Application of rice chitinase OsCHIT5 in resistance to rice blast

Abstract

The invention discloses an application of rice chitinase OsCHIT5 in resistance to rice blast. It is found for the first time that a transgenic plant with the Oschit5 gene knocked out has remarkable resistance to magnaporthe oryzae, and the resistance of a transgenic plant with the OsCHIT5 overexpressed to the magnaporthe oryzae is reduced. The invention can be used for cultivating rice blast resistant rice plants, thereby having wide application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of rice chitinase OsCHIT5 in resistance to rice blast. Background Technology

[0002] Rice is one of the most important food crops, with more than half of the world's population relying on it as a staple food. In my country, rice is also the staple food source for over 60% of the population. In major rice-producing areas such as South China, Central China, and Northeast China, the annual planting area exceeds 30 million hectares, and the annual output accounts for more than 40% of the country's total grain output, making it a core pillar for strengthening the national food security defense line. Rice blast, one of the three major diseases of rice, is a devastating global disease caused by the filamentous fungus *Magnaporthe oryzae*, often referred to as "rice cancer" (Pennisi E. Armed and dangerous. Science. 2010 Feb 12;327(5967):804-5.). This disease ravages major rice-producing areas year-round, causing approximately 10% yield loss globally, seriously threatening food security and agricultural production stability. This disease can occur throughout the entire growth period of rice and in all parts of the plant. Depending on the location of the disease, rice blast can be classified into leaf blast, node blast, and panicle blast, etc., and it is extremely prone to large-scale outbreaks under suitable temperature (24~26℃) and high humidity (relative humidity above 90%). Faced with the environmental challenges of global warming and the pressure of global population growth, how to effectively control rice blast and ensure stable, high-yield, and high-quality grain production is an urgent problem to be solved in agricultural production. Currently, agricultural production mainly controls rice blast by planting disease-resistant varieties and spraying chemical agents. However, due to the complex and diverse physiology of the rice blast pathogen and its rapid mutation, disease-resistant varieties gradually lose their resistance after 3-5 years of planting. Using preventative pesticides such as tricyclazole and kasugamycin to control rice blast is often ineffective due to the short window of opportunity during the rice heading stage. Furthermore, long-term, excessive use of a single pesticide can lead to pesticide resistance in the rice blast fungus (Zhang H, Zheng X, Zhang Z. The Magnaporthe grisea species complex and plant pathogenesis. Mol Plant Pathol. 2016 Aug;17(6):796-804.). Therefore, exploring the genetic resources of rice's own disease resistance, elucidating its molecular mechanisms, and cultivating high-yielding, high-quality, and disease-resistant new rice varieties are the most economical and effective fundamental strategies for achieving green and sustainable agricultural development.

[0003] Through long-term co-evolution with pathogens, rice has developed a complex and sophisticated immune system. This system mainly includes basal resistance (PTI) triggered by cell surface pattern recognition receptors (PRRs) and specific resistance (ETI) mediated by intracellular nucleotide-binding oligomerization domain-like receptors (NLRs) (Zhang, X., Wang, Z., Jiang, C. etal. Regulation of biotic interactions and responses to abiotic stresses by MAP kinase pathways in plant pathogenic fungi. Stress Biology 1, 5 (2021).; Bigeard J, Colcombet J and Hirt H. Signaling mechanisms in pattern-triggered immunity (PTI). Molecular Plant. 2015, 8: 521-539). In recent years, with the rapid development of molecular biology and omics technologies, the research focus has shifted from cloning single disease-resistant genes to understanding how disease-resistant proteins sense pathogen signals, how they transmit immune signals through protein-protein interactions and post-translational modifications, and how they coordinate disease resistance with the balance of growth and development.

[0004] Studies have shown that plants do not synthesize chitin themselves; therefore, their chitinase genes are typically strongly induced only under biotic or abiotic stress. Chitinases, as an important class of pathogenesis-related proteins, play a central role in plant defense. They hydrolyze chitin, a major component of the cell wall of pathogenic fungi, thereby directly inhibiting hyphal growth. Simultaneously, their degradation product, chitin oligosaccharides, can act as elicitors, activating a broader immune response in plants (rathi, NB, Durga Rani, CV, Prakasam, V. et al. Oschib1 gene encoding a GH18 chitinaseconfers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA. Plant Mol Biol 114, 41 (2024)). Chitin is a skeletal component of the cell wall of most pathogenic fungi. As an important part of the plant's innate immune system, chitinases can catalyze the hydrolysis of the β-1,4 glycosidic bonds of chitin, thereby directly disrupting the hyphal structure of invading pathogenic fungi and inhibiting their growth and reproduction. Studies have shown that chitinase activity is an important marker of rice disease resistance (JDG Jones, JL Dangl, The plant immune system, Nature, 444 (2006), pp. 323-329; Kumar KK, Poovannan K, Nandakumar R, Thamilarasi K, Geetha C, Jayashree N, Kokiladevi E, Raja JA, Samiyappan R, Sudhakar D, Balasubramanian P (2003) A high throughput functional expression assay system for a defence gene conferring transgenic resistance on rice against the sheath blight pathogen, Rhizoctonia solani. Plant Sci 165(5):969–976).For example, in rice monogenic lines carrying the broad-spectrum resistance gene Pik-h and the narrow-spectrum resistance gene Pik-s, respectively, after inoculation with rice blast fungus, the chitinase activity of the broad-spectrum resistant material increased faster and reached a higher peak, and the enzyme activity level was positively correlated with the resistance ability of the resistance gene (Q. Li, et al. Perception of damaged self in plants, Plant Physiol., 182 (2020), pp. 1545-1565). This demonstrates that chitinase may play an active role in rice-specific disease resistance responses. Activation of the plant immune system requires a large amount of energy and resources, which may inhibit normal growth and development; this phenomenon is known as the "growth-defense tradeoff." Chitinase may maintain this balance by regulating the intensity of the immune response. The immune receptor OsCERK1 on the surface of rice cells recognizes long-chain chitin and activates the immune response, but this process is precisely regulated by the negative regulator OsCIE1. Studies suggest that certain chitinases may maintain the inhibitory effect of OsCIE1 on OsCERK1 and prevent excessive immune activation by degrading long-chain chitin (an immune activation signal) to generate short-chain chitin (a symbiotic signal). (Wang, G., Chen, X., Yu, C. et al. Release of a ubiquitin brake activates, OsCERK1-triggered immunity in rice. Nature 629, 1158–1164 (2024).)

[0005] However, there are many chitinase genes in rice, and their functions are also different. Accurate identification of the specific classification and unique function of the chitinase genes studied in rice is of great importance, and has important guiding value for in-depth analysis of rice disease resistance mechanisms and exploration of rice disease resistance gene resources. Summary of the Invention

[0006] This invention discovers that transgenic rice with OsCHIT5 gene knockout mutation exhibits significant resistance to rice blast fungus, while rice overexpressing the OsCHIT5 gene displays a susceptible phenotype. This leads to the completion of this invention.

[0007] The technical solution adopted by this invention to solve its technical problem is as follows:

[0008] In a first aspect, the present invention provides a rice chitinase OsCHIT5, wherein the protein is selected from any one of (A1)-(A3):

[0009] (A1) A protein with the amino acid sequence shown in SEQ ID NO:3;

[0010] (A2) Proteins derived from 1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO.3;

[0011] (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (A1) or (A2).

[0012] Secondly, the present invention provides a rice chitinase OsCHIT5 gene, which is selected from any one of the following (A1)-(A3):

[0013] (A1) The DNA sequence is shown in SEQ ID NO:1;

[0014] (A2) The CDS sequence is shown in SEQ ID NO:2;

[0015] (A3) A nucleotide sequence that hybridizes with a DNA sequence defined by (A2) under strict conditions.

[0016] The DNA sequence in (A1) is a nucleotide sequence containing a promoter, exons, introns and terminators.

[0017] The CDS sequence in (A2) is the coding sequence of the OsCHIT5 gene.

[0018] Thirdly, the present invention provides a reagent for resisting rice blast, wherein the active ingredient of the reagent is a substance that inhibits the expression of the OsCHIT5 gene as described in claim 2 and / or knocks out the OsCHIT5 gene.

[0019] In a specific implementation, the substance contains any one of the following (A1)-(A3):

[0020] (A1) sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the OsCHIT5 gene described above;

[0021] (A2) Generate DNA molecules that generate sgRNA targeting the OsCHIT5 gene described above, generate DNA molecules that generate siRNA targeting the OsCHIT5 gene described above, generate DNA molecules that generate shRNA targeting the OsCHIT5 gene described in Claim 2, generate DNA molecules that generate miRNA targeting the OsCHIT5 gene described above, or generate DNA molecules that generate antisense RNA targeting the OsCHIT5 gene described above.

[0022] (A3) An expression vector that generates sgRNA targeting the OsCHIT5 gene described above, an expression vector that generates siRNA targeting the OsCHIT5 gene described above, an expression vector that generates shRNA targeting the OsCHIT5 gene described above, an expression vector that generates miRNA targeting the OsCHIT5 gene described above, or an expression vector that generates antisense RNA targeting the OsCHIT5 gene described above.

[0023] Fourthly, the present invention provides a method for improving rice resistance to rice blast by inhibiting the expression of the OsCHIT5 gene described above in the recipient rice, thereby obtaining target rice with higher resistance to rice blast than the recipient rice.

[0024] In a specific implementation, the rice chitinase OsCHIT5 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:3.

[0025] In a specific implementation, the CDS sequence of the chitinase OsCHIT5 gene is shown in SEQ ID NO:2.

[0026] In a specific implementation scheme, the inhibition of OsCHIT5 gene expression in recipient rice is achieved by gene editing of the OsCHIT5 gene in recipient rice; the gene editing is achieved using a CRISPR / Cas9 system.

[0027] In a specific implementation, the CRISPR / Cas9 system includes sgRNA.

[0028] In a more specific implementation, the sgRNA is as shown in SEQ ID NO: 4.

[0029] In a specific implementation plan, the rice chitinase OsCHIT5 mutant gene is constructed into a CRISPR-Cas9 recombinant vector, the plant expression vector is transformed into Agrobacterium, and then rice is inoculated. Knockout mutant lines or their stable genetic offspring with rice blast resistance are selected.

[0030] In a specific implementation plan, the Agrobacterium is EHA105.

[0031] The genetically modified rice obtained by the above methods does not affect normal growth and grain production.

[0032] Fifthly, the present invention provides a CRISPR / Cas9 system, wherein the CRISPR / Cas9 system includes sgRNA, preferably as shown in SEQ ID NO: 4.

[0033] In a sixth aspect, the present invention provides the application of the rice chitinase OsCHIT5, the rice chitinase OsCHIT5 gene, the reagents, or the system described above in the cultivation of rice blast-resistant rice.

[0034] Beneficial effects

[0035] This invention, through innovative research and development, knocks out the rice chitinase OsCHIT5 gene, significantly improving rice blast resistance and has broad application prospects. Attached Figure Description

[0036] Figure 1 To detect the disease resistance of rice with overexpression and knockout of the rice chitinase OsCHIT5 gene. Figure 1 Figure A in the figure represents the Western blot analysis of OsCHIT5 protein overexpression plants; Figure 1 Figure B in the diagram represents the OsCHIT5 gene sequence and the sequencing results of the target gene knockout. Figure 1 Figure C in the figure represents the resistance and susceptibility of OsCHIT5 gene knockout rice and overexpression rice to rice blast fungus; Figure 1 Figure D in the figure shows the statistical results of the length of lesions formed by rice blast fungus infection in the leaves shown in Figure C; Figure 1 Figure E in the figure represents the relative biomass of rice blast fungus on the leaves as shown in Figure C. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0038] Example 1: Obtaining rice with overexpression of the rice chitinase OsCHIT5 gene

[0039] The coding region of the target gene OsCHIT5 (sequence shown in SEQ ID NO:2) was constructed into the plant expression vector pCAMBIA2300 (or pXQ) using the homologous recombination method (Zheng Luping, Lin Chen, Xie Liyan, et al. Breeding and disease resistance analysis of transgenic rice with co-interference of rice stripe virus NS2 and NS3 genes [J]. Acta Virologica Sinica, 2014, 30(6)). Based on the complete coding region sequence of the OsCHIT5 gene, specific upstream and downstream primers were designed. When designing primers, homologous arms near the SmaI restriction site in the pXQ vector were added to the upstream and downstream primers of OsCHIT5 to amplify the OsCHIT5 gene. The primers were: F: TAGGTAGAAGAGGTACCCGGGATGAAAGCCACGACGAC; R: ATCCTTGTAATCCATCCCGGGCTGCGCCGCCGTCAAC. The amplification parameters were: 95℃ for 3 min, 95℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, and 72℃ for 5 min, with 34 cycles of denaturation, annealing, and extension to obtain the target gene. The target gene and the linearized pXQ vector digested with SmaI were then ligated using a homologous recombinase to form the pXQ-OsCHIT5-Flag vector, which was introduced into *E. coli* competent cells JM109. Single colonies were amplified, and plasmids were extracted. Because the vector carries a Flag tag, it can be used for later validation. The pXQ-OsCHIT5 plasmid was chemically transformed into *Agrobacterium* EHA105, and positive single colonies were selected to prepare OD. 600 ZH11 rice callus was infected with a 0.2% inoculum solution for 15 min, followed by incubation at 20 ℃ for 48-72 h. Single-clone callus tissue was then picked and cultured on selection medium at 26 ℃ in the dark. After 25 days, positive single-clone callus tissue was selected and subcultured at 26 ℃ in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto differentiation medium and cultured at 27 ℃ under light for 15 days. Then, 3-5 cm differentiated shoots were inoculated onto rooting medium and cultured at 30 ℃ under light for 7 days. When the seedlings reached approximately 8 cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. Through two consecutive field propagation processes, sufficient OsCHIT5 overexpression transgenic rice seeds were obtained, while simultaneously stabilizing the plant's genetic background. Two healthy independent overexpression lines were selected from the transgenic plants obtained from seed propagation. Total protein was extracted from rice leaves and detected by Western blot using a Flag tag-specific antibody (the results are shown in Figure A of Figure 1).

[0040] The test results showed that the specific OsCHIT5-Flag fusion protein band could be detected in both transgenic lines, while the band was not detected in the wild-type control plants. This indicates that the OsCHIT5 gene was successfully overexpressed in the selected transgenic lines, and these positive plants can be used for subsequent functional verification experiments.

[0041] Example 2: Obtaining rice with OsCHIT5 gene knockout of chitinase

[0042] This invention utilizes CRISPR / Cas9 technology to construct an OsCHIT5 knockout vector. The vector backbone is BGK03 (Jialin L, Linjuan O, Jiali Z, et al. CRISPR / Cas9-mediated Editing of the Rice Catalase Gene OsCAT2[J]. Molecular Plant Breeding, 2018.), and the sgRNA sequence is: GGCTCCACTTCCGACTACTG (as shown in SEQ ID NO: 4). The target site is Chr5:19435559. The knockout vector is then chemically transformed into Agrobacterium EHA105, and positive single colonies are selected to prepare OD. 600 Rice callus was infected with a 0.2% inoculum solution for 15 min, then cultured at 20 ℃ for 48-72 h. Single-clone callus tissue was then picked and cultured on selection medium at 26 ℃ in the dark. After 25 days, positive single-clone callus tissue was selected and subcultured at 26 ℃ in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto differentiation medium and cultured at 27 ℃ under light for 15 days. Then, 3-5 cm differentiated shoots were inoculated onto rooting medium and cultured at 30 ℃ under light for 7 days. When the seedlings reached about 8 cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. After two propagation cycles, a knockout mutant of the OsCHIT5 gene was obtained. Sequencing confirmed that the gene had been knocked out and that two different mutations existed. Figure 1 (Figure B) Identification primers: F: GTAGTCGCCGAGCAGTGTG; R: AAGAGCTCCTTCGACACGAC.

[0043] Example 3: Analysis of resistance and susceptibility of rice chitinase OsCHIT5 gene knockout plants and overexpression plants

[0044] To evaluate the rice blast resistance of transgenic rice, this invention employs a wound inoculation method to conduct rice blast fungus inoculation experiments on wild-type and transgenic rice lines. The specific operational procedure is as follows: First, rice blast fungus spore suspension is prepared. Mature rice blast fungus SDC plates are used, and spores on the surface of sterile horizontal plates are collected. The filtered spore suspension is then counted under a microscope using a hemocytometer, ultimately achieving a spore suspension concentration of 5 × 10⁻⁶. 4 The samples were collected at a concentration of 1000 spores / ml and kept for later use. Next, rice leaves were pretreated and inoculated. Rice plants with a growth cycle of approximately 4 weeks were selected, including wild-type rice ZH11, knockout mutant rice, and overexpression rice plants. At least 3 healthy leaves of consistent growth status were selected from each line. The selected leaves were laid flat in 25 cm culture dishes, ensuring the leaves remained in a moist environment throughout the culture process. Before inoculation, the leaves needed to be wounded. Two wound points were gently made on each leaf using a sterile needle to facilitate infection by the rice blast fungus. Then, 20 μl of the prepared rice blast fungus spore solution was pipetted and slowly added to the wound points on the leaves. The inoculated culture dishes were placed in a 28°C dark environment for 24 h to promote spore germination and initial infection. Afterward, the culture was transferred to alternating light and dark conditions of 12 h light and 12 h dark for 5-7 hours. d. During the cultivation period, regularly observe the disease incidence on the leaves. After the lesions stabilize, take photos of the leaf phenotypic pattern and measure the length of the lesions on the leaves of each strain. Figure 1 (Figure D in the middle), and samples were taken for determination of rice blast pathogen biomass (Figure D in the middle). Figure 1 (Figure E). It was found that the lesions in rice plants overexpressing OsCHIT5 were significantly larger than those in wild-type rice ZH11, while the lesions in oschit5 knockout mutant plants were significantly smaller than those in wild-type rice ZH11. Figure 1 Figure C shows that OsCHIT5 negatively regulates rice resistance to rice blast, and knocking out CHIT5 can improve rice's disease resistance.

[0045] SEQ ID NO:1

[0046]

[0047] SEQ ID NO:2

[0048]

[0049] SEQ ID NO:3

[0050] MKATTTAVALLVAAAAMAAQVVAEQCGSQAGGALCPNCLCCSSYGWCGSTSDYCGDGCQSQCDGCGGGGGGGGGGGGGGGGGGAVEAVVSKELFEQLLLHRNDAACPARGFYTYDALVTAAAAFPDFAATGDDEARKREVAAFLGQTSHETTGGWATAPDGPYSWG YCFKEEIGATASYCVPSAEWPCAPDKKYFGRGPIQLSYNYNYGPAGEAIGEDLLNNPELVASDPVVSFKTALWFWMTPQSPKPSCHDVITGQWTPSSGDIAAGRVPGYGVITNIINGGLECGFGPDDRVANRIGFYQRYCDVLGIGYGSNLDCYDQRPFNSGLTAAQ

[0051] SEQ ID NO:4

[0052] GGCTCCACTTCCGACTACTG

[0053] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A rice chitinase OsCHIT5, characterized in that, It is selected from any one of the following (A1)-(A3): (A1) A protein with the amino acid sequence shown in SEQ ID NO:3; (A2) Proteins derived from 1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO.3; (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (A1) or (A2).

2. The rice chitinase OsCHIT5 gene, selected from any one of the following (A1)-(A3): (A1) The DNA sequence is shown in SEQ ID NO:1; (A2) The CDS sequence is shown in SEQ ID NO:2; (A3) A nucleotide sequence that hybridizes with a DNA sequence defined by (A2) under strict conditions.

3. A reagent for resisting rice blast, characterized in that, The active ingredient of the reagent is a substance that inhibits the expression of the OsCHIT5 gene as described in claim 2 and / or knocks out the OsCHIT5 gene.

4. The reagent according to claim 3, characterized in that, The substance contains any one of the following (A1)-(A3): (A1) Targeting the sgRNA, siRNA, shRNA, miRNA or antisense RNA of the OsCHIT5 gene as described in claim 2; (A2) Generate a DNA molecule that targets the sgRNA of the OsCHIT5 gene as described in claim 2, generate a DNA molecule that targets the siRNA of the OsCHIT5 gene as described in claim 2, generate a DNA molecule that targets the shRNA of the OsCHIT5 gene as described in claim 2, generate a DNA molecule that targets the miRNA of the OsCHIT5 gene as described in claim 2, or generate a DNA molecule that targets the antisense RNA of the OsCHIT5 gene as described in claim 2. (A3) Generate an expression vector for sgRNA targeting the OsCHIT5 gene of claim 2, an expression vector for siRNA targeting the OsCHIT5 gene of claim 2, an expression vector for shRNA targeting the OsCHIT5 gene of claim 2, an expression vector for miRNA targeting the OsCHIT5 gene of claim 2, or an expression vector for antisense RNA targeting the OsCHIT5 gene of claim 2.

5. A method for improving rice resistance to rice blast, characterized in that, By inhibiting the expression of the OsCHIT5 gene as described in claim 2 in the recipient rice, a target rice variety with higher resistance to rice blast than the recipient rice is obtained.

6. The method according to claim 5, characterized in that, The inhibition of OsCHIT5 gene expression in recipient rice was achieved by gene editing of the OsCHIT5 gene in recipient rice; the gene editing was performed using a CRISPR / Cas9 system.

7. The method according to claim 6, characterized in that, The CRISPR / Cas9 system includes sgRNA.

8. The method according to claim 7, characterized in that, The sgRNA is shown in SEQ ID NO:

4.

9. The Crisper / Cas9 system, characterized in that, The CRISPR / Cas9 system includes sgRNA, preferably as shown in SEQ ID NO:

4.

10. The application of the rice chitinase OsCHIT5 of claim 1, the rice chitinase OsCHIT5 gene of claim 2, the reagent of claim 3, or the system of claim 9 in the cultivation of rice blast-resistant rice.

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