Application of OsCSLE6 gene in regulating drought resistance of rice

Abstract

The application discloses application of an OsCSLE6 gene in regulating drought resistance of rice, relates to the technical field of genetic engineering, and has the technical points that the sequence of the OsCSLE6 gene is a DNA sequence as shown in SEQ ID NO:1; or a DNA sequence homologous to SEQ ID NO:1 by at least 90%; or a subfragment functionally equivalent to the sequence shown in SEQ ID NO:1. Mutant plants generated by gene editing can significantly improve drought resistance of rice seedlings and adult plants, indicating that the gene is a gene negatively regulating drought resistance of rice, and can be used for improving drought resistance of cultivated rice.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the OsCSLE6 gene in regulating drought resistance in rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of China's most important food crops, and its yield is crucial to China's food security. Rice grows in flooded conditions, consuming a significant amount of water during its growth and development. However, China's per capita water resources are only one-quarter of the world average, classifying it as a country with absolute water scarcity. China's rice production is facing a severe water shortage crisis. Discovering drought-resistant genes in rice and improving its drought resistance is of great significance for ensuring stable rice yields under drought conditions. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems and provide the application of the OsCSLE6 gene in regulating rice drought resistance, with the aim of improving rice drought resistance.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows: an application of the rice OsCSLE6 gene, wherein the sequence of the OsCSLE6 gene is as shown in the DNA fragment of SEQ ID NO:1; or

[0005] DNA fragments that are at least 90% homologous to SEQ ID NO:1; or

[0006] It functions as a subfraction of the nucleotide sequence shown in SEQ ID NO:1.

[0007] In some implementation cases, the application refers to the use of proteins encoded by the OsCSLE6 gene in regulating drought resistance in rice.

[0008] In some embodiments, the amino acid sequence of the protein encoded by the OsCSLE6 gene is shown in SEQ ID NO: 11; or

[0009] The amino acid sequence of the protein encoded by the OsCSLE6 gene is selected from one of the homologous sequences of SEQ ID NO:11, conserved variants, allelic variants, natural mutants, and induced mutants.

[0010] This invention also provides an application of the above-mentioned gene or protein-related biomaterials in the drought resistance of rice, wherein the biomaterials are any of the following:

[0011] a) An expression cassette containing a DNA fragment with a nucleotide sequence such as SEQ ID NO:1, or an expression cassette containing a DNA fragment that is at least 90% homologous to SEQ ID NO:1;

[0012] b) A recombinant vector containing a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:1, or a recombinant vector containing a DNA fragment that is at least 90% homologous to SEQ ID NO:1; preferably, the recombinant vector is a gene knockout vector, a knockdown vector, a knock-in vector, or an overexpression vector;

[0013] c) Recombinant microorganisms, recombinant cells, transgenic plant tissues or transgenic plants containing the recombinant vector described in b);

[0014] d) Tissue cultures or protoplasts of regenerative cells from the transgenic plants described in c).

[0015] In some embodiments, the plasmid used to construct the recombinant vector is a Ti plasmid or a plant virus vector.

[0016] This invention also provides an OsCSLE6 gene knockout mutant based on CRISPR / Cas9 technology. The OsCSLE6 gene knockout mutant is prepared by the following steps: designing an sgRNA and a corresponding target sequence primer, wherein the nucleotide sequence of the sgRNA target is shown in SEQ ID NO:2; constructing an sgRNA expression cassette for the OsCSLE6 gene to be used in the production of drought-resistant transgenic plants using PCR amplification;

[0017] The sgRNA expression cassette was loaded into a CRISPR / Cas9 vector to obtain a CRISPR / Cas9-sgRNA vector containing the target sequence.

[0018] The CRISPR / Cas9-sgRNA vector containing the target sequence was transformed into Agrobacterium, and then transformed into rice callus tissue to obtain the rice OsCSLE6 gene mutant KO plant.

[0019] The present invention also provides a breeding method for improving the drought resistance of rice, by screening rice varieties containing the OsCSLE6 gene as described in claim 1.

[0020] In some embodiments, the breeding method is as follows: constructing the recombinant gene editing vector as described above, and cultivating rice plants with mutations in the OsCSLE6 gene.

[0021] The following are the sequences involved in this invention:

[0022] SEQ ID NO: 1 (OsCSLE6 nucleotide sequence):

[0023]

[0024] SEQ ID NO: 2 (sgRNA target sequence):

[0025] 5'-GATTCCGAGAAGTAAGCAGCTGG-3'

[0026] SEQ ID NO: 3 (casOsCSLE6-F primer sequence):

[0027] 5'-CCACCAAGCCTTGTCATCTC-3'

[0028] SEQ ID NO: 4 (casOsCSLE6-R primer sequence):

[0029] 5'-TACCGAAACCTAGGGGAACC-3'

[0030] SEQ ID NO: 5 (OsCSLE6ko1 nucleotide sequence):

[0031] ATGGAGACGACGACGACGGAGAGGCGGCGGCTGTTCGCGACGGAGAAGGTGGGCGGCAGGGCGGTGTACAGGCTGCAGGCGGCGACGGTGGCCGCCGGGATACTGCTGGTGCTCTACTACAGGGCGACGCGCGTGCCGGCCGCCGGCGAAGGGCGGGCGGCGTGGCTGGGGATGGCGGCGGCGGAGCTCTGGTTCGCCGTCTACTGGGTCATCACGCAGTCCGTCCGGTGGTGCCCCGTCCGCCGCCGCACCTTCAAGAACAGGCTCGCCGAGAGATACAAAGAAAATCTACCTGGTGTGGATGTTTTTGTATGCACTGCAGACCCACACGCAGAGCCACCAAGCCTTGTCATCTCTACCATCCTATCAGTCATGGCATACAATTACCCATCAGAAAAAATAAGTGTGTATCTTTCTGATGATGGTGGTTCAATTCTTACTTTCTACGCTCTATGGGAGGCATCCATGTTTGCAAAGAAATGGCTACCATTCTGCAGAAGATATAACATTGAGCCAAGGTCCCCAGCTAGCTTACTTCTCGGAATCAGAAGGGCATCACAATCTGTGCTCCCCAAAAGAATGGTCATTTATCAAGAACCTGTATGA

[0032] SEQ ID NO: 6 (Truncated amino acid sequence of OsCSLE6ko1):

[0033] METTTTERRRLFATEKVGGRAVYRLQAATVAAGILLVLYYRATRVPAAGEGRAAWLGMAAAELWF AVYWVITQSVRWCPVRRRTFKNRLAERYKENLPGVDVFVCTADPHAEPPSLVISTILSVMAYNYPSEKI SVYLSDDGGSILTFYALWEASMFAKKWLPFCRRYNIEPRSPASLLLGIRRASQSVLPKRMVIYQEPV*

[0034] SEQ ID NO: 7 (Nucleotide sequence of OsCSLE6ko2):

[0035] ATGGAGACGACGACGACGGAGAGGCGGCGGCTGTTCGCGACGGAGAAGGTGGGCGGCAGGGCGGTGTACAGGCTGCAGGCGGCGACGGTGGCCGCCGGGATACTGCTGGTGCTCTACTACAGGGCGACGCGCGTGCCGGCCGCCGGCGAAGGGCGGGCGGCGTGGCTGGGGATGGCGGCGGCGGAGCTCTGGTTCGCCGTCTACTGGGTCATCACGCAGTCCGTCCGGTGGTGCCCCGTCCGCCGCCGCACCTTCAAGAACAGGCTCGCCGAGAGATACAAAGAAAATCTACCTGGTGTGGATGTTTTTGTATGCACTGCAGACCCACACGCAGAGCCACCAAGCCTTGTCATCTCTACCATCCTATCAGTCATGGCATACAATTACCCATCAGAAAAAATAAGTGTGTATCTTTCTGATGATGGTGGTTCAATTCTTACTTTCTACGCTCTATGGGAGGCATCCATGTTTGCAAAGAAATGGCTACCATTCTGCAGAAGATATAACATTGAGCCAAGGTCCCCAGCTTGCTTACTTCTCGGAATCAGAAGGGCATCACAATCTGTGCTCCCCAAAAGAATGGTCATTTATCAAGAACCTGTATGA

[0036] SEQ ID NO: 8 (Truncated amino acid sequence of OsCSLE6ko2):

[0037] METTTTERRRLFATEKVGGRAVYRLQAATVAAGILLVLYYRATRVPAAGEGRAAWLGMAAAELWF AVYWVITQSVRWCPVRRRTFKNRLAERYKENLPGVDVFVCTADPHAEPPSLVISTILSVMAYNYPSEKI SVYLSDDGGSILTFYALWEASMFAKKWLPFCRRYNIEPRSPACLLLGIRRASQSVLPKRMVIYQEPV*

[0038] SEQ ID NO: 9 (Nucleotide sequence of OsCSLE6ko3):

[0039] ATGGAGACGACGACGACGGAGAGGCGGCGGCTGTTCGCGACGGAGAAGGTGGGCGGCAGGGCGGTGTACAGGCTGCAGGCGGCGACGGTGGCCGCCGGGATACTGCTGGTGCTCTACTACAGGGCGACGCGCGTGCCGGCCGCCGGCGAAGGGCGGGCGGCGTGGCTGGGGATGGCGGCGGCGGAGCTCTGGTTCGCCGTCTACTGGGTCATCACGCAGTCCGTCCGGTGGTGCCCCGTCCGCCGCCGCACCTTCAAGAACAGGCTCGCCGAGAGATACAAAGAAAATCTACCTGGTGTGGATGTTTTTGTATGCACTGCAGACCCACACGCAGAGCCACCAAGCCTTGTCATCTCTACCATCCTATCAGTCATGGCATACAATTACCCATCAGAAAAAATAAGTGTGTATCTTTCTGATGATGGTGGTTCAATTCTTACTTTCTACGCTCTATGGGAGGCATCCATGTTTGCAAAGAAATGGCTACCATTCTGCAGAAGATATAACATTGAGCCAAGGTCCCCAGCTGGCTTACTTCTCGGAATCAGAAGGGCATCACAATCTGTGCTCCCCAAAAGAATGGTCATTTATCAAGAACCTGTATGA

[0040] SEQ ID NO: 10 (Truncated amino acid sequence of OsCSLE6ko3):

[0041] METTTTERRRLFATEKVGGRAVYRLQAATVAAGILLVLYYRATRVPAAGEGRAAWLGMAAAELWF AVYWVITQSVRWCPVRRRTFKNRLAERYKENLPGVDVFVCTADPHAEPPSLVISTILSVMAYNYPSEKI SVYLSDDGGSILTFYALWEASMFAKKWLPFCRRYNIEPRSPAGLLLGIRRASQSVLPKRMVIYQEPV*

[0042] SEQ ID NO: 11 (Amino acid sequence of normally expressed OsCSLE6):

[0043] *

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] This invention utilizes CRISPR / Cas9 technology to create OsCSLE6 loss-of-function mutant plants, which can improve the seedling survival rate of transgenic plants under drought stress and enhance drought resistance in mature plants. It can be applied to improve the drought resistance of cultivated rice. Attached Figure Description

[0046] Figure 1 This is the knockout mutant gene sequence of the OsCSLE6 gene created based on CRISPR / Cas9 technology in the embodiments of the present invention;

[0047] Figure 2 This is an example of the performance of the OsCSLE6 gene knockout mutant and wild type under 20% PEG6000 simulated drought stress in this invention embodiment;

[0048] Figure 3 This invention relates to the drought resistance performance of the OsCSLE6 gene knockout mutant and the wild-type drought-resistant mature plants in a greenhouse. Detailed Implementation

[0049] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0050] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used reagents used in the examples are commercially available products.

[0051] In this article, the terms "isolated" or "purified" DNA refer to DNA or fragments that have been separated from the sequences flanking them in their native state, and also to DNA or fragments that have been separated from the components that accompany nucleic acids in their native state, and from the proteins that accompany them in the cell.

[0052] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0053] The present invention also includes variants of the open reading frame sequence in SEQ ID NO:1 that encode a protein having the same function as OsCSLE6. 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.

[0054] This invention also includes variants of the SEQ ID NO:1 sequence having the same function as OsCSLE6. These variants include (but are not limited to): deletions, insertions, and / or substitutions 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.

[0055] The percentage of protein homology was determined by GAP (Needleman and Wunsh, 1970) analysis (GCG procedure), with parameters gap creation penalty = 5 and gap extension penalty = 0.3. When the analyzed sequence is at least 15 amino acids long, the GAP analysis is performed on regions of at least 15 amino acids in both sequences being tested. More preferably, when the analyzed sequence is at least 50 amino acids long, the GAP analysis is performed on regions of at least 50 amino acids in both sequences being tested. More preferably, when the analyzed sequence is at least 100 amino acids long, the GAP analysis is performed on regions of at least 100 amino acids in both sequences being tested. More preferably, when the analyzed sequence is at least 250 amino acids long, the GAP analysis is performed on regions of at least 250 amino acids in both sequences being tested. Even more preferably, when the analyzed sequence is at least 500 amino acids long, the GAP analysis is performed on regions of at least 500 amino acids in both sequences being tested.

[0056] Polynucleotides (DNA or RNA), vectors, transformants, and organisms can be isolated and purified using methods known in the art.

[0057] The polynucleotides isolated in this invention include, but are not limited to: the nucleotide sequence of SEQ ID NO:1 encoding the OsCSLE6 gene; or the nucleotide sequence that can hybridize with the nucleotide sequence from position 1 to 2187 in SEQ ID NO:1; or the subfraction that functions equivalent to the sequence shown in SEQ ID NO:1.

[0058] The cloned OsCSLE6 gene can be used as a probe to screen for the gene or homologous gene of this invention from cDNA and genomic libraries, or it can be synthesized directly using gene synthesis methods. Similarly, PCR (polymerase chain reaction) technology can be used to amplify the OsCSLE6 gene and any DNA segment or its homologous segment from the genome and cDNA.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 methods 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.

[0066] In this invention, the term "transformant" refers to a host cell or organism carrying a foreign DNA molecule.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The present invention will be described in detail below through embodiments:

[0071] Example 1: Construction and genetic transformation of the rice gene OsCSLE6 gene editing vector

[0072] 1) Construction of target gene editing vectors:

[0073] For the construction of the OsCSLE6 gene vector for gene editing using the CRISPR / Cas9 system, the sgRNA target was designed as 5'-GATTCCGAGAAGTAAGCAGCTGG-3' (SEQ ID NO:2) using the CRISPR-P 2.0 tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Then, the gene editing vector was constructed according to the methods provided by published CRISPR / Cas9 gene editing systems. Finally, the U6 promoter and sgRNA expression cassette were loaded into the expression vector pYLCRISPR / Cas9Pubi-H. The constructed plasmid was transformed into Agrobacterium EHA105 for rice callus transformation experiments.

[0074] 2) Rice genetic transformation

[0075] 2.1 Seed disinfection

[0076] After removing the husks from mature Nipponbare rice seeds, place them in sterile Erlenmeyer flasks and soak them in 75% alcohol for 1-2 minutes, then rinse twice with sterile water. Next, sterilize them with 3% NaClO for 30 minutes, shaking frequently during this time. Then rinse them 3-4 times with sterile water, blot off excess water with sterile filter paper, and inoculate approximately 30 seeds onto callus induction medium (NB + 2,4-D 3.0 mg / L) per dish. Incubate in the dark at 28°C.

[0077] 2.2 Subculture

[0078] After nearly a month of induction, rice plants developed yellow, swollen callus tissue. The scutellum was removed, and the callus was transferred to fresh callus induction medium (NB + 2,4-D 2.0 mg / L) for subculture. Subculture was performed every two weeks, and generally 2-4 subcultures were sufficient to obtain pale yellow, granular embryogenic callus tissue suitable for transgenic transformation. Two weeks after subculture, embryogenic granules were selected for genetic transformation.

[0079] 2.3 Culture of Agrobacterium

[0080] Pick a single colony from the transformation plate and culture it in 1 ml of Agrobacterium tumefaciens medium. Add 1 ml of the above culture to 50 ml of Agrobacterium tumefaciens medium (containing the appropriate antibiotic), and incubate at 200 rpm and 28°C for 5-6 hours until the OD600 reaches 0.6-1.0. Two hours before the end of the incubation, add acetosringone (AS, final concentration 100 uM). Take the above bacterial suspension, incubate at 4000 rpm for 10 minutes at room temperature, discard the supernatant, and resuspend the bacterial cells in MS liquid medium (containing 100 uM AS). Incubate under the same conditions as above for 2 hours until the OD600 of the bacterial suspension is 0.5-1. At this point, it can be used to transform callus tissue.

[0081] 2.4 Co-culture

[0082] Rice embryogenic callus was immersed in Agrobacterium tumefaciens solution for 20-30 minutes, then the moisture was absorbed with sterile absorbent paper. The infected callus was then placed on co-culture medium (MS + 2,4-D 2.0 mg / L + AS 100 uM) and incubated in the dark at 28°C for three days.

[0083] 2.5 Washing with bacteria

[0084] The co-cultured callus tissue was first rinsed with sterile water 3-5 times, then soaked in MS liquid medium containing 400 mg / L Cef for 20-30 minutes, and then transferred to sterile filter paper to dry.

[0085] 2.6 Screening and Culture

[0086] The dried callus tissue was inoculated onto selective medium (NB + 2,4-D 2.0 mg / L + Hyg 30 mg / L + Cef 400 mg / L). After 3 weeks, newly grown callus tissue was selected and inoculated onto selective medium (NB + 2,4-D 2.0 mg / L + Hyg 50 mg / L + Cef 250 mg / L), and then selected for another 2 weeks.

[0087] 2.7 Differentiation Culture

[0088] The resistant callus tissues obtained after two rounds of screening were transferred to pre-differentiation medium (N6 + KT 2.0 mg / L + NAA 0.2 mg / L + 6-BA 2.0 mg / L + Hyg 30 mg / L + Cef 200 mg / L + agar 9 g / L + sucrose 45 g / L) and cultured in the dark for about 10 days. Then, they were transferred to differentiation medium (N6 + KT 2.0 mg / L + NAA 0.2 mg / L + 6-BA 2.0 mg / L + Hyg 30 mg / L + agar 4.5 g / L + sucrose 30 g / L) and cultured under light.

[0089] 2.8 Rooting Culture

[0090] After about 1-2 months, the seedlings, which are about 2cm tall, are transferred to rooting medium (1 / 2 MS + Hyg 15mg / L + Agar 4.5g / L + Sucrose 20g / L) to induce the formation of adventitious roots.

[0091] 2.9 Transplantation of transgenic seedlings

[0092] 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. Cover them with a glass cover for a few days at first, and remove the glass cover after the plants are strong. Cultivate them in a greenhouse.

[0093] Example 2: Screening of OsCSLE6 gene-editing mutants

[0094] For T0 generation single plants with CRISPR / Cas9-edited OsCSLE6 gene, first-generation sequencing was used for identification. Genomic DNA was extracted from T0 generation single plants using a rapid DNA extraction method. Primers casOsCSLE6-F:5'-CCACCAAGCCTTGTCATCTC-3' (SEQ ID NO:3) and casOsCSLE6-R:5'-TACCGAAACCTAGGGGAACC-3' (SEQ ID NO:4) were designed targeting the sgRNA editing site to amplify fragments covering the editing site region. The PCR products were sequenced. Multiple sequence alignment was performed using the Nipponbare target site sequence as a reference sequence and all amplified editing site regions to identify homozygous single plants that underwent genome editing. Based on the sequencing results, such as Figure 1The multiple sequence alignment results shown indicate the following mutation types: insertion of one base A at the target site of sgRNA (OsCSLE6ko1, SEQ ID NO:5), leading to a frameshift mutation and premature transcript termination, resulting in a 201-amino acid polypeptide (SEQ ID NO:6); insertion of one base T (OsCSLE6ko2, SEQ ID NO:7), leading to a frameshift mutation and premature transcript termination, resulting in a 201-amino acid polypeptide (SEQ ID NO:8); and insertion of one base G (OsCSLE6ko3, SEQ ID NO:9), leading to a frameshift mutation and premature transcript termination, resulting in a 201-amino acid polypeptide (SEQ ID NO:10). The amino acid sequences resulting from these three mutations differ by one amino acid. These sequences are inconsistent with the protein sequence obtained from normal expression of the OsCSLE6 gene (SEQ ID NO:1) (SEQ ID NO:11).

[0095] Example 3: OsCSLE6 gene regulates drought resistance in rice seedlings

[0096] The T2 generation lines OsCSLE6ko1, OsCSLE6ko2, and OsCSLE6ko3, homozygous mutants of the OsCSLE6 gene editing from Example 2, were selected. Drought stress was simulated using PEG-6000 (polyethylene glycol) to identify the seedling drought resistance of the mutants and wild-type materials. The specific steps were as follows: rice seeds were soaked, germinated, planted on cultivation racks, and hydroponically cultured in rice nutrient solution until the three-leaf stage. Then, rice nutrient solution containing 20% ​​PEG-6000 was added for 10 days, followed by rehydration for 2 days, and the survival rate was then calculated. The results showed that after drought stress treatment (… Figure 2 The survival rate of wild-type plants was 49.9%; the survival rates of the three mutant lines were 85.4%, 89.6%, and 85.4%, respectively. The survival rate of OsCSLE6 mutant plants was significantly higher than that of wild-type plants, indicating that knocking out the OsCSLE6 gene can significantly improve the drought resistance of rice seedlings.

[0097] Example 4: OsCSLE6 gene regulates drought resistance in adult rice plants.

[0098] The T2 generation lines OsCSLE6ko1, OsCSLE6ko2, and OsCSLE6ko3, homozygous mutants of the OsCSLE6 gene editing from Example 2, were selected and planted at the Lingshui transgenic base in Hainan. Two treatments, water and drought, were implemented. Transplanting began one month after seedling raising, and the drought-treated materials were subjected to drought stress 20 days after transplanting. After maturity, the yields under water and drought conditions were measured to evaluate drought resistance. The study found that under normal paddy field conditions, the yield per plant of the OsCSLE6 mutant was not different from that of the wild-type plant; however, under drought conditions, the yield per plant of the OsCSLE6 mutant was significantly higher than that of the wild-type plant, indicating that OsCSLE6 negatively regulates rice drought resistance. Figure 3 ).

[0099] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The application of the OsCSLE6 gene in regulating drought resistance in rice, characterized by: The OsCSLE6 gene in rice was knocked out using the CRISPR / Cas9 gene editing system, thereby causing the OsCSLE6 protein to lose its function and thus improving the drought resistance of rice; the nucleotide sequence of the OsCSLE6 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The knockout of the OsCSLE6 gene is achieved by site-specific editing of the OsCSLE6 gene using a CRISPR / Cas9 system guided by sgRNA; the target sequence of the sgRNA is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that: The OsCSLE6 gene knockout mutant is mutant transcript 1, mutant transcript 2, or mutant transcript 3; The mutant transcript 1 gene sequence is shown in SEQ ID NO:5, the mutant transcript 2 gene sequence is shown in SEQ ID NO:7, and the mutant transcript 3 gene sequence is shown in SEQ ID NO:9.

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