Application of glutamate decarboxylase protein in regulating GABA content in plants

Abstract

The present invention discloses the use of a glutamate decarboxylase protein for regulating GABA content in plants. The glutamate decarboxylase protein is selected from the following proteins: a1) a protein having the amino acid sequence of SEQ ID No. 2 in the sequence listing; a2) a protein having the same function as the protein in a1) by substitution, deletion, and / or addition of one or more amino acid residues; and a3) a protein having at least 75% identity with the amino acid sequence defined in a1) or a2) and having the same function. When expressed in plants, the glutamate decarboxylase protein can regulate the γ-aminobutyric acid content in the plants without affecting the phenotype of the offspring.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to application of glutamate decarboxylase protein in regulating plant GABA content. Background Art

[0002] Watermelon (Citrullus lanatus), a member of the genus Citrullus in the Cucurbitaceae family, is a widely cultivated and consumed horticultural crop worldwide. Its cultivated area accounts for 7% of the world's vegetable crop area, with an annual global production exceeding 100 million tons. As one of the world's major producers and consumers of watermelon, the development of the watermelon industry has played a significant role in agricultural development and increasing farmers' incomes. Gamma-aminobutyric acid (GABA) has an inhibitory effect on the central nervous system, promoting relaxation and calmness, thereby helping to alleviate anxiety and improve sleep quality. The GABA content in watermelon is negligible in nature. Increasing the activity of GABA synthase (also known as glutamate decarboxylase (GAD)) can promote GABA synthesis. However, increasing GABA content in watermelon by altering GAD activity has not been reported.

[0003] In recent years, the emergence of gene-editing technologies like CRISPR / Cas9 has made the application of genetic engineering in agriculture more efficient and precise. Using these technologies, researchers can target or activate genes related to GABA synthesis and metabolism, significantly increasing GABA levels in plants. GAD is a key enzyme in GABA synthesis.

[0004] The effects of genetic engineering techniques vary across different plants. Different plants respond differently to gene editing and transgenics, resulting in inconsistent increases in GABA content or phenotypic changes in subsequent generations. Therefore, it is necessary to develop specific genetic engineering strategies for each plant to achieve plants with unchanged phenotypes and increased GABA content.

[0005] Therefore, providing a method for increasing the GABA content in a new germplasm of a plant such as watermelon is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] To solve the above problems, the present invention provides a use of a glutamate decarboxylase protein in regulating the GABA content in plants. The glutamate decarboxylase protein of the present invention can regulate the GABA content when expressed in Cucurbitaceae plants, and the phenotype of the plant offspring remains unchanged.

[0007] One aspect of the present invention provides a glutamate decarboxylase protein, which is any one of the following:

[0008] a1) a protein having the amino acid sequence of SEQ ID No. 2;

[0009] a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are substituted and / or deleted and / or added;

[0010] a3) a protein that has an amino acid sequence identity of at least 75% with that described in a1) or a2) and has the same function;

[0011] a4) A fusion protein obtained by connecting a tag to the end of any protein described in a1) to a3).

[0012] In the present invention, the glutamate decarboxylase protein (GAD) can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0013] In the present invention, the glutamate decarboxylase protein is derived from watermelon. In a specific embodiment of the present invention, the glutamate decarboxylase protein is derived from Citrullus lanatus.

[0014] In the present invention, the above-mentioned 75% or more identity may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0015] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence or nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, you can calculate the identity of a pair of amino acid or nucleotide sequences by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be calculated.

[0016] In the present invention, those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the glutamate decarboxylase protein of the present invention by known methods, such as directed evolution or point mutation. Artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the isolated glutamate decarboxylase protein of the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as they encode the glutamate decarboxylase protein and have the function of the glutamate decarboxylase protein.

[0017] In the present invention, in order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0018] In the present invention, the tag protein can be conventional in the art, including but not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0019] Another aspect of the present invention provides a nucleic acid molecule encoding the glutamate decarboxylase protein.

[0020] In the present invention, the nucleic acid molecule may be any of the following:

[0021] b1) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No. 1;

[0022] b2) the coding sequence is the nucleic acid molecule shown in SEQ ID No. 3;

[0023] b3) has 90% or more identity with the nucleotide sequence described in b1) or the coding sequence described in b2);

[0024] b4) A nucleic acid molecule that hybridizes under stringent conditions to the nucleotide sequence described in b1) or the coding sequence described in b2).

[0025] Another aspect of the present invention provides a glutamate decarboxylase protein mutant, which is any one of the following:

[0026] c1) obtained by knocking out the inhibitory domain of the glutamate decarboxylase protein;

[0027] c2) a protein having an amino acid sequence of SEQ ID No. 9;

[0028] c3) a protein having the same function as that of the amino acid sequence shown in SEQ ID No. 9, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0029] c4) Proteins that have an amino acid sequence identity of at least 75% with that specified in c2) or c3) and have the same function;

[0030] c5) A fusion protein obtained by connecting a tag to the end of any protein described in c1) to c4).

[0031] In the present invention, the glutamate decarboxylase protein mutant is ClGAD2.

[0032] In the present invention, the glutamate decarboxylase protein mutant can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0033] In the present invention, the glutamate decarboxylase protein mutant is derived from watermelon. In a specific embodiment of the present invention, the wild-type sequence of the glutamate decarboxylase protein mutant is derived from Citrullus lanatus.

[0034] In the present invention, the above-mentioned 75% or more identity may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0035] In the present invention, those skilled in the art can readily mutate the nucleotide sequence encoding the ClGAD2 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence of the isolated ClGAD2 protein of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the ClGAD2 protein and possess the function of the ClGAD2 protein.

[0036] In the present invention, in order to facilitate purification or detection of the protein in c2), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 9 in the sequence listing.

[0037] Another aspect of the present invention provides a nucleic acid molecule encoding the glutamate decarboxylase protein mutant.

[0038] In the present invention, the nucleic acid molecule may be any of the following:

[0039] d1) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No. 7;

[0040] d2) the coding sequence is the nucleic acid molecule shown in SEQ ID No. 8;

[0041] d3) having 90% or more identity with the nucleotide sequence described in d1) or the coding sequence described in d2);

[0042] d4) hybridizes with the nucleotide sequence described in d1) or the coding sequence described in d2) under stringent conditions.

[0043] In a specific implementation of the present invention, the nucleic acid molecule shown in SEQ ID No. 8 is obtained by gene knockout of the nucleic acid molecule shown in SEQ ID No. 3.

[0044] Gene knockout refers to the inactivation of a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence. The gene editing technology preferably utilizes the CRISPR / Cas9 gene editing system.

[0045] The target gene described in a preferred embodiment of the present invention is located in the exon region of the nucleic acid molecule shown in SEQ ID No. 3.

[0046] In a preferred embodiment of the present invention, the target gene comprises the sequence shown in SEQ ID No. 4 and / or the sequence shown in SEQ ID No. 5. Specifically, the gene knockout comprises mutating the sequence shown in SEQ ID No. 4 to 5'-GATATCAAAACGACGGGC-3' and / or mutating the sequence shown in SEQ ID No. 5 to 5'-GACACAGAAGAACGATGG-3'.

[0047] Another aspect of the present invention provides a biomaterial, wherein the biomaterial contains the nucleic acid molecule; the biomaterial is any one of the following:

[0048] e1) an expression cassette containing the nucleic acid molecule;

[0049] e2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in e1);

[0050] e3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in e1), or a recombinant microorganism containing the recombinant vector described in e2);

[0051] e4) a transgenic plant cell line containing the nucleic acid molecule, or a transgenic plant cell line containing the expression cassette described in e1);

[0052] e5) transgenic plant tissue containing the nucleic acid molecule, or transgenic plant tissue containing the expression cassette described in e1);

[0053] e6) a transgenic plant organ containing the nucleic acid molecule, or a transgenic plant organ containing the expression cassette described in e1);

[0054] g1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the glutamate decarboxylase protein;

[0055] g2) an expression cassette containing the nucleic acid molecule described in g1);

[0056] g3) a recombinant vector containing the nucleic acid molecule described in g1), or a recombinant vector containing the expression cassette described in g2);

[0057] g4) a recombinant microorganism containing the nucleic acid molecule described in g1), or a recombinant microorganism containing the expression cassette described in g2), or a recombinant microorganism containing the recombinant vector described in g3);

[0058] g5) a transgenic plant cell line containing the nucleic acid molecule described in g1) or a transgenic plant cell line containing the expression cassette described in g2);

[0059] g6) transgenic plant tissue containing the nucleic acid molecule described in g1) or transgenic plant tissue containing the expression cassette described in g2);

[0060] g7) A transgenic plant organ containing the nucleic acid molecule described in g1) or a transgenic plant organ containing the expression cassette described in g2).

[0061] Optionally, the expression cassette described in g2) is an expression cassette having a DNA molecule shown as SEQ ID No. 6.

[0062] In the present invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0063] The vectors described herein are well known to those skilled in the art, including but not limited to plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector pHSE401 may be used.

[0064] A recombinant expression vector containing the ClGAD2 gene is constructed using existing plant expression vectors. Such plant expression vectors include, but are not limited to, binary Agrobacterium vectors or vectors suitable for plant microprojectile bombardment. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, namely, a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples of such untranslated regions include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the rouge synthase Nos gene) and plant genes (such as the soybean storage protein gene), which have similar functions.

[0065] When using the ClGAD2 gene to construct a recombinant plant expression vector, any enhancing or constitutive promoter can be added before its transcriptional initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when using the gene of the present invention to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be the ATG start codon or an adjacent region start codon, but must be in frame with the coding sequence to ensure correct translation of the entire sequence. The sources of translational control signals and initiation codons are diverse and can be either natural or synthetic. The translation initiation region can be derived from the transcriptional initiation region or a structural gene.

[0066] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For safety reasons, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0067] Transgenic cell lines and transgenic plants with altered fertility can be obtained by introducing the ClGAD2 gene or a fragment thereof into plant cells or recipient plants using any vector capable of directing exogenous gene expression in plants. Expression vectors carrying the ClGAD2 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0068] Another aspect of the present invention further provides a use of the glutamate decarboxylase protein or the glutamate decarboxylase protein mutant or the nucleic acid molecule or the biomaterial in any of the following:

[0069] 1) Application in regulating GABA content in plants;

[0070] 2) Application in the preparation of products for regulating plant GABA content;

[0071] 3) Application in the cultivation of plants with altered GABA content;

[0072] 4) Use in the preparation of products for cultivating plants with altered GABA content;

[0073] 5) Application in plant breeding.

[0074] In the present invention, the regulation may be increasing or decreasing the GABA content in the plant.

[0075] Another aspect of the present invention further provides a use of a substance for regulating gene expression or a substance for regulating the activity or content of the glutamate decarboxylase protein (or the glutamate decarboxylase protein mutant) in any of the following:

[0076] 1) Application in regulating GABA content in plants;

[0077] 2) Application in the preparation of products for regulating plant GABA content;

[0078] 3) Application in cultivating plants with increased GABA content;

[0079] 4) Application in the preparation of products for cultivating plants with increased GABA content;

[0080] 5) Application in plant breeding;

[0081] Wherein, the gene encodes the glutamate decarboxylase protein or the glutamate decarboxylase protein mutant.

[0082] In the present invention, the substance that regulates the activity or content of the glutamate decarboxylase protein (or the glutamate decarboxylase protein mutant) may be a substance that regulates gene expression.

[0083] In the present invention, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation:

[0084] 1) Regulation at the transcriptional level of the gene;

[0085] 2) post-transcriptional regulation of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene);

[0086] 3) Regulation of RNA transport of the gene (i.e., regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm);

[0087] 4) regulation of the translation of the gene;

[0088] 5) regulation of mRNA degradation of the gene;

[0089] 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0090] In the present invention, the regulation and control may be upward regulation, enhancement or improvement. The upward regulation, enhancement or improvement of the expression level of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the recipient plant, or / and the upward regulation, enhancement or improvement of the activity and / or content of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant are achieved by introducing the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant into the recipient plant.

[0091] In the present invention, the regulation may also be down-regulation, weakening or reduction of the expression of the coding gene. Down-regulation, weakening or reduction of the expression of the coding gene may be achieved by gene knockout or gene silencing.

[0092] In the present invention, the substance that regulates gene expression or the substance that regulates the activity or content of the glutamate decarboxylase protein (or the glutamate decarboxylase protein mutant) may be the biomaterial.

[0093] Another aspect of the present invention further provides a method for increasing the GABA content in plants, which comprises expressing the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant.

[0094] In the present invention, the expression can be to suppress, reduce, enhance or increase the expression of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the plant, and / or, can be to suppress, reduce, enhance or increase the activity and / or content of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the plant to increase the GABA content of the plant.

[0095] Another aspect of the present invention provides a method for cultivating plants with increased GABA content, comprising expressing the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the plant to obtain plants with increased GABA content.

[0096] In one embodiment of the present invention, the method for cultivating a plant with altered GABA content comprises the following steps:

[0097] (1) constructing a recombinant expression vector of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant;

[0098] (2) The recombinant expression vector constructed in step (1) is transferred into a recipient plant to obtain a plant with altered GABA content.

[0099] In the present invention, the GABA content may be specifically expressed as: the GABA content is increased by more than 10%, for example, more than 300%.

[0100] In the present invention, the plant may be as follows:

[0101] f1) monocots or dicots;

[0102] f2) Cucurbitales;

[0103] f3) Cucurbitaceae;

[0104] f4) Citrullus;

[0105] f5) Watermelon.

[0106] Positive progress: The present invention deletes a specific site in the gene encoding the glutamate decarboxylase protein and introduces it into plants for expression, resulting in plants with significantly increased GABA content. Compared with wild-type plants, the phenotype is normal and stable, with no visible phenotypic changes in female flowers, leaves, tendrils, stems, roots, or growth potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG1 is a schematic diagram of the ClGAD2 gene structure and target location in Example 1; in the ClGAD2 gene structure diagram, Target1 and Target2 are both located in the last exon (indicated by the red line).

[0108] Figure 2 is a map of the pHSE401 vector in Example 1.

[0109] Figure 3 shows the alignment results of the glutamate decarboxylase protein gene at two target sites between the WT and positive ClGAD2 gene-edited strains in Example 1; where - indicates deletion; the target site sequence and PAM sequence are marked with black and red letters, respectively.

[0110] Figure 4 shows the watermelon fruit phenotypes of the wild-type WT and positive ClGAD2 gene-edited lines in Example 2.

[0111] FIG5 is a phenotypic comparison of GABA content in wild-type WT and positive ClGAD2 gene-edited watermelon lines in Example 2. DETAILED DESCRIPTION

[0112] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0113] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques and conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples, unless otherwise noted, were commercially available.

[0114] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0115] The pCBC-DT1T2 used in the following examples is described in: Tian, ​​S., Jiang, L., Gao, Q. et al. Efficient CRISPR / Cas9-based gene knockout in watermelon. Plant Cell Rep 36, 399–406 (2017). https: / / doi.org / 10.1007 / s00299-016-2089-5. The public may obtain this biological material from the applicant for use solely for replicating the experiments described herein and not for any other purpose.

[0116] pHSE401 in the following examples was purchased from addgene: https: / / www.addgene.org / 62201 / .

[0117] The watermelon germplasm material 'WT' used in the following examples is described in Ren et al., Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits, THE PLANT CELL 2021: 33: 1554–1573. The public may obtain this biological material from the applicant for use solely for replicating the experiments described herein and not for any other purpose.

[0118] Example 1. Obtaining ClGAD2 gene knockout mutants

[0119] 1. Design of editing sites for GAD (Cla009138)

[0120] Target gene sites for gene editing were designed based on the GAD genomic DNA sequence (SEQ ID No. 1) and the online target gene site design website CRISPR P (http: / / crispr.hzau.edu.cn / CRISPR2 / news.php, V2). Two sets of editing sites, Target1 and Target2, were designed. A schematic diagram of the GAD gene structure is shown in Figure 1.

[0121] The genomic sequence encoding the GAD protein is shown in SEQ ID No. 1 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID No. 2 in the sequence listing. The CDS encoding the GAD protein is shown in SEQ ID No. 3 in the sequence listing. The target gene sites Target1 and Target2 are located in the last exon of the GAD gene. Target-1 sequence: 5'-GATATCAAAACGACGGTGGC-3' (SEQ ID No. 4);

[0122] Target-2 sequence: 5'-GACACAGAAGAACGACGTGG-3' (SEQ ID No. 5).

[0123] 2. Obtaining the target fragment

[0124] PCR amplification was performed using the intermediate vector pCBC-DT1T2 as a template and Target2-BsF / Target2-F0 / Target1-R0 / Target1-BsR (containing Target1 and Target2, respectively) as primers using Vazyme P505 high-fidelity enzyme (Phanta Max Super-Fidelity DNA Polymerase).

[0125] Amplification system (50 μL): 2x PhantaMax Buffer 25 μL, dNTP Mix (10 mM) 1 μL, Target2-BsF / Target1-BsR primers (10 μM) 2 μL each, Target2-F0 / Target1-R0 (0.5 μM) 2 μL each, PhantaMax Super-Fidelity DNA Polymerase 1 μL, template DNA 2 μL, ddH2O 13 μL.

[0126] The PCR reaction procedure was as follows: initial denaturation at 95°C for 3 minutes; 35 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 30 seconds; and final extension at 72°C for 5 minutes. The target fragment (Target-1)-(gRNA-Sc)-(U6-26t)-(U6-29p)-(Target-2) (SEQ ID No. 6) was recovered by 1% agarose gel electrophoresis. The primer sequences for Target2-BsF, Target2-F0, Target1-R0, and Target1-BsR are as follows:

[0127] Target2-BsF: 5'-AACCCACGTCGTTCTTCTGTGTCAATCTCTTAGTCGACTCTAC -3';

[0128] Target2-F0: 5'-ATTATTGGTCTCGAAACCCACGTCGTTCTTCTGTGTC -3';

[0129] Target1-R0: 5'-ATATATGGTCTCGATTGATATCAAAACGACGGTGGCGTT -3';

[0130] Target1-BsR: 5'-TGATATCAAAACGACGGTGGCGTTTTAGAGCTAGAAATAGC-3'.

[0131] 3. Construction of CRISPR / Cas9 editing vector

[0132] The CRISPR / Cas9 vector pHSE401 (see Figure 2 for a vector map) was digested and ligated using the restriction endonucleases BsaI-HF (NEB) and T4 Ligase (NEB). The digestion and recombination system (15 μL) consisted of 1.5 μL of 10xBSA, 2 μL of pHSE401 (1 μg / μL), 1 μL of BsaI-HF (NEB), 1 μL of T4 Ligase (NEB), 1.5 μL of 10xNEB T4 Buffer, 2 μL of the target fragment obtained above, and 6 μL of ddH2O. The reaction was incubated at 37°C for 5 h, 50°C for 5 min, and 80°C for 10 min. Transformation was then performed into DH5α competent cells.

[0133] Colony PCR was performed on the transformed DH5α competent cells using identification primers U626-IDF and U629-IDR. The primer sequences are as follows:

[0134] U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3';

[0135] U629-IDR: 5'-AGCCCTCTTCTTCGATCCATCAAC-3'.

[0136] After confirming that the band size was correct, the bacteria were inoculated and shaken. After sequencing, sequence alignment was performed to obtain the recombinant plasmid pHSE401-sgRNA1-sgRNA2.

[0137] The structure of pHSE401-sgRNA1-sgRNA2 is described as follows: a recombinant vector is obtained by replacing the small fragment between the restriction endonuclease BsaI-HF recognition site and the T4 Ligase recognition site of the starting vector pHSE401 with the DNA molecule of SEQ ID No. 6, while keeping the other sequences of the starting vector unchanged.

[0138] The sequenced recombinant plasmid pHSE401-sgRNA1-sgRNA2 was transformed into competent Agrobacterium tumefaciens EHA105. PCR detection of competent Agrobacterium tumefaciens EHA105 was performed using primers U626-IDF and U626-IDR, and the positive Agrobacterium clone EHA105 / pHSE401-sgRNA1-sgRNA2 was obtained.

[0139] 4. Watermelon genetic transformation

[0140] Sowing: Take 50 seeds of the laboratory watermelon germplasm 'WT' and soak them in a 55°C waterbath for 30 minutes. Then remove the husks. In a clean bench, disinfect the peeled seed kernels with 75% alcohol for 1 minute, then soak them in 3% sodium hypochlorite for 15 minutes. Rinse five times with sterile water. Spread the kernels onto a breeding medium (BM; H2O, 6g / L Agar) and incubate them in the dark at 25°C for approximately 3 days.

[0141] Inoculation: Pick a single colony of the Agrobacterium-positive clone EHA105 / pHSE401-sgRNA1-sgRNA2 verified by colony PCR into LB liquid culture medium containing 50 mg / L kanamycin and 25 mg / L rifampicin. When the bacterial solution concentration reaches OD600 0.6-0.8, centrifuge at 5000 rpm for 5 minutes to collect the bacteria. Discard the supernatant and resuspend the bacteria in MS liquid culture medium (MS + 30% sucrose + 6-BA) to a final concentration of OD600 = 0.4.

[0142] Dip Infection: When the seed kernel radicle grows to approximately 1 cm, cut off the ends of the cotyledons and cut the explant into small pieces for dipping. Mix the cut cotyledons in a 20 mL syringe containing 10 mL of resuspended bacterial solution and dip under negative pressure for 15 minutes. Remove the explant and air dry on sterile filter paper. Transfer the explant to co-culture medium (CM; MS + 30% sucrose + Phytagel + 6-BA) lined with filter paper and incubate at 28°C in the dark for 3 days. MS was purchased from Phytotech, and 6-BA from Yuanye.

[0143] Recovery culture: After 3 days of co-cultivation, the cotyledon pieces were transferred to recovery medium (RM; MS + 30% sucrose + phytagel + 6-BA + timentin) and cultured at 28°C, with a light intensity of 20,000 lux and a light duration of 16 h / d for 7 days.

[0144] Selective culture: After the recovery culture is completed, the explants are transferred to selective medium (SM; MS + 30% sucrose + agar + 6-BA + timentin + Basta) for selective culture and subcultured at 28°C for 3-4 weeks, with subculture every 12 days.

[0145] Shoot elongation culture: GFP-tagged explants with obvious buds were transferred to seedling elongation medium (SE; MS + 30% sucrose + agar + 6-BA + Timentin) for culture.

[0146] Rooting culture: The selected buds were transferred to MS medium containing IAA and timentin for rooting culture and cultured at 28℃ until roots formed.

[0147] Transplanting: When the regenerated seedlings take root and grow 4-5 true leaves, take them out of the culture bottle, carefully remove the culture medium at the roots, and transplant them into a flowerpot with a substrate: vermiculite ratio of 1:3. Water thoroughly to keep warm and moisturize, and then manage normally after cultivation.

[0148] The MS medium used in the experiment was PhytoTech M519.

[0149] Finally, a positive ClGAD2 gene-edited strain was obtained.

[0150] 5. Detection of editing in positive ClGAD2 gene plant lines

[0151] Regenerated seedlings of the watermelon ClGAD2 gene-edited line containing the pHSE401-sgRNA1-sgRNA2 vector were sampled, and genomic DNA was extracted using the CTAB method.

[0152] Specific steps: Take a small amount of young watermelon leaves and quickly grind them into powder in liquid nitrogen, and place them in a 1.5mL centrifuge tube; add preheated 800μL CTAB extraction buffer and incubate in a 65℃ water bath for 30min; add an equal volume of chloroform and isopropanol (the volume ratio of chloroform to isopropanol is 24:1), mix well, and centrifuge at 8000r / min for 10min; transfer the supernatant to a new 1.5mL centrifuge tube, add 2 / 3 volume of isopropanol, mix gently by inverting, and place in a -20℃ refrigerator to precipitate for 1h; centrifuge at 10000r / min for 10min; discard the supernatant, rinse the precipitate twice with 75% volume ethanol, pour it out, absorb the remaining liquid, dry it at room temperature, dissolve it with 100μL ddH2O (containing 0.1% RNase), and store it at 4℃ for later use.

[0153] The extracted genomic DNA was used as a template and primers ClGAD2-F / ClGAD2-R were used to amplify the sequence containing the two target sites by PCR.

[0154] The specific primer sequences for ClGAD2-IDF1 / ClGAD2-IDR1 and ClGAD2-IDF2 / ClGAD2-IDR2 are as follows:

[0155] ClGAD2-F: 5'-ACGTGATGGAAAACTGCAAAGA-3';

[0156] ClGAD2-R: 5'-TTTCCATCACAAACTTCCTCCA -3';

[0157] Amplification system (50 μL) and reaction procedure: 2×T5 SuperMIX 25 μL, 2 μL of each primer (10 μM), 1 μL of template DNA, and 20 μL of ddH2O.

[0158] PCR reaction procedure: initial denaturation at 98°C for 3 minutes; 35 cycles of denaturation at 98°C for 10 seconds, annealing at 58°C for 10 seconds, and extension at 72°C for 15 seconds; and final extension at 72°C for 5 minutes. Band size was determined by electrophoresis on a 1% agarose gel. The remaining PCR product was sequenced using primers ClGAD2-F / ClGAD2-R.

[0159] The target editing comparison results of the ClGAD2 mutant are shown in Figure 3, where ClGAD2 has a 2bp deletion at the Target1 target site and a 2bp deletion at the Target2 target site.

[0160] Compared with the wild type (WT), the mutant ClGAD2 gene in the two homologous chromosomes was obtained by the following mutations: the "5'-GATATCAAAACGACGGTGGC-3'" in the GAD gene of the wild type WT was mutated to "5'-GATATCAAAACGACGGGC-3'", which was deleted by two nucleotides; the "5'-GACACAGAAGAACGACGTGG-3'" was mutated to "5'-GACACAGAAGAACGATGG-3'", which was deleted by two nucleotides;

[0161] Deletion of the above nucleotides results in ClGAD2 with enhanced protein function.

[0162] Example 2 Phenotypic Observation of ClGAD2 Gene-Edited Plants

[0163] The positive ClGAD2 gene-edited strains obtained in Example 1 (the ClGAD2 genes obtained from two repeated experiments in the same batch were recorded as glgad2-1 and glgad2-2, respectively) and wild-type watermelon WT were planted in a greenhouse at the Beijing base in the spring of 2024 and managed normally. After the fruits matured, their phenotypes were observed. As shown in Figure 4, there was no significant difference in the morphology of plants containing the ClGAD2 gene compared with the wild type.

[0164] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to measure GABA content in plants harboring the ClGAD2 gene and wild-type watermelon (WT). The results are shown in Figure 5. gad2-1 and gad2-2 represent the glgad2-1 and glgad2-2 gene-edited lines, respectively. The results showed that compared to the WT watermelon (WT), plants harboring the ClGAD2 gene had a threefold increase in GABA content. The strains were stable, and repeated experiments yielded consistent results.

[0165] The specific experimental conditions for liquid chromatography tandem mass spectrometry (LC-MS / MS) are as follows:

[0166] Sample pretreatment: (1) Weigh 50 mg (± 2.5 mg) of sample (unless otherwise specified, plant samples are assumed to be freeze-dried and animal samples are assumed to be fresh) into a 2 mL centrifuge tube and record the weight of each sample; (2) Immediately add 500 μL of -20°C pre-cooled 70% methanol-water extract to the weighed sample and vortex for 3 min; (3) Centrifuge at 12,000 r / min for 10 min at 4°C and aspirate 300 μL of the supernatant into a 1.5 mL centrifuge tube; (4) Place in a -20°C refrigerator for 30 min and centrifuge again at 4°C, 12,000 r / min for 10 min; (5) Take 200 μL of the supernatant after centrifugation and pass it through a protein precipitation plate for analysis on the machine. The supernatant was stored at -20°C.

[0167] Chromatography and mass spectrometry acquisition conditions: The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP® 6500+, https: / / sciex.com.cn / ). The liquid phase conditions mainly include: 1) chromatographic column: ACQUITY BEH Amide column (1.7 µm, 100 mm × 2.1 mm id); 2) mobile phase: phase A, ultrapure water (containing 2 mM ammonium acetate, 0.04% formic acid); phase B, acetonitrile (containing 2 mM ammonium acetate, 0.04% formic acid); 3) gradient elution program: A / B ratio of 10:90 (V / V) from 0 to 1.2 min, 40:60 (V / V) from 9 min, 60:40 (V / V) from 10 to 11 min, and 10:90 (V / V) from 11.01 to 15 min; 4) flow rate, 0.4 mL / min; column temperature, 40°C; injection volume, 2 μL.

[0168] Mass spectrometry conditions included an electrospray ionization (ESI) source temperature of 550°C, a mass spectrometer voltage of 5500 V in positive ion mode, a mass spectrometer voltage of -4500 V in negative ion mode, and a curtain gas (CUR) of 35 psi. In a Q-Trap 6500+, each ion transition was scanned based on optimized declustering potential (DP) and collision energy (CE).

[0169] Quantitation is accomplished using a triple quadrupole mass spectrometer in Multiple Reaction Monitoring (MRM) mode. In MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, eliminating ions corresponding to other molecular weight substances to preliminarily eliminate interference. The precursor ions are ionized by the collision chamber and fragmented into multiple fragment ions. These fragment ions are then filtered by the triple quadrupole to select the desired characteristic fragment ions, eliminating non-target ion interference and ensuring more accurate and reproducible quantitation. After obtaining mass spectrometry data for different samples, the chromatographic peaks of all target substances are integrated, and quantitative analysis is performed using a standard curve.

[0170] In summary, the present invention provides a method for creating new watermelon germplasm with increased GABA content through gene editing technology. By editing the GABA content regulatory gene GAD, varieties with increased GABA content can be quickly obtained, which has important application potential in the improvement of high-quality new watermelon varieties.

[0171] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A glutamate decarboxylase protein mutant, characterized in that The amino acid sequence of the protein is SEQ ID No.

8.

2. A nucleic acid molecule encoding the glutamate decarboxylase protein mutant according to claim 1; wherein the nucleic acid molecule is a nucleic acid molecule whose coding sequence is shown in SEQ ID No.

7.

3. The biological material containing the nucleic acid molecule according to claim 2, characterized in that The biological material is any one of the following: e1) an expression cassette containing the nucleic acid molecule; e2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in e1); e3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette of e1), or a recombinant microorganism containing the recombinant vector of e2).

4. Use of the glutamate decarboxylase protein mutant according to claim 1, the nucleic acid molecule according to claim 2, or the biomaterial according to claim 3 in any of the following: 1) Application in increasing GABA content in watermelon; 2) Application in the preparation of products with increased GABA content in watermelon; 3) Application in cultivating watermelons with increased GABA content; 4) Application in the preparation of products for cultivating watermelons with increased GABA content; 5) Application in watermelon breeding.

5. A method for increasing the GABA content of watermelon, characterized in that: The method comprises expressing the coding gene of the glutamate decarboxylase protein mutant as claimed in claim 1 to increase the GABA content of the watermelon.

6. A method for cultivating watermelon with increased GABA content, characterized in that: The method comprises expressing the coding gene of the glutamate decarboxylase protein mutant as claimed in claim 1, thereby obtaining a watermelon with increased GABA content.

7. The method for cultivating watermelon with increased GABA content as claimed in claim 6, wherein: The method comprises the following steps: (1) constructing a recombinant expression vector of the gene encoding the glutamate decarboxylase protein mutant according to claim 1; and (2) transferring the recombinant expression vector constructed in step (1) into a recipient watermelon to obtain a watermelon with increased GABA content.

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