Application of wild rice C2H2-24 gene in regulating rice salt tolerance
By cloning the C2H2-24 gene from common wild rice and overexpressing it in rice, the problem of poor salt tolerance in rice varieties was solved, and salt tolerance was significantly improved. This provides efficient gene resources and operational methods, and ensures the utilization of saline-alkali land.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SANYA NATIONAL INSTITUTE OF SOUTHERN BREEDING CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-04-27
- Publication Date
- 2026-07-17
AI Technical Summary
Existing rice varieties are sensitive to salt stress and have poor salt tolerance, making them difficult to utilize effectively in saline-alkali land. Salt-tolerant gene resources for cultivated rice are scarce, and traditional breeding methods are inefficient.
The C2H2-24 gene was cloned and identified from common wild rice. This gene was overexpressed in rice using transgenic technology. Salt tolerance was improved by utilizing the zinc finger protein transcription factor encoded by the gene. Recombinant vectors and recombinant microorganisms were constructed to achieve efficient and targeted gene transfer.
It significantly improved the salt tolerance of rice, provided important genetic resources and operational methods, achieved a breakthrough in salt-tolerant breeding, and ensured stable and increased rice yields in saline-alkali land.
Smart Images
Figure CN122104794B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to wild rice. C2H2-24 Application of genes in regulating salt tolerance in rice. Background Technology
[0002] Soil salinization is one of the major abiotic stressors limiting crop production, and the loss of arable land and agricultural yield reduction due to salinization are becoming increasingly prominent globally each year. Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, but it is highly sensitive to salt stress, especially during the seedling and reproductive growth stages, where salt damage significantly affects tillering, panicle development, and final yield. Currently, rice yield increases have reached a plateau. Therefore, exploring and utilizing salt-tolerant gene resources to cultivate salt-tolerant rice varieties is not only an effective way to manage and improve saline-alkali land, but also an important direction for ensuring stable and increased grain production.
[0003] During its long domestication process, cultivated rice has lost or weakened many genes related to stress resistance. In contrast, the ancestral species of cultivated rice—common wild rice (…) Oryza rufipogon Wild rice retains rich genetic diversity and exhibits strong tolerance to environmental stresses such as drought, high salinity, and low temperature. Therefore, discovering and cloning key salt-tolerant genes from wild rice and introducing them into cultivated rice is an important means to broaden the genetic base of cultivated rice and achieve breakthroughs in salt-tolerant breeding.
[0004] Existing research has reported some salt tolerance-related genes in rice, but most of them originate from cultivated rice and have limited salt tolerance effects; while functional salt tolerance gene resources derived from wild rice remain very scarce. Genetic engineering technologies such as transgenics can overcome reproductive isolation limitations between species and achieve efficient targeted transfer of target genes, providing a direct and effective approach to improving rice salt tolerance.
[0005] This invention identifies a novel salt tolerance-related gene in common wild rice. C2H2-24 ( LOC_ Os02g08510 This gene contains a characteristic 18 bp insertion sequence in its coding region, which encodes a zinc finger protein transcription factor that significantly enhances salt tolerance in rice. Near-isogenic lines were constructed using the Indica 93-11 background, and wild rice was overexpressed in the Zhonghua 11 background. C2H2-24 The transgenic lines expressing the gene all showed a significant improvement in salt tolerance; however, overexpression of the homologous allele in cultivated rice did not produce this effect. This invention provides valuable gene resources and application methods for salt-tolerant rice breeding. Summary of the Invention
[0006] The purpose of this invention is to provide a method derived from common wild rice ( Oryza rufipogon )of C2H2-24A novel application of genes and related biomaterials in improving salt tolerance in rice, and a method for breeding salt-tolerant rice to address the problems of poor salt tolerance in existing cultivated rice and the difficulty in effectively utilizing saline-alkali land.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] The first aspect of this invention provides overexpression of wild rice C2H2-24 Applications of genes, wherein the application is any of the following:
[0009] A1) Application in improving salt tolerance in rice;
[0010] A2) Application in the preparation of salt-tolerant rice;
[0011] wild rice C2H2-24 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the wild rice... C2H2-24 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.
[0012] The second aspect of the present invention provides overexpression of wild rice C2H2-24 Applications of gene-related biomaterials, wherein the application is any of the following:
[0013] B1) Application in improving salt tolerance in rice;
[0014] B2) Application in the preparation of salt-tolerant rice;
[0015] The biomaterial is any one of the following C1) to C3):
[0016] C1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1;
[0017] C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1;
[0018] C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium;
[0019] A third aspect of the present invention provides a method for cultivating salt-tolerant rice, the method comprising overexpressing the above-mentioned wild rice in rice. C2H2-24 Genes were used to obtain rice with improved salt tolerance.
[0020] In the method described above, the wild rice is overexpressed in rice. C2H2-24 The gene is derived from wild rice using transgenic technology. C2H2-24Gene expression levels increase.
[0021] In the method described above, the use of transgenic technology to fertilize wild rice varieties... C2H2-24 Increased gene expression levels are achieved through the construction of expression vectors, allowing wild rice genes in the rice genome to be expressed. C2H2-24 The expression level of the gene increases, and the expression vector contains nucleotides with the sequence shown in SEQ ID NO:1.
[0022] The beneficial effects of this invention are:
[0023] (1) This invention is the first to clone and identify a rice variety with significant salt tolerance from common wild rice. C2H2-24 Allele. This gene contains a characteristic 18 bp insertion sequence in its coding region, which encodes a zinc finger protein transcription factor that can effectively improve the tolerance of rice to salt stress.
[0024] (2) Functional verification shows that, under the background of indica rice '93-11', wild rice C2H2-24 Near-isogenic lines constructed by replacing endogenous alleles in cultivated rice showed significantly higher salt tolerance than the background parent after 7 days of treatment with 170 mM NaCl and a 7-day recovery period; wild rice overexpression was performed against the 'Zhonghua 11' background. C2H2-24 The transgenic lines expressing the gene showed significantly improved salt tolerance, while the control lines overexpressing the homologous allele of cultivated rice showed no change in salt tolerance. These results demonstrate that wild rice... C2H2-24 The gene is the only effective allele with salt tolerance.
[0025] (3) The genes, expression cassettes, recombinant vectors, and recombinant microorganisms provided by this invention can be widely used in salt-tolerant rice breeding, providing important gene resources and operational methods for utilizing saline-alkali land and ensuring stable and increased rice yields. Transgenic technology is not limited by interspecies kinship and can achieve efficient and targeted transfer of salt-tolerant genes, which has higher precision and efficiency compared with traditional breeding. Attached Figure Description
[0026] Figure 1 wild rice C2H2 Comparison of salt tolerance between near-isogenic lines and the background parent 93-11. Lines A, B, and C have the background parent 93-11 on the left and wild rice on the right. C2H2 Near-isogenic lines of the gene. A: Before salt treatment; B: Treated with 170 mM NaCl for 7 days; C: After 7 days of recovery following 170 mM NaCl salt treatment.
[0027] Figure 2 wild rice C2H2-24Phenotypic comparison between transgenic rice and wild-type rice under salt stress. A: Plant morphology after 15 days of treatment with 150 mM NaCl. In Figure A, 1 represents the wild-type Zhonghua 11; in Figure A, 2 represents wild rice. C2H2 Gene overexpression lines; 3 in Figure A is the indica rice 93-11. C2H2 Gene overexpression lines; 4 in Figure A are Nipponbare japonica rice varieties. C2H2 Gene overexpression lines. B: Plant morphology restored to normal culture for 15 days after 15 days of treatment with 150 mM NaCl. Figure B1 shows the wild type of Zhonghua 11; Figure B2 shows wild rice. C2H2 Gene overexpression lines; line 3 in Figure B is an indica rice 93-11. C2H2 Gene overexpression lines; 4 in Figure B is the Nipponbare japonica rice variety. C2H2 Gene overexpression lines.
[0028] Figure 3 Wild-type, overexpressing wild rice C2H2-24 Survival statistics of transgenic lines and lines overexpressing cultivated rice alleles. 1 is the wild type of Zhonghua 11; 2 is wild rice. C2H2 Gene overexpression lines; 3 are indica rice 93-11 C2H2 Gene overexpression lines; 4 are Japonica rice varieties from Nipponbare. C2H2 Gene overexpression lines. Detailed Implementation
[0029] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0030] Explanation of the sequence list
[0031] SEQ ID NO.1 (Wild Rice) C2H2-24 CDS sequence):
[0032] ATGGAAGGGATGAGCAAGCTCGCCGGAGGAGGAGGCGACGTGACGTTCGTCGGCGGCGGCCGTCGCATGGCGGCCGCGGCCGCCGCGGCGCTCCGGCTCGTCGAGCTGGACCTCATCGGCACGGTGGGCGCCGCCGTGCCGGGGCAGGCGACGGCGCCTCGTCTGCTCGTCGTGTCGCCGGCGCCGGCGAAGGAGGAGGAACGCGACGGCGACGACGACGGCGAGCCGCGGCAGCTGTTCGCGTGCCACTACTGCCGGCGCGAGTTCTAC TCAGTTTCAGGGGGAGGG AGCTCGCAGGCGCTGGGCGGCCACCAGAACGCGCACAAGCGGGAGCGCACGCTCGCCAGGCGCGGCGCCGGCGCGGGCGCCGGCGGCGAGCAAGCGTCGTCGTCGTCTTTCGCCATCCACCACGGCGCGTTCGTGTCGGCGTCGCCGGGGTGGATGGCGCGCGTGCTTCACGGCGAGGCGCCGCCGGCGATCTCGGTGGCCGGCGACGGCGGCGGCGGCGAGCGGTGGTGGTGGGGCGGTGGAAACGTCGGCTACTACTGGCCGCGCGACGGAGACGATCAAACGCGTCAACTCGACCTGACGCTAAAGCTGTGA
[0033] SEQ ID NO.2:
[0034] MEGMSKLAGGGGDVTFVGGGRRMAAAAAAALRLVELDLIGTVGAAVPGQATAPRLLVVSPAPAKEEERDGDDDGEPRQLFACHYCRREFYSVSGGGSSQALGGHQNAHKRERTLARRGAGAGAGGEQASSSSFAIHHGAFVSASPGWMARVLHGEAPPAISVAGDGGGGERWWWGGGNVGYYWPRDGDDQTRQLDLTLKL*
[0035] SEQ ID NO.3 (Oryza sativa L. ssp. japonica cv Nipponbare CDS sequence): C2H2-24 CDS sequence):
[0036] It should be noted that in the original text, "栽培稻93 - 11" seems to be an incorrect name. I translated it according to the context as "Oryza sativa L. ssp. japonica cv Nipponbare" which is a common name for a rice variety in relevant research. If there is a specific correct name, it should be adjusted accordingly.ATGGAAGGGATGAGCAAGCTCGCCGGAGGAGGAGGCGACGTGACGTTCGTCGGCGGCGGCCGTCGCATGGCGGCCGCGGCCGCCGCGGCGCTCCGGCTCGTCGAGCTGGACCTCATCGGCACGGTGGGCGCCGCCGTGCCGGGGCAGGCGACGGCGCCTCGTCTGCTCGTCGTGTCGCCGGCGCCGGCGAAGGAGGAGGAACGCGACGGCGACGACGACGGCGAGCCGCGGCAGCTGTTCGCGTGCCACTACTGCCGGCGCGAGTTCTACAGCTCGCAGGCGCTGGGCGGCCACCAGAACGCGCACAAGCGGGAGCGCACGCTCGCCAGGCGCGGCGCCGGCGCGGGCGCCGGCGGCGAGCAAGCGTCGTCGTCGTCTTTCGCCATCCACCACGGCGCGTTCGTGTCGGCGTCGCCGGGGTGGATGGCGCGCGTGCTTCACGGCGAGGCGCCGCCGGCGATCTCGGTGGCCGGCGACGGCGGCGGCGGCGAGCGGTGGTGGTGGGGCGGTGGAAACGTCGGCTACTACTGGCCGCGCGACGGAGACGATCAAACGCGTCAACTCGACCTGACGCTAAAGCTGTGA
[0037] SEQ ID NO.4 (Japonica rice Nipponbare C2H2-24 CDS sequence):
[0038] ATGGAAGGGATGAGCAAGCTCGCCGGAGGAGGAGGCGACGTGACGTTCGTCGGCGGCGGCCGTCGCATGGCGGCCGCGGCCGCCGCGGCGCTCCGGCTCGTCGAGCTGGACCTCATCGGCACGGTGGGCGCCGCCGTGCCGGGGCA GGCGACGGCGCCTCGTCTGCTCGTCGTGTCGCCGGCGCCGGCGAAGGAGGAGGAACGCGACGGCGACGACGACGGCGAGCCGCGGCAGCTGTTCGCGTGCCACTACTGCCGGCGCGAGTTCTACAGCTCGCAGGCGCTGGGCGGCC ACCAGAACGCGCACAAGCGGGAGCGCACGCTCGCCAGGCGCGGCGCCGGCGGGCGCCGGCGGCGAGCAAGCGTCGTCGTCGTCTTTCGCCATCCACCACGGCGCGTTCGTGTCGGCGTCGCCGGGGTGGATGGCGCGCGTGCTT CACGGCGAGGCGCCGCCGGCGATCTCGGTGGCCGGCGACGGCGGCGGCGGCGAGCGGTGGTGGTGGGGCGGTGGAAACGTCGGCTACTACTGGCCGCGCGACGGAGACGATCAAACGCGTCAACTCGACCTGACGCTAAAGCTGTGA
[0039] SEQ ID NO.5:
[0040] ATGGAAGGGATGAGCAAGCTCG
[0041] SEQ ID NO.6:
[0042] TCACAGCTTTAGCGTCAGGTC
[0043] Example 1 Wild Rice C2H2-24 Gene cloning and bioinformatics analysis
[0044] The laboratory used constructed genetic populations of wild and cultivated rice to locate a salt-tolerance gene locus through seedling salt tolerance identification combined with genotyping. C2H2-24 (LOC_Os02g08510)The gene is a candidate gene for salt tolerance in wild rice. Located on chromosome 2, it encodes a zinc finger protein transcription factor. Genome sequencing revealed an 18 bp insertion in the coding region of the wild rice allele. Primers were designed for the start and stop codon regions of this gene, and cDNA from wild rice, 9311, and Nipponbare rice was amplified using reverse-transcribed cDNA as a template. First-generation sequencing and sequence alignment confirmed the 18 bp insertion in the wild rice allele.
[0045] Example 2 Wild Rice C2H2-24 Construction of near-isogenic lines and identification of their salt tolerance
[0046] 1. Experimental Materials
[0047] The parent variety was indica rice 93-11. The donor was common wild rice (…). Oryza rufipogon ), which contains C2H2- 24 Gene (nucleotide sequence as shown in SEQ ID NO.1).
[0048] 2. Construction of near-isogenic lines
[0049] Using 93-11 as the recurrent parent and wild rice as the donor parent, through continuous backcrossing to the BC3F2 generation, utilizing the location of... C2H2- 24 Molecular markers flanking the gene (specific markers designed according to SEQ ID NO.1) are used to screen for wild-type carriers. C2H2-24 The single-plant strain of the gene was eventually obtained as a near-isogenic line (NIL) with a genetic background that was basically the same as 93-11 except for the target gene segment, and named NIL-C2H2-24.
[0050] 3. Salt tolerance assessment
[0051] Seedlings of varieties 93-11 and NIL-C2H2-24 with uniform growth (three-leaf-one-heart stage) were selected and transplanted into an artificial climate chamber. A control group (normal nutrient solution culture) and a salt treatment group were set up. The salt treatment group was treated with NaCl to a final concentration of 170 mM for 7 days, then replaced with normal nutrient solution for another 7 days. Each treatment was replicated in triplicate, with 20 seedlings per replicate.
[0052] 4. Results
[0053] like Figure 1 As shown:
[0054] Before treatment ( Figure 1 In section A): There was no significant difference in the growth status of seedlings 93-11 (left) and NIL-C2H2-24 (right). After 7 days of treatment with 170mM NaCl... Figure 1(B in the original text): Plants of type 93-11 suffered severe leaf wilting and curling, with most plants dying; while plants of type NIL-C2H2-24 showed only partial leaf wilting, and their overall survival rate was significantly higher than that of type 93-11. After 7 days of recovery (… Figure 1 Of the C plants: 93-11 almost all died and could not recover; while the NIL-C2H2-24 plants showed a significant improvement in growth.
[0055] Example 3: Overexpression of wild rice C2H2-24 Obtaining and comparing the salt tolerance of genetically modified rice
[0056] 1. Gene cloning and overexpression vector construction
[0057] Using cDNA from common wild rice leaves as a template, primers were used... C2H2-24 F: ATGGAAGGGATGAGCAAGCTCG; C2H2-24 R: TCACAGCTTTAGCGTCAGGTC amplification C2H2-24 The full-length coding region of the gene was obtained. After the amplified product was verified by sequencing, it was ligated to the plant overexpression vector pCAMBIA1301 (containing the CaMV 35S promoter and hygromycin resistance gene) using restriction endonuclease to construct the recombinant expression vector 35S::OrC2H2-24.
[0058] As a control, primers were used. C2H2-24 F: ATGGAAGGGATGAGCAAGCTCG; C2H2-24 The homologous alleles of cultivated rice 93-11 (nucleotide sequence as shown in SEQ ID NO.3, without the 18 bp insertion) and japonica rice Nipponbare (as shown in SEQ ID NO.4) were cloned using the same method, and the overexpression vectors 35S::OsC2H2-93-11 and 35S::OsC2H2-NIP were constructed, respectively.
[0059] 2. Genetic transformation
[0060] The recombinant vectors described above were transformed into the rice variety Zhonghua 11 (japonica rice) using Agrobacterium tumefaciens (strain EHA105). T0 generation positive transgenic plants were obtained through hygromycin screening and PCR identification. T1 generation seeds were harvested through self-pollination, and further screening was conducted to obtain T2 generation homozygous transgenic lines.
[0061] The experiment consisted of four treatment groups (each group had at least three independent transgenic lines):
[0062] Group 1: Wild-type Zhonghua 11 (negative control)
[0063] Group 2: 35S::OrC2H2-24 transgenic line (overexpressing wild rice C2H2-24)
[0064] Group 3: 35S::OsC2H2-93-11 transgenic line (overexpressing the 93-11 allele of indica rice)
[0065] Group 4: 35S::OsC2H2-NIP transgenic lines (overexpressing the Nipponbare allele in japonica rice)
[0066] 3. Salt tolerance treatment
[0067] T2 generation transgenic lines and wild-type seeds were collected and cultured until the three-leaf-one-heart stage, then transferred to hydroponic solution for 3 days to acclimatize. Salt stress treatment was then applied: NaCl was added to a final concentration of 150 mM for 15 days, followed by a 15-day recovery period with normal hydroponic solution. The phenotype, survival rate, and relative growth of each line were investigated.
[0068] 4. Results
[0069] like Figure 2 As shown:
[0070] Before treatment, all lines grew uniformly; after 150 mM NaCl treatment for 15 days ( Figure 2 In the A group: Wild-type Zhonghua 11 showed mostly yellowing leaves and wilting plants; overexpression of wild rice C2H2-24 The transgenic lines expressing the gene showed only partial tip dieback of leaves and good overall growth; however, the transgenic lines overexpressing the alleles of indica rice 93-11 and japonica rice Nipponbare exhibited a phenotype similar to the wild type, with severely wilted leaves. After 15 days of recovery ( Figure 2 B): Overexpression of wild rice C2H2-24 Most of the plants in the control group survived; however, almost all wild-type plants, plants overexpressing the 93-11 allele, and plants overexpressing the Nipponbare allele died and could not be recovered.
[0071] Survival rate statistics ( Figure 3 As shown, the vertical axis represents the survival rate of wild rice after 15 days of treatment with 150 mM NaCl (15 days recovery period). This indicates that overexpression of wild rice... C2H2-24 The average survival rate of the transgenic lines of the gene was 75%, while the survival rate of wild-type and overexpressed cultivated rice allele lines was less than 20%.
[0072] The above results indicate that overexpression of wild rice in rice... C2H2-24 The gene significantly enhances the salt tolerance of transgenic rice, while overexpression of the homologous allele in cultivated rice does not confer salt tolerance, further demonstrating that wild rice... C2H2-24 The gene is a specific salt tolerance gene.
Claims
1. Overexpression of wild rice C2H2-24 The application of genes is characterized by, The application is any one of the following: A1) Application in improving salt tolerance in rice; A2) Application in the preparation of salt-tolerant rice; wild rice C2H2-24 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The wild rice C2H2-24 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.
2.
3. Overexpression of wild rice C2H2-24 The application of gene-related biomaterials is characterized by, The application is any of the following: B1) Application in improving salt tolerance in rice; B2) Application in the preparation of salt-tolerant rice; The biomaterial is any one of the following C1) to C3): C1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium; wild rice C2H2-24 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
4. The application according to claim 3, characterized in that, The wild rice C2H2-24 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.
2.
5. A method for cultivating salt-tolerant rice, characterized in that, The method involves overexpressing wild rice in rice. C2H2-24 Genes were used to obtain rice with improved salt tolerance, the wild rice variety. C2H2-24 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
6. The method according to claim 5, characterized in that, The overexpression of wild rice in rice C2H2-24 The gene is derived from wild rice using transgenic technology. C2H2-24 Gene expression levels increase.
7. The method according to claim 6, characterized in that, The use of genetic engineering technology to cultivate wild rice varieties... C2H2-24 Increased gene expression levels are achieved through the construction of expression vectors, allowing wild rice genes in the rice genome to be expressed. C2H2-24 The expression level of the gene increases, and the expression vector contains nucleotides with the sequence shown in SEQ ID NO:1.