MdtDTL protein and its phosphorylation site mutant, encoding gene in improving the application of malic acid content

Abstract

This invention discloses the application of the MdtDTL protein and its phosphorylation site mutants, as well as the encoding gene, in increasing malic acid content, belonging to the field of plant genetic engineering technology. This invention utilizes the apple vacuolar membrane dicarboxylic acid transport gene. MdtDTL As a regulatory target, it was found that overexpression of this gene can promote the transport and accumulation of malic acid in cell vacuoles, thereby significantly increasing the malic acid content in apple fruits or apple cells. This provides a new functional gene resource for the regulation of malic acid content, improvement of fruit acidity, and enhancement of apple flavor and quality. Site-directed mutagenesis of the phosphorylation site of the MdtDTL protein can enhance or optimize the protein's promoting effect on malic acid accumulation, resulting in a higher malic acid content. MdtDTL It is further improved based on overexpression.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of an MdtDTL protein and its phosphorylation site mutant, and the encoding gene in increasing malic acid content. Background Technology

[0002] apple( Malus domestica (Borkh.) Fruit quality encompasses intrinsic quality centered on organic acids and sugars, extrinsic quality centered on color, and extended quality centered on aromatic substances and bioactive compounds. Organic acids are a crucial component of apple fruit quality, not only influencing flavor but also serving as precursors for the synthesis of other quality substances, significantly impacting the fruit's market competitiveness and economic benefits. Malic acid is the dominant organic acid in apples, accounting for over 80% of the total acid content. The accumulation of malic acid in the fruit is influenced by both malic acid metabolism and transport. As a metabolic storage depot within plant cells, the transport of malic acid from the cytoplasm to the vacuoles largely determines malic acid accumulation and fruit acidity.

[0003] Current research indicates that MdALMT9 / Today1 It is the major gene controlling malic acid content, and its activity is regulated by alleles, alternative splicing, and upstream transcription factors. Arabidopsis thaliana (… Arabidopsis thaliana AttDT is one of the earliest discovered proteins with malic acid transport properties. It mainly participates in the transmembrane transport of malic acid between the vacuoles and cytoplasm, and also participates in regulating the dynamic pH balance of plant cells. AttDT in Arabidopsis thaliana is involved in the transport of malic acid in sweet orange (…). Citrus sinensis The homologous protein CsCit1 in ) belongs to the citrate / H + Co-transporter, mainly mediating the outflow of citric acid from vacuole.

[0004] With rising living standards, consumers are increasingly demanding higher quality and flavor from apples, making fruit flavor improvement a crucial direction for the high-quality development of the apple industry. Therefore, elucidating the molecular mechanisms of malic acid accumulation and regulation is essential for improving fruit flavor. Current research on malic acid regulation focuses primarily on the transcriptional level, but in-depth exploration of post-translational modifications of vacuolar membrane transport proteins, especially phosphorylation, remains lacking. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of MdtDTL protein and its phosphorylation site mutants and encoding genes in increasing malic acid content.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of MdtDTL protein in increasing malic acid content, said MdtDTL protein being a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0007] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0008] In the above applications, overexpression of the MdtDTL protein in apple callus or apple fruit can significantly enhance the accumulation of malic acid.

[0009] A second aspect of the invention provides the use of the gene encoding the MdtDTL protein in either (1) or (2) below: (1) Increase malic acid content; (2) Cultivate apple varieties with high malic acid content; The gene encoding the MdtDTL protein is MdtDTL A gene is a nucleic acid molecule as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0010] In the above applications, by promoting MdtDTL Gene expression is used to increase malic acid content.

[0011] Preferred, promoting MdtDTL The substance used for gene expression is any one of the following: C1) contains MdtDTL Gene expression cassettes; C2) contains MdtDTL Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdtDTL Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

[0012] A third aspect of the present invention provides the application of a phosphorylation site mutant of the MdtDTL protein in increasing malic acid content, wherein the phosphorylation site mutant of the MdtDTL protein is MdtDTL(D); the amino acid sequence of the MdtDTL(D) is shown in SEQ ID NO.4.

[0013] In the above applications, overexpressing a phosphorylation site mutant of the MdtDTL protein in apples can significantly increase the malic acid content in apple fruits.

[0014] This invention reveals that sorbitol signaling can enhance the malic acid transport capacity of the MdtDTL protein by inducing phosphorylation modification. Mass spectrometry analysis shows that threonine (T) at position 12 of the MdtDTL protein is its phosphorylation site. The phosphorylation site of the MdtDTL protein was mutated to a non-phosphorylated mutant form (alanine, A) and a phosphorylated mimic mutant form (aspartic acid, D), respectively, yielding the non-phosphorylated mutant MdtDTL(A) and the phosphorylated mutant MdtDTL(D). The amino acid sequence of MdtDTL(A) is shown in SEQ ID NO. 3; the amino acid sequence of MdtDTL(D) is shown in SEQ ID NO. 4.

[0015] The results showed that mutating the phosphorylation site of the MdtDTL protein could further increase the malic acid content.

[0016] In a fourth aspect, the invention provides the use of the gene encoding the MdtDTL protein phosphorylation site mutant in either (1) or (2) below: (1) Increase malic acid content; (2) Cultivate apple varieties with high malic acid content; The gene encoding the MdtDTL protein phosphorylation site mutant is: MdtDTL(D) Genes; the stated MdtDTL(D) The nucleotide sequence of the gene is shown in SEQ ID NO.6.

[0017] In the above applications, by promoting MdtDTL(D) Gene expression is used to increase malic acid content.

[0018] Preferred, promoting MdtDTL(D) The substance used for gene expression is any one of the following: C1) contains MdtDTL(D) Gene expression cassettes; C2) contains MdtDTL(D) Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdtDTL(D)Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

[0019] The beneficial effects of this invention are: This invention utilizes the apple vacuolar membrane dicarboxylic acid transport gene. MdtDTL As a regulatory target, it was found that overexpression of this gene can promote the transport and accumulation of malic acid in cell vacuoles, thereby significantly increasing the malic acid content in apple fruits or apple cells, providing a new functional gene resource for the regulation of malic acid content, improvement of fruit acidity, and enhancement of apple flavor and quality.

[0020] This invention further discovers that the phosphorylation state of the MdtDTL protein is closely related to its ability to regulate malate accumulation. By performing site-directed mutagenesis on the phosphorylation sites of the MdtDTL protein, the promoting effect of this protein on malate accumulation can be enhanced or optimized, resulting in malate levels within a certain range. MdtDTL The expression level was further enhanced based on overexpression. This result indicates that, in addition to regulating gene expression levels, fine-grained regulation of malic acid content can be achieved through modification of protein phosphorylation sites, thus expanding a new pathway for regulating apple organic acid metabolism.

[0021] Compared with simply relying on traditional breeding to screen for high-acid materials, this invention clarifies that... MdtDTL The functional role of gene and its phosphorylation site mutations in increasing malic acid content has the advantages of clear target, clear regulatory direction, and strong operability. It can be used in apple germplasm resource improvement, molecular breeding, gene editing breeding or transgenic breeding, and provide a technical basis for breeding apple varieties with high malic acid content, balanced sweet and sour flavor or higher processing suitability. Attached Figure Description

[0022] Figure 1 Apple dicarboxylic acid transporter gene MdtDTL Chromosomal location and genomic structure of apple dicarboxylic acid transporter gene; MdtDTL It is located near the end of chromosome 10; it consists of 7 exons (black region) and 8 introns (gray region).

[0023] Figure 2 : MdtDTL With unit point mutant MdtDTL(A) and MdtDTL(D) Detection of expression levels in overexpressed transgene callus.

[0024] Figure 3 Overexpression MdtDTL and its point mutants MdtDTL(A) and MdtDTL(D) Determination of malic acid content in transgenic apple callus.

[0025] Figure 4 Transient overexpression in apple fruit MdtDTL , MdtDTL(A) , MdtDTL(D) Malic acid content determination after three genes; In the figure, A represents the phenotype of apple fruit injected; B represents the result of malic acid content determination; The numbers 1-4 on the horizontal axis of A and B represent injection of empty vector pIR, injection of transient overexpression vector MdtDTL-pIR, MdtDTL(A)-pIR and MdtDTL(D)-pIR, respectively. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] As mentioned earlier, elucidating the molecular mechanisms of malic acid accumulation and regulation is crucial for improving fruit flavor. Current research on the regulation of malic acid content largely focuses on the transcriptional level, but in-depth exploration of post-translational modifications of vacuolar membrane transport proteins, especially phosphorylation, is still lacking.

[0028] Therefore, this invention utilizes homologous cloning to obtain the gene encoding the apple dicarboxylic acid transporter. MdtDTL Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0029] To investigate the function of the MdtDTL protein, this invention employed Agrobacterium-mediated transformation to transfer the gene encoding the MdtDTL protein into apple callus tissue. Analysis of the malic acid content in the obtained transgenic apple callus revealed overexpression of the protein. MdtDTL The gene significantly increases malic acid accumulation. Transient transfection in apple fruit... MdtDTL Genes can also increase the malic acid content in the injection area.

[0030] The study found that sorbitol signaling can enhance the malic acid transport capacity of MdtDTL by inducing phosphorylation modification; mass spectrometry analysis identified the 12th threonine residue of the MdtDTL protein as its phosphorylation site.

[0031] In this invention, the 12th threonine residue of the MdtDTL protein was mutated to obtain one non-phosphorylated mutant MdtDTL (A) and one phosphorylated mutant MdtDTL (D).

[0032] MdtDTL(A) is a protein with a 12th threonine mutation to alanine, and its amino acid sequence is shown in SEQ ID NO.3; the encoding gene MdtDTL(A) The nucleotide sequence is shown in SEQ ID NO.5.

[0033] MdtDTL(D) is a protein with the 12th threonine residue mutated to aspartic acid, and its amino acid sequence is shown in SEQ ID NO.4; the encoding gene MdtDTL(D) The nucleotide sequence is shown in SEQ ID NO.6.

[0034] Overexpression in apple callus or fruit using the same method. MdtDTL(D) Genes, and overexpression of unmutated genes MdtDTL Compared to genes, it can further increase malic acid content.

[0035] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, conventional conditions are generally followed, such as those described in J. Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual* (3rd Edition), Science Press, 2002; and D.L. Spector et al., eds., *Cellular Laboratory Experiments*, Science Press, 2001; or the conditions recommended by the manufacturer. Wherein: Callus medium: MS + 1.0 mg / L 2,4-D + 1.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, adjust pH to 5.8.

[0036] Callus selection medium: MS + 1.0 mg / L 2,4-D + 1.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar + 250 mg / L cephalosporin + 30 mg / L kanamycin.

[0037] The pIR carrier is described in patent CN122235370A.

[0038] Example 1: Apple MdtDTL Gene chromosome location and genome structure Using the nucleotide sequence of AttDT in Arabidopsis thaliana published in the Arabidopsis genome database, a homologous sequence (sequence number MD10G1333000) was found in the Apple Genome Database BLAST. MdtDTLChromosomal mapping of genes was performed using the Apple website (http: / / genomics.research.iasma.it / ) genome database; while genome structure prediction was performed using the database (http: / / gsds.cbi.pku.edu.cn / index.php).

[0039] Apple dicarboxylic acid transport gene MdtDTL It is located on chromosome 10 near the centromere; it consists of 7 exons and 8 introns. Figure 1 ).

[0040] Example 2: MdtDTL and MdtDTL(A) , MdtDTL(D) Construction of gene expression vectors RNA was extracted from tissue culture leaves of Gala apples using the RNAplant plus Reagent kit, and cDNA was synthesized by reverse transcription. Primers MdtDTL-F and MdtDTL-R were designed and used as templates for PCR amplification using the synthesized cDNA.

[0041] MdtDTL-F: 5'-ATGGATAATCATCCAATAT-3'; (SEQ ID NO.7) MdtDTL-R: 5'-TTAAACTGGTTCACTTGTCTTAA-3'. (SEQ ID NO.8) After the PCR reaction, the PCR products were recovered, ligated into a vector, transformed, and then sequenced at Beijing BGI Genomics Co., Ltd. to confirm the presence of the desired product. MdtDTL The full-length CDS sequence of the gene is shown in SEQ ID NO.1.

[0042] Unit point mutations were obtained using a two-round bridging PCR method. MdtDTL(A) Genes and MdtDTL(D) Genes. Specifically: According to the obtained MdtDTL The full-length CDS sequence of the gene was obtained, and bridging primers were designed for PCR amplification. The first round of PCR amplification used the cDNA synthesized via reverse transcription as a template, employing a pair of primers with 3' end mutations (MdtDTL(A / D)-F1 / R1) and a pair of primers with 5' end mutations (MdtDTL(A / D)-F2 / R2), respectively. The second round of PCR amplification used the two PCR products from the first round as templates, employing primers MdtDTL-F1 and MdtDTL-R2.

[0043] The amplification reaction programs for both the first and second rounds of PCR were as follows: pre-denaturation at 94℃ for 5 minutes; cycling parameters were 94℃ denaturation for 40 seconds, 56℃ annealing for 40 seconds, 72℃ extension for 90 seconds, for 35 cycles; and a full extension at 72℃ for 10 minutes.

[0044] MdtDTL(A)-F1:5'-ATGGATAATCATCCAATATC-3'; (SEQ ID NO.9) MdtDTL(A)-R1: 5'-GAGAGGGAGAAGTGGGGCTT-3'; (SEQ ID NO.10) MdtDTL(A)-F2: 5'-CCCAAAGCCCCACTTCTCC-3'; (SEQ ID NO.11) MdtDTL(A)-R2: 5'-AACTGGTTCACTTGTCTTA-3'; (SEQ ID NO. 12) MdtDTL(D)-F1:5'-ATGGATAATCATCCAATATC-3'; (SEQ ID NO.13) MdtDTL(D)-R1: 5'-GAGAGGGAGAAGTGGCTGTT-3'; (SEQ ID NO. 14) MdtDTL(D)-F2: 5'-CCCAAAGACCCACTTCTCC-3'; (SEQ ID NO. 15) MdtDTL(D)-R2: 5'-AACTGGTTCACTTGTCTTA-3'; (SEQ ID NO. 16) MdtDTL-F1: 5'-ATGGATAATCATCCAATATC-3'; (SEQ ID NO.17) MdtDTL-R2: 5'-AACTGGTTCACTTGTCTTA-3'. (SEQ ID NO.18) After the PCR reaction, 1.0% agarose gel electrophoresis, PCR product recovery, vector ligation, transformation, and sequencing were performed to obtain the gene encoding the mutation of threonine at position 12 of the MdtDTL protein to alanine. MdtDTL(A) The gene, whose nucleotide sequence is shown in SEQ ID NO.5; and the gene encoding the mutation of threonine at position 12 of the MdtDTL protein to aspartic acid. MdtDTL (D) The gene, whose nucleotide sequence is shown in SEQ ID NO.6.

[0045] Each containing MdtDTL Gene, MdtDTL(A) Gene, MdtDTL(D) The gene fragment was ligated between the XbaI and BamHI restriction sites of the pBI121 vector, and the expression was successfully constructed after sequencing verification. MdtDTL Recombinant gene expression vector pBI121-MdtDTL、 Express MdtDTL(A) Recombinant gene expression vector pBI121-MdtDTL(A) and expression MdtDTL(D) Recombinant gene expression vector pBI121- MdtDTL(D) .

[0046] Example 3: Obtaining callus from transgenic apples and detecting malic acid 1. Obtaining callus from genetically modified apples: The recombinant expression vector constructed in Example 2 pBI121-MdtDTL、pBI121- MdtDTL(A) and pBI121- MdtDTL(D) Agrobacterium LB4404 competent cells were transformed. PCR identification was performed, and positive colonies were selected for sequencing. Recombinant Agrobacterium cells with correct sequencing results were selected for transformation of apple callus.

[0047] The recombinant Agrobacterium cells to be infected were precipitated and suspended in MS liquid medium to a final concentration of OD0.05. 600 The concentration was 0.6. Callus obtained from *Agrobacterium tumefaciens* fruit was soaked in the above bacterial solution for about 20 minutes. The surface bacterial solution was then removed with sterile filter paper, and the callus was transferred to *Agrobacterium tumefaciens* callus culture medium and incubated in the dark at room temperature for 3 days. The infected *Agrobacterium tumefaciens* callus was washed 3-5 times with sterile water (containing 250 mg / L cephalosporin) to remove residual *Agrobacterium*. The washed callus was then spread on *Agrobacterium tumefaciens* callus selection medium. Newly grown callus was selected at least 3 times using *Agrobacterium tumefaciens* callus selection medium. The RNA from the selected resistant callus was used to obtain cDNA using a reverse transcription kit, and then qPCR was used to detect whether it was a transgenic *Agrobacterium tumefaciens* callus. Figure 2 ). Identified MdtDTL and MdtDTL (A / D) The callus from the apple tree was used for subsequent phenotypic identification.

[0048] 2. Malic acid detection in callus tissue of genetically modified apples: Take the overexpression of non-mutated cells with good growth status. MdtDTL and overexpression of unit point mutations MdtDTL (A) Gene, MdtDTL(D)0.1 g of transgenic and wild-type apple callus was ground into powder using liquid nitrogen and transferred to a 10 mL centrifuge tube. Then, 5 mL of 80% ethanol was added, and the mixture was incubated in a 75 °C water bath for 30 mins with shaking every 10 mins. After centrifugation at 4000 rpm for 10 mins, the supernatant was transferred to a 10 mL centrifuge tube and dried at 65 °C for 2 days. After drying, 1 mL of double-distilled water was added to dissolve the callus completely, and the solution was filtered through a 0.22 μm aqueous filter into a high-performance liquid chromatography (HPLC) vial. The malic acid content in the apple callus was determined using HPLC.

[0049] The results are as follows Figure 3 As shown, the results revealed that compared to wild-type (WT), callus tissue overexpressing the unmutated MdtDTL-OE showed significantly higher malic acid content; callus tissue overexpressing the non-phosphorylated mutant MdtDTL(A)-OE showed significantly lower malic acid content than MdtDTL-OE, but higher than wild-type; while callus tissue overexpressing the phosphorylated mimic mutant MdtDTL(D)-OE showed significantly higher malic acid content than the control, and even exceeded that of the MdtDTL-OE group. These results indicate that phosphorylation modification of MdtDTL can effectively enhance its biological function of promoting malic acid accumulation.

[0050] Example 4: Instantaneous transformation experiment of apple fruit To further verify the phosphorylation function of MdtDTL in apple fruit, this invention employed an apple fruit transient rotation experiment, the method of which is as follows: 1. Construction of transient overexpression vectors: Each MdtDTL Gene, MdtDTL(A) Gene, MdtDTL(D) The gene and pIR vector were homologously recombinated using the Uniclone One Step Seamless Cloning Kit (Sinopec China Ltd., Beijing) to construct transient overexpression vectors MdtDTL-pIR, MdtDTL(A)-pIR, and MdtDTL(D)-pIR.

[0051] 2. Instantaneous injection into apple fruit: The concentrations of transient overexpression vectors MdtDTL-pIR, MdtDTL(A)-pIR, and MdtDTL(D)-pIR were adjusted to 15 ng / μL, respectively. Magnesium chloride (MgCl2), 2-morpholinoethanesulfonic acid (MES), and acetylsyleugenone (ACE) were added to a final concentration of 10 mmol / L to prepare infection solutions. The pIR vector was used as a control, and infection solutions were prepared using the same method.

[0052] Apple fruit injection was performed on mature Gala apples, with 200 μL injected into each injection well.

[0053] After injection, the fruit was placed in an incubator at a constant temperature of 19°C in the dark for 7 days, and the malic acid content was then measured. Subsequently, a 1 cm cube of pulp near the injection hole was taken, and the malic acid content was determined by HPLC according to the method in Example 3.

[0054] The results are as follows Figure 4 As shown, the results revealed that compared to the control (pIR), the malic acid content in the region injected with the MdtDTL-pIR plasmid was significantly increased; the region injected with MdtDTL(A)-pIR had significantly higher malic acid content than the control but lower than that of MdtDTL(D)-pIR; and the region injected with MdtDTL(D)-pIR not only had significantly higher malic acid content than the control but also exceeded that of the MdtDTL-pIR treatment group. These results indicate that the regulatory effect of MdtDTL on malic acid accumulation is phosphorylation-dependent, meaning that the phosphorylated form of MdtDTL can significantly promote the accumulation of malic acid in apple fruits.

[0055] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of MdtDTL protein in increasing malic acid content, characterized in that, The MdtDTL protein is the protein shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The application according to claim 1, characterized in that, Increase malic acid content by overexpressing MdtDTL protein.

3. The application of the gene encoding the MdtDTL protein in the following (1) or (2): (1) Increase malic acid content; (2) Cultivate apple varieties with high malic acid content; The gene encoding the MdtDTL protein is MdtDTL A gene is a nucleic acid molecule as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, By promoting MdtDTL Gene expression is used to increase malic acid content.

5. The application according to claim 4, characterized in that, Promote MdtDTL The substance used for gene expression is any one of the following: C1) contains MdtDTL Gene expression cassettes; C2) contains MdtDTL Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdtDTL Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

6. The application of a phosphorylation site mutant of the MdtDTL protein in increasing malic acid content, characterized in that... The phosphorylation site mutant of the MdtDTL protein is MdtDTL(D). The amino acid sequence of MdtDTL(D) is shown in SEQ ID NO.

4.

7. The application according to claim 6, characterized in that, Increase malic acid content by overexpressing a phosphorylation site mutant of the MdtDTL protein.

8. Application of the gene encoding the MdtDTL protein phosphorylation site mutant in the following (1) or (2): (1) Increase malic acid content; (2) Cultivate apple varieties with high malic acid content; The gene encoding the MdtDTL protein phosphorylation site mutant is: MdtDTL(D) Genes; the stated MdtDTL(D) The nucleotide sequence of the gene is shown in SEQ ID NO.

6.

9. The application according to claim 8, characterized in that, By promoting MdtDTL(D) Gene expression is used to increase malic acid content.

10. The application according to claim 9, characterized in that, Promote MdtDTL(D) The substance used for gene expression is any one of the following: C1) contains MdtDTL(D) Gene expression cassettes; C2) contains MdtDTL(D) Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdtDTL(D) Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

Images