Application of OsSRT1 gene in creating new germplasm of direct seeding rice

Abstract

This invention discloses OsSRT1 The application of genes in creating new germplasm suitable for direct-seeding rice involves the field of plant genetic engineering technology. OsSRT1 The nucleotide sequence of the gene is shown in SEQ ID NO:1. A breeding method for direct-seeded rice with high germination and emergence rates includes: knocking out the gene using CRISPR / Cas9 gene editing technology. OsSRT1 Genes. This invention discloses for the first time... OsSRT1 The gene has a negative regulatory effect on rice seed germination rate and direct seeding rate; knocking out OsSRT1 The partial exon sequences of the gene can significantly improve the germination rate and direct seeding rate of rice seeds. This invention not only provides a novel molecular target for breeding direct-seeded rice with high germination and seedling emergence rates, but also provides a theoretical basis and technical support for the breeding of new germplasm suitable for direct-seeded rice. At the same time, it can effectively solve the problem of missing seedlings and broken rows in production, and provide a guarantee for achieving increased and stable rice yields.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering and molecular breeding technology, specifically to... OsSRT1 Application of genes in creating new germplasm suitable for direct-seeded rice. Background Technology

[0002] Rice, as the world's most important staple food crop, is a vital food source for more than half of the world's population. Rapid and uniform seed germination is essential for robust seedling growth. Seeds with rapid and uniform germination characteristics are called high-vitality seeds, which are crucial for cultivating strong seedlings, enhancing stress tolerance, and achieving high yields. Meanwhile, direct-seeding rice cultivation has seen its application area expanding year by year in southern rice-growing regions due to its advantages such as labor saving, water conservation, and high efficiency. However, under direct-seeding conditions, seeds are located in deep soil, creating a relatively harsh germination environment, resulting in low emergence rates, which has become a key bottleneck restricting the widespread adoption of this technology. Therefore, there is an urgent need to cultivate a rice variety with excellent germination ability, capable of achieving rapid and uniform germination and emergence under field conditions.

[0003] Seed germination is a crucial step in the establishment of direct-seeded rice, and is finely regulated by the balance of endogenous hormones (abscisic acid ABA and gibberellin GA) and epigenetic modifications. Histone deacetylases (HDACs) regulate the expression of germination-related genes by removing acetyl groups from histone lysine residues, thereby altering chromatin structure. OsSRT1 The gene is a member of the rice histone deacetylase SIR2 family, but regarding OsSRT1 The role of this gene in regulating seed germination, ABA / GA metabolism, and direct seeding has not been reported, and there are no application examples of using this gene to create new germplasm suitable for direct seeding rice.

[0004] Gene editing technology centered on the CRISPR / Cas9 system has become an important tool in plant functional genomics research due to its advantages such as ease of operation, high editing efficiency, and strong specificity. Originating from the prokaryotic immune system, this technology uses sgRNA to guide the Cas9 enzyme to precisely cut target DNA, relying on cellular repair mechanisms to achieve gene knockout or replacement, significantly improving the precision of gene regulation. Currently, this technology is widely used in gene function verification and crop genetic improvement, providing an efficient solution for elucidating plant growth and development mechanisms and breeding high-yielding, high-quality, and stress-resistant crop varieties. Summary of the Invention

[0005] This invention aims to provide OsSRT1The application of genes in creating new rice germplasm suitable for direct seeding involves deleting the function of a gene to improve seed germination rate, direct seeding emergence rate, and early seedling growth vigor, thereby creating new rice germplasm suitable for direct seeding cultivation. This solves the technical problems of low emergence rate and uneven emergence in existing direct seeding rice, and provides new gene resources and technical approaches for the genetic improvement of related traits in creating new rice germplasm suitable for direct seeding.

[0006] Therefore, in a first aspect, the present invention provides OsSRT1 The application of genes in creating new germplasm suitable for direct-seeding rice, the aforementioned OsSRT1 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] Furthermore, the aforementioned OsSRT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:3.

[0008] Furthermore, the application is achieved by knocking out the rice's... OsSRT1 Genes can improve the germination rate of rice seeds and / or the seedling emergence rate after direct seeding.

[0009] Furthermore, the knockout of rice OsSRT1 Genes can reduce the ratio of abscisic acid to gibberellin in rice seeds, thereby increasing the germination rate and / or direct seeding rate of rice seeds.

[0010] A second aspect of the present invention provides a breeding method for direct-seeded rice with high germination and high emergence rates, the method comprising: knocking out the gene in the application using CRISPR / Cas9 gene editing technology. OsSRT1 Gene.

[0011] Furthermore, the knockout OsSRT1 Gene-based methods include: designing the... OsSRT1 The target site for CRISPR / Cas9 gene editing knockout was identified; a recombinant vector was constructed based on the target site; the recombinant vector was used to infect callus tissue of recipient rice using Agrobacterium-mediated transformation to obtain mutant seed material; the mutant seed material was planted, and successful knockout of the target site was screened. OsSRT1 Homozygous mutant strains of the gene.

[0012] Furthermore, the nucleotide sequence of the target is shown in SEQ ID NO:4 and SEQ ID NO:5.

[0013] Furthermore, the homozygous mutant line described above... OsSRT1 The mutation types of the gene include: the deletion of an A base at the 16th nucleotide of the target site shown in SEQ ID NO:4; or the insertion of a T base between the 15th and 16th nucleotides of the target site shown in SEQ ID NO:5.

[0014] Furthermore, the screening includes using primers to screen plants grown from the seeds of the mutant material. OsSRT1 The gene was amplified by PCR and then detected.

[0015] Furthermore, the primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO:6 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:7.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention discloses for the first time OsSRT1 Genes and their encoded proteins can regulate rice seed germination and direct seeding, and provide a method for constructing... OsSRT1 Gene knockout mutant vectors were transformed into japonica rice varieties, and successful results were obtained. OsSRT1 Methods for developing gene mutant plants. Experimental results show that... OsSRT1 The gene has a negative regulatory effect on rice seed germination rate and direct seeding rate; knocking out OsSRT1 The partial exon sequences of the gene can significantly improve the germination rate and direct seeding rate of rice seeds. This invention not only provides a novel molecular target for breeding direct-seeded rice with high germination and seeding rates, but also provides a theoretical basis and technical support for the breeding of new germplasm suitable for direct-seeded rice. At the same time, it can effectively solve the problem of missing seedlings and broken rows in production, save seed costs, and provide a guarantee for achieving increased and stable rice yields. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1 Provided for embodiments of the present invention OsSRT1 Gene knockout target design and mutant material identification diagram; Figure 2 Wild-type Nipponbare (NIP) and the wild-type Nipponbare (NIP) provided for embodiments of the present invention OsSRT1 Gene mutant materials ( ossrt1-1 and ossrt1-2 Plumule phenotype of the plant 96 hours after seed germination experiment; Figure 3 Wild-type Nipponbare (NIP) and the wild-type Nipponbare (NIP) provided for embodiments of the present invention OsSRT1 Gene mutant materials ( ossrt1-1 and ossrt1-2 The image shows the results of a seed germination experiment on the plant. Figure 3 A shows the length of the seed embryo after 96 hours. Figure 3 B shows the seed germination rate within 96 hours. Figure 4 Wild-type Nipponbare (NIP) and the wild-type Nipponbare (NIP) provided for embodiments of the present invention OsSRT1 Gene mutant materials ( ossrt1-1 and ossrt1-2 Phenotypic images of seedling emergence from simulated direct seeding experiments of plants, among which, Figure 4 A shows the phenotypic diagram of seedling emergence on day 11. Figure 4 B shows the seedling phenotype on day 14; Figure 5 Wild-type Nipponbare (NIP) and the wild-type Nipponbare (NIP) provided for embodiments of the present invention OsSRT1 Gene mutant materials ( ossrt1-1 and ossrt1-2 The results of the simulated direct seeding experiment of the plants are shown in the figure. Figure 5 A is a chart showing the seedling growth on day 14. Figure 5 B shows the seed germination rate within 14 days; Figure 6 Wild-type Nipponbare (NIP) and the wild-type Nipponbare (NIP) provided for embodiments of the present invention OsSRT1 Gene mutant materials ( ossrt1-1 and ossrt1-2 A graph showing the ABA / GA ratio of seed embryo hormones in plants after 96 hours. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques and conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0020] A first aspect of the present invention provides OsSRT1 The application of genes in creating new germplasm suitable for direct-seeding rice. OsSRT1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, its CDS sequence is shown in SEQ ID NO:2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:3.

[0021] By knocking out rice OsSRT1 The gene can reduce the ratio of abscisic acid to gibberellin in rice seeds, thereby increasing the germination rate and / or direct seeding rate of rice seeds.

[0022] The embodiments of the present invention are studied OsSRT1The application of this gene in regulating rice seed germination has revealed that intervening in this gene can directionally improve the germination characteristics of rice seeds, increase seed germination rate, direct seeding rate, and early seedling vigor, solve the technical problems of low seedling emergence and uneven emergence in current direct seeding rice, provide a new molecular target for rice breeding, and provide new germplasm resources for breeding suitable direct seeding rice.

[0023] A second aspect of this invention provides a breeding method for direct-seeded rice with high germination and emergence rates, comprising: knocking out [a specific gene] using CRISPR / Cas9 gene editing technology. OsSRT1 Gene.

[0024] Because of the high efficiency and specificity of the CRISPR / Cas9 system, it can precisely target... OsSRT1 Gene editing significantly shortens the breeding cycle compared to traditional mutation breeding, and preserves the genetic background of the recipient's superior varieties to the greatest extent, effectively avoiding linkage burden problems.

[0025] In some embodiments, knockout OsSRT1 Genetic methods include: design OsSRT1 Targets for CRISPR / Cas9 gene editing knockout; Construct recombinant vectors based on targets; The recombinant vector was used to infect the callus tissue of recipient rice with Agrobacterium-mediated infection to obtain mutant seed material. Seeds of mutant materials were planted and successfully screened for knockout. OsSRT1 Homozygous mutant strains of the gene.

[0026] The method provided in this invention is simple to operate, has a short cycle time, and is highly efficient, precisely knocking out gene editing technology. OsSRT1 This method avoids the drawbacks of traditional breeding, such as long cycles and trait linkage, and can quickly obtain new rice materials with stable inheritance and high germination rates, which have broad application prospects and important production value.

[0027] The nucleotide sequences of the above targets are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0028] In some embodiments, the above homozygous mutant lines OsSRT1 Gene mutation types include: The A base is deleted at nucleotide position 16 of the target site shown in SEQ ID NO:4; or Insert a T base between the 15th and 16th nucleotides of the target site as shown in SEQ ID NO:5.

[0029] In some embodiments, screening includes screening plants grown from seeds of mutant material via primer pairs. OsSRT1 The gene was amplified by PCR and then detected.

[0030] In some embodiments, the primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO:6 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:7.

[0031] The recipient rice mentioned above is japonica rice, and optionally, the recipient rice is Nipponbare, derived from a common cultivar.

[0032] Japonica rice is an important subspecies of rice globally, but some varieties suffer from slow germination. This solution validates and optimizes Japonica rice, directly addressing practical pain points in its production and providing strong experimental evidence and technical reference for its application to indica rice or other gramineous crops.

[0033] Example 1 Rice OsSRT1 Gene structure analysis and gene editing target design rice OsSRT1 Gene structure analysis results as follows Figure 1 As shown, this gene contains 13 exons. gRNA target site sequences were designed on exons 8 and 12, allowing the Cas9 protein to be mutated at specific gene sequences to obtain two variants. OsSRT1 The mutant type of the gene is rice. The target sequence is shown in SEQ ID NO:4 and SEQ ID NO:5.

[0034] Example 2 Rice OsSRT1 Gene knockout mutation acquisition and homozygous mutant screening and sequencing 1. Vector construction and transformation: Based on the target design of Example 1, the corresponding sgRNA was synthesized, and then the obtained mutant vector was introduced into the callus tissue of wild-type rice variety by Agrobacterium-mediated transformation to obtain mutant material seeds.

[0035] 2. T1 generation plant culture, DNA extraction, and PCR amplification: T1 generation plants were obtained by planting mutant material seeds. DNA was extracted from leaves of the T1 generation plants and then... OsSRT1 The gene's nucleotide sequence was designed with forward primer sequence SEQ ID NO:6 and reverse primer SEQ ID NO:7. Specific amplification was achieved using a standard Taq polymerase PCR reaction. OsSRT1 The full-length segment of a gene.

[0036] 3. Sequencing and Alignment Analysis: The amplified fragments were sent to Zhejiang Shangya Company for sequencing. The sequencing results were aligned using SnapGene (v6.0.2) software.

[0037] 4. Mutant Screening and Identification: Two homozygous mutants with mutation sites located at the target site were finally screened out and named as follows: ossrt1-1 and ossrt1-2 , OsSRT1 A schematic diagram of the gene structure and target is shown below. Figure 1 As shown, where ossrt1-1 The A base is missing in exon 8. ossrt1-2 A T base was inserted into exon 12.

[0038] Example 3 Rice OsSRT1 Phenotypic analysis of gene knockout mutant lines in seed germination experiments 1. Seed pretreatment: Select plump and uniform seeds and an appropriate amount of wild-type Nipponbare seeds (NIP) from the homozygous T2 generation mutant seeds obtained in Example 2. After manually removing the husks, place the whole rice seeds in a 50℃ oven to dry for 5 days to reduce seed moisture content, reduce dormancy effects, and ensure relatively uniform germination.

[0039] 2. Disinfection and Cultivation: The dried, hulled rice seeds were soaked in 70% ethanol for 5 minutes each time, for a total of two washes. Then, they were rinsed twice with ultrapure water. The washed seeds were placed in round petri dishes lined with two layers of sterile filter paper. The petri dishes were then placed in an artificial climate incubator at 70% relative humidity and 26°C for cultivation in the dark.

[0040] 3. Trait Measurement and Statistics: Seed germination traits were measured and recorded at specific times, with at least 30 seeds measured. A plumule length ≥2mm was used as the germination standard. Germination rate was calculated at 96 hours after germination. Germination rate = (Number of germinated seeds on day n / Total number of seeds) × 100%. Each experimental group was repeated at least 3 times. Seed germination phenotypes were observed and photographed. The final seed germination plumule phenotype, sprout length, and germination rate results are shown below. Figure 2 and Figure 3 As shown.

[0041] 4. Conclusion Analysis: During the germination process, such as Figure 2 and Figure 3 As shown, ossrt1-1 and ossrt1-2 The embryo length is longer than that of the wild type; ossrt1-1 and ossrt1-2 The seed germination rates of the [type name] and [type name] were increased by 22.5% and 24.6% respectively compared to the wild type. This significant phenotypic difference confirms [the following]. OsSRT1 There is a direct causal relationship between gene knockout and increased germination rate. In rice, gene knockout... OsSRT1 Genes can improve the germination rate of rice seeds, i.e., rice OsSRT1 Genes negatively regulate rice seed germination rate.

[0042] Example 4 Rice OsSRT1 Phenotypic analysis of gene knockout mutant lines in simulated direct-seeding rice experiments 1. Seed pretreatment: Select plump and uniform seeds and an appropriate amount of wild-type Nipponbare seeds (NIP) from the homozygous T2 generation mutant seeds obtained in Example 2. Place the rice seeds in a 37°C oven for 48 hours to break dormancy and unify the initial moisture content of the seeds.

[0043] 2. Simulated direct seeding: Dry-treated seeds were sown in seedling trays filled with ordinary field soil at a depth of 3 cm. After sowing, the trays were placed in a walk-in artificial climate chamber with the following conditions: 30℃ during the light phase (14h) / 28℃ during the dark phase (10h). Appropriate water was added daily to ensure the soil remained moist.

[0044] 3. Phenotypic Measurement and Statistics: Emergence was recorded every 24 hours after sowing, with the embryo breaking through the soil layer to 2mm as the emergence standard. This was repeated for 14 consecutive days. After the experiment, 20 seedlings were randomly selected to measure their length, and the average value was used for statistical analysis. Each experimental group was repeated at least three times. The phenotype of the direct-seeded rice seedlings was observed and photographed. The final phenotypic chart, seedling length, and emergence rate of the direct-seeded rice seedlings are shown below. Figure 4 and Figure 5 As shown.

[0045] 4. Conclusion and Analysis: During the simulated live broadcast, if... Figure 4 and Figure 5 As shown, ossrt1-1 and ossrt1-2 The seedlings are longer than the wild type; ossrt1-1 and ossrt1-2 The seed germination rate of the [type] was increased by 26.1% and 24.9% respectively compared to the wild type. This significant phenotypic difference confirms [the following]. OsSRT1 There is a direct causal relationship between gene knockout and increased seedling emergence rate in rice. OsSRT1 Genes can improve the emergence rate of direct-seeded rice, i.e., rice OsSRT1 Negative gene regulation of rice seedling emergence rate in direct seeding.

[0046] Example 5 Rice OsSRT1 Gene knockout mutant line detection and ABA / GA analysis of germinating seed plumules 1. Seed pretreatment: Same as in Example 3.

[0047] 2. Disinfection and incubation: Same as in Example 3.

[0048] 3. Hormone Detection and Statistics: 96 hours after germination, seed embryos were harvested using a clean blade, and the levels of hormones ABA and GA within the embryos were measured. Each experimental group was repeated at least three times. The ABA / GA ratio of the measured results was calculated, and the final ABA / GA ratio of the seed embryos was obtained as follows: Figure 6 As shown.

[0049] 4. Conclusion Analysis: During the germination process, such as Figure 6 As shown, ossrt1-1 and ossrt1-2 The mutant lines resulted in a decreased ABA / GA ratio. This significant difference confirms... OsSRT1 Gene knockout can cause seeds to transition from a dormant state to a germination state, increasing seed germination rate and direct seeding rate, indicating that rice... OsSRT1 Genes negatively regulate rice seed germination rate and direct seeding rate.

[0050] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. OsSRT1 The application of genes in creating new germplasm suitable for direct-seeding rice is characterized by, The OsSRT1 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The OsSRT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

3.

3. The application according to claim 1, characterized in that, The application is achieved by knocking out the rice... OsSRT1 Genes can improve the germination rate of rice seeds and / or the seedling emergence rate after direct seeding.

4. The application according to claim 3, characterized in that, The knockout rice OsSRT1 Genes can reduce the ratio of abscisic acid to gibberellin in rice seeds, thereby increasing the germination rate and / or direct seeding rate of rice seeds.

5. A breeding method for direct-seeded rice with high germination and emergence rates, characterized in that, The method includes: knocking out the gene in the application of claim 1 using CRISPR / Cas9 gene editing technology. OsSRT1 Gene.

6. The method according to claim 5, characterized in that, The knockout OsSRT1 Genetic methods include: The design described OsSRT1 Targets for CRISPR / Cas9 gene editing knockout; Construct a recombinant vector based on the target; The recombinant vector was used to infect the callus tissue of recipient rice through Agrobacterium-mediated infection to obtain mutant seed material. Seeds of the mutant material were planted, and successful screening was performed to remove the mutant material. OsSRT1 Homozygous mutant strains of the gene.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the target is shown in SEQ ID NO:4 and SEQ ID NO:

5.

8. The method according to claim 6 or 7, characterized in that, The homozygous mutant line described OsSRT1 Gene mutation types include: The nucleotide at position 16 of the target site shown in SEQ ID NO:4 is missing an A base; or A T base is inserted between the 15th and 16th nucleotides of the target site as shown in SEQ ID NO:

5.

9. The method according to claim 6, characterized in that, The screening includes using primers to screen plants grown from the seeds of the mutant material. OsSRT1 The gene was amplified by PCR and then detected.

10. The method according to claim 9, characterized in that, The primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO:6 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:7.

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