Application of protein OsDIF in regulating drought resistance and Azospirillum brasilense colonization of rice root system

Abstract

This invention discloses the application of the protein OsDIF in regulating drought resistance and root colonization of *Azotobacter brasiliensis* in rice. The amino acid sequence of the protein OsDIF is shown in SEQ ID No. 1. Experiments have demonstrated that it improves homozygous mutant strains. Osdif‑1 The expression level of the protein OsDIF can improve drought resistance and root colonization of *Azotobacter brasiliensis*; while using a protein containing... OsDIF Recombinant Agrobacterium with a gene knockout vector transformed the rice variety Ishikari Shiro-mae, enabling it to... OsDIF Gene editing occurs when two homologous chromosomes... OsDIF When all genes mutate, the activity of the protein OsDIF is lost; this loss of OsDIF activity results in transgenic rice with reduced root colonization of *Azotomyces brasiliensis* and decreased drought resistance. Therefore, the protein OsDIF can regulate drought resistance and root colonization of *Azotomyces brasiliensis* in rice. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of protein OsDIF in regulating drought resistance in rice and the colonization of Azotobacter brasiliensis in the roots. Background Technology

[0002] Drought is the most significant environmental factor affecting plant growth and crop yield. It has become a serious problem impacting agricultural production. Improving crop drought resistance and enhancing the adaptability of crops and cash crops to adverse conditions through genetic engineering are crucial and critical issues that urgently need to be addressed in new variety breeding. In recent years, extensive research has been conducted on the mechanisms of plant responses to drought and other abiotic stresses from physiological, biochemical, metabolic, ecological, genetic, and evolutionary perspectives, accumulating a wealth of data. In particular, with the development of molecular biology, researchers have been able to understand the mechanisms of plant resistance to drought stress at the molecular level, including gene composition, expression regulation, and signal transduction, opening new avenues for improving plant stress resistance through genetic engineering. Due to the complexity of plant stress resistance traits, improving plant stress resistance using traditional breeding methods is extremely difficult. While genetic engineering has opened new avenues for plant stress resistance breeding with the development of molecular biology, the isolation of highly efficient stress-resistant genes remains a major limiting factor in plant stress resistance genetic engineering.

[0003] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, with more than half of the global population relying on it as their staple food. Developing high-yield and drought-resistant rice varieties is an important measure to ensure national food security, and the discovery of drought-resistant genes is the cornerstone of drought-resistant molecular breeding. Summary of the Invention

[0004] The purpose of this invention is to improve plant drought resistance and / or root microbial colonization.

[0005] This invention first protects the application of the protein OsDIF, which may be at least one of S1)-S3):

[0006] S1) Regulates plant drought resistance; S2) Regulates the colonization of plant root microorganisms; S3) Cultivate transgenic plants with altered drought resistance and / or altered root microbial colonization.

[0007] In the above applications, the protein OsDIF can be a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; a2) A protein that has more than 90% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; a3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of a1) or a2).

[0008] SEQ ID No.1 consists of 471 amino acid residues.

[0009] To facilitate the purification of the protein in a1), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein shown in SEQ ID No. 1.

[0010] Table 1. Sequence of Labels

[0011] The protein in a2) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0012] The proteins mentioned in a2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The gene encoding the protein in a2) above can be obtained by deleting one or more amino acid residues from the codons of the DNA sequence shown in SEQ ID No. 2 or SEQ ID No. 3, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0014] This invention also protects the application of nucleic acid molecules encoding any of the aforementioned proteins OsDIF or biological materials containing said nucleic acid molecules, which may be at least one of S1)-S3): S1) Regulates plant drought resistance; S2) Regulates the colonization of plant root microorganisms; S3) Cultivate transgenic plants with altered drought resistance and / or altered root microbial colonization.

[0015] In the above applications, the nucleic acid molecule encoding the protein OsDIF can be a DNA molecule as shown in b1), b2), b3), or b4): b1) The coding region is the DNA molecule shown in SEQ ID No. 3; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3 or SEQ ID No. 2; b3) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the protein OsDIF; b4) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes the protein OsDIF.

[0016] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0017] SEQ ID No. 2 consists of 2331 nucleotides, SEQ ID No. 3 consists of 1416 nucleotides, and the nucleotides shown in SEQ ID No. 3 or SEQ ID No. 2 encode the amino acid sequence shown in SEQ ID No. 1.

[0018] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein OsDIF of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the protein OsDIF isolated according to this invention, as long as they encode the protein OsDIF, are derived from and equivalent to the nucleotide sequence of this invention.

[0019] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0020] In the above applications, the biomaterial may be at least one of the following: D1) An expression cassette containing the nucleic acid molecule; D2) A recombinant vector containing the nucleic acid molecule or a recombinant vector containing the expression cassette; D3) Recombinant microorganisms containing the nucleic acid molecule, recombinant microorganisms containing the expression cassette, or recombinant microorganisms containing the recombinant vector; D4) A plant containing the nucleic acid molecule, a plant containing the expression cassette, or a plant containing the recombinant vector; the plant is a plant cell, plant tissue, and / or plant organ.

[0021] In any of the above-described applications, the regulation of plant drought resistance can be used to increase or decrease plant drought resistance.

[0022] In any of the above applications, the regulation of plant root microbial colonization can be used to promote or inhibit plant root microbial colonization.

[0023] The present invention also protects a method for cultivating transgenic plant A, which may include the following steps: increasing the expression level and / or activity of any of the proteins OsDIF described above in the starting plant to obtain transgenic plant A; compared with the starting plant, the transgenic plant A has improved drought resistance and / or improved root microbial colonization.

[0024] In the above method, the "increasing the expression level and / or activity of any of the above-mentioned proteins OsDIF in the starting plant" can be achieved by methods well known in the art, such as transgenic technology, multiple copying, altering promoters, and regulatory factors, to increase the expression level and / or activity of any of the above-mentioned proteins OsDIF in the starting plant.

[0025] In the above method, the "increasing the expression level and / or activity of any of the above-mentioned proteins OsDIF in the starting plant" can be specifically achieved by introducing a nucleic acid molecule encoding any of the above-mentioned proteins OsDIF into the starting plant.

[0026] In the above method, the "introduction of a nucleic acid molecule encoding any of the above-mentioned proteins OsDIF into the starting plant" can be achieved by introducing a recombinant vector into the starting plant; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any of the above-mentioned proteins OsDIF into an expression vector.

[0027] The recombinant vector may specifically be the recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF mentioned in the embodiments.

[0028] The genetically modified plant A mentioned in Example 2 may specifically be... Co#1 and Co#5 The starting plant at this time is rice, specifically the homozygous mutant strain mentioned in the examples. Osdif-1 .

[0029] The present invention also protects a method for cultivating transgenic plant B, comprising the following steps: reducing the expression level and / or activity of any of the proteins OsDIF described above in the starting plant to obtain transgenic plant B; compared with the starting plant, the drought resistance and / or root microbial colonization of transgenic plant B are reduced.

[0030] In the above method, the reduction of the expression level and / or activity of any of the proteins OsDIF in the starting plant can be achieved by methods well known in the art, such as DNA insertion, RNA interference, homologous recombination, and site-directed gene editing.

[0031] Specifically, the reduction of the expression level and / or activity of any of the aforementioned proteins OsDIF in the starting plant can be achieved by mutating the encoding gene of any of the aforementioned proteins OsDIF in the starting plant.

[0032] The gene encoding any of the aforementioned proteins OsDIF can be a DNA molecule as shown in b1), b2), b3), or b4): b1) The coding region is the DNA molecule shown in SEQ ID No. 3; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3 or SEQ ID No. 2; b3) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the protein OsDIF; b4) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes the protein OsDIF.

[0033] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0034] SEQ ID No. 2 consists of 2331 nucleotides, SEQ ID No. 3 consists of 1416 nucleotides, and the nucleotides shown in SEQ ID No. 3 or SEQ ID No. 2 encode the amino acid sequence shown in SEQ ID No. 1.

[0035] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein OsDIF of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the protein OsDIF isolated according to this invention, as long as they encode the protein OsDIF, are derived from and equivalent to the nucleotide sequence of this invention.

[0036] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0037] In the above method, the gene encoding the protein OsDIF in the mutant-initiating plant can be the one shown in SEQ ID No. 2. OsDIF Gene mutation OsDIF / -4bp or OsDIF / -818bp.

[0038] The OsDIF / -4bp is a DNA molecule obtained by deleting four nucleotides from position 28 to 31 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged.

[0039] The OsDIF / -818bp is a DNA molecule obtained by deleting 818 nucleotides from position 30 to 847 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged.

[0040] In the above method, the gene encoding the protein OsDIF in the mutant starting plant is introduced into the starting plant via a CRISPR / Cas9 system. The CRISPR / Cas9 system may include a recombinant expression vector containing a DNA molecule expressing gRNA targeting the gene encoding any of the aforementioned proteins OsDIF.

[0041] In the above method, the target sequence of the gRNA may be as shown in SEQ ID No. 2, positions 15-37 from the 5' end.

[0042] This invention also protects the application of substances encoding genes for any of the aforementioned proteins OsDIF in mutant plants, which may be at least one of S1)-S3): S1) Regulates plant drought resistance; S2) Regulates the colonization of plant root microorganisms; S3) Cultivate transgenic plants with altered drought resistance and / or altered root microbial colonization.

[0043] In the above applications, the gene encoding any of the aforementioned proteins OsDIF in the mutant-initiating plant can be the one shown in SEQ ID No. 2. OsDIF Gene mutation OsDIF / -4bp or OsDIF / -818bp.

[0044] The OsDIF / -4bp is a DNA molecule obtained by deleting four nucleotides from position 28 to 31 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged.

[0045] The OsDIF / -818bp is a DNA molecule obtained by deleting 818 nucleotides from position 30 to 847 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged.

[0046] In the above applications, the material encoding the gene for any of the aforementioned proteins OsDIF in the mutant-originating plant can be a CRISPR / Cas9 system. The CRISPR / Cas9 system may include a recombinant expression vector; the recombinant expression vector contains a DNA molecule expressing gRNA targeting the gene encoding any of the aforementioned proteins OsDIF. Preferably, the target sequence of the gRNA may be as shown in SEQ ID No. 2, positions 15-37 from the 5' end.

[0047] The plant described above may be any one of the following c1) to c5): c1) a dicotyledonous plant; c2) a monocotyledonous plant; c3) a grass; c4) rice; c5) the rice variety Ishikari Shiroge.

[0048] Any of the microorganisms mentioned above may be *Azotobacter brasiliensis* (… Azospirillum brasilense ).

[0049] The improved drought resistance mentioned above can be manifested as a reduction in leaf curling after drought treatment.

[0050] The improved drought resistance mentioned above can be manifested as an increase in biomass and / or root fresh weight after drought treatment.

[0051] The reduced drought resistance mentioned above can be manifested as increased leaf curling after drought treatment.

[0052] The reduction in drought resistance mentioned above can be manifested as a decrease in SPAD value, biomass, root fresh weight, and / or number of adventitious roots after drought treatment.

[0053] Experiments have shown that using ingredients containing OsDIFRecombinant Agrobacterium with a gene knockout vector transformed the rice variety Ishikari Shiro-mae, enabling it to... OsDIF Gene editing, OsDIF After genes are edited using the CRISPR / Cas9 restriction enzyme, it can cause... OsDIF Gene mutation, when two homologous chromosomes... OsDIF When all genes mutate, it can lead to the loss of OsDIF protein activity; the loss of OsDIF protein activity results in the formation of root-borne Azotobacter brasiliensis (…). Azospirillum brasilense Transgenic rice with reduced colonization and drought resistance. Simultaneously, it increases homozygous mutant strains. Osdif-1 The expression level of the protein OsDIF in the medium can improve drought resistance and the root system of *Azotobacter brasiliensis* (…). Azospirillum brasilense Colonization. This demonstrates that the protein OsDIF can regulate drought resistance in rice and the colonization of *Azotrophus brasiliensis* (a type of bacteria) in the roots. Azospirillum brasilense Colonization. This invention has significant application value.

[0054] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0055] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid sequence or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence can be calculated by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residuegap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search, thus obtaining the identity value (%). Alternatively, sequence analysis software such as CLC MainWorkbench and MegAlign can be used. TM The determination can be performed, for example, using a computer program BLAST with default parameters, especially BLASTP or TBLASTN. The 90% or higher identity mentioned herein can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.

[0056] The term "conservative substitution" generally refers to the replacement of one amino acid residue with another amino acid residue in a side chain that has similar physicochemical properties. For example, conservative substitutions can occur between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conserved substitutions generally do not cause significant changes in protein conformation and structure, and essentially do not alter the protein's biological activity. Conservative substitutions in the protein sequence that are expected to have only a minimal or no effect on protein structure or function can be readily designed by those skilled in the art.

[0057] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "comprising" and "including" are used interchangeably.

[0058] "Transgenic plant" refers to a plant whose genome has been altered by integrating or inserting recombinant DNA molecules, constructs, cassettes, or sequences for the expression of non-coding RNA molecules, mRNA, and / or proteins. Transgenic plants include R0 generation plants developed or regenerated from initially transformed plant cells and their offspring, or plants obtained by crossing with R0 generation transgenic plants containing recombinant DNA molecules, constructs, cassettes, or sequences. Plants with integrated or inserted recombinant DNA molecules, constructs, cassettes, or sequences are considered transgenic plants, even if the plant also has other mutations or edits that are not themselves considered transgenic.

[0059] "Plant cell" is the biological cell of a plant, which is taken from the plant or derived from a culture obtained by culturing cells taken from the plant.

[0060] "Transgenic plant cell" refers to any plant cell transformed with a stably integrated recombinant DNA molecule, construct, cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0061] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0062] The drought resistance comparisons are conducted under comparable conditions. "Comparable conditions" refer to the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly promote or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus). Attached Figure Description

[0063] Figure 1 homozygous mutant strain Osdif-1 The mutation type.

[0064] Figure 2 To detect homozygous mutant strains Osdif-1 and homozygous mutants Osdif-2 Azotobacter brasiliensis (root) Azospirillum brasilense The colonization status of (n=6) was analyzed. Data are presented as mean ± SD, and student's t-test was used for difference analysis.

[0065] Figure 3 To detect homozygous mutant strains Osdif-1 Drought resistance. Data are presented as mean ± SD (n=5), and student's t-test was used for difference analysis.

[0066] Figure 4 For qRT-PCR detection Co#1 and Co#5 middle OsDIF The relative expression level of genes.

[0067] Figure 5 For testing Co#1 and Co#5 Azotobacter brasiliensis (root) ​ The colonization status of (n=6) was analyzed. Data are presented as mean ± SD, and student's t-test was used for difference analysis.

[0068] ​ For testing ​ and ​ Drought resistance. Data are presented as mean ± SD (n=5), and student's t-test was used for difference analysis. Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0072] In rice genomic DNA, ​ The nucleotide sequence of the gene is shown in SEQ ID No. 2. In rice cDNA, ​ The nucleotide sequence of the gene is shown in SEQ ID No. 3. ​ The gene encodes the protein OsDIF. The amino acid sequence of protein OsDIF is shown in SEQ ID No. 1.

[0073] Example 1: Inhibiting the expression of the protein OsDIF can reduce the drought resistance of rice and the root-borne *Azotobacter brasiliensis* (…). ​ colonization one, ​ Construction of gene knockout vector 1. Using the formula shown in SEQ ID No. 2 ​ The gene's nucleotide sequence was used for target design on the website, and suitable targets were selected based on their location and targeting specificity. The final target sequence obtained was: 5'-GCCGGCTTCCCACGCCGCCGCGG-3' (i.e., positions 15-37 from the 5' end of SEQ ID No. 2), corresponding to the target gene. ​ Gene.

[0074] 2. Using 5'-GCCGGCTTCCCACGCCGCCGCGG-3' as the target, the following method was described in the literature (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, Guo J, Chen L, Zhao X, Dong Z, Liu YG. (2015) A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Mol Plant 8:1274-1284). ​ Gene knockout vector.

[0075] II. Obtaining OsDIF homozygous mutants Because rice is a diploid plant, when Cas9 begins to edit specific genes, both alleles on two homologous chromosomes within the same cell can potentially be edited, resulting in the same or different types of mutations. Therefore, two alleles in a single plant are considered two gene editing events. A homozygous mutant refers to a plant whose two homologous chromosomes... ​ The genes have undergone the same mutation. A biallelic mutant refers to a plant whose two homologous chromosomes have undergone the same mutation. ​ All genes underwent mutations, but the forms of mutation differed. The number of heterozygous mutant strains refers to the number of homozygous chromosomes on one of the plant's two homozygous chromosomes. ​ A gene mutation occurred, on another homologous chromosome ​ No gene mutation occurred. Wild type refers to the type of chromosome between the two homologous chromosomes of this plant. ​ No gene mutations were observed.

[0076] 1. ​Gene knockout vectors were introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Subsequently, using Agrobacterium-mediated genetic transformation (described in the following literature: Deng, M., Hu, B., Xu, L., Liu, Y., Wang, F., Zhao, H., Wei, X., Wang, J., and Yi, K. (2014). OsCYCP1;1, a PHO80 homologous protein, negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.). Plant Mol Biol 86, 655-669.), the recombinant Agrobacterium was transferred into the rice variety Ishikari Shiramo to obtain T0 generation transgenic rice.

[0077] 2. Genomic DNA was extracted from the leaves of T0 generation transgenic rice and used as a template. PCR amplification was performed using a CRISPR-Cas9 vector-specific primer pair consisting of 5'-GCCAGATCTCCCCTCCCCTT-3' and 5'-AGGCTATGCACGCCGAGGAA-3', yielding the PCR amplification product. The following determination was then made: if the PCR amplification product of a T0 generation transgenic rice contained a DNA fragment of 440 bp, then the T0 generation transgenic rice was a transgenic positive seedling.

[0078] 3. Self-pollinate the transgenic positive seedlings obtained in step 2. The resulting seeds are T1 generation seeds, and the plants grown from T1 generation seeds are T1 generation plants.

[0079] 4. Using genomic DNA from leaves of rice cultivars Ishikari Hakumo or T1 generation plants as templates, PCR amplification was performed using primer pairs consisting of 5'-GCCAGATCTCCCCTCCCCTT-3' and 5'-AGGCTATGCACGCCGAGGAA-3', respectively, to obtain the corresponding PCR amplification products. The PCR amplification products were then sequenced. Sequencing results were compared with... ​ The Cas9 target sequence of the gene is compared, and the mutation types are counted.

[0080] Some test results can be found ​ (WT stands for Ishikari Hakuge rice variety) ​ homozygous mutant strain ​ , ​ homozygous mutant strain ​ The results showed that two homozygous OsDIF mutant strains were obtained, and they were named homozygous mutant strains. ​ and homozygous mutants ​Homozygous mutant strain ​ and homozygous mutants ​ Two homologous chromosomes ​ All genes have undergone mutations. Specifically: ​ On two homologous chromosomes ​ The gene has a 4-nucleotide deletion of “GCCG” (i.e., a 4-nucleotide deletion between positions 28 and 31 from the 5' end of SEQ ID No. 2), which causes a frameshift, premature termination of the encoded protein, and loss of function of the OsDIF protein; ​ On two homologous chromosomes ​ The gene has a deletion of 818 nucleotides (i.e., 818 nucleotides between positions 30 and 847 from the 5' end of SEQ ID No. 2), resulting in the loss of function of the OsDIF protein.

[0081] III. Detection of homozygous mutant strains ​ and homozygous mutants ​ Azotobacter brasiliensis (root) ​ colonization status The experiment was repeated three times, and the average value was taken. Six rice seedlings were tested each time. The steps for each repetition were as follows: 1. Select plump rice plants (Ishikari Shiro-mae rice variety, homozygous mutant strain). ​ or homozygous mutant ​ 2 After peeling the seeds, add clean water and soak them at 37℃ until they show signs of sprouting (2-3 days), changing the water morning and evening during this period. Sow the sprouted rice seeds on nylon mesh containing rice nutrient solution and culture them at 30℃ / 22℃ with alternating light and dark conditions (i.e., alternating 12h light culture and 12h dark culture, with a light intensity of 3000 Lux) for 7 days to obtain rice seedlings.

[0082] The complete nutrient solution for rice is described in the following literature: Lei Xu, Hongyu Zhao, Wenyuan Ruan, Minjuan Deng, Fang Wang, Jinrong Peng, Jie Luo, Zhixiang Chen, and Keke Yi. (2017) ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice. The Plant Cell, Vol. 29: 560–574. The pH of the complete nutrient solution for rice is approximately 5.5.

[0083] 2. After completing step 1, transfer rice seedlings with basically uniform growth to soil and randomly divide them into four groups. Each group will undergo the following treatment: Drought treatment group: After transplanting into soil, the plants were irrigated normally for 14 days, after which watering was stopped and drought treatment was carried out until the drought phenotype appeared.

[0084] Drought treatment + inoculation group: After transplanting into the soil, the plants were irrigated and cultured normally for 7 days; then inoculated with *Azotomyces brasiliensis* (…). ​ Continue culturing for 7 days, then inoculate each rice seedling with *Azotomyces brasiliensis* (…). ​ Approximately 10 5 CFU; then stop watering and allow the plant to dry until the drought phenotype appears.

[0085] Conventional treatment group: During the cultivation period, the rice seedlings were irrigated normally and kept submerged.

[0086] Standard treatment + inoculated group: After transplanting the soil, normal irrigation and culture were carried out for 7 days; then inoculated with *Azotomyces brasiliensis* (…). ​ Continue culturing for 7 days, then inoculate each rice seedling with *Azotomyces brasiliensis* (…). ​ Approximately 10 5 CFU; then continue normal irrigation, keeping the seedlings submerged throughout the process.

[0087] Brazilian azospira ( ​ This is a product of BNCC (BeNa Culture Collection), with product catalog number BNCC361938.

[0088] 3. Detection of *Azotobacter brasiliensis* in rice roots (…) ​ Colonization status After completing step 2, the roots of each rice seedling were first immersed in a 75% (v / v) ethanol aqueous solution for 1 minute (for surface sterilization), and then washed 4 times with sterile double-distilled water. Then, they were washed with a 0.45% (m / v) NaCl aqueous solution and continuously diluted. The diluted solution was inoculated onto LB solid medium and incubated at 37°C for 24 hours. The bacterial colonies were then counted and expressed as CFUs / g FW.

[0089] Test results are shown ​ (SSBM refers to the rice variety Ishikari Shiroge) ​ homozygous mutant strain ​ , ​ homozygous mutant strain ​ CK represents those not inoculated with Azotobacter brasiliensis ( ​ ), + ​ To inoculate with Azotobacter brasiliensis ( ​ The results showed that inhibiting the expression of the protein OsDIF significantly reduced the expression of *Azotobacter brasiliensis* in rice roots. ​ ) colonization.

[0090] IV. Detection of homozygous mutant strains ​ drought resistance The experiment was repeated three times and the average value was taken. Six rice seedlings were tested each time. The steps for each repetition were as follows: When the drought phenotype appeared in the drought treatment group in step 3 (i.e., the leaves of the rice seedlings began to curl), the SPAD value of each rice seedling was measured with a SPAD instrument. The biomass and fresh weight of the rice seedlings and the number of indeterminate roots were weighed and counted. Then the average value was calculated for each group.

[0091] Test results are shown ​ (SSBM refers to the rice variety Ishikari Shiroge) ​ homozygous mutant strain ​ In A and D, CK (-) or (-) indicates no inoculation with *Azotocinobacter brasiliensis*. ​ ), 10 5 (+) or (+) indicates inoculation with *Azotomyces brasiliensis* ( ​ Of B, C, E, and F, CK represents the uninoculated strain of *Azotomyces brasiliensis*. ​ ), 10 5 To inoculate with Azotobacter brasiliensis ( ​ (A and D represent growth phenotypes, B represents biomass, C represents SPAD value, E represents root fresh weight, and F represents adventitious root number). The results showed that inhibiting the expression of the protein OsDIF significantly reduced the drought resistance of rice. The reduced drought resistance was manifested in more severe leaf curling after drought treatment, and varying degrees of reduction in SPAD value, biomass, root fresh weight, and adventitious root number.

[0092] Example 2: Overexpression of protein OsDIF improves drought resistance and root-derived *Azotobacter brasiliensis* (Azotobacter spp.) ​ colonization I. Construction of recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF 1. Extract genomic DNA from the leaves of the rice variety Nipponbare, i.e., Nipponbare genomic DNA.

[0093] 2. Using Nipponbare genomic DNA as a template, 5'-CGC was employed. ​ ATGGACGCGGCGTGGCGCGGCG-3' (underlined is the recognition site of the restriction endonuclease BamHI) and 5'-ACGC ​ PCR amplification was performed using a primer pair consisting of GAATTCGGGCAGCTTCTGGAGG-3' (the underlined part is the recognition site of the restriction endonuclease SalI), and the approximately 4690 bp PCR amplification product A was recovered using a gel extraction kit (QIAGEN).

[0094] 3. Digest the PCR amplification product A recovered in step 2 with restriction endonucleases BamHI and SalI, and recover the digested fragment 1.

[0095] 4. Using the vector p35S-GFP (NovoPro product, catalog number V009750) as a template, 5'-ACGC was used. ​ atggtgagcaagggcgagga-3' (underlined is the recognition site of the restriction endonuclease SalI) and 5'-AA ​ PCR amplification was performed using a primer pair consisting of TCACTTGTACAGCTCGTCCATG-3' (the underlined part indicates the recognition site of the restriction endonuclease PstI). The approximately 717 bp PCR amplification product B was recovered using a gel extraction kit. PCR amplification product B is the GFP sequence.

[0096] 5. Digest the PCR amplification product B recovered in step 4 with restriction endonucleases SalI and PstI, and recover the digested fragment 2.

[0097] 6. The pCAMBIA1300 vector (a product of Abcam, catalog number ab275754) was digested with restriction endonucleases SalI and PstI to recover approximately 9000 bp of the vector backbone.

[0098] 7. Use T4 DNA ligase (NEB) to ligate the enzyme digestion product 2 recovered in step 5 and the vector backbone 1 recovered in step 6 to obtain the recombinant plasmid pCAMBIA1300-EGFP.

[0099] 8. The recombinant plasmid pCAMBIA1300-EGFP was digested with restriction endonucleases BamHI and SalI, and the vector backbone of approximately 9000 bp was recovered.

[0100] 9. The enzyme fragment 1 recovered in step 3 and the vector backbone 2 recovered in step 8 were ligated using T4 DNA ligase (NEB) to obtain the recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF.

[0101] The recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF was sequenced. Sequencing results showed that the recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF was obtained by replacing a small DNA fragment between the restriction endonucleases BamHI and SalI in the pCAMBIA1300-EGFP vector with a DNA fragment whose nucleotide sequence is shown in SEQ ID No. 4.

[0102] The recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF expresses the protein OsDIF with the amino acid sequence shown in SEQ ID No. 1.

[0103] II. Transfer ​ The acquisition of genetically modified rice 1. The recombinant plasmid pCAMBIA1300-proOsDIF-OsDIF was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Then, Agrobacterium-mediated transformation was performed (described in the following literature: Deng, M., Hu, B., Xu, L., Liu, Y., Wang, F., Zhao, H., Wei, X., Wang, J., and Yi, K. (2014). OsCYCP1;1, a PHO80 homologous protein, negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.). Plant Mol Biol 86, 655-669.) to transform the recombinant Agrobacterium into a homozygous mutant strain. ​ Obtain T0 transfer ​ Genetically modified rice.

[0104] 2. Extract T0 transfer ​Genomic DNA from rice leaves was used as a template, and PCR amplification was performed using primer pairs consisting of 5'-CGTAAACGGCCACAAGTTCAG-3' and 5'-TCACTTGTACAGCTCGTCCAT-3' to obtain the PCR amplification product. Then, the following judgment was made: If a certain T0 generation... ​ If the PCR amplification product of genetically modified rice contains a 650bp DNA fragment, then the T0 generation of transgenic rice... ​ The genetically modified rice is a positive transgenic seedling.

[0105] A total of 10 transgenic positive seedlings were obtained through testing.

[0106] 3. qRT-PCR detection of transgenic positive seedlings ​ Relative expression level of genes (1) Total RNA was extracted from the roots or aboveground parts of 10 transgenic positive seedlings and rice variety Ishikari Shiro, and then reverse transcribed to obtain cDNA.

[0107] (2) Using the cDNA obtained in step (1) as a template, qRT-PCR was performed to detect the cDNA. ​ Relative gene expression levels (in terms of) Actin (Genes are used as internal references).

[0108] Detection OsDIF The primers for the gene are: 5'-CCAAGCTTGTGGCTTATCCTT-3' and 5'-AAGAACTGACCTGCCACAGC-3'.

[0109] Detection Actin The primers for the gene are 5'-CTGGTATCGTGTTGGACTCTGG-3' and 5'-CCCGTTCAGCAGTGGTAGT-3'.

[0110] Some test results can be found Figure 4 (WT stands for the rice variety Ishikari Shiroge). Results showed that, compared to the rice variety Ishikari Shiroge, 4 out of 10 transgenic positive seedlings... OsDIF The relative expression level of the gene was significantly increased. Two of these four plants were randomly selected and named... Co#1 and Co#5 That is, turn OsDIF Genetically modified rice.

[0111] III. Testing Co#1 and Co#5 Azotobacter brasiliensis (root) Azospirillum brasilense colonization status The experiment was repeated three times, and the average value was taken. Six rice seedlings were tested each time. The steps for each repetition were as follows: 1. Select plump rice (rice variety Ishikari Hakuge, etc.) Co#1 or Co#5 After peeling the seeds, add clean water and soak them at 37℃ until they show signs of sprouting (2-3 days), changing the water morning and evening during this period. Sow the sprouted rice seeds on nylon mesh containing rice nutrient solution and culture them at 30℃ / 22℃ with alternating light and dark conditions (i.e., alternating 12h light culture and 12h dark culture, with a light intensity of 3000 Lux) for 7 days to obtain rice seedlings.

[0112] 2. After completing step 1, transfer rice seedlings with basically uniform growth to soil and randomly divide them into four groups. Each group will undergo the following treatment: Drought treatment group: After transplanting into soil, the plants were irrigated normally for 14 days, after which watering was stopped and drought treatment was carried out until the drought phenotype appeared.

[0113] Drought treatment + inoculation group: After transplanting into the soil, the plants were irrigated and cultured normally for 7 days; then inoculated with *Azotomyces brasiliensis* (…). Azospirillum brasilense Continue culturing for 7 days, then inoculate each rice seedling with *Azotomyces brasiliensis* (…). Azospirillum brasilense Approximately 10 5 CFU; then stop watering and allow the plant to dry until the drought phenotype appears.

[0114] Conventional treatment group: During the cultivation period, the rice seedlings were irrigated normally and kept submerged.

[0115] Standard treatment + inoculated group: After transplanting the soil, normal irrigation and culture were carried out for 7 days; then inoculated with *Azotomyces brasiliensis* (…). Azospirillum brasilense Continue culturing for 7 days, then inoculate each rice seedling with *Azotomyces brasiliensis* (…). Azospirillum brasilense Approximately 10 5 CFU; then continue normal irrigation, keeping the seedlings submerged throughout the process.

[0116] 3. Detection of *Azotobacter brasiliensis* in rice roots (…) Azospirillum brasilense Colonization status After completing step 2, the roots of each rice seedling were first immersed in a 75% (v / v) ethanol aqueous solution for 1 minute (for surface sterilization), and then washed 4 times with sterile double-distilled water. Then, they were washed with a 0.45% (m / v) NaCl aqueous solution and continuously diluted. The diluted solution was inoculated onto LB solid medium and incubated at 37°C for 24 hours. The bacterial colonies were then counted and expressed as CFUs / g FW.

[0117] Test results are shown Figure 5 (SSBM is the rice variety Ishikari Shirofumi, CK is rice without inoculation with Azotobacter brasiliensis) Azospirillum brasilense ), + Azospirillum To inoculate with Azotobacter brasiliensis ( Azospirillum brasilenseThe results showed that overexpression of the protein OsDIF significantly increased the levels of *Azotobacter brasiliensis* in rice roots. Azospirillum brasilense ) colonization.

[0118] IV. Testing Co#1 and Co#5 drought resistance The experiment was repeated three times and the average value was taken. Five rice seedlings were tested each time. The steps for each repetition were as follows: When the drought phenotype appeared in the drought treatment group in step 3 (i.e., the leaves of the rice seedlings began to curl), the biomass and root fresh weight of the rice seedlings were weighed; then the average value was calculated for each group.

[0119] Test results are shown Figure 6 (SSBM is the rice variety Ishikari Shiroge, Co is...) Co#1 In A and C, CK represents the uninoculated strain of *Azotobacter brasiliensis* (…). Azospirillum brasilense ), + Azospirillum To inoculate with Azotobacter brasiliensis ( Azospirillum brasilense In B and D, CK represents the uninoculated strain of *Azotomyces brasiliensis* (…). Azospirillum brasilense ), + Azospirillum To inoculate with Azotobacter brasiliensis ( Azospirillum brasilense (A and C represent growth phenotypes, B represents biomass, and D represents root fresh weight). The results showed that overexpression of the protein OsDIF significantly improved the drought resistance of rice. The improved drought resistance was manifested in reduced leaf curling and varying degrees of increase in biomass and root fresh weight after drought treatment.

[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of protein OsDIF is at least one of S1)-S3): S1) Regulates plant drought resistance; S2) Regulates the colonization of plant root microorganisms; S3) Cultivate transgenic plants with altered drought resistance and / or altered root microbial colonization; The protein OsDIF is a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; a2) A protein that has more than 90% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; a3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of a1) or a2).

2. The application of a nucleic acid molecule encoding the protein OsDIF of claim 1 or a biological material containing said nucleic acid molecule, comprising at least one of S1)-S3): S1) Regulates plant drought resistance; S2) Regulates the colonization of plant root microorganisms; S3) Cultivate transgenic plants with altered drought resistance and / or altered root microbial colonization.

3. The application according to claim 1 or 2, characterized in that: The regulation of plant drought resistance refers to either increasing or decreasing plant drought resistance. The regulation of plant root microbial colonization refers to either promoting or inhibiting plant root microbial colonization.

4. A method for cultivating transgenic plant A, comprising the following steps: increasing the expression level and / or activity of the protein OsDIF as described in claim 1 in the starting plant to obtain transgenic plant A; compared with the starting plant, the transgenic plant A has improved drought resistance and / or improved root microbial colonization.

5. The method according to claim 4, characterized in that: The increase in the expression level and / or activity of the protein OsDIF of claim 1 in the starting plant is achieved by introducing a nucleic acid molecule encoding the protein OsDIF of claim 1 into the starting plant.

6. A method for cultivating transgenic plant B, comprising the following steps: reducing the expression level and / or activity of the protein OsDIF as described in claim 1 in the starting plant to obtain transgenic plant B; compared with the starting plant, the transgenic plant B has reduced drought resistance and / or reduced root microbial colonization.

7. The method according to claim 6, characterized in that: The reduction of the expression level and / or activity of the protein OsDIF in claim 1 in the starting plant is achieved by mutating the gene encoding the protein OsDIF in the starting plant; The gene encoding the protein OsDIF in the mutant plant is shown in SEQ ID No.

2. OsDIF Gene mutation OsDIF / -4bp or OsDIF / -818bp; The OsDIF / -4bp is a DNA molecule obtained by deleting four nucleotides from position 28 to 31 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged; The OsDIF / -818bp is a DNA molecule obtained by deleting 818 nucleotides from position 30 to 847 starting from the 5' end of SEQ ID No.2, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged.

8. The method according to claim 7, characterized in that: The gene encoding the protein OsDIF in the mutant starting plant was introduced into the starting plant via the CRISPR / Cas9 system. The CRISPR / Cas9 system includes a recombinant expression vector containing a DNA molecule that expresses gRNA encoding a gene that targets the protein OsDIF. Preferably, the target sequence of the gRNA is shown in positions 15-37 from the 5' end of SEQ ID No.

2.

9. The application according to any one of claims 1 to 3 or the method according to any one of claims 4 to 8, characterized in that: The plant is any one of the following c1) to c5): c1) dicotyledonous plants; c2) monocotyledonous plants; c3) grasses; c4) rice; c5) rice variety Ishikari Shiroge.

10. The application according to any one of claims 1 to 3 or the method according to any one of claims 4 to 8, characterized in that: The microorganism is *Azotobacter brasiliensis* (… Azospirillum brasilense ).

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