Application of OsGRX6 gene in improving plant tolerance to ammonium nitrogen toxicity

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to OsGRX6 Application of a gene in improving tolerance of plants to ammonium nitrogen toxicity. OsGRX6 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. OsGRX6 The gene can be used for regulating the tolerance of plants to ammonium nitrogen toxicity: by CRISPR / Cas9 gene editing technology OsGRX6 Gene deletion rice plants; by OsGRX6 Gene overexpression, obtain OsGRX6 Gene overexpression rice plants; plant experiment results show that deletion OsGRX6 The gene can improve the tolerance of rice root system to high ammonium, and overexpression OsGRX6 The gene reduces the tolerance of rice root system to high ammonium; the application has important significance for cultivating new rice varieties with tolerance to ammonium nitrogen toxicity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to OsGRX6 Application of a gene in improving the tolerance of plants to ammonium nitrogen toxicity. BACKGROUND

[0002] Rice is one of the most important food crops in the world and is the staple food of about half of the world's population. Its yield and quality are of great significance to global food security and agricultural production. At present, the overuse of ammonium nitrogen fertilizer in agricultural activities is common, leading to the increasingly prominent problem of ammonium nitrogen toxicity in rice planting. Excessive application of ammonium nitrogen fertilizer can cause the concentration of ammonium ions in the soil solution of the paddy field to be too high, thereby causing toxic effects on the rice roots, inhibiting root growth and development, making the roots shorter and thicker, hindering the growth of lateral roots, and reducing the absorption capacity of the roots for nutrients and water. At the same time, excessive ammonium nitrogen can also cause soil acidification and compaction in the paddy field, affecting soil fertility and microbial community structure, and further adversely affecting the entire ecological environment, such as water body eutrophication and increased greenhouse gas emissions.

[0003] At present, the main means to deal with ammonium nitrogen toxicity in rice include optimizing fertilization techniques, using microorganisms to improve the soil, and screening ammonium-tolerant varieties. Although optimizing fertilization techniques can reduce the amount of ammonium nitrogen fertilizer to some extent, it is difficult to completely eliminate the effects of ammonium nitrogen toxicity and is difficult to accurately control the amount of fertilizer in actual operation. Using microorganisms to improve the soil requires a long time to take effect and the effect is affected by factors such as the soil environment and microbial species in the paddy field, and is unstable. Screening ammonium-tolerant varieties requires a large amount of time and resources for screening and breeding, and it is difficult to genetically modify ammonium tolerance traits. Therefore, it is of great significance to identify key genes in the response of rice to ammonium nitrogen toxicity in order to improve the tolerance of rice to ammonium nitrogen toxicity and cultivate new ammonium-tolerant varieties, which can provide new ideas and methods for solving this agricultural problem. SUMMARY

[0004] To solve the above problems, the application provides OsGRX6 Application of a gene in improving the tolerance of plants to ammonium nitrogen toxicity, which finds that silencing or knocking out OsGRX6 The gene can improve the tolerance of plants to ammonium nitrogen toxicity. OsGRX6

[0005] To achieve the above purpose, the specific technical solutions of the application are as follows:

[0006] The first aspect of the application provides a OsGRX6 Application of a gene in improving the tolerance of plants to ammonium nitrogen toxicity, wherein OsGRX6 ​The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0007] Further, the application is specifically: knocking out or silencing the above-mentioned OsGRX6 gene in plants to improve the tolerance of plants to ammonium nitrogen toxicity; or inhibiting the expression of the protein encoded by the above-mentioned OsGRX6 gene to improve the tolerance of plants to ammonium nitrogen toxicity.

[0008] Further, the application is specifically: knocking out or silencing the above-mentioned OsGRX6 gene in plants to improve the tolerance of plants to ammonium nitrogen toxicity; or inhibiting the expression of the protein encoded by the above-mentioned

[0009] Further, the plant is rice.

[0010] The second aspect of the present application provides an application of the above-mentioned OsGRX6 gene in rice breeding, and the application is: obtaining a rice strain resistant to ammonium nitrogen toxicity by screening rice plants in which the above-mentioned OsGRX6 gene is knocked out; or obtaining a rice strain resistant to ammonium nitrogen toxicity by inhibiting the expression of the protein encoded by the above-mentioned OsGRX6 gene.

[0011] The third aspect of the present application provides a method for breeding a rice strain resistant to ammonium nitrogen toxicity, and the method is: knocking out the above-mentioned OsGRX6 gene in rice.

[0012] Further, the method specifically comprises the following steps:

[0013] Constructing a gene knockout vector, wherein the gene knockout vector is a plant expression vector carrying a sequence for knocking out the gene with the nucleotide sequence shown as SEQ ID NO. 1; OsGRX6

[0014] Introducing the gene knockout vector into cells of the rice, knocking out the gene with the nucleotide sequence shown as SEQ ID NO. 1, and culturing to obtain a transgenic rice strain resistant to ammonium nitrogen toxicity. OsGRX6

[0015] Further, the plant expression vector is pYLCRISPR / Cas9-MH.

[0016] Compared with the prior art, the present application has the beneficial effects that:

[0017] The present application first proposes and verifies that the OsGRX6 gene can regulate the tolerance of plants to ammonium nitrogen toxicity, OsGRX6 ​​The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The gene is obtained by CRISPR / Cas9 gene editing technology OsGRX6 The gene deletion rice plant is obtained by OsGRX6 The gene overexpression rice plant is obtained by OsGRX6 The gene overexpression rice plant is obtained by OsGRX6 The gene deletion can improve the tolerance of rice root to high ammonium, and the overexpression OsGRX6 The gene can reduce the tolerance of rice root to high ammonium; the gene OsGRX6 The gene can genetically improve the tolerance of plants to ammonium nitrogen toxicity, and the present application has important significance for cultivating new rice varieties with tolerance to ammonium nitrogen toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0019] Figure 1 Fig. 1 is a structural schematic diagram of the intermediate vectors pYLgRNA-OsU6a / LacZ and pYLgRNA-OsU6b, Figure 1 Fig. 1A is a schematic diagram of the overall structure of the pYLgRNA-OsU6a / LacZ vector; Figure 1 Fig. 1B shows the connection mode of OsU6a / LacZ and gRNA in Fig. 1A; Figure 1 Fig. 1C is a schematic diagram of the overall structure of the pYLgRNA-OsU6b vector; Figure 1 Fig. 1D shows the connection mode of OsU6a / LacZ and gRNA in Fig. 1C. Figure 1 Fig. 1D shows the connection mode of OsU6a / LacZ and gRNA in Fig. 1C.

[0020] Figure 2 Fig. 2 is a schematic diagram for constructing a deletion mutant strain by using CRISPR / Cas9 gene editing technology.

[0021] Figure 3 Fig. 3 is a wild type rice plant and OsGRX6 Fig. 4 is the analysis result of the tolerance of the root system of the gene deletion rice plant to high ammonium, WT represents the wild type rice plant, grx6-2 Fig. 5 is a schematic diagram of the construction of two grx6-4 Fig. 5 is a schematic diagram of the construction of two OsGRX6 Fig. 5 is a schematic diagram of the construction of two + Fig. 5A is a schematic diagram of the construction of two Figure 3 Fig. 5A is a schematic diagram of the construction of twogrx6-2 Mutations of the mutant gene, the triangle indicates the mutation position of the gene, the single underlined base sequence is the PAM site of the sgRNA recognition position, and the double underlined base sequence is the start codon of the gene GRX6 Mutations of the mutant gene, the triangle indicates the mutation position of the gene, the single underlined base sequence is the PAM site of the sgRNA recognition position, and the double underlined base sequence is the start codon of the gene Figure 3 Figure B of grx6-4 Mutations of the mutant gene, the triangle indicates the mutation position of the gene, the single underlined base sequence is the PAM site of the sgRNA recognition position, and the double underlined base sequence is the start codon of the gene GRX6 Mutations of the mutant gene, the triangle indicates the mutation position of the gene, the single underlined base sequence is the PAM site of the sgRNA recognition position, and the double underlined base sequence is the start codon of the gene Figure 3 Figure C of wild type rice plants and OsGRX6 Figure D of wild type rice plants, Figure 3 Figure D of wild type rice plants, grx6-2 Figure D of wild type rice plants, grx6-4 Figure D of wild type rice plants, Figure 3 Figure E of wild type rice plants, grx6-2 Figure E of wild type rice plants, grx6-4 Figure E of wild type rice plants,

[0022] Figure 4 Figure F of wild type rice plants and OsGRX6 Figure G of wild type rice plants and GRX6-OE6 Figure G of wild type rice plants, GRX6-OE7 Figure G of wild type rice plants, GRX6-OE8 Figure G of wild type rice plants, OsGRX6 Figure G of wild type rice plants, + Figure G of wild type rice plants, Figure 4 Figure A of wild type rice plants and overexpression OsGRX6 Figure A of wild type rice plants and overexpression Figure 4 Figure B of overexpression OsGRX6 Figure B of overexpression OsGRX6 Figure B of overexpression Figure 4 Figure C of wild type rice plants and overexpression OsGRX6 Figure C of wild type rice plants and overexpression DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0024] The vectors pYLgRNA-OsU6a / LacZ, pYLgRNA-OsU6b and pYLCRISPR / Cas9-MH in the present application are all donated by Professor Liu Yaoguang of South China Agricultural University. The rice variety used in the embodiments of the present application is Wuyunjing 27.

[0025] Rice is an important food crop worldwide, and the problem of ammonium toxicity caused by excessive application of ammonium nitrogen fertilizer is increasingly serious in rice cultivation. Excessive ammonium nitrogen fertilizer can cause high ammonium ion concentration in paddy soil, inhibit rice root growth, make it shorter and thicker, hinder lateral root growth, and reduce root absorption capacity. At the same time, it can also cause soil acidification and compaction, affect soil fertility and microbial community, and further lead to environmental problems such as water eutrophication and increased greenhouse gas emissions. Current measures to address ammonium toxicity in rice include optimizing fertilization techniques, using microorganisms to improve soil, and screening for ammonium-tolerant varieties. Optimizing fertilization techniques is difficult to precisely control the amount of fertilizer and cannot completely eliminate ammonium toxicity, and using microorganisms to improve soil is slow-acting and unstable. Screening for ammonium-tolerant varieties is time-consuming and labor-intensive, and genetic improvement is difficult. Therefore, it is of great significance to identify key genes in the response of rice to ammonium toxicity to improve the tolerance of rice to ammonium toxicity and breed new varieties tolerant to ammonium toxicity, which can provide new ideas for solving this agricultural problem.

[0026] The present application provides a kind of OsGRX6 The application of the gene in improving the tolerance of plants to ammonium toxicity, OsGRX6 The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2. The present application obtains OsGRX6 Ammonium-tolerant rice plants lacking the gene; by OsGRX6 The gene overexpression, obtains OsGRX6 Ammonium-tolerant rice plants overexpressing the gene; the experimental results show that: OsGRX6 The deletion of the gene can improve the tolerance of rice roots to high ammonium, and the overexpression of OsGRX6 The gene can reduce the tolerance of rice roots to high ammonium. Thus, OsGRX6 The gene can regulate the tolerance of plants to ammonium toxicity.

[0027] Example 1:OsGRX6 Breeding of gene deletion rice plants

[0028] OsGRX6 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the PAM site is marked with an underline, AGG The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2.

[0029] SEQ ID NO. 1:

[0030] ATGTACC AGG CGATCCCGTACAGCAGCACCCGGCCGTGGCTCAGGCCGGAGCCGGCGGCGAGCGTGGTCGACGTCGTGA CCG TGGAGACGACGACGG CGG TCG OsGRX6 GTCGGGGCGGTGAGGCGGAGGTCGTGGGGGAGGAGGAGGCGGCGGAGGTGCGGAGGGCGGTGGCGGAGAGCCCGGTGCTGGTGGTGGGGAGGCGCGGGTGCTGCCTCATCCACGTGGTGAAGCGGCTGCTGCAGGGGCTCGGGGTCAACCCGGCCGTGCACGAGGTCGCCGGCGAGGCCGCGCTCAAGGGGGTTGTGCCGGCCGGTGGGGAGGCCGCGGCGCTCCCCGCCGTGTTCGTCGGGGGGAAGCTCCTCGGCGGGCTCGACCGCCTCATGGCCGTCCACATCTCCGGCGAGCTCGTGCCCATCCTCAAGAAGGCCGGTGCCCTCTGGCTTTAA.

[0031] SEQ ID NO. 2:

[0032] MYQAIPYSSTRPWLRPEPAASVVDVVKVETTTAVAGRGGEAEVVGEEEAAEVRRAVAESPVLVVGRRGCCLIHVVKRLLQGLGVNPAVHEVAGEAALKGVVPAGGEAAALPAVFVGGKLLGGLDRLMAVHISGELVPILKKAGALWL.

[0033] The present application utilizes CRISPR / Cas9 gene editing technology to edit the genome DNA of the rice plant to obtain a rice plant with a gene deletion. OsGRX6 The present application utilizes CRISPR / Cas9 gene editing technology to edit the genome DNA of the rice plant to obtain a rice plant with a gene deletion. OsGRX6Rice plants with gene deletion, so as to explore OsGRX6 the function of the gene. The specific steps are as follows.

[0034] S1, gene editing target site design and primer synthesis

[0035] According to Figure 1 the PAM site in the genomic sequence, a suitable gene editing target spacer is designed, and then an enzyme cutting site is added at the C terminal and N terminal of the spacer respectively, and the spacer primer sequence is synthesized, as shown in Table 1.

[0036] Table 1 Spacer primer sequence

[0037]

[0038] Dilute the Spacer primers in Table 1 to 10 nM, mix 10 μL grx6-1-F and 10 μL grx6-1-R, 10 μL grx6-2-F and 10 μL grx6-2-R, 10 μL grx6-3-F and 10 μL grx6-3-R, and 10 μL grx6-4-F and 10 μL grx6-4-R respectively; then denature at 90°C for 30S, and anneal at room temperature to form double-stranded Spacers.

[0039] S2, sgRNA expression cassette construction

[0040] The double-stranded grx6-1-F / grx6-1-R and grx6-2-F / grx6-2-R are connected to the intermediate vector pYLgRNA-OsU6a / LacZ by the method of enzyme cutting and ligation; the grx6-3-F / grx6-3-R and grx6-4-F / grx6-4-R are connected to the intermediate vector pYLgRNA-OsU6b, to construct the sgRNA expression cassette, and the structure of the intermediate vector is as shown in Bsa .

[0041] The reaction system is: 1 μL 10x Bsa I Buffer + 1 μL 10x Takara T4 DNA ligase buffer, 20 ng pYLgRNA-OsU6a / LacZ or 20 ng pYLgRNA-OsU6b, 0.5 μL double-stranded Spacer, 5 U Figure 2 I, 35 U T4 DNA ligase.

[0042] Cyclic reaction with a temperature cycler: 37°C for 5 min, 20°C for 5 min, 5 cycles.

[0043] After the connection is completed, the sgRNA expression cassette is amplified with the connection product as a template. Among them, the reaction product of grx6-1 / grx6-3 is amplified by using the upstream primer Uctcg-B1' shown in SEQ ID NO. 11 and the downstream primer gRcggt-B2 shown in SEQ ID NO. 12, and the reaction product of Spacer grx6-2 / grx6-4 is amplified by using the upstream primer Uctcg-B2' shown in SEQ ID NO. 13 and the downstream primer gRcggt-BL shown in SEQ ID NO. 14;

[0044] SEQ ID NO. 11:

[0045] 5'-TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG-3';

[0046] SEQ ID NO. 12:

[0047] 5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3';

[0048] SEQ ID NO. 13:

[0049] 5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3';

[0050] SEQ ID NO. 14:

[0051] 5'-AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC-3'.

[0052] The reaction system is: 0.2 μL template, 1 μL upstream primer, 1 μL downstream primer, 2 μL Buffer, 4 μL 2.5 mM dNTP, 0.2 μL DNA polymerase, 11.6 μL ddH2O.

[0053] The reaction procedure is: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 45 s, 30 cycles; 72°C for 5 min; 12°C for 5 min.

[0054] S3, vector construction

[0055] After the amplified product is purified, it is connected to the expression vector pYLCRISPR / Cas9-MH, and the structural schematic diagram is as shown in Bsa .

[0056] 50 ng gRNA expression cassette fragment product, 10 U OsGRX6 I, 1.5 μL 10x NEB endonuclease Buffer, 70 ng uncut pYLCRISPR / Cas9-MH, 1.5 μL 10x Takara T4 DNA ligase buffer and 35 U ligase.

[0057] Cycling the reaction with a temperature cycler for 15 cycles: 37℃ 2 min; 10℃ 3 min, 20℃ 5 min; finally 37℃ 2 min.

[0058] S4, Transformation and positive clone screening

[0059] 5 μL of the vector constructed in step S3 was transferred into E. coli DH5α competent cells, which were spread on LB plates containing 50 μg / mL kanamycin and cultured at 37℃ for 24 h.

[0060] Single colonies were picked and positive clones were identified by colony PCR using the upstream primer SPL shown in SEQ ID NO. 15 and the downstream primer SPR shown in SEQ ID NO. 16.

[0061] SEQ ID NO. 15: 5'-GCGCGGTGTCATCTATGTTACT-3';

[0062] SEQ ID NO. 16: 5'-CCCGACATAGATGCAATAACTTC-3';

[0063] The reaction system was: SanTaq Mix 5 μL, SPL 0.5 μL, SPR 0.5 μL, ddH2O 4 μL, and single colonies were added to the reaction system for reaction. The reaction program was 98℃ 5 min; 98℃ 30 s, 58℃ 30 s, 72℃ 90 s, 35 cycles; 72℃ 5 min.

[0064] The plasmid of the positive clone was extracted and sent to the company for infection of rice callus, so as to cultivate transgenic rice materials. The obtained transgenic rice lines were T0 generation.

[0065] S5, Identification of transgenic rice

[0066] Firstly, PCR was used to identify whether the T0 generation of transgenic rice lines contained Cas9 gene, so as to determine whether the vector plasmid was successfully inserted into the rice genomic DNA. The amplification primers for identifying transgenic rice were the upstream primer Cas9-F shown in SEQ ID NO. 17 and the downstream primer Cas9-R shown in SEQ ID NO. 18.

[0067] SEQ ID NO. 17: 5'-CTGACGCTAACCTCGACAAG-3';

[0068] SEQ ID NO. 18: 5'-CCGATCTAGTAACATAGATGACACC-3';

[0069] The reaction system is: 0.2 μL template, 1 μL upstream primer, 1 μL downstream primer, 2 μL Buffer, 4 μL 2.5 mM dNTP, 0.2 μL DNA polymerase, 11.6 μL ddH2O;

[0070] The reaction procedure is: 94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 45s, 30 cycles; 72℃ 5min; 12℃ 5min.

[0071] Then, the genomic sequence containing Spacer is used as a template, and the primer is designed as the upstream primer Cas9-F shown in SEQ ID NO. 19 and the downstream primer Cas9-R shown in SEQ ID NO. 20 to amplify the template, and sequencing is used to determine whether the gene editing exists.

[0072] SEQ ID NO. 19: 5'-AGCCATCACGCCAAGACG-3';

[0073] SEQ ID NO. 20: 5'-GATGTGGCAAATGTCACTGTCC-3';

[0074] The reaction system is: 0.2 μL template, 1 μL upstream primer, 1 μL downstream primer, 2 μL Buffer, 4 μL 2.5 mM dNTP, 0.2 μL DNA polymerase, 11.6 μL ddH2O;

[0075] The reaction procedure is: 94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 45s, 30 cycles; 72℃ 5min; 12℃ 5min.

[0076] The T0 generation of rice transgenic lines is planted in the field, and the T1 generation seeds are harvested. After the T1 generation seeds germinate, the plants of the T0 generation with mutations but without the Cas9 gene of the T1 generation are screened, that is grx6-2 the gene deletion rice plants, and finally grx6-4 and OsGRX6 two OsGRX6 gene deletion rice plants are obtained.

[0077] Example 2: OsGRX6Cultivation of rice plants with overexpressed genes

[0078] The OsGRX6 gene CDS was connected to the CaMV35S-driven vector plasmid by homologous recombination to obtain the recombinant plasmid CaMV35S- OsGRX6 The CaMV35S- OsGRX6 was introduced into Agrobacterium to obtain the recombinant bacteria containing CaMV35S- OsGRX6 Then, the Agrobacterium-mediated method was used to obtain the transgenic rice lines with overexpressed genes. OsGRX6 The specific operation is as follows:

[0079] S1, the seed coat of rice was peeled off, and then the rice was sequentially sterilized with 75v / v% ethanol for 2 min, 30v / v% sodium hypochlorite solution for 30 min, and sterilized water for washing and air drying, and then inoculated into the induction medium and cultured at 28°C in the dark for 3 weeks. The naturally divided embryogenic callus was picked out and placed into the subculture medium and subcultured at 28°C under illumination for 1 week.

[0080] S2, 500 μL of the cultured Agrobacterium liquid containing CaMV35S- OsGRX6 was centrifuged at 4°C and 4000 rpm for 2 min, and the supernatant was discarded. The bacterial liquid was resuspended with 300 μL of AAM solution containing 200 μM acetyl-syringone, and the bacterial liquid was diluted to an OD 600 value of 0.1.

[0081] S3, the well-grown rice callus was taken and immersed in the Agrobacterium suspension for 5 min. The callus was picked out, drained on a sterile filter paper, and then placed on the co-culture medium and cultured at 25°C in the dark until Agrobacterium grew on the surface of the callus. Then, the Agrobacterium on the surface of the callus was washed off with sterile water, and the callus was washed twice with a sterile aqueous solution containing 250 mg / L kanamycin sulfate and 50 mg / L hygromycin. After air drying, the callus was placed on the selection medium containing 250 mg / L kanamycin sulfate and 50 mg / L hygromycin for the first round of selection, and cultured at 28°C under illumination for 14 days.

[0082] S4, the initial callus with resistance was transferred to a new selection medium for the second round of selection, and cultured at 28°C under illumination until the resistant callus grew.

[0083] S5, the yellow resistant callus was picked and transferred to the differentiation medium, and the rice seedlings were grown and then planted in the field for breeding.

[0084] The expression level of the OsGRX6 gene was detected by qPCR, and finally OsGRX6 the rice plant samples with high expression level of the gene GRX6-OE6 were obtained GRX6-OE7 , GRX6-OE8 andOsGRX6 .

[0085] Example 3: OsGRX6 The Influence of Genes on Plant Tolerance to High Ammonium

[0086] As the main source of rhizosphere nutrition, the root system can quickly and sensitively sense nutrients in the environment by changing the configuration of taproot length, lateral root length, density and spatial distribution, which is crucial for plants to efficiently obtain nutrients. Therefore, root system configuration is a key agronomic trait that affects the efficient use of nutrients in crops.

[0087] To clarify OsGRX6 Does the gene affect the tolerance of rice roots to high ammonium levels? This study compared wild-type rice seeds with... Figure 3 Rice seeds from gene-deleted plants germinated simultaneously. Rice seedlings with root lengths of 2-3 cm were selected and cultured for 4 days at 28°C under nitrogen-deficient and nutrient solutions containing 1.25 mM ammonium sulfate, respectively, with 16 hours of light and 8 hours of darkness. The nutrient solution was changed every two days. The rice seedlings were then removed, and the root phenotype of the rice was recorded using a plant root scanner. The root length of the rice was measured using ImageJ software.

[0088] The results are as follows OsGRX As shown, compared to wild-type rice, the mutant rice exhibits a significant increase in root elongation, indicating that... OsGRX6 Deletion of gene 6 can improve the tolerance of rice roots to high ammonium.

[0089] Select plump wild-type rice seeds and Figure 4 Rice seeds with overexpressed genes were used. After germination, rice seedlings with roots of equal length were selected and cultured for 4 days in a nitrogen-deficient nutrient solution containing 1.25 mM ammonium sulfate at 28°C under conditions of 16 hours of light and 8 hours of darkness. The nutrient solution was changed every two days. The rice seedlings were then removed, and the root phenotype of the rice was recorded using a plant root scanner. The root length of the rice was measured using ImageJ software.

[0090] The results are as follows OsGRX6 As shown, compared to wild-type rice, overexpression ​ The gene can significantly increase the sensitivity of rice roots to high ammonium levels.

[0091] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0092] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the application can be practiced otherwise than as specifically described herein.

Claims

1. A kind OsGRX6 The application of genes in improving plant tolerance to ammonium nitrogen toxicity is characterized by, The OsGRX6 The nucleotide sequence of the gene is shown in SEQ ID NO.1; [The following appears to be a separate, unrelated sentence:] In knockout or silenced plants... OsGRX6 Genes were developed to enhance the plant's tolerance to ammonium nitrogen toxicity; the plant in question is rice.

2. The one described in claim 1 OsGRX6 The application of gene-encoded proteins in improving plant tolerance to ammonium nitrogen toxicity is characterized by, Suppress the OsGRX6 The expression of gene-encoded proteins is used to enhance plant tolerance to ammonium nitrogen toxicity; OsGRX6 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2; the plant is rice.

3. The one described in claim 1 OsGRX6 The application of genes in rice breeding is characterized by, By filtering and knocking out the above OsGRX6 Rice plants that produce the gene, thereby obtaining rice lines tolerant to ammonium nitrogen toxicity; or rice plants that inhibit the gene... OsGRX6 The expression of gene-encoded proteins is used to obtain rice lines tolerant to ammonium nitrogen toxicity.

4. A method for cultivating rice tolerant to ammonium nitrogen toxicity, characterized in that, The rice as described in claim 1 OsGRX6 Gene knockout.

5. The method for cultivating rice tolerant to ammonium nitrogen toxicity according to claim 4, characterized in that, Includes the following steps: Construct a gene knockout vector, wherein the gene knockout vector contains a nucleotide sequence as shown in SEQ ID NO.

1. OsGRX6 Plant expression vectors for gene knockout; The gene knockout vector was introduced into rice cells, and the nucleotide sequence was as shown in SEQ ID NO.

1. OsGRX6 Gene knockout and cultivation yielded rice varieties tolerant to ammonium nitrogen poisoning.

6. The method for cultivating rice tolerant to ammonium nitrogen toxicity according to claim 5, characterized in that, The plant expression vector is pYLCRISPR / Cas9-MH.

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