Application of Rice OsGLN1;1 Gene in Plant Breeding for Arsenic Stress Tolerance

Abstract

The invention discloses the application of rice OsGLN1;1 gene in cultivating plant varieties with high tolerance to arsenic stress, and belongs to the field of genetic engineering technology. The OsGLN1;1 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO.4. The invention can improve the ability of plants to resist arsenic stress by increasing the expression of OsGLN1;1 or increasing the acetylation modification degree of the 112th amino acid of the protein encoded by the gene, and can be applied to plant genetic engineering breeding, providing a new method for the creation or improvement of new germplasm for plants to resist arsenic stress.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to application of rice OsGLN1;1 gene and the protein encoded by it in regulating plant arsenic stress ability or arsenic stress-related breeding. Background Art

[0002] Arsenic is a type of metalloid substance widely found in the natural environment. Arsenic is toxic to humans, animals, and plants. Since rice is cultivated by flooding, it has a very high absorption efficiency of arsenic. As rice is the staple food of half of the world's population, the arsenic enriched in its body will enter the human body through the food chain and threaten human health. Exploring and applying genetic resources related to resistance to arsenic stress in plants is of great significance for improving rice quality and increasing food security.

[0003] However, the genetic regulatory network of plant response to arsenic stress is intricate, and the molecular regulatory mechanisms of plants with different genetic backgrounds vary greatly. Although some genes that respond to arsenic stress in plants have been reported, discovering and cloning more new genes that have the function of regulating plant resistance to arsenic stress, discovering new regulatory mechanisms, and developing new methods for cultivating plant varieties that resist arsenic stress are still technical issues that need to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a rice OsGLN1;1 gene or its orthologous gene in regulating the response of a plant to arsenic stress and / or cultivating a plant variety resistant to arsenic stress.

[0005] The present invention creates a rice OsGLN1; 1 full-length gene and a full-length gene containing an acetylation site mutation (simulating deacetylated OsGLN1; 1) by genetic engineering technology. 112K-112R and simulated sustained acetylation of OsGLN1;1 112K-112Q ) was overexpressed in the model plant Arabidopsis thaliana and a stable transgenic strain was obtained. The performance of the transgenic strain under arsenic stress was analyzed to determine that enhancing the expression level of the rice OsGLN1;1 gene can improve the ability of the model plant to resist arsenic stress, and this ability is affected by acetylation modification. This gene is expected to be applied to genetic engineering breeding of plants, providing a theoretical basis for the creation or improvement of new germplasms that can resist arsenic stress and plants that are sensitive to arsenic. Taking into account the degeneracy of codons, the bases of the nucleotide sequence of the present invention are modified without changing the amino acid sequence, which also falls within the scope of protection of the present invention.

[0006] In one aspect, the present invention provides a gene capable of improving the ability of plants to resist arsenic stress, wherein the amino acid sequence encoded by the gene is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0007] In a preferred embodiment, the nucleotide sequence of the coding region of the gene capable of improving the ability of plants to resist arsenic stress is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] In another aspect, the present invention provides the use of the aforementioned gene in regulating plant resistance to arsenic stress.

[0009] Furthermore, the regulation of plant resistance to arsenic stress is to improve the ability of plants to resist arsenic stress by overexpressing any gene or its orthologous gene, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous genes.

[0010] In a preferred embodiment, the plant is a monocotyledonous plant.

[0011] Furthermore, the plant is a plant of the Poaceae family.

[0012] Furthermore, the plant is a rice plant.

[0013] Furthermore, the plant is rice.

[0014] In a preferred embodiment, the plant is a dicotyledonous plant.

[0015] Furthermore, the plant is a cruciferous plant.

[0016] Furthermore, the plant is of the genus Arabidopsis.

[0017] Furthermore, the plant is Arabidopsis thaliana.

[0018] On the other hand, the present invention provides the use of a protein encoded by any of the aforementioned genes or its orthologous genes in regulating plant resistance to arsenic stress.

[0019] Furthermore, the regulation of plant resistance to arsenic stress is to improve the ability of plants to resist arsenic stress by overexpressing a protein encoded by any gene or its orthologous gene, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous gene.

[0020] In a preferred embodiment, the plant is a monocotyledonous plant.

[0021] Furthermore, the plant is a plant of the Poaceae family.

[0022] Furthermore, the plant is a rice plant.

[0023] Furthermore, the plant is rice.

[0024] In a preferred embodiment, the plant is a dicotyledonous plant.

[0025] Furthermore, the plant is a cruciferous plant.

[0026] Furthermore, the plant is of the genus Arabidopsis.

[0027] Furthermore, the plant is Arabidopsis thaliana.

[0028] In another aspect, the present invention provides the use of any of the aforementioned genes or their orthologous genes in breeding plant varieties with high resistance to arsenic stress.

[0029] Furthermore, plant varieties with high resistance to arsenic stress are cultivated by overexpressing any gene or its orthologous gene, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous gene.

[0030] In a preferred embodiment, the plant is a monocotyledonous plant.

[0031] Furthermore, the plant is a plant of the Poaceae family.

[0032] Furthermore, the plant is a rice plant.

[0033] Furthermore, the plant is rice.

[0034] In a preferred embodiment, the plant is a dicotyledonous plant.

[0035] Furthermore, the plant is a cruciferous plant.

[0036] Furthermore, the plant is of the genus Arabidopsis.

[0037] Furthermore, the plant is Arabidopsis thaliana.

[0038] On the other hand, the present invention provides the use of a protein encoded by any of the aforementioned genes or its orthologous genes in breeding plant varieties with high resistance to arsenic stress.

[0039] Furthermore, plant varieties with high resistance to arsenic stress are cultivated by overexpressing the protein encoded by any gene or its orthologous gene, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous gene.

[0040] In a preferred embodiment, the plant is a monocotyledonous plant.

[0041] Furthermore, the plant is a plant of the Poaceae family.

[0042] Furthermore, the plant is a rice plant.

[0043] Furthermore, the plant is rice.

[0044] In a preferred embodiment, the plant is a dicotyledonous plant.

[0045] Furthermore, the plant is a cruciferous plant.

[0046] Furthermore, the plant is of the genus Arabidopsis.

[0047] Furthermore, the plant is Arabidopsis thaliana.

[0048] In one aspect, the present invention provides a gene with high glutamine synthetase activity, wherein the amino acid sequence encoded by the gene is shown in SEQ ID NO.5.

[0049] In a preferred embodiment, the nucleotide sequence of the coding region of the aforementioned gene is shown as SEQ ID NO.2.

[0050] In another aspect, the present invention provides use of the aforementioned gene in regulating glutamine synthetase activity.

[0051] Furthermore, the regulating plant glutamine synthetase activity is to increase the plant glutamine synthetase activity by overexpressing any of the aforementioned genes or their orthologous genes, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or their orthologous genes.

[0052] In a preferred embodiment, the plant is a monocotyledonous plant.

[0053] Furthermore, the plant is a plant of the Poaceae family.

[0054] Furthermore, the plant is a rice plant.

[0055] Furthermore, the plant is rice.

[0056] In a preferred embodiment, the plant is a dicotyledonous plant.

[0057] Furthermore, the plant is a cruciferous plant.

[0058] Furthermore, the plant is of the genus Arabidopsis.

[0059] Furthermore, the plant is Arabidopsis thaliana.

[0060] On the other hand, the present invention provides use of a protein encoded by any of the aforementioned genes or its orthologous genes in regulating glutamine synthetase activity.

[0061] Furthermore, the regulating plant glutamine synthetase activity is to increase the plant glutamine synthetase activity by overexpressing a protein encoded by any of the aforementioned genes or its orthologous genes, or by increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous genes.

[0062] In a preferred embodiment, the plant is a monocotyledonous plant.

[0063] Furthermore, the plant is a plant of the Poaceae family.

[0064] Furthermore, the plant is a rice plant.

[0065] Furthermore, the plant is rice.

[0066] In a preferred embodiment, the plant is a dicotyledonous plant.

[0067] Furthermore, the plant is a cruciferous plant.

[0068] Furthermore, the plant is of the genus Arabidopsis.

[0069] Furthermore, the plant is Arabidopsis thaliana.

[0070] In another aspect, the present invention provides use of any of the aforementioned genes or their orthologous genes in breeding plant varieties with high glutamine synthetase activity.

[0071] Furthermore, plant varieties with high glutamine synthetase activity are cultivated by overexpressing any of the aforementioned genes or their orthologous genes, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or their orthologous genes.

[0072] In a preferred embodiment, the plant is a monocotyledonous plant.

[0073] Furthermore, the plant is a plant of the Poaceae family.

[0074] Furthermore, the plant is a rice plant.

[0075] Furthermore, the plant is rice.

[0076] In a preferred embodiment, the plant is a dicotyledonous plant.

[0077] Furthermore, the plant is a cruciferous plant.

[0078] Furthermore, the plant is of the genus Arabidopsis.

[0079] Furthermore, the plant is Arabidopsis thaliana.

[0080] In another aspect, the present invention provides the use of a protein encoded by any of the aforementioned genes or its orthologous genes in breeding plant varieties with high glutamine synthetase activity.

[0081] Furthermore, plant varieties with high glutamine synthetase activity are cultivated by overexpressing a protein encoded by any gene or its orthologous gene, or increasing the degree of acetylation modification of the 112th amino acid of the protein encoded by any of the aforementioned genes or its orthologous gene.

[0082] In a preferred embodiment, the plant is a monocotyledonous plant.

[0083] Furthermore, the plant is a plant of the Poaceae family.

[0084] Furthermore, the plant is a rice plant.

[0085] Furthermore, the plant is rice.

[0086] In a preferred embodiment, the plant is a dicotyledonous plant.

[0087] Furthermore, the plant is a cruciferous plant.

[0088] Furthermore, the plant is of the genus Arabidopsis.

[0089] Furthermore, the plant is Arabidopsis thaliana.

[0090] Compared with the prior art, the present invention has the following advantages:

[0091] 1) The present invention provides a new method for regulating plant resistance to arsenic stress or improving glutamine synthetase activity, namely, improving the ability of plants to resist arsenic stress or improving glutamine synthetase activity by overexpressing the OsGLN1;1 gene or the protein encoded by it.

[0092] 2) The present invention provides a new method for cultivating plant varieties that are resistant to arsenic stress, namely, cultivating plant varieties with high resistance to arsenic stress or increased glutamine synthetase activity by overexpressing the OsGLN1;1 gene or the protein encoded by it.

[0093] 3) The present invention can also regulate the resistance of plants to arsenic stress or increase the activity of glutamine synthetase or cultivate new plant varieties by regulating the acetylation modification degree of the 112th amino acid of the OsGLN1;1 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The method for regulating plant resistance to arsenic stress and / or cultivating plant varieties resistant to arsenic stress and its beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0095] Figure 1 Shown is the protein expression level analysis of Arabidopsis overexpression lines.

[0096] Figure 2 Shown are OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Analysis of the sensitivity of overexpression lines to arsenic stress. (A) Wild type (WT) and OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Phenotypes of overexpression lines under arsenic stress; (B) Wild type (WT) and OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Phenotypes of overexpression lines under normal conditions.

[0097] Figure 3 Shown are OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Analysis of enzyme activity of overexpression strains under arsenic stress. T-test analysis, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. (n=3) DETAILED DESCRIPTION

[0098] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0099] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0100] Example 1

[0101] Whole-protein acetylation analysis revealed that lysine deacetylation occurred in OsGLN1;1 protein of rice seedlings after arsenic treatment

[0102] In order to explore the effect of heavy metal arsenic on lysine acetylation modification of rice proteins, we used whole-protein acetylomics technology to identify the proteins and sites that underwent acetylation modification changes in rice seedlings before and after arsenic treatment. The specific method is:

[0103] 1. Rice materials and treatment. After germination, rice seeds were transferred to Kimura B rice culture medium ((NH4)2SO4 (48.2 mg / L), KH2PO4 (24.8 mg / L), KNO3 (18.5 mg / L), K2SO4 (14.9 mg / L), MgSO4·7H2O (135.1 mg / L), Ca(NO3)2·4H2O (86.4 mg / L), MnCl2·4H2O (1.81 mg / L), H2MoO4·H2O (0.09 mg / L), H3BO3 (2.86 mg / L), ZnSO4·7H2O (0.22 mg / L), CuSO4·5H2O (0.08 mg / L), Na2EDTA (7.45 mg / L), and FeSO4·7H2O (0.5 mg / L, pH 5.7), and were divided into a control group and a treatment group. After culturing for 2 weeks, the treatment group was transferred to Kimura B culture medium supplemented with 100 μM NaAsO2 [As(III)], while the control group continued to grow in the original culture medium. After 6 hours of treatment, the materials were collected and stored in a -80°C refrigerator.

[0104] 2. Whole-protein acetylomics analysis. The collected rice seedling materials were sent to Hangzhou Jingjie Biological Co., Ltd. for whole-protein acetylomics analysis. Through the organic combination of a series of cutting-edge technologies such as protein extraction, enzyme digestion, modified peptide enrichment (lysine acetylation modified pan-antibody), liquid chromatography-mass spectrometry tandem analysis, and bioinformatics analysis, the samples were studied by modification quantitative omics. The final results showed that compared with the control group, the acetylation levels of 621 lysine sites on 535 proteins in the arsenic treatment group were increased, while the acetylation levels of 230 sites on 211 proteins were decreased.

[0105] 3. Among the proteins identified to be acetylated, we found that the 112th lysine site of glutamine synthetase OsGLN1;1 (Protein accession: P14656) was significantly deacetylated (Table 1). Since glutamine synthetase plays an important role in anti-oxidative stress, we speculated that deacetylation of OsGLN1;1 may affect the antioxidant response of rice seedlings after arsenic stress.

[0106] Table 1. Site information of acetylation modification of OsGLN1;1

[0107]

[0108] Example 2

[0109] OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Construction and identification of

[0110] To investigate the functions of acetylation and deacetylation of OsGLN1;1, we constructed OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q carrier.

[0111] First, we cloned the cDNA sequence of the OsGLN1;1 gene. The specific method is as follows:

[0112] Young rice leaves were collected and RNA was extracted using the Magen Plant RNA Extraction Kit (R4151-02). The cDNA was synthesized according to the instructions of the Vazyme 1st Strand Reverse Transcription Kit (R211-01). The full-length sequence of the OsGLN1;1 gene was obtained by PCR amplification using the cDNA as a template, the F primer (GGTAGATCTGACTAGTATGGCTTCTCTCACCGATCTC) and the R primer (CTTCTCCTTTACTAGTGGGCTTCCAGATGATGGTG), and the KOD FX high-fidelity enzyme (ToYoBo). The reaction system was: 2×KOD buffer 25μL, 2mM dNTPs 8μL, F primer and R primer 1.5μL each, cDNA template 1μL, KOD FX (1U / μl) 0.5μL, and water was added to 50μL. The reaction procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing and extension at 68°C for 1 min 10 sec, 32 cycles; final extension at 68°C for 5 min. After the reaction, the PCR product was recovered using a gel recovery kit (Magen, catalog number D2111-01).

[0113] Secondly, the pCAMBIA1302 binary vector with GFP tag was linearized using restriction endonuclease SpeI (NEB, catalog number R3133V). The enzyme digestion system was: 10 μg of vector, 2.5 μL of SpeI endonuclease, 5 μL of 10×buffer, and water was added to 50 μL. The digestion conditions were: 37°C, 2.5 hours. After the reaction, the linearized vector was recovered using the Magen gel recovery kit. Finally, the OsGLN1;1 gene was inserted into the pCAMBIA1302 binary vector by homologous recombination. The recombination system was: 43 ng of target fragment, 211 ng of vector fragment, 2×MultiFSeamless Assembly Mix (Abclonal, catalog number RK21020) 2.5 μL, and the total volume was 5 μL. The recombination conditions were: 50°C, 15 min. After the reaction, the product was transformed into 50 μL of E. coli competent cells DH5α and spread on LB solid culture medium containing kanamycin (working concentration of 50 mg / L), inverted, and cultured overnight in a 37°C incubator. Single clones were picked and identified by PCR. The PCR-positive single clones were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequencing primers were GFP-R: TCACCTTCACCCTCTCCACT and GLN1;1-R1: GGCAGCATTGTGCCT. The two sequencing results were spliced ​​and compared with the reference sequence. The recombinant plasmid with completely consistent sequences was the correct one.

[0114] OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q The construction process of is basically the same as the above steps, except that we mutated the acetylation site in the OsGLN1;1 gene. The specific method is: we introduced the mutation site into the OsGLN1;1 gene sequence by bridge PCR technology, so as to mutate the lysine (K) that undergoes acetylation modification to glutamine (KQ) to simulate acetylation, and mutate the lysine site to arginine (KR) to simulate deacetylation. Finally, the simulated deacetylated OsGLN1;1 was obtained. 112K-112R and simulated sustained acetylation of OsGLN1;1 112K-112Q The bridging primer sequences are OsGLN1; 1 112K-112R -F(AGGCACAATGCTGCCAGGATCTTTCAGCTCCCCT) and OsGLN1;1 112K-112R -R(AGGGGAGCTGAAGATCCTGGCAGCATTGTGCCT), OsGLN1;1 112K-112Q -F(AGGCACAATGCTGCCCAGATCTTCAGCTCCCCT) and OsGLN1;1112K-112Q -R(AGGGGAGCTGAAGATCTGGGCAGCATTGTGCCT) insert the obtained gene into pCambia-1302-GFP, and obtain the correct recombinant vector after sequencing. We will get p35S-OsGLN1;1-GFP, p35S-OsGLN1;1 112K-112R -GFP and p35S-OsGLN1;1 112K-112Q -GFP binary expression vector was used to transform Agrobacterium competent cells GV3101.

[0115] Finally, the above vectors were transferred into Arabidopsis thaliana through Agrobacterium-mediated transformation. The T1 generation seeds were evenly spread on a 1 / 2MS solid culture medium containing 50μg / L hygromycin, and the transgenic positive seedlings (positive seedlings with obvious growth advantages) were selected. The positive seedlings were transferred to nutrient soil for continued cultivation until the T2 generation seeds were harvested. Take the T2 generation seeds (about 100 seeds) and spot them one by one on a 1 / 2MS solid culture medium containing 50μg / L hygromycin. After growing for a certain period of time, the separation ratio was counted. If the number of resistant plants and non-resistant plants meets the ratio of 3:1, it means that the plant has a single copy insertion, and the seeds collected from a single plant are T3 generation seeds. The T3 generation seeds are continued to be spotted on a 1 / 2MS solid culture medium containing hygromycin. If all the seeds can grow normally, it proves that the strain is a homozygous plant. We used Western Blot technology to detect OsGLN1;1 and OsGLN1;1 in the transgenic strains. 112K-112R and OsGLN1;1 112K-112Q The protein expression level of the transgenic line was used to determine whether the obtained transgenic line was overexpressed ( Figure 1 ).

[0116] Example 3

[0117] OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Analysis of sensitivity of overexpression lines to arsenic stress

[0118] The wild-type WT, OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Arabidopsis seeds were sterilized with 1% NaClO and treated at 4°C for 3 days, then sown in 1 / 2MS solid medium. After 2 days, they were transferred to 1 / 2MS medium containing 50μM NaAsO2[As(III)] and cultured for 14 days to observe their phenotypes. 112K -112R and OsGLN1;1 112K-112Q There was no significant difference in the growth of the overexpression lines under normal conditions ( Figure 2 B); Under arsenic stress conditions, the taproot of the OsGLN1;1 overexpressing strain was significantly longer than that of the wild type, indicating that overexpression of OsGLN1;1 can improve Arabidopsis' tolerance to arsenic stress. 112K-112Q The taproot of the overexpression strain was also longer than that of the wild type under arsenic stress, while the deacetylated OsGLN1;1 112K-112R There was no significant difference in taproot length between the overexpression strain and the wild type under arsenic stress. In conclusion, overexpression of OsGLN1;1 can improve the resistance of the model plant Arabidopsis to arsenic stress, and acetylation modification has a certain effect on the resistance function of this gene.

[0119] Example 4: OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Analysis of enzyme activities of overexpression strains under arsenic stress

[0120] The OsGLN1;1 gene can catalyze the condensation of glutamate and ammonia to form glutamine, playing an important role in nitrogen metabolism. At the same time, under heavy metal stress conditions, it can regulate the expression of some peroxidases to reduce the oxidation level of cell membranes and thus reduce oxidative damage. Therefore, we further analyzed the OOsGLN1;1 and OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q Enzyme activity of overexpression strains under arsenic stress. First, the wild-type WT, OsGLN1;1, OsGLN1;1 112K-112R and OsGLN1;1 112K-112Q The seeds of the overexpression strain Arabidopsis thaliana were sterilized with 1% NaClO and treated at 4°C for 3 days, then sown in 1 / 2MS solid medium, cultured vertically for 7 days, and then transferred to 500μM NaAsO2[As(III)] for 9 hours. The materials were collected and quickly frozen with liquid nitrogen. The enzyme activity was determined according to the operating instructions of the glutamine synthetase (GS) activity detection kit produced by Shanghai Shenggong Biotechnology Co., Ltd. The specific results are as follows Figure 3 As shown, compared with wild type (GFP), OsGLN1;1 and OsGLN1;1 112K-112Q The enzyme activity of the overexpression strain was significantly higher than that of the wild type after arsenic stress treatment for 9 hours, among which OsGLN1;1 112K-112Q The overexpression strain had the highest enzyme activity, while OsGLN1;1 112K-112R The enzyme activity of the overexpression strain was slightly higher than that of the wild type, but lower than that of OsGLN1;1 and OsGLN1;1 112K-112QOverexpression strain. Based on the above results, it is confirmed that the OsGLN1;1 gene can improve the resistance of plants to arsenic stress, and the increase in resistance is caused by the enhancement of its enzyme activity. At the same time, its enzyme activity is also affected by acetylation modification, that is, the increase in acetylation level can increase the enzyme activity of OsGLN1;1. Therefore, we can develop some small molecule compounds to specifically promote the activity of this enzyme to improve the resistance of plants to arsenic stress without resorting to transgenic means, eliminating the doubts of the general public about the safety of transgenic plants.

[0121] Based on the above results, it is confirmed that the GLN1;1 gene is involved in the response to arsenic stress, so genetic engineering technology can be used to improve the tolerance of plants to arsenic, such as by overexpressing GLN1;1. Therefore, GLN1;1 can be used in genetic breeding to improve the tolerance of plants to arsenic stress.

[0122] The nucleotide sequences of the rice GLN1;1 gene and its mutants are shown below:

[0123] SEQ ID NO.1 (OsGLN1; 1)

[0124] ATGGCTTCTCTCACCGATCTCGTCAACCTCAACCTCTCCGACACCACGGAGAAGATCATC

[0125] GCCGAGTACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAGGCTAGGACTCTC

[0126] TCCGGCCCTGTGACTGATCCCAGCAAGCTGCCCAAGTGGAACTACGATGGCTCCAGCACC

[0127] GGCCAGGCCCCCGGCGAGGACAGTGAGGTCATCCTGTACCCACAGGCTATCTTCAAGGAC

[0128] CCATTCAGGAAGGGAAACAACATCCTTGTCATGTGCGATTGCTACACGCCAGCCGGAGAA

[0129] CCGATCCCCACCAACAAGAGGCACAATGCTGCCAAGATCTTCAGCTCCCCTGAGGTTGCT

[0130] TCTGAGGAGCCCTGGTACGGTATTGAGCAAGAGTACACCCTCCTCCAGAAGGACATCAAC

[0131] TGGCCCCTTGGCTGGCCTGTTGGTGGCTTCCCTGGTCCTCAGGGTCCTTACTACTGTGGT

[0132] ATCGGTGCTGACAAGTCTTTTGGGCGTGATATTGTTGACTCCCACTACAAGGCTTGCCTC

[0133] TATGCCGGCATCAACATCAGTGGAATCAACGGCGAGGTCATGCCAGGACAGTGGGAGTTC

[0134] CAAGTTGGCCCGTCTGTCGGCATTTCTGCCGGTGATCAGGTGTGGGTTGCTCGCTACATT

[0135] CTTGAGAGGATCACCGAGATCGCCGGAGTCGTCGTCTCATTTGACCCCAAGCCCATCCCG

[0136] GGAGACTGGAACGGTGCTGGTGCTCACACCAACTACAGCACCAAGTCGATGAGGAACGAT

[0137] GGTGGCTACGAGATCATCAAGTCCGCCATTGAGAAGCTCAAGCTCAGGCACAAGGAGCAC

[0138] ATCTCCGCCTACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGAC

[0139] ATCAACACCTTCAGCTGGGGAGTTGCCAACCGCGGCGCCTCGGTCCGCGTCGGCCGGGAG

[0140] ACGGAGCAGAACGGCAAGGGCTACTTCGAGGATCGCCGGCCGGCGTCCAACATGGACCCT

[0141] TACATCGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA

[0142] SEQ ID NO.2(OsGLN1;1 112K-112Q )

[0143] ATGGCTTCTCTCACCGATCTCGTCAACCTCAACCTCTCCGACACCACGGAGAAGATCATC

[0144] GCCGAGTACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAGGCTAGGACTCTC

[0145] TCCGGCCCTGTGACTGATCCCAGCAAGCTGCCCAAGTGGAACTACGATGGCTCCAGCACC

[0146] GGCCAGGCCCCCGGCGAGGACAGTGAGGTCATCCTGTACCCACAGGCTATCTTCAAGGAC

[0147] CCATTCAGGAAGGGAAACAACATCCTTGTCATGTGCGATTGCTACACGCCAGCCGGAGAA

[0148] CCGATCCCCACCAACAAGAGGCACAATGCTGCCCAGATCTTCAGCTCCCCTGAGGTTGCT

[0149] TCTGAGGAGCCCTGGTACGGTATTGAGCAAGAGTACACCCTCCTCCAGAAGGACATCAAC

[0150] TGGCCCCTTGGCTGGCCTGTTGGTGGCTTCCCTGGTCCTCAGGGTCCTTACTACTGTGGT

[0151] ATCGGTGCTGACAAGTCTTTTGGGCGTGATATTGTTGACTCCCACTACAAGGCTTGCCTC

[0152] TATGCCGGCATCAACATCAGTGGAATCAACGGCGAGGTCATGCCAGGACAGTGGGAGTTC

[0153] CAAGTTGGCCCGTCTGTCGGCATTTCTGCCGGTGATCAGGTGTGGGTTGCTCGCTACATT

[0154] CTTGAGAGGATCACCGAGATCGCCGGAGTCGTCGTCTCATTTGACCCCAAGCCCATCCCG

[0155] GGAGACTGGAACGGTGCTGGTGCTCACACCAACTACAGCACCAAGTCGATGAGGAACGAT

[0156] GGTGGCTACGAGATCATCAAGTCCGCCATTGAGAAGCTCAAGCTCAGGCACAAGGAGCAC

[0157] ATCTCCGCCTACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGAC

[0158] ATCAACACCTTCAGCTGGGGAGTTGCCAACCGCGGCGCCTCGGTCCGCGTCGGCCGGGAG

[0159] ACGGAGCAGAACGGCAAGGGCTACTTCGAGGATCGCCGGCCGGCGTCCAACATGGACCCT

[0160] TACATCGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGASEQ ID NO.3(OsGLN1;1 112K-112R )

[0161] ATGGCTTCTCTCACCGATCTCGTCAACCTCAACCTCTCCGACACCACGGAGAAGATCATC

[0162] GCCGAGTACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAGGCTAGGACTCTC

[0163] TCCGGCCCTGTGACTGATCCCAGCAAGCTGCCCAAGTGGAACTACGATGGCTCCAGCACC

[0164] GGCCAGGCCCCCGGCGAGGACAGTGAGGTCATCCTGTACCCACAGGCTATCTTCAAGGAC

[0165] CCATTCAGGAAGGGAAACAACATCCTTGTCATGTGCGATTGCTACACGCCAGCCGGAGAA

[0166] CCGATCCCCACCAACAAGAGGCACAATGCTGCCAGGATCTTCAGCTCCCCTGAGGTTGCT

[0167] TCTGAGGAGCCCTGGTACGGTATTGAGCAAGAGTACACCCTCCTCCAGAAGGACATCAAC

[0168] TGGCCCCTTGGCTGGCCTGTTGGTGGCTTCCCTGGTCCTCAGGGTCCTTACTACTGTGGT

[0169] ATCGGTGCTGACAAGTCTTTTGGGCGTGATATTGTTGACTCCCACTACAAGGCTTGCCTC

[0170] TATGCCGGCATCAACATCAGTGGAATCAACGGCGAGGTCATGCCAGGACAGTGGGAGTTC

[0171] CAAGTTGGCCCGTCTGTCGGCATTTCTGCCGGTGATCAGGTGTGGGTTGCTCGCTACATT

[0172] CTTGAGAGGATCACCGAGATCGCCGGAGTCGTCGTCTCATTTGACCCCAAGCCCATCCCG

[0173] GGAGACTGGAACGGTGCTGGTGCTCACACCAACTACAGCACCAAGTCGATGAGGAACGAT

[0174] GGTGGCTACGAGATCATCAAGTCCGCCATTGAGAAGCTCAAGCTCAGGCACAAGGAGCAC

[0175] ATCTCCGCCTACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGAC

[0176] ATCAACACCTTCAGCTGGGGAGTTGCCAACCGCGGCGCCTCGGTCCGCGTCGGCCGGGAG

[0177] ACGGAGCAGAACGGCAAGGGCTACTTCGAGGATCGCCGGCCGGCGTCCAACATGGACCCT

[0178] TACATCGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA

[0179] The amino acid sequences encoded by the rice GLN1;1 gene and its mutants are as follows:

[0180] SEQ ID NO.4 (OsGLN1;1)

[0181] MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSST

[0182] GQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVA

[0183] SEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACL

[0184] YAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIP

[0185] GDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETAD

[0186] INTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP

[0187] SEQ ID NO.5 (OsGLN1;1 112K-112Q )

[0188] MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSST

[0189] GQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAQIFSSPEVA

[0190] SEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACL

[0191] YAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIP

[0192] GDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETAD

[0193] INTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP

[0194] SEQ ID NO.6(OsGLN1;1 112K-112R )

[0195] MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSST

[0196] GQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAARIFSSPEVA

[0197] SEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACL

[0198] YAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIP

[0199] GDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETAD

[0200] INTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP

[0201] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gene capable of improving a plant's ability to resist arsenic stress, characterized in that: The amino acid sequence encoded by the gene is shown in SEQ ID NO.5, and the plant is rice or Arabidopsis thaliana.

2. Use of a gene in regulating plant resistance to arsenic stress, characterized in that: The amino acid sequence encoded by the gene is shown in SEQ ID NO.4 or SEQ ID NO.5, and the regulation of plant resistance to arsenic stress is to improve the ability of the plant to resist arsenic stress by overexpressing the gene, and the plant is rice or Arabidopsis thaliana.

3. A use of a protein in regulating plant resistance to arsenic stress, wherein the amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5, wherein the regulation of plant resistance to arsenic stress is to improve the ability of the plant to resist arsenic stress by overexpressing the protein, and the plant is rice or Arabidopsis thaliana.

4. A use of a gene or a protein encoded by the gene in cultivating a plant variety with high resistance to arsenic stress, wherein the use is to cultivate a plant variety with high resistance to arsenic stress by overexpressing the gene or overexpressing the protein, the amino acid sequence encoded by the gene is shown in SEQ ID NO.4 or SEQ ID NO.5, and the plant is rice or Arabidopsis thaliana.

5. Use of the gene according to claim 1 or the protein encoded by the gene or the protein encoded by the gene in claim 1 for improving the glutamine synthetase activity of plants under arsenic stress or cultivating plant varieties with high glutamine synthetase activity under arsenic stress, wherein the plant is rice or Arabidopsis thaliana.

Images