Rice blast resistance related gene OsCBSX4 and application thereof

Abstract

The invention relates to the technical field of rice disease resistance breeding, in particular to a rice blast resistance related gene OsCBSX4 and application thereof. The CDS of the OsCBSX4 gene is 693bp, and cystathionine beta synthetase consisting of 231 amino acids is coded; the invention discovers and proves that the OsCBSX4 positively regulates the resistance of rice to rice blast for the first time, the resistance of rice to rice blast can be reduced by knocking out the OsCBSX4 gene through a CRISPR / Cas9 method, the resistance of rice to rice blast can be remarkably enhanced by improving the expression level of OsCBSX4, and the gene can be applied to genetic improvement of resistance and yield of related agricultural crops such as rice.

Description

Technical Field

[0001] The invention relates to the technical field of rice disease resistance breeding, and in particular to a rice blast resistance-related gene OsCBSX4 and an application thereof. Background Art

[0002] Rice (Oryza sativa) is one of the most important food crops in the world, but its stable production and yield increase have always been threatened by a variety of diseases. Among them, rice blast caused by Magnaporthe oryzae is one of the main rice diseases. It occurs in rice-growing areas all over the world and generally causes a 10-35% reduction in yield. Practice has proved that breeding and promoting new rice varieties with long-lasting and broad-spectrum resistance is one of the most effective ways to prevent and control rice blast. By studying the mechanism of rice immune system to resist pathogen invasion, finding broad-spectrum resistance genes and applying them to agricultural production will provide important theoretical guidance and gene resources for rice disease resistance breeding, greatly accelerating the genetic improvement of rice resistance and the process of disease resistance molecular breeding.

[0003] Under the threat of pathogens for a long time, plants have evolved a complex and sophisticated immune system to identify and defend against the invasion of pathogens. This immune system can be divided into two levels. The immune response stimulated by the conservative components of pathogens, PAMPs (pathogen-associated molecular patterns), is called PTI (PAMP-triggered immunity), which is the first level of plant innate immunity and has the characteristics of broad spectrum and persistence. However, after some pathogens successfully infect the host, they will release effectors into the host cells to inhibit PTI. Faced with new challenges, plants have evolved a second-level defense system, which specifically recognizes effectors through disease-resistant proteins (R proteins) inside cells, activates downstream disease-resistant responses, and regains the ability to resist pathogen infection. This second level of immune response is also called ETI (effector triggered immunity), which has the characteristics of specificity and strong resistance.

[0004] CBS (Cystathionine-beta-synthase) protein is cystathionine synthase. Its function in plants has been rarely reported. CBS protein is generally believed to regulate the redox homeostasis of plants by interacting with Trx proteins. For example, Arabidopsis CBSX1, CBSX2 and CBSX3 are reported to participate in the regulation of anther development by interacting with Trx proteins. After Ritesh Kumar et al. overexpressed OsCBSCBSPB4 in Nicotiana benthamiana, the overexpressed tobacco delayed leaf senescence, root growth was vigorous, and biomass increased under salt, dehydration, oxidation and extreme temperature treatments. Syed Adeel Zafar et al. found that the DPS1 gene containing the CBS domain is related to the development of rice flower organs and the accumulation of reactive oxygen. The dps1 mutant showed degeneration of the apical spikelet, sterile middle spikelet, small and white anthers, altered cuticle morphology, missing pollen grains, increased accumulation of reactive oxygen species (ROS) in the panicle, decreased antioxidant activity, increased programmed cell death, and DPS1 interacted with Trx1 and Trx20, suggesting that DPS1 may participate in ROS scavenging by interacting with Trx1 and Trx20, thereby regulating ROS homeostasis. Wang Xuelu's team found that soybean CBS proteins GmNAS1 and GmNAP1 regulate nodule nitrogen fixation capacity by sensing nodule energy status. Mou et al. found that overexpression of rice OsCBSX3 can significantly enhance rice resistance to rice blast. This shows that CBS proteins play an important role in plant growth and development, biotic and abiotic stresses. Hemant R Kushwaha et al. analyzed the proteins containing CBS domains in the Arabidopsis and rice genomes and found that there are 34 CBS proteins in Arabidopsis and 59 CBS proteins in rice, but the functions of most plant CBS proteins are still unclear.

[0005] The release of the rice genome sequence and the rapid development of modern molecular technology and genetic engineering have provided effective means for the discovery and utilization of disease-resistant genes. In the early stage of the experiment, the inventors found that a cystathionine synthase protein OsCBSX4 was induced to express by rice blast infection. After knocking out the gene using CRISPR / Cas9 technology, the rice resistance to rice blast was significantly reduced, while increasing its expression level significantly enhanced the resistance of rice to rice blast. Therefore, the OsCBSX4 gene has an application basis for improving rice resistance to rice blast. Summary of the invention

[0006] The rice blast resistance-related gene OsCBSX4 provided by the present invention has a regulatory effect on the rice blast resistance reaction. The deletion of the OsCBSX4 gene reduces the disease resistance of rice to rice blast, while the overexpression of the OsCBSX4 gene can enhance the disease resistance of rice to rice blast. The OsCBSX4 gene can be applied to the genetic improvement of improving the resistance of rice and other related agricultural crops.

[0007] The technical solution of the present invention is as follows:

[0008] One of the purposes of the present invention is to provide a rice blast resistance-related gene OsCBSX4, the nucleotide sequence of the OsCBSX4 gene is shown in SEQ ID NO: 1, and a gene sequence having more than 90% homology with the DNA sequence shown in SEQ ID NO: 1, and also including mutant alleles or derivatives produced by insertion, substitution or deletion of one or more bases, and the full length of the CDS sequence is 4005 bp.

[0009] Another object of the present invention is to provide a protein encoded by the rice blast resistance-related gene OsCBSX4, wherein the amino acid sequence of the protein encoded by the OsCBSX4 gene is as shown in SEQ ID NO.2, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2.

[0010] The third object of the present invention is to provide a use of a rice blast resistance-related gene OsCBSX4 or a protein encoded by the rice blast resistance-related gene OsCBSX4 in improving plant disease resistance.

[0011] The fourth object of the present invention is to provide a rice blast resistance related gene OsCBSX4 or a protein encoded by a rice blast resistance related gene OsCBSX4 in improving rice blast resistance, thereby improving crop resistance to rice blast fungus by increasing the expression level of the OsCBSX4 gene.

[0012] The beneficial effects of the present invention are:

[0013] 1. The present invention screened the interacting proteins of rice blast regulatory protein OsBSK1-2, found its interacting protein OsCBSX4, and identified the function of the protein, and discovered and proved for the first time the positive regulatory role of OsCBSX4 gene in rice resistance to rice blast; and proved through various methods that the deletion of OsCBSX4 gene reduced the resistance of rice to rice blast, while the overexpression of OsCBSX4 gene could enhance the resistance of rice to rice blast, thus indicating that OsEDC4 gene positively regulates the resistance of rice to rice blast; the above results all indicate that OsEDC4 gene is an excellent candidate gene for creating transgenic rice with enhanced resistance to rice blast and for crop molecular design, and has important theoretical value and broad application prospects;

[0014] 2. The present invention reduces the resistance of rice to rice blast by knocking out the OsCBSX4 gene in the rice variety Zhonghua 11 (ZH11) through a genetic engineering method. Compared with the wild type, the oscbsx4 mutant has more lesions and rice blast fungus growth, less hydrogen peroxide accumulation, and a decreased expression level of rice disease resistance-related genes. Increasing the expression level of the OsCBSX4 gene can significantly enhance the resistance of rice to rice blast. The rice blast resistance gene OsCBSX4 provided by the present invention can be used for genetic improvement of improving the resistance of rice and other related agricultural crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram for verifying the interaction between OsBSK1-2 and OsCBSX4 in Example 2 of the present invention;

[0016] Figure 2 This is a diagram for identifying the disease resistance phenotype of the OsCBSX4 gene knockout allele mutant 1 plant in Example 3 of the present invention;

[0017] Figure 3 This is a diagram for identifying the disease resistance phenotype of the OsCBSX4 gene knockout allele mutant 2 plant in Example 4 of the present invention;

[0018] Figure 4 This is a diagram for identifying the disease resistance phenotype of OsCBSX4 overexpressing plants in Example 5 of the present invention. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited thereto. In the present invention, rice variety ZH11 and rice blast fungus Guy11 are taken as research objects.

[0020] Example 1

[0021] This embodiment provides a rice blast resistance-related gene OsCBSX4, wherein the nucleotide sequence of the OsCBSX4 gene is shown in SEQ ID NO: 1, and the full length of the sequence is 693 bp, as well as a gene sequence having more than 90% homology with the DNA sequence shown in SEQ ID NO: 1, and also includes mutant alleles or derivatives generated by insertion, substitution or deletion of one or more bases; the amino acid sequence of the protein encoded by the OsCBSX4 gene is shown in SEQ ID NO. 2, encoding 231 amino acids, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

[0022] Example 2

[0023] OsCBSX4 protein interacts with OsBSK1-2;

[0024] (1) Materials and methods

[0025] Bimolecular fluorescence complementation (BiFC) experiment: The full-length cDNA sequences of OsCBSX4 and OsBSK1-2 (CDS see SEQ ID NO: 3, protein sequence see SEQ ID NO: 4) were constructed into pSCYNE-35S and pSCYCE-35S vectors, respectively, to form OsCBSX4-YN and OsBSK1-2-YC fusion expression vectors. The recombinant plasmids were then transformed into Agrobacterium GV3101 competent cells by electroporation. Agrobacterium containing OsCBSX4-YN and OsBSK1-2-YC were mixed at a ratio of 1:1 and injected into tobacco leaves for transient expression. After 2 days, the leaves were observed under a laser confocal microscope and the corresponding images were saved.

[0026] Luciferase complementation assay (LCI): The full-length cDNAs of OsCBSX4 and OsBSK1-2 were cloned into Nluc and Cluc vectors, respectively, and the recombinant plasmids were transformed into Agrobacterium GV3101 competent cells by electroporation. Agrobacterium containing Nluc-OsCBSX4 and OsBSK1-2-Cluc were mixed at a ratio of 1:1 and injected into tobacco leaves for transient expression. At the same time, Nluc-OsCBSX4 and YFP-Cluc, as well as OsBSK1-2-Cluc and YFP-Nluc were co-injected into tobacco leaves as negative controls. Two days after injection, 1 mM luciferin was applied and observed and photographed using a low-light cooled CCD imaging device.

[0027] Co-immunoprecipitation (Co-IP): The full-length cDNAs of OsCBSX4 and OsBSK1-2 were cloned into pCMBIA-1300-GFP and pYBA1143-HA, respectively. The recombinant plasmids were transformed into Agrobacterium GV3101 by electroporation. The OsCBSX4-GFP and OsBSK1-2-HA fusion proteins were co-expressed in tobacco by Agrobacterium-mediated tobacco transient expression. The co-expression of GFP and OsBSK1-2-HA in tobacco was used as a control. The total protein of tobacco leaves in the experimental and control groups was extracted with plant protein extract, and then incubated with GFP-beads at 4°C for 2h. After that, protein loading buffer was added to the samples. After heating at 95°C for 10min, the Input and IP samples were analyzed by immunoblotting with anti-GFP and HA antibodies, respectively.

[0028] (2) Results and analysis

[0029] See also Figure 1 , Figure 1 A: BiFC experiment was used to verify the interaction between OsCBSX4 and OsBSK1-2; Figure 1B: The interaction between OsCBSX4 and OsBSK1-2 was verified by LCI method; Figure 1 C is the use of Co-IP method to verify the interaction between OsCBSX4 and OsBSK1-2; Figure 1 The results showed that the interaction between OsCBSX4 and OsBSK1-2 could be detected using BiFC, LCI and Co-IP methods.

[0030] Example 3

[0031] The rice blast resistance of oscbsx4 allele 1 (oscbsx4-1) was significantly reduced compared with the wild type;

[0032] (1) Materials and methods

[0033] Construction and planting of oscbsx4-1 mutant: The oscbsx4-1 mutant was constructed in the rice variety Zhonghua 11 (ZH11) background using CRISPR-Cas9 technology; the mutant seeds were soaked and germinated, then planted in a mixture of nutrient soil and vermiculite at a ratio of 2:1 and cultured in an artificial climate chamber for 15-20 days;

[0034] Culture of rice blast fungus: Inoculate the filter paper with rice blast fungus on CM culture medium and place it in a dark incubator at 28℃ for about one week. Then cut the culture medium with mycelium into small pieces and inoculate it on rice bran culture medium. Place it in a dark incubator at 28℃ for 10 days. Then use a sterile glass slide to scrape off the mycelium on the surface of the culture medium on a clean bench. Then place it in a light incubator at 28℃ to stimulate spore production. After 3-4 days, the rice blast fungus spores can be used for subsequent inoculation experiments.

[0035] Spray inoculation method: suspend the spores of rice blast fungus in 0.02% Tween-20 solution and filter with lens paper to adjust the spore concentration to 1×10 5 Spray the spores of rice blast fungus evenly on the leaves of 15-20-day-old rice seedlings with a sprayer, place them in a 26°C high humidity environment, darken them for 24 hours, and then continue to culture them at 26°C, 12 hours of light and 12 hours of darkness, keeping the environment high humidity, and observe the disease phenotype after 3-5 days.

[0036] Punch hole inoculation method: When the rice is 5-6 weeks old, use a mouse ear punch to make a wound at 1 / 3 of the distance from the leaf tip, and add 10 μL of 2×10 5 100 / ml of rice blast fungus spore solution, and seal the wound and spore solution with tape, incubate in a dark environment at 26 degrees and high humidity for 24 hours, then transfer to an inoculation room at 26 degrees, high humidity, and a photoperiod of 14 hours light / 10 hours dark, and observe the phenotype after 7-8 days of inoculation;

[0037] Determination of rice blast fungus biomass: Total DNA from diseased leaves was extracted, and then quantitative PCR analysis at the DNA level was performed using a quantitative PCR kit. The relative rice blast fungus biomass was calculated as the amount of Pot2 gene in rice blast fungus relative to the amount of ubiquitin gene in rice.

[0038] Quantitative PCR analysis: Total RNA of the samples was extracted using TRIzol reagent, and the first-strand complementary DNA was synthesized using a reverse transcription kit according to the manufacturer's instructions, and then quantitative PCR analysis was performed using a quantitative PCR kit according to the operating instructions;

[0039] ROS burst assay: 100 μL of sterile water was added to the wells of a 96-well ELISA plate, and samples were taken from the leaves of 2-week-old rice seedlings using a hole puncher. Two circular leaves were placed in each well, and the plates were kept away from light at room temperature overnight. On the next day, the sterile water in the wells of the ELISA plate was removed, and 50 μL of active oxygen determination reaction solution (containing 20 μM Luminol, 2.5 μg / mL Peroxidase) was added to each well. After being kept away from light for 30 minutes, 50 μL of active oxygen determination reaction solution was added to the control group, and 50 μL of active oxygen determination reaction solution containing 0.32 μmol / mL chitin was added to the experimental group, and the plates were quickly placed in a full-wavelength multifunctional ELISA reader to determine the chemiluminescence value.

[0040] DAB staining method for detecting H2O2: Cut the rice leaves 2 days after spray inoculation and immerse them in DAB solution at 0.8MPa, evacuate in the dark for 30 minutes, and leave them at room temperature in the dark overnight; the next day, move the leaves from the DAB solution to anhydrous ethanol and decolorize them in a 95℃ water bath until the green color of the leaves disappears completely, then observe and photograph the leaves.

[0041] (2) Results and analysis

[0042] See also Figure 2 , Figure 2 A is the genotype identification of the oscbsx4-1 mutant; Figure 2 B and 2C are the identification of the rice blast resistance phenotype of the oscbsx4-1 mutant by the spray inoculation method, bar = 1 cm; Figure 2 D and 2E are the identification of the rice blast resistance phenotype of the oscbsx4-1 mutant by the punch inoculation method, bar = 1 cm; Figure 2 F is the detection of rice blast fungus biomass in the punch-hole inoculated sample; Figure 2 G and 2H are quantitative PCR detection of the expression levels of the pathogenesis-related genes OsPR5 and OsPR10 in oscbsx4-1 synaptosomes and wild-type rice before and after spray inoculation with rice blast fungus; Figure 2 I is the detection of chitin-induced ROS burst in oscbsx4-1 synaptosomes and wild type; Figure 2J is DAB staining of H2O2 in the leaves of oscbsx4-1 synapses and wild-type rice 2 days after spray inoculation with rice blast fungus;

[0043] The results showed that after spray inoculation with rice blast fungus, the number of lesions in oscbsx4-1 was greater than that in the wild type ( Figure 2 B and 2C); After inoculation with rice blast fungus by punching method, the lesions of oscbsx4-1 were larger than those of wild type ( Figure 2 D and 2E), the biomass of rice blast fungus in diseased leaves is higher ( Figure 2 F); In addition, after inoculation with rice blast fungus, the expression levels of pathogenesis-related genes OsPR5 and OsPR10 in oscbsx4-1 blastocysts were lower than those in the wild type ( Figure 2 G and 2H); chitin-induced ROS burst and rice blast-induced H2O2 accumulation in oscbsx4-1 synaptosomes were also lower than those in the wild type ( Figure 2 I and 2J), the above results indicate that the mutation of OsCBSX4 reduces the resistance of rice to rice blast.

[0044] Example 4

[0045] The rice blast resistance of oscbsx4 allele 2 (oscbsx4-2) was also significantly reduced compared with the wild type;

[0046] (1) Materials and methods

[0047] The construction of the oscbsx4-2 mutant, spray inoculation, punch inoculation and rice blast fungus biomass determination were the same as in Example 3.

[0048] (2) Results and analysis

[0049] See also Figure 3 , Figure 3 A is the genotype of the oscbsx4-2 mutant; Figure 3 B shows the phenotype of diseased leaves of the wild type and oscbsx4-2 mutant 3 days after spray inoculation with rice blast fungus, bar=1 cm. Figure 3 C is the statistics of the number of lesions on the diseased leaves of the wild type and oscbsx4-2 mutant 3 days after spray inoculation with rice blast fungus; Figure 3 D is the phenotype of diseased leaves of the wild type and oscbsx4-2 mutant 8 days after inoculation with rice blast fungus by punch inoculation method; Figure 3 E is the statistics of the lesion area and the growth of rice blast fungus on the diseased leaves of the wild type and oscbsx4-2 mutant 8 days after inoculation with rice blast fungus by punch inoculation method, bar=1cm.

[0050] The results showed that 3 days after spray inoculation with rice blast fungus, the oscbsx4-2 mutant had more lesions than the wild type ( Figure 3 B and 3C); 8 days after punch inoculation, the lesion area of ​​the oscbsx4-2 mutant was larger than that of the wild type ( Figure 3 The above results indicate that knocking out OsCBSX4 at different target sites will also reduce the resistance of rice to rice blast.

[0051] Example 5

[0052] Overexpression of OsCBSX4 can significantly improve rice resistance to rice blast fungus.

[0053] (1) Materials and methods

[0054] Construction and detection of OsCBSX4 overexpressing plants: The full-length CDS of OsCBSX4 was connected to the downstream of the 35S promoter of the pYBA1143-HA vector, and the constructed OsCBSX4 overexpression vector was transformed into the wild-type variety ZH11 through Agrobacterium-mediated genetic transformation; the expression of OsCBSX4-HA fusion protein in different T0 generation strains was detected by immunoblotting.

[0055] Identification of rice blast resistance of OsCBSX4 overexpressing plants: The rice wild-type material ZH11 and two OsCBSX4 overexpressing lines OsCBSX4-OE-11 and OsCBSX4-OE-12 were identified for rice blast resistance. The rice planting and inoculation methods were the same as those in Example 3.

[0056] (2) Results and analysis

[0057] See also Figure 4 , Figure 4 A is the immunoblot detection of OsCBSX4-HA fusion protein in different strains of the T0 generation with successful transformation and overexpression of OsEDC4; Figure 4 B and 4C are the inoculation of wild type ZH11 and OsCBSX4 overexpression lines OsCBSX4-OE-11 and OsCBSX4-OE-12 with rice blast fungus strain Guy11 by punch inoculation method, phenotypic observation and rice blast fungus growth statistics 7 days after inoculation, bar = 1 cm.

[0058] from Figure 4 It can be seen that the OsCBSX4-HA fusion protein is relatively more expressed in the overexpression strains OsCBSX4-OE-11 and OsCBSX4-OE-12 ( Figure 4 A); Inoculated with rice blast fungus by punching method, the lesion area of ​​OsCBSX4-OE overexpressing plants was smaller than that of wild type ( Figure 4 B), the biomass of rice blast fungus is less ( Figure 4C), indicating that overexpression of OsCBSX4 can improve rice resistance to rice blast. The above phenotypes prove that OsCBSX4 positively regulates rice resistance to rice blast.

[0059] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A rice blast resistance-related gene OsCBSX4, characterized in that: The nucleotide sequence of the CDS region of the OsCBSX4 gene is shown in SEQ ID NO:

1.

2. A protein encoded by the rice blast resistance-related gene OsCBSX4, characterized in that: The amino acid sequence of the protein encoded by the OsCBSX4 gene is shown in SEQ ID NO.

2.

3. Use of the rice blast resistance-related gene OsCBSX4 according to claim 1 or the protein encoded by the rice blast resistance-related gene OsCBSX4 according to claim 2 in improving plant disease resistance.

4. Use of a rice blast resistance-related gene OsCBSX4 according to claim 1 or a protein encoded by a rice blast resistance-related gene OsCBSX4 according to claim 2 in improving rice blast resistance, characterized in that: Improving crop resistance to rice blast fungus by increasing the expression level of the OsCBSX4 gene.

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