Application of glutamic acid transaminase in high yield and stress tolerance of rice

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of glutamic acid transaminase in high yield and stress tolerance of rice. Experiments discover that rice plant grains overexpressing the glutamic acid transaminase CkTA are enlarged, the thousand grain weight is increased, the tiller number is increased, the total weight of single plant grains is increased, and the biomass is increased. Overexpression of glutamic acid transaminase CkTA can improve the ammonium salt absorption efficiency of rice. Through overexpression of glutamic acid transaminase CkTA, the growth speed of rice under a waterflooding condition can be increased. It is found for the first time that glutamic acid transaminase CkTA can improve the yield, nitrogen utilization efficiency and flooding resistance of rice, and an important theoretical basis is provided for breeding of high-yield and stable-yield rice varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to application of glutamate aminotransferase in rice high-yield and stress-resistance. Background Art

[0002] Using synthetic biology to explore novel metabolic pathways for photorespiration in C3 plants, such as rice, could create a novel photorespiratory metabolic pathway that could metabolize the plant-toxic glyoxylate produced during rice photorespiration while also reducing the energy waste and carbon loss caused by photorespiration. This represents a new approach to achieving high rice yield and stress tolerance. Transaminases, particularly glutamate transaminase, play a key role in the design of photorespiratory metabolic pathways. This enzyme transfers the amino group of glutamate to other keto acids (such as pyruvate and oxaloacetate) to produce α-ketoglutaric acid (2-oxoglutaric acid, 2OG), crucial for the reuse of ammonia produced by photorespiration. During the screening of transaminases, it was discovered that the glutamate-alanine aminotransferase CkTA from Citrobacter krusei has high glutamate catalytic activity. Previous articles have reported that CkTA exhibits high activity towards various amine donors, including L-Glu, L-Ala, L-Asp, L-Pro, L-Lys, L-His, L-asn, L-Val, and L-Arg. L-Glu has been shown to be the optimal amine donor for CKTA (https: / / doi.org / 10.1016 / j.jbiotec.2019.06.008). CKTA catalyzes the reaction: GLU + glyoxylate Gly+2OG, GLU+OAA Asp+2OG and GLU+pyruvate Ala+2OG.

[0003] However, there are no reports in the prior art on the use of glutamate aminotransferase CkTA in improving rice yield and waterlogging tolerance. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of glutamate aminotransferase in rice high yield and stress tolerance.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] First, the invention provides an application of glutamate aminotransferase in increasing rice yield. Rice plants overexpressing glutamate aminotransferase CkTA have larger grains, higher thousand-grain weight, more tillers, higher total grain weight per plant, and higher biomass.

[0007] Secondly, the application of glutamate aminotransferase in improving the nitrogen utilization efficiency of rice is provided. Overexpression of glutamate aminotransferase CkTA can improve the ammonium absorption efficiency of rice.

[0008] Thirdly, the application of glutamate aminotransferase in improving the waterlogging resistance of rice is provided. Overexpression of glutamate aminotransferase CkTA can increase the growth rate of rice under waterlogging conditions.

[0009] The beneficial effects of the present invention are:

[0010] The present invention discovered for the first time that glutamate aminotransferase CkTA can improve rice yield, nitrogen utilization efficiency and waterlogging resistance, providing an important theoretical basis for the breeding of high-yield and stress-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the pZmUbi::CkTA::pGN2201 vector; LB: left border of T-DNA; PZmUbi: maize Ubi promoter; CkTA: full-length CDS fragment of CkTA after codon optimization; RB: right border of T-DNA;

[0012] Figure 2 Phenotypic and expression analysis of pZmUbi::CkTA transgenic plants; A. PCR detection of the CkTA gene in T0 generation overexpressing transgenic plants; B. RT-PCR analysis of cDNA reverse-transcribed from total RNA extracted from leaves of WT and three pZmUbi::CkTA plants, with rice β-actin (β-ACTIN) as a control; C, D. Phenotypes of overexpressing transgenic plants (C: seedling stage, D: maturity stage); EJ. Agronomic trait evaluation of WT and pZmUbi::CkTA transgenic plants (n=9, values ​​are mean ± SD, significant differences (P < 0.05) are indicated by letters); WT: Nipponbare; OE1 / OE2: two pZmUbi::CkTA overexpressing lines;

[0013] Figure 3 for 15 Analysis of N accumulation and total nitrogen content and expression of nitrogen-related genes; A. Expression of some genes related to nitrogen absorption, transport and metabolism; B, C. 15 N-nitrate (B) and 15 N-ammonium (C) labeled shoots of WT and pZmUbi::CkTA transgenic plants 15 N accumulation determination; D. Nitrogen content of flag leaves of WT and pZmUbi::CkTA transgenic plants grown in the field in Langfang in 2024 at the heading stage;

[0014] Figure 4Light response curves of WT and pZmUbi::CkTA transgenic plants; values ​​are mean ± standard deviation (n=3);

[0015] Figure 5 The phenotypes of wild-type and overexpressing plants treated with waterlogging; A. Growth status of seedlings after being waterlogged for 4 days (left) and 6 days (right); B. Statistical results of seedling bud length; WT: Nipponbare; OE1 and OE2: two overexpression lines of pZmUbi::CkTA, Bar=1cm. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0017] The Agrobacterium and rice used in the test examples of the present invention are from:

[0018] Agrobacterium AGL1: The strain was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZK296.

[0019] The wild type is Nipponbare rice (Oryza sativa L. ssp. Japonica; hereinafter also referred to as wild-type rice WT).

[0020] Data processing: GraphPad Prism 8 statistical software was used for data analysis. The significance of a single group of data was analyzed using the Student's test. P < 0.05 (*) indicates a significant difference, P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****) indicate an extremely significant difference. The significance of multiple groups of data was determined by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. The largest mean value is marked with the letter a in the results. If the second group of data shows a significant difference, it is marked with b, if there is no significant difference, it is marked with a, and so on.

[0021] Experimental Example 1 Overexpression vector construction

[0022] Construction of CkTA overexpression vector

[0023] 1. The protein sequence of the target gene CkTA aminotransferase (SEQ ID NO. 1) was searched through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The protein sequence was then codon-optimized to improve its expression efficiency in rice (the optimized and synthesized nucleic acid sequence is shown in SEQ ID NO. 2). The base sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0024] 2. PCR amplification was performed using the synthetic gene as a template to obtain the CDS sequence of CkTA. The ZmUbi promoter sequence was also amplified using extracted maize DNA as a template. The stop codon was amplified using the TNOS sequence (template pCAMBIA1300-NOS, retained in the laboratory). The PCR amplification program was: 95°C for 1 min; 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min 30 s, for 30 cycles. The high-fidelity DNA polymerase used was Phanta-Mix (Vazyme, Nanjing). When designing primers for CkTA, the stop codon was removed and the 1xFLAG sequence was added.

[0025] 3. Perform gel electrophoresis on the PCR amplified fragments and purify and recover them if they are correct.

[0026] 4. The recovered products were ligated to T vectors (pBM16A, pBM27, purchased from Biomed Biotech) to obtain pBM16A::pZmUbi, pBM16A::TNOS, and pBM27A::CkTA recombinant plasmids. The three plasmids were mixed at a ratio of 1:1:1, 50 ng of each vector was added, BasI endonuclease and T4 ligase were added, and the cells were ligated to the expression vector pGN2201 (DOI:10.3390 / plants11040488). E. coli was then transformed, and positive clones were identified by colony PCR. The clones were sent to a biological company for sequencing and plasmid extraction, and the overexpression vector ( Figure 1 ).

[0027] Table 1: Primer sequences for vector construction

[0028]

[0029]

[0030] Experimental Example 2: Acquisition and identification of transgenic plants

[0031] 1. Obtaining transgenic plants

[0032] The recombinant plasmid was transformed into Agrobacterium (AGL1), and the CkTA overexpressing plant driven by ZmUbi was obtained by genetic transformation of Agrobacterium. The transformation method is as follows:

[0033] Preparation of competent Agrobacterium tumefaciens (AGL1):

[0034] 1. Use a toothpick to pick up the Agrobacterium tumefaciens strain AGL1 kept in the laboratory and streak it onto LB solid medium containing rifampicin antibiotics, and culture it upside down at 28°C overnight;

[0035] 2. Pick a single colony with a toothpick and inoculate it into 5 mL of liquid LB medium containing Rif. Culture overnight at 28°C and 220 × g.

[0036] 3. Pipette 2 mL of overnight culture solution into 500 mL of LB culture medium containing Rif and culture overnight at 28°C and 220 × g;

[0037] 4. Bacterial liquid OD 600 =0.5, and the bacterial solution was transferred to a 50 mL round-bottom centrifuge tube and centrifuged at 2,500 × g for 10 min;

[0038] 5. Discard the supernatant and resuspend the cells in 20 mL of pre-chilled 0.15 M NaCl solution. Centrifuge at 2,500 × g for 10 min at 4°C and discard the supernatant.

[0039] 6. Repeat step 5;

[0040] 7. Add an appropriate amount of pre-cooled 10% glycerol according to the bacterial count, repeatedly pipette to resuspend the bacteria, and dispense into 1.5mL sterile centrifuge tubes (50μL per tube). Quickly freeze in liquid nitrogen and store in a -80℃ refrigerator.

[0041] Rice genetic transformation:

[0042] (1) Callus induction:

[0043] 1. Select about 200 full rice seeds and remove the husks;

[0044] 2. Place the seeds in a 50 mL sterile centrifuge tube, wash the seeds with sterile water, and then sterilize them upside down in about 40 mL of 75% alcohol for 1 minute. Pour out the alcohol and repeat this step once;

[0045] 3. Add 40 mL of 50% NaClO solution to the centrifuge tube and sterilize for 10 minutes. Repeat once. Then, wash the seeds 5-6 times with 40 mL of sterile water until the solution is completely clear.

[0046] 4. Use an alcohol burner to heat a spoon in a clean bench and spread the seeds on a Petri dish lined with filter paper to dry. Then, use sterilized tweezers to place the seeds on MS+ culture medium, approximately 20 seeds per dish. Incubate in a dark incubator at 28°C for approximately 30 days.

[0047] (2) Wound healing subculture:

[0048] 1. Open the MS solid culture medium in advance, blow dry it thoroughly in a clean bench, and sterilize the tweezers with an alcohol lamp at high temperature;

[0049] 2. Select embryonic calli that are creamy yellow, hard, and naturally detached and place them on MS medium, with about 100 calli placed per dish. Seal the dish and culture at 28°C for 5-7 days.

[0050] (3) Infection and co-culture

[0051] 1. The day before infection, activate the Agrobacterium by spreading it onto fresh Rif+Kan dual-antibody solid medium. The next day, scrape the Agrobacterium off with a pipette in a clean bench and place it into AAM liquid medium. Shake on a shaker at 200 rpm at 28°C for 0.5-1 hour. Adjust the OD value to 0.12-0.15 with AAM.

[0052] 2. Place the selected calli into a 50mL centrifuge tube. Add Agrobacterium to the tube and infect at 90 rpm at 28°C for 30 minutes. Discard the infection solution and blot the calli with filter paper to remove any residual Agrobacterium. Place the calli in an empty Petri dish lined with filter paper to air dry. Transfer the calli to NBCO+AS co-culture medium and incubate in the dark at 22°C for 3-4 days.

[0053] (4) Resistance screening

[0054] First screening: Use a spoon to transfer the callus tissue to a sterile 50ml centrifuge tube and rinse 5-7 times with sterile water until the supernatant is clear. Discard the supernatant and add 30ml of NBL + Tim (200mg / ml). Gently shake on a shaker at 100rpm for 1 hour. Discard the supernatant and air dry on filter paper. Use a spoon to transfer the calli to pre-dried selective medium and use forceps to evenly distribute the calli to prevent contact inhibition and widespread contamination. Incubate in the dark at 28°C for 10-15 days. Second screening: Transfer the calli from the first screening to the same medium under a clean bench for further screening, using approximately 25 calli per dish. Incubate for approximately 15-20 days. During the incubation process, distinct yellow particles the size of millet grains will naturally fall off.

[0055] (5) Differentiation

[0056] Transfer the above-mentioned naturally fallen yellow particles the size of millet grains to the differentiation medium, pick one newly fallen callus from each callus, do not pick repeatedly, about 20 grains per dish, seal it and culture it in the light on a 28℃ culture rack for one month.

[0057] (6) Strong seedlings

[0058] Transfer the differentiated healthy seedlings to 1 / 2MS seedling medium in a clean bench and culture in the light at 28°C for 2-3 weeks.

[0059] Culture medium used in the experiment:

[0060] 1. Callus Induction: MS+: MS macronutrients 25 mL / L, MS micronutrients 2 mL / L, MS organic 2 mL / L, ferric salts 5 mL / L, 2,4-D 5 mg / L, inositol 0.12 g / L, sucrose 33 g / L, casein CH 0.28 g / L, proline 2.8 g / L, phytagel 3.67 g / L, pH 5.82. Culture conditions: 28-30°C, dark, 4 weeks. (Pour 25 dishes per liter and allow the medium to dry out before use).

[0061] 2. Callus Subculture MS: MS macronutrients 25 mL / L, MS micronutrients 2 mL / L, MS organic 2 mL / L, ferric salts 5 mL / L, 2,4-D 2.3 mg / L, inositol 0.12 g / L, sucrose 33 g / L, acid-hydrolyzed casein CH 0.28 g / L, Phytagel (plant gel) 3.67 g / L, pH 5.82. Culture conditions: 28°C, dark, 5-7 days. (Pour 30 dishes per liter and allow the medium to dry out before use.)

[0062] 3. LB resistance medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar. No pH adjustment is required. Add 1 mL of kanamycin and rifampicin. (Pour 60 plates per liter of medium. Pour a small amount first and shake the plates until the bottom is completely covered.)

[0063] 4. Bacterial liquid medium (AAM): AA macronutrient 25 mL / L, AA micronutrient 2 mL / L, MS organic 2 mL / L, iron salt 5 mL / L, inositol 0.12 g / L, acid hydrolyzed casein 0.48 g / L, glucose 35 g / L, sucrose 68.7 g / L, aspartic acid 267 mg / L, glutamine 877 mg / L, L-arginine 177 mg / L, glycine 77 mg / L, pH 5.22. Add 40 mg / L AS after sterilization.

[0064] 5. Co-culture NBCO: 25 mL / L N6 macronutrient, 2 mL / L B5 micronutrient, 2 mL / L B5 organic, 5 mL / L iron salt, 2.3 mg / L 2,4-D, 0.12 g / L inositol, 33 g / L sucrose, 10 g / L glucose, 3.67 g / L Phytagel, pH 5.22. Add 20 mg / L As after sterilization. Culture conditions: 20-22°C, dark, 3-4 days. (Pour 25 dishes per liter. Dry the culture medium and add filter paper before use.)

[0065] 6. Recovery culture (NBL liquid screening): N6 macronutrient 25 mL / L, B5 micronutrient 2 mL / L, B5 organic 1 mL / L, iron salt 5 mL / L, 2,4-D 2.3 mg / L, inositol 0.12 g / L, sucrose 33 g / L, acid-hydrolyzed casein CH 0.32 g / L, proline 0.52 g / L, pH 5.72. Add 200 mg / L of Timentin after sterilization.

[0066] 7. Selective medium NBS: N6 macronutrient 25 mL / L, B5 micronutrient 2 mL / L, B5 organic 2 mL / L, iron salt 5 mL / L, 2,4-D 2.3 mg / L, inositol 0.12 g / L, sucrose 33 g / L, casein CH 0.27 g / L, proline 0.47 g / L, phytagel 3.67 g / L, pH 5.72. After sterilization, add ① 500 mg / L cephalosporin or 200 mg / L Timentin, and ② 50 mg / L hygromycin or 150 mg / L G418. Culture conditions: 28°C, dark, 14 days. (Pour 25 dishes per liter and air dry the medium before use).

[0067] 8. NBD differentiation culture: MS macro 25 mL / L, MS micro 2 mL / L, MS organic 2 mL / L, iron salt 5 mL / L, NAA 0.04 mg / L, KT 2.2 mg / L, inositol 0.12 g / L, sucrose 33 g / L, sorbitol 33 g / L, acid-hydrolyzed casein CH 0.32 g / L, Phytagel 4.77 g / L, pH = 5.82. Culture conditions: 28°C, 16 hr / day light, 3-4 weeks. (Pour 25 dishes per liter and allow the medium to dry out before use).

[0068] 9. Seedling Growth Medium: (1 / 2 MS bulk) MS bulk 12.5 mL / L, MS trace 2 mL / L, MS organic 2 mL / L, iron salt 5 mL / L, inositol 0.12 g / L, sucrose 33 g / L, agar 6.3 g / L, pH = 5.82. Culture conditions: 28°C, 14-16 hours / day light, 2-3 weeks (pour 25-30 dishes per liter).

[0069] After all culture media are prepared, they need to be placed for 3 to 4 days to observe whether they are contaminated before use.

[0070] 2. Screening of positive plants

[0071] The recombinant plasmid was transformed into Nipponbare using Agrobacterium-mediated genetic transformation, and 22 ZmUbi-activated CkTA overexpressing plants were obtained. Figure 2WT) and CkTA overexpressing transgenic plants, and 17 positive lines were screened ( Figure 2 A), total RNA was extracted from wild-type rice and four transgenic plants overexpressing CkTA, and reverse transcribed to obtain cDNA as a template. RT-PCR was used to analyze the expression of CkTA in four independent lines. The results showed that CkTA was successfully expressed in rice ( Figure 2 B).

[0072] Semi-quantitative PCR amplification was performed using CkTA-QRT1 primer and CkTA-QRT2 primer, followed by nucleic acid gel electrophoresis detection (DNA identification primers are the same).

[0073] CkTA-QRT1: 5'-TCAAGGCTACTGTGCGACGC-3';

[0074] CkTA-QRT2: 5'-CGTTTCCAGAGTTTGAGTCCGG-3'.

[0075] The internal reference primers used were actin-F and actin-R:

[0076] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0077] actin-R: 5'-AATGAGTAACCACGCTCCGC-3'.

[0078] Experimental methods:

[0079] 1. Extraction of Rice Genomic DNA

[0080] The simple CTAB method was used for extraction. The specific steps are as follows:

[0081] 1) Take approximately 400 mg of fresh young rice leaves, freeze them in liquid nitrogen, grind them into powder, and quickly transfer them to a 2.0 mL Eppendorf tube;

[0082] 2) Add 800 μL of CTAB extraction buffer (100 mM Tris-HCl pH = 8, 20 mM EDTA, 1.4 M NaCl, 2.0% CTAB, 1% PVP) preheated at 65°C and incubate in a 65°C water bath for 30 min, shaking every 15 min to mix thoroughly.

[0083] 3) Add an equal volume of chloroform and isoamyl alcohol mixture (24:1), shake to mix, and let stand for 5 minutes;

[0084] 4) Centrifuge at 10,000 rpm for 5 min and transfer the supernatant to a new 2.0 mL centrifuge tube;

[0085] 5) Add an equal volume of isopropanol, mix vigorously, and place at -20°C for 30 min;

[0086] 6) Centrifuge at 10,000 rpm for 5 min, discard the supernatant and collect the precipitate;

[0087] 7) Add 800 μL of 75% ethanol to wash the precipitate;

[0088] 8) Centrifuge at 13,000 rpm for 10 min and discard the supernatant;

[0089] 9) Blow dry the precipitate on a clean bench and add 50 μL ddH2O to dissolve it.

[0090] 2. Extraction of total RNA from rice

[0091] Total RNA was extracted from wild-type rice plants using the RNAprep pure plant kit from Tiangen Biochemical. Tissue samples for testing were collected from the field, placed in tin foil, quickly frozen in liquid nitrogen, and brought back to the Beijing laboratory for storage in an ultra-low temperature freezer. Tissue samples used to test tissue expression levels included leaves, sheaths, stems, flowers, and roots of mature wild-type plants, while seedling tissues included leaves, sheaths, and roots. The RNA extracted here was from field-grown rice leaf tissue. The specific steps for RNA extraction are as follows:

[0092] (1) Grind 100 mg of sample under liquid nitrogen cryogenic conditions, then add 500 μL of lysis buffer SL. Add β-mercaptoethanol before use and vortex vigorously to mix.

[0093] (2) Centrifugation at 12,500 rpm for 5 min;

[0094] (3) Transfer the supernatant to a CS filter column, centrifuge at 12,500 rpm for 2 min, and transfer the supernatant to a new tube.

[0095] (4) Add 0.4 times the volume of the supernatant liquid anhydrous ethanol, mix well and a precipitate will precipitate. Transfer it to the CR3 adsorption column and centrifuge at 12,500 rpm for 30 seconds. Discard the waste liquid and return the CR3 to the original tube.

[0096] (5) Carefully add 400 μL of RW1 to CR3, incubate at 13,000 rpm for 1 min, discard the lower solution, and return CR3 to the tube.

[0097] (6) Preparation of DNase I working solution: Take 10 μL of DNase I stock solution, add 70 μL of RDD solution, and gently tap to mix.

[0098] (7) Add 80 μL of DNase I working solution to the center of the CR3 adsorption column and digest at room temperature for 15 min.

[0099] (8) Add 350 μL of RW1 to CR3, centrifuge at 12,500 rpm for 30 seconds, discard the waste liquid, and return CR3 to the tube.

[0100] (9) Add 500 μL of RW to CR3, centrifuge at 12,500 rpm for 30 seconds, discard the waste liquid, and return CR3 to the tube.

[0101] (10) Repeat step (9).

[0102] (11) Centrifuge at 12,500 rpm for 3 min, transfer CR3 to a clean RNase-free centrifuge tube, add 70 μL RNase-free ddH2O to the center of the membrane, incubate at room temperature for 5 min, and centrifuge at 13,000 rpm for 2 min to obtain the RNA extraction product.

[0103] Note: The elution buffer volume should be greater than 30 μL; a smaller volume will affect recovery efficiency. Store RNA samples in a -80°C freezer. To increase RNA yield, add the resulting RNA solution to CR3, incubate at room temperature for 1-2 minutes, and centrifuge at 13,000 rpm for 2 minutes to obtain a higher yield and more concentrated RNA solution.

[0104] 3. RNA reverse transcription

[0105] First-strand cDNAs were synthesized using a reverse transcription kit IIIFirst Strand Synthesis Kit (Invitrogen, USA). Rice genes have a high GC content, so the high GC reverse transcription method recommended by the kit was used, and Oligo(dT)20 (50 μM) was selected for synthesis. The specific method is as follows:

[0106] (1) Preparation of RNA-primer mixture: 2 μg total RNA: n μL (volume n is determined based on RNA concentration), Oligo(dT)20 (50 μM): 1 μL, 10 mM dNTP mix: 2.5 μL, RNase-free ddH2O to 25 μL;

[0107] (2) Incubate the RNA sample at 65°C for 3 min in a PCR instrument and then immediately transfer to 55°C;

[0108] (3) Preparation of cDNA-Synthesis Mix (25 μL): RNase-Free ddH2O 3 μL, 10×RT buffer 5 μL, 25 mM MgCl2 10 μL, 0.1 M DTT 5 μL, RNase OUT Recombinant RNaseI inhibitor 1 μL, Super Script III RT 1 μL, mix well and set aside;

[0109] (4) Gently and thoroughly mix the RNA-primer mixture and cDNA-Synthesis Mix, and react on a PCR instrument at 55°C for 1 h and terminate at 85°C for 3 min.

[0110] (5) Add 1-2 μL of RNase H and digest in a PCR instrument at 37°C for 30 min. Store at -20°C until use.

[0111] Test Example 3 Agronomic Trait Statistics High Yield and Efficient Nitrogen Utilization

[0112] All materials were planted in the experimental fields of the Institute of Biology, Chinese Academy of Agricultural Sciences. After collecting T0 transgenic plant seeds, two positive overexpression lines were selected and planted in the field to obtain T1 and T2 transgenic lines and observe their phenotypes. Compared with the wild type, the transgenic plants had more tillers ( Figure 3 C (seedling stage), D (maturity stage), where WT: Nipponbare; OE1 and OE2: two overexpression lines of pZmUbi::CkTA. Further agronomic trait statistics showed that compared with Nipponbare, the pZmUbi::CkTA overexpression plants had larger grains, 1000-grain weight increased by 9.8-11.8%, the number of tillers increased significantly, the total grain weight per plant increased by 35.48%-64%, and the biomass increased by 53.8%-71.9% ( Figure 2 E-2J) (n=9, values ​​are mean ± SD, significant differences (P<0.05) are indicated by letters). These results indicate that the two transgenic plants can accumulate more biomass during growth, thus laying the foundation for improving overall yield.

[0113] Citrobacter koseri TA (transaminase of Citrobacter koseri) is a glutamate-dependent transaminase (TA). Its function mainly revolves around the transamination reaction. With glutamate (Glutamate) as the core amino donor, it can transfer the amino group of glutamate to other keto acids (such as pyruvate or oxaloacetate) to generate corresponding amino acids (such as alanine or aspartic acid) and α-ketoglutarate. This process plays a key role in amino acid synthesis and catabolism, maintaining intracellular nitrogen balance. Therefore, in this experimental example, QRT-PCR was used to detect the expression levels of genes related to nitrogen absorption, transport and assimilation in pZmUbi::CkTA plants. The results showed that compared with the wild type, the expression levels of some nitrogen absorption, transport and metabolism-related genes were higher ( Figure 3 A, n=3), Figure 3 The names and sequences of the quantitative primers for nitrogen-related genes involved in A are shown in Table 2. 15 N-nitrate and 15 The N-ammonium salt supply experiment was used to test nitrogen utilization efficiency. The experiment showed that compared with WT, there was no significant change in nitrate in the pZmUbi::CkTA overexpressing plants, but the ammonium salt absorption efficiency increased ( Figure 3 B), the flag leaves of wild-type and transgenic plants were collected in the experimental field of the Institute of Biology, Chinese Academy of Agricultural Sciences for total nitrogen content analysis. The results showed that compared with WT, the total nitrogen content of pZmUbi::CkTA overexpressing plants was higher ( Figure 3 C and D).

[0114] Table 2: Names and sequences of primers for nitrogen-related gene quantification

[0115]

[0116] The keto acids (such as α-ketoglutarate and oxaloacetate) produced by the transamination reaction can enter the tricarboxylic acid cycle (TCA cycle), providing energy (ATP) and carbon skeleton for rice, supporting growth (such as root development and tillering). To evaluate the effect of introducing CkTA on plant photosynthesis, this experiment used a portable photosynthesis measurement system (LI-6800, LI-COR) to measure gas exchange. The results showed that when the photon flux density (PFD) was lower than 400 μmol·m -2 ·s -1 When the PFD exceeded 400 μmol·m -2· s -1 When the pZmUbi::CkTA had a higher net photosynthetic rate than WT ( Figure 4The ambient CO2 concentration (Ca)ΦCO2 was maintained at 400 μmol·mol -1 , the leaf temperature was set at 28°C.

[0117] Test Example 4: Water Flooding Resistance

[0118] Glutamate + pyruvate reaction catalyzed by glutamate-alanine aminotransferase Alanine + α-ketoglutarate can participate in the recycling of nitrogen in rice and the hypoxia response of rice. Under flooding conditions, transaminases work together with the fermentation pathway to help rice maintain energy supply through anaerobic respiration. It is speculated that CkTA may be related to tolerance to flooding, so this experimental example will observe the phenotype of pZmUbi::CkTA transgenic plants using flooding stress treatment. First, wild-type and transgenic seeds were germinated in the dark at 37°C. After 3 days, seeds with consistent germination were selected and placed in glass bottles with 6 cm of tap water. The results showed that at 4 and 6 days after treatment, the coleoptiles of pZmUbi::CkTA overexpressing transgenic seedlings were significantly longer than those of wild-type control plants ( Figure 5 A and B) show that pZmUbi::CkTA overexpressing transgenic plants grew faster than wild-type controls under flooding conditions.

[0119] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0120] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An application of glutamate aminotransferase in rice high yield and stress tolerance, characterized in that: Rice plants overexpressing the glutamate aminotransferase CkTA had larger grains, higher 1000-grain weight, more tillers, higher total grain weight per plant, and higher biomass.

2. An application of glutamate aminotransferase in rice high yield and stress tolerance, characterized in that: Overexpression of glutamate aminotransferase CkTA can improve the ammonium absorption efficiency of rice.

3. An application of glutamate aminotransferase in rice high yield and stress tolerance, characterized in that: Overexpression of glutamate aminotransferase CkTA can increase the growth rate of rice under flooding conditions.

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