Drought-resistant and salt-tolerant rice gene osmate6 and application of encoded protein thereof

Abstract

The application discloses a rice drought-resistant and salt-tolerant gene OsMATE6 and application thereof, which comprises deleting the OsMATE6 gene in rice, and compared with a wild type, can significantly improve the drought resistance of rice seedlings under drought and high-salt stress conditions, and significantly improve the drought resistance of plants and yield under field soil drought treatment conditions. The drought-resistant and salt-tolerant function of the OsMATE6 gene provides a new gene resource for plant drought resistance and salt tolerance genetic breeding, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the rice OsMATE6 gene and its encoded protein in improving the drought resistance and salt tolerance of rice. Background Technology

[0002] Rice (Oryza sativa L.) is an important food crop, with about 60% of my country's population relying on it as their staple food. However, rice is also a water-intensive crop, accounting for over 65% of my country's total agricultural water consumption, making it the largest water consumer in agriculture. With global climate change, the increasing frequency and intensity of extreme weather events such as droughts pose a serious threat to rice growth, development, and yield. Developing water-saving and drought-resistant rice varieties and enhancing their drought resistance is an effective way to alleviate my country's water shortage and promote sustainable agricultural development. Furthermore, improper irrigation leading to secondary soil salinization severely restricts rice production, and my country has 2.34 million hectares of such soil salinization. 2 Coastal mudflats and 100 million hectares 2 Inland saline-alkali land represents a vast potential resource. Rice, as a moderately salt-sensitive crop, is considered the preferred food crop for developing coastal mudflats and saline-alkali land. Therefore, exploring drought- and salt-tolerant genes to improve rice's resistance to drought and salt stress is of great significance for promoting sustainable agricultural development and safeguarding national food security.

[0003] Drought and high salt concentrations both lead to osmotic and oxidative stress in plants. Furthermore, high salt concentrations can cause secondary stresses such as ion toxicity. Drought resistance and salt tolerance in rice are both quantitative traits controlled by multiple genes, resulting in a complex genetic basis. Hundreds of QTLs (Quantitative Trait Loci) or genes associated with drought resistance have been cloned in rice. For example, the DRO1 gene, cloned using map-based cloning technology, participates in regulating the development of deeper roots under drought stress, thereby improving rice drought resistance. OsbZIP23 regulates rice drought resistance through an ABA (abscisic acid)-dependent signaling pathway. The zinc finger transcription factor DST negatively regulates rice drought resistance by modulating the expression of reactive oxygen species scavenging-related genes. Currently, hundreds of QTLs related to rice salt tolerance have been reported, but most of these QTLs have a small phenotypic contribution, making fine mapping and cloning difficult. Among them, SKC1 has been cloned and its function analyzed, specifically mapped to an HKT (High Affinity K) gene. + The Transporter family (high-affinity K+ transporters) encodes Na+. + The specific transporter gene OsHKT1;5, transcription factors OsMYB106 and OsSUVH7 participate in resistance to salt stress by regulating the expression of OsHKT1;5.

[0004] The multidrug and toxic compound extrusion (MATE) family is a widespread family of secondary transport proteins found in prokaryotes and eukaryotes. MATE transporters are widely involved in various processes of plant growth and development. Reported functions of MATE transporters include the efflux of foreign substances, accumulation of secondary metabolites (such as alkaloids and flavonoids), iron transport, aluminum detoxification, pathogen resistance, and plant hormone signal transduction. In rice, the MATE family comprises 46 members, but only a few genes have been reported in functional studies. For example, DG1 mediates long-distance transport of leaf-derived ABA to the spikelet and regulates starch synthesis gene expression in a temperature-dependent manner, thereby regulating grain filling. BIRG1 is located in the cell membrane and participates in chloride ion (Cl) ion exchange. - OsMATE6 has the ability to efflux, negatively regulate rice grain size, and positively regulate tolerance to salt stress. However, there are currently no reports on the research and application of the OsMATE6 gene, especially in regulating drought resistance and salt tolerance. Summary of the Invention

[0005] One objective of this invention is to identify the rice drought and salt tolerance-related gene OsMATE6 and its encoded protein, the loss of function of which enhances rice's resistance to drought and salt stress. In the art, "salt tolerance" generally refers to a plant's ability to withstand salt damage during growth; "drought resistance" generally refers to a plant's adaptation to and resistance to water loss caused by drought during growth. Another objective of this invention is to determine the application of the identified rice drought and salt tolerance-related gene OsMATE6 in improving rice's drought and salt tolerance, and to provide a method for breeding drought-resistant and salt-tolerant rice using the identified OsMATE6 gene. This invention has significant reference value and potential application value for researching how to improve rice's resistance to drought and salt stress.

[0006] Therefore, in a first aspect, the present invention has identified a rice drought and salt tolerance related gene OsMATE6, which encodes the amino acid sequence shown in SEQ ID NO.2.

[0007] In a preferred aspect, the nucleotide sequence of the rice drought-resistance and salt-tolerance-related gene OsMATE6 can be selected from:

[0008] 1) The DNA sequence shown in SEQ ID NO.1; or

[0009] 2) A nucleotide sequence that can hybridize with the DNA sequence shown in SEQ ID NO.1 under highly stringent conditions.

[0010] In a second aspect, the present invention provides a protein encoded by the rice drought and salt tolerance-related gene OsMATE6, wherein the OsMATE6 protein is one of the following (a), (b), or (c):

[0011] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.2;

[0012] (b) A protein derived from (a) having the same activity as (a) by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0013] (c) Other genes encode proteins that have more than 50% amino acid identity with the amino acid sequence shown in SEQ ID NO.2 and have the same activity as (a).

[0014] Of these, SEQ ID NO.2 consists of 549 amino acid residues.

[0015] In a third aspect, the present invention relates to the use of gene knockout vectors or gene knockout expression cassettes comprising a gene as shown in SEQ ID NO.1 or a gene comprising a gene encoding a polypeptide as shown in SEQ ID NO.2 in the breeding of rice with drought resistance and salt tolerance.

[0016] In a fourth aspect, the present invention relates to the application of the rice OsMATE6 gene in the breeding of rice with drought resistance and salt tolerance, wherein the gene encodes a polypeptide with the amino acid sequence of SEQ ID NO.2. The inventors have discovered that the deletion of the OsMATE6 gene in rice can improve the drought resistance and salt tolerance of rice.

[0017] In a fifth aspect, the present invention relates to a method for breeding rice with drought resistance and salt tolerance, the method comprising knocking out the OsMATE6 gene in rice to obtain transgenic rice with drought resistance and salt tolerance, or obtaining offspring rice with OsMATE6 gene loss of function by hybridization.

[0018] In a preferred embodiment of the present invention, a method for cultivating rice with drought resistance and salt tolerance is provided, the method comprising the following steps:

[0019] (1) Select the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 and construct it into the gene knockout vector;

[0020] (2) Transform rice cells or tissues (e.g., electrical conductivity, gene gun) with the gene knockout vector (e.g., CRISPR / Cas9 vector) constructed in step (1) to obtain transgenic rice;

[0021] (3) The obtained transgenic rice was subjected to resistance screening and the expression level at the transcriptional level to determine that the target gene had been knocked out, thereby obtaining transgenic knockout rice.

[0022] The transgenic rice is a knockout mutant osmate6-1 and osmate6-2. In osmate6-1, a 1 bp deletion occurs at position 476 bp in the coding region of the OsMATE6 gene, corresponding to the nucleotide sequence of SEQ ID NO:1. In osmate6-2, a 57 bp deletion occurs at positions 458-514 bp in the coding region of the OsMATE6 gene, corresponding to the nucleotide sequence of SEQ ID NO:1.

[0023] In another implementation, rice varieties with OsMATE6 gene loss of function are crossed with another type of rice to obtain offspring rice with OsMATE6 gene loss of function. Compared with uncrossed rice, the hybrid offspring rice exhibits stronger drought resistance and salt tolerance. For example, crossing with the mutant ptm97-1 to obtain offspring rice with OsMATE6 gene loss of function results in hybrid rice with stronger drought resistance and salt tolerance compared with uncrossed rice.

[0024] Specifically, the technical problem of the present invention is solved by the following technical solution.

[0025] 1. Application of the rice OsMATE6 gene in the breeding of rice with drought resistance and salt tolerance, wherein the gene encodes a polypeptide with the amino acid sequence of SEQ ID NO.2.

[0026] 2. The application according to item 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0027] 3. The application of gene knockout vectors or gene knockout expression cassettes containing genes as shown in SEQ ID NO.1 or containing genes encoding polypeptides as shown in SEQ ID NO.2 in the breeding of drought-resistant and salt-tolerant rice.

[0028] 4. A method for breeding rice with drought resistance and salt tolerance, comprising obtaining rice with loss of function of the OsMATE6 gene, thereby breeding rice with stronger drought resistance and salt tolerance compared with wild type, the nucleotide sequence of said gene being shown in SEQ ID NO.1.

[0029] 5. The method according to item 4, wherein the gene encodes a polypeptide with the amino acid sequence of SEQ ID NO.2.

[0030] 6. The method according to item 4 or 5, wherein obtaining rice with OsMATE6 gene loss of function is achieved by crossing an OsMATE6 gene loss of function mutant with another rice variety, and the hybrid offspring rice exhibits stronger drought resistance and salt tolerance compared to the uncrossed rice.

[0031] 7. The method according to item 6, wherein the OsMATE6 gene loss-of-function mutant is ptm97-1, wherein the guanine at position 1000 of the nucleotide sequence corresponding to SEQ ID NO:1 in the coding region of the OsMATE6 gene in ptm97-1 is mutated to adenine, or the glycine at position 334 of the amino acid sequence corresponding to SEQ ID NO:2 in the protein encoded by the OsMATE6 gene is mutated to arginine.

[0032] 8. The method according to item 4 or 5, wherein obtaining rice with OsMATE6 gene loss of function is achieved by knocking out the rice OsMATE6 gene to obtain a mutant through CRISPR-Cas9, zinc finger nucleases (ZFNs), TALENs or RNAi gene silencing technology.

[0033] 9. The method according to item 8, wherein the mutant is osmate6-1 or osmate6-2, wherein osmate6-1 has a 1 bp deletion at position 476 bp of the nucleotide sequence corresponding to SEQ ID NO:1 in the coding region of the OsMATE6 gene, and wherein osmate6-2 has a 57 bp deletion at positions 458-514 bp of the nucleotide sequence corresponding to SEQ ID NO:1 in the coding region of the OsMATE6 gene.

[0034] 10. The application according to any one of items 1-3 or the method according to any one of items 4-9, wherein the rice is Longjing 31 or ZH11. Attached Figure Description

[0035] Figure 1 Identification of drought-resistant rice mutants: A. Phenotype of wild-type (LG31) and M3 generation mutant (ptm97-1) after 12 days of hydroponics followed by treatment with 25% PEG4000; B. Phenotype of wild-type (LG31) and M4 generation mutant (ptm97-1) after treatment with 25% PEG4000.

[0036] Figure 2The drought resistance phenotypes of mutant ptm97-1 during the seedling stage and in the field were analyzed, including: A. Phenotypes of wild-type (LG31) and M5 generation mutant (ptm97-1) before and after treatment with 25% PEG4000 12 days after hydroponics; B. Survival statistics; C. Phenotypes of LG31 and ptm97-1 seedlings before and after soil drought treatment; D. Survival statistics; E. Phenotypes of LG31 and ptm97-1 in field drought trials; and F. Yield statistics.

[0037] Figure 3 Salt tolerance phenotype of mutant ptm97-1 seedlings, including A. Phenotypes of wild type (LG31) and M5 generation mutant (ptm97-1) before and after treatment with 150mM NaCl 12 days after hydroponics, and B. Survival statistics.

[0038] Figure 4 The candidate mutant gene ptm97-1 was validated by first-generation sequencing and obtained through homozygous knockout. Among them, A. First-generation sequencing validation results of the candidate mutant gene LOC_Os02g45380 (OsMATE6) of ptm97-1; B. Two homozygous knockout mutants (osmate6-1, osmate6-2) of OsMATE6 and their editing types were obtained by CRISPR technology in the Zhonghua 11 (ZH11) background.

[0039] Figure 5 The drought-resistant phenotypes of OsMATE6 mutant seedlings are shown in the following figures: A. Phenotypes of wild-type (ZH11) and OsMATE6 mutants (osmate6-1, osmate6-2) before and after treatment with 25% PEG4000 12 days after hydroponics, and B. Survival statistics; C. Phenotypes of wild-type (ZH11) and mutant osmate6-1 seedlings before and after soil drought treatment, and D. Survival statistics; E. Phenotypes of wild-type (ZH11) and mutant osmate6-2 seedlings before and after soil drought treatment, and F. Survival statistics.

[0040] Figure 6 Salt-tolerant phenotypes of OsMATE6 mutant seedlings during the seedling stage, including A. Phenotypes of wild type (ZH11) and OsMATE6 mutants (osmate6-1, osmate6-2) before and after treatment with 150mM NaCl 12 days after hydroponics, and B. Survival statistics. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. However, it should be understood that the embodiments described are merely illustrative and are not intended to limit the scope and spirit of the invention.

[0042] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0043] Example 1: Screening process and identification of drought-resistant rice mutants

[0044] Wild-type seeds of the rice variety Longjing 31 (LG31) were induced to mutate using ethyl methyl sulfonate (EMS) (CAS: 62-50-0) (provided by Pan Guojun, Rice Research Institute, Heilongjiang Academy of Agricultural Sciences). The specific method of mutagenesis was as follows: Longjing 31 seeds were soaked in a 0.5% EMS solution for 16 hours, repeatedly rinsed, and then sown in an experimental field (M1 generation). After self-pollination, the M1 generation plants were harvested to create an M2 generation rice mutant library [approximately 320,000 M1 seeds, mixed in pools of 1000-1200 M1 plants, for a total of 103 pools]. The mutant library was screened using a 25% PEG4000 (Lotte, Korea, PEG) solution. The specific procedure was as follows: Approximately 10,000 seeds (M2 generation) were selected from each pool, soaked and germinated for 2-3 days, then evenly spread in seedling trays and hydroponically cultured with Yoshida nutrient solution (nutrient solution formula below) for 12 days (with fresh nutrient solution prepared every 2 days). Then, the seedlings were treated with 25% PEG4000 (prepared with Yoshida nutrient solution). Seedlings that survived for 8 days were selected as initial screening PEG-resistant positive seedlings for subsequent research. One mutant with excellent PEG-resistant phenotype was selected from pool number 97 and named ptm97-1. P EG t olerance m utant 97-1 The mutants were then transplanted to the field and allowed to grow normally until flowering and seed production. Further screening of the harvested M3 and M4 generation mutant seeds was conducted using the same method. The results showed that, compared to the wild-type LG31, the mutant ptm97-1 still exhibited excellent PEG resistance. Figure 1 (AB) confirmed that the mutant indeed has a PEG-resistant phenotype and can be stably inherited. Drought and high-salt environments can cause osmotic stress in plants. PEG, as an osmotic regulator, can simulate the osmotic stress caused by drought and salt stress with a certain concentration of PEG solution. It is widely used as a commonly used and relatively ideal system in the study of plant drought resistance, salt tolerance and other stress resistance (Zhang Lijun, Fan Jinjuan, Ruan Yanye, Guan Yixin. Application of polyethylene glycol in the physiological study of plant osmotic stress [J]. Plant Physiology Communications, 2004(03):361-364.).

[0045] Yoshida nutrient solution formula: NH4NO3 (1mM), NaH2PO4·2H2O (0.6mM), K2SO4 (0.3mM), CaCl2 (0.3mM), MgCl2·6H2O (0.6mM), Fe(II)-EDTA (10μM), H3BO3 (48.7μM), MnSO4 (9μM), CuSO4·5H2O (0.3μM), ZnSO4·7H2O (0.7μM), NaMoO4·2H2O (0.1μM), with the final pH adjusted to 5.8.

[0046] Example 2: Seedling and field drought-resistant phenotypes of mutant ptm97-1

[0047] Seedling hydroponic experiment: Wild-type (LG31) and M5 mutant (ptm97-1) seeds were soaked in distilled water and germinated in a 37°C oven for 2-3 days. Seeds with consistent germination were transferred to a PCR plate (with the bottom removed) and hydroponically grown in Yoshida nutrient solution for 12 days (with fresh nutrient solution changed every 2 days). Then, 25% PEG4000 (prepared with Yoshida nutrient solution) was applied. Leaf curling and gradual drying symptoms were observed within hours of treatment. After a significant difference in drying symptoms between the control LG31 and the mutant ptm97-1 (approximately 1 day), the normal nutrient solution was restored for 4 days, and the survival rate was recorded. The entire rice growth process was conducted in an artificial climate chamber with growth conditions of 14 hours of light / 10 hours of darkness and a light intensity of 300 μmol / m². 2 / s, temperature was 30 degrees Celsius under light and 28 degrees Celsius under darkness, and humidity was set to 70%. Experimental results showed that after treatment with 25% PEG4000, the mutant ptm97-1 exhibited less leaf curling, slower leaf water loss and drying, and a significantly higher survival rate after restoring normal nutrient solution compared to the control LG31. Figure 2 AB).

[0048] Seedling stage soil drought experiment: After soaking and germinating LG31 and PTM97-1 seeds, seeds with consistent germination were evenly distributed in the same flowerpot filled with nutrient soil. The pots were placed in a water trough with an appropriate amount of water and grown in a greenhouse for 12 days. The flowerpots were then removed and placed on a growing rack to drain. After several days, the soil gradually dried, and rice seedlings began to show symptoms of leaf curling and drying. Once the drying symptoms of the control LG31 and the mutant PTM97-1 showed a significant difference (approximately 12 days), the flowerpots were returned to the water trough for rehydration. The survival rate was recorded one day after rehydration. Figure 2 CD). Experimental results showed that after soil drought treatment during the seedling stage, the mutant ptm97-1 exhibited leaf curling later than the control LG31, with less leaf curling and slower leaf drying. Its survival rate after rehydration was significantly higher than that of the control LG31. Figure 2CD).

[0049] Field drought experiment: The field drought experiment was completed in Bengbu City, Anhui Province during the peak season of 2022. LG31 and PTM97-1 seeds were soaked and germinated before being sown in a nursery in paddy fields. Seedlings were transplanted at the 3-leaf stage (approximately 25 days after sowing). Each material was divided into three independent plots, with 12 rows and 10 plants per row in each plot, with a row spacing of 20 cm and a plant spacing of 17 cm. Irrigation was stopped in the drought treatment group after the seedlings turned green, while the paddy field control group was managed normally. After the rice matured, edge rows were removed, and 30 plants from each plot were selected for harvesting and yield measurement. The results showed that the leaves of the rice in the drought treatment group curled and dried during the flowering and grain-filling stages. The mutant PTM97-1 showed leaf curling later than the control LG31, with less curling and slower leaf drying, and its final yield was significantly higher than the control LG31 (17.7% increase). In contrast, there was no significant difference in yield between the mutant PTM97-1 and LG31 in the paddy field control group. Figure 2 EF).

[0050] Example 3: Salt-tolerant phenotype of mutant ptm97-1 seedlings

[0051] The hydroponic cultivation of rice seedlings was conducted as described in the "Seedling Hydroponic Experiment" section of Example 2. After 12 days of hydroponics in normal nutrient solution, the seedlings were treated with 150 mM NaCl (prepared with Yoshida nutrient solution). Several hours after treatment, leaf curling and gradual drying symptoms were observed. After approximately 3 days (when the drying symptoms of the control LG31 and the mutant ptm97-1 showed a significant difference), the normal nutrient solution was restored for 3 days, and the survival rate was recorded. The experimental results showed that after treatment with 150 mM NaCl, the mutant ptm97-1 experienced slower water loss and drying of its leaves, and its survival rate after restoring normal nutrient solution was significantly higher than that of the control LG31. Figure 3 AB).

[0052] Example 4: First-generation sequencing verification and homozygous knockout of the mutant ptm97-1 candidate mutant gene

[0053] Seeds of the M3 generation of the ptm97-1 mutant were germinated in the field and grown to the flowering stage. The ptm97-1 mutant (as the male parent) was crossed with LG31 (as the female parent) to harvest the BC1F1 generation hybrid seeds. The BC1F1 generation hybrid seeds were then transplanted back into the field to allow self-pollination, and the BC1F2 generation seeds were harvested to construct a BC1F2 segregating population.

[0054] The BC1F2 populations of control LG31 and ptm97-1 were treated with 25% PEG4000. After all the control LG31 plants died, a very small number of resistant individual plants in the segregating population of ptm97-1 BC1F2 still survived (2-3 days after treatment). The normal nutrient solution was restored for 4 days. Genomic DNA was extracted from 30 surviving and best-growing seedlings selected from the BC1F2 segregating population of ptm97-1. Genomic DNA from each of these 30 samples was then mixed in equal volumes. Genome resequencing and analysis were performed using the MutMap method (Abe, A., Kosugi, S., Yoshida, K., Natsume, S., Takagi, H., Kanzaki, H., Matsumura, H., Yoshida, K., Mitsuoka, C., Tamiru, M., Innan, H., Cano, L., Kamoun, S., and Terauchi, R. (2012). Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 30, 174-178) (Pasenno Biotechnology Co., Ltd.). Candidate mutation sites involved in regulating PEG resistance were obtained and further validated using first-generation sequencing. The results showed that the mutant ptm97-1 had a G->A mutation at the Chr2:27592162 site on chromosome 2, and the gene containing this mutation site was annotated as LOC_Os02g45380(OsMATE6). In the coding region of the LOC_Os02g45380 / OsMATE6 gene (the nucleotide sequence of the coding region of the LOC_Os02g45380 / OsMATE6 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2), guanine at position 1000 was mutated to adenine (G1000A), resulting in a glycine at position 334 of the encoded protein being arginine (G334R). Figure 4As shown in Figure A, this enhanced the ptm97-1 mutant's resistance to PEG. Additionally, we obtained two knockout mutants, osmate6-1 and osmate6-2, in the ZH11 background (provided by Professor Li Shigui's research group at Sichuan Agricultural University). The construction of these knockout mutants was completed by Baige Gene Technology (Jiangsu) Co., Ltd. The steps included using the company's high-throughput CRISPR-Cas9 target design program to design targets in the CDS region of OsMATE6, selecting targets with high scores, low off-target rates, and suitable locations as the final targets. The target sequences were constructed into the BGK03 vector, and then transformed into ZH11 via Agrobacterium-mediated genetic transformation. Subsequent resistance screening and target sequencing yielded T0 generation positive seedlings. The specific implementation method for obtaining mutants is described in the reference (Lu, Y., Ye, X., Guo, R., Huang, J., Wang, W., Tang, J., Tan, L., Zhu, JK, Chu, C., and Qian, Y. (2017). Genome-wide Targeted Mutagenesis in Rice Using the CRISPR / Cas9 System. Mol Plant 10, 1242-1245). First-generation sequencing confirmed that osmate6-1 has a 1 bp deletion at position 476 bp in the CDS, and osmate6-2 has a 57 bp deletion at positions 458-514 bp in the CDS. Figure 4 B).

[0055] Example 5: Drought-resistant phenotype of OsMATE6 mutant seedlings

[0056] The hydroponic cultivation of wild-type (ZH11) and OsMATE6 knockout mutants (osmate6-1 and osmate6-2) seedlings was as described in the "Seedling Hydroponic Experiment" section of Example 2. After 12 days of hydroponics in normal nutrient solution, the seedlings were treated with 25% PEG4000 (prepared with Yoshida nutrient solution). Leaf curling and gradual drying symptoms were observed within hours of treatment. After a significant difference in drying symptoms between the control ZH11 and the knockout mutants osmate6-1 / osmate6-2 (approximately 2 days), normal nutrient solution was restored for 3 days, and survival rates were recorded. The experimental results show that after 25% PEG4000 treatment, the leaves of the knockout mutants osmate6-1 / osmate6-2 lost water and dried out more slowly, and their survival rate after restoring normal nutrient solution was significantly higher than that of the control ZH11. Figure 5 AB).

[0057] The hydroponic cultivation of wild-type (ZH11) and OsMATE6 knockout mutants (osmate6-1 and osmate6-2) seedlings was as described in the "Seedling Soil Drought Experiment" section of Example 2. The results showed that after soil drought treatment during the seedling stage, the mutants osmate6-1 / osmate6-2 exhibited leaf curling later than the control ZH11, with less severe leaf curling and slower leaf drying. Their survival rate after rehydration was significantly higher than that of the control ZH11. Figure 5 CF).

[0058] Example 6: Salt-tolerant phenotype of OsMATE6 mutant seedlings

[0059] The hydroponic cultivation of wild-type (ZH11) and OsMATE6 knockout mutants (osmate6-1 and osmate6-2) seedlings was as described in the "Seedling Hydroponic Experiment" section of Example 2. After 12 days of hydroponics in normal nutrient solution, the seedlings were treated with 150 mM NaCl (prepared with Yoshida nutrient solution). Several hours after treatment, leaf curling and gradual drying symptoms were observed. After approximately 3 days (when the drying symptoms of the control ZH11 and the knockout mutants osmate6-1 / osmate6-2 showed a significant difference), the normal nutrient solution was restored for 2 days, and the survival rate was recorded. The experimental results showed that after treatment with 150 mM NaCl, the leaves of the knockout mutants osmate6-1 / osmate6-2 lost water and dried out more slowly, and their survival rate after restoring normal nutrient solution was significantly higher than that of the control ZH11. Figure 6 AB).

[0060] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0061] sequence

[0062] SEQ ID NO:1 OsMATE6 nucleotide sequence

[0063]

[0064] SEQ ID NO: 2 Amino acid sequence of OsMATE6

[0065] MSSPRRDGRGAVDDLTASLLHKGDGGEAVFVVVVVPPVAEEEEPPPVLTCKPPGRFARAVKEAWSVPFPMMPSMSAGAAGAEARSILGLALPMILTGLLLYLRSMISMLFLGRLGGLALAGGSLAIGFANITGYSVLSGLAMGMEPICGQAFGAGHYDLLGVTMQRTVLLLVAASVPIAGLWVHMRPLLLLCGQDAAIAAVAETYILASLPDLLLQAFLHPVRIYLRTQSINLPLTVCAALAIALHLPINYVAVSVLGLGIKGVALASVLANLNLVLFLFGYIWFKGVHKRTGGFALSADCLRGWGELVSLALPSCISVCLEWWWYEIMILLCGLLANPQATVASMGILIQTTSLIYIFPSSLGFGVSTRVSNELGANRPERACRAATVGLMLGFAFGGVASAFACHVRGAWATMFTADPAIVALTASVLPILGACELGNCPQTTGCGVLRGSARPKDAASINLRSFYLVGTPVALILAFWYHYDFRGLWLGLLAAQATCVVRMLLVIGETDWTAEAKRAQQLTGAADIKDCGGKGDHVAVIEQPDEQC。

Claims

1. A method for cultivating rice plants that are drought-resistant and / or salt-tolerant compared to wild-type rice, comprising reducing the expression level of the OsMATE6 gene in the rice plant, wherein the nucleotide sequence of the coding region of the OsMATE6 gene is shown in SEQ ID NO:

1.

2. The method according to claim 1, wherein the reduction is gene knockout.

3. The method of claim 1, wherein the reduction is achieved by CRISPR-Cas9, zinc finger nucleases (ZFNs), TALENs, or RNAi gene silencing technology.

4. The method according to claim 2, wherein gene knockout is achieved by a 1 bp deletion at the 476 bp position of the nucleotide sequence of the coding region of the OsMATE6 gene.

5. The method according to claim 2, wherein gene knockout is achieved by a 57 bp deletion at position 458-514 bp of the nucleotide sequence of the coding region of the OsMATE6 gene.

6. A method of breeding a rice plant having drought resistance and / or salt tolerance as compared to a wild type rice, characterized in that, This is achieved by mutating guanine to adenine at position 1000 of the coding region of the OsMATE6 gene in the rice plant, as shown in SEQ ID NO:

1.

7. The method according to any one of claims 1-6, wherein the rice is Longjing 31 or ZH11.

Images