEQ ID NO.19: 5'-CAGCTCGAGCTCGATGGATCCCTTGACGTAGGGCGGATCGATA-3'.

[0085] The nucleotide sequence of AD-DPE1 primer F is shown in SEQ ID NO.20: 5'-CCATGGAGGCCAGTGAATTCATGGCCTTGAAACTGAATAGCT-3'.

[0086] The nucleotide sequence of AD-DPE1 primer R is shown in SEQ ID NO.21: 5'-CAGCTCGAGCTCGATGGATCCCAGCCGCCCATACATCGACAAC-3'.

[0087] The nucleotide sequence of AD-PHS2 primer F is shown in SEQ ID NO.22: 5'-CCATGGAGGCCAGTGAATTCATGGCACCTACTGCGAAATCGG-3.

[0088] The nucleotide sequence of AD-PHS2 primer R is shown in SEQ ID NO.23: 5'-CAGCTCGAGCTCGATGGATCCTGGTACGCGGCACTCTTCGATG-3'.

[0089] Y2H competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., product number YC1002S.

[0090] 2.7. Bimolecular Fluorescence Complementarity Using YFP N -TRX-M4 primers F and YFP N -TRX-M4 primers amplify the linker-linked amplified pSPYNE-35S bimolecular fluorescent complementary vector. TRX-M4 The CDS sequence of the gene will include the adapter. TRX-M4 The CDS sequence of the gene was inserted into the bimolecular fluorescent complementary vector pSPYNE-35S to obtain YFP. N -TRX-M4 recombinant plasmid; using YFP C -APS1 primers F and YFP C -APS1 primers R, YFP C -SS1 primers F and YFP C -SS1 primers R, YFP C -DPE1 primers F and YFP C -DPE1 primers R, YFP C -PHS2 primers F and YFP C -PHS2 primers R amplify the linker-containing bimolecular fluorescent complementary vector pSPYCE-35S. APS1 , SS1 , DPE1 and PHS2 The CDS sequence of the gene will include the adapter. APS1 , SS1 , DPE1 and PHS2The CDS sequence of the gene was inserted into the bimolecular fluorescent complementary vector pSPYCE-35S to obtain YFP. C -APS1, YFP C -SS1、YFP C -DPE1 and YFP C -PHS2 recombinant plasmid. YFP C -EV, i.e., pSPYCE-35S empty vector plasmid; YFP N -TRX-M4, YFP C -APS1, YFP C -SS1、YFP C -DPE1 and YFP C - PHS2 recombinant plasmid was transformed into GV3101 Agrobacterium competent cells to obtain YFP C -EV, YFP N -TRX-M4, YFP C -APS1, YFP C -SS1、YFP C -DPE1 and YFP C -PHS2 Agrobacterium. After shaking and resuspending the Agrobacterium, let it stand for 30 minutes. The resuspended YFP N -TRX-M4 and YFP respectively C -APS1, YFP C -SS1、YFP C -DPE1 and YFP C -PHS2 was co-injected with tobacco as the experimental group; simultaneously, resuspended YFP was used. N -TRX-M4 and YFP C Tobacco was co-injected with EVs as a control group. Tobacco from both the experimental and control groups was cultured in the dark for 1 day and then in the light for 2 days, and then observed using a confocal microscope.

[0091] Among them, YFP N The nucleotide sequence of the TRX-M4 primer F is shown in SEQ ID NO.24: 5'-GCCTGGCGCGCCACTAGTGGATCCATGGCTACAGTCCTCGACTCCG-3'.

[0092] YFP N The nucleotide sequence of the TRX-M4 primer R is shown in SEQ ID NO.25: 5'-GGGAGCGGTACCCTCGAGGTCGACCAAGAACTTCTCTATGCTGGTG-3'.

[0093] YFP CThe nucleotide sequence of APS1 primer F is shown in SEQ ID NO.26: 5'-GCCTGGCGCGCCACTAGTGGATCCATGGCGTCCTCTTCGATGTCAG-3'.

[0094] YFP C The nucleotide sequence of APS1 primer R is shown in SEQ ID NO.27: 5'-GGGAGCGGTACCCTCGAGGTCGACGATTACTGTTCCACTAGGAATC-3'.

[0095] YFP C The nucleotide sequence of primer F for SS1 is shown in SEQ ID NO.28: 5'-GCCTGGCGCGCCACTAGTGGATCCATGGGGTCTCTGAAATTCACTA-3'.

[0096] YFP C The nucleotide sequence of primer R for SS1 is shown in SEQ ID NO.29: 5'-GGGAGCGGTACCCTCGAGGTCGACCTTGACGTAGGGCGGATCGATA-3'.

[0097] YFP C The nucleotide sequence of primer F for DPE1 is shown in SEQ ID NO.30: 5'-GCCTGGCGCGCCACTAGTGGATCCATGGCCTTGAAACTGAATAGCT-3'.

[0098] YFP C The nucleotide sequence of primer R for DPE1 is shown in SEQ ID NO.31: 5'-GGGAGCGGTACCCTCGAGGTCGACCAGCCGCCCATACATCGACAAC-3'.

[0099] YFP C The nucleotide sequence of the PHS2 primer F is shown in SEQ ID NO.32: 5'-GCCTGGCGCGCCACTAGTGGATCCATGGCACCTACTGCGAAATCGG-3'.

[0100] YFP C The nucleotide sequence of primer R for PHS2 is shown in SEQ ID NO.33: 5'-GGGAGCGGTACCCTCGAGGTCGACTGGTACGCGGCACTCTTCGATG-3'.

[0101] 2.8 Detection of AGPase, SS, DPE and PHS enzyme activities TRX-M4 Enzymatic activities of TRX-M4 interacting proteins in leaf tissues of apple seedlings with gene overexpression and RNA interference were detected using the following kits: AGPase (Suzhou Greens Biotechnology Co., Ltd., catalog number: G0536W), SSS (Jiangsu Adison Biotechnology Co., Ltd., catalog number: ADS-W-DF006), DPE1 (Vancoway Mall, catalog number: F50363-A), and PHS2 (Suzhou Greens Biotechnology Co., Ltd., catalog number: G0570W). All procedures were performed according to the manufacturer's protocols.

[0102] 2.9. Tomatoes are like apples TRX-M4 Identification of homologous genes Apple in Tomato TRX-M4 Homologous genes were identified using NCBI protein alignment. TRX-M4 After submitting the protein sequence of the gene (SEQ ID NO.34) to the Protein BLAST module of NCBI and clicking BLAST, the result with the highest confidence level was selected as the apple in the tomato. TRX-M4 Homologous genes.

[0103] apple TRX-M4 The protein sequence of the gene is shown in SEQ ID NO.34: MATVLDSVVVPRSSALPSSAALSRIPAPSSLSSLSAGARLPRYRGLKVGSALRTRSVGSLSYGPASGQASRRGGLVVCEAQDTAVEVPAVSDATWQSLVLDSEYPVLVEFWAPWCGPCRMIHPIIDELAKQYAGKLKCYKVNTDESASIATRYGIRSIPTVIIFKGGEKKDAVIGAVPKSTLTTSIEKFL.

[0104] 2.10. Tomatoes are like apples TRX-M4 Construction of homologous gene CRISPR gene knockout vector Reference Tomato TRX-M4Based on the DNA sequence of the gene (SEQ ID NO.35), two gRNAs were designed: gRNA 1 (SEQ ID NO.36) and gRNA 2 (SEQ ID NO.37). Following the methods described in the literature, tomato plants with adapters for constructing the CRISPR gene knockout pKSE401 vector were amplified using pKSE401-tomato-TRX-M4 primers F and R. TRX-M4 A dual-target CRISPR gene knockout sequence will knock out the linker in tomatoes. TRX-M4 The dual-target CRISPR gene knockout sequence was inserted into the CRISPR gene knockout vector pKSE401 to obtain the pKSE401-TRX-M4 recombinant plasmid.

[0105] Among them, tomatoes TRX-M4 The DNA sequence of the gene is shown in SEQ ID NO.35:

[0106] Apple in Tomato TRX-M4 The gRNA 1 sequence of the homologous CRISPR gene knockout vector is shown in SEQ ID NO.36: GAGAAGCTAACAGAGAAACG.

[0107] Apple in Tomato TRX-M4 The gRNA 2 sequence of the homologous CRISPR gene knockout vector is shown in SEQ ID NO.37: GTTAGCTTCTCTCAATTCAG.

[0108] The nucleotide sequence of primer F for pKSE401-tomato-TRX-M4 is shown in SEQ ID NO.38: 5'-ATATATGGTCTCGATTGGAGAAGCTAACAGAGAAACGGTTTTAGAGCTAGAAATAGC-3'.

[0109] The nucleotide sequence of primer R for pKSE401-tomato-TRX-M4 is shown in SEQ ID NO.39: 5'-ATATATGGTCTCGATTGGAGAAGCTAACAGAGAAACGGTTTTAGAGCTAGAAATAGC-3'.

[0110] The pKSE401 vector (see authorized patent: CN120366376B) is preserved by the Apple Stress Resistance and Quality Improvement Innovation Team of the College of Horticulture, Northwest A&F University.

[0111] 2.11 Genetic transformation of tomato leaves The constructed recombinant plasmids p2300-TRX-M4 and pKSE401-TRX-M4 were transformed into GV3101 Agrobacterium competent cells.

[0112] Tomato transformation was performed using a published protocol with slight modifications: cotyledons of 7-day-old tomato ('AilsaCraig') were cut into small pieces and pre-cultured in the dark for 24 hours, infected with Agrobacterium (OD 0.4, 4 minutes), then co-cultured in the dark for 2 days, and screened under light conditions. The resulting resistant shoots rooted before being transplanted into the soil.

[0113] The tomato ('Ailsa Craig') seeds were purchased from Chongqing Baoguang Biotechnology Co., Ltd.

[0114] 3. Data Processing Experimental data were processed using IBM SPSS 16.0 software, and differences between groups were assessed using t-tests and one-way ANOVA. P <0.05 is considered significant), and the results are presented as mean ± standard deviation (SD) of three independent biological replicates.

[0115] 4. Results and Analysis 4.1 TRX-M4 Identification of transgenic apple strains To verify TRX-M4 The function of genes in regulating the soluble sugar content of apples will TRX-M4 The complete CDS coding region sequence of the gene was cloned into the pCAMBIA2300 vector to obtain the p2300-TRX-M4 overexpression vector; simultaneously, TRX-M4 A 240bp CDS coding region sequence of the gene was cloned into pK7GWIWG2D(Ⅱ) to obtain the PK7-TRX-M4 RNA interference vector. Apple tissue culture seedlings were infected according to established experimental methods. After obtaining differentiated resistant shoots, the resistant shoots were propagated and rooted. DNA and mRNA levels of the transplanted lines were identified, resulting in 3 overexpression lines and 3 RNA interference lines. Figure 1 ).

[0116] 4.2 TRX-M4 Analysis of soluble sugar content in transgenic apple leaves To verify TRX-M4 The function of the gene in regulating the soluble sugar content of apples was investigated, and the total reducing sugar, fructose, glucose, and sucrose content in transgenic apple leaves were analyzed. The results showed that... TRX-M4 Genes negatively regulate the content of total reducing sugar, fructose, glucose, and sucrose in apple leaves. TRX-M4 When the gene is overexpressed, the contents of total reducing sugar, fructose, glucose, and sucrose are all significantly reduced, while TRX-M4 The opposite is true when there is gene RNA interference. Figure 2 ).

[0117] 4.3 TRX-M4 Analysis of soluble sugar content in genetically modified apples To further verify TRX-M4 To investigate whether a gene has a regulatory function on the soluble sugar content in apple fruit, transient overexpression and silencing were performed in apple fruit using VIGS technology. TRX-M4 Genes were analyzed, and the content of several major soluble sugars was determined. The results showed that when... TRX-M4 When the gene is overexpressed in apple fruit, the content of fructose, glucose, and sucrose in the fruit all decreases; while when TRX-M4When genes are silenced, the content of fructose, glucose, and sucrose in the fruit is significantly increased. Figure 3 ).

[0118] The above results indicate that TRX-M4 The gene also has a negative regulatory function on the content of fructose, glucose and sucrose in apple fruit.

[0119] 4.4 TRX-M4 Analysis of redox properties in transgenic apple leaves now that TRX-M4 The gene has similar functions in apple leaves and fruits, and further analysis was conducted. TRX-M4 The potential molecular mechanisms by which genes regulate fructose, glucose, and sucrose content. Based on previous research, TRX-M Genes exert their functions through redox pathways. Therefore, for TRX-M4 The contents of total free sulfhydryl groups, reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione in transgenic apple leaves, as well as their ratio (GSH / GSSG), were analyzed. The results showed that when... TRX-M4 When the gene is overexpressed, the content of total free sulfhydryl groups and reduced glutathione (GSH) in apple leaves increases significantly, while the content of oxidized glutathione (GSSG) decreases significantly, ultimately leading to an increase in the GSH / GSSG ratio. TRX- M4 When gene RNA interference occurs, the results are the opposite ( Figure 4 ).

[0120] The above results indicate that TRX-M4 Gene overexpression enhanced the reduced state within cells of apple leaves, suggesting TRX-M4 Genes perform their molecular functions through redox pathways.

[0121] 4.5 Identification of TRX-M4 interacting proteins To further reveal TRX-M4The intrinsic mechanism by which genes exert their molecular functions through redox pathways. This invention screened interacting proteins of TRX-M4. Preliminary IP-MS analysis confirmed that several coding genes involved in starch synthesis and cleavage pathways may be interacting proteins of TRX-M4. Further yeast two-hybrid assays confirmed that the small subunit of ADP-glucose pyrophosphorylase (APS1), starch synthase 1 (SS1), disproportionating enzyme 1 (DPE1), and α-glucan phosphorylase 2 (PHS2) are interacting proteins of TRX-M4. Figure 5 Subsequent bimolecular fluorescence complementation assays further confirmed the interactions between the aforementioned proteins. Figure 5 ).

[0122] 4.6. Study on the regulation of TRX-M4 on the activity of its interacting proteases To further elucidate the potential impact of TRX-M4's interaction with starch synthesis and cleavage-related proteins on target enzyme activity, this invention... TRX-M4 The enzyme activities of the four starch metabolism-related proteins mentioned above were further measured in the leaves of transgenic apples with gene overexpression and RNA interference. The results showed that... TRX-M4 When the gene is overexpressed, the activities of adenosine diphosphate glucose pyrophosphorylase and amylase are significantly enhanced. TRX-M4 The opposite result is obtained when gene RNA interference is performed. Figure 6 );and TRX-M4 The activities of superoxide dismutase and α-glucan phosphorylase did not change significantly during gene overexpression and RNA interference.

[0123] The above results indicate that TRX-M4 The regulation of soluble sugar content in apple leaves and fruits by genes may be achieved by promoting the enzymatic activity of adenosine diphosphate glucose pyrophosphorylase and starch synthase.

[0124] 4.7 TRX-M4 Analysis of soluble sugar content in genetically modified tomato fruits To further clarify the practical application of this invention, it is necessary to obtain... TRX-M4 Genetically modified apple fruits were used for verification. TRX-M4 Genes regulate the content of fructose, glucose, and sucrose in apple fruits, but due to the long juvenile stage of apple trees, researchers often use transgenic tomato fruits as a substitute. Therefore, genetic transformation of tomatoes has yielded... TRX-M4 Overexpression ( Figure 7) and CRISPR gene knockout ( Figure 8 ; Figure 9 The genetically modified seedlings were harvested. TRX-M4 Tomato fruits with overexpression and CRISPR gene knockout ( Figure 10 ), used for verification TRX-M4 Practical application of its function in regulating fructose, glucose, and sucrose content in fruits. Results showed that... TRX-M4 Genes can negatively regulate the content of fructose, glucose, and sucrose in tomato fruits. Figure 11 This confirms the practical application value of the present invention.

[0125] In summary, this invention obtains TRX-M4 Transgenic apple lines with gene overexpression and RNA interference demonstrate that when TRX-M4 When the gene is overexpressed, the contents of fructose, glucose, and sucrose all decrease significantly, and the reducing state of cells in leaves is enhanced; when TRX-M4 When gene RNA interference occurs, the results are the opposite. This was achieved through transient injection of VIGS into apple fruits and obtaining... TRX-M4 The transgenic tomato fruits with gene overexpression and CRISPR gene knockout further validated... TRX-M4 The gene's negative regulatory function on fructose, glucose, and sucrose content in fruit was also analyzed. TRX-M4 The gene promotes the allocation of assimilates in apple fruit toward starch synthesis through interaction with the proteins APS1, SS1, DPE1, and PHS2, and enhances the activity of adenosine diphosphate glucose pyrophosphorylase and starch synthase, thereby reducing the accumulation of soluble sugars.

[0126] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0127] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. TRX-M4 The application of gene-related biomaterials in the preparation of products that regulate the soluble sugar content of fruits, characterized by: The TRX-M4 The nucleotide sequence of the gene is shown in SEQ ID NO.3; The TRX-M4 Gene-related biomaterials can improve TRX-M4 The substances and energy of gene expression are reduced. TRX-M4 Substances involved in gene expression: The ability to improve TRX-M4 The gene-expressed substance is used to reduce the content of soluble sugars in fruits, which can reduce... TRX- M4 The gene-expressed substance is used to increase the content of soluble sugars in fruits; The soluble sugars include fructose, glucose, and sucrose.

2. The application according to claim 1, characterized in that, The fruit includes apples.

3. The application according to claim 1, characterized in that, The energy can reduce TRX-M4 The substances expressed by genes include the above. TRX-M4 RNA interference vectors or CRISPR gene knockout vectors for genes.

4. The application according to claim 3, characterized in that, The TRX-M4 RNA interference vectors for genes contain TRX-M4 Recombinant expression vectors for gene interference sequences; The TRX-M4 The CRISPR gene knockout vector contains a target gene. TRX-M4 CRISPR / Cas9 gene knockout vector containing the sgRNA sequence of the gene.

5. The application according to claim 1, characterized in that, The ability to improve TRX-M4 The substances expressed by genes include the above. TRX-M4 Gene overexpression vectors.

6. The application according to claim 5, characterized in that, The TRX-M4 Gene overexpression vectors are used to express the gene. TRX-M4 It was constructed by cloning the gene into a plant expression vector.

7. A method for increasing the fructose, glucose, and sucrose content in apples, characterized in that, The method involves reducing the content of the substance described in claim 1 in apples through genetic engineering. TRX-M4 Gene expression levels.

8. The method according to claim 7, characterized in that, Using genetic engineering to reduce the [content] in apples TRX-M4 The process of gene expression level is as follows: The TRX-M4 Introducing an RNA interference vector or CRISPR gene knockout vector into an apple reduces the... TRX-M4 Gene expression, thereby increasing the content of fructose, glucose and sucrose in apple fruits.

9. A method for reducing the fructose, glucose, and sucrose content in apples, characterized in that, The method involves using genetic engineering to improve the quality of apples as described in claim 1. TRX-M4 Gene expression levels.

10. The method according to claim 9, characterized in that, Improving the quality of apples through genetic engineering TRX- M4 The process of gene expression level is as follows: The TRX-M4 Gene overexpression vectors were introduced into apples to enhance the expression of the gene. TRX-M4 Gene expression is reduced, thereby decreasing the content of fructose, glucose, and sucrose in apples.

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