Application of glutamate decarboxylase protein in regulating and controlling GABA (gamma-aminobutyric acid) content of plants

Abstract

The invention discloses an application of a glutamate decarboxylase protein in regulating and controlling the GABA content of a plant. The glutamate decarboxylase protein is the following protein a1), a2) or a3): a1) the protein of which the amino acid sequence is SEQ ID No.2 in a sequence table; a2) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues to the protein in a1) and has the same function; a3) protein which has more than 75% of identity with the amino acid sequence limited in a1) or a2) and has the same function. Expression of the glutamate decarboxylase protein in plants can regulate and control the content of gamma-aminobutyric acid in the plants and does not affect phenotypes of offspring.

Description

Technical Field

[0001] The 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 crop of the genus Citrullus in the Cucurbitaceae family, is a widely cultivated and edible horticultural crop worldwide. Its planting area accounts for 7% of the world's vegetable crop planting area, and the global annual output exceeds 100 million tons. As one of the world's important watermelon producers and consumers, the development of the watermelon industry has played an important role in my country's agricultural development and the increase of farmers' income. γ-Aminobutyric acid (GABA) has an inhibitory effect on the central nervous system, which can make people feel relaxed and calm, thereby helping to reduce anxiety and improve sleep quality to a certain extent. The GABA content of watermelon in nature is negligible. Increasing the activity of γ-aminobutyric acid synthase (GABA synthase), also known as glutamate decarboxylase (GAD), can promote the synthesis of GABA. There has been no report on increasing the GABA content in watermelon by changing the activity of glutamate decarboxylase.

[0003] In recent years, the emergence of gene editing technologies such as CRISPR / Cas9 has made the application of genetic engineering technology in agriculture more efficient and precise. Through these technologies, researchers can target or activate genes related to GABA synthesis and metabolism, thereby significantly increasing the GABA content in plants. GAD is the key enzyme for the synthesis of GABA.

[0004] The effects of genetic engineering technology on different plants vary. Different plants respond differently to gene editing and transgenics, resulting in different effects on the increase of GABA content or affecting the phenotype of plant offspring. Therefore, it is necessary to develop specific genetic engineering strategies for different plants to obtain plants with no obvious changes in phenotype and 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] In order to solve the above problems, the present invention provides an application of glutamate decarboxylase protein in regulating the GABA content of plants. The glutamate decarboxylase protein of the present invention can regulate the GABA content when expressed in Cucurbitaceae plants, and the phenotype of the offspring of the plants remains unchanged.

[0007] One aspect of the present invention provides a glutamate decarboxylase protein, which is any one of the following: a1) a protein having an amino acid sequence of SEQ ID No. 2; a2) a protein having the same function as the one shown in SEQ ID No. 2 after one or more amino acid residues are replaced and / or deleted and / or added; a3) a protein that has an amino acid sequence identity of more than 75% with that of a1) or a2) and has the same function; a4) A fusion protein obtained by connecting a tag to the end of any protein described in a1) to a3).

[0008] In the present invention, the glutamate decarboxylase protein (GAD) can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

[0009] 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.

[0010] In the present invention, the above 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.

[0011] In this article, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences or nucleotide sequences, the identity value (%) can be obtained.

[0012] In the present invention, those of ordinary skill in the art can easily adopt known methods, such as directed evolution or point mutation methods, to mutate the nucleotide sequence encoding the glutamate decarboxylase protein of the present invention. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the glutamate decarboxylase protein isolated by the present invention, as long as they encode the glutamate decarboxylase protein and have the function of the glutamate decarboxylase protein, are all derived from the nucleotide sequence of the present invention and are equal to the sequence of the present invention.

[0013] In the present invention, in order to facilitate the purification or detection of the protein in a1), a tag protein may be connected to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0014] In the present invention, the tag protein may 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.

[0015] Another aspect of the present invention provides a nucleic acid molecule encoding the glutamate decarboxylase protein.

[0016] In the present invention, the nucleic acid molecule may be any of the following: b1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID No. 1; b2) the coding sequence is the nucleic acid molecule shown in SEQ ID No. 3; b3) has 90% or more identity with the nucleotide sequence described in b1) or with the coding sequence described in b2); b4) A nucleic acid molecule that hybridizes to the nucleotide sequence described in b1) or the coding sequence described in b2) under stringent conditions.

[0017] Another aspect of the present invention provides a glutamate decarboxylase protein mutant, which is any one of the following: c1) obtained by knocking out the inhibitory domain of the glutamate decarboxylase protein; c2) a protein having an amino acid sequence of SEQ ID No. 9; c3) a protein having the same function as that of SEQ ID No. 9, wherein one or more amino acid residues are substituted and / or deleted and / or added; c4) Proteins that have an amino acid sequence identity of more than 75% with that specified in c2) or c3) and have the same function; c5) A fusion protein obtained by connecting a tag to the end of any protein described in c1) to c4).

[0018] In the present invention, the glutamate decarboxylase protein mutant is ClGAD2.

[0019] In the present invention, the glutamate decarboxylase protein mutant can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

[0020] 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.

[0021] In the present invention, the above 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.

[0022] In the present invention, a person skilled in the art can easily mutate the nucleotide sequence encoding protein ClGAD2 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of protein ClGAD2 isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode protein ClGAD2 and have the function of protein ClGAD2.

[0023] 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.

[0024] Another aspect of the present invention provides a nucleic acid molecule encoding the glutamate decarboxylase protein mutant.

[0025] In the present invention, the nucleic acid molecule may be any of the following: d1) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No.7; d2) the coding sequence is the nucleic acid molecule shown in SEQ ID No.8; d3) has 90% or more identity with the nucleotide sequence described in d1) or with the coding sequence described in d2); d4) hybridizes with the nucleotide sequence described in d1) or the coding sequence described in d2) under stringent conditions.

[0026] 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.

[0027] Wherein, the gene knockout refers to the phenomenon of inactivating a specific target gene by gene editing technology. The gene knockout inactivates a specific target gene by changing the DNA sequence. The gene editing technology is preferably a technology using the CRISPR / Cas9 gene editing system.

[0028] The target gene described in the preferred embodiment of the present invention is located in the exon region of the nucleic acid molecule shown in SEQ ID No.3.

[0029] In a preferred embodiment of the present invention, the target gene includes the sequence shown in SEQ ID No. 4 and / or the sequence shown in SEQ ID No. 5. Specifically, the gene knockout is to mutate the sequence shown in SEQ ID No. 4 to 5'-GATATCAAAACGACGGGC-3'; and / or, to mutate the sequence shown in SEQ ID No. 5 to 5'-GACACAGAAGAACGATGG-3'.

[0030] 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: 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 described in e1), or a recombinant microorganism containing the recombinant vector described in e2); e4) a transgenic plant cell line containing the nucleic acid molecule, or a transgenic plant cell line containing the expression cassette described in e1); e5) transgenic plant tissue containing the nucleic acid molecule, or transgenic plant tissue containing the expression cassette described in e1); e6) a transgenic plant organ containing the nucleic acid molecule, or a transgenic plant organ containing the expression cassette described in e1); g1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the glutamate decarboxylase protein; g2) an expression cassette containing the nucleic acid molecule described in g1); g3) a recombinant vector containing the nucleic acid molecule described in g1), or a recombinant vector containing the expression cassette described in g2); 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); 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); g6) transgenic plant tissue containing the nucleic acid molecule described in g1) or transgenic plant tissue containing the expression cassette described in g2); 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).

[0031] Optionally, the expression cassette described in g2) is an expression cassette having a DNA molecule shown in SEQ ID No.6.

[0032] 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.

[0033] 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, etc.), cosmids (ie, cosmids), Ti plasmids or viral vectors. Specifically, the vector may be pHSE401.

[0034] A recombinant expression vector containing the ClGAD2 gene is constructed using an existing plant expression vector. The plant expression vector includes, but is not limited to, a binary Agrobacterium vector or a vector that can be used for plant microprojectile bombardment. The plant expression vector may also contain a 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as rouge synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0035] When using the ClGAD2 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0036] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes including but not limited to enzymes or luminescent compounds that can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0037] By using any vector that can guide the expression of foreign genes in plants, the ClGAD2 gene or a fragment of its gene is introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered fertility. The expression vector carrying the ClGAD2 gene can be used to transform plant cells or tissues by 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.

[0038] Another aspect of the present invention also provides an application of the glutamate decarboxylase protein or the glutamate decarboxylase protein mutant or the nucleic acid molecule or the biomaterial in any of the following: 1) Application in regulating plant GABA content; 2) Application in the preparation of products for regulating plant GABA content; 3) Application in the cultivation of plants with altered GABA content; 4) Use in the preparation of products for cultivating plants with altered GABA content; 5) Application in plant breeding.

[0039] In the present invention, the regulation may be increasing or decreasing the GABA content in the plant.

[0040] 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: 1) Application in regulating plant GABA content; 2) Application in the preparation of products for regulating plant GABA content; 3) Application in breeding plants with increased GABA content; 4) Application in the preparation of products for cultivating plants with increased GABA content; 5) Application in plant breeding; Wherein, the gene encodes the glutamate decarboxylase protein or the glutamate decarboxylase protein mutant.

[0041] 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.

[0042] In the present invention, the substance regulating gene expression may be a substance that performs at least one of the following six types of regulation: 1) Regulation at the transcriptional level of the gene; 2) regulation after the transcription of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm); 4) Regulation of the translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0043] In the present invention, the regulation can be up-regulation, enhancement or improvement. The up-regulation, enhancement or improvement of the expression amount of the coding gene of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the recipient plant, or / and the up-regulation, enhancement or improvement of the activity and / or content of the coding gene of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant is achieved by introducing the coding gene of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant into the recipient plant.

[0044] In the present invention, the regulation may also be down-regulation, weakening or reducing the expression of the coding gene. Down-regulation, weakening or reducing the expression of the coding gene may be achieved by gene knockout or gene silencing.

[0045] In the present invention, the substance regulating the expression of the gene or the substance regulating the activity or content of the glutamate decarboxylase protein (or the glutamate decarboxylase protein mutant) may be the biological material.

[0046] Another aspect of the present invention also provides a method for increasing the GABA content in plants, which comprises expressing the coding gene of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant.

[0047] In the present invention, the expression may be to suppress, reduce, enhance or up-regulate the expression of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the plant, and / or, may be to suppress, reduce, enhance or up-regulate 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.

[0048] Another aspect of the present invention also provides a method for cultivating plants with increased GABA content, which comprises expressing the coding gene of the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant in the plant to obtain plants with increased GABA content.

[0049] In one embodiment of the present invention, the method for cultivating a plant with altered GABA content comprises the following steps: (1) constructing a recombinant expression vector of the gene encoding the glutamate decarboxylase protein and / or the glutamate decarboxylase protein mutant; (2) The recombinant expression vector constructed in step (1) is transferred into a recipient plant to obtain a plant with altered GABA content.

[0050] 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%.

[0051] In the present invention, the plant may be as follows: f1) monocots or dicots; f2) Cucurbitales; f3) Cucurbitaceae; f4) Citrullus; f5) Watermelon.

[0052] Positive improvement effect: The present invention deletes the coding gene of glutamate decarboxylase protein at a specific site, introduces it into plants for expression, and obtains plants with significantly increased GABA content. Compared with the wild-type plant varieties, the phenotype is normal and stable, such as no visible phenotypic changes in female flowers, leaves, tendrils, stems, roots, growth potential, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] 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 in the last exon (indicated by the red line).

[0054] FIG. 2 is a map of the pHSE401 carrier in Example 1.

[0055] FIG3 is the comparison result of glutamate decarboxylase protein gene at two target sites of WT and positive ClGAD2 gene-edited strains in Example 1; wherein, - indicates deletion; Target sequence and PAM sequence are marked with black and red letters, respectively.

[0056] FIG. 4 shows the watermelon fruit phenotypes of the wild-type WT and positive ClGAD2 gene-edited strains in Example 2.

[0057] FIG. 5 is a phenotypic comparison of GABA content in wild-type WT and positive ClGAD2 gene-edited watermelon strains in Example 2. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0060] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0061] The pCBC-DT1T2 in the following examples has been described in: Tian, ​​S., Jiang, L., Gao, Q. et al. Efficient CRISPR / Cas9-based gene knockout in watermelon. Plant Cell Rep36, 399–406 (2017). https: / / doi.org / 10.1007 / s00299-016-2089-5. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0062] The pHSE401 in the following examples was purchased from addgene: https: / / www.addgene.org / 62201 / .

[0063] The watermelon germplasm material 'WT' in the following example has been recorded 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 can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0064] Example 1. Obtaining ClGAD2 gene knockout mutants 1. Design of editing site of GAD (Cla009138) The target gene sites for gene editing were designed according to the genomic DNA sequence of GAD (SEQ ID No. 1) and the online design website for target gene sites CRISPR P (http: / / crispr.hzau.edu.cn / CRISPR2 / news.php, V2). Two sets of editing sites, Target1 and Target2, were designed. The schematic diagram of the GAD gene structure is shown in Figure 1.

[0065] The genomic sequence encoding the GAD protein is shown in SEQ ID No. 1 in the sequence list, and the encoded amino acid sequence is shown in SEQ ID No. 2 in the sequence list. The CDS encoding the GAD protein is shown in SEQ ID No. 3 in the sequence list. Among them, the target gene sites Target1 and Target2 are at the last exon of the GAD gene. Target-1 sequence: 5'-GATATCAAAACGACGGTGGC-3' (SEQ ID No. 4); Target-2 sequence: 5'-GACACAGAAGAACGACGTGG-3' (SEQ ID No. 5).

[0066] 2. Obtaining the target fragment 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).

[0067] Amplification system (50μL): 2xPhantaMax Buffer 25μL, dNTP Mix (10mM) 1μL, Target2-BsF / Target1-BsR primers (10μM) 2μL each, Target2-F0 / Target1-R0 (0.5μM) 2μL each, PhantaMax Super-Fidelity DNA Polymerase 1μL, template DNA 2μL, ddH2O 13μL.

[0068] PCR reaction procedure: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 55℃ for 15s, extension at 72℃ for 30s, 35 cycles; complete extension at 72℃ for 5min. 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 of Target2-BsF, Target2-F0, Target1-R0, and Target1-BsR are as follows: Target2-BsF: 5'-AACCCACGTCGTTCTTCTGTGTCAATCTCTTAGTCGACTCTAC -3'; Target2-F0: 5'-ATTATTGGTCTCGAAACCCACGTCGTTCTTCTGTGTC -3'; Target1-R0: 5'-ATATATGGTCTCGATTGATATCAAAACGACGGTGGCGTT -3'; Target1-BsR: 5'-TGATATCAAAACGACGGTGGCGTTTTAGAGCTAGAAATAGC-3'.

[0069] 3. Construction of CRISPR / Cas9 editing vector The CRISPR / Cas9 vector pHSE401 (see Figure 2 for the vector map) was digested and recombined using restriction endonucleases BsaI-HF (NEB) and T4 Ligase (NEB). Restriction digestion and recombination system (15μL): 10xBSA 1.5μL, pHSE401 (1μg / μl) 2μL, BsaI-HF (NEB) 1μL, T4 Ligase (NEB) 1μL, 10xNEB T4 Buffer 1.5μL, the target fragment obtained above 2μL, ddH2O 6μL. Reaction procedure: 37℃ 5 h, 50℃ 5min, 80℃ 10min, transform DH5α competent cells.

[0070] The transformed DH5α competent cells were tested by colony PCR using identification primers U626-IDF and U629-IDR. The primer sequences are as follows: U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3'; U629-IDR: 5'-AGCCCTCTTCTTCGATCCATCAAC-3'.

[0071] After the correct band size was confirmed, the bacteria were inoculated and shaken, and after sequencing, sequence alignment was performed to obtain the recombinant plasmid pHSE401-sgRNA1-sgRNA2.

[0072] 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 a DNA molecule of SEQ ID No.6, and keeping the other sequences of the starting vector unchanged.

[0073] The recombinant plasmid pHSE401-sgRNA1-sgRNA2 with correct sequencing was transformed into Agrobacterium EHA105 competent cells. The competent cells of Agrobacterium EHA105 were tested by PCR using primers U626-IDF and U626-IDR to obtain the Agrobacterium positive clone EHA105 / pHSE401-sgRNA1-sgRNA2.

[0074] 4. Genetic transformation of watermelon Sowing: Take 50 seeds of the laboratory watermelon germplasm material 'WT', soak them in a 55℃ water bath for 30 minutes, and then peel off the seed shells. In the clean bench, disinfect the peeled seed kernels with 75% alcohol for 1 minute, soak them in 3% sodium hypochlorite for 15 minutes, and then wash them with sterile water for 5 times. Spread the seed kernels on the sowing medium (BM, breeding medium; H2O, Agar 6g / L), and culture them in the dark at 25℃ for about 3 days.

[0075] 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, collect the bacteria by centrifugation at 5000rpm for 5 minutes. 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.

[0076] Dip infection: When the radicle of the seed kernel grows to about 1 cm, cut off the two ends of the cotyledon and cut the explant into small pieces for dipping. Mix the cut cotyledon in a 20 mL syringe containing 10 mL of resuspended bacterial solution, and then dip it under negative pressure for 15 minutes. Take out the explant and dry it on sterile filter paper, transfer it to co-culture medium (CM, MS+30% sucrose+plant gel+6-BA) padded with filter paper for co-culture, and culture it in the dark at 28℃ for 3 days; MS was purchased from Phytotech and 6-BA was purchased from Yuanye Company.

[0077] Recovery culture: After 3 days of co-cultivation, the cotyledon blocks were transferred to recovery medium (RM; MS + 30% sucrose + phytagel + 6-BA + timentin) and cultured for 7 days at 28°C, with a light intensity of 20,000 lux and a light duration of 16 h / d.

[0078] Selective culture: After the recovery culture is completed, the explants are transferred to selective medium (SM, selective medium; 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.

[0079] Shoot elongation culture: The explants with obvious buds and GFP tags were transferred to seedling elongation medium (SE; MS+30% sucrose+agar+6-BA+timentin) for culture.

[0080] Rooting culture: The selected buds were transferred to MS medium containing IAA and timentin for rooting culture and cultured at 28°C until roots formed.

[0081] 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 manage normally after cultivation.

[0082] The MS medium used in the experiment was PhytoTech M519.

[0083] Finally, a positive ClGAD2 gene-edited strain was obtained.

[0084] 5. Editing detection of positive ClGAD2 gene plant lines The 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.

[0085] Specific steps: Take a small amount of young watermelon leaves and quickly grind them into powder in liquid nitrogen, and put them in a 1.5mL centrifuge tube; add preheated 800μL CTAB extraction buffer and place 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 turning upside down 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 and 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.

[0086] The extracted genomic DNA was used as a template and the primers ClGAD2-F / ClGAD2-R were used to amplify the sequence containing the two target sites by PCR.

[0087] The specific primer sequences of ClGAD2-IDF1 / ClGAD2-IDR1 and ClGAD2-IDF2 / ClGAD2-IDR2 are as follows: ClGAD2-F: 5'-ACGTGATGGAAAACTGCAAAGA-3'; ClGAD2-R: 5'-TTTCCATCACAAACTTCCTCCA -3'; Amplification system (50 μL) and reaction procedure: 2×T5 SuperMIX 25 μL, primers (10 μM) 2 μL each, template DNA 1 μL, ddH2O 20 μL.

[0088] PCR reaction procedure: pre-denaturation at 98℃ for 3min; denaturation at 98℃ for 10s, annealing at 58℃ for 10s, extension at 72℃ for 15s, 35 cycles; complete extension at 72℃ for 5min. Band size was detected by 1% agarose gel electrophoresis. The remaining PCR product was sequenced using the ClGAD2-F / ClGAD2-R primers.

[0089] The target editing comparison results of ClGAD2 mutants are shown in Figure 3, where ClGAD2 lacks 2bp at the Target1 target site and 2bp at the Target2 target site.

[0090] Compared with the wild type (WT), for the mutant ClGAD2 gene, the mutant ClGAD2 gene in the 2 homologous chromosomes was obtained by the following mutations: “5′-GATATCAAAACGACGGTGGC-3′” in the GAD gene of the wild type WT was mutated to “5′-GATATCAAAACGACGGGC-3′”, which was deleted for two nucleotides; “5′-GACACAGAAGAACGACGTGG-3′” was mutated to “5′-GACACAGAAGAACGATGG-3′”, which was deleted for two nucleotides; Deletion of the above nucleotides results in ClGAD2 with enhanced protein function.

[0091] Example 2 Phenotypic observation of ClGAD2 gene-edited plants 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 the wild-type watermelon WT were planted in the greenhouse of 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 obvious difference in the morphology of plants containing the ClGAD2 gene compared with the wild type.

[0092] Liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to detect the GABA content of plants containing the ClGAD2 gene and wild-type watermelon WT. The test results are shown in Figure 5. In Figure 5, gad2-1 and gad2-2 are positive glgad2-1 gene-edited lines and positive glgad2-2 gene-edited lines, respectively. The results showed that compared with the wild-type watermelon WT, the GABA content of plants containing the ClGAD2 gene increased by 3 times, and the strains were stable, and the same results could be obtained after repeated experiments.

[0093] The specific experimental conditions of liquid chromatography tandem mass spectrometry (LC-MS / MS) are as follows: Sample pretreatment: (1) Weigh 50 mg (±2.5 mg) of sample (unless there are special requirements, plant samples are assumed to be freeze-dried samples, and animal samples are assumed to be fresh samples) 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 12000 r / min for 10 min at 4°C, and pipette 300 μL of supernatant into a 1.5 mL centrifuge tube; (4) Place in a -20°C refrigerator for 30 min, and centrifuge again at 4°C, 12000 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 should be stored at -20°C.

[0094] Chromatography and mass spectrometry acquisition conditions: The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UltraPerformance Liquid Chromatography, UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem Mass Spectrometry, MS / MS) (QTRAP® 6500+, https: / / sciex.com.cn / ). The liquid phase conditions mainly include: 1) chromatographic column: ACQUITY BEH Amide column (1.7µm, 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: 0-1.2 min A / B is 10:90 (V / V), 9minA / B is 40:60 (V / V), 10-11 min is 60:40 (V / V), 11.01-15min is 10:90 (V / V); 4) flow rate 0.4 mL / min; column temperature 40°C; injection volume 2μL.

[0095] The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 550°C, mass spectrometry voltage 5500 V in positive ion mode, mass spectrometry voltage -4500 V in negative ion mode, and curtain gas (CUR) 35 psi. In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0096] Quantification is accomplished using the multiple reaction monitoring (MRM) analysis of a triple quadrupole mass spectrometer. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, and excludes ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced by the collision chamber and then broken to form multiple fragment ions, which are then filtered by the triple quadrupole to select the required characteristic fragment ions, eliminating non-target ion interference, making quantification more accurate and more repeatable. After obtaining the mass spectrometry data of different samples, the chromatographic peaks of all target substances are integrated and quantitative analysis is performed using the standard curve.

[0097] In summary, the present invention provides a method for creating new watermelon germplasm with increased GABA content by gene editing technology. By editing the GABA content regulating 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.

[0098] 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 the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A glutamate decarboxylase protein, characterized in that It is a protein with an amino acid sequence of SEQ ID No. 2; The glutamate decarboxylase protein is derived from Citrullus lanatus.

2. A nucleic acid molecule encoding the glutamate decarboxylase protein according to claim 1; the nucleic acid molecule may be any of the following: b1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 1; b2) The coding sequence is the nucleic acid molecule shown in SEQ ID No.

3.

3. A glutamate decarboxylase protein mutant, characterized in that: It is any of the following: c1) obtained by knocking out the inhibitory domain of the glutamate decarboxylase protein according to claim 1; c2) A protein having an amino acid sequence of SEQ ID No.

9.

4. A nucleic acid molecule encoding the glutamate decarboxylase protein mutant according to claim 3; the nucleic acid molecule may be any of the following: d1) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No.7; d2) The coding sequence is the nucleic acid molecule shown in SEQ ID No.

8.

5. A biological material containing the nucleic acid molecule according to claim 2 or 4, characterized in that: The biological material is any 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 described in e1), or a recombinant microorganism containing the recombinant vector described in e2); e4) a transgenic plant cell line containing the nucleic acid molecule, or a transgenic plant cell line containing the expression cassette described in e1); e5) transgenic plant tissue containing the nucleic acid molecule, or transgenic plant tissue containing the expression cassette described in e1); e6) A transgenic plant organ containing the nucleic acid molecule, or a transgenic plant organ containing the expression cassette of e1).

6. Use of the glutamate decarboxylase protein according to claim 1 or the glutamate decarboxylase protein mutant according to claim 3 or the nucleic acid molecule according to claim 2 or 4 or the biomaterial according to claim 5 in any of the following: 1) Application in regulating plant GABA content; 2) Application in the preparation of products for regulating plant GABA content; 3) Application in the cultivation of plants with altered GABA content; 4) Use in the preparation of products for cultivating plants with altered GABA content; 5) Application in plant breeding.

7. A method for increasing the GABA content in plants, characterized in that: The method comprises expressing the coding gene of the glutamate decarboxylase protein according to claim 1 and / or the coding gene of the glutamate decarboxylase protein mutant according to claim 3 to increase the GABA content of the plant.

8. A method for cultivating a plant with increased GABA content, characterized in that: The method comprises expressing the coding gene of the glutamate decarboxylase protein according to claim 1 and / or the coding gene of the glutamate decarboxylase protein mutant according to claim 3 to obtain a plant with increased GABA content.

9. The method for cultivating a plant with increased GABA content according to claim 8, characterized in that: The method comprises the following steps: (1) constructing a recombinant expression vector of the gene encoding the glutamate decarboxylase protein as claimed in claim 1 and / or the glutamate decarboxylase protein mutant as claimed in claim 3; (2) transferring the recombinant expression vector constructed in step (1) into a recipient plant to obtain a plant with increased GABA content.

10. The use according to claim 6, and / or the method according to any one of claims 7 to 9, characterized in that: The plant is any of the following: f1) monocots or dicots; f2) Cucurbitales; f3) Cucurbitaceae; f4) Citrullus; f5) Watermelon.

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