Rice cold and salt tolerance regulation gene sop17 and application thereof

Abstract

This invention belongs to the field of rice molecular breeding technology and discloses genes regulating cold and salt tolerance in rice seedlings. SOP17 Its applications. This gene has the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. This invention obtained a seedling-stage cold-resistant mutant of indica rice 9311 through EMS mutagenesis. sop17-1 The clone was obtained by analyzing Mutmap. SOP17 Genes. Constructed using CRISPR / Cas9 gene editing technology. SOP17 Gene knockout vector, to obtain SOP17 Homozygous mutant lines. Functional validation showed that... SOP17 SOP17 This gene simultaneously negatively regulates cold and salt tolerance in rice seedlings. Knocking out this gene significantly enhances the tolerance of rice seedlings to low temperature (8°C) and salt (150mM NaCl). This invention provides new gene resources and molecular targets for breeding stress-resistant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding technology, specifically relating to genes regulating cold and salt tolerance in rice seedlings. SOP17 And its applications. Background Technology

[0002] Rice originated in tropical and subtropical regions and is a warm-climate crop, extremely sensitive to low temperatures. Low temperatures are one of the important environmental factors limiting rice production and affecting its geographical distribution. In rice production, with rising planting costs and changes in planting methods, direct seeding has become the main development direction for rice cultivation, replacing transplanting. However, direct seeding often encounters cold snaps in spring, leading to widespread seedling death and reduced yields. Therefore, improving rice's cold tolerance and cultivating new cold-resistant rice varieties are key to addressing the impact of low temperatures on rice production.

[0003] Low-temperature tolerance in rice is a complex quantitative trait with a complex genetic basis and is regulated by multiple genes, making it extremely difficult to study. Currently, only 5 finely mapped seedling-stage low-temperature tolerance QTLs have been identified in rice. qCTS4, qCtss11, qSCT1, qSCT11 and qLOP2 / qPSR2-1 The number of cloned and functionally identified cold-resistant genes for seedlings is still very limited, including... COLD1, qCTS-9 GSTZ2, RGA4L, ALA4, COLD6, CTF1, CTP-1, SOP10, COLD8 Etc. Overexpression COLD1 The gene significantly enhanced the cold resistance of rice. qCTS-9 The overexpression lines showed significantly enhanced tolerance to low temperatures. OsGSTZ2 Phenylalanine at position 99 plays an important role in improving the low-temperature tolerance of rice seedlings. RGA4L The protein encodes an NLR protein that specifically regulates abiotic stress. By interacting with OsHSP90 and OsLEA5, it positively regulates cold tolerance during the seedling, tillering, and flowering stages of rice without affecting yield. OsALA4 Encoding a phospholipid invertase, COLD6 regulates intracellular homeostasis by modulating cell membrane fluidity, thus modulating the cold tolerance of rice seedlings. COLD6 interacts with the G protein α subunit RGA1, negatively regulating cold tolerance through the 2′,3′-cAMP signaling pathway; its knockout or natural mutation can enhance cold tolerance. CTP-1 It is a recessive cold-resistant gene discovered in the tropical japonica rice variety Sileawah. COLD8 Encoding chloroplast membrane triglyceride lipase, this gene negatively regulates seedling cold tolerance by modulating the accumulation of unsaturated fatty acids. Although the aforementioned cold tolerance genes have been cloned and functionally verified, the genetic resources available for genetic improvement of cold-tolerant rice remain limited, far from meeting production needs.

[0004] Currently, the total area of ​​saline-alkali soil worldwide is over 1 billion hectares, and it is increasing at a rate of 1 to 1.5 million hectares annually. Therefore, breeding salt-tolerant crops and effectively utilizing saline-alkali land resources is an important new direction for improving crop productivity in my country, and it also aligns with the strategy of "shifting from improving plants adapted to saline-alkali land to breeding salt-tolerant plants adapted to saline-alkali land." Domestic and international scholars have conducted extensive research on salt-tolerant rice, and cloning salt-tolerant genes in rice will help accelerate the breeding of salt-tolerant rice. Existing research has found... OsMDH1 Mutations increase intracellular vitamin B6 content in rice cells, thereby improving rice's salt tolerance. Recent studies have identified a novel AP2 family transcription factor, OsSAE1, which directly binds to the promoter of OsABI5, a key gene in the ABA signaling pathway, and inhibits its expression, thus promoting rice seed germination and improving the salt tolerance of rice seedlings. OsRMT1, an E3 ubiquitin ligase, degrades the OsMBL1 protein through ubiquitination, forming a negative regulatory module for salt tolerance. SIT2, a receptor-like kinase, negatively regulates rice salt tolerance; its overexpression increases plant sensitivity to salt. STRK1 positively regulates rice salt tolerance and has been reported by the National Salt-Tolerant Rice Technology Innovation Center as a key gene that can significantly improve rice salt tolerance. Despite these advancements, the genetic resources available for the genetic improvement of salt-tolerant rice remain insufficient, hindering the development of new salt-tolerant rice varieties.

[0005] It is particularly important to note that there is currently a lack of rice germplasm resources that simultaneously possess both cold and salt tolerance characteristics in production. In actual rice production, low temperature and salt stress often coexist or alternate, such as when early rice is directly sown and encounters a late spring frost while the soil salinity is high. Therefore, it is urgent to create new genetic resources that simultaneously improve rice's cold and salt tolerance in order to enhance rice's overall resistance to multiple adversities and achieve the goals of stable yield and stress resistance.

[0006] With the rapid development of gene editing technology, especially the widespread application of the CRISPR / Cas9 system, a powerful tool has been provided for the precise improvement of crop stress resistance traits. By knocking out or causing loss-of-function mutations in target genes through gene editing technology, superior germplasm resources can be created in a short period of time, accelerating the breeding of new stress-resistant rice varieties. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a gene that simultaneously negatively regulates cold and salt tolerance in rice, along with its applications. This invention experimentally identifies and confirms that this gene in rice... SOP17 Deletion or inactivation of genes can significantly enhance both cold and salt tolerance in rice.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for simultaneously improving the cold and salt tolerance of rice, by using a CRISPR / Cas9 gene editing system to knock out the gene in rice. SOP17 The gene improves the cold and salt tolerance of rice; the nucleotide sequence of the SOP17 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the aforementioned SOP17 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0010] Furthermore, the method of using the CRISPR / Cas9 gene editing system to knock out the gene in rice... SOP17 Gene editing was achieved by constructing CRISPR / Cas9 gene editing vectors.

[0011] Furthermore, the CRISPR / Cas9 gene editing vector contains a target for the gene editing vector. SOP17 The target sequence of the gene, wherein the target sequence is the nucleotide sequence shown in SEQ ID NO.5.

[0012] Furthermore, the CRISPR / Cas9 gene editing vector also contains a U6a promoter-driven sgRNA expression cassette.

[0013] Secondly, the present invention also provides a method for knocking out rice SOP17 Applications of genes, wherein the application is any of the following: A1) Application in simultaneously improving the cold and salt tolerance of rice; A2) Application in the preparation of cold- and salt-tolerant rice; The SOP17 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Furthermore, the knockout of rice SOP17 Gene editing is achieved through the CRISPR / Cas9 gene editing system.

[0015] Thirdly, the present invention also provides a method for knocking out rice SOP17 Applications of gene-related biomaterials, wherein the application is any of the following: B1) Application in simultaneously improving the cold and salt tolerance of rice; B2) Application in the preparation of cold- and salt-tolerant rice; The biomaterial is any one of the following C1) to C2): C1) contains the aforementioned gene editing vector; C2) Recombinant Agrobacterium containing the above-mentioned gene-editing vector; The SOP17The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses and verifies SOP17 This gene is a key gene that negatively regulates cold and salt tolerance in rice seedlings. Loss-of-function mutations can significantly enhance the tolerance of rice seedlings to low temperatures and salt, providing a novel gene target for genetic improvement of rice stress resistance. The obtained... SOP17 Loss-of-function mutants exhibit stronger tolerance (such as higher survival rate) under low temperature and salt stress conditions, and can be directly used as excellent germplasm resources for molecular design breeding of cold- and salt-tolerant rice varieties, with high application value. Attached Figure Description

[0017] Figure 1 For the present invention sop17-1 Mutant screening flowchart.

[0018] Figure 2 This is a flowchart of the technical process of the present invention, showing the route of SOP17 gene cloning.

[0019] Figure 3 This describes the SOP17 gene location and SOP17 gene structure of the present invention. Figure 3 'a' represents a schematic diagram illustrating the location of the SOP17 gene using the Mutmap method. Figure 3 b represents the structure diagram of the SOP17 gene and SOP17 protein.

[0020] Figure 4 Created using gene editing methods in this invention SOP17 Information on homozygous mutant strains. This includes... SOP17 The location of gene editing targets on the genome, and SOP17 Sequence of a homozygous mutant strain.

[0021] Figure 5 For 9311 and SOP17 Results of cold tolerance assessment of seedlings of homozygous mutant strains. Figure 5 'a' indicates the phenotype after low-temperature treatment at 8°C. Figure 5 b represents the survival rate after treatment at 8°C.

[0022] Figure 6 For 9311 and SOP17 Results of salt tolerance identification of seedling homozygous mutant lines. Figure 6 'a' represents the phenotype of seedlings grown under normal conditions for 12 days before salt treatment. Figure 6 b represents the phenotype after 7 days of treatment with 150 mM NaCl followed by 5 days of recovery. Figure 6c. Survival rate statistics of seedlings after 7 days of treatment with 150 mM NaCl and 5 days of recovery. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0024] Figure 1 This is a flowchart of the mutant screening process of the present invention. Figure 2 This is a flowchart illustrating the technical process of the present invention. SOP17 The technical approach to gene cloning.

[0025] Example 1: Mutant sop17-1 The acquisition Take 10,000 rice 9311 seeds and soak them in tap water at room temperature for 12 hours, gently shaking them every 2 hours to ensure they fully absorb water. After absorbing water, rinse them with tap water, place them in a petri dish lined with moistened filter paper, let them air dry at room temperature for 6 hours, and then soak them for another 12 hours.

[0026] After draining the treated seeds, place them in a 0.75% EMS (ethyl methanesulfonate) solution and treat at room temperature for 12 hours. Gently shake the seeds several times during treatment to ensure thorough contact between the seeds and the EMS solution. After mutagenesis treatment, rinse the seeds six times with tap water for 10 minutes each time to remove any residual EMS from the seed surface. Finally, rinse with running water for 1.5 hours to remove any remaining EMS, obtaining the mutagenized M0 seeds.

[0027] The mutagenized M0 seeds were sown in May 2015 at the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, in Changping District, Beijing. Twenty-five days after sowing, the seedlings were transplanted and managed normally in the field. Before heading, the rice plants were isolated with plastic film to prevent cross-pollination. In October 2015, approximately 4,000 well-developed M1 individual seeds were harvested and stored in separate bags.

[0028] In December 2015, approximately 4,000 seeds of the M1 family were sown at the experimental base in Lingshui, Hainan, and in May 2016, seeds of more than 3,700 M2 families were harvested.

[0029] The harvested M2 family seeds were sown in an artificial climate chamber, with 20 seedlings planted per family. After 3 weeks of growth under normal conditions (14 hours light (28°C) / 10 hours darkness (24°C)), a low-temperature treatment was administered at 8°C for 48 hours. Following treatment, the seedlings were transferred to normal growing conditions for 5 days to recover. By observing the growth phenotypes and survival rates of different materials after treatment, a low-temperature resistant mutant was screened and named [name missing]. sop17-1 .

[0030] Example 2: Candidate Genes SOP17 Location and Cloning The results obtained in Example 1 sop17-1 The mutant was crossed with the wild-type 9311 to obtain F1 seeds. After sowing, the F1 plants were self-pollinated to harvest the F2 population. Cold tolerance was assessed in the F2 population, and the results showed a cold tolerance segregation ratio of 168 cold-intolerant plants to 55 cold-tolerant plants (χ²). 2 =0.694), which meets the separation ratio of 3:1, indicating that sop17-1 It is a recessive mutant controlled by a single gene.

[0031] Leaves from 25 cold-tolerant individual plants were mixed in equal amounts to obtain a pool, and genomic DNA was extracted from the pool. Genome resequencing was then performed. Mutmap analysis revealed a significant SNP-index peak on chromosome 10. Figure 3 a) The peak region is located in the 11.61-19.74 Mb region of chromosome 10. Comparison with the Rice MSU database (http: / / rice.uga.edu / ) revealed a SNP mutation in the second exon of the LOC_Os10g32970 gene within this region (the G base at 1494 bp in SEQ ID NO.1 was mutated to an A base). This SNP mutation caused the codon encoding the 109th amino acid in the LOC_Os10g32970 gene to change from TGG to TAG, resulting in premature termination of tryptophan at position 109 (W109*, Figure 4 (b) in the middle.

[0032] SOP17 The gene (LOC_Os10g32970) encodes a translation initiation factor (eIF4E-2), which has 5 exons and 4 introns, with a coding region length of 2690 bp. Figure 3 b). Using genomic DNA from 9311 as a template, amplification was performed using primers CF8443 (5'-GGTCGTAGCCCAGGTGAAA-3', SEQ ID NO.3) and CF8446 (5'-AACACTAGCTGAGGCTGTAAAGAA-3', SEQ ID NO.4). SOP17 The genome sequence was obtained and ligated into the pEasy-Blunt-Zero-Cloning Kit vector (TransGen Biotech Ltd., CB501-01) for first-generation sequencing to obtain... SOP17 The exact sequence (SEQ ID NO.1). The SOP17 protein (SEQ ID NO.2) encoded by this gene has typical domain features of eIF4E family proteins.

[0033] Example 3: SOP17Obtaining gene-edited lines Obtained in Example 2 SOP17 Using the precise sequence as a template, CRISPR target design was performed using the http: / / crispr.dbcls.jp database, and a single-target gene editing vector driven by the U6a promoter was constructed. The corresponding target sequence (SEQ ID NO.5) is shown in Table 1. The construction process is as follows: First, a pair of complementary oligonucleotide chains were designed and synthesized targeting the target, and double-stranded DNA adapters with sticky ends were formed by annealing. 1 μg of the pYLsgRNA intermediate vector plasmid carrying the U6a promoter was digested with restriction endonuclease BsaI at 37℃ for 20 min, followed by inactivation at 70℃ for 10 min. The annealed double-stranded adapter was then ligated to the digested intermediate vector containing the promoter. Using the ligation product as a template, the promoter-driven sgRNA expression cassette was amplified using the corresponding primer pair. The resulting PCR product was recovered and purified, then digested with BsaI at 37℃ for 30 min and inactivated at 75℃ for 5 min. The digested sgRNA expression cassette was mixed with the BsaI-linearized pYLCRISPR / Cas9-MH backbone vector at a molar ratio of 3:1, and T4 DNA ligase was added for ligation at 20℃ for 2-4 h. 5-10 μL of the ligation product was transformed into *E. coli* DH5α competent cells and plated on a substrate containing 50 μL of... After incubating kanamycin on LB agar plates at 37°C overnight, single colonies were picked and sequenced for identification. Positive clones with correct sequencing were considered successfully constructed gene editing vectors.

[0034] Table 1

[0035] The constructed CRISPR vector was transferred into Agrobacterium tumefaciens strain EHA105 via electroporation, and positive transgenic single plants were obtained by infecting callus tissue of 9311. Transgenic T0 generation plants were planted at the Lingshui base in Hainan. Positive transgenic plants were obtained by detecting the T0 generation material using CF2601 (5'-CCCGACATAGATGCAATAACTTC-3', SEQ ID NO.7) and CF5304 (5'-CTTTGTTGTCGTCCAATTGC-3', SEQ ID NO.8). Simultaneously, using the genomic DNA of the positive transgenic plants as a template, DNA fragments including the editing site were amplified and sequenced using primers CF5032 (5'-AAGCCGAAACCTTCTTCCC-3', SEQ ID NO.9) and CF5033 (5'-CCAGACAATCCCTAAACATCAGTA-3', SEQ ID NO.10) for screening. SOP17 Harvesting T1 generation seeds from homozygous mutant material.

[0036] In the T1 generation plants, screening was conducted to identify those that did not contain the Cas9 transgene and SOP17 Plants with homozygous gene mutations were selected, and their T2 generation seeds were harvested. Two plants were confirmed to have been obtained. SOP17 The homozygous mutant strains were named as follows: SOP17 CR #1 and SOP17 CR #2, both involved base deletions at the sgRNA1 target site, among which SOP17 CR #1 is a 4bp deletion of bases 22 bp downstream of the start codon ATG (the four ATTG bases from 224 bp to 227 bp in SEQ ID NO.1). SOP17 CR #2 involves a 1 bp deletion 24 bp downstream of the start codon ATG (the 227 bp T base in SEQ ID NO.1). Figure 4 This leads to a frameshift mutation.

[0037] Example 4: SOP17 Identification of cold tolerance in homozygous mutant strains The two obtained in Example 3 SOP17 Homozygous mutant strains ( SOP17 CR #1 and SOP17 CR #2) and wild-type 9311 were planted in an artificial climate chamber under normal growing conditions of 14 hours of light (28°C) / 10 hours of darkness (24°C). After 3 weeks of growth, the seedlings were transferred to an 8°C artificial climate chamber for 48 hours. After the treatment, the seedlings were transferred back to normal conditions to resume growth.

[0038] Observe the phenotype of seedlings after low-temperature treatment and calculate the survival rate. For example... Figure 5 As shown, SOP17 CR ( SOP17 CR #1 and SOP17 CR #2) After treatment at 8°C, its survival rate was significantly higher than that of wild-type 9311 (P<0.01), proving that SOP17 Genes negatively regulate cold tolerance in rice seedlings, and loss-of-function mutations can enhance cold tolerance in rice seedlings. Simultaneously, cold tolerance gene mutants previously discovered in the laboratory... sop10 As a control, the results showed that SOP17 CR Survival rate higher after low-temperature treatment sop10 ,illustrate SOP17 Comparison SOP10 It has stronger cold resistance.

[0039] Example 5: SOP17 Salt tolerance identification of homozygous mutant lines The two obtained in Example 3 SOP17 Homozygous mutant strains ( SOP17 CR #1 and SOP17 CR #2) and wild-type 9311 were planted in hydroponic containers filled with Kimura medium (Coolaber, NS1050). The hydroponic containers were placed in an artificial climate chamber with growth conditions of 14 hours light (28°C) / 10 hours darkness (24°C). The Kimura medium was changed every 3 days. After 10 days of growth, sodium chloride (final concentration 150 mM, Sigma, 401676) was added to the Kimura medium. After 7 days of salt treatment, the seedling roots were rinsed with water, and the medium was replaced with normal Kimura medium. The seedlings were cultured for another 5 days. The survival rate of each treatment was recorded.

[0040] like Figure 6 As shown, SOP17 Homozygous mutant strains ( SOP17 CR #1 and SOP17 CR #2) After 7 days of growth in 150 mM sodium chloride culture medium, its survival rate reached 58%, significantly higher than the 7% of the wild-type 9311. The results indicate that... SOP17 Gene mutations significantly enhance rice's salt tolerance, proving SOP17 It also participates in the regulation of cold and salt tolerance in rice seedlings.

[0041] In summary, this invention has isolated and cloned a gene that regulates cold and salt tolerance in rice seedlings. SOP17 This invention provides new genes and germplasm resources for breeding cold- and salt-tolerant rice, and is of great significance to the field of rice molecular breeding.

Claims

1. A method for simultaneously improving the cold tolerance and salt tolerance of rice, characterized in that, Using the CRISPR / Cas9 gene editing system to knock out rice SOP17 Genes that enhance the cold and salt tolerance of rice; SOP17 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The SOP17 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The method of using the CRISPR / Cas9 gene editing system to knock out rice SOP17 Gene editing was achieved by constructing CRISPR / Cas9 gene editing vectors.

4. The method according to claim 3, characterized in that, The CRISPR / Cas9 gene editing vector contains a target for the... SOP17 The target sequence of the gene is the nucleotide sequence shown in SEQ ID NO.5; the CRISPR / Cas9 gene editing vector also contains an sgRNA expression cassette driven by the U6a promoter.

5. Knock out the rice SOP17 The application of genes is characterized by, The application is any one of the following: A1) Application in simultaneously improving the cold and salt tolerance of rice; A2) Application in the preparation of cold- and salt-tolerant rice; The SOP17 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, The knockout of rice SOP17 Gene editing is achieved through the CRISPR / Cas9 gene editing system.

7. Knock out rice SOP17 The application of gene-related biomaterials is characterized by, The application is any one of the following: B1) Application in simultaneously improving the cold and salt tolerance of rice; B2) Application in the preparation of cold- and salt-tolerant rice; The biomaterial is any one of the following C1) to C2): C1) contains the gene editing vector as described in claim 4; C2) contains recombinant Agrobacterium tumefaciens containing the gene-editing vector in C1); The SOP17 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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