Rice transcription factor OsGATA16 and application of rice transcription factor OsGATA16 in regulation and control of rice blast resistance of rice

Abstract

The invention is suitable for the technical field of molecular biology, and provides a rice transcription factor OsGATA16 and application thereof in regulation and control of rice blast resistance of rice. The method comprises the following steps: firstly, cloning a rice OsGATA16 gene, then constructing an overexpression vector and a mutant vector, and transforming rice to obtain a corresponding strain. Research finds that after a mutant strain with OsGATA16 knocked out is inoculated with germs, the number of scabs is obviously smaller than that of a wild type control group, the disease resistance of rice can be accurately regulated and controlled, and the rice blast resistance is obviously improved. The regulation and control mode can effectively reduce pesticide use and reduce environmental pollution and residual risk. Meanwhile, the successfully constructed stable genetic regulation material can be stored for a long time and used for multiple times in molecular and phenotype levels through verification. Besides, the OsGATA16 gene can be used as a disease-resistant molecular marker or a functional gene to be applied to various breeding systems, a new target is provided for breeding new rice materials with high rice blast resistance, and the OsGATA16 gene has a wide practical application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a rice transcription factor OsGATA16 and an application thereof in regulating rice resistance to rice blast. Background Art

[0002] Rice blast, a disease caused by the blast fungus Pyricularia oryzae ( Magnaporthe oryzae Rice diseases caused by the genus Rhizoctonia solani (Rhizoctonia solani) have long been a major challenge for global rice production. Known for their rapid spread, strong mutation, and devastating damage, the disease can quickly spread across vast tracts of rice fields, causing severe yield losses or even complete crop failure, thus seriously threatening the safety and stability of global rice production.

[0003] In recent years, with the rapid development of molecular biology and genomics technologies, scientists have gradually recognized that transcription factors play a key role in crop disease resistance, achieving rapid disease resistance by regulating a series of downstream defense-related genes. Among them, GATA zinc-finger transcription factors, which bind to W-GATA-R (W=T / A, R=G / A) sequences in the promoters of their target genes, are widely present in plant growth and development and environmental responses, but their function in rice blast resistance has not been reported.

[0004] In existing rice blast control technologies, agricultural production relies heavily on chemical pesticides to control the disease due to a lack of effective disease-resistant materials. However, the long-term use of chemical pesticides not only increases production costs but also leads to the development of drug resistance in pathogens, thereby compromising control effectiveness. More seriously, the overuse of chemical pesticides poses a potential threat to the ecological environment and human health, sparking widespread concern and worry from all sectors of society. Therefore, the development of green and sustainable rice blast control technologies has become an urgent need in the fields of rice breeding and disease control. In the field of transcription factor research, although studies have revealed that families such as WRKY and MYB are involved in plant disease defense responses, the mechanism of action of rice GATA transcription factors in pathogen stress, particularly the function of OsGATA16 in regulating rice blast resistance, remains unclear. In light of this, the present invention proposes a rice transcription factor, OsGATA16, and its use in regulating rice blast resistance. Summary of the Invention

[0005] The purpose of the present invention is to provide a rice transcription factor OsGATA16 and its application in regulating rice blast resistance, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions: A rice transcription factor OsGATA16 or its encoding gene is used in regulating rice resistance to rice blast or breeding for rice blast resistance. The amino acid sequence of the rice transcription factor OsGATA16 is shown in SEQ ID No. 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1.

[0007] Furthermore, by knocking out the rice transcription factor gene OsGATA16 , to improve rice's resistance to rice blast.

[0008] Furthermore, the knockout is achieved by CRISPR / Cas9 method.

[0009] Application of a recombinant vector in regulating rice resistance to rice blast or breeding for rice blast resistance, the recombinant vector comprising a rice transcription factor gene OsGATA16 , whose nucleotide sequence is shown in SEQ ID No.1.

[0010] The invention discloses an application of a recombinant bacterium containing a recombinant vector in regulating the resistance of rice to rice blast or breeding rice blast-resistant varieties.

[0011] A method for regulating rice resistance to rice blast disease by regulating rice transcription factor genes in the following manner OsGATA16 Expression: Knockout or reduction OsGATA16 Gene expression to improve rice resistance to rice blast disease; or overexpression OsGATA16 genes to reduce rice's resistance to blast disease; The rice transcription factor gene OsGATA16 The nucleotide sequence is shown in SEQ ID No.1.

[0012] Furthermore, the knockout or reduction OsGATA16 Gene expression is achieved through CRISPR / Cas9 method; or overexpression OsGATA16 The gene is transferred into rice recipient material by constructing a recombinant expression vector.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly improve rice blast resistance: by constructing OsGATA16 Overexpression transgenic lines and knockout mutant lines were tested for rice blast infection, and the results showed that knockout OsGATA16 The mutant strain had significantly fewer lesions after inoculation with rice blast fungus than the wild-type control, indicating that knocking out this gene significantly enhanced rice's resistance to rice blast and achieved precise regulation of rice's disease resistance.

[0014] 2. Effectively reduce the use of pesticides, green and environmentally friendly: This invention uses the regulation of endogenous genes OsGATA16By regulating the expression level of the gene, rice disease resistance can be enhanced, and the occurrence of rice blast can be controlled without relying on exogenous chemical pesticides. This approach reduces the frequency of pesticide use in agricultural production, reduces environmental pollution and the risk of pesticide residues, and produces significant ecological benefits.

[0015] 3. Construction of stable and reliable genetic regulation materials: The present invention successfully constructed OsGATA16 Overexpression transgenic lines and CRISPR mutant lines, among which the overexpression transgenic lines OsGATA16 Expression levels are significantly higher than those of wild-type controls, and knockout mutant lines exhibit base deletions or insertions at the target site, leading to gene function ablation. These lines have been validated at both the molecular and phenotypic levels, with clear genetic backgrounds and stable traits, making them suitable for long-term storage and multiple uses.

[0016] 4. Ease of application in practical breeding: The target gene of the present invention OsGATA16 This gene can be used as a disease resistance molecular marker or as a functional gene introduced into other rice varieties or cultivated materials. It is applicable to conventional breeding, molecular breeding, and gene-editing breeding systems, and has broad practical application prospects. It provides a new molecular breeding target for the selection of new rice materials with broad-spectrum, long-lasting, and high resistance to rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 for OsGATA16 Knockout mutant lines ( GATA16 -CR1, GATA16 -CR2) sequencing results.

[0018] Figure 2 for OsGATA16 Overexpression transgenic lines ( GATA16 -OE1, GATA16- OE2) expression level identification results.

[0019] Figure 3 for OsGATA16 The blast infection phenotype of the knockout mutant line; where: a is OsGATA16 Scanned photos of leaves of knockout mutant lines, b is OsGATA16 Statistics of lesion area in knockout mutant lines. Lesion area = mean ± SD (n = 12). Asterisks indicate significant differences compared with wild-type ZH11. ****: p < 0.0001 (Student's t-test).

[0020] Figure 4 for OsGATA16 The blast infection phenotype of the overexpressing transgenic lines; where: a is OsGATA16 Scanning photos of leaves of overexpressing transgenic lines, b is OsGATA16Lesion area statistics for overexpressing transgenic lines. Lesion area = mean ± SD (n = 12). Asterisks indicate significant differences compared with wild-type KT. ***: p < 0.001, ****: p < 0.0001 (Student's t-test). DETAILED DESCRIPTION

[0021] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0022] The present invention provides a rice GATA transcription factor, OsGATA16 (LOC_Os06g37450), and its use in rice blast resistance. The OsGATA16 transcription factor is localized in the cell nucleus and can regulate the expression of downstream target genes, thereby affecting the rice immune response to the rice blast fungus.

[0023] OsGATA16 The gene contains 1,173 bases and encodes a 390-amino acid protein, OsGATA16. This protein contains a typical GATA zinc-finger domain and possesses typical DNA binding and transcriptional regulatory functions. OsGATA16 is localized in the nucleus and, in rice, belongs to the class II, group A, GATA transcription factor family.

[0024] OsGATA16 Gene nucleotide sequence:

[0025] OsGATA16 protein amino acid sequence: MSTIYMSQLSAALPLMEGEHHHHHQDHHQGHFQAFSLQPKDPPVLFPFVISRRSSSSSPSDSTTLSYGSDHHLTQQQQHQHQAMLEPQNMIGGSSAGIFATPFPTVKSIRDDMIERSQFDPYDTEKLQASCGLAKVVAGGKWSAVPAAKMKITRKMGEPSSGVTGGAATTVAPKKPRRRPAQAYEDHGHGGAMGQAF GVIRVCSDCNTTKTPLWRSGPCGPKSLCNACGIRQRKARRAMMASGLPASPNAAGPKAAAHSGAAAVAAAQPKVKKEKRADVDRSSLPFKKRCKVVQVEDHQTLPAATNAAAAAAMEETAESATVAPPPAPTTRGGTLVDSIGLSWSKTHAAATASCSFRPSPPVAPGFAAAVQDEITDAAMLLMTLSCGLVRS (as SEQ ID NO.2 shown).

[0026] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0027] Example 1: OsGATA16 Gene cloning; 1. RNA extraction; Wild-type ZH11 rice was hydroponically cultured with Kimura nutrient solution until the three-leaf stage at 28°C with 12h light and 25°C with 12h dark. RNA was extracted using the ReverTraAce qPCR RT Master Mix. The specific steps are as follows: (1) Take an appropriate amount of fresh rice material, put it into a liquid nitrogen pre-cooled mortar, add liquid nitrogen and quickly grind it into powder, then quickly transfer the powder to a pre-cooled 2 mL centrifuge tube; (2) Add 1 mL of TRIzol, mix thoroughly using a vortex shaker, and place in an ice box for 10 minutes; (3) Add 500 μL of chloroform, mix thoroughly using a vortex shaker, and let stand at room temperature for 10 min; (4) Centrifuge at 4°C, 12,000 rpm for 10 min. After centrifugation, remove 500 μL of the supernatant and transfer it to a 1.5 mL centrifuge tube. (5) Add 500 μL of isopropanol, mix by inversion, place on ice for 30 min, and centrifuge at 12,000 rpm at 4°C for 10 min. Discard the supernatant, wash the precipitate with 75% ethanol (prepared with DEPC water), and centrifuge again at 12,000 rpm at 4°C. (6) Aspirate the supernatant as much as possible, dry it at room temperature, and then dissolve it in 100 μL of DEPC water. Measure the RNA concentration using a UV spectrophotometer and store it in a -80°C refrigerator until use.

[0028] 2. mRNA reverse transcription; (1) Using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit, remove the RNA stored in a -80°C refrigerator and place it on ice to thaw. Calculate the volume required for 2 μg of total RNA based on the RNA concentration, add it to a PCR tube, and make up to 12 μL with water from the kit. React on a 65°C PCR instrument for 5 minutes and then quickly cool on ice for 2 minutes. (2) Add 4 μL of 4×DN Master Mix (mixed with gDNA Remover), mix well, and place in a 37°C PCR instrument for 5 minutes, then quickly cool on ice for 2 minutes; (3) Add 4 μL of 5×RT Master Mix II, mix well, and place on a PCR instrument for reaction. The reaction program is set as follows: 37°C, 30 min; 50°C, 5 min; 98°C, 5 min; 16°C, 5 min.

[0029] (4) After the reaction is completed, store the cDNA at -20°C for later use.

[0030] Example 2: OsGATA16 Construction of overexpression vector; Gene material source: RNA was extracted from rice variety ZH11 and reverse transcribed to obtain cDNA, and the target gene was amplified using specific primers. OsGATA16 CDS region; Vector construction: using infusion homologous recombination technology, OsGATA- The PCR product was homologously recombined with the linearized vector pc1300-35S-FLAG to construct the recombinant vector 35S- OsGATA16 ; Transformation process: The recombinant vector was transferred into Agrobacterium EHA105, and Kitaake rice callus was infected through Agrobacterium-mediated method to obtain resistant transformants, which were cultured to T3 generation to form stable genetic material.

[0031] 1. Design primers; According to the pc1300-35S-FLAG vector information, the KpnI+BamHI double restriction enzyme site was selected and the primers were designed as follows: GATA16-flag-F: ACGATGATAAGGGCGGTACCATGTCTACCATCTACATGAGTCAGC (as shown in SEQ IDNO.3); GATA16-flag-R: AGGCTACGTAGGATCCTCAGCTCCGGACAAGCCC (as shown in SEQ ID NO. 4).

[0032] 2. PCR Reaction and Vector Linearization: Perform PCR using the ZH11 cDNA template using the high-fidelity KOD enzyme protocol. Verify the PCR product by agarose gel electrophoresis. Simultaneously, double-digest the pc1300-35S-FLAG vector with KpnI and BamHI, and verify the product by agarose gel electrophoresis.

[0033] 3. Recovery of target fragment: Cut out the specific band obtained in the previous step and purify the DNA according to the steps of the gel recovery kit.

[0034] 4. Infusion reaction: According to the infusion product system, the purified target DNA fragment product is connected with the linearized vector.

[0035] 5. Transformation: Remove the ligation solution and store it at -20°C. Perform the transformation as follows: (1) Mix the ligation solution and DH5α competent cells and place them on ice for 30 minutes.

[0036] (2) Heat in a 42°C metal bath for 90 seconds and cool for 2 minutes.

[0037] (3) Add 500 μl of LB liquid culture medium and shake at 37°C and 220 rpm / min for 1 h.

[0038] (4) Centrifuge at 6000 rpm / min for 5 minutes, remove the supernatant and keep a small amount to mix well, then spread it on solid LB medium with corresponding resistance, and culture it upside down at 37°C overnight.

[0039] 6. Identification of positive clones: Pick single clones and perform colony PCR using GATA16-flag-F and GATA16-flag-R primers. Streak the colonies on kanamycin-resistant culture plates. Select colony PCR-positive clones for expansion and extract plasmids for sequencing.

[0040] 7. Select the correctly sequenced plasmid to transform Agrobacterium EHA105 and transform the rice variety Kitaake.

[0041] The results of identification of overexpression transgenic rice are shown in Figure 2 , overexpressing transgenic lines ( GATA16 -OE1, GATA16- OE2) OsGATA16 The expression level was significantly higher than that of wild-type KT control, which indicated that the overexpression transgenic lines OsGATA16 The expression level increased significantly, further proving that the overexpression transgenic line was successfully constructed.

[0042] Example 3: OsGATA16 Construction of mutant vectors; 1. Target site design: CRISPR-GE (http: / / skl.scau.edu.cn / ) was used to design spacers (target sites), with target sites containing 7 bp deletions (ATG starting at 37-43 bp) and 2 bp deletions (ATG starting at 44-45 bp).

[0043] 2. Editing vector construction: Using pEASY-gRNA-U6c as a template, amplify the target gRNA expression cassette. After purification, the PCR product is homologously recombined with the pCRISPR-zero vector linearized with the BsaI restriction endonuclease. Competent E. coli is then transformed to screen for recombinant clones. Two gRNA expression cassettes, driven by the U3 and U6a promoters, are constructed. These are then amplified by PCR and cloned sequentially into the pYLCRISPR / Cas9-MH master vector to complete the construction of the CRISPR / Cas9 gene editing vector.

[0044] 3. Genetic transformation: Rice variety ZH11 was transformed by Agrobacterium EHA105, and T2 generation homozygous mutant materials were obtained by screening.

[0045] 4. Mutation verification: Sanger sequencing OsGATA16 Knockout mutant lines ( GATA16 -CR1, GATA16 -CR2) sequencing, the results showed that base deletion or insertion occurred at the target site, resulting in a frame shift in the reading frame, thus successfully knocking out the function of the target gene. The specific results of mutant sequencing are shown in Figure 1 .

[0046] Example 4: Rice blast fungus infection experiment; 1. Experimental materials; Experimental group 1: wild type KT, OsGATA16 Overexpression transgenic lines ( GATA16- OE1, GATA16- OE2); Experimental group 2: wild-type ZH11, OsGATA16 Knockout mutant lines ( GATA16-CR1, [[ID= -CR2).

[0047] 2. Preparation of rice blast fungus; (1) Rice blast fungus ​ Strain 70-15 was inoculated onto PDA medium (potato dextrose agar) and cultured at 28°C for 7 days. The cultured strain was harvested and cultured in CM medium with shaking at 28°C and 200 rpm for 24–48 hours.

[0048] (2) Collect the mycelium and transfer it to conidia induction medium. Cultivate in the dark for 48 hours, then treat with light (12 hours) to induce spore formation. Rinse the surface of the strain with sterile water and collect the conidia suspension.

[0049] (3) Dilute the spores to a concentration of about 1×10 5 spores / mL. Add 0.02% Tween-20 to promote even spore distribution. Observe the spore concentration under a microscope to ensure uniformity.

[0050] 3. Rice leaf inoculation; (1) Inoculate rice plants at the 4-5 leaf stage and grow them in a greenhouse (temperature 28°C, humidity 80%, 12 h light / 12 h dark).

[0051] (2) Use a sprayer to evenly spray the spore suspension on the leaf surface, ensuring that the leaves of each plant are covered with the suspension.

[0052] (3) Spray an equal amount of sterile water (with 0.02% Tween-20) on the leaves of the control group.

[0053] 4. Cultivation and lesion observation; (1) After inoculation, place the plants in a high humidity (>90%) growth environment in the dark for 24 hours to allow spore germination. Humidity control: Rice blast fungus spore germination and infection require high humidity. Ensure that the humidity is greater than 90% after inoculation. Then, return to normal growth conditions (28°C, 80% humidity, 12 hours light / 12 hours dark).

[0054] (2) Lesion observation: Observe the phenotype of leaf lesions 5 to 7 days after inoculation. Record the number, size, distribution, and color of the lesions.

[0055] (3) Photographic records: Use a high-definition camera to capture images of leaf lesions and save representative phenotypes for each group. Measure the lesion area and quantify the phenotype using Graphpad.

[0056] 5. Data analysis; (1) Statistical analysis: Compare the lesion area and disease index between different genotypes. Use significance test to evaluate phenotypic differences.

[0057] (2) Visualization results: Draw a bar graph to show the distribution of lesion area and disease index of different genotypes.

[0058] ​ The blast infection phenotypes of knockout mutant lines and overexpression transgenic lines are shown in ​ and ​ , it can be clearly seen ​ The knockout mutant lines had significantly fewer lesions after inoculation with rice blast fungus than the wild-type ZH11 control (see ​ a and b), ​ The overexpressing transgenic lines had significantly more lesions after inoculation with rice blast fungus than the wild-type KT control (see ​ a and b), indicating ​ Negative regulation of rice resistance to rice blast.

[0059] Summarize: The present invention has verified different ​ Regulatory effect of transgenic lines on rice blast resistance. ​ In the transgenic lines, the resistance of rice to rice blast was significantly reduced, as shown by a significant increase in the area of ​​lesions after inoculation; ​ The disease resistance of the knockout mutant strains was significantly enhanced, and the area of ​​lesions was significantly reduced. In terms of practical breeding applications, the results of this invention are of great significance. Breeders can use gene editing technology to accurately knock out ​ This molecular-based precision breeding strategy provides a solid theoretical foundation and feasible technical solutions for developing new blast-resistant rice varieties, and has a positive impact on promoting the development of rice disease-resistant breeding.

[0060] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A use of a rice transcription factor OsGATA16 or its encoding gene in regulating rice resistance to rice blast or breeding for rice blast resistance, characterized in that: The amino acid sequence of the rice transcription factor OsGATA16 is shown in SEQ ID No. 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that By knocking out the rice transcription factor gene OsGATA16 , to improve rice's resistance to rice blast.

3. The use according to claim 2, characterized in that The knockout was achieved by the CRISPR / Cas9 method.

4. Use of a recombinant vector in regulating rice resistance to rice blast or breeding for rice blast resistance, characterized in that: The recombinant vector contains a rice transcription factor gene OsGATA16 , whose nucleotide sequence is shown in SEQ ID No.

1.

5. Use of a recombinant bacterium comprising a recombinant vector in regulating rice resistance to rice blast or in breeding rice blast-resistant varieties.

6. A method for regulating rice resistance to rice blast, characterized in that: Regulation of rice transcription factor genes by OsGATA16 Expression: Knockout or reduction OsGATA16 Gene expression to improve rice resistance to rice blast disease; or overexpression OsGATA16 genes to reduce rice's resistance to blast disease; The rice transcription factor gene OsGATA16 The nucleotide sequence is shown in SEQ ID No.

1.

7. The method according to claim 6, characterized in that The knockout or reduction OsGATA16 Gene expression is achieved through CRISPR / Cas9 method; or overexpression OsGATA16 The gene is transferred into rice recipient material by constructing a recombinant expression vector.

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