Application of OsRST31 gene in enhancing salt tolerance in rice
The OsRST31 gene and its protein obtained through EMS mutagenesis and map cloning technology have successfully improved the tolerance of rice to salt stress, solved the problem of rice growth restriction under salt stress, and has important breeding application value.
Patent Information
- Application Number
- CN202410193599.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-21
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2044-02-21
AI Technical Summary
Rice is limited in growth under salt stress, resulting in a decrease in yield and quality. It is difficult for the prior art to effectively improve the salt tolerance of rice.
The mutant osrst31 with enhanced salt tolerance in rice was obtained through EMS mutagenesis, and the OsRST31 gene and its encoded protein were successfully isolated and cloned by map cloning technology. The negative regulatory role in regulating salt tolerance in rice was verified through transgenic technology.
The OsRST31 mutant significantly improves tolerance to salt stress, can maintain high growth performance under high salt conditions, and provides an effective genetic means to improve the salt tolerance of rice.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to application of OsRST31 gene in enhancing salt tolerance of rice. Background Art
[0002] Rice is an important food crop and the main staple food in my country. The production of rice is very susceptible to salt stress, which directly affects the yield and quality of rice. How to improve the resistance of rice to stress in the form of global environmental changes is an effective means to ensure my country's food security. With the acceleration of modern industrial and agricultural progress, the problem of salinization of paddy soil is becoming increasingly serious, seriously threatening the normal growth of rice, resulting in reduced yield and deterioration of quality. On the other hand, in order to ensure my country's food security, rice needs to be planted in the southeast coastal tidal flats and northwest saline-alkali lands.
[0003] Therefore, it is urgent to breed salt-tolerant rice varieties. Therefore, it is urgent to explore and apply rice salt-tolerant gene resources and apply them to improve rice salt tolerance breeding. Summary of the invention
[0004] In view of the above-mentioned prior art, the present invention proposes a gene and a protein thereof that can enhance the salt tolerance of rice.
[0005] The present invention uses map-based cloning to obtain the OsRST31 gene and the protein it encodes, and the specific steps are as follows:
[0006] 1. Isolation and genetic analysis of osrst31 mutants:
[0007] The rice salt stress-tolerant osrst31 mutant of the invention is a mutation produced by EMS (Ethyl Methyl Sulfonate) mutagenesis of the japonica rice variety Zhonghua 11. Through a forward and reciprocal cross experiment with a wild type, it is proved that the mutant is controlled by a recessive single gene.
[0008] 2. Growth status of osrst31 mutant and wild type under salt treatment:
[0009] After seven days of treatment with 120 mM NaCl, the wild type dried up and died at the second leaf stage, while the mutant grew a third leaf. The length and dry weight of the aboveground part of the mutant were significantly higher than those of the wild type. The above experiments proved that the osrst31 mutant tolerated salt stress.
[0010] 3. Cloning of OsRST31 gene:
[0011] (1) Localization of OsRST31:
[0012] In order to isolate the OsRST31 gene, the present invention first constructed a segregation population, hybridized the osrst31 mutant with the indica rice variety TN1 to obtain F1 offspring, and self-pollinated the F1 offspring to obtain an F2 segregation population. Individual plants with the same phenotype as osrst31 were selected from the F2 segregation population for gene cloning. Using 232 pairs of SSR and InDel primers evenly distributed on 12 rice chromosomes, the candidate gene was preliminarily located between chromosome 8 B8-3 and B8-4. Further using the sequence difference between osrst31 and TN1, 7 pairs of SSR and InDel primers were developed to finely locate the candidate gene within the range of 87.15kb between H8-7 and H8-17. By sequencing and analyzing the open reading frame (ORF) of this segment, it was found that the insertion of a base (T) on LOC_Os08g07010 caused a frameshift mutation. Therefore, the candidate gene was determined as the target gene of OsRST31.
[0013] (2) Identification and functional analysis of the OsRST31 gene:
[0014] Through the transgenic technology, the results show that the present invention obtains transgenic rice that restores the salt tolerance of the osrst31 mutant to the wild-type phenotype, proving that the present invention correctly clones the OsRST31 gene.
[0015] Based on the above research results, the present invention develops corresponding applications.
[0016] In a first aspect, the present invention provides the use of the OsRST31 gene in regulating the salt tolerance of rice.
[0017] Furthermore, the regulation is negative regulation.
[0018] Among them, the OsRST31 gene is:
[0019] i) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2; or
[0020] ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and the nucleotide sequence expresses a protein with the same function; or
[0021] iii) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i) or ii) and expresses a protein with the same function.
[0022] Note: SEQ ID NO: 1 is the full length of gDNA, and SEQ ID NO: 2 is the full length of cDNA.
[0023] On the other hand, the present invention provides an application of a protein encoded by a salt tolerance regulatory gene OsRST31, wherein the protein is used to regulate rice salt tolerance, and the protein has a sequence as shown in (A) or (B):
[0024] (A) the amino acid sequence shown in SEQ ID No: 3;
[0025] (B) A protein derived from (A) in which one or more amino acids are added and / or substituted and / or deleted in the amino acid sequence defined in (A) and which has the same function.
[0026] In another aspect, the present invention provides a mutant gene of the salt tolerance regulatory gene OsRST31, wherein the mutant gene sequence is: based on the coding nucleotide sequence shown in SEQ ID No: 1, a T base is inserted at position 3483.
[0027] On the other hand, the present invention provides a mutant protein encoded by the mutant gene, wherein the amino acid sequence of the mutant protein is a frameshift mutation caused on the basis of the encoded amino acid sequence shown in SEQ ID No: 3.
[0028] In another aspect, the present invention provides an application of the mutant gene and mutant protein in improving the salt tolerance of rice. For example, in practical applications, gene mutation can be used to improve the salt tolerance of rice; or mutant materials containing mutant genes can be used to improve other rice materials through traditional breeding methods such as backcrossing.
[0029] The technical effect of the present invention is as follows: the present invention obtains a rice salt tolerance enhanced mutant osrst31 through EMS mutagenesis, and successfully isolates a rice salt tolerance negative regulatory gene, OsRST31, through map-based cloning and complementary transgenics. The experimental results show that the mutant with loss of function of OsRST31 has significantly improved tolerance to salt stress compared with the wild type, and can be effectively applied to the molecular improvement of rice salt tolerance, and has great development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The phenotype (A) and the comparison of plant height and dry weight (BC) of the osrst31 mutant and the wild-type material after 7 days of 120 mM NaCl treatment in the example of the present invention;
[0031] Figure 2 is a cloning map of the OsRST31 gene in the embodiment of the present invention;
[0032] Figure 3 This is the vector map of pCAMBIA2300-OsRST31 in the embodiment of the present invention;
[0033] Figure 4 The phenotypes of the transgenic overexpression complemented T1 rice and osrst31 mutant after 7 days of 120 mM NaCl treatment in the examples of the present invention (A) and the comparison of plant height and dry weight (BC). DETAILED DESCRIPTION
[0034] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.
[0035] Example 1: Cloning of OsRST31:
[0036] 1. Rice material: rice salt tolerance enhanced mutant osrst31, whose wild type material is japonica rice variety Zhonghua 11. Rice (Oryza sativa L.cv Zhonghua 11). Figure 1 As shown, after 7 days of 120 mM NaCl treatment, the wild-type plants withered and died at the 2-leaf stage; the osrst31 mutant grew a third leaf ( Figure 1 A). Compared with the wild type, the plant height and dry weight of the mutant were significantly higher than those of the wild type under salt treatment ( Figure 1 B, Figure 1 C).
[0037] 2. Positioning population: The osrst31 mutant was hybridized with the indica rice variety TN1, and the F1 generation was self-pollinated in bags to obtain the F2 population, with a total of 3606 plants in the F2 population. After salt treatment, 2701 plants were consistent with the wild-type phenotype, and 905 plants were consistent with the osrst31 mutant phenotype. 21 plants with the osrst31 mutant phenotype were selected as the initial positioning population, and the remaining 884 plants with the osrst31 mutant phenotype were selected as the fine positioning population. Leaves of about 3 cm were taken from each plant for extraction of genomic DNA.
[0038] 3. SSR and Indel markers locate the OsRST31 gene: The CTAB method was used to extract genomic DNA from rice leaves. Take 3 cm rice leaves, put them in a 2.0 ml centrifuge tube, add 1 steel ball, 800 μl of extraction solution, put them in the TissueLyser II system tissue grinder, grind for 50 seconds, put them in a 65°C water bath and incubate for 30 minutes, shake them once during the period, then add 500 μl of chloroform and shake them up and down vigorously, let them stand for 10 minutes and then centrifuge, take 500 μl of supernatant, add 1000 μl of anhydrous ethanol, centrifuge, and add 350 μl of ultrapure water as DNA samples after the precipitate is dried. Use 1 μl of DNA sample for each PCR reaction.
[0039] Preliminary positioning of the OsRST31 gene: 21 plants with osrst31 mutant phenotypes were selected from the F2 population of osrst31 and TN1 for preliminary positioning. Based on the published molecular genetic maps created for japonica and indica rice, 232 pairs of SSR and Indel primers that were approximately evenly distributed on each chromosome were selected, and OsRST31 was ultimately preliminarily positioned between the two molecular markers B8-3 and B8-4 on chromosome 8.
[0040] PCR amplification program: 95℃5min
[0041]
[0042] Fine positioning of OsRST31 gene: In order to narrow the positioning interval of OsRST31 gene, the present invention selected the remaining 884 plants with osrst31 mutant phenotype from the F2 population of osrst31 and TN1 for fine positioning. Based on the initial positioning, 7 new molecular markers were developed by sequence comparison analysis of indica rice variety 9311 and japonica rice variety Nipponbare, and finally OsRST31 was accurately positioned within the range of 87.15kb between molecular markers H8-7 and H8-17.
[0043] 4. OsRST31 gene prediction, acquisition of full-length cDNA gene and prediction of function: According to the results of fine positioning, 10 candidate genes were found within the 87.15kb range according to the prediction of the Rice Genome Annotation Project (http: / / rice.uga.edu / index.shtml). The present invention designed sequencing primers for these 10 candidate genes, and amplified these 10 candidate genes from the genomes of osrst31 and wild-type japonica rice variety Zhonghua 11 by PCR for sequencing analysis. It was found that a T base was inserted into the DNA fragment of the LOC_Os08g07010 gene, resulting in a frameshift mutation. According to the gene annotation information on the website of The Rice Annotation Project (https: / / rapdb.dna.affrc.go.jp), the gene is predicted to encode an ABCG transporter.
[0044] Example 2: Construction of pCAMBIA1300-OsRST31 plant expression vector:
[0045] According to the whole genome sequence of rice variety Oryza sativa Lcv. Nipponbare provided on the website of The Rice Annotation Project, specific primers for amplifying the full-length sequence of OsRST31 gene were designed ( Figure 2 ). The primer sequences are as follows:
[0046] OsRST31C-F primer: 5'-gctcggtacccggggatccCTATGCGCAATGGTGCTACA-3'
[0047] OsRST31C-R primer: 5′-aggtcgactctagaggatccGGGCCACTCGAATTGAGACA-3′;
[0048] Then, the genomic DNA of rice variety Nipponbare was extracted, and the genomic cDNA of Zhonghua 11 was used as a template to amplify the OsRST31 genomic DNA fragment of 8459 bp using the primers designed above (OsRST31C-F and OsRST31C-R). The following amplification program was used: 94℃ pre-denaturation for 4min; 98℃ denaturation for 10 seconds, 55℃ annealing for 30 seconds, 68℃ extension for 9min, 33 cycles; and finally 68℃ extension for another 10min. The target fragment amplified by PCR was recovered; at the same time, the pCAMBIA1300 empty vector was linearized with BamHI endonuclease, and the pCAMBIA1300 backbone was recovered. Then, the reaction was prepared using Infusion enzyme, the recovered OsRST31 gene fragment and the pCAMBIA2300 empty vector after enzyme digestion. Specific reaction system: 5× In-fusion mix 4μl; linearized vector 2μl; inserted target fragment 2μl; ultrapure water 12μl; incubate at 37℃ for 30min, ice bath for 5min, then transform E. coli DH5a competent cells, and then sequence verified by Nanjing Qingke Company, finally obtain OsRST31 complementary expression vector pCAMBIA1300-OsRST31( Figure 3 ), and the pCAMBIA1300-OsRST31 expression vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation.
[0049] Example 3: Transformation of rice with the pCAMBIA1300-OsRST31 expression vector:
[0050] The recombinant expression vector pCAMBIA1300-OsRST31 was transformed into rice mature embryos using Agrobacterium-mediated callus infection of rice mature embryos. The transformation method is as follows:
[0051] (1) Induction of mature embryonic callus in rice: The mature osrst31 mutant seeds were shelled, and then surface-sterilized with 70% alcohol for 30 seconds, repeated 3 times, and then soaked in 30% NaClO solution for 20 minutes, repeated once, and then washed with sterile water 7-10 times. The seeds were then cultured on an induction medium at 28°C in the dark to induce callus for transformation.
[0052] (2) Co-cultivation of rice callus and Agrobacterium: The EHA105 strain identified in Example 2 as containing the pCAMBIA1300-OsRST31 expression vector was activated and resuspended to adjust OD600 to 0.5-0.7. The callus was collected in a 250 ml sterile conical flask, and the resuspended Agrobacterium suspension was poured into the callus to infect the callus. After soaking for 15-20 minutes, the suspension was poured out, and the infecting callus was placed on sterile filter paper to absorb the excess Agrobacterium bacterial liquid. The callus was then placed in a culture dish covered with sterile filter paper and cultured at 29° C. in the dark for 3 days.
[0053] (3) Screening of resistant calli: After the co-cultivation was completed, the calli were transferred to a screening medium containing 50 mg / ml hygromycin, and resistant calli were screened at 28°C.
[0054] (4) Differentiation of resistant calli: Calli with good growth status in the screening medium were placed in differentiation medium and cultured at 28°C until seedlings were differentiated and grown.
[0055] (5) Rooting of differentiated seedlings: When the differentiated seedlings are about 2 cm or larger, they are transferred to a rooting medium for rooting culture. The seedlings with roots are transplanted to a transgenic nursery for growth.
[0056] (6) Plant identification: After 7 days of treatment with 120 mM NaCl, compared with the osrst31 mutant, the transgenic complemented T1 generation plants withered and died at the two-leaf stage ( Figure 4 A), plant height and dry weight returned to the same level as the wild type ( Figure 4 B, Figure 4 C).
[0057] The above are only implementation modes of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure made using the contents of the present invention specification, directly or indirectly used in other related technical fields, is also within the patent protection scope of the present invention.
Claims
1. Application of OsRST31 gene in regulating salt tolerance of rice, characterized in that: The nucleotide sequence of the OsRST31 gene is shown in SEQ ID NO:
2.
2. Use of the protein encoded by the OsRST31 gene as claimed in claim 1 in regulating the salt tolerance of rice.
3. The use according to claim 2, characterized in that The amino acid sequence of the protein encoded by the OsRST31 gene is shown in SEQ ID NO:3.