Application and method of gamma-aminobutyric acid (GABA) transporter gene OsGAT3 in improving rice yield and breeding

Abstract

The invention relates to the technical field of rice molecular breeding, in particular to application of a gamma-aminobutyric acid (GABA) transporter gene OsGAT3 in improving rice yield breeding and a method. After overexpression of the cloned OsGAT3 gene, the yield, salt resistance and drought resistance of the rice are remarkably improved, which shows that the OsGAT3 gene has obvious influence on the yield, salt resistance and drought resistance of the rice, so that the yield, salt resistance and drought resistance of the rice can be genetically improved by improving the expression of the OsGAT3 gene through a genetic engineering technology. Based on the functions of the OsGAT3 gene found by the inventor, the OsGAT3 gene can be used for improving the rice yield and breeding with salt resistance and drought resistance. The purpose of rice salt-resistant and drought-resistant breeding is to cultivate high-yield rice plants with relatively strong salt-resistant and drought-resistant capabilities. Expression of the OsGAT3 gene can be improved through an overexpression technology, and rice plants with high yield, salt resistance and high drought resistance are obtained.

Description

Technical Field

[0001] This application relates to the field of rice molecular breeding technology, specifically to the application and method of the γ-aminobutyric acid (GABA) transporter gene OsGAT3 in breeding to improve rice yield. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and China is the world's largest producer and consumer of rice. Therefore, rice production is undoubtedly related to my country's food security. However, rice is a monocotyledonous crop that is sensitive to salt and drought. The absorption and accumulation of salt in the soil will seriously affect its yield (Hussain S, Zhang J, Zhong C, et al. Effects of salt stress on rice growth, development characteristics, and the regulating ways: A review [J]. Journal of integrative agriculture, 2017, 16(11): 2357-2374.). Drought, salinity, and extreme temperatures are the most significant abiotic stresses affecting stable rice yield (Yadav C, Bahuguna RN, Dhankher OP, et al. Physiological and molecular signatures reveal differential response of rice genotypes to drought and drought combination with heat and salinity stress[J]. Physiology and Molecular Biology of Plants, 2022, 28(4):899-910.), with salinity and drought stress being the most severe limiting factors. Therefore, identifying genes related to rice's salt tolerance, drought resistance, and ability to increase yield under stress, exploring the mechanisms of rice's salt and drought resistance, cultivating new salt- and drought-tolerant rice varieties, and improving rice's salt tolerance, drought resistance, and yield are of great significance for ensuring my country's food security.

[0003] If we want to improve rice's tolerance to salt and drought while also increasing its yield, the most important thing is to screen and cultivate new rice varieties that can improve salt resistance, drought resistance, and yield.

[0004] Molecular breeding applies molecular biology techniques to breeding, conducting breeding at the molecular level. It is a breeding method distinct from traditional hybridization breeding. Molecular breeding includes transgenic breeding, which applies genetic engineering to breeding work, using gene introduction to cultivate new varieties that meet specific requirements. Transgenic breeding of rice to improve rice yield and its tolerance to salt and drought is of great significance for increasing rice yield and stress resistance. In the practice of transgenic breeding, the selection of the target gene is crucial. There is an urgent need to screen and study genes related to rice development to obtain new rice varieties that can significantly increase yield and enhance stress resistance. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this application provides the application and method of the γ-aminobutyric acid (GABA) transporter gene OsGAT3 in breeding to improve rice yield, specifically achieved through the following technical solution:

[0006] Application of the γ-aminobutyric acid (GABA) transporter gene OsGAT3 in breeding to improve rice yield. The amino acid sequence of the OsGAT3 gene is shown in SEQ ID NO.1 below:

[0007] MGAPSREDEEAKKMEAGGDTVGQKLDAGALFVLQSKGSWLHCGYHLTTSIVAPPLLSLPFAFASLGWAAGLICLVIGAAVTFYSYNLISLVLEHHAQQGRRQLRFRDMATDILG PGWGRFYIGPIQFLVCFGAVVACTLLAGQSMKAIYLIANPGGTIKLYVFVAIFGVFMMILAQMPSFHSLRHVNLISLVLCLAYSFCAVAACIYLGSSKGAPEKDYSIAGANTRDR VFGVFNAIAVIATTYGNGIIPEIQATVAAPVTGKMFKGLCLCYAVVVTTFFSVAISGYWAFGNQSQGTLLSNFMVGGRAVIPEWLLLIIELFTLLQLSAVAVVYLQPTNEVLEGL LSDPKAGQYAARNVAPRVLSRTAAVALGTTIAAMVPFFGDMNALIGAFGFLPLDFAVPAVFYNVTFKPSKKGAVFWLNTTIAVVFSALAVVASVAAVRQIILDANSYKLFANV*.

[0008] Furthermore, the nucleotide sequence of the γ-aminobutyric acid (GABA) transporter OsGAT3 is shown in SEQ ID NO.2 below:

[0009]

[0010] Furthermore, the aforementioned breeding aspect for improving rice yield is specifically about breeding to increase rice yield under adverse conditions.

[0011] Furthermore, the adversity mentioned refers to the stress of salt or drought.

[0012] A breeding method to increase rice yield by increasing the protein expression level of the OsGAT3 gene in rice plants.

[0013] Furthermore, the aforementioned increase in rice yield refers to increasing the yield of rice under adverse conditions.

[0014] Furthermore, the method of increasing the protein expression level of the OsGAT3 gene in rice plants involves introducing an overexpression vector of the OsGAT3 gene into rice.

[0015] Furthermore, the introduction of the OsGAT3 gene overexpression vector into rice specifically involves linking the OsGAT3 gene cDNA to the pCAMBIA-1306 vector to obtain the overexpression vector OsGAT3-p1306; introducing the overexpression vector OsGAT3-p1306 into rice, and after screening and cultivation, obtaining OsGAT3 gene overexpressing plants.

[0016] Furthermore, the cDNA of the OsGAT3 gene is obtained from rice using RT-PCR.

[0017] Furthermore, the introduction of the overexpression vector OsGAT3-p1306 into rice is specifically carried out using Agrobacterium-mediated genetic transformation.

[0018] Compared with the prior art, the technical effects created by this application are reflected in:

[0019] (1) Increasing the protein expression level of the OsGAT3 gene in rice plants resulted in slower water loss rates, smaller changes in fresh weight, higher survival rates, increased POD (peroxidase) and CAT (catalase) enzyme activities, lower MDA (malondialdehyde) content, increased number of tillers per plant, and increased number of grains per plant under salt and drought stress, thereby improving the salt and drought resistance and yield of rice plants. After knocking out the OsGAT3 gene, rice experienced faster water loss rates, larger changes in fresh weight, lower survival rates, no significant increase in POD (peroxidase) and CAT (catalase) enzyme activities, higher MDA (malondialdehyde) content, reduced number of tillers per plant, and reduced number of grains per plant under salt and drought stress, thereby reducing the salt and drought resistance and yield of rice plants. Without affecting the activity of the OsGAT3 protein (i.e., not at the active site of the protein), those skilled in the art can substitute, add, and / or delete one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 to obtain amino acid sequences with equivalent functions. Therefore, the OsGAT3 protein also includes proteins with equivalent activity obtained by substituting, replacing, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1. Furthermore, considering codon degeneracy and codon preferences among different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0020] (2) The inventors cloned the OsGAT3 gene cDNA sequence from rice Zhonghua 11 (ZH11). By constructing an OsGAT3 gene overexpression vector, the overexpression vector was introduced into Zhonghua 11 (ZH11) to obtain OsGAT3 gene overexpressing plants. Compared with Zhonghua 11 (ZH11), these plants had a slower rate of water loss and smaller changes in fresh weight under salt and drought stress, higher survival rate, increased POD (peroxidase) and CAT (catalase) enzyme activities, lower MDA (malondialdehyde) content, increased number of tillers per plant, increased number of grains per plant, and significantly improved salt and drought resistance as well as yield. By constructing a gene knockout vector for the OsGAT3 gene and introducing it into Zhonghua 11 (ZH11), OsGAT3 gene knockout plants were obtained. Compared with Zhonghua 11 (ZH11), these plants exhibited faster water loss rates, greater fresh weight variations, and lower survival rates under salt and drought stress. POD (peroxidase) and CAT (catalase) enzyme activities were not significantly increased, but MDA (malondialdehyde) content was higher, and the number of tillers and grains per plant was reduced. Salt and drought resistance and yield were significantly decreased. Furthermore, under exogenous GABA application, the salt and drought resistance of OsGAT3 gene overexpression plants were significantly enhanced. The above studies show that overexpression of the OsGAT3 gene in rice plants can improve rice yield, salt tolerance, and drought resistance. Specifically, it improves rice yield, salt tolerance, and drought resistance by increasing the number of tillers and grains per plant, enhancing the water retention capacity of rice plants, increasing the activity of POD and CAT enzymes, and enhancing the absorption of GABA.

[0021] (3) Overexpression of the cloned OsGAT3 gene significantly improved the yield, salt resistance and drought resistance of rice, indicating that the OsGAT3 gene has a significant effect on the yield, salt resistance and drought resistance of rice. Therefore, improving the expression of the OsGAT3 gene through genetic engineering technology can genetically improve the yield, salt resistance and drought resistance of rice.

[0022] (4) The successful cloning of the OsGAT3 gene confirms that the GATs family of γ-aminobutyric acid (GABA) transporters are involved in the transport of γ-aminobutyric acid (GABA), which plays a vital role in plant growth, development and stress resistance. It can enrich our understanding of plant GATs and greatly promote the genetic improvement of plant yield, salt resistance and drought resistance.

[0023] (5) Based on the function of the OsGAT3 gene discovered by the inventors, it can be used in breeding to improve rice yield, salt tolerance, and drought resistance. The purpose of rice salt tolerance and drought resistance breeding is to cultivate rice plants with high yield, strong salt tolerance, and strong drought resistance. The expression of the OsGAT3 gene can be increased through overexpression technology to obtain rice plants with high yield, strong salt tolerance, and strong drought resistance. Attached Figure Description

[0024] Figure 1 This is a statistical bar chart of real-time quantitative PCR in Example 1 of the present invention. Figure 1 A), Comparison of mutant material identification diagrams ( Figure 1 B)(mean±SD, n=3).

[0025] Figure 2 The GABA content of different parts of the field plants in Example 2 of this invention ( Figure 2 AC) and total nitrogen content (2D-F), the samples were: wild-type control Zhonghua 11 (ZH11), three lines of OsGAT3 gene overexpressing plants OE1, OE2 and OE3 (mean±SD, n=3), and three lines of OsGAT3 gene mutant plants C1, C2 and C3.

[0026] Figure 3 The fresh weight of the seedlings before salt treatment in Example 3 of this invention ( Figure 3 G), fresh weight of seedlings before salt and GABA compound treatment ( Figure 3 H) and taking photos ( Figure 3 AB); Fresh weight of seedlings after salt treatment (AB); Figure 3 I), Fresh weight of seedlings after salt and GABA compound treatment ( Figure 3 M) and taking photos ( Figure 3 CD); Survival rate of seedlings after recovery treatment in salt-treated groups (CD); Figure 3 N), survival rate of seedlings after recovery treatment in the salt and GABA combined treatment group (N), Figure 3 O); leaf NBT after 0h and 48h of seedling salt treatment or seedling salt and GABA combined treatment. Figure 3 E) and DAB Figure 3 F) staining diagram; catalase activity in salt-treated leaves of seedlings (F) Figure 3 J), peroxidase activity (3K), malondialdehyde content (J), peroxidase activity (3K), malondialdehyde content ( Figure 3 Figure L); Catalase activity in leaves of seedlings treated with GABA compound salt (L) Figure 3 P), peroxidase activity (P), Figure 3 Q), Malondialdehyde content ( Figure 3 R) bar chart; the samples were: control wild-type Zhonghua 11 (ZH11), three OsGAT3 gene overexpressing plants OE1, OE2 and OE3 (mean±SD, G, H, I, M, N, O (n=30), J, K, L, P, Q, R (n=3)), and three OsGAT3 gene mutant plants C1, C2 and C3.

[0027] Figure 4 The fresh weight of seedlings before 15% PEG treatment in Example 4 of this invention ( Figure 4G), the fresh weight of seedlings before treatment with 15% PEG and GABA (G) Figure 4 H) and taking photos ( Figure 4 AB); Fresh weight of seedlings treated with 15% PEG (AB); Figure 4 I), the fresh weight of seedlings after treatment with 15% PEG and GABA ( Figure 4 M) and taking photos ( Figure 4 CD); Survival rate of seedlings after recovery treatment in the 15% PEG treatment group (CD); Figure 4 N), survival rate of seedlings after recovery treatment in the 15% PEG and GABA combined treatment group (N), Figure 4 O); NBT in leaves after 0h and 48h of seedling treatment with 15% PEG or seedlings treated with a combination of 15% PEG and GABA. Figure 4 E) and DAB Figure 4 F) staining diagram; catalase activity in leaves treated with 15% PEG in seedlings ( Figure 4 J), peroxidase activity ( Figure 4 K), malondialdehyde content ( Figure 4 Figure L); Catalase activity in leaves of seedlings treated with a combination of 15% PEG and GABA (L) Figure 4 P), peroxidase activity (P), Figure 4 Q), Malondialdehyde content ( Figure 4 R) bar chart; the samples were: control wild-type Zhonghua 11 (ZH11), three OsGAT3 gene overexpressing plants OE1, OE2 and OE3 (mean±SD, G, H, I, M, N, O (n=30), J, K, L, P, Q, R (n=3)), and three OsGAT3 gene mutant plants C1, C2 and C3.

[0028] Figure 5 , Figure 6 , Figure 7 For the OsGAT3-related transgenic plants of Example 5 of this invention, at 2mM

[0029] NH4NO3 ( Figure 5 AB), 2mM NH4NO3 + 30mM NaCl ( Figure 6 AB) and 2mM

[0030] NH4NO3 + 5% PEG Figure 7 AB) Overall plant phenotype under treatment throughout the entire growth period. OsGAT3-related transgenic plants under 2mM NH4NO3 ( ) Figure 5 C) 2mM NH4NO3 + 30mM NaCl Figure 6 C) and 2mM NH4NO3 + 5% PEG ( Figure 7C) Number of tillers per plant under treatment throughout the entire growth period. OsGAT3-related transgenic plants under 2mM NH4NO3 ( Figure 5 D) 2mM NH4NO3 + 30mM NaCl ( Figure 6 D) and 2mM NH4NO3 + 5% PEG ( Figure 7 D) Number of seeds per plant under treatment throughout the entire growth period. OsGAT3-related transgenic plants under 2mM NH4NO3 ( Figure 5 E), 2mM NH4NO3 + 30mM NaCl ( Figure 6 E) and 2mM NH4NO3 + 5% PEG ( Figure 7 E) Yield per plant under treatment throughout the entire growth period. The samples were: control wild-type Zhonghua 11 (ZH11), three lines of OsGAT3 gene overexpressing plants OE1, OE2 and OE3 (mean±SD, n=30), and three lines of OsGAT3 gene mutant plants C1, C2 and C3. Detailed Implementation

[0031] The technical solution of this application will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the descriptions provided. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0032] Example 1: Construction of OsGAT3 gene transgenic material

[0033] (1) Construction of OsGAT3 gene overexpression plants

[0034] RNA was extracted from rice cultivar ZH11 and reverse transcribed into cDNA. The cDNA of the OsGAT3 gene was amplified by PCR using primer pairs F1 and R1 (the protein sequence of the OsGAT3 gene is shown in SEQ ID NO. 1, and the gene sequence of the OsGAT3 gene is shown in SEQ ID NO. 2). Primer pairs F1 and R1 are as follows:

[0035] F1: 5'-GGATCCATGGGGGCGCCGAGCAGGGA-3' (SEQ ID NO.3, containing restriction site BamH I);

[0036] R1: 5'-TCTAGACACGTTCGCGAACAGTTTAT-3' (SEQ ID NO.4, containing the restriction enzyme site Xba I).

[0037] The OsGAT3 gene overexpression vector OsGAT3-p1306 was constructed by ligating BamHI and XbaI into the pCAMBIA-1306 vector (purchased from Cambia). The overexpression vector was then introduced into the normal rice variety Zhonghua 11 (ZH11) using Agrobacterium-mediated genetic transformation.

[0038] All the obtained transgenic seedlings were cultured in a normal nutrient solution with added hygromycin for one week. If the seedlings grew normally, the transgenic plants were considered positive. All the positive transgenic plants were transplanted into baskets with soil, and watered and fertilized regularly. When the seedlings grew to about 15cm in height, they were planted in the field. After the seedlings grew larger, the transgenic rice plants were harvested individually and planted until homozygous transgenic plants were identified in the T2 generation, which yielded OsGAT3 gene overexpression plants, namely OsGAT3-OE1, OsGAT3-OE2, and OsGAT3-OE3.

[0039] Leaves of plants overexpressing the OsGAT3 gene were collected, RNA was extracted and reverse transcribed into cDNA, and the expression level of the OsGAT3 gene in the overexpressing plants was detected by real-time quantitative PCR. The results showed that ( Figure 1 The expression level of the OsGAT3 gene in the A)(mean±SD, n=3) overexpression plants was higher than that in the control plant, Zhonghua 11 (ZH11) (the expression level of wild-type ZH11 in the figure is defined as "1"). The experimental results indicate that the transgenic transformation was successful, and the gene was successfully overexpressed, and the overexpression plants were successfully constructed. The primer pairs F2 and R2 used for real-time quantitative PCR have the following sequences:

[0040] F2: 5'-TCGTGGCAATCTTCAGGCGTCTTCA-3' (SEQ ID NO.5);

[0041] R2: 5'-TCCTTCTCGGGCCGCTCCCTTTGAG-3' (SEQ ID NO. 6).

[0042] Figure 1 The data were analyzed using SPSS software for one-way ANOVA, and Duncan's method was used to perform significance analysis at the 0.05 level. Different lowercase letters represent the level of difference.

[0043] (2) Construction of OsGAT3 gene mutant plants (gene knockout)

[0044] The gene knockout vector OsGAT3-C of the OsGAT3 gene was constructed using primer pairs F3 and R3, where F3 and R3 are respectively:

[0045] F3: 5'-CCTGAGCAACTTCATGGGTTTTAGAGCTAGAAATAGCAAGTTA-3' (SEQ ID NO. 7);

[0046] R3: 5'-CCACCATGAAGTTGCTCGCCACGGATCATCTGCACAACTC-3' (SEQ ID NO. 8).

[0047] Gene knockout was performed using the CRISPR / Cas9 system, and the method is described in the existing technical literature: MaXetal, Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genomeediting in monocot and dicot plants. MolPlant. 2015, 8(8):1274-1284. The gene knockout expression vector was introduced into the normal japonica rice variety Zhonghua 11 (ZH11) using Agrobacterium-mediated genetic transformation. The mutant plants were sequenced at generation T0, confirming that five gene knockout lines had been identified. Figure 1 B) Continue independent propagation to the T1 generation to obtain independent mutant plant lines of the OsGAT3 gene (all homozygous mutants), namely: OsGAT3-C1 (1 bp deletion), OsGAT3-C2 (7 bp increase), OsGAT3-C3 (2 bp increase), OsGAT3-C5 (C→G), and OsGAT3-C7 (3 bp deletion). The primer pairs F4 and R4 used to identify the mutant plants have the following sequences:

[0048] F4: 5'-TCCCTCGTGCTGTGCCTCGCCTAC-3' (SEQ ID NO.9);

[0049] R4: 5'-AGCGAAGTCGAGCGGGAGGAAGCC-3' (SEQ ID NO. 10).

[0050] Example 2: Determination of GABA and total nitrogen content in OsGAT3 gene-related materials from the field

[0051] (1) Determination of GABA content in plants

[0052] Overexpression plants (Example 1), Zhonghua 11, and gene knockout plants (Example 1) were used for field planting. Three lines of plants overexpressing the OsGAT3 gene, three lines of plants knocked out of the OsGAT3 gene, and one control plant (ZH11) were randomly selected from the field. Stems, leaves, and seeds of each plant were cut off with scissors, placed in kraft paper bags, and dried at 42℃ in a drying oven. Once the samples reached constant weight, each part was pulverized using a grinder, and the powder was passed through an 80-mesh sieve. 0.1g of the sample was accurately weighed into a 1.5ml EP tube using a 0.01g electronic balance. The GABA content was measured using a γ-aminobutyric acid (GABA) content kit (purchased from Suzhou Grees Biotechnology Co., Ltd.). 1ml of extraction buffer was added to the 1.5ml EP tube containing 0.1g of plant tissue powder, homogenized on ice, and centrifuged at 12000rpm, 4℃ or room temperature for 10min. 100ul of the supernatant (or 100ul of distilled water added to the control tube) was transferred to a new 1.5ml EP tube. Add 60 μL of reagent 1 (660 μL for control), 200 μL of reagent 2 (0 μL for control), and 400 μL of reagent 3 (0 μL for control) to EP tubes in sequence and mix well. Incubate in a boiling water bath (95-100℃) for 10 min, then in an ice bath to room temperature. When a blue-green color appears, transfer all the supernatant to a 1 mL glass cuvette (1 cm optical path). Read the absorbance value (A) of each tube at 645 nm. Then ΔA = A(determined) - A(control). Finally, according to the formula, GABA content (ug / g weight) = {(ΔA + 0.0033) / 0.0086} / (W * V1 / V) * D (Note: V - extraction volume, 1 mL; V1 - sample volume added, 0.1 mL; D - dilution factor, undiluted is 1; W - sample mass). Finally, the GABA content in each tissue of the OsGAT3 overexpression material was measured. Figure 2 The contents of AC (mean ± SD, n = 3) were significantly higher than those of other materials. The GABA content in all parts of the OsGAT3 mutant material was lower than that in the control wild-type ZH11 material.

[0053] The above results indicate that increased OsGAT3 expression significantly increases the GABA content in rice plants.

[0054] (2) Determination of total nitrogen content in plants

[0055] Overexpressing plants (Example 1), Zhonghua 11, and gene knockout plants (Example 1) were used for field cultivation. Three lines of OsGAT3 gene overexpressing plants, three lines of OsGAT3 gene knockout plants, and one control Zhonghua 11 (ZH11) were randomly selected from the field. Stems, leaves, and seeds of each plant were cut off with scissors, placed in kraft paper bags, and dried at 42°C in a drying oven. Once the samples reached constant weight, each part was pulverized using a grinder. 0.5XXX g of the sample (dry sample) was weighed using a 0.01% balance and placed in a digestion tube. Then, 0.5X g of copper sulfate pentahydrate and 4.5X g of potassium sulfate were weighed using a 1 / 1000 balance and placed in the digestion tube. 8 ml of concentrated sulfuric acid was accurately pipetted into the digestion tube (Note: X represents any number). Next, turn on the digestion furnace and begin digestion. First, digest at 280°C for about 20 minutes, then at 350°C for about 20 minutes, and finally at 450°C. Once the liquid in the digestion tube turns emerald green, continue digesting for another 30-40 minutes. After digestion is complete, allow it to cool to room temperature for later use.

[0056] Methyl red-bromocresol green mixed indicator: Accurately weigh 0.1g of methyl red and dilute to 100ml with anhydrous ethanol to prepare reagent A; accurately weigh 0.5g of bromocresol green and dilute to 100ml with anhydrous ethanol to prepare reagent B; mix reagents A and B to obtain methyl red-bromocresol green mixed indicator.

[0057] Boric acid indicator solution: Weigh 20g of boric acid and dilute to 1000ml with ultrapure water (adjust the pH to 4.5 with dilute acid or dilute alkali).

[0058] 10 mol / L sodium hydroxide: 400 g of sodium hydroxide dissolved in 1 L of water.

[0059] Preparation and standardization of 0.2N (0.1mol / L) sulfuric acid standard solution (stock solution): Pipette 6ml of concentrated sulfuric acid into a 1L volumetric flask and dilute to volume with water. Label the flask as 0.2N sulfuric acid. Then standardize the solution by weighing 0.2XXXg of anhydrous sodium carbonate (dried at 160℃ for two hours) into three 150ml Erlenmeyer flasks. Dissolve each flask in 30ml of water, add 2 drops of methyl red-bromocresol green mixed indicator (titrate with an acid burette without a glass bulb at the tip), and titrate with 0.2N sulfuric acid solution until the solution changes from green to purple-red. Boil for 2-3 minutes (gentle boiling) to remove CO2, cool, and continue titrating until the solution suddenly turns wine-red. Simultaneously perform a blank experiment: Blank: Add 30ml of water to one 150ml Erlenmeyer flask, add 2 drops of methyl red-bromocresol green mixed indicator, and titrate until the calculated concentration is 0.2279N. Label the flask as 0.2279N sulfuric acid nitrogen standard solution.

[0060] 0.01139N sulfuric acid nitrogen standard solution: Pipette 50 ml of 0.2279N sulfuric acid nitrogen standard solution into a 1L volumetric flask and dilute to volume with distilled water.

[0061] The formula for calculating the standard solution is: N = M / (V1 - V2) * 0.05299 (Note: N - equivalent concentration of sulfuric acid; M - mass of anhydrous sodium carbonate (g); V1 - volume of sulfuric acid standard solution used (ml); V2 - volume of sulfuric acid standard solution used for blank solution (ml), this item is generally 0).

[0062] Operating procedures for the Kjeldahl nitrogen analyzer: The digested sample solution should be diluted to 100 ml with distilled water. Then, pipette 5 ml of the test solution into the long glass tube of the nitrogen analyzer. Pipe 5 ml of boric acid indicator solution into a wide-mouthed Erlenmeyer flask. Alkali solution time: 4 seconds. After distillation for 6 minutes, remove the flask. Titrate with a semi-micro burette, the titration changing from green to white to pink (the laboratory has purchased an electronic burette, which is convenient, accurate, and reliable).

[0063] Finally, according to the formula: Total nitrogen of plants (%) = Equivalent concentration of H2SO4 standard solution * (Volume of H2SO4 standard solution consumed during sample titration - Volume of H2SO4 standard solution in blank test) * Dividend factor (20 according to the above method, because 5mL was drawn from 100mL) * 0.014 * 100 / Sample weight (0.5XXXg here according to the above method). Finally, the total nitrogen of each tissue of the OsGAT3 overexpression material was measured. Figure 2 The DF (mean ± SD, n = 3) content was significantly higher than that of other materials. The total nitrogen content in all parts of the OsGAT3 mutant material was lower than that of the control wild-type ZH11 material.

[0064] The above results indicate that increased OsGAT3 expression significantly increases the total nitrogen content of rice plants.

[0065] Example 3: Salt tolerance test of OsGAT3 gene-related materials

[0066] (1) Soaking of rice seeds: Rice seeds were soaked in a 0.6% dilute nitric acid solution for 8 hours. The rice seeds used were ZH11, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3 (all seeds were obtained from the Plant Hormone and Nutrition Molecular Regulation Laboratory of Guizhou University).

[0067] (2) Cultivation of rice seeds: After soaking, rinse the seeds 3-5 times, divide them into petri dishes, add appropriate pure water, and place them in a constant temperature incubator at 37℃ for cultivation. Change the water 2-3 times a day.

[0068] (3) Hydroponics: When the rice plants were uniformly cultured to the 3-leaf stage, 3L of ordinary rice culture solution was added to each large black box. The rice plants were then transferred to the large black boxes for further cultivation. Two large black boxes were set up, one for the salt treatment group and the other for the salt and GABA combined treatment group. The preparation of the ordinary culture solution is a routine technique for those skilled in the art, and the method is referenced (Yoshida S, Fomo DA, Cock JH, etc. Routine procedure for growing rice plants in culture solution. In: Laboratory manual for physiological studies of rice[J], International Rice Research Institute, 61-66.).

[0069] (4) GABA treatment and salt stress (NaCl) treatment: After 3 days of hydroponics, the seedlings had basically adapted to the large black box culture. To reduce workload and facilitate subsequent work, GABA stock solution and NaCl stock solution were prepared with concentrations of 1 mol / L and 4 mol / L, respectively. 0 ml of GABA stock solution and 90 ml of NaCl stock solution were added to one large black box (3 L) of rice culture medium in the salt treatment group, resulting in GABA and NaCl concentrations of 0 mmol / L and 120 mmol / L, respectively. 90 mL of NaCl stock solution was added to one large black box (3 L) of rice culture medium in the salt and GABA combined treatment group, resulting in a NaCl concentration of 120 mmol / L. Subsequently, 0.6 ml of GABA stock solution was added to one small black box (3 L) of rice culture medium in the salt and GABA combined treatment group, bringing the GABA concentration in the small black box to 0.2 mmol / L. After treating the rice with the above nutrient solution for 7 days, the rice culture medium for each treatment was replaced with ordinary nutrient solution (rice culture medium containing 0 mmol / L GABA and 0 mmol / L NaCl) for a 7-day recovery treatment. Note: The rice nutrient solution was changed daily to ensure the stability of the concentrations of GABA and NaCl.

[0070] (5) Biomass determination: After all treatments were subjected to salt stress or combined salt and GABA treatment, the fresh weight of seedlings related to the OsGAT3 gene in each treatment was calculated. In addition, photos of each treatment were taken, and the seedlings were evenly spread out in a row in a large black box. The order of the materials was ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3. The photos are shown below. Figure 3 As shown in AB, the seedling biomass after each treatment is as follows: Figure 3 G (salt treatment group) and Figure 3 H (salt and GABA combined treatment group) is shown. After each treatment underwent recovery treatment with ordinary nutrient solution, the seedling biomass of each treatment was as follows. Figure 3 I (Salt Treatment Group) and Figure 3 M (salt and GABA combined treatment group) is shown.

[0071] (6) Survival rate determination: After all treatments underwent recovery treatment with ordinary nutrient solution, the number of dead and surviving seedlings in each treatment was counted, and the survival rate was calculated. In addition, photographs were taken of each treatment, and the seedlings were evenly spread out in a row in a large black box. The material arrangement order was ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3. The photographs are shown below. Figure 3 As shown in CD, the seedling survival rates after each treatment are as follows: Figure 3 N (salt treatment group) and Figure 3 As shown in O (salt and GABA combined treatment group).

[0072] (7) Determination of NBT and DAB: Preparation of NBT staining solution: Accurately weigh 50 mg of nitrotetrazolium chloride (NBT) and dissolve it in 100 ml of Tris buffer (pH 7.4) to obtain NBT staining working solution. Store at 4°C protected from light for up to one week.

[0073] Preparation of DAB staining solution: Accurately weigh 0.04 g of diaminobenzidine (DAB) and dissolve it in 76 mL of ultrapure water. Then adjust the pH to 3.8 with HCl to promote the dissolution of DAB and obtain the DAB staining working solution. Prepare and use immediately.

[0074] NBT staining: Prepare NBT staining solution (see solution formula) and dispense into 10mL centrifuge tubes; cut rice leaves from the same location after treatment with NaCl or NaCl and GABA for 0h and 48h, and immediately place them into 10mL centrifuge tubes containing NBT staining solution; place the centrifuge tubes in a vacuum pump to create a vacuum; incubate overnight at 37℃; discard the NBT staining solution, add 95% ethanol to submerge the sample, and place the centrifuge tubes in an 80℃ water bath for decolorization, changing the 95% ethanol every 10 minutes. After the green color of the leaves has completely faded, remove the leaves and photograph the staining results as shown in the image. Figure 3 As shown in E.

[0075] DAB staining: Prepare DAB staining solution (see solution formula) and dispense into 10mL centrifuge tubes; cut rice leaves from the same location after treatment with NaCl or NaCl and GABA for 0h and 48h, and immediately place them into 10mL centrifuge tubes containing DAB staining solution; place the centrifuge tubes in a vacuum pump to create a vacuum; incubate overnight at 37℃; discard the DAB staining solution, add 95% ethanol to submerge the sample, and place the centrifuge tubes in an 80℃ water bath for decolorization, changing the 95% ethanol every 10 minutes. After the green color of the leaves has completely faded, remove the leaves and photograph the staining results as shown in the image. Figure 3 As shown in F.

[0076] (8) Determination of stress resistance indicators: After all treatments had undergone salt stress treatment or combined salt and GABA treatment for 48 hours, 0.1g of sword leaves from 5 seedlings of each treatment line (ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3) were taken for determination of POD (peroxidase) enzyme activity, CAT (catalase) enzyme activity, and MDA (malondialdehyde) content. The stress resistance indicators were determined using kit methods. The POD enzyme activity assay kit (50T), CAT enzyme activity assay kit (50T), and MDA (96T) content assay kit were all purchased from Nanjing Dulai Biotechnology Co., Ltd. The test results are shown in [link to test results]. Figure 3 , Figure 3 J is a statistical graph of CAT enzyme activity in various materials of the salt treatment group (mean±SD, n=3); Figure 3 P is a statistical graph of CAT enzyme activity in various materials of the salt and GABA combined treatment group (mean±SD, n=3); Figure 3 K is a statistical graph of POD enzyme activity in various materials of the salt treatment group (mean±SD, n=3); Figure 3 Q is a statistical graph of POD enzyme activity (mean±SD, n=3) of various materials in the salt and GABA combined treatment group; Figure 3L is a statistical graph of MDA content in various materials of the salt treatment group (mean±SD, n=3); Figure 3 R is a statistical graph of MDA content (mean ± SD, n = 3) of various materials in the salt and GABA combined treatment group.

[0077] The above results indicate that increasing the expression level of the OsGAT3 gene in rice can alleviate the harmful effects of salt stress on rice growth and development, and the salt resistance effect is more pronounced after GABA application. Under salt stress, exogenous GABA application can increase the activity of CAT and POD enzymes, reduce the accumulation of MDA, and increase the distribution area of ​​superoxide anions and superoxide enzymes, thereby alleviating the damage caused by salt stress to rice seedlings and increasing the survival rate of rice under salt stress.

[0078] Example 4: Drought resistance test of OsGAT3 gene-related materials

[0079] (1) Soaking of rice seeds: Rice seeds were soaked in a 0.6% dilute nitric acid solution for 8 hours. The rice seeds used were ZH11, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3 (all seeds were obtained from the Plant Hormone and Nutrition Molecular Regulation Laboratory of Guizhou University).

[0080] (2) Cultivation of rice seeds: After soaking, rinse the seeds 3-5 times, divide them into petri dishes, add appropriate pure water, and place them in a constant temperature incubator at 37℃ for cultivation. Change the water 2-3 times a day.

[0081] (3) Hydroponics: When the rice plants were uniformly cultured to the 3-leaf stage, 3L of ordinary rice culture solution was added to each of the large black boxes. The rice plants were then transferred to the large black boxes for further cultivation. Two large black boxes were set up, one for the 15% PEG treatment group and the other for the 15% PEG and GABA combined treatment group. The preparation of the ordinary culture solution is a routine technique for those skilled in the art, and the method is referenced (Yoshida S, Fomo DA, Cock JH, etc. Routine procedure for growing rice plants in culture solution. In: Laboratory manual for physiological studies of rice [J], International Rice Research Institute, 61-66.).

[0082] (4) GABA treatment and drought stress (PEG) treatment: After 3 days of hydroponics, the seedlings had basically adapted to the large black box culture. To reduce workload and facilitate subsequent work, GABA stock solution and NaCl stock solution were prepared, with concentrations of 1 mol / L for GABA and 15% PEG for NaCl. 0 ml of GABA stock solution and 3 L of 15% PEG stock solution were added to one large black box of rice culture medium in the PEG treatment group, resulting in concentrations of 0 mmol / L for GABA and 15% PEG for PEG in one large black box. 3 L of 15% PEG stock solution was added to one large black box of rice culture medium in the PEG and GABA combined treatment group, resulting in a PEG concentration of 15% PEG in one large black box. Subsequently, 1.5 ml of GABA stock solution was added to one small black box (3 L) of rice culture medium in the PEG and GABA combined treatment group, bringing the GABA concentration in the small black box to 0.5 mmol / L. After 0 h and 48 h of rice treatment with the above nutrient solution, a small amount of leaves from the same part of each treatment were taken and stained with NBT and DAB. After 7 days of rice treatment with the above nutrient solution, the rice culture medium of the two treatments was replaced with ordinary nutrient solution (rice culture medium containing 0 mmol / L GABA and 0% PEG) for a 9-day recovery treatment. Note: The rice nutrient solution was changed daily to ensure the stability of the concentrations of GABA and PEG.

[0083] (5) Biomass determination: After all treatments were subjected to drought stress or PEG and GABA combined treatment, the fresh weight of seedlings of OsGAT3 gene-related materials for each treatment was counted. In addition, photos of each treatment were taken, and the seedlings were evenly spread out and connected in a row in a large black box. The material arrangement order was ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, OsGAT3-C3. The photos are shown below. Figure 4 As shown in AB, the seedling biomass after each treatment is as follows: Figure 4 G (15% PEG treatment group) and Figure 4 The results for group H (15% PEG and GABA combined treatment) are shown below. After all treatments underwent recovery treatment with standard nutrient solution, the seedling biomass for each treatment was as follows: Figure 4 I (15% PEG treatment group) and Figure 4 M (15% PEG and GABA combined treatment group) is shown.

[0084] (6) Survival rate determination: After all treatments underwent recovery treatment with ordinary nutrient solution, the number of dead and surviving seedlings in each treatment was counted, and the survival rate was calculated. In addition, photographs were taken of each treatment, and the seedlings were evenly spread out in a row in a large black box. The material arrangement order was ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3. The photographs are shown below. Figure 4 As shown in CD, the seedling survival rates after each treatment are as follows: Figure 4 N (15% PEG-treated group) and Figure 4 The results are shown in group O (15% PEG and GABA combined treatment group).

[0085] (7) Determination of NBT and DAB: Preparation of NBT staining solution: Accurately weigh 50 mg of nitrotetrazolium chloride (NBT) and dissolve it in 100 ml of Tris buffer (pH 7.4) to obtain NBT staining working solution. Store at 4°C protected from light for up to one week.

[0086] Preparation of DAB staining solution: Accurately weigh 0.04 g of diaminobenzidine (DAB) and dissolve it in 76 mL of ultrapure water. Then adjust the pH to 3.8 with HCl to promote the dissolution of DAB and obtain the DAB staining working solution. Prepare and use immediately.

[0087] NBT staining: Prepare NBT staining solution (see solution formula) and dispense into 10mL centrifuge tubes; cut rice leaves from the same location after treatment with 15% PEG or 15% PEG and GABA for 0h and 48h, and immediately place them into 10mL centrifuge tubes containing NBT staining solution; place the centrifuge tubes in a vacuum pump to create a vacuum; incubate overnight at 37℃; discard the NBT staining solution, add 95% ethanol to submerge the sample, and place the centrifuge tubes in an 80℃ water bath for decolorization, changing the 95% ethanol every 10 minutes. After the green color of the leaves has completely faded, remove the leaves and photograph the staining results as shown in the image. Figure 4 As shown in E.

[0088] DAB staining: Prepare DAB staining solution (see solution formula) and dispense into 10mL centrifuge tubes; cut rice leaves from the same location after treatment with NaCl or NaCl and GABA for 0h and 48h, and immediately place them into 10mL centrifuge tubes containing DAB staining solution; place the centrifuge tubes in a vacuum pump to create a vacuum; incubate overnight at 37℃; discard the DAB staining solution, add 95% ethanol to submerge the sample, and place the centrifuge tubes in an 80℃ water bath for decolorization, changing the 95% ethanol every 10 minutes. After the green color of the leaves has completely faded, remove the leaves and photograph the staining results as shown in the image. Figure 4 As shown in F.

[0089] (8) Determination of stress resistance indicators: After all treatments had undergone drought stress treatment or 15% PEG and GABA combined treatment for 48 hours, 0.1g of flag leaf samples from 5 seedlings of each treatment line (ZH11-1, ZH11-2, ZH11-3, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3) were taken for determination of POD (peroxidase) enzyme activity, CAT (catalase) enzyme activity, and MDA (malondialdehyde) content. The stress resistance indicators were determined using kit methods. The POD enzyme activity assay kit (50T), CAT enzyme activity assay kit (50T), and MDA (96T) content assay kit were all purchased from Nanjing Dulai Biotechnology Co., Ltd. The test results are shown in [link to test results]. Figure 4 , Figure 4 J is a statistical graph of CAT enzyme activity in various materials in the 15% PEG treatment group (mean ± SD, n = 3); Figure 4 P is a statistical graph of CAT enzyme activity in various materials of the 15% PEG and GABA combined treatment group (mean ± SD, n = 3); Figure 4 J is a statistical graph of POD enzyme activity in various materials of the 15% PEG-treated group (mean ± SD, n = 3); Figure 4 Q is a statistical graph of POD enzyme activity (mean±SD, n=3) of various materials in the 15% PEG and GABA combined treatment group; Figure 4 L is a statistical graph of MDA content (mean±SD, n=3) of various materials in the 15% PEG treatment group; Figure 4 R is a statistical graph of MDA content (mean ± SD, n = 3) in various materials of the 15% PEG and GABA composite treatment group.

[0090] The above results indicate that increasing the expression level of the OsGAT3 gene in rice can alleviate the damage of drought stress to rice growth and development, and the drought resistance effect is more significant after applying GABA. Under drought stress, exogenous application of GABA can increase the activity of CAT and POD enzymes, reduce the accumulation of MDA, and increase the distribution area of ​​superoxide anions and superoxide enzymes, thereby alleviating the damage caused by drought stress to rice seedlings and increasing the survival rate of rice under drought stress.

[0091] Example 5: Salt and simulated drought hydroponics experiments on OsGAT3 gene-related materials throughout their entire growth period

[0092] (1) Soaking of rice seeds: Rice seeds were soaked in a 0.6% dilute nitric acid solution for 8 hours. The rice seeds used were ZH11, OsGAT3-OE1, OsGAT3-OE2, OsGAT3-OE3, OsGAT3-C1, OsGAT3-C2, and OsGAT3-C3 (all seeds were obtained from the Plant Hormone and Nutrition Molecular Regulation Laboratory of Guizhou University).

[0093] (2) Cultivation of rice seeds: After soaking, rinse the seeds 3-5 times, divide them into petri dishes, add appropriate pure water, and place them in a constant temperature incubator at 37℃ for cultivation. Change the water 2-3 times a day.

[0094] (3) Hydroponics: When the rice plants were uniformly cultured to the 3-leaf stage, 10L of ordinary rice culture solution was added to each blue pot. Then, the rice plants were transferred to the blue pots for further cultivation. Nine blue pots were set up, with three blue pots as the NH4NO3 treatment group, three blue pots as the NH4NO3 and NaCl combined treatment group, and three blue pots as the NH4NO3 and PEG combined treatment group. The preparation of ordinary culture solution is a routine technique for those skilled in the art, and the method is referenced (Yoshida S, Fomo DA, Cock JH, etc. Routine procedure for growing rice plants in culture solution. In: Laboratory manual for physiological studies of rice[J], International Rice Research Institute, 61-66.).

[0095] (4) NH4NO3 treatment, NH4NO3+NaCl treatment, and NH4NO3+PEG treatment: After 3 days of hydroponics, the seedlings had basically adapted to the rice culture in the blue pots. To reduce workload and facilitate subsequent work, NH4NO3 stock solution and NaCl stock solution were prepared. The concentration of the NH4NO3 stock solution was 1 mol / L, and the concentration of the NaCl stock solution was 4 mol / L. 20 ml of NH4NO3 stock solution was added to each of the three blue pots in the 10 L rice culture medium of the NH4NO3 treatment group, so that the NH4NO3 concentration in the three blue pots in the NH4NO3 treatment group was 2 mmol / L. 20 ml of NH4NO3 stock solution and 75 mL of NaCl stock solution were added to each of the three blue pots in the 10 L rice culture medium of the NH4NO3 and NaCl combined treatment group, so that the concentrations of NH4NO3 and NaCl in the three blue pots in the NH4NO3 and NaCl combined treatment group were 2 mmol / L and 30 mmol / L, respectively. In the NH4NO3 and PEG combined treatment group, 19 ml of NH4NO3 stock solution and 500 g of PEG6000 were added to 9.5 L of rice culture medium in three blue pots, resulting in NH4NO3 and PEG concentrations of 2 mmol / L and 5% PEG, respectively. Phenotypic analysis was performed after the rice reached grain-filling maturity. Note: The rice nutrient solution was changed every 7 days to ensure the stability of the NH4NO3, NH4NO3+NaCl, and NH4NO3+PEG treatment concentrations.

[0096] (5) Phenotypic photography: After rice plants that had undergone NH4NO3 treatment, NH4NO3+NaCl treatment, and NH4NO3+PEG treatment to maturity, the overall plant phenotypes of the OsGAT3 gene-related materials for each treatment were photographed: such as Figure 5 AB (treated with 2mM NH4NO3), Figure 6 AB (treated with 2mM NH4NO3 + 30mM NaCl) Figure 7 As shown in AB (2mM NH4NO3 + 5% PEG treatment).

[0097] (6) Biomass determination: After rice plants underwent NH4NO3 treatment, NH4NO3+NaCl treatment, and NH4NO3+PEG treatment until maturity, the yields of OsGAT3 gene-related materials for each treatment were statistically analyzed. Figure 5 E (treated with 2mM NH4NO3), Figure 5 E(treated with 2mM NH4NO3 + 30mM NaCl) Figure 5 E(2mM NH4NO3 + 5% PEG treatment) is shown.

[0098] (7) Determination of tiller number and grain number per plant: After rice plants underwent NH4NO3 treatment, NH4NO3+NaCl treatment, and NH4NO3+PEG treatment until maturity, the tiller number per plant of OsGAT3 gene-related materials for each treatment was counted: e.g. Figure 5 C(2mM NH4NO3 treatment) (mean±SD, n=30) Figure 6 C(2mM NH4NO3 + 30mM NaCl treatment) (mean ± SD, n = 30) Figure 7 C(2mM NH4NO3 + 5% PEG treatment) (mean ± SD, n = 30) is shown. Additionally, the number of seeds per plant in OsGAT3 gene-related materials for each treatment was statistically analyzed: [e.g., ...] Figure 5 D(2mM NH4NO3 treatment) (mean±SD, n=30) Figure 6 D(treatment with 2mMNH4NO3 + 30mM NaCl) (mean ± SD, n = 30) Figure 7 The results are shown as D(2mM NH4NO3 + 5% PEG treatment) (mean ± SD, n = 30).

[0099] The above results indicate that increasing the expression level of the OsGAT3 gene in rice can enhance rice's resistance to salt and drought, as well as increase rice tillering and yield.

[0100] Finally, it should be noted that the above embodiments are merely representative examples of this application. Obviously, the technical solutions of this application are not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this application should be considered within the scope of protection of this application.

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