Application of OsIAA29 gene or protein encoded by OsIAA29 gene in regulation and control of salt tolerance of rice

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an OsIAA29 gene or a protein encoded by the OsIAA29 gene in regulation and control of rice salt tolerance, and the sequence of the rice OsIAA29 gene is selected from a nucleotide sequence as shown in SEQ ID NO.1; or a sequence which encodes the protein sequence as shown in SEQ ID NO.2 and has more than 90% of homology with the nucleotide sequence as shown in SEQ ID NO.1. The rice OsIAA29 gene is knocked out, so that the salt tolerance of rice is improved, and the survival rate of rice in a salt stress environment is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of the OsIAA29 gene or its encoded protein in regulating the salt tolerance of rice. Background Art

[0002] Land salinization refers to the process by which salts in the soil substratum or groundwater rise to the surface with capillary water and accumulate in the surface layer after evaporation. This process is also known as salinization or soil salination. Soluble salts include sulfates, chlorides, and carbonates of sodium, potassium, calcium, and magnesium. Soil salinization is considered saline when its salt content exceeds 0.3%. Factors contributing to land salinization include both natural and human factors. Natural factors include the influence of climatic conditions: during droughts, evaporation in arid areas exceeds precipitation, causing salt to migrate to the surface with water and accumulate. Topographical factors include poor drainage in low-lying areas, which can lead to salt accumulation and retention in runoff. Groundwater influences include shallow, highly mineralized groundwater transporting salt to the surface through capillary action. In coastal environments, seawater intrusion or erosion can increase soil salinity. Human factors include inappropriate irrigation, sewage disposal, development, and improper farm management.

[0003] Rice (Oryza sativa L.) is one of China's most important staple crops, and rice production is crucial to China's food security. However, soil salinization is a major threat to agricultural development. During rice production, salt stress severely impacts rice growth and development, and consequently, rice yield. Therefore, exploring pathways to regulate salt tolerance in rice is of great significance. Summary of the Invention

[0004] To explore new approaches to regulating rice salt tolerance, the present invention provides the use of the OsIAA29 gene or its encoded protein in regulating rice salt tolerance. By knocking out the OsIAA29 gene in rice, the present invention improves rice salt tolerance and increases rice survival rate under salt stress.

[0005] The present invention provides a use of a rice OsIAA29 gene or a protein encoded thereof in regulating salt tolerance in rice, wherein the sequence of the rice OsIAA29 gene is selected from:

[0006] The nucleotide sequence shown in SEQ ID NO.1;

[0007] or, a sequence encoding the protein sequence shown in SEQ ID NO.2 and having a homology of more than 90% with the nucleotide sequence shown in SEQ ID NO.1;

[0008] The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0009] The present invention improves the salt tolerance of rice by knocking out the OsIAA29 gene of rice, thereby increasing the survival rate of rice under salt stress environment.

[0010] Furthermore, the application approach is: improving the salt tolerance of rice by knocking out the OsIAA29 gene or down-regulating the expression of the rice OsIAA29 gene.

[0011] Furthermore, knockout of the OsIAA29 gene was achieved by designing sgRNA and constructing a CRISPR / Cas9 gene editing vector.

[0012] Furthermore, the sgRNA target sequence is shown as SEQ ID NO.3.

[0013] The present invention also provides a CRISPR / Cas9 gene editing vector, which is obtained by loading the sgRNA target sequence expression cassette shown in SEQ ID NO.3 into the expression vector pYLCRISPR / Cas9Pubi-H.

[0014] The present invention also provides a genetically engineered bacterium containing the CRISPR / Cas9 gene editing vector.

[0015] The present invention also provides a use of the CRISPR / Cas9 gene editing vector or the genetically engineered bacteria in regulating rice salt tolerance, wherein the CRISPR / Cas9 gene editing vector is used to knock out the OsIAA29 gene.

[0016] The present invention also provides a method for improving the salt tolerance of rice, comprising the following steps:

[0017] Design the sgRNA target sequence for the OsIAA29 gene and construct a CRISPR / Cas9 gene editing vector;

[0018] Construction of genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors;

[0019] The genetically engineered bacteria containing the CRISPR / Cas9 gene editing vector were transformed into rice to obtain a rice mutant strain with the OsIAA29 gene knockout.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This study screened and identified a rice gene, OsIAA29. Mutant plants generated by gene editing to knock out OsIAA29 showed significantly improved salt tolerance at the seedling stage, demonstrating that this gene negatively regulates salt tolerance in rice and can be used to improve salt tolerance in cultivated rice. The OsIAA29 loss-of-function mutant plants, created using CRISPR / Cas9 technology, can improve the survival rate of transgenic plants at the seedling stage under salt stress conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The performance of the OsIAA29 gene knockout mutant and the wild type under 150 mM NaCl stress;

[0024] In the figure, A shows the survival of wild-type and mutant rice before NaCl stress treatment;

[0025] B shows the survival of wild-type and mutant rice after NaCl stress treatment;

[0026] C is the effect of NaCl stress treatment on the survival rate of wild-type and mutant rice. DETAILED DESCRIPTION

[0027] 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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention, unless otherwise specified, 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 according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0028] In the present invention, the terms "isolated" and "purified" DNA mean that the DNA or fragment has been separated from the sequences on both sides of it in its natural state, and also mean that the DNA or fragment has been separated from the components that accompany the nucleic acid in its natural state and has been separated from the proteins that accompany it in the cell.

[0029] The present invention also encompasses variants of the open reading frame sequence that encode proteins with the same functions as OsIAA29. These variants include, but are not limited to, deletions, insertions, and / or substitutions of several nucleotides (generally 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10), and additions of several nucleotides (generally within 60, preferably within 30, more preferably within 10, and most preferably within 5) to the 5' and / or 3' ends.

[0030] The present invention also encompasses variants of sequences with the same function as OsIAA29. 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, and most preferably 1-10), as well as the addition of one or more amino acids (typically within 20, preferably within 10, and more preferably within 5) to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function.

[0031] The vector used in the present invention can be, for example, a phage, plasmid, cosmid, minichromosome, virus, or retroviral vector. Vectors that can be used to clone and / or express the polynucleotides of the present invention are vectors that can replicate and / or express the polynucleotides in the host cells in which the polynucleotides are to be replicated and / or expressed. Generally speaking, recombinant expression vectors carrying the nucleic acid sequences of the present invention can be introduced into plant cells using conventional biotechnology 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).

[0032] A variety of methods have been developed for operatively linking polynucleotides to vectors via complementary cohesive ends. For example, complementary homopolymeric sequence fragments can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then linked by hydrogen bonding between the complementary homopolymeric tails to form a recombinant DNA molecule.

[0033] Synthetic linkers containing one or more restriction sites offer another method for ligating DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or Escherichia coli DNA polymerase I. These polymerases use their 3' and 5' exonucleolytic activities to remove protruding gamma-single-stranded ends and their polymerization activities to fill in 3'-recessed ends. The combined activities thus produce blunt-ended DNA segments. These blunt-ended segments are then incubated with a molar excess of linker molecules in the presence of an enzyme that catalyzes the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. The resulting reaction products are DNA segments with polymerase linker sequences at their ends. These DNA segments are then cleaved with appropriate restriction enzymes that produce ends compatible with the DNA segments and ligated into expression vectors that have been cleaved with enzymes that produce ends compatible with the DNA segments. Synthetic linkers containing multiple restriction endonuclease sites are commercially available from various vendors.

[0034] Other newly developed technologies utilize homologous recombination methods to homologously recombine a polynucleotide carrying a specific sequence linker or a homologous sequence linker with a vector, and to form a recombinant DNA molecule by combining the DNA segment to be inserted into the vector DNA with the vector also carrying the specific sequence or homologous sequence through the action of recombinase.

[0035] The polynucleotide insert should be operably linked to an appropriate promoter compatible with the host cell in which the polynucleotide is expressed. The promoter may be a strong promoter and / or an inducible promoter. Examples of some promoters include the bacteriophage PL promoter, the E. coli lac, trP, phoA, tac promoters, the SV40 early and late promoters, and the 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 a ribosome binding site for translation in the transcribed region. The coding portion of the transcript expressed by the recombinant vector may include a translation initiation codon at the start and a termination codon (UAA, UGA, or UAG) appropriately located at the end of the translated polypeptide.

[0036] As described above, the expression vector can include at least one selectable marker. Such markers include genes encoding antibiotic resistance, such as the neomycin phosphotransferase gene nptII, the hygromycin phosphotransferase gene hpt, and the dihydrofolate reductase gene dhfr; and 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, protoplasts and plant cells. Appropriate culture media and culture conditions for the above-mentioned host cells are known in the art.

[0037] Transformation methods for target genes or polynucleotides: One type is vector-mediated transformation, in which the target gene is inserted into a vector molecule such as an Agrobacterium plasmid or viral DNA. Transfer of the vector DNA then introduces the target gene into the plant genome; Agrobacterium-mediated and viral-mediated methods fall under this method. The second type is direct gene transfer, which involves directly introducing the exogenous target gene into the plant genome through physical or chemical methods. Physical methods include gene gun transformation, electroporation, ultrasound, microinjection, and laser microbeam; chemical methods include PEG-mediated transformation and liposome-mediated transformation. The third type is germplasm system methods, which include pollen tube channel, germ cell infection, and embryo sac and ovary injection.

[0038] In the present invention, the term "transformant" is used to refer to a host cell or organism carrying a heterologous DNA molecule.

[0039] The present invention also includes host cells containing the nucleotide sequences of the present invention operably linked to one or more heterologous control regions (e.g., promoters and / or enhancers) using techniques known in the art. Host strains can be selected that are capable of regulating the expression of the inserted gene sequence or modifying and processing the gene product in a specific manner as desired. Expression from certain promoters is increased in the presence of certain inducers.

[0040] Successfully transformed cells, ie, cells or organisms containing a recombinant vector containing the nucleotide sequence of the present invention, can be identified by well-known techniques.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the related listed items. The present invention is described in detail below by way of examples:

[0042] Example 1: Construction of gene editing vector and genetic transformation of rice OsIAA29 gene.

[0043] 1. Construction of gene editing vector

[0044] The nucleotide sequence of the rice OsIAA29 gene is shown in SEQ ID NO.1, and the protein sequence encoded by the gene is shown in SEQ ID NO.2.

[0045] SEQ ID NO.1:

[0046] ATGAAGGATAGGAATGCTTCTGCTGAGCCTGTGGTGAAGCCAGGATTATCACCATCAAGATTTGTGAAGGTTTTCATGCATGGGGAACCCTTTGGGAGGAAGATCAATTTGGCTCTCCATAACAACTAT GACTCTTTGTCCTTCACTTTAAAAAAATTGGGCAACAACTATTCCATGTCACCATTTGAACTTGAAGGCTTGGTGAACAAGGAAGAAGATGGTGCAATAGACAGCGACTTTGACCTCTTGTATGATGAT ATGGATGGTGTTCGTTACTTCCTTGGTGATGTCCCATGGGAGGTTTTCACCACTACAGTGAAAAAGATCTACATTGTCCCTGCAGAACAACAGAATGAAAATGATTATCAGGAGGAGGAAGAGGACAAC GCGGCTGCTGCTGCTACTGCTGACGAAGATGGAGATGGAGCTGCTGCTGACGATGGCGTTGCTGCTGCAGCTGACGACGTTGACGATGTTGCTGGATACACGAGCAACGATGACCCAAGCTTCGACTGA.

[0047] SEQ ID NO.2:

[0048] MKDRNASAEPVVKPGLSPSRFVKVFMHGEPFGRKINLALHNNYDSLSFTLKKLGNNYSMSPFELEGLVNKEEDGAIDSDFDLLYDDMDGVRYFLGDVPWEVFTTTVKKIYIVPAEQQNENDYQEEEEDNAAAAATADEDGDGAAADDGVAAAADDVDDVAGYTSNDDPSFD.

[0049] The sgRNA target was designed using the CRISPR-P 2.0 tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ): 5'-GCTGAGCCTGTGGTGAAGCCAGG-3' (SEQ ID NO. 3). The gene editing vector was then constructed according to the published methods of the 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 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 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 in rice callus.

[0050] 2. Rice genetic transformation

[0051] (1) Seed disinfection

[0052] Mature Nipponbare rice seeds were shelled and placed in a sterile Erlenmeyer flask. The seeds were soaked in 75% alcohol for 2 min and rinsed twice with sterile water. They were then sterilized with 3% NaClO for 30 min with frequent shaking. The seeds were then washed four times with sterile water. Excess water was absorbed with sterile filter paper. The seeds were inoculated onto the first callus induction medium for induction culture, with approximately 30 seeds per dish, and cultured in the dark at 28°C.

[0053] The first callus induction medium formula is: NB+2,4-D 3.0 mg / L.

[0054] (2) Subculture

[0055] After nearly a month of induction culture, the rice plants develop yellow, swollen calli. The scutellum is removed and the calli are transferred to a second callus induction medium for subculture. Subculture occurs every two weeks. After three subcultures, bright yellow, granular embryonic calli suitable for genetic transformation are obtained.

[0056] The formula of the second callus induction medium is: NB+2,4-D 2.0 mg / L.

[0057] (3) Cultivation of Agrobacterium

[0058] A single colony was picked from the transformation plate and cultured in 1 mL of LB medium containing 50 mg / L Kan and 20 mg / L rifampicin at 200 rpm and 28°C to obtain a culture. 1 mL of the culture was inoculated into 50 mL of LB medium containing 50 mg / L Kan and 20 mg / L rifampicin and cultured at 200 rpm and 28°C until the OD 600 The bacterial solution was 0.7, and acetosyringone (AS, acetosringone, final concentration 100 μM) was added 2 h before the end of the culture. The above bacterial solution was centrifuged at 4000 rpm for 10 min at room temperature, the supernatant was discarded, and the bacteria were resuspended in MS liquid medium containing AS (AS final concentration 100 μM), and cultured at 200 rpm and 28 ° C for 2 h to make the OD of the bacterial solution 600 =1, obtain Agrobacterium culture solution.

[0059] (4) Co-culture

[0060] Rice embryonic callus was immersed in OD 600 = 1 Agrobacterium solution for 25 minutes, then dry the water with sterile absorbent paper, place the infected rice embryonic callus on the co-cultivation medium, and culture it in the dark at 28℃ for three days.

[0061] The co-culture medium formula is: MS+2,4-D 2.0 mg / L+AS 100 μM.

[0062] (5) Sterilization

[0063] After co-cultivation, the rice embryonic callus was first washed four times with sterile water and then immersed in MS liquid medium containing 400 mg / L cephalosporin (Cef) for 25 minutes. The callus was then transferred to sterile filter paper and dried.

[0064] (6) Screening and culture

[0065] The dried callus was inoculated on the first selection medium. After 3 weeks, the newly grown callus was selected and inoculated on the second selection medium and selected for another 2 weeks to obtain resistant callus.

[0066] The first selection culture medium formula is: NB+2,4-D 2.0 mg / L+Hyg 30 mg / L+Cef 400 mg / L.

[0067] The second selection culture medium formula is: NB+2,4-D 2.0 mg / L+Hyg 50 mg / L+Cef 250 mg / L.

[0068] (7) Differentiation culture

[0069] The resistant callus obtained after the second screening was transferred to the pre-differentiation medium and cultured in the dark at 26°C for about 10 days, and then transferred to the differentiation medium and cultured in the light at 24°C for 1 month to obtain seedlings. The light culture conditions were 12 hours / 12 hours in the dark, and the light intensity was 7000 lμx.

[0070] The formula of the pre-differentiation medium is: 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.

[0071] The differentiation medium formula is: 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.

[0072] (8) Rooting culture

[0073] The seedlings, about 2 cm in height, were transferred to rooting medium to induce the formation of adventitious roots.

[0074] Rooting medium formula: 1 / 2MS + Hyg 15 mg / L + agar 4.5 g / L + sucrose 20 g / L.

[0075] (9) Transplantation of transgenic seedlings

[0076] When the seedlings grew to 10 cm in height, they were taken out, the attached solid culture medium was washed with sterile water, and they were transplanted into soil for planting to obtain T0 generation plants.

[0077] Example 2: Screening of OsIAA29 gene-edited mutants.

[0078] First-generation sequencing was used to identify T0-generation individuals of the OsIAA29 gene that had been edited with CRISPR / Cas9. Genomic DNA from T0-generation individuals was extracted using a rapid DNA extraction method. The cas OsIAA29-F primer (SEQ ID NO. 4) and the cas OsIAA29-R primer (SEQ ID NO. 5) were designed to flank the sgRNA editing site shown in SEQ ID NO. 3. Amplified fragments encompassing the editing site were then sequenced.

[0079] cas OsIAA29-F: 5'-CCCGCTTACTTCCATCATGT-3' (SEQ ID NO.4);

[0080] cas OsIAA29-R: 5'-CCAAAATGCATCATTATTTCTCC-3' (SEQ ID NO. 5).

[0081] The target site sequence of Nipponbare rice was used as a reference sequence to perform multiple sequence alignment with all amplified editing site regions to identify homozygous plants that underwent genome editing.

[0082] According to the sequencing results, the mutation types are as follows:

[0083] (1) A base A is inserted into the target site of the sgRNA (position 37 of the nucleotide sequence shown in SEQ ID NO. 1), forming the mutant OsIAA29ko1 shown in SEQ ID NO. 6, abbreviated as KO1. This mutation causes a frameshift mutation, leading to premature termination of the transcript, and forming a 40-amino acid polypeptide shown in SEQ ID NO. 7.

[0084] (2) Three T bases are deleted from positions 35 to 37 of the nucleotide sequence shown in SEQ ID NO. 1, forming the OsIAA29ko2 mutant shown in SEQ ID NO. 8, abbreviated as KO2. This mutation results in one amino acid deletion and one amino acid mutation, forming a 170-amino acid polypeptide shown in SEQ ID NO. 9, which is inconsistent with the protein sequence (SEQ ID NO. 2) obtained by normal expression of the OsIAA29 gene coding region sequence (SEQ ID NO. 1).

[0085] SEQ ID NO.6:

[0086] ATGAAGGATAGGAATGCTTCTGCTGAGCCTGTGGTGAAAGCCAGGATTATCACCATCAAGATTTGTGAAGGTTTTCATGCATGGGGAACCCTTTGGGAGGAAGATCAATTTGGCTCTCCATAACAACTATGACTCTTTGTCCTTCACTTTAAAAAAATTGGGCAACAACTATTCCATGTCACCATTTGAACTTGAAGGCTTGGTGAACAAGGAAGAAGATGGTGCAATAGACAGCGACTTTGACCTCTTGTATGATGATATGGATGGTGTTCGTTACTTCCTTGGTGATGTCCCATGGGAGGTTTTCACCACTACAGTGAAAAAGATCTACATTGTCCCTGCAGAACAACAGAATGAAAATGATTATCAGGAGGAGGAAGAGGACAACGCGGCTGCTGCTGCTACTGCTGACGAAGATGGAGATGGAGCTGCTGCTGACGATGGCGTTGCTGCTGCAGCTGACGACGTTGACGATGTTGCTGGATACACGAGCAACGATGACCCAAGCTTCGACTGA。

[0087] SEQ ID NO.7:

[0088] MKDRNASAEPVVKARIITIKICEGFHAWGTLWEEDQFGSP。

[0089] SEQ ID NO.8:

[0090] ATGAAGGATAGGAATGCTTCTGCTGAGCCTGTGGAGCCAGGATTATCACCATCAAGATTTGTGAAGGTTTTCATGCATGGGGAACCCTTTGGGAGGAAGATCAATTTGGCTCTCCATAACAACTATGA CTCTTTGTCCTTCACTTTAAAAAAATTGGGCAACAACTATTCCATGTCACCATTTGAACTTGAAGGCTTGGTGAACAAGGAAGAAGATGGTGCAATAGACAGCGACTTTGACCTCTTGTATGATGATAT GGATGGTGTTCGTTACTTCCTTGGTGATGTCCCATGGGAGGTTTTCACCACTACAGTGAAAAAGATCTACATTGTCCCTGCAGAACAACAGAATGAAAATGATTATCAGGAGGAGGAAGAGGACAACG CGGCTGCTGCTGCTACTGCTGACGAAGATGGAGATGGAGCTGCTGCTGACGATGGCGTTGCTGCTGCAGCTGACGACGTTGACGATGTTGCTGGATACACGAGCAACGATGACCCAAGCTTCGACTGA.

[0091] SEQ ID NO.9:

[0092] MKDRNASAEPVEPGLSPSRFVKVFMHGEPFGRKINLALHNNYDSLSFTLKKLGNNYSMSPFELEGLVNKEEDGAIDSDFDLLYDDMDGVRYFLGDVPWEVFTTTVKKIYIVPAEQQNENDYQEEEEDNAAAAATADEDGDGAAADDGVAAAADDVDDVAGYTSNDDPSFD.

[0093] Example 3: Application study of OsIAA29 gene in regulating salt tolerance at the rice seedling stage.

[0094] In this example, the T2 generation strains of the OsIAA29 gene-edited homozygous mutants OsIAA29ko1 and OsIAA29ko2 in Example 2 were selected, and salt stress was carried out using NaCl to identify the salt tolerance of the mutants and the wild-type materials at the seedling stage.

[0095] The specific steps are as follows: soak the rice seeds in pure water, germinate at 30°C for 40 hours, plant them on a culture rack, and use Yoshida rice nutrient solution for hydroculture until the three-leaf and one-heart stage. Add rice nutrient solution containing 150mM NaCl for 13 days, and then change to normal Yoshida rice nutrient solution for 10 days before calculating the survival rate.

[0096] The results are as follows Figure 1 As shown, after NaCl stress treatment, the survival rate of wild-type plants (WT) was 5.56%; the survival rate of transgenic non-mutated plants (WT) was 8.33%; and the survival rates of the two mutant lines were 37.50% (KO1) and 36.11% (KO2), respectively. Therefore, the survival rate of OsIAA29 mutant plants was significantly higher than that of wild-type plants, indicating that knocking out the OsIAA29 gene can significantly improve salt tolerance in rice seedlings.

[0097] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art understand the basic inventive concepts.

[0098] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Use of the OsIAA29 gene or its encoded protein in regulating salt tolerance in rice, characterized in that: The sequence of the rice OsIAA29 gene is selected from: The nucleotide sequence shown in SEQ ID NO.1; or, a sequence encoding the protein shown in SEQ ID NO. 2 and having a homology of more than 90% with the nucleotide sequence shown in SEQ ID NO. 1; The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The use of the OsIAA29 gene or its encoded protein in regulating salt tolerance in rice according to claim 1, characterized in that: The application approach is: improving the salt tolerance of rice by knocking out the OsIAA29 gene or down-regulating the expression of the rice OsIAA29 gene.

3. The use of the OsIAA29 gene or its encoded protein in regulating salt tolerance in rice according to claim 1, characterized in that: Knockout of the OsIAA29 gene was achieved by designing sgRNA and constructing a CRISPR / Cas9 gene editing vector.

4. The use of the OsIAA29 gene or its encoded protein in regulating salt tolerance in rice according to claim 3, characterized in that: The sgRNA target sequence is shown in SEQ ID NO.

3.

5. A CRISPR / Cas9 gene editing vector, characterized in that: The sgRNA target sequence expression cassette shown in SEQ ID NO. 3 of claim 4 is loaded into the expression vector pYLCRISPR / Cas9Pubi-H.

6. A genetically engineered bacterium containing the CRISPR / Cas9 gene editing vector according to claim 5.

7. Use of the CRISPR / Cas9 gene editing vector according to claim 5 or the genetically engineered bacteria according to claim 6 in regulating salt tolerance of rice, characterized in that: The CRISPR / Cas9 gene editing vector is used to knock out the OsIAA29 gene.

8. A method for improving salt tolerance of rice, characterized in that: The steps include: Designing the sgRNA target sequence of the OsIAA29 gene and constructing the CRISPR / Cas9 gene editing vector according to claim 5; Construction of genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors; The genetically engineered bacteria containing the CRISPR / Cas9 gene editing vector were transformed into rice to obtain a rice mutant strain with the OsIAA29 gene knockout.

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