Application of OsSAUR5 gene in regulation and control of drought tolerance of rice

Abstract

The invention discloses application of an OsSAUR5 gene in regulation and control of rice drought tolerance, and relates to the technical field of gene engineering, and an amino acid sequence coded by the OsSAUR5 gene is shown as SEQ ID NO.2. The rice gene OsSAUR5 is screened and identified, and a mutant plant generated by knocking out the OsSAUR5 gene through gene editing can significantly improve the drought tolerance of rice in the seedling stage, which indicates that the gene is a gene for negatively regulating the drought tolerance of rice and can be used for improving the drought tolerance of cultivated rice. According to the OsSAUR5 functional deletion mutant plant created by utilizing a CRISPR / Cas9 technology, the seedling survival rate of a transgenic plant under a drought stress condition can be improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the rice OsSAUR5 gene in regulating rice drought resistance. Background Technology

[0002] Drought resistance refers to the ability of crops to resist damage caused by drought stress through their physiological and biochemical reactions, morphological structures, and molecular mechanisms. Rice (Oryza sativa L.) is one of the most important food crops; however, global climate change is leading to a continuous increase in the frequency, intensity, and scope of drought disasters, posing an unprecedented threat to rice production. Therefore, in-depth research on rice drought resistance is not only urgent but also of profound strategic significance. 1) Addressing climate change and ensuring food security: Drought has become one of the most serious abiotic stress factors limiting rice production. Frequent droughts lead to large-scale reductions in rice yields or even crop failure, directly jeopardizing food supply. Studying the drought resistance mechanism of rice and cultivating drought-resistant varieties is an inevitable choice for stabilizing rice yields, coping with extreme climates, and ensuring "absolute food security." 2) Conserving water resources and achieving sustainable agricultural development: Rice is a typical water-intensive crop, and traditional flood irrigation methods consume a large amount of global freshwater resources. Against the backdrop of increasingly scarce water resources, cultivating drought-resistant and water-saving rice varieties can significantly reduce agricultural water use, improve water use efficiency, and promote the transformation of agricultural production methods towards a resource-saving and environmentally friendly sustainable direction. 3) Expanding planting areas and ensuring farmers' livelihoods: Many regions possess vast land resources but are unable to grow rice due to water scarcity or inadequate irrigation facilities. The successful cultivation of highly drought-resistant rice varieties is expected to transform these arid or semi-arid areas into arable land, not only expanding the rice planting area but also providing new sources of livelihood for local farmers. 4) Deepening basic biological research and promoting technological self-reliance: Rice is the model plant for research on grasses. Analyzing its molecular, physiological, and genetic mechanisms of drought resistance can not only be directly applied to breeding practices but also provide valuable insights for stress resistance research on other important crops such as wheat and corn.

[0003] Therefore, developing drought-resistant rice resources is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides the application of the rice OsSAUR5 gene in regulating rice drought resistance. The present invention improves rice drought resistance and increases rice survival rate under drought stress by knocking out the rice OsSAUR5 gene.

[0005] The present invention provides the OsSAUR5 gene, the amino acid sequence encoded by the OsSAUR5 gene is shown in SEQ ID NO.2.

[0006] Another object of the present invention is to provide an OsSAUR5 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide the application of knocking out or knocking down the OsSAUR5 gene in improving the drought resistance of rice, wherein the amino acid sequence encoded by the OsSAUR5 gene is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide the application of sgRNA that knocks out the OsSAUR5 gene in improving the drought resistance of rice, wherein the target sequence of the sgRNA is shown in SEQ ID NO.3.

[0009] Another object of the present invention is to provide a CRISPR / Cas9 gene editing vector, wherein the CRISPR / Cas9 gene editing vector is obtained by loading the expression cassette of the sgRNA target sequence shown in SEQ ID NO.3 into the expression vector pYLCRISPR / Cas9Pubi-H.

[0010] Another object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned CRISPR / Cas9 gene editing vector.

[0011] Another object of the present invention is to provide the application of the above-mentioned CRISPR / Cas9 gene editing vector or the above-mentioned genetically engineered bacteria in improving the drought resistance of rice.

[0012] Another object of the present invention is to provide a method for improving the drought resistance of rice, comprising the following steps:

[0013] The sgRNA target sequence of the OsSAUR5 gene was designed, and a CRISPR / Cas9 gene editing vector was constructed. The sgRNA target sequence is shown in SEQ ID NO.3.

[0014] Construct genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors;

[0015] Genetically engineered bacteria containing a CRISPR / Cas9 gene editing vector were transformed into rice to obtain rice mutants with the OsSAUR5 gene knocked out.

[0016] Another object of the present invention is a rice mutant, wherein the nucleotide sequence of the OsSAUR5 encoding gene of the mutant is SEQ ID NO6, SEQ ID NO.8 or SEQ ID NO.10.

[0017] Another object of the present invention is to provide the application of the above-mentioned rice mutants in rice hybridization breeding.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention screened and identified a rice gene, OsSAUR5. Mutant plants generated by gene editing to knock out the OsSAUR5 gene significantly improved drought resistance in rice seedlings, indicating that this gene negatively regulates rice drought resistance and can be used to improve the drought resistance of cultivated rice. The OsSAUR5 loss-of-function mutant plants created using CRISPR / Cas9 technology in this invention can improve the seedling survival rate of transgenic plants under drought stress conditions. Attached Figure Description

[0020] 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.

[0021] Figure 1 The results show the performance of the OsSAUR5 gene knockout mutant and wild type under drought stress. Among them, A represents the survival of wild type and mutant rice before drought stress treatment; B represents the survival of wild type and mutant rice after drought stress treatment.

[0022] Figure 2 This invention relates to the effects of drought stress treatment on the survival rates of wild-type and OsSAUR5 gene knockout mutant rice. Detailed Implementation

[0023] 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 the conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0024] 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.

[0025] The present invention also includes variants that encode open reading frame sequences in proteins having the same function as OsSAUR5. These variants include, but are not limited to, deletions, insertions, and / or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10), and additions of several nucleotides (typically up to 60, preferably up to 30, more preferably up to 10, most preferably up to 5) at the 5 and / or 3 ends.

[0026] This invention also includes variations of the sequence having the same function as OsSAUR5. These variations include, but are not limited to, the deletion, insertion, and / or substitution of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to the C-terminus and / or N-terminus. 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 to the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0027] The vectors used in this invention can be, for example, bacteriophages, plasmids, granules, mini-chromosomes, viruses, or retroviral vectors. 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).

[0028] 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.

[0029] Synthetic adapters containing one or more restriction sites provide another method for ligating DNA segments to vectors. DNA segments resulting from restriction digestion by an endonuclease 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.

[0030] 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.

[0031] 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 bacteriophage 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 (UAA, UGA, or UAG) appropriately located at the end of the translated polypeptide.

[0032] As described above, the expression vector may include at least one selection marker. These markers include genes encoding antibiotic resistance, such as the neomycin phosphotransferase gene nptⅡ, the hygromycin phosphotransferase gene hpt, and the dihydrofolate reductase gene dhfr; another class is genes encoding herbicide resistance, such as the phosphinothricin acetyltransferase gene Bar and the 5-enolpyruvate shikimatr-3-phosphate synthase gene epsps. Representative examples of suitable hosts include, but are not limited to, protoplast cells and plant cells. Suitable culture media and conditions for the aforementioned host cells are known in the art.

[0033] 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.

[0034] In this invention, the term "transformant" refers to a host cell or organism carrying a foreign DNA molecule.

[0035] 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.

[0036] 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.

[0037] 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 through embodiments:

[0038] Example 1: Construction and genetic transformation of gene editing vector for rice OsSAUR5 gene

[0039] 1. Construction of gene editing vectors

[0040] The nucleotide sequence of the rice OsSAUR5 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0041] ATGGGAGAGCAAGGAGGCAGGGCAAGCAGCAACAAGATCAGGGACATTGTGAGGCTGCACCAGCTTCTCAAGAGGTGGAAGAGGGCTGCACTTGCACCAAAGGCCGGCAAGAACAACAATGGCGGCGGTGCATCGGTCCCGAAAGGGTTCTTCGCGGTGTGCGTCGGGGAGGAGATGAGGAGGTTTGTCATCCCCACA GAGTATCTTGGCCACTGGGCATTTGAGCAGCTACTCAGGAAGGCAGAGGAGGAGTTTGGGTTCCAGCATGAGGGAGCTCTGAGGATTCCATGTGATGTTGAGGTGTTTGAGGGTATCTTGAGGCTGGTTGGCAGGAAGGATGAGAAGGCAGCAATGTGCTACTCTTCTTCAGAGCATGAGATCTTGTGCAGATGA, SEQ. ID NO.1.

[0042] MGEQGGRASSNKIRDIVRLHQLLKRWKRAALAPKAGKNNNGGGASVP KGFFAVCVGEEMRRFVIPTEYLGHWAFEQLLRKAEEEFGFQHEGALRIPCDV EVFEGILRLVGRKDEKAAMCYSSSEHEILCR, SEQ ID NO.2.

[0043] The sgRNA target was designed using the CRISPR-P 2.0 tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ) as 5'-GAGGGCTGCACTTGCACCAAAGG-3' (SEQ ID NO.3). Then, the gene editing vector was constructed according to the method 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 (MaX, 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 YA Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 2015, 8:1274-1284). The constructed gene-editing vector was transformed into Agrobacterium tumefaciens EHA105 for genetic transformation experiments on rice callus.

[0044] 2. Genetic transformation of rice

[0045] (1) Seed disinfection

[0046] After removing the husks, mature Nipponbare rice seeds were placed in sterile Erlenmeyer flasks, soaked in 75% alcohol for 2 minutes, and rinsed twice with sterile water. Then, they were sterilized with 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.

[0047] The first callus induction medium formula is: (NB + 2,4-D 3.0 mg / L).

[0048] (2) Subculture

[0049] 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. After another two weeks of subculture, rice embryogenic callus granules were selected for genetic transformation.

[0050] The second callus induction medium formula is: (NB + 2,4-D 2.0 mg / L).

[0051] (3) Culture of Agrobacterium

[0052] A single colony was picked from the transformation plate and transferred to 1 mL of Agrobacterium tumefaciens medium for culture. 1 mL of the culture was then added to 50 mL of Agrobacterium tumefaciens medium (containing the appropriate antibiotic), and incubated at 200 rpm and 28°C until OD reached [the desired growth rate]. 600 The OD value was set at 0.7. Acetylsringone (AS, final concentration 100 μM) was added 2 hours before the end of the culture. The bacterial culture was then incubated at 4000 rpm for 10 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in MS liquid medium (containing 100 μM AS). The culture was then continued at 200 rpm and 28°C for 2 hours to allow the OD value of the bacterial culture to adjust. 600 =1, at which point it can be used to transform callus tissue.

[0053] The Agrobacterium culture medium formula is: yeast extract 10.0 g / L + peptone 10.0 g / L + sodium chloride 5.0 g / L.

[0054] (4) Co-cultivation

[0055] Rice embryogenic callus tissue was immersed in OD 600 The infected rice embryogenic callus was kept in the Agrobacterium tumefaciens solution at 1 for 25 minutes, then the moisture was absorbed with sterile absorbent paper. The infected callus was then placed on a co-culture medium and incubated in the dark at 28°C for three days.

[0056] The co-culture medium formula is: MS + 2,4-D 2.0 mg / L + AS 100 uM.

[0057] (5) Washing with disinfectant

[0058] After co-culture, the rice embryogenic callus was rinsed four times with sterile water, then soaked in MS liquid medium containing 400 mg / L cephalosporin (Cef) for 25 min, and then transferred to sterile filter paper to dry.

[0059] (6) Screening and Cultivation

[0060] 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.

[0061] The first selective culture medium formula is: NB + 2,4-D 2.0 mg / L + Hyg 30 mg / L + Cef 400 mg / L.

[0062] The second selective culture medium formula is: NB + 2,4-D 2.0 mg / L + Hyg 50 mg / L + Cef 250 mg / L.

[0063] (7) Differentiation culture

[0064] The resistant callus tissues obtained after two screenings were transferred to predifferentiation medium and cultured in the dark (culture temperature 25-28℃) for about 10 days, and then transferred to differentiation medium and cultured under light (light culture temperature 23-25℃, 12 hours of light / 12 hours of darkness, light intensity 6000-8000 lux) for 1 month to obtain seedlings.

[0065] The predifferentiation medium formula is: N6+KT 2.0mg / L+NAA 0.2mg / L+6-BA 2.0 mg / L+Hyg 30mg / L+Cef 200 mg / L+Agar 9g / L+Sucrose 45g / L.

[0066] The differentiation medium formula is: N6+KT 2.0mg / L+NAA 0.2mg / L+6-BA 2.0mg / L+Hyg 30mg / L+Agar 4.5g / L+Sucrose 30g / L.

[0067] (8) Rooting culture

[0068] Transplant seedlings that are about 2cm tall onto a rooting medium to induce the formation of adventitious roots.

[0069] Rooting medium formula: 1 / 2 MS + Hyg 15 mg / L + Agar 4.5 g / L + Sucrose 20 g / L.

[0070] (9) Transplantation of transgenic seedlings

[0071] 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.

[0072] Example 2: Screening of OsSAUR5 gene-editing mutants

[0073] The T0 generation of single plants with CRISPR / Cas9-edited OsSAUR5 gene were identified using first-generation sequencing. Genomic DNA was extracted from the T0 generation single plants using a rapid DNA extraction method. Primers cas OsSAUR5-F (SEQ ID NO.4) and cas OsSAUR5-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.

[0074] cas OsSAUR5-F: 5'-AACAATGGGAGAGCAAGGAG-3' (SEQ ID NO. 4);

[0075] cas OsSAUR5-R: 5'-CTCATGCTGGAACCCAAACT-3' (SEQ ID NO. 5).

[0076] 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.

[0077] Based on the sequencing results, the mutation types are as follows:

[0078] (1) A single base C is deleted at the target site of sgRNA (i.e., position 97 of SEQ ID NO.1), forming the mutant OsSAUR5ko1 shown in SEQ ID NO.6, abbreviated as KO1. This mutation leads to a frameshift mutation, causing premature termination of the transcript and forming a 58-amino acid polypeptide shown in SEQ ID NO.7.

[0079] (2) A single T base is inserted at the target site of the sgRNA (i.e., position 98 of SEQ ID NO.1), forming the OsSAUR5ko2 mutant shown in SEQ ID NO.8, abbreviated as KO2. This mutation results in a frameshift mutation, causing premature termination of the transcript and forming a 74-amino acid polypeptide shown in SEQ ID NO.9.

[0080] (3) One base A is inserted at the target site of sgRNA (i.e., position 98 of SEQ ID NO.1), forming the OsSAUR5ko3 mutant shown in SEQ ID NO.10, abbreviated as KO3. This mutation leads to a frameshift mutation, causing premature termination of the transcript and forming a 74-amino acid polypeptide shown in SEQ ID NO.11.

[0081] ATGGGAGAGCAAGGAGGCAGGGCAAGCAGCAACAAGATCAGGGACATTGTGAGGCTGCACCAGCTTCTCAAGAGGTGGAAGAGGGCTGCACTTGCACAAAGGCCGGCAAGAACAACAATGGCGGCGGTGCATCGGTCCCGAAAGGGTTCTTCGCGGTGTGCGTCGGGGAGGAGATGAGGAGGTTTGTCATCCCCACAGAGTATCTTGGCCACTGGGCATTTGAGCAGCTACTCAGGAAGGCAGAGGAGGAGTTTGGGTTCCAGCATGAGGGAGCTCTGAGGATTCCATGTGATGTTGAGGTGTTTGAGGGTATCTTGAGGCTGGTTGGCAGGAAGGATGAGAAGGCAGCAATGTGCTACTCTTCTTCAGAGCATGAGATCTTGTGCAGATGA,SEQ ID NO.6。

[0082] MGEQGGRASSNKIRDIVRLHQLLKRWKRAALAQRPARTTMAAVHRSR KGSSRCASGRR,SEQ IDNO.7。

[0083] ATGGGAGAGCAAGGAGGCAGGGCAAGCAGCAACAAGATCAGGGACATTGTGAGGCTGCACCAGCTTCTCAAGAGGTGGAAGAGGGCTGCACTTGCACTCAAAGGCCGGCAAGAACAACAATGGCGGCGGTGCATCGGTCCCGAAAGGGTTCTTCGCGGTGTGCGTCGGGGAGGAGATGAGGAGGTTTGTCATCCCCACAGAGTATCTTGGCCACTGGGCATTTGAGCAGCTACTCAGGAAGGCAGAGGAGGAGTTTGGGTTCCAGCATGAGGGAGCTCTGAGGATTCCATGTGATGTTGAGGTGTTTGAGGGTATCTTGAGGCTGGTTGGCAGGAAGGATGAGAAGGCAGCAATGTGCTACTCTTCTTCAGAGCATGAGATCTTGTGCAGATGA,SEQ ID NO.8。

[0084] MGEQGGRASSNKIRDIVRLHQLLKRWKRAALALKGRQEQQWRRCIGPERVLRGVRRGGDEEVCHPHRVSWPLGI, SEQ ID NO.9.

[0085] ATGGGAGAGCAAGGAGGCAGGGCAAGCAGCAACAAGATCAGGGACATTGTGAGGCTGCACCAGCTTCTCAAGAGGTGGAAGAGGGCTGCACTTGCACACAAAGGCCGGCAAGAACAACAATGGCGGCGGTGCATCGGTCCCGAAAGGGTTCTTCGCGGTGTGCGTCGGGGAGGAGATGAGGAGGTTTGTCATCCCCACA GAGTATCTTGGCCACTGGGCATTTGAGCAGCTACTCAGGAAGGCAGAGGAGGAGTTTGGGTTCCAGCATGAGGGAGCTCTGAGGATTCCATGTGATGTTGAGGTGTTTGAGGGTATCTTGAGGCTGGTTGGCAGGAAGGATGAGAAGGCAGCAATGTGCTACTCTTCTTCAGAGCATGAGATCTTGTGCAGATGA, SEQ. ID NO.10.

[0086] MGEQGGRASSNKIRDIVRLHQLLKRWKRAALAHKGRQEQQWRRCIGPERVLRGVRRGGDEEVCHPHRVSWPLGI, SEQ ID NO.11.

[0087] Example 3: Application Study of OsSAUR5 Gene in Regulating Drought Tolerance in Rice Seedlings

[0088] In this embodiment, the T2 generation lines of the OsSAUR5 gene-edited homozygous mutants OsSAUR5ko1, OsSAUR5ko2 and OsSAUR5ko3 from Example 2 were selected. Drought treatment was carried out at the three-leaf-one-heart stage to conduct drought stress and identify the drought resistance of the mutants and wild-type materials at the seedling stage.

[0089] 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 sow them in small buckets. Among them, OsSAUR5ko1, OsSAUR5ko2 and OsSAUR5ko3 are planted in the same small bucket as wild-type plants, half on each side, and kept in a saturated state. When the three leaves and one heart stage are reached, watering is stopped. Watering is resumed after four days, and the survival rate is counted.

[0090] The results are attached. Figure 1 and attached Figure 2 As shown, after drought stress treatment, the survival rate of wild-type plants (WT) was 21.1%; the survival rates of the three mutant lines were 48.9% (KO1), 44.5% (KO2), and 57.8% (KO3), respectively. It is evident that the survival rate of the OsSAUR5 gene mutant plants was significantly higher than that of the wild-type plants. The results of this invention indicate that knocking out the OsSAUR5 gene can significantly improve drought tolerance in rice seedlings.

[0091] 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.

[0092] 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. The OsSAUR5 gene, characterized by, The amino acid sequence encoded by the OsSAUR5 gene is shown in SEQ ID NO.

2.

2. OsSAUR5 protein, characterized by, The amino acid sequence of the OsSAUR5 protein is shown in SEQ ID NO.

2.

3. The application of knockout or knockdown of the OsSAUR5 gene in improving drought resistance in rice, characterized in that... The amino acid sequence encoded by the OsSAUR5 gene is shown in SEQ ID NO.

2.

4. The application of sgRNA knockout of the OsSAUR5 gene in improving drought resistance in rice, characterized by: The target sequence of the sgRNA is shown in SEQ ID NO.

3.

5. A CRISPR / Cas9 gene editing vector, characterized in that, The CRISPR / Cas9 gene editing vector was obtained by loading the expression cassette containing the sgRNA target sequence shown in SEQ ID NO.3 into the expression vector pYLCRISPR / Cas9Pubi-H.

6. A genetically engineered bacterium containing the CRISPR / Cas9 gene editing vector as described in claim 5.

7. The application of the CRISPR / Cas9 gene editing vector of claim 5 or the genetically engineered bacteria of claim 6 in improving the drought resistance of rice.

8. A method for improving the drought resistance of rice, characterized in that, Includes the following steps: The sgRNA target sequence of the OsSAUR5 gene was designed, and a CRISPR / Cas9 gene editing vector was constructed. The sgRNA target sequence is shown in SEQ ID NO.

3. Construct genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors; Genetically engineered bacteria containing a CRISPR / Cas9 gene editing vector were transformed into rice to obtain rice mutants with the OsSAUR5 gene knocked out.

9. A rice mutant, characterized in that, The nucleotide sequence of the OsSAUR5 encoding gene of the mutant is SEQ ID NO6, SEQ ID NO.8 or SEQ ID NO.

10.

10. The application of the rice mutant according to claim 6 in rice hybridization breeding.

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