Application of rice OsSAUR4 gene in regulation and control of rice drought tolerance

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a rice OsSAUR4 gene in regulation and control of rice drought tolerance, and the sequence of the rice OsSAUR4 gene is selected from any one of the following: 1) a nucleotide sequence as shown in SEQ ID NO.1; 2) a protein sequence as shown in SEQ ID NO.2, and 3) a sequence of which the homology with the nucleotide sequence as shown in SEQ ID NO.1 is more than 90%. The rice OsSAUR4 gene is knocked out, so that the drought tolerance of rice is improved, and the survival rate of rice in a drought stress environment is improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a type of rice. OsSAUR4 Application of genes in regulating drought resistance in rice. Background Technology

[0002] Drought resistance is the ability of crops to adapt to or resist drought stress, and it is formed by plants in the process of long-term adaptation to arid environments. Drought resistance is mainly divided into four types: drought escape, drought avoidance, drought tolerance, and recovery resistance. Drought tolerance refers to the level at which plants can maintain survival, growth, and development under drought conditions. China's per capita water resources are only about one-quarter of the world average, and agricultural water use accounts for 62.3% of total water use. China's water resource use faces a triple challenge: total water scarcity, uneven distribution in time and space, and low efficiency. Therefore, improving water resource utilization efficiency and exploring drought-resistant genes in agricultural production are inevitable choices.

[0003] Rice ( Oryza sativa. Rice (L) is one of my country's most important food crops, and stable and high yields are crucial for ensuring food security in my country and the world. In recent years, global warming and frequent natural disasters such as droughts and high temperatures have severely impacted agricultural production. Therefore, the discovery of drought-resistant genes is of great significance; it also lays the theoretical foundation for cultivating drought-resistant varieties. Summary of the Invention

[0004] To explore new ways to regulate drought resistance in rice, this invention provides rice OsSAUR4 Application of genes in regulating drought resistance in rice. This invention involves constructing rice genes. OsSAUR4 The mutant material was used to verify that this gene negatively regulates drought resistance in rice, and knocking out this gene improved the survival rate of rice.

[0005] This invention provides rice OsSAUR4 Application of genes in regulating drought resistance in rice, the rice OsSAUR4 The gene sequence is selected from any of the following: 1) The nucleotide sequence shown in SEQ ID NO.1; 2) The protein sequence shown in SEQ ID NO.2; 3) A sequence that shares more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1; The regulation refers to: knocking out rice OsSAUR4 Genes or downregulation in rice OsSAUR4 This invention uses gene expression levels to prepare mutant rice to improve the drought resistance of rice. The invention involves knocking out gene expression levels in rice. OsSAUR4 Genes have improved the drought resistance of rice and increased the survival rate of rice under drought stress.

[0006] Furthermore, knock out rice OsSAUR4 Gene editing is achieved by synthesizing sgRNA and then constructing a CRISPR / Cas9 gene editing vector.

[0007] Furthermore, the sequence of the sgRNA is shown in SEQ ID NO.3.

[0008] Furthermore, the method for constructing the CRISPR / Cas9 gene editing vector is as follows: The expression cassette containing sgRNA is loaded into the expression vector to obtain the CRISPR / Cas9 gene editing vector.

[0009] Furthermore, the expression vector is pYLCRISPR / Cas9Pubi-H.

[0010] Furthermore, the method for preparing mutant rice is as follows: The CRISPR / Cas9 gene editing vector was transformed into Agrobacterium to obtain genetically engineered bacteria; By infecting rice with genetically engineered bacteria and culturing it, mutant rice can be obtained.

[0011] Furthermore, the process of infecting rice with genetically engineered bacteria is as follows: Genetically engineered bacteria were cultured on a large scale to prepare an infection solution; Rice embryogenic callus was infected with an infecting solution.

[0012] Furthermore, the culture conditions were expanded to 200 rpm and 28°C, with acetylsyl syringone added to the culture medium at a final concentration of 100 μM.

[0013] Furthermore, the OD of the dyeing solution 600 The value is 1.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a rice OsSAUR4 Application of genes in regulating drought resistance in rice, the rice OsSAUR4 The gene sequence is selected from any one of the following: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) the protein sequence shown in SEQ ID NO.2; 3) a sequence with more than 90% homology to the nucleotide sequence shown in SEQ ID NO.1; the regulation refers to: knocking out rice OsSAUR4 Genes or downregulation in rice OsSAUR4 This invention uses gene expression levels to create mutant rice varieties to improve drought resistance. It involves gene editing to knock out genes. OsSAUR4The obtained mutant material significantly improved drought resistance in rice seedlings, indicating that this gene negatively regulates rice salt tolerance and can be used to improve the drought resistance of cultivated rice. This invention utilizes CRISPR / Cas9 technology to create... OsSAUR4 The loss-of-function mutant plant can improve the seedling survival rate of transgenic plants under drought conditions. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 For the present invention OsSAUR4 Drought resistance of gene knockout mutants and wild-type rice. A shows the survival of wild-type and mutant rice before drought stress treatment; B shows the survival of wild-type and mutant rice after drought stress treatment; C shows the statistical results of the survival rate of wild-type and mutant rice after drought stress treatment. Detailed Implementation

[0017] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, The conditions described in 1989, or as recommended by the manufacturer. Unless otherwise specified, all materials, reagents, etc., used in the following examples are commercially available.

[0018] In this invention, the terms "isolated" or "purified" DNA refer to DNA or fragments that have been isolated from sequences flanking them in their natural state, and also to DNA or fragments that have been separated from components that accompany nucleic acids in their natural state, and from proteins that accompany them in cells.

[0019] The present invention also includes variants that encode open reading frame sequences in proteins having the same function as OsSAUR4. These variants include, but are not limited to, several deletions, insertions, and / or substitutions of nucleotides, typically 1 to 90, preferably 1 to 60, more preferably 1 to 20, and most preferably 1 to 10, and the addition of several nucleotides at the 5' and / or 3' ends, typically up to 60, preferably up to 30, more preferably up to 10, and most preferably up to 5.

[0020] This invention also includes variations of the sequence having the same function as OsSAUR4. These variations include, but are not limited to, deletions, insertions, and / or substitutions of several, typically 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10 amino acids, as well as the addition of one or more amino acids at the C-terminus and / or N-terminus, typically up to 20, preferably up to 10, and more preferably up to 5. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0021] The vectors used in this invention can be, for example, bacteriophages, plasmids, granules, mini-chromosomes, viruses, or retroviruses. Vectors that can be used to clone and / or express the polynucleotides of this invention are vectors capable of replicating and / or expressing the polynucleotides in host cells where replication and / or expression of the polynucleotides are required. Generally, recombinant expression vectors carrying the nucleic acid sequences of this invention can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

[0022] Several methods have been developed for operatively linking polynucleotides to vectors via complementary sticky ends. For example, complementary homomeric sequence fragments can be added to a DNA segment to be inserted into the vector DNA. The vector and the DNA segment are then linked by hydrogen bonds between the complementary homomeric tails to form a recombinant DNA molecule.

[0023] Synthetic adapters containing one or more restriction sites provide another method for ligating DNA segments to vectors. DNA segments produced by restriction digestion with endonucleases are treated with phage T4 DNA polymerase or E. coli DNA polymerase I. Both polymerases remove protruding γ-single-stranded ends with their 3', 5'-exonuclease activities and fill in 3'-concave ends with their polymerization activities. Thus, the combination of these activities produces blunt-ended DNA segments, which are then incubated with a molar excess of adapter molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as phage T4 DNA ligase. The reaction product is a DNA segment with polymerase sequences at its ends. These DNA segments are then lysed with a suitable restriction enzyme and ligated into an enzyme-lysed expression vector that produces ends compatible with the DNA segments. Synthetic adapters containing multiple restriction endonuclease sites are available from various vendors.

[0024] Other newly developed technologies utilize homologous recombination, which involves homologous recombination of a vector with a polynucleotide carrying a specific or homologous sequence adapter. The DNA segment to be inserted into the vector DNA is then combined with the vector, which also carries a specific or homologous sequence, to form a recombinant DNA molecule through the action of recombinase.

[0025] The polynucleotide insert should be operatively linked to a suitable promoter compatible with the host cell expressing the polynucleotide. The promoter can be a strong promoter and / or an inducible promoter. Some examples of promoters include the phage PL promoter, E. coli lac, trP, phoA, tac promoter, SV40 early and late promoters, and retroviral LTR promoters; other suitable promoters are known to those skilled in the art. The expression recombinant vector further contains transcription initiation and termination sites, and ribosome-binding sites for translation in the transcription region. The coding portion of the transcript expressed by the recombinant vector may include a translation initiation codon located at the initiation site and a stop codon appropriately located at the end of the translated polypeptide.

[0026] As described above, the expression vector may include at least one selection marker. The marker may include a gene encoding antibiotic resistance, such as neomycin phosphotransferase (NMT). Neomycin phosphotransferase )Gene nptⅡ Hygromycin phosphotransferase ( Hygromycin phosphotransferase ) gene hpt and dihydrofolate reductase (Dihydrofolate reductase )Gene dhfr Another type encodes herbicide resistance genes, such as glyphosate acetyltransferase (AST). Phosphinothricin acetyltransferase )Gene Bar 5-Enolpyruvyloxalate-3-phosphate synthase ( 5- Enoylpyruvate shikimatr-3-phosphate )Geneepsps Representative examples of suitable hosts include, but are not limited to, protoplast cells and plant cells. Appropriate culture media and conditions for the aforementioned host cells are known in the art.

[0027] Transformation methods for target genes or polynucleotides can be categorized into three types: First, vector-mediated transformation, where the target gene is inserted into a vector molecule such as an Agrobacterium plasmid or viral DNA. The target gene is then introduced into the plant genome via the transfer of the vector DNA. Agrobacterium-mediated and virus-mediated transformations fall into this category. Second, direct gene introduction methods, which involve directly introducing exogenous target genes into the plant genome using physical or chemical methods. Physical methods include gene gun transformation, electroporation transformation, ultrasound transformation, microinjection, and laser microbeam transformation; chemical methods include PEG-mediated transformation and liposome transformation. Third, germplasm system methods, including pollen tube pathway methods, germ cell staining methods, and embryo sac and ovary injection methods.

[0028] In this invention, the term "transformer" refers to a host cell or organism carrying a foreign DNA molecule.

[0029] The present invention also includes host cells containing the nucleotide sequences of the present invention, said nucleotide sequences being operatively linked to one or more heterologous control regions (such as promoters and / or enhancers) using techniques known in the art. Host strains capable of regulating the expression of the inserted gene sequence, or capable of modifying and processing the gene product in a desired specific manner, can be selected. In the presence of certain inducers, the expression initiated by certain promoters may be increased.

[0030] Cells that have been successfully transformed can be identified using well-known techniques; that is, cells or organisms containing the recombinant vector with the nucleotide sequence described in this invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. The invention is described in detail below by way of examples.

[0032] The list of abbreviations for this invention is shown in Table 1.

[0033] Table 1. List of abbreviations for this invention Example 1 rice OsSAUR4 The application of genes in regulating drought resistance in rice is as follows: 1. Rice OsSAUR4Construction of gene editing vectors and genetic transformation.

[0034] (1) Construction of gene editing vectors.

[0035] rice OsSAUR4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1: ATGGGGGTACTTCCGGGCGCCGAGGAGGAAGGCGGCGGCGGCGGCGGAGGGGGAGAGCGTGCGGGCGGCGCTGCTCGTCGGCGGCGGCGGCGAGGAGGCGGCGGTGCCGAAGGGGTACTTCGCGGTGTACGTGGGCGCGGAGGCGAGGCGGTTCGTGGTGCCGACGAGCTACCTCCGGCAGCC GGCGTTCCGGGGCCTCATGGAGCTCGCCGCCGACGAGTTCGGCTTCGCCCAGGAGGGCGGCCTCCGCCTCCCCTGCCGCGAGGAGGACTTCCAGGCCACCGTCGCCGCGCTCGACGCGCGCCGCCGCCCGGCGAGCGGCGGCGCGATCATGAGCACCATGGTCAAGGCCAGGTCGTTGTAG.

[0037] SEQ ID NO.2: MGYFRAPRRKAAAAAEGESVRAALLVGGGGEEAAVPKGYFAVYVGAEARRFVVPTSYLRQPAFRGLMELAADEFGFAQEGGLRLPCREEDFQATVAALDARRRPASGGAIMSTMVKARSL.

[0038] sgRNA targets were designed using CRISPR-P 2.0 tools, and the sgRNA target sequence is shown in SEQ ID NO.3. Then, the gene editing vector was constructed according to the methods provided by the published CRISPR / Cas9 gene editing system. Finally, the U6 promoter and sgRNA expression cassette were loaded into the expression vector pYLCRISPR / Cas9Pubi-H to obtain the gene editing vector. For detailed operational steps, please refer to the reference Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Liu Y. A RobustCRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editingin Monocot and Dicot Plants. Molecular Plant, 2015, 8: 1274-1284. The constructed gene editing vector was transformed into Agrobacterium EHA105 to obtain genetically engineered bacteria, which were then used for genetic transformation experiments on rice callus tissue.

[0039] CRISPR-P 2.0: http: / / crispr.hzau.edu.cn / CRISPR2 / .

[0040] SEQ ID NO. 3: 5'-GAGGCGGAGGCCGCCCTCCTGGG-3'.

[0041] (2) Genetic transformation of rice.

[0042] 1) Seed disinfection.

[0043] After removing the husks, mature Nipponbare rice seeds were placed in sterile Erlenmeyer flasks and soaked in v / v 75% alcohol for 2 minutes, then rinsed twice with sterile water. Next, they were sterilized with v / v 3% NaClO for 30 minutes, shaking frequently during the process. They were then rinsed four times with sterile water, and excess water was absorbed with sterile filter paper. The seeds were then inoculated onto the first callus induction medium for induction culture, with approximately 30 seeds per dish, and cultured in the dark at 28°C.

[0044] The first callus induction medium formula is: NB medium + 3 mg / L 2,4-D.

[0045] 2) Subgeneration training.

[0046] After nearly a month of induction culture, rice grew yellow, swollen callus tissue. The scutellum was removed, and the callus tissue was transferred to a second callus induction medium for subculture. Subculture was performed every two weeks, and after three subcultures, pale yellow, granular embryogenic callus tissue suitable for transgenic transformation was obtained. Two weeks after subculture, rice embryogenic callus granules were selected for genetic transformation.

[0047] The second callus induction medium formula is: NB medium + 2 mg / L 2,4-D.

[0048] 3) Culture of Agrobacterium.

[0049] Single colonies containing the gene-editing vector were picked from transformation plates and cultured in 1 mL of Agrobacterium tumefaciens medium to obtain a culture. 1 mL of the culture was added to 50 mL of Agrobacterium tumefaciens medium containing the appropriate antibiotic, and the culture was incubated at 200 rpm and 28°C until OD reached [the desired growth rate]. 600 The OD value was set at 0.7. Two hours before the end of the culture, acetylsyl syringone was added to a final concentration of 100 μM. The bacterial culture was then incubated at room temperature for 10 minutes at 4000 rpm, the supernatant was discarded, and the cells were resuspended in MS liquid medium containing 100 μM AS. The culture was then incubated for 2 hours under the same conditions as above, allowing the OD value of the bacterial culture to reach a certain level. 600 =1, obtain the infection solution, which can then be used to transform embryogenic callus tissue.

[0050] 4) Co-cultivation.

[0051] Rice embryogenic callus tissue was immersed in OD 600 After incubating the Agrobacterium tumefaciens solution at 1 for 25 minutes, the water was then absorbed with sterile absorbent paper. The infected rice embryogenic callus was then placed on a co-culture medium and incubated in the dark at 28°C for three days.

[0052] The co-culture medium formula is: MS + 2,4-D 2 mg / L + AS 100 μM.

[0053] 5) Wash with bacteria.

[0054] After co-culture, the rice embryogenic callus was rinsed four times with sterile water, then soaked in MS liquid medium containing 400 mg / L Cef for 25 min, and finally transferred to sterile filter paper to dry.

[0055] 6) Screening and cultivation.

[0056] The dried callus tissue was inoculated onto a first selective medium. After 3 weeks, newly grown callus tissue was selected and inoculated onto a second selective medium. After another 2 weeks, resistant callus tissue was obtained.

[0057] The first selective culture medium formula is: NB + 2,4-D 2mg / L + Hyg 30mg / L + Cef 400mg / L.

[0058] The second selective culture medium formula is: NB + 2,4-D 2mg / L + Hyg 50mg / L + Cef 250mg / L.

[0059] 7) Differentiation culture.

[0060] The resistant callus tissues obtained after two rounds of screening were transferred to pre-differentiation medium and cultured in the dark at 28°C for about 10 days, and then transferred to differentiation medium and cultured under light for 1 month to obtain seedlings. The light culture temperature was 23°C, with 12 hours of light and 12 hours of darkness, and the light intensity was 8000 lux.

[0061] The predifferentiation medium formula is: N6+KT 2mg / L+NAA 0.2mg / L+6-BA 2mg / L+Hyg 30mg / L+Cef 200mg / L+Agar 9g / L+Sucrose 45g / L.

[0062] The differentiation medium formula is: N6+KT 2mg / L+NAA 0.2mg / L+6-BA 2mg / L+Hyg 30mg / L+Agar 4.5g / L+Sucrose 30g / L.

[0063] 8) Rooting culture.

[0064] Transplant seedlings that are about 2cm tall onto a rooting medium to induce the formation of adventitious roots.

[0065] Rooting medium formula: 1 / 2 MS + Hyg 15 mg / L + Agar 4.5 g / L + Sucrose 20 g / L.

[0066] 9) Transplanting of genetically modified seedlings.

[0067] When the seedlings grow to 10cm in height, remove them, wash off the attached solid culture medium with sterile water, and transplant them into the soil for planting.

[0068] 2. OsSAUR4 Screening for gene-edited mutants.

[0069] For CRISPR / Cas9 editing OsSAUR4 The T0 generation single plants were identified using first-generation sequencing. Genomic DNA was extracted from the T0 generation single plants using a rapid DNA extraction method. Primers casSAUR4-F (SEQ ID NO.4) and casSAUR4-R (SEQ ID NO.5) were designed flanking the sgRNA editing site shown in SEQ ID NO.3 to amplify the fragment covering the editing site region. The PCR products were then sequenced.

[0070] SEQ ID NO. 4, casSAUR4-F: 5'-GGTACTTCGCGGTGTACGTG-3'.

[0071] SEQ ID NO. 5, casSAUR4-R: 5'-CTCTCCTATGGCGGGCTAGT-3'.

[0072] Using the target site sequence of Nipponbare rice as a reference sequence, multiple sequence alignment was performed with all amplified editing site regions to identify homozygous single plants that underwent genome editing.

[0073] Based on the sequencing results, the mutation types are as follows: (1) A single base A is deleted at position 236 of the target site of sgRNA, forming the mutant shown in SEQ ID NO.6, Ossaur4ko1, abbreviated as KO1. This mutation leads to a frameshift mutation, causing premature termination of the transcript and forming a polypeptide of 110 amino acids shown in SEQ ID NO.7.

[0074] (2) The deletion of two bases AG at positions 236 and 237 forms the Ossaur4ko2 mutant shown in SEQ ID NO.8, abbreviated as KO2. This mutation results in a frameshift, forming a 125-amino acid polypeptide shown in SEQ ID NO.9, which is similar to... OsSAUR4 The protein sequence obtained by normal expression of the gene coding region sequence SEQ ID NO.1 is inconsistent with SEQ ID NO.2.

[0075] SEQ ID NO.6: ATGGGGGTACTTCCGGGCGCCGAGGAGGAAGGCGGCGGCGCCGGCGGAGGGGGAGAGCGTGCGGGCGGCGCTGCTCGTCGGCGGCGGCGGCGAGGAGGCGGCGGTGCCGAAGGGGTACTTCGCGGTGTACGTGGGCGCGGAGGCCAGGGCGGTTCGTGGTGCCGACGAG CTACCTCCGGCAGCCGGCGTTCCGGGGCCTCATGGAGCTCGCCGCCGACGAGTTCGGCTTCGCCCAGGGGGCGGCCTCCGCCTCCCCTGCCGCGAGGAGGACTTCCAGGCCACCGTCGCCGCGCTCGACGCGCGCCGCCGCCCGGCGAGCGGCGGCGCGATCATGA.

[0076] SEQ ID NO.7: MGYFRAPRRKAAAAAEGESVRAALLVGGGGEEAAVPKGYFAVYVGAEARRFVVPTSYLRQPAFRGLMELAADEFGFAQGAASASPAARRTSRPPSPRSTRAAARRAAARS.

[0077] SEQ ID NO.8: ATGGGGGTACTTCCGGGCGCCGAGGAGGAAGGCGGCGGCGGCGGCGGAGGGGGAGAGCGTGCGGGCGGCGCTGCTCGTCGGCGGCGGCGGCGAGGAGGCGGCGGTGCCGAAGGGGTACTTCGCGGTGTACGTGGGCGCGGAGGGCGAGGCGGTTCGTGGTGCCGACGAGCTACCTCCGGCAGCCGGCGTTC CGGGGCCTCATGGAGCTCGCCGCCGACGAGTTCGGCTTCGCCCAGGGGCGGCCTCCGCCTCCCCTGCCGCGAGGAGGACTTCCAGGCCACCGTCGCCGCGCTCGACGCGCGCCGCCGCCCGGCGAGCGGCGGCGCGATCATGAGCACCATGGTCAAGGCCAGGTCGTTGTAGCCACTAGCCCGCCATAG.

[0078] SEQ ID NO.9: MGYFRAPRRKAAAAAEGESVRAALLVGGGGEEAAVPKGYFAVYVGAEARRFVVPTSYLRQPAFRGLMELAADEFGFAQGRPPPPLPRGGLPGHRRRARRAPPPGERRRDHEHHGQGQVVVATSPP.

[0079] 3. OsSAUR4 Application research on gene regulation of drought resistance in rice seedlings.

[0080] This invention selects OsSAUR4 T2 generation lines of the gene-edited homozygous mutants Ossaur4ko1 and Ossaur4ko2 were used. Water withholding was applied at the three-leaf-one-heart stage to induce drought stress and to identify the seedling drought tolerance of the mutants and wild-type plants.

[0081] The specific steps are as follows: Soak rice seeds in purified water and germinate them at 30℃ for 48 hours. Select plants that show white sprouts and grow uniformly and plant them in small plastic buckets. Among them, Ossaur4ko1 and Ossaur4ko2 are planted in the same small plastic bucket containing soil, with half on each side, and three biological replicates. When the plants grow to the three-leaf-one-heart stage, they are subjected to drought treatment. After water is withheld for 4 days, they are re-watered, and the survival rate of the seedlings is counted.

[0082] The results are as follows Figure 1 As shown, after drought stress treatment, the survival rates of wild-type plants WT, KO1, and KO2 were 21.6%, 54.1%, and 47.4%, respectively, indicating that the survival rates of the two mutant plants after drought stress were significantly higher than those of the wild-type plants. The results indicate... OsSAUR4 Negative regulation of drought resistance in rice.

[0083] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0084] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Oryza sativa OsSAUR4 application of the gene in regulating drought tolerance in rice, characterized in that, The rice OsSAUR4 the sequence of the gene is selected from any one of the following: 1) the nucleotide sequence shown in SEQ ID NO. 1; 2) the protein sequence shown in SEQ ID NO. 2; 3) a sequence with more than 90% homology to the nucleotide sequence shown in SEQ ID NO. 1; The regulation refers to: preparing mutant rice by knocking out the rice OsSAUR4 gene or down-regulating the expression level of the rice OsSAUR4 gene to improve the drought tolerance of rice.

2. Use according to claim 1, characterized in that, Knockout of rice OsSAUR4 The gene is realized by synthesizing sgRNA, and further constructing CRISPR / Cas9 gene editing vector.

3. Use according to claim 2, characterized in that, The sequence of the sgRNA is shown in SEQ ID NO.

3.

4. Use according to claim 2, characterized in that, The construction method of the CRISPR / Cas9 gene editing vector is as follows: The expression cassette containing the sgRNA is loaded into an expression vector, and the CRISPR / Cas9 gene editing vector is obtained.

5. Use according to claim 4, characterized in that, The expression vector is pYLCRISPR / Cas9Pubi-H.

6. Use according to claim 2, characterized in that, The method for preparing the mutant rice is as follows: The CRISPR / Cas9 gene editing vector is transformed into Agrobacterium to obtain a genetically engineered bacterium; The genetically engineered bacterium is used to infect rice, and the mutant rice is obtained by cultivation.

7. Use according to claim 6, characterized in that, The process of using the genetically engineered bacterium to infect rice is as follows: The genetically engineered bacterium is cultured to prepare an infection solution; The infection solution is used to infect the embryogenic callus of rice.

8. Use according to claim 7, characterized in that, The culture condition is 200 rpm, 28℃, and acetyl cinnamone is added to the culture medium at a final concentration of 100 μM.

9. Use according to claim 7, characterized in that, OD of the inoculum 600 was 1.

Images