Rice submergence tolerance gene ST4 and its application
By overexpressing the rice flood-resistant gene ST4 in rice plants, the problem of insufficient flood-resistant resistance during the germination period of rice seeds was solved, the germination rate and seedling rate were significantly improved, the adaptability of rice to the flooded environment was enhanced, and the foundation for flood-resistant rice breeding was provided.
Patent Information
- Application Number
- CN202510200385.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2045-02-24
AI Technical Summary
The prior art has challenges in improving the flood tolerance of rice, especially in the insufficient flood tolerance during the seed germination period, which affects rice breeding and production.
By overexpressing the rice flood-resistant gene ST4 in rice plants, the ST4 gene is introduced into the rice plants by recombinant vector technology to achieve its overexpression, thereby improving the germination rate and seedling rate of rice.
The germination rate and seedling rate of rice have been significantly improved, the adaptability of rice to flooded environment has been enhanced, and the foundation has been laid for cultivating new varieties of flood-resistant rice.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of gene engineering, in particular to a rice flood-tolerant gene ST4 and an application thereof. Background Art
[0002] The flatness of mechanized fields cannot meet the standards of nursery fields. The importance of flood resistance during the seed germination period has become prominent and is receiving increasing attention. Improving rice's flood resistance has become an important goal of rice breeding.
[0003] Rice flooding tolerance is a quantitative trait that is regulated by multiple genetic factors and the environment. The study of the genetic basis of rice flooding tolerance is still in its infancy. Many quantitative trait loci (QTLs) have been located and cloned, such as Sub1, CIPK15, OsTPP7, SK1 / 2, SnRK1A and OsUGT75A. Natural variation in these genes has been shown to enhance rice's adaptability to flooding. However, natural alleles with flooding tolerance are still rare. Dongxiang wild rice (DY) in Jiangxi Province has extremely strong flooding tolerance. Discovering new flooding tolerance genes has important application value for breeding rice varieties suitable for direct seeding and studying the molecular basis of flooding tolerance. Summary of the invention
[0004] The purpose of the present invention is to provide a rice flood-tolerant gene ST4 and its application. Overexpressing the rice flood-tolerant gene ST4 in rice plants can significantly improve the germination rate and seedling rate of rice, laying a foundation for cultivating flood-tolerant rice.
[0005] To achieve the above objectives, in a first aspect, the present invention provides a rice flooding tolerance gene ST4, the nucleotide sequence of the rice flooding tolerance gene ST4 is shown in SEQ ID NO.1, and the coding sequence of the rice flooding tolerance gene ST4 is shown in SEQ ID NO.2.
[0006] In a second aspect, the present invention provides a rice flooding tolerance protein, the amino acid sequence of the rice flooding tolerance protein is shown as SEQ ID NO.3, and the coding gene of the rice flooding tolerance protein is shown as SEQ ID NO.1.
[0007] In a third aspect, the present invention provides a recombinant vector comprising the rice flooding tolerance gene ST4.
[0008] Furthermore, the recombinant vector is a plant overexpression vector.
[0009] In a fourth aspect, the present invention provides the use of the rice flood tolerance gene ST4 or a recombinant vector comprising the rice flood tolerance gene ST4 in improving the flood tolerance of rice.
[0010] Furthermore, when used, the rice flood-tolerant gene ST4 or a recombinant vector containing the rice flood-tolerant gene ST4 is introduced into the rice plant, so that the rice flood-tolerant gene ST4 is overexpressed in the rice plant.
[0011] In a fifth aspect, the present invention provides the use of rice flooding tolerance protein in improving rice flooding tolerance.
[0012] In a sixth aspect, the present invention provides a method for breeding flood-tolerant rice, wherein the rice flood-tolerant gene ST4 or a recombinant vector comprising the rice flood-tolerant gene ST4 is introduced into rice plants to overexpress the rice flood-tolerant gene ST4 in the rice plants.
[0013] In a seventh aspect, the present invention provides flood-tolerant rice cultivated by the above-mentioned cultivation method.
[0014] The advantages and positive effects of the rice flooding tolerance gene ST4 and its application described in the present invention are:
[0015] 1. The present invention discloses a rice flood-tolerant gene ST4 and its application. By overexpressing the rice flood-tolerant gene ST4 in rice plants, the germination rate and seedling rate of rice can be significantly improved, laying a foundation for cultivating new flood-tolerant rice varieties.
[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The results of the identification of the expression amount of the ST4 overexpression material in the embodiment of the present invention;
[0018] Figure 2 This is a phenotype diagram of ST4 overexpression germination in waterlogging in the embodiment of the present invention;
[0019] Figure 3 is the seedling rate of the ST4 overexpression material in the embodiment of the present invention after flooding and germination;
[0020] Figure 4 It is the target site mutation form of the ST4 knockout material in the embodiment of the present invention;
[0021] Figure 5 This is a phenotype diagram of the ST4 knockout material germination after flooding in the embodiment of the present invention;
[0022] Figure 6 It is the seedling rate of the ST4 knockout material in the embodiment of the present invention after flooding and germination. DETAILED DESCRIPTION
[0023] The technical solution of the present invention is further described below through the accompanying drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the common meanings understood by people with ordinary skills in the field to which the present invention belongs.
[0024] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental instruments, equipment and reagents in the following examples without specifying the source are all commercially available raw materials. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the contents described can be applied to the method of the present invention.
[0025] The nucleotide sequence of gene ST4 is as follows (SEQ ID NO.1):
[0026] ATGAGCCTGAGGTACGCGAGCAGGGTGTTGCTGAGAGCGGCGGCGCAGGCCATGAGGGCCGCGAGGCAGCCGGCGCCTGCGGTGGCCAAGCCGGTGCCAGCGGCGAAGCCATCGGCGGTGGCGCCAGGCACGGCTC AGCGGCAGCAGGCGAGGCGGCTCTCCGGTGGCGTGGCTGAGCCGGCGGAGAAGGCGGCCGTGGCGGAGAGGATGAGGCGGCGGCGCAGGGAAGTCCGAGAATGTGATGCAGCTCGTGTGCTGGGGACCCAGTTAG
[0027] The CDS sequence of gene ST4 is as follows (SEQ ID NO.2):
[0028] ATGAGCCTGAGGTACGCGAGCAGGGTGTTGCTGAGAGCGGCGGCGCAGGCCATGAGGGCCGCGAGGCAGCCGGCGCCTGCGGTGGCCAAGCCGGTGCCAGCGGCGAAGCCATCGGCGGTGGCGCCAGGCACGGCTC AGCGGCAGCAGGCGAGGCGGCTCTCCGGTGGCGTGGCTGAGCCGGCGGAGAAGGCGGCCGTGGCGGAGAGGATGAGGCGGCGGCGCAGGGAAGTCCGAGAATGTGATGCAGCTCGTGTGCTGGGGACCCAGTTAG
[0029] The protein sequence encoded by gene ST4 is as follows (SEQ ID NO.3):
[0030] MSLRYASRVLLRAAAQAMRAARQPAPAVAKPVPAAKPSAVAPGTAQRQQARRLSGGVAEPAEKAAVAERMRRRRREKSENVMQLVCWGPS
[0031] Example 1 Cloning of gene ST4
[0032] 1. Extract genomic DNA from rice variety Dongye, use primers 5'-ATGAGCCTGAGGTACGCGAGCA-3' (SEQ ID NO.4) and 5'-CTAACTGGGTCCCCAGCACA-3' (SEQ ID NO.5) for polymerase chain reaction (PCR), sequence the obtained PCR product, and obtain the gene sequence of rice gene ST4. Gene ST4 consists of 273 bases, and the nucleotide sequence is SEQ ID NO.1. The PCR program is as follows: 94℃ pre-denaturation for 5 minutes; 35 cycles (94℃ denaturation for 30 seconds; 55℃ annealing for 30 seconds; 72℃ extension for 6 minutes), 72℃ extension for 10 minutes.
[0033] 2. RNA was extracted from the leaves of rice variety Dongye, reverse transcribed into cDNA, and polymerase chain reaction (PCR) was performed using primers 5'-ATGAGCCTGAGGTACGCGAGCA-3' (SEQ ID NO.6) and 5'-CTAACTGGGTCCCCAGCACA -3' (SEQ ID NO.7). The amplified product size was 273 bp. The obtained PCR product was sequenced and analyzed to obtain the CDS coding sequence of gene ST4, which consists of 273 bases, and the nucleotide sequence is shown in SEQ ID NO.2. The PCR program is as follows: 94℃ pre-denaturation for 5 minutes; 30 cycles (94℃ denaturation for 30 seconds; 55℃ annealing for 30 seconds; 72℃ extension for 1 minute), 72℃ extension for 7 minutes.
[0034] 3. The CDS coding sequence was translated using Primer 3 software to obtain the amino acid sequence, which encodes 90 amino acids and is the amino acid sequence shown in SEQ ID NO.3.
[0035] The above primers were synthesized by Shanghai Bioengineering and the sequences were determined by Shanghai Bioengineering. DNA and RNA extraction, PCR and reagent formulations refer to the Molecular Cloning Experiment Guide (J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Jin Dongyan et al. (translation), Science Press, 2002).
[0036] Example 2 Overexpression of ST4 gene improves rice germination and seedling rate under flooding
[0037] 1. Construction of overexpression vector:
[0038] According to the cDNA sequence of rice gene ST4, see the full-length coding sequence shown in SEQ ID NO.2, homologous recombination linkers on both sides of the Kpn I and BamH I double restriction sites of pU1301 vector were introduced into the upstream and downstream primers respectively, and the primers were designed as follows:
[0039] OX-F:5'-TTACGAACGATAGCCGGTACCATGAGCCTGAGGTACGCGAGCA-3' (SEQ ID NO.8),
[0040] OX-R: 5'-TTTTGCGGACTCTAGAGGATCCCTAACTGGGTCCCCAGCACA-3' (SEQ ID NO. 9).
[0041] The rice variety Dongye genome obtained in Example 1 was used as a template, and primers OX-F and OX-R were used for polymerase chain reaction (PCR), and the product size was 316 bp. The PCR program was as follows: 94°C pre-denaturation for 5 minutes; 30 cycles (94°C denaturation for 30 seconds; 55°C annealing for 30 seconds; 72°C extension for 1 minute), 72°C extension for 7 minutes. The ST4 gene fragment separated by PCR amplification was connected to the pu1301 vector after double digestion with Kpn I and BamH I by homologous recombination. After sequencing and comparison, it was transformed into Agrobacterium EHA105 to obtain the ST4 gene overexpression vector.
[0042] 2. Obtaining overexpression transgenic plants:
[0043] The ST4 gene overexpression vector was transferred into the indica rice variety Ganzaoxian 49 (GZX49) through Agrobacterium-mediated genetic transformation. T0 transgenic plants were obtained after selection culture, differentiation, rooting and seedling hardening. All transgenic materials were propagated to obtain stable inherited T1 materials. For a certain T1 transgenic line, if all the T2 plants sampled were transgenic plants, the T1 transgenic line was a homozygous transgenic line. Three homozygous transgenic lines OX1, OX2 and OX3 were selected for subsequent analysis.
[0044] 3. Identification of overexpressing transgenic plants:
[0045] (1) Expression level identification:
[0046] The seedlings at the 2-leaf stage were taken, total RNA was extracted, and cDNA was obtained by reverse transcription, and fluorescent real-time quantitative PCR was performed. The primers used to detect the expression of the ST4 gene were:
[0047] RT-F: 5'-GAGCAGAGTGACTAGTGTGATT-3' (SEQ ID NO. 10);
[0048] RT-R: 5'-CTTGCAGAATACAATACCGTCA-3' (SEQ ID NO. 11).
[0049] The reagent used for quantitative analysis was FastStart Universal SYBR Green Master (ROX). The instrument used was the real-time fluorescence quantitative PCR instrument ViiA7 from Applied Biosystems, USA. Ubqtin gene was used as an internal reference.
[0050] The relative expression of ST4 gene in the overexpression material is as follows Figure 1 shown. Figure 1 In the above, GZX49 represents the wild type of the receptor of the overexpression material, and OX1, OX2 and OX3 represent the ST4 gene overexpression transgenic strains. Figure 1 It can be seen that in the ST4 gene overexpression transgenic lines, the relative expression level of the ST4 gene was significantly higher than that of rice GZX49.
[0051] (2) Identification of rice flooding tolerance phenotype:
[0052] First, the seed germination of the test materials was tested under normal conditions, and the germination rate of the test materials was counted after 7 days to ensure that there was no significant difference in the seed vitality of the test materials and that the germination rate was above 95%. Then 30 dry and plump fresh seeds were placed in a colorless transparent glass bottle of the same size (3.5 cm in diameter and 16 cm in height). To prevent the seeds from floating, the seeds were covered with a mesh sheet with a pore size of 3 mm. The glass bottle was then filled with distilled water and placed under conditions suitable for rice growth. The test was repeated three times for each material, and the germination and seedling formation of the rice seeds were continuously observed for 21 days, and the seedling formation rate was counted. Photos after flooding germination are as follows: Figure 2 shown. Figure 2 In the figure, OX1, OX2 and OX3 represent transgenic plants overexpressing ST4 gene, and the scale bar is 2 cm. Figure 2 It can be seen that under flooding conditions, the seedling rate of ST4 gene overexpressing transgenic plants is significantly better than that of rice GZX49.
[0053] The results of seedling rate are as follows Figure 3 shown. Figure 3 In the figure, OX1, OX2 and OX3 represent transgenic plants overexpressing the ST4 gene. Figure 3 It can be seen that the seedling rates of OX1, OX2 and OX3 plants were significantly higher than that of the wild-type rice GZX49.
[0054] Example 3 Crispr knockout of ST4 gene reduces rice's ability to withstand flooding
[0055] 1. Construction of CRISPR knockout vector:
[0056] According to the gene sequence of rice gene ST4, such as the full-length sequence shown in SEQ ID NO.1, the target site for ST4 gene knockout was designed through the website http: / / skl.scau.edu.cn / targetdesign / . The designed target site is: 5'-ATCGGCGGTGGCGCCAGGCA-3' (SEQ ID NO.12). Homologous recombination linkers on both sides of the KpnI restriction site of pCXUN_CAS9 vector were introduced into the upstream and downstream primers respectively. The designed primers are:
[0057] U3-F: 5'-GTCGTTTCCCGCCTTCAGTTTgtaattcatccaggtctccaag -3' (SEQ IDNO.13),
[0058] U3-R: 5'-CTGTCAAACACTGATAGTTTAAACgctgtgccgtacgacggtacg-3' (SEQ IDNO.14),
[0059] Crispr-F:5'-ATCGGCGGTGGCGCCAGGCAgttttagagctagaaatagcaagtta-3' (SEQ IDNO.15),
[0060] Crispr-R: 5'- TGCCTGGCGCCACCGCCGATgccacggatcatctgcacaactc-3' (SEQ ID NO. 16).
[0061] Using OsU3 as a template, the primers U3-F and Crispr-R were used to perform polymerase chain reaction (PCR) to obtain PCR product 1, with a product size of 476 bp. The PCR program was as follows: 94°C pre-denaturation for 5 minutes; 30 cycles (94°C denaturation for 30 seconds; 55°C annealing for 30 seconds; 72°C extension for 30 seconds), and 72°C extension for 7 minutes. Using OsU3 as a template, the primers U3-R and Crispr-F were used to perform polymerase chain reaction (PCR) to obtain PCR product 2, with a product size of 368 bp. The PCR program was as follows: 94°C pre-denaturation for 5 minutes; 30 cycles (94°C denaturation for 30 seconds; 55°C annealing for 30 seconds; 72°C extension for 30 seconds), and 72°C extension for 7 minutes. Finally, the mixture of PCR product 1 and PCR product 2 was used as a template, and primers U3-F and U3-R were used for polymerase chain reaction (PCR) to obtain the final PCR product with a size of 824 bp. The PCR program was as follows: 94°C pre-denaturation for 5 minutes; 30 cycles (94°C denaturation for 30 seconds; 55°C annealing for 30 seconds; 72°C extension for 1 minute), and 72°C extension for 7 minutes. The final product separated by PCR amplification was connected to the pCXUN_CAS9 vector after KpnI digestion by homologous recombination. After sequencing and alignment, it was transformed into Agrobacterium EHA105 to obtain the ST4 gene Crispr knockout vector.
[0062] 2. Obtaining Crispr knockout plants:
[0063] The ST4 gene CRISPR knockout vector was transferred into the ST4-carrying strain by Agrobacterium-mediated genetic transformation. dy The material WD133 (the material was obtained by hybridizing Dongxiang wild rice with GZX49, backcrossing with GZX49 as the recurrent parent for three generations, and finally self-pollination, combined with molecular marker-assisted selection, with GZX49 as the background, carrying ST4 dy The transgenic line material was named WD133). After selection culture, differentiation, rooting, and seedling hardening, T0 generation transgenic plants were obtained. All transgenic materials were propagated to obtain T1 generation materials with homozygous mutations and no cas9 protein residues. Three homozygous transgenic lines CR1, CR2, and CR3 were selected for subsequent analysis.
[0064] 3. Identification of Crispr knockout plants:
[0065] 1) Genotype identification:
[0066] Seedlings at the 2-leaf stage were taken and DNA was extracted. Primers sq-F and sq-R were used for polymerase chain reaction (PCR), and the amplified product size was 429 bp. The obtained PCR product was sequenced to analyze the editing of the ST4 gene target site. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 32 cycles (94℃ denaturation for 30 seconds; 55℃ annealing for 30 seconds; 72℃ extension for 30 seconds), 72℃ extension for 7 minutes. The primer sequences used are as follows:
[0067] sq-F: 5'-ATGAGCCTGAGGTACGCGAGCA-3' (SEQ ID NO. 17),
[0068] sq-R: 5'-CTAACTGGGTCCCCAGCACA-3' (SEQ ID NO. 18).
[0069] The sequencing results of the target sites of the CRISPR knockout materials are as follows Figure 4 shown. Figure 4 In the figure, WT represents the knockout material wild type WD133 (CGAAGCC ATCGGCGGTGGCGCCAGGCACGGCTCAGCGGCA (SEQ ID NO.19)), CR1 (CGAAGCCATCGGCGGTGGAGGCACGGCTCAGCGGCA (SEQ ID NO.20)), CR2 (CGAAGCC ATCGGCGGTGGGCCAGGCACGGCTCAGCGGCA (SEQ ID NO.21)) and CR3 (CGAAGCC ATCGGCGGTGGCGCCTGGCACGGCTCAGCGGCA (SEQ ID NO.22)) represent the ST4 gene Crispr knockout strain. In the ST4 gene Crispr knockout strain, homozygous mutations occurred in the target site of the ST4 gene.
[0070] 2) Identification of flood tolerance phenotype:
[0071] First, the seed germination of the test materials was tested under normal conditions, and the germination rate of the test materials was counted after 7 days to ensure that there was no significant difference in the seed vitality of the test materials and the germination rate was above 95%. Then 30 dry and plump seeds were placed in a colorless transparent glass bottle of the same size (3.5 cm in diameter and 16 cm in height). To prevent the seeds from floating, the seeds were covered with a mesh sheet with a pore size of 3 mm. Then the glass bottle was filled with distilled water and placed under conditions suitable for rice growth. The test was repeated three times for each material, and the germination and seedling formation of rice seeds were observed for 21 days, and the seedling formation rate was counted.
[0072] Photos after 21 days of flooding Figure 5 shown. Figure 5 In the figure, WT represents WD133, CR1, CR2 and CR3 represent ST4 gene Crispr knockout plants, and the scale bar is 2 cm. Figure 5 It can be seen that after flooding stress, the growth conditions of ST4 gene Crispr knockout plants (CR1, CR2 and CR3) were significantly worse than those of WD133.
[0073] The survival rate results are as follows Figure 6 shown. Figure 6 WT indicates carrying ST4 dy WD133, CR1, CR2 and CR3 represent ST4 gene Crispr knockout plants. Figure 6 It can be seen that 21 days after flooding and germination, the seedling rate of ST4 gene Crispr knockout plants (CR1, CR2 and CR3) was significantly lower than that of WD133.
[0074] Therefore, the present invention adopts the above-mentioned rice flood-tolerant gene ST4 and its application, and by overexpressing the rice flood-tolerant gene ST4 in rice plants, can significantly improve the germination rate and seedling rate of rice, laying a foundation for cultivating flood-tolerant rice.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.
Claims
1. Application of rice flood tolerance gene ST4 in improving rice flood tolerance, characterized in that: The rice flood tolerance gene ST4 is overexpressed in rice plants to improve the flood tolerance of rice. The nucleotide sequence of the rice flood tolerance gene ST4 is shown in SEQ ID NO.1, and the coding sequence of the rice flood tolerance gene ST4 is shown in SEQ ID NO.
2.
2. The application of rice flooding tolerance protein in improving rice flooding tolerance is characterized by: The amino acid sequence of the rice flooding tolerance protein is shown in SEQ ID NO.3, and the coding gene of the rice flooding tolerance protein is shown in SEQ ID NO.
1.
3. Use of a recombinant vector comprising the rice flood tolerance gene ST4 as claimed in claim 1 in improving rice flood tolerance, characterized in that: The recombinant vector is a plant overexpression vector.
4. The use according to claim 3, characterized in that: When used, the rice flood-tolerant gene ST4 or a recombinant vector containing the rice flood-tolerant gene ST4 is introduced into rice plants to overexpress the rice flood-tolerant gene ST4 in the rice plants.
5. A method for cultivating flood-tolerant rice, characterized in that: The rice flood tolerance gene ST4 or a recombinant vector containing the rice flood tolerance gene ST4 is introduced into rice plants to overexpress the rice flood tolerance gene ST4 in the rice plants. The nucleotide sequence of the rice flood tolerance gene ST4 is shown in SEQ ID NO.1.