A rice transcription factor, OsGATA16, and its application in regulating rice resistance to rice blast.

Abstract

This invention relates to the field of molecular biology and provides a rice transcription factor OsGATA16 and its application in regulating rice resistance to rice blast. Firstly, rice was cloned. OsGATA16 The gene was then used to construct overexpression and mutant vectors, which were then transformed into rice to obtain the corresponding lines. The study found that knocking out… OsGATA16 The mutant lines, after inoculation with the pathogen, showed significantly fewer lesions than the wild-type control, demonstrating precise regulation of rice disease resistance and a significant improvement in rice blast resistance. This regulatory approach can effectively reduce pesticide use, lowering environmental pollution and residue risks. Furthermore, the successfully constructed stable genetic regulatory material has been validated at both the molecular and phenotypic levels, allowing for long-term storage and repeated use. OsGATA16 Genes can be used as molecular markers or functional genes for disease resistance in various breeding systems, providing new targets for breeding new rice materials with high resistance to rice blast, and have broad practical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a rice transcription factor OsGATA16 and its application in regulating rice resistance to rice blast. Background Technology

[0002] Rice blast, a disease caused by the rice blast fungus (… Magnaporthe oryzae Rice diseases caused by SARS-CoV-2 have long been a major problem in global rice production. These diseases are known for their rapid spread, high variability, and severe damage, capable of rapidly spreading to large areas of rice fields in a short period, causing serious yield losses or even total 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 crucial role in crop disease resistance responses. They achieve rapid disease resistance responses by regulating a series of downstream defense-related genes. Among them, GATA zinc finger transcription factors can bind to the W-GATA-R (W=T / A, R=G / A) sequence on the promoters of their target genes and are widely present in plant growth, development, and environmental responses. However, their function in the mechanism of rice blast resistance has not yet been reported.

[0004] In existing rice blast control technologies, due to the lack of effective resistant materials, agricultural production heavily relies on chemical pesticides to control rice blast. However, long-term use of chemical pesticides not only increases production costs but also leads to drug resistance in pathogens, thus affecting control efficacy. More seriously, the overuse of chemical pesticides poses a potential threat to the ecological environment and human health, arousing widespread concern and anxiety across society. Therefore, developing green and sustainable rice blast control technologies has become an urgent need in the fields of rice breeding and disease control. Regarding transcription factor research, although existing studies have revealed the involvement of families such as WRKY and MYB in plant disease defense responses, the mechanism of action of rice GATA transcription factors under pathogen stress, especially the function of OsGATA16 in regulating rice blast resistance, remains unclear. Therefore, this invention proposes a rice transcription factor, OsGATA16, and its application in regulating rice blast resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a rice transcription factor OsGATA16 and its application in regulating rice resistance to rice blast, thereby addressing the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The application of a rice transcription factor OsGATA16 or its encoding gene in regulating rice resistance to rice blast or in rice blast resistance breeding, wherein 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.

[0008] Furthermore, by knocking out the rice transcription factor gene OsGATA16 To improve the resistance of rice to rice blast.

[0009] Furthermore, the knockout is achieved using the CRISPR / Cas9 method.

[0010] Application of a recombinant vector in regulating rice resistance to rice blast or in rice blast resistance breeding, wherein the recombinant vector contains rice transcription factor genes. OsGATA16 Its nucleotide sequence is shown in SEQ ID No. 1.

[0011] Application of a recombinant strain containing a recombinant vector in regulating rice resistance to rice blast or in rice blast resistance breeding.

[0012] A method for regulating rice resistance to rice blast involves regulating rice transcription factor genes in the following ways. OsGATA16 Expression:

[0013] Knock out or reduce OsGATA16 Gene expression to enhance rice resistance to rice blast; or overexpression OsGATA16 Genes to reduce rice's resistance to rice blast;

[0014] The rice transcription factor gene OsGATA16 The nucleotide sequence is shown in SEQ ID No. 1.

[0015] Furthermore, the knockout or reduction OsGATA16 Gene expression is achieved via the CRISPR / Cas9 method; or overexpression. OsGATA16 The gene was transferred into the rice receptor material by constructing a recombinant expression vector.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. Significantly enhances rice blast resistance: by constructing OsGATA16 Overexpression transgenic lines and knockout mutant lines were used, and rice blast fungus infection experiments were conducted. The results showed that the knockout mutants... OsGATA16 The mutant lines showed significantly fewer lesions than the wild-type control after inoculation with rice blast fungus, indicating that knocking out this gene significantly enhanced rice resistance to rice blast, achieving precise regulation of rice's disease resistance.

[0018] 2. Effectively reduces pesticide use, environmentally friendly: This invention utilizes the regulation of endogenous genes OsGATA16 By adjusting the expression level of [a specific substance], rice disease resistance can be enhanced, controlling rice blast without relying on exogenous chemical pesticides. This approach reduces the frequency of pesticide use in agricultural production, decreases environmental pollution and pesticide residue risks, and generates significant ecological benefits.

[0019] 3. Construction of stable and reliable genetic regulatory materials: This invention successfully constructed... OsGATA16 Overexpression transgenic lines and CRISPR mutant lines, among which the overexpression transgenic lines OsGATA16 The expression levels were significantly higher than those of the wild-type control. The knockout mutant lines showed base deletions or insertions at the target site, resulting in gene knockout. These lines were validated at both the molecular and phenotypic levels, with a clear genetic background and stable traits, making them suitable for long-term preservation and repeated use.

[0020] 4. Facilitates practical application in breeding: The target gene of this invention OsGATA16 It can be used as a molecular marker for disease resistance, or as a functional gene introduced into other rice varieties or cultivated materials. This gene is applicable to conventional breeding, molecular breeding, and gene editing breeding systems, and has broad practical application prospects. It provides a novel molecular breeding target for breeding new rice materials with broad-spectrum, durable, and highly resistant rice blast. Attached Figure Description

[0021] Figure 1 for OsGATA16 Knockout mutant lines ( GATA16 -CR1、 GATA16 -CR2) sequencing results.

[0022] Figure 2 for OsGATA16 Overexpression transgenic lines ( GATA16 -OE1、 GATA16- OE2 expression level identification results.

[0023] Figure 3 for OsGATA16 Knockout mutant lines exhibit rice blast infection phenotype; where: a represents OsGATA16 Leaf scan images of knockout mutant lines, b is OsGATA16 Statistical data on lesion area of ​​knockout mutant lines. Lesion area = mean ± standard deviation (n=12), asterisk indicates significant difference compared with wild-type ZH11. ****: p<0.0001 (Student's t-test).

[0024] Figure 4 for OsGATA16 Overexpression of transgenic rice blast infection phenotype; where: a is OsGATA16Leaf scan images of overexpressing transgenic lines, b is OsGATA16 Statistical data on lesion area of ​​overexpressing transgenic lines. Lesion area = mean ± standard deviation (n=12), asterisks indicate significant differences compared to wild-type KT. ***: p<0.001, ****: p<0.0001 (Student's t-test). Detailed Implementation

[0025] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0026] This invention provides a rice GATA transcription factor OsGATA16 (LOC_Os06g37450) and its application in rice resistance to rice blast. The OsGATA16 transcription factor is located in the cell nucleus and can regulate the expression of downstream target genes, thereby affecting the immune response of rice to rice blast fungus.

[0027] OsGATA16 The gene contains 1173 bases and encodes a 390-amino acid protein, OsGATA16. This protein contains a typical GATA zinc finger domain and exhibits typical DNA binding and transcriptional regulatory functions. OsGATA16 is located in the cell nucleus and belongs to group IIA of the GATA transcription factor family in rice.

[0028] OsGATA16 Gene nucleotide sequence:

[0029]

[0030] OsGATA16 protein amino acid sequence:

[0031] MSTIYMSQLSAALPLMEGEHHHHHQDHHQGHFQAFSLQPKDPPVLFPFVISRRSSSSSPSDSTTLSYGSDHHLTQQQQHQHQAMLEPQNMIGGSSAGIFATPFPTVKSIRDDMIERSQFDPYDTEKLQASCGLAKVVAGGKWSAVPAAKMKITRKMGEPSSGVTGGAATTVAPKKPRRRPAQAYEDHGHGGAMGQAF GVIRVCSDCNTTKTPLWRSGPCGPKSLCNACGIRQRKARRAMMASGLPASPNAAGPKAAAHSGAAAVAAAQPKVKKEKRADVDRSSLPFKKRCKVVQVEDHQTLPAATNAAAAAAMEETAESATVAPPPAPTTRGGTLVDSIGLSWSKTHAAATASCSFRPSPPVAPGFAAAVQDEITDAAMLLMTLSCGLVRS (as SEQ ID NO.2 shown).

[0032] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: OsGATA16 Gene cloning;

[0034] 1. RNA extraction;

[0035] Wild-type ZH11 rice was selected and cultured hydroponically in Kimura nutrient solution until the three-leaf stage under alternating conditions of 28℃, 12h light and 25℃, 12h darkness. RNA extraction was performed using a reverse transcription kit (ReverTraAce qPCR RT Master Mix). The specific steps are as follows:

[0036] (1) Take an appropriate amount of fresh rice material, put it into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen and grind it into powder quickly, then quickly transfer the powder into a pre-cooled 2mL centrifuge tube;

[0037] (2) Add 1 mL of TRIzol, mix thoroughly with a vortex mixer, and place in an ice box to stand for 10 min;

[0038] (3) Add 500 μL of chloroform, mix thoroughly with a vortex mixer, and let stand at room temperature for 10 min;

[0039] (4) The centrifuge is set to 4°C, 12000 rpm, and centrifuged for 10 min. After centrifugation, 500 μL of the supernatant is taken and transferred to a 1.5 mL centrifuge tube;

[0040] (5) Add 500 μL of isopropanol, mix by inversion, place on ice for 30 min, and centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant, wash the precipitate with 75% ethanol (prepared with DEPC water), and centrifuge again at 12000 rpm at 4°C;

[0041] (6) Aspirate the supernatant as cleanly as possible, air dry at room temperature, add 100 μL of DEPC water to dissolve, measure the RNA concentration using a UV spectrophotometer, and store at -80°C for later use.

[0042] 2. mRNA reverse transcription;

[0043] (1) Using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit, take out the RNA stored in the -80℃ freezer, place it on ice to thaw, calculate the volume required for 2μg of total RNA based on the RNA concentration, add it to the PCR tube, and add water from the kit to make up to 12μL. React on a PCR instrument at 65°C for 5min, and then quickly place it on ice to cool for 2min.

[0044] (2) Add 4 μL of 4×DN Master Mix (containing gDNA Remover), mix well, and place in a 37°C PCR instrument for 5 min, then quickly cool on ice for 2 min;

[0045] (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.

[0046] (4) After the reaction is complete, store the cDNA at -20°C for later use.

[0047] Example 2: OsGATA16 Construction of overexpression vectors;

[0048] Source of genetic material: RNA was extracted from rice variety ZH11 and cDNA was obtained by reverse transcription. The target gene was amplified using specific primers. OsGATA16 The CDS region; vector construction: using infusion homologous recombination technology, ... OsGATA16 The PCR product was homologously recombinated with the linearized vector pc1300-35S-FLAG to construct the recombinant vector 35S- OsGATA16Transformation process: The recombinant vector was transferred into Agrobacterium EHA105, and Kitaake rice callus was infected by Agrobacterium-mediated transformation to obtain resistant transformants. The transformants were cultured to the T3 generation to form stable genetic material.

[0049] 1. Design primers;

[0050] Based on the pc1300-35S-FLAG vector information, the KpnI+BamHI double restriction site was selected, and primers were designed as follows:

[0051] GATA16-flag-F: ACGATGATAAGGGCGGTACCATGTCTACCATCTACATGAGTCAGC (as shown in SEQ IDNO.3);

[0052] GATA16-flag-R: AGGCTACGTAGGATCCTCAGCTCCGGACAAGCCC (as shown in SEQ ID NO.4).

[0053] 2. PCR reaction and vector linearization by enzyme digestion: PCR was performed using ZH11 cDNA as a template according to a high-fidelity KOD enzyme program, and the PCR products were verified by agarose gel electrophoresis. Simultaneously, the pc1300-35S-FLAG vector was digested with KpnI and BamHI, and the digestion was verified by agarose gel electrophoresis after the reaction.

[0054] 3. Target fragment recovery: Cut off the specific band obtained in the previous step and purify the DNA according to the gel extraction kit steps.

[0055] 4. Infusion reaction: Following the infusion product system provided, the purified DNA fragment of the target fragment is ligated with the linearized vector.

[0056] 5. Conversion: Remove the ligation solution and store it at -20℃. Perform the conversion according to the following steps:

[0057] (1) Mix the ligation solution with DH5α competent cells and place them on ice for 30 min.

[0058] (2) Heat in a 42℃ metal bath for 90 seconds and cool for 2 minutes.

[0059] (3) Add 500 μl of LB liquid culture medium and mix at 37°C and 220 rpm / min for 1 h.

[0060] (4) Centrifuge at 6000 rpm / min for 5 min, remove the supernatant and leave a small amount to mix and spread on solid LB medium with the corresponding resistance, and incubate overnight at 37°C with the medium inverted.

[0061] 6. Identification of positive clones: Single colonies were picked and colony PCR was performed using GATA16-flag-F and GATA16-flag-R primers. At the same time, the colonies were streaked on kanamycin-resistant medium plates. Colony PCR positive clones were selected for propagation, and plasmids were extracted for sequencing.

[0062] 7. Select plasmids with correct sequencing to transform Agrobacterium EHA105 and then transform the rice variety Kitaake.

[0063] The results of the identification of overexpression transgenic rice are shown below. Figure 2 overexpression transgenic lines ( GATA16 -OE1、 GATA16- OE2) OsGATA16 The expression level was significantly higher than that of the wild-type KT control, indicating that the overexpression transgenic lines showed significantly higher expression levels. OsGATA16 The expression level increased significantly, further proving that the overexpression transgenic line was successfully constructed.

[0064] Example 3: OsGATA16 Construction of mutant vectors;

[0065] 1. Target design: Spacers (target sites) were designed using CRISPR-GE (http: / / skl.scau.edu.cn / ), with deletions of 7bp (ATG starting, 37-43bp) and 2bp (ATG starting, 44-45bp) respectively.

[0066] 2. Construction of editing vectors: The target gRNA expression cassette was amplified using pEASY-gRNA-U6c as a template. After purification, the PCR product was homologously recombinated with the pCRISPR-zero vector linearized with BsaI restriction endonuclease. The resulting product was transformed into competent E. coli to screen for recombinant clones. Two gRNA expression cassettes were constructed using the U3 and U6a promoters, respectively, and then sequentially cloned into the pYLCRISPR / Cas9-MH master vector by PCR amplification, thus completing the construction of the CRISPR / Cas9 gene editing vector.

[0067] 3. Genetic transformation: Rice variety ZH11 was transformed with Agrobacterium tumefaciens EHA105, and T2 generation homozygous mutant materials were obtained by screening.

[0068] 4. Mutation verification: Mutation was verified using Sanger sequencing. OsGATA16 Knockout mutant lines ( GATA16 -CR1、 GATA16 Sequencing of the mutant (CR2) revealed a base deletion or insertion at the target site, resulting in a frameshift and successfully knocking out the target gene's function. Detailed sequencing results of the mutant can be found in [link to relevant documentation]. Figure 1 .

[0069] Example 4: Rice blast fungus infection experiment;

[0070] 1. Experimental materials;

[0071] Experimental group 1: Wild-type KT, OsGATA16 Overexpression transgenic lines ( GATA16- OE1, GATA16- OE2);

[0072] Experimental group 2: Wild-type ZH11, OsGATA16 Knockout mutant lines ( GATA16 -CR1、 GATA16 -CR2).

[0073] 2. Preparation of rice blast fungus;

[0074] (1) Rice blast fungus Magnaporthe oryzae Strain 70-15 was inoculated onto PDA medium (potato dextrose agar) and cultured at 28°C for 7 days. The cultured strains were then collected and cultured in CM medium with shaking at 28°C and 200 rpm for 24–48 hours.

[0075] (2) After collecting the mycelium, transfer it to the conidia induction medium. Incubate in the dark for 48 h, then treat with light (12 h) to induce conidia formation. Rinse the surface of the strain with sterile water and collect the conidia suspension.

[0076] (3) Dilute the spore concentration with sterile water to approximately 1×10⁻⁶. 5 Spores / mL. Add 0.02% Tween-20 to promote uniform spore distribution. Observe the spore concentration under a microscope to ensure uniformity.

[0077] 3. Rice leaf inoculation;

[0078] (1) Inoculate rice plants at the 4-5 leaf stage and grow them in a greenhouse (temperature 28℃, humidity 80%, 12h light / 12h darkness).

[0079] (2) Use a sprayer to spray the spore suspension evenly on the leaf surface, ensuring that the leaves of each plant are covered with the suspension.

[0080] (3) Spray an equal amount of sterile water (with 0.02% Tween-20 added) on the leaves of the control group.

[0081] 4. Culture and observation of lesions;

[0082] (1) After inoculation, place the plants in a high humidity (humidity > 90%) culture environment and allow spores to germinate in the dark for 24 hours. Humidity control: Rice blast fungus spores require high humidity for germination and infection, so ensure that the humidity of the environment is greater than 90% after inoculation. Then restore normal growth conditions (28℃, 80% humidity, 12h light / 12h dark).

[0083] (2) Observation of lesions: Observe the phenotype of leaf lesions 5-7 days after inoculation. Record the number, size, distribution and color differences of lesions.

[0084] (3) Photo recording: Use a high-definition camera to take images of leaf lesions and save the representative phenotype of each group. Measure the lesion area and quantify the phenotype using Graphpad.

[0085] 5. Data analysis;

[0086] (1) Statistical analysis: Compare the lesion area and disease index among different genotypes. Use significance test to assess phenotypic differences.

[0087] (2) Visualization results: A bar chart is drawn to show the distribution of lesion area and disease index of different genotypes.

[0088] OsGATA16 The knockout mutant lines and overexpression transgenic lines show the rice blast infection phenotypes. Figure 3 and Figure 4 It can be clearly seen OsGATA16 After inoculation with rice blast fungus, the knockout mutant lines showed significantly fewer lesions than the wild-type ZH11 control (see [link]). Figure 3 (a and b) OsGATA16 The transgenic lines overexpressing the strain showed significantly more lesions than the wild-type KT control after inoculation with rice blast fungus (see [link]). Figure 4 (a and b) Explanation OsGATA16 Negative regulation of rice resistance to rice blast.

[0089] Summarize:

[0090] Through the above-described embodiments, this invention verifies different OsGATA16 The regulatory effect of transgenic lines on rice blast resistance. In overexpression OsGATA16 In the transgenic lines, rice resistance to rice blast was significantly reduced, manifested by a substantial increase in lesion area after inoculation; while OsGATA16 The knockout mutant lines exhibited significantly enhanced disease resistance, with a marked reduction in lesion area. This invention is of great significance in practical breeding applications. Breeders can use gene editing technology to precisely knock out mutants according to specific needs. OsGATA16By modifying genes or regulating their expression levels, targeted improvements in rice blast resistance can be achieved. This precision breeding strategy based on molecular mechanisms provides a solid theoretical foundation and feasible technical solutions for cultivating new rice varieties resistant to rice blast, and plays a positive role in promoting the development of rice disease-resistant breeding.

[0091] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A use of a rice transcription factor OsGATA16 or a coding gene thereof in regulating rice resistance to rice blast or breeding rice plants resistant to rice blast, characterized in that, The amino acid sequence of the rice transcription factor OsGATA16 is shown as SEQ ID No. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 1; By knocking out the rice transcription factor gene OsGATA16 to improve the resistance of rice to rice blast; the knockout is achieved by CRISPR / Cas9 method.

2. A method of modulating rice resistance to rice blast, characterized by, Regulation of expression of rice transcription factor genes by the following means: OsGATA16 ​ knockout or reduction OsGATA16 expression of a gene to increase resistance of rice to blast disease; The nucleotide sequence of the rice transcription factor gene OsGATA16 is shown as SEQ ID No.

1.

3. The method of claim 2, wherein, the knock-out or reduction OsGATA16 The expression of the genes is achieved by CRISPR / Cas9 method.

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