Application of rice OSDHAR1 gene in breeding of plants tolerant to arsenic stress
By overexpressing the rice OsDHAR1 gene and its acetylation site mutant in the model plant Arabidopsis thaliana, the molecular regulatory differences in plant resistance to arsenic stress were solved, enhancing the plant's tolerance to arsenic stress and antioxidant response, and providing a theoretical basis for breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-16
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies are insufficient to effectively discover and clone genes that regulate plant resistance to arsenic stress, resulting in significant differences in the molecular regulatory mechanisms of plants to arsenic stress, which affects food security.
We created the full-length OsDHAR1 gene of rice and its full-length gene containing acetylation site mutations using genetic engineering technology, and overexpressed it in the model plant Arabidopsis thaliana. We analyzed its performance under arsenic stress, determined that enhancing the expression level of the rice OsDHAR1 gene can improve the plant's ability to resist arsenic stress, and that this ability can be regulated by acetylation modification.
It improved the plant's tolerance to arsenic stress, enhanced the plant's antioxidant capacity, and provided a theoretical basis for breeding arsenic-resistant plant varieties.
Smart Images
Figure CN118147162B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically relating to rice. OSDHAR1 Application of genes and their encoded proteins in regulating plant arsenic stress capacity or in arsenic stress-related breeding. Background Technology
[0002] Arsenic is a metalloid substance widely found in the natural environment, and it is toxic to humans, animals, and plants. Because rice is cultivated using flooded farming methods, it has an extremely high absorption efficiency for arsenic. As rice is the staple food for half the world's population, the arsenic accumulated in rice will enter the human body through the food chain, threatening human health. Discovering and applying genetic resources from plants related to resisting arsenic stress is of great significance for improving rice quality and enhancing food security.
[0003] However, the genetic regulatory network of plant response to arsenic stress is complex, and the molecular regulatory mechanisms vary considerably among plants with different genetic backgrounds. Although some genes that respond to arsenic stress in plants have been reported, discovering and cloning more new genes that regulate plant resistance to arsenic stress, exploring new regulatory mechanisms, and developing new methods for breeding plant varieties resistant to arsenic stress remain urgent technical challenges. Summary of the Invention
[0004] The purpose of this invention is to provide a rice OSDHAR1 Application of genes or their orthologous genes in regulating plant responses to arsenic stress and / or in breeding arsenic-resistant plant varieties.
[0005] This invention creates rice using genetic engineering technology. OsDHAR1 Full-length genes and their full-length genes containing acetylation site mutations (mimicking deacetylation) OsDHAR1 8K-8R and simulated continuous acetylation OsDHAR1 8K-8Q Stable transgenic lines were obtained by overexpressing the gene in the model plant Arabidopsis thaliana. The performance of the transgenic lines under arsenic stress was analyzed to determine the enhancement effect on rice. OsDHAR1 The gene expression level can enhance the ability of model plants to resist arsenic stress, and this ability is affected by acetylation modification. This gene holds promise for application in plant genetic engineering breeding, providing a theoretical basis for the creation or improvement of new arsenic-sensitive germplasm in plants. Considering the degeneracy of codons, modifications to the nucleotide sequence of this invention without altering the amino acid sequence also fall within the scope of protection of this invention.
[0006] On the one hand, the present invention provides a gene that can enhance 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 coding region nucleotide sequence of the gene that enhances the plant's ability to resist arsenic stress is as shown in SEQ ID NO.1 or SEQ ID NO.2.
[0008] On the other hand, the present invention provides the application of the aforementioned gene in regulating the ability of plants to resist arsenic stress.
[0009] Furthermore, the regulation of plant resistance to arsenic stress is achieved by overexpressing any of the aforementioned genes or their orthologs, or by increasing the degree of acetylation modification of the 8th amino acid of the protein encoded by any of the aforementioned genes or their orthologs.
[0010] In the preferred embodiment, the plant is a monocotyledonous plant.
[0011] Furthermore, the plant in question belongs to the Poaceae family.
[0012] Furthermore, the plant in question is a member of the genus *Oryza*.
[0013] Furthermore, the plant in question is rice.
[0014] In the preferred embodiment, the plant is a dicotyledonous plant.
[0015] Furthermore, the plant in question is a member of the Brassicaceae family.
[0016] Furthermore, the plant in question is a species of the Arabidopsis genus.
[0017] Furthermore, the plant in question is Arabidopsis thaliana.
[0018] On the other hand, the present invention provides the application of proteins encoded by any of the aforementioned genes or their orthologous genes in regulating the ability of plants to resist arsenic stress.
[0019] Furthermore, the regulation of plant resistance to arsenic stress is achieved by overexpressing the protein encoded by any of the aforementioned genes or their orthologous genes, or by increasing the degree of acetylation modification of the 8th amino acid of the protein encoded by any of the aforementioned genes or their orthologous genes.
[0020] In the preferred embodiment, the plant is a monocotyledonous plant.
[0021] Furthermore, the plant in question belongs to the Poaceae family.
[0022] Furthermore, the plant in question is a member of the genus *Oryza*.
[0023] Furthermore, the plant in question is rice.
[0024] In the preferred embodiment, the plant is a dicotyledonous plant.
[0025] Furthermore, the plant in question is a member of the Brassicaceae family.
[0026] Furthermore, the plant in question is a species of the Arabidopsis genus.
[0027] Furthermore, the plant in question is Arabidopsis thaliana.
[0028] On the other hand, the present invention provides the application of any of the aforementioned genes or their orthologous genes in the breeding of plant varieties with high resistance to arsenic stress.
[0029] Furthermore, plant varieties with high resistance to arsenic stress can be cultivated by overexpressing any of the aforementioned genes or their orthologs, or by increasing the degree of acetylation modification of the 8th amino acid of the protein encoded by any of the aforementioned genes or their orthologs.
[0030] In the preferred embodiment, the plant is a monocotyledonous plant.
[0031] Furthermore, the plant in question belongs to the Poaceae family.
[0032] Furthermore, the plant in question is a member of the genus *Oryza*.
[0033] Furthermore, the plant in question is rice.
[0034] In the preferred embodiment, the plant is a dicotyledonous plant.
[0035] Furthermore, the plant in question is a member of the Brassicaceae family.
[0036] Furthermore, the plant in question is a species of the Arabidopsis genus.
[0037] Furthermore, the plant in question is Arabidopsis thaliana.
[0038] On the other hand, the present invention provides the application of proteins encoded by any of the aforementioned genes or their orthologous genes in the breeding of plant varieties with high resistance to arsenic stress.
[0039] Furthermore, plant varieties with high resistance to arsenic stress can be cultivated by overexpressing the protein encoded by any of the aforementioned genes or their orthologous genes, or by increasing the degree of acetylation modification of the 8th amino acid of the protein encoded by any of the aforementioned genes or their orthologous genes.
[0040] In the preferred embodiment, the plant is a monocotyledonous plant.
[0041] Furthermore, the plant in question belongs to the Poaceae family.
[0042] Furthermore, the plant in question is a member of the genus *Oryza*.
[0043] Furthermore, the plant in question is rice.
[0044] In the preferred embodiment, the plant is a dicotyledonous plant.
[0045] Furthermore, the plant in question is a member of the Brassicaceae family.
[0046] Furthermore, the plant in question is a species of the Arabidopsis genus.
[0047] Furthermore, the plant in question is Arabidopsis thaliana.
[0048] Compared with the prior art, the present invention has the following advantages:
[0049] 1) This invention provides a novel method for regulating the ability of plants to resist arsenic stress, namely by overexpressing OSDHAR1 Genes or the proteins they encode enhance a plant's ability to resist arsenic stress.
[0050] 2) This invention provides a novel method for cultivating plant varieties resistant to arsenic stress, namely, through overexpression OSDHAR1 Genes or their encoded proteins are used to cultivate plant varieties with high resistance to arsenic stress.
[0051] 3) This invention can also be controlled by adjusting... OSDHAR1 The degree of acetylation modification of the 8th amino acid in a gene can be used to regulate a plant's ability to resist arsenic stress or to cultivate plant varieties that are tolerant or sensitive to arsenic stress. Attached Figure Description
[0052] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, illustrates the method of the present invention for regulating the ability of plants to resist arsenic stress and / or cultivating plant varieties resistant to arsenic stress, and its beneficial effects.
[0053] Figure 1 Analysis of protein expression levels in Arabidopsis thaliana overexpression lines.
[0054] Figure 2 OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Sensitivity analysis of overexpression lines to arsenic stress. (A) Wild type (WT), OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Phenotype of overexpressing lines under arsenic stress; (B) Wild type (WT) OsDHAR1 , OsDHAR1 8K-8R andOsDHAR1 8K-8Q Phenotype of overexpression lines under normal conditions.
[0055] Figure 3 As shown OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Analysis of enzyme activity of overexpression lines under arsenic stress.
[0056] T -test analysis, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. (n=3) Detailed Implementation
[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0058] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0059] Experimental Example 1: Rice seedlings after arsenic treatment OsDHAR1 Protein undergoes lysine deacetylation
[0060] To explore the effects of heavy metal arsenic on lysine acetylation modification of rice proteins, we used whole-protein acetylationomics to identify proteins and sites in rice seedlings that underwent acetylation modification changes before and after arsenic treatment. The specific methods were as follows: 1. Rice materials and treatments. After rice seeds germinated, they 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). The samples were cultured in a medium (mg / L), pH 5.7, and divided into a control group and a treatment group. After 2 weeks of culture, the treatment group was transferred to Kimura B medium supplemented with 100 μM NaAsO2 [As(III)], while the control group continued to grow in the original medium. After 6 h of treatment, the material was harvested and stored at -80°C. o 1. Refrigerator C. 2. Whole-protein acetylation analysis. The collected rice seedlings were sent to Hangzhou Jingjie Biotechnology Co., Ltd. for whole-protein acetylation analysis. A series of cutting-edge technologies, including protein extraction, enzyme digestion, modified peptide enrichment (lysine acetylation modification pan-antibody), liquid chromatography-mass spectrometry tandem analysis, and bioinformatics analysis, were combined to conduct quantitative omics research on the modified proteins. The final results showed that, compared to the control group, the acetylation level of 621 lysine sites on 535 proteins was increased in the arsenic-treated group, while the acetylation level of 230 sites on 211 proteins was decreased. 3. Among the proteins identified as undergoing acetylation modification, we found that dehydroascorbic acid reductase... OsDHAR1 Significant deacetylation occurred at the 8th lysine residue on (Protein accession Q65XA0) (Table 1). Since dehydroascorbic acid reductase plays an important role in antioxidant stress, we hypothesize that OsDHAR1 deacetylation may affect the antioxidant response of rice seedlings after arsenic stress.
[0061] Table 1. Site information for OsDHAR1 acetylation modification.
[0062]
[0063] Example 2 OsDHAR1 ,OsDHAR1 8K-8R and OsDHAR1 8K-8Q Construction and identification
[0064] To investigate the function of OsDHAR1 acetylation and deacetylation modifications, we constructed... OsDHAR1 , OsDHAR1 8K -8R and OsDHAR1 8K-8Q The carrier.
[0065] First, we cloned OsDHAR1 The cDNA sequence of the gene was obtained. The specific method is as follows: Young rice leaves were collected, and RNA was extracted using the Magen Plant RNA Extraction Kit (R4151-02). cDNA synthesis was performed according to the instructions of the Vazyme First-Strand Reverse Transcription Kit (R211-01). Using this cDNA as a template, PCR amplification was performed using primers F (GGTAGATCTGACTAGTATGGGCGTGGAGGTGTGC) and R (CTTCTCCTTTACTAGTCGCATTCACTTTTGGTGCCC) with KODFX high-fidelity enzyme (ToYoBo). OsDHAR1 The full-length sequence of the gene was obtained. The reaction mixture consisted of: 25 μL of 2×KOD buffer, 8 μL of 2 mM dNTPs, 1.5 μL each of F and R primers, 1 μL of cDNA template, 0.5 μL of KOD FX (1 U / μL), and water to a final volume of 50 μL. The reaction program 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 60 sec, for 32 cycles; and final extension at 68 °C for 5 min. After the reaction, the PCR products were recovered using a gel extraction kit (Magen, catalog number D2111-01).
[0066] Next, the GFP-tagged pCAMBIA1302 binary vector was linearized using the restriction endonuclease SpeI (NEB, catalog number R3133V). The digestion system consisted of 10 μg vector, 2.5 μL SpeI endonuclease, 5 μL 10× buffer, and water to a final volume of 50 μL. The digestion conditions were 37°C for 2.5 hours. After the reaction, the linearized vector was recovered using a Magen gel extraction kit. Finally, homologous recombination was used to... OsDHAR1The gene was inserted into the pCAMBIA1302 binary vector. The recombination system consisted of 26 ng of the target fragment, 211 ng of the vector fragment, and 2.5 μL of 2×MultiFSeamless Assembly Mix (Abclonal, catalog number RK21020), for a total volume of 5 μL. The recombination conditions were 50℃ for 15 min. After the reaction, the product was transformed into 50 μL of *E. coli* competent cells (DH5α) and plated on LB agar containing kanamycin (working concentration 50 mg / L). The plates were incubated overnight at 37℃. Single clones were picked and identified by PCR. PCR-positive single clones were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing primers were GFP-R: TCACCTTCACCCTCTCCACT. The sequencing results were compared with the reference sequence; those with completely identical sequences were considered the correct recombinant plasmids. OsDHAR1 8K-8R and OsDHAR1 8K-8Q The construction process is largely the same as the steps described above, the difference being that we will... OsDHAR1 The acetylation sites in the gene were mutated. Specifically, bridging PCR was used to introduce mutations into the OsDHAR1 gene sequence, thereby mutating lysine (K) to glutamine (KQ) to simulate acetylation and lysine to arginine (KR) to simulate deacetylation. This resulted in a simulated deacetylation. OsDHAR1 8K-8R and simulated continuous acetylation OsDHAR1 8K-8Q The full-length gene. The upstream primer sequences used are OsDHAR1 and OsDHAR2, respectively. 8K-8R -F (GGTAGATCTGACTAGTATGGGCGTGGAGGTGTGCGTCAGGGCCGCCGTCGGCC) and OsDHAR1 8K-8Q -F (GGTAGATCTGACTAGTATGGGCGTGGAGGTGTGCGTCCAGGCCGCCGTCGGCC), with the downstream primer remaining the R primer (CTTCTCCTTTACTAGTCGCATTCACTTTTGGTGCCC), the resulting gene was inserted into pCambia-1302-GFP, and sequencing yielded the correct recombinant vector. We will obtain p35S - OsDHAR1 - GFP, p35S - OsDHAR1 8K-8R -GFP and p35S - OsDHAR1 8K-8Q -GFP The binary expression vector was transformed into Agrobacterium competent cells GV3101.
[0067] Finally, the above vectors were transformed into Arabidopsis thaliana using Agrobacterium-mediated transformation. T1 generation seeds were evenly spread on 1 / 2 MS solid medium containing 50 μg / L hygromycin, and transgenic positive seedlings (showing significant growth advantage) were selected. These positive seedlings were transferred to nutrient soil for further cultivation until T2 generation seeds were harvested. Approximately 100 T2 generation seeds were individually spotted on 1 / 2 MS solid medium containing 50 μg / L hygromycin. After a certain growth period, the segregation ratio was calculated. If the ratio of resistant to non-resistant plants was 3:1, it indicated a single-copy insertion, and seeds were collected from each plant, becoming T3 generation seeds. T3 generation seeds were then spotted on 1 / 2 MS solid medium containing hygromycin. If all seeds grew normally, the line was considered homozygous. We used Western blotting to detect the transgenic line... OsDHAR1, OsDHAR1 8K-8R and OsDHAR1 8K-8Q The protein expression level is used to determine whether the obtained transgenic lines are overexpressing. Figure 1 ).
[0068] SEQ ID NO.1 ( OsDHAR1 )
[0069] ATGGGCGTGGAGGTGTGCGTCAAGGCCGCCGTCGGCCACCCGGACACGCTCGGCGACTGTCCATTCTCGCAGAGGGTGCTGCTGACTCTGGAGGAGAAGAAGGTGCCCTACGAGATGAAGCTCATCGACGTCCAGAACAAGCCCGACTGGTTTCTGAAGATCAGCCCAGAGGGGAAGGTGCCTGTGTTTAACGGTGGTGATGGCAAATGGATTCCTGATTCTGATGTGATCACTCAAGTCATTGAGGAGAAGTACCCAACCCCGTCTCTTGTCACCCCTCCTGAGTATGCATCAGTGGGATCAAAAATTTTCTCATGCTTCACAACGTTCTTGAAGAGCAAGGATCCAAATGATGGTTCAGAGAAGGCACTTCTTACTGAACTGCAGGCACTCGAGGAGCATCTGAAAGCTCATGGCCCCTTTATCAACGGGCAGAACATTTCAGCTGCTGACCTTAGCCTGGCACCAAAGCTCTACCATCTCCAGGTTGCTCTGGAGCATTTCAAAGGCTGGAAGATCCCGGAAGACCTAACCAATGTTCATGCTTACACAGAGGCTCTGTTTAGCCGCGAATCTTTCATCAAGACGAAGGCAGCTAAGGAGCACCTGATTGCTGGATGGGCACCAAAAGTGAATGCGTAA
[0070] SEQ ID NO.2( OsDHAR1 8K-8Q )
[0071] ATGGGCGTGGAGGTGTGCGTCCAGGCCGCCGTCGGCCACCCGGACACGCTCGGCGACTGTCCATTCTCGCAGAGGGTGCTGCTGACTCTGGAGGAGAAGAAGGTGCCCTACGAGATGAAGCTCATCGACGTCCAGAACAAGCCCGACTGGTTTCTGAAGATCAGCCCAGAGGGGAAGGTGCCTGTGTTTAACGGTGGTGATGGCAAATGGATTCCTGATTCTGATGTGATCACTCAAGTCATTGAGGAGAAGTACCCAACCCCGTCTCTTGTCACCCCTCCTGAGTATGCATCAGTGGGATCAAAAATTTTCTCATGCTTCACAACGTTCTTGAAGAGCAAGGATCCAAATGATGGTTCAGAGAAGGCACTTCTTACTGAACTGCAGGCACTCGAGGAGCATCTGAAAGCTCATGGCCCCTTTATCAACGGGCAGAACATTTCAGCTGCTGACCTTAGCCTGGCACCAAAGCTCTACCATCTCCAGGTTGCTCTGGAGCATTTCAAAGGCTGGAAGATCCCGGAAGACCTAACCAATGTTCATGCTTACACAGAGGCTCTGTTTAGCCGCGAATCTTTCATCAAGACGAAGGCAGCTAAGGAGCACCTGATTGCTGGATGGGCACCAAAAGTGAATGCGTAA
[0072] SEQ ID NO.3( OsDHAR1 8K-8R )
[0073] ATGGGCGTGGAGGTGTGCGTCAGGGCCGCCGTCGGCCACCCGGACACGCTCGGCGACTGTCCATTCTCGCAGAGGGTGCTGCTGACTCTGGAGGAGAAGAAGGTGCCCTACGAGATGAAGCTCATCGACGTCCAGAACAAGCCCGACTGGTTTCTGAAGATCAGCCCAGAGGGGAAGGTGCCTGTGTTTAACGGTGGTGATGGCAAATGGATTCCTGATTCTGATGTGATCACTCAAGTCATTGAGGAGAAGTACCCAACCCCGTCTCTTGTCACCCCTCCTGAGTATGCATCAGTGGGATCAAAAATTTTCTCATGCTTCACAACGTTCTTGAAGAGCAAGGATCCAAATGATGGTTCAGAGAAGGCACTTCTTACTGAACTGCAGGCACTCGAGGAGCATCTGAAAGCTCATGGCCCCTTTATCAACGGGCAGAACATTTCAGCTGCTGACCTTAGCCTGGCACCAAAGCTCTACCATCTCCAGGTTGCTCTGGAGCATTTCAAAGGCTGGAAGATCCCGGAAGACCTAACCAATGTTCATGCTTACACAGAGGCTCTGTTTAGCCGCGAATCTTTCATCAAGACGAAGGCAGCTAAGGAGCACCTGATTGCTGGATGGGCACCAAAAGTGAATGCGTAA
[0074] SEQ ID NO.4( OsDHAR1 )
[0075] MGVEVCVKAAVGHPDTLGDCPFSQRVLLTLEEKKVPYEMKLIDVQNKPDWFLKISPEGKVPVFNGGDGKWIPDSDVITQVIEEKYPTPSLVTPPEYASVGSKIFSCFTTFLKSKDPNDGSEKALLTELQALEEHLKAHGPFINGQNISAADLSLAPKLYHLQVALEHFKGWKIPEDLTNVHAYTEALFSRESFIKTKAAKEHLIAGWAPKVNA
[0076] SEQ ID NO.5( OsDHAR1 8K-8Q )
[0077] MGVEVCVQAAVGHPDTLGDCPFSQRVLLTLEEKKVPYEMKLIDVQNKPDWFLKISPEGKVPVFNGGDGKWIPDSDVITQVIEEKYPTPSLVTPPEYASVGSKIFSC FTTFLKSKDPNDGSEKALLTELQALEEHLKAHGPFINGQNISAADLSLAPKLYHLQVALEHFKGWKIPEDLTNVHAYTEALFSRESFIKTKAAKEHLIAGWAPKVNA
[0078] SEQ ID NO.6 ( OsDHAR1 8K-8R )
[0079] MGVEVCVRAAVGHPDTLGDCPFSQRVLLTLEEKKVPYEMKLIDVQNKPDWFLKISPEGKVPVFNGGDGKWIPDSDVITQVIEEKYPTPSLVTPPEYASVGSKIFSC FTTFLKSKDPNDGSEKALLTELQALEEHLKAHGPFINGQNISAADLSLAPKLYHLQVALEHFKGWKIPEDLTNVHAYTEALFSRESFIKTKAAKEHLIAGWAPKVNA
[0080] Example 3 OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Sensitivity analysis of overexpression lines to arsenic stress
[0081] We will wild-type WT, OsDHAR1, OsDHAR1 8K-8R and OsDHAR1 8K-8Q Arabidopsis seeds were sterilized with 1% NaClO and treated at 4°C for 3 days, then sown on 1 / 2 MS solid medium. Two days later, they were transferred to a medium containing 50 μM NaAsO.
[0082] 2. The phenotypes of WT and [As(III)] were observed after 14 days of incubation on 1 / 2 MS medium. The results showed that WT, OsDHAR1, OsDHAR1 8K-8R and OsDHAR1 8K-8Q Overexpression lines showed no significant difference in growth under normal conditions. Figure 2 B); while under arsenic stress conditions... OsDHAR1The taproot of the overexpression line was significantly longer than that of the wild type, indicating that overexpression... OsDHAR1 It can improve the tolerance of Arabidopsis thaliana to arsenic stress. Meanwhile, it simulates continuous acetylation. OsDHAR1 8K-8Q Overexpression lines also exhibited longer taproots under arsenic stress compared to wild-type lines, while simulated deacetylation... OsDHAR1 8K-8R The overexpression lines showed no significant difference in taproot length compared to the wild type under arsenic stress. In conclusion, overexpression... OsDHAR1 It can improve the resistance of the model plant Arabidopsis thaliana to arsenic stress, and acetylation modification has a certain impact on the function of this gene in resisting arsenic stress.
[0083] Example 4: OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Analysis of enzyme activity of overexpression lines under arsenic stress
[0084] OsDHAR1 The gene can promote ascorbic acid cycling and protein folding, improving field adaptability by enhancing AsA accumulation and redox balance, thereby increasing yield and biomass. Therefore, we further analyzed... OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Enzyme activity of overexpression lines under arsenic stress. First, the GFP empty vector, OsDHAR1 , OsDHAR1 8K-8R and OsDHAR1 8K-8Q Overexpression line Arabidopsis seeds were sterilized with 1% NaClO and treated at 4°C for 3 days, then sown in 1 / 2 MS solid medium and vertically cultured for 7 days. Afterwards, they were transferred to a medium containing 500 μM NaAsO2 [As(III)] for 6 hours, and the collected materials were rapidly frozen in liquid nitrogen. Enzyme activity was determined according to the instructions of the dehydroascorbic acid reductase (DHAR) activity assay kit produced by Shanghai Sangon Biotech Co., Ltd. The specific results are as follows: Figure 3 As shown, compared to the empty GFP vector, OsDHAR1 and OsDHAR1 8K-8Q The overexpression lines showed significantly higher enzyme activity than the GFP empty vector after 6 hours of arsenic stress treatment. OsDHAR1 8K-8R The enzyme activity of the overexpression lines was not significantly different from that of the GFP empty vector, a result consistent with their phenotype under arsenic stress. These results confirm that… OsDHAR1The gene can enhance plant resistance to arsenic stress, and this increase in resistance is due to enhanced enzyme activity. This enzyme activity is also affected by acetylation modification; specifically, increased acetylation levels enhance the activity of the OsDHAR1 enzyme. Therefore, we can develop small molecule compounds to specifically promote the activity of this enzyme, thereby improving plant resistance to arsenic stress, without resorting to genetic engineering, thus alleviating public concerns about the safety of genetically modified plants.
[0085] Based on the above results, it is confirmed that... OSDHAR1 The gene is involved in the response to arsenic stress, and its expression level can be increased to enhance the plant's tolerance to arsenic stress.
[0086] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A gene capable of enhancing the ability of plants to resist arsenic stress, wherein the amino acid sequence encoded by the gene is shown in SEQ ID NO.5, and the plant is Arabidopsis thaliana and / or rice.
2. The application of the rice OSDHAR1 gene in regulating plant resistance to arsenic stress, characterized by, The amino acid sequence encoded by the OSDHAR1 gene is shown in SEQ ID NO.4 or SEQ ID NO.5, and the plant is Arabidopsis thaliana or rice; the regulation of plant resistance to arsenic stress is achieved by overexpressing the OSDHAR1 gene to enhance the plant's ability to resist arsenic stress.
3. The application of the protein encoded by the rice OSDHAR1 gene in regulating plant resistance to arsenic stress, characterized by: The amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5, and the plant is Arabidopsis thaliana or rice; the regulation of plant resistance to arsenic stress is achieved by overexpressing the protein to enhance the plant's ability to resist arsenic stress.
4. The application of the rice OSDHAR1 gene in breeding plant varieties with high resistance to arsenic stress, characterized in that... The amino acid sequence encoded by the OSDHAR1 gene is shown in SEQ ID NO.4 or SEQ ID NO.5, and the plant is Arabidopsis thaliana or rice; the cultivation of plant varieties with high resistance to arsenic stress is achieved by overexpressing the OSDHAR1 gene.
5. The application of the protein encoded by the rice OSDHAR1 gene in breeding plant varieties with high resistance to arsenic stress, characterized in that... The amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5, and the plant is Arabidopsis thaliana or rice; the cultivation of plant varieties with high resistance to arsenic stress is achieved by overexpressing the protein.