Application of TaGSH1 protein in regulating wheat growth rate and stress resistance

Abstract

The application discloses application of TaGSH1 protein or a fusion protein thereof or a coding gene thereof or biological material containing the coding gene in negative regulation of wheat growth speed, positive regulation of oxidation-reduction homeostasis in the wheat, positive regulation of stress resistance of the wheat and wheat variety improvement. TaGSH1 A preparation method of mutant wheat. TaGSH1-1A 、 TaGSH1-1B 、 TaGSH1-1D The application also discloses a wheat mutant material with three genes simultaneously homozygous knockout. The application also first finds that TaGSH1-1A protein, TaGSH1-1B protein and TaGSH1-1D protein play an important role in negative regulation of wheat growth speed, positive regulation of oxidation-reduction homeostasis in the wheat and stress resistance of the wheat.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of TaGSH1 protein in regulating wheat growth rate and stress resistance. Background Technology

[0002] Wheat is one of the world's most important and widely cultivated food crops, providing humans with a vital source of energy and protein. Therefore, high and stable wheat yields have always been a research focus worldwide. Wheat's relatively long growth period further increases its risk of environmental stress. As an important food crop, the discovery and utilization of wheat stress resistance-related genes play a crucial role in variety improvement and yield enhancement. Abiotic stresses such as salinity, drought, high temperature, and heavy metals can induce excessive accumulation of reactive oxygen species (ROS) in plants. ROS have high reactivity and cytotoxicity, and must be promptly removed to protect cells from oxidative damage.

[0003] Glutathione (GSH) is the most abundant thiol-containing low-molecular-weight peptide in plants. It exists in the cytoplasm, chloroplasts, and mitochondria in both reduced (GSH) and oxidized (GSSG) forms, participating in various biochemical processes within plants, including metabolism, detoxification, antioxidant responses, and maintenance of redox homeostasis. GSH also maintains redox homeostasis in plants under oxidative stress by modifying specific cysteine ​​residues of target proteins with S-glutathioneylation. GSH1 The gene encodes γ-glutamylcysteine ​​synthetase (γ-GCS), a key rate-limiting enzyme in the GSH synthesis pathway. Currently, regarding... GSH1 The function, regulatory network, and molecular mechanism of this gene in wheat stress resistance formation are still unclear, which limits its application in wheat molecular breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of TaGSH1 protein in regulating wheat growth rate and stress resistance, so as to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides the application of TaGSH1 protein or its fusion protein or its encoding gene or biological material containing said encoding gene in the negative regulation of wheat growth rate, wherein the TaGSH1 protein is TaGSH1-1A protein, TaGSH1-1B protein and TaGSH1-1D protein, the amino acid sequence of TaGSH1-1A protein is shown in Seq ID NO.1, the amino acid sequence of TaGSH1-1B protein is shown in Seq ID NO.2, and the amino acid sequence of TaGSH1-1D protein is shown in Seq ID NO.3.

[0007] Furthermore, the biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, non-renewable plant cells, or non-renewable plant tissues.

[0008] The second aspect of this invention provides the application of TaGSH1 protein or its fusion protein or its encoding gene or biological material containing the encoding gene in the positive regulation of redox homeostasis in wheat, wherein the TaGSH1 protein is TaGSH1-1A protein, TaGSH1-1B protein and TaGSH1-1D protein, the amino acid sequence of TaGSH1-1A protein is shown in Seq ID NO.1, the amino acid sequence of TaGSH1-1B protein is shown in Seq ID NO.2, and the amino acid sequence of TaGSH1-1D protein is shown in Seq ID NO.3.

[0009] Furthermore, the biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, non-renewable plant cells, or non-renewable plant tissues.

[0010] A third aspect of this invention provides the application of TaGSH1 protein or its fusion protein or its encoding gene or biological material containing said encoding gene in the positive regulation of wheat stress resistance, wherein the TaGSH1 protein is TaGSH1-1A protein, TaGSH1-1B protein and TaGSH1-1D protein, the amino acid sequence of TaGSH1-1A protein is shown in Seq ID NO.1, the amino acid sequence of TaGSH1-1B protein is shown in Seq ID NO.2, and the amino acid sequence of TaGSH1-1D protein is shown in Seq ID NO.3; the stress resistance includes resistance to salt stress.

[0011] Furthermore, the biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, non-renewable plant cells, or non-renewable plant tissues.

[0012] The fourth aspect of this invention provides the application of TaGSH1 protein or its fusion protein or its encoding gene or biological materials containing the encoding gene in wheat variety improvement, wherein the improvement includes accelerating wheat growth rate, maintaining redox homeostasis in wheat, or improving wheat stress resistance; wherein the TaGSH1 protein is TaGSH1-1A protein, TaGSH1-1B protein, and TaGSH1-1D protein, the amino acid sequence of TaGSH1-1A protein is shown in Seq ID NO.1, the amino acid sequence of TaGSH1-1B protein is shown in Seq ID NO.2, and the amino acid sequence of TaGSH1-1D protein is shown in Seq ID NO.3; the stress resistance includes resistance to salt stress.

[0013] Furthermore, the biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, non-renewable plant cells, or non-renewable plant tissues.

[0014] The fifth aspect of the present invention provides TaGSH1 The method for preparing mutant wheat includes the following steps:

[0015] 1) Based on wheat TaGSH1 Gene sequence selection for CRISPR-Cas9 knockout targets, with TS1 and TS2 as the CRISPR-Cas9 knockout targets. TaGSH1 Genes are TaGSH1-1A Gene, TaGSH1-1B Genes and TaGSH1-1D Genes, the ones mentioned TaGSH1-1A The gene sequence is shown in Seq ID NO.4. TaGSH1-1B The gene sequence is shown in Seq ID NO.5. TaGSH1-1D The gene sequence is shown in Seq ID NO.6, the sequence of target TS1 is shown in SEQ ID NO.7, and the sequence of target TS2 is shown in SEQ ID NO.8;

[0016] 2) Construct a CRISPR-Cas9-TaGSH1 target knockout vector, transform wheat, and obtain transgenic wheat material;

[0017] 3) Design specific primers to identify and screen the transgenic wheat materials from step 3). TaGSH1-1A , TaGSH1-1B , TaGSH1-1D Wheat mutant material with simultaneous homozygous knockout of three genes, i.e. TaGSH1 Mutant wheat.

[0018] Further, the sequences of the specific primers described in step 3) are shown in SEQ ID NO.9-14.

[0019] The beneficial effects of this invention are:

[0020] This invention is based on wheat TaGSH1 Gene( TaGSH1-1A Gene, TaGSH1-1B Genes and TaGSH1-1D Gene sequence selection for CRISPR-Cas9 knockout targets, using TS1 and TS2 as CRISPR-Cas9 knockout targets, successfully prepared TaGSH1-1A , TaGSH1-1B , TaGSH1-1D Wheat mutants with simultaneous homozygous knockout of three genes. Based on this, this invention is the first to discover that TaGSH1-1A, TaGSH1-1B, and TaGSH1-1D proteins play important roles in negatively regulating wheat growth rate (but not affecting maturity phenotype or yield traits), positively regulating redox homeostasis in wheat, and wheat stress resistance (such as salt stress resistance). This invention provides a practical method for rapid wheat breeding and the cultivation of stress-resistant wheat using genetic engineering technology, and has significant breeding application value. Attached Figure Description

[0021] Figure 1 for Tagsh1-1-1-11 , Tagsh1-13-1-22 A schematic diagram of genome sequence alignment at mutation sites.

[0022] Figure 2 For wild-type wheat Fielder and Tagsh1-13-1-22 The results of the growth of mutant wheat at the same time point are shown in the figure.

[0023] Figure 3 For wild-type wheat Fielder and Tagsh1-1-1-11 The results of the growth of mutant wheat at the same time point are shown in the figure.

[0024] Figure 4 For wild-type wheat Fielder and Tagsh1-13-1-22 A schematic diagram of the mature phenotype of mutant wheat.

[0025] Figure 5 For wild-type wheat Fielder and Tagsh1-1-1-11 A schematic diagram of the mature phenotype of mutant wheat.

[0026] Figure 6 For wild-type wheat Fielder and TaGSH1 Figure showing the height of mutant wheat plants.

[0027] Figure 7 For wild-type wheat Fielder and TaGSH1 Figure showing the number of grains per ear in mutant wheat.

[0028] Figure 8 For wild-type wheat Fielder and TaGSH1 Phenotypic diagram of mutant wheat seeds.

[0029] Figure 9 For wild-type wheat Fielder and TaGSH1 The results of the mutant wheat grain length (left) and grain width (right) are shown in the figure.

[0030] Figure 10 For wild-type wheat Fielder and TaGSH1 Statistical results of wheat grain length in mutant strains.

[0031] Figure 11 For wild-type wheat Fielder and TaGSH1 Statistical results of mutant wheat grain width.

[0032] Figure 12 For wild-type wheat Fielder and TaGSH1 Statistical results of the thousand-grain weight of mutant wheat.

[0033] Figure 13 For wild-type wheat Fielder and TaGSH1 Figure showing the results of salt stress on mutant wheat.

[0034] Figure 14 For wild-type wheat Fielder and TaGSH1 Statistical results of leaf length in mutant wheat under salt stress.

[0035] Figure 15 For wild-type wheat Fielder and TaGSH1 Statistical results of total root count under salt stress in mutant wheat.

[0036] Figure 16 For wild-type wheat Fielder and TaGSH1 Statistical results of root length in mutant wheat under salt stress.

[0037] Figure 17 For wild-type wheat Fielder and TaGSH1 Statistical results of leaf weight of mutant wheat under salt stress.

[0038] Figure 18 For wild-type wheat Fielder and TaGSH1 Statistical results of root weight of mutant wheat under salt stress.

[0039] Figure 19 For wild-type wheat Fielder and Tagsh1-13-1-22The figure shows the statistical results of GSH and related metabolite content in mutant wheat under normal growth conditions. In the figure, GSH, γ-Glu-Cys, GSSG, and Cys are reduced glutathione, γ-glutamylcysteine, oxidized glutathione, and cysteine, respectively, and the same applies below.

[0040] Figure 20 For wild-type wheat Fielder and Tagsh1-1-1-11 Statistical results of GSH and related metabolite content in mutant wheat under normal growth conditions.

[0041] Figure 21 For wild-type wheat Fielder and Tagsh1-13-1-22 Statistical results of GSH and related metabolite content in mutant wheat under salt stress treatment.

[0042] Figure 22 For wild-type wheat Fielder and Tagsh1-1-1-11 Statistical results of GSH and related metabolite content in mutant wheat under salt stress treatment.

[0043] Figure 23 For wild-type wheat Fielder and Tagsh1-1-1-11 Figure showing the amount of S-glutathioneized modified protein in mutant wheat. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise specified, all experimental and detection methods in the following examples are conventional methods; the yellow scales in the accompanying figures are all 20 cm. Unless otherwise specified, all reagents and materials are commercially available.

[0046] Example 1 TaGSH1 Preparation and identification of mutant wheat

[0047] 1. TaGSH1 Preparation of mutant wheat

[0048] TaGSH1 The gene has one copy each on chromosomes A, B, and D of wheat chromosome 1. TaGSH1-1A , TaGSH1-1B , TaGSH1-1D Since common wheat is an allohexaploid (AABBDD), we used Arabidopsis thaliana as a knockout agent when designing the knockout scheme. GSH1 Using the AT4G23100 gene as a reference sequence, homologous gene sequences in Chinese spring wheat were first obtained through BLAST alignment from the Ensembl Plants database; then, these sequences were further BLAST aligned using the WheatOmics 1.0 database to obtain... TaGSH1 Homologous sequences of the gene in the wheat Fielder A, B, and D subgenomes. TaGSH1 The nucleotide sequence of the gene in the wheat FielderA subgenome has 100% similarity to the gene TraesFLD1A01G094600.1 in the WheatOmics 1.0 database; TaGSH1 The nucleotide sequence of the gene in the wheat Fielder B subgenome has 100% similarity to the gene TraesFLD1B01G105400.1 in the WheatOmics 1.0 database; TaGSH1 The nucleotide sequence of the gene in the wheat Fielder D subgenome showed 100% similarity to the gene TraesFLD1D01G087700.1 in the WheatOmics 1.0 database. The amino acid sequence of wild-type wheat TaGSH1-1A is shown in Seq ID NO.1, and the gene sequence is shown in Seq ID NO.4; the amino acid sequence of TaGSH1-1B is shown in Seq ID NO.2, and the gene sequence is shown in Seq ID NO.5; the amino acid sequence of TaGSH1-1D is shown in Seq ID NO.3, and the gene sequence is shown in Seq ID NO.6.

[0049] according to TaGSH1 The subgenomic sequences of genes A, B, and D were used to screen for CRISPR-Cas9 knockout targets, and their off-target probabilities were evaluated. This was done to design a system capable of... TaGSH1 For sgRNAs edited in the coding region, we searched for protospacer neighbor motifs (PAMs) conforming to 5'-N(20 bp)-NGG-3' in conserved regions. To avoid off-target effects, the specificity of candidate targets was evaluated using online tools CRISPRdirect and CHOPCHOP. The screening criteria included: (1) the target sequence was present and completely matched in all three subgenomes; (2) there were at least 3 mismatches with other potential sites in the wheat genome to ensure a low off-target rate; (3) the GC content was between 40% and 70%, and it did not contain consecutive Ts (≥4) to prevent transcription termination. To ensure knockout efficiency, this invention designed and constructed two target editing vectors to transform wheat Fielder, and constructed... TaGSH1 After knocking out the material, we ultimately selected the two highest-scoring sequences as the final targets, as follows:

[0050] The target TS1 sequence is: 5'-GATGGCGGTCGCGTCGCGCCTGG-3' (as shown in SEQ ID NO.7);

[0051] The target TS2 sequence is: 5'-CGCGTCCGGCTGCAAGCCCAAGG-3' (as shown in SEQ ID NO.8).

[0052] Using the pOPGR-TS1 vector (Li J, Wang Z, He G, Ma L, Deng XW. (2020). CRISPR / Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat. J Genet Genomics. 47, 263-272.) as a template, two rounds of PCR were performed to obtain the TaU3p-target-optimized sgRNA expression cassette, which was then inserted into the StuI site of the pCas9T plasmid (CN 118834900 A) to generate the expression vectors pCas9T-TS1 and pCas9T-TS2, respectively.

[0053] Expression vectors pCas9T-TS1 and pCas9T-TS2 were transformed into Agrobacterium EHA105 competent cells using a freeze-thaw method to obtain Agrobacterium culture. Wheat Fielder seeds, approximately 15 days post-pollination, were harvested and embryos were extracted. 1 mL of Agrobacterium culture was transferred to a 1.5 mL centrifuge tube, and 1.4 μL of acetylsylgenin (0.1 M) was added and mixed thoroughly. The prepared Agrobacterium culture was added to the embryos for 5 minutes of incubation. The embryos were then placed on co-culture medium and incubated in the dark at 23°C for 3 days. After co-culture, the embryos were placed on resting medium and incubated in the dark at 25°C for 5 days to obtain callus tissue. The callus tissue was transferred to selection medium, the culture dishes were sealed with sealing film, and incubated in the dark at 25.5°C for 2 weeks. The callus tissue was then cut and transferred to a new selection medium, the culture dishes were sealed with sealing film, and incubated in the dark at 25.5°C for another 2 weeks. Two weeks after callus tissue was cut and screened, resistant callus tissue exhibiting green buds was transferred to regeneration medium, the culture dishes were sealed with sealing film, and cultured in a 25℃ incubator under light / dark conditions (16 h / 8 h) for two weeks. After two weeks of regeneration, healthy seedlings were transferred to new regeneration medium and cultured in a 25℃ incubator under light / dark conditions (16 h / 8 h) until they reached a certain size, at which point samples could be taken for testing.

[0054] The co-culture medium was MS medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 2.0 mg / L dicamba + 30 g / L sucrose + 0.7 w / v% agar + 100 μmol / L acetosyringone (AS), pH 5.8.

[0055] The resting medium was MS medium containing 2.0 mg / L 2,4-D, 30 g / L sucrose, 0.7 w / v% agar, and 300 mg / L cephalosporin, pH 5.8.

[0056] The selection medium was prepared by adding 5 mg / L glufosinate to the resting medium.

[0057] The regeneration medium was MS medium containing 3.0 mg / L zeatin, 30 g / L sucrose, and 0.7 w / v agar, pH 5.8.

[0058] 2. TaGSH1 Identification of mutant wheat

[0059] Primers designed to specifically amplify the target sequence from three homologous genes were used to amplify the gene-editing material. TaGSH1 The primer sequences for TaGSH1-1A F / R, TaGSH1-1B F / R, and TaGSH1-1D F / R of the transgenic materials are shown in Table 1.

[0060] Table 1

[0061]

[0062] TaGSH1For genotyping of transgenic materials, wheat leaves approximately 1 cm in length were taken and placed in a 2.0 mL round-bottom grinding tube with two 3 mm sterile steel beads. After rapid freezing in liquid nitrogen, the tubes were quickly placed in a homogenizer and pulverized at 40 Hz for 1 min to obtain a uniform powder. 700 µL of preheated CTAB extraction buffer (65°C) was added to the sample, and the mixture was inverted and incubated at 65°C for 1 h. Then, 700 µL of a mixture of phenol, chloroform, and isopropanol (25:24:1 v / v) was added, and the mixture was inverted until homogeneous. The mixture was centrifuged at 12000 rpm for 10 min at room temperature. The supernatant was transferred to a new 1.5 mL centrifuge tube, and 700 µL of isopropanol was added. After inverting and mixing, the mixture was frozen at -20°C for 30 min and centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and the tubes were washed with 1 mL of 75 v / v ethanol, centrifuged at 12000 rpm for 1 min, and the washing process was repeated three times. After opening the lid and allowing it to air dry, add 60 µL of ddH2O and gently mix. The PCR detection system used is shown in Table 2. 5 µL of PCR product was subjected to agarose gel electrophoresis. Samples with a clear single band and correct target fragment were selected for sequencing. After sequence alignment, TaGSH1-1A / B / D lines with base editing were selected. Tagsh1-1-1-11 and Tagsh1-13-1-22 The genome sequence alignment of its mutation sites is as follows: Figure 1 As shown. The selected triprost homozygous plants were then multiplied and then planted in an artificial climate chamber for phenotypic observation.

[0063] Table 2

[0064]

[0065] Example 2 TaGSH1 Phenotypic observation and statistical analysis of agronomic traits of mutant wheat

[0066] Phenotypic observation

[0067] Will TaGSH1 Mutant wheat ( Tagsh1-13-1-22 , Tagsh1-1-1-11 ) and wild-type wheat Fielder were planted in experimental fields (under consistent conditions for each treatment) and TaGSH1 Recording the heading time of mutant wheat TaGSH1 Growth status of mutant wheat and wild-type wheat Fielder. The phenotype of wheat after full maturity was observed, and its plant height was measured and statistically analyzed.

[0068] Experimental results are as follows Figures 2-3 As shown, compared to wild-type wheat Fielder, TaGSH1 Mutant wheat grows faster. For example... Figures 4-6As shown, compared to wild-type wheat Fielder, TaGSH1 The mutant wheat showed no significant difference in maturity phenotype.

[0069] Statistics on agronomic traits

[0070] TaGSH1 After the mutant wheat and wild-type wheat Fielder were fully mature, they were harvested as single plants and single ears, dried in an oven at 37℃ for 3 days, and the main ears of each wheat plant were selected for threshing. The number of grains per ear, grain length, and grain width were measured and recorded. Finally, the thousand-grain weight was determined after threshing all ears of the entire plant.

[0071] Experimental results are as follows Figures 7-12 As shown, compared to wild-type wheat Fielder, TaGSH1 The number of grains per ear, grain length, grain width, and thousand-grain weight of the mutant wheat showed no significant changes.

[0072] In summary, TaGSH1 negatively regulates wheat growth rate but does not directly affect maturity phenotype or yield traits.

[0073] Example 3 TaGSH1 Observation of salt stress phenotypes and statistical analysis of stress traits in mutant wheat

[0074] Phenotypic observation

[0075] Will TaGSH1 Mutant wheat ( Tagsh1-13-1-22 , Tagsh1-1-1-11 Both wheat (Cellular wheat) and wild-type wheat (Fielder) were placed in germination boxes and cultured in a light incubator at 22℃ and 43% relative humidity for 8 hours in darkness and 16 hours in light. During culture, the normal growth group (CK) was supplemented with 10 mL of ddH2O, and the salt stress treatment group (200 mM NaCl treatment) was supplemented with 10 mL of 200 mM NaCl. After 7 days of culture, the leaf length, root length, and total number of roots of wheat were observed.

[0076] Experimental results are as follows Figure 13 As shown, compared to wild-type wheat Fielder, TaGSH1 Mutant wheat is more significantly affected by salt stress.

[0077] Statistics on stress traits

[0078] T aGSH1 Mutant wheat ( Tagsh1-13-1-22 , Tagsh1-1-1-11After culturing the wheat (including wild-type wheat Fielder) in a light incubator for 7 days, seedlings with abnormal growth (i.e., individual seedlings whose growth differed significantly from other seedlings) were removed. The leaf length, root length, and total number of roots of the remaining seedlings were measured and recorded. Finally, the leaves and roots were dried at 75℃ to constant weight and then weighed to determine the leaf weight and root weight.

[0079] Experimental results are as follows Figures 14-18 As shown, compared to wild-type wheat Fielder TaGSH1 Mutant wheat is more significantly affected by salt stress.

[0080] In conclusion, TaGSH1 positively regulates wheat stress resistance.

[0081] Example 4 TaGSH1 Detection of GSH and related metabolite content in mutant wheat

[0082] Using mass spectrometry TaGSH1 Mutant wheat ( Tagsh1-13-1-22 , Tagsh1-1-1-11 The contents of GSH and its related metabolites in wild-type wheat (Fielder) under salt stress and normal growth conditions were detected. TaGSH1 Mutant wheat ( Tagsh1-13-1-22 , Tagsh1-1-1-11 Wheat (Fielder variety) and wild-type wheat were placed in germination boxes (containing 10 mL ddH2O) and incubated in a 22℃, 43% relative humidity light incubator for 8 h in darkness and 16 h light. After 6 days of cultivation, seedlings in the normal growth group were transferred to germination boxes containing 10 mL ddH2O for 24 h, while seedlings in the salt stress treatment group were transferred to germination boxes containing 10 mL 200 mM NaCl for 24 h. A suitable amount of sample (wheat leaves) was taken and placed in a liquid nitrogen-pre-cooled grinding tube. After rapid freezing in liquid nitrogen, the sample was ground into a homogeneous powder using a grinder at 40 Hz for 1 min. Approximately 50 mg of sample powder was weighed from each sample. On ice, 0.1 v / v% formic acid solution was added to each group to achieve a material-to-liquid ratio of 50 mg / mL. After vortexing and sonication at 4℃ for 20 min, the mixture was centrifuged at 12700 rpm at 4℃ for 15 min. The mixed solution was drawn up with a disposable syringe and passed through a 0.22 μm organic membrane to obtain the extract. The extract was diluted with 0.1 v / v% formic acid solution to a solid-liquid ratio of 20 mg / mL. The extract was then centrifuged at 4°C and 12700 rpm for 15 min. 200 μL of the supernatant was collected in a sample vial and analyzed by mass spectrometry.

[0083] The results are as follows Figures 19-22 As shown, the normal growth group and the salt stress treatment group, compared with wild-type wheat Fielder, TaGSH1The mutant wheat had lower levels of GSH and its related metabolites, and after salt stress treatment, TaGSH1 Mutant wheat showed less increase in GSH and related metabolites compared to wild-type wheat Fielder.

[0084] Example 5 TaGSH1 Detection of S-glutathioneyltransferase modified protein content in mutant wheat

[0085] The treatments were as shown in Example 4 (the normal growth group was designated CK, and the salt stress treatment group was designated NaCl). TaGSH1 Mutant wheat ( Tagsh1-1-1-11 Because in the phenotypic identification of Example 3, it was found that Tagsh1-1-1-11 The effects of stress are more pronounced, and this was found in the metabolic study of Example 4. Tagsh1-1-1-11 The content of GSH is also lower, therefore this embodiment uses... Tagsh1- 1-1-11Western blot analysis was performed on samples of wild-type wheat (Fielder) and wheat leaves. After grinding, 300 μL of RIPA lysis buffer containing 1 mM PMSF was added per 40 mg of plant tissue. The mixture was incubated on ice for 10 min, inverting the container during this time. After complete lysis, the mixture was centrifuged at 12700 rpm for 5 min, and the supernatant was collected to determine the protein concentration. A solution of the same protein concentration was pipetted and standardized to 300 μL to ensure equal protein concentration in each tube. 75 μL of 5× Protein Loading Buffer was added, and the tubes were incubated in a 25°C water bath for 30 min, followed by an ice bath for 2 min. 50 μg of protein from each group was then subjected to SDS-PAGE electrophoresis: the protein was first concentrated at 100 V, 400 mA, for 10 min, then separated at 150 V, 400 mA, for 40 min. After electrophoresis, the separated proteins were transferred to a PVDF membrane and transferred at 100 V, 400 mA for 90 min. After transfer, the membrane was placed in TBST buffer containing 5 w / v% skim milk powder and blocked at 25°C for 120 min, with gentle shaking on a shaker during blocking. After blocking, the membrane was washed 5 times with 10 mL of TBST buffer each time, for 6 min each time, at a speed of 80 rpm. After washing, mouse monoclonal anti-S-glutathioneyl modified antibody IgG2a (primary antibody) was diluted 1:500 with 5 w / v% skim milk solution to obtain a primary antibody dilution buffer. The membrane was placed in the primary antibody dilution buffer and incubated overnight at 4°C with gentle shaking. After primary antibody incubation, the membrane was washed 5 times with TBST buffer. After washing, horseradish peroxidase-labeled goat anti-mouse IgG (secondary antibody) was diluted 1:2000 with 3 w / v% skim milk solution to obtain a secondary antibody dilution buffer. The membrane was placed in the secondary antibody dilution buffer and incubated at room temperature with gentle shaking for 1 h. After the secondary antibody incubation, the membrane was washed five times with TBST buffer. After washing, the membrane was placed in ECL luminescence solution (solution A and solution B were mixed at a volume ratio of 1:1) to fully bind the membrane to the developing substrate ECL, and then developed using a multicolor fluorescence imaging system.

[0086] The results are as follows Figure 23 As shown, compared to wild-type wheat Fielder, TaGSH1 The mutant wheat contains lower levels of S-glutathione-modified proteins.

Claims

1. The application of TaGSH1 protein or its fusion protein or its encoding gene or biological material containing said encoding gene in the negative regulation of wheat growth rate, wherein, The TaGSH1 protein is TaGSH1-1A, TaGSH1-1B, and TaGSH1-1D. The amino acid sequence of TaGSH1-1A is shown in Seq ID NO.1, the amino acid sequence of TaGSH1-1B is shown in Seq ID NO.2, and the amino acid sequence of TaGSH1-1D is shown in Seq ID NO.

3.

2. The application according to claim 1, characterized in that, The biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, non-renewable plant cells, or non-renewable plant tissues.

3. A kind TaGSH1 A method for preparing mutant wheat, characterized in that, Includes the following steps: 1) Based on wheat TaGSH1 Gene sequence selection for CRISPR-Cas9 knockout targets, with TS1 and TS2 as the CRISPR-Cas9 knockout targets. TaGSH1 Genes are TaGSH1-1A Gene, TaGSH1-1B Genes and TaGSH1-1D Genes, the ones mentioned TaGSH1-1A The gene sequence is shown in Seq ID NO.

4. TaGSH1-1B The gene sequence is shown in Seq ID NO.

5. TaGSH1-1D The gene sequence is shown in Seq ID NO.6, the sequence of target TS1 is shown in SEQ ID NO.7, and the sequence of target TS2 is shown in SEQ ID NO.8; 2) Construct a CRISPR-Cas9-TaGSH1 target knockout vector, transform wheat, and obtain transgenic wheat material; 3) Design specific primers to identify and screen the transgenic wheat materials from step 3). TaGSH1-1A , TaGSH1- 1B , TaGSH1-1D Wheat mutant materials with simultaneous homozygous knockout of three genes, i.e. TaGSH1 Mutant wheat.

4. The one according to claim 3 TaGSH1 A method for preparing mutant wheat, characterized in that, The sequences of the specific primers described in step 3) are shown in SEQ ID NO.9-14.

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