Application of rice OsmPHBc1 gene in improving rice blast resistance

Abstract

The application relates to the field of genetic engineering and crop breeding technology, in particular to a rice OsmPHBc1 gene in improving the rice resistance to rice blast. The application overexpresses OsmPHBc1 gene in rice, and it is found through disease resistance detection that: OsmPHBc1 The overexpression of the gene significantly improves the resistance of the rice to the rice blast fungus. Therefore, OsmPHBc1 The gene can be introduced into the rice as a target gene to improve the disease resistance of the rice and improve the rice varieties. The application also proves that the protein expressed by the introduced OsmPHBc1 gene regulates the PTI response process by promoting the expression of defense-related genes OsPBZ1 and OsPAL1 effectively improves the defense ability of the rice. The application clearly defines the mechanism of the pathogenic bacteria invasion and the influence on the function of the mitochondria, and has important significance for the improvement and breeding of the rice.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and crop breeding technology, specifically to rice. OsmPHBc1 Application of genes in improving rice resistance to rice blast. Background Technology

[0002] Rice blast is a major fungal disease of rice caused by the rice blast fungus. It occurs widely in rice-growing areas worldwide and can lead to significant yield reductions or even crop failure in severe cases, making it one of the key bottlenecks restricting high and stable rice yields. Over its long evolutionary process, rice has developed a complex disease resistance and defense system. By recognizing the invasion signals of the rice blast fungus, it activates the expression of a series of defense-related genes, initiating an immune response to resist the pathogen's infection.

[0003] Mitochondria, as the "energy factories" of plant cells, are not only the core site of energy metabolism but also an indispensable signaling regulatory center in plant immune responses, playing a crucial role in rice's resistance to rice blast fungus infection. When rice is infected by rice blast fungus, mitochondria sense the pathogen's invasion signals and initiate a series of stress responses, including regulating the production and clearance of mitochondrial reactive oxygen species, maintaining the homeostasis of the mitochondrial electron transport chain, and regulating mitochondrial membrane potential and permeability. This, in turn, activates the expression of downstream defense-related genes, initiating the basal and specific immune responses in rice, forming an effective defense against rice blast fungus. Current research has confirmed that various mitochondrial-localized proteins are involved in the regulation of rice blast resistance, such as the mitochondrial-localized pentapeptide repeat protein OsNBL3, the cytochrome c oxidase assembly protein OsCOX11, and the glutoreductin OsGRXS15, which has dual mitochondrial and nuclear localization. These proteins affect rice's disease resistance by regulating mitochondrial function. However, the functions of most proteins in rice mitochondria remain unclear, especially those related to mitochondrial metabolism and signal transduction, whose specific mechanisms of action in rice resistance to rice blast are still ambiguous. Meanwhile, effector factors secreted by the rice blast fungus can specifically target rice mitochondria, weakening the rice's immune defenses by interfering with mitochondrial electron transport, disrupting mitochondrial structural integrity, and inhibiting the normal production of reactive oxygen species, thereby achieving successful infection. Currently, research on rice blast resistance targeting mitochondrial genes still has many gaps, urgently requiring the discovery of new mitochondrial-localized rice blast resistance genes and the clarification of their mechanisms of action to provide new genetic resources and theoretical basis for molecular breeding of rice blast resistance. Summary of the Invention

[0004] The purpose of this invention is to provide rice OsmPHBc1This invention addresses the limitations of existing technologies, such as the limited genetic resources for rice blast resistance and the lack of clarity regarding some resistance mechanisms, by applying genes to enhance rice blast resistance. It provides new genetic resources and technical pathways for breeding rice varieties resistant to rice blast.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of the OsmPHBc1 protein in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The amino acid sequence of the OsmPHBc1 protein is shown in SEQ ID NO.2.

[0006] Optionally, by increasing the activity or expression level of the OsmPHBc1 protein in the rice, the rice's resistance to rice blast can be improved or new rice varieties with high resistance to rice blast can be bred.

[0007] This invention provides OsmPHBc1 The application of genes in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Optionally, by increasing the amount of rice... OsmPHBc1 The expression level of genes can be increased to improve the resistance of rice to rice blast or to cultivate new rice varieties with high resistance to rice blast.

[0009] This invention provides a method comprising OsmPHBc1 Application of genetic biomaterials in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] Optionally, the biological material includes a recombinant vector or recombinant bacteria.

[0011] This invention provides a method for improving rice blast resistance, comprising overexpressing in the rice OsmPHBc1 The steps of gene generation; the described OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] Optionally, the overexpression includes... OsmPHBc1 The steps include: connecting a gene to an expression vector to construct a recombinant vector; transferring the recombinant vector into Agrobacterium to construct a recombinant engineered bacterium; and then inoculating the recombinant engineered bacterium into rice.

[0013] This invention provides a method for breeding transgenic rice with high resistance to rice blast, comprising overexpressing in the rice... OsmPHBc1 The steps of gene generation; the described OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Optionally, the overexpression includes... OsmPHBc1 The steps include: ligating a gene to an expression vector to construct a recombinant vector; transforming the recombinant vector into Agrobacterium to construct a recombinant engineered bacterium; and then inoculating the recombinant engineered bacterium into rice. The present invention discloses the following technical effects: This invention will OsmPHBc1 Transgenic rice was constructed by overexpressing the gene in rice. Disease resistance testing revealed that overexpression of this gene enhanced the rice's resistance to rice blast fungus. Therefore, OsmPHBc1 The gene can be introduced into rice as a target gene, which can improve the rice's disease resistance and can be used for rice variety improvement. This invention also confirms the effectiveness of the introduced gene. OsmPHBc1 The proteins expressed by genes promote defense-related genes. OsPBZ1 and OsPAL1 Expressing and regulating the PTI response process effectively enhances rice's defense capabilities, thereby improving its resistance to rice blast. This invention clarifies the mechanism by which pathogens invade and affect mitochondrial function, which is of great significance for rice breeding and improvement. Furthermore, this invention provides new gene targets and technical support for breeding rice blast resistance. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Map of the pRHVcGFP expression vector; Figure 2 In genetically modified rice OsmPHBc1 Expression level analysis; NPB represents wild-type Nipponbare rice variety, OX- OsmPHBc1 -GFP-#2 and OX- OsmPHBc1 -GFP-#8 represents two overexpression lines transformed with NPB as the background. Figure 3For OX- OsmPHBc1 The results of the resistance test of transgenic plants to the physiological race RB22 of rice blast fungus (A) and the number of lesions after transgenic plants and wild-type plants were inoculated with NPB of rice blast fungus race RB22 (B). Figure 4 For OX- OsmPHBc1 In transgenic plants OsPBZ1 Genes and OsPAL1 A graph showing the gene expression levels of a gene. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] The sequence involved in this invention is as follows: rice OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.1: ATGGCCGGCGGCGGGCAGGCGGCGGCGTCGCTGCTGACGAAGCTGGCGCAGGCGGCGGCCGGGCTGGGCATCGCGGCGTCGGCGGCGTCGACGGCGCTGTACACGGTGGACGGCGGGCAGCGCGCCGTGATCTTCGACCGGTTCCGCGGCGTGCTCCCGGAGACCTCGTCGGAGGGCACCCACTTCATCGTCCCCTGGCTCCAGAAGCCCTTCATCTTCGACATCCGCACCCGCCCGCACAGCTTCTCCTCCACCTCTGGGACCAAGGACCTGCAGATGGTCAGCCTCACCCTCCGCGTCCTCGCGCGCCCCGACATCGACCGCCTCCCGGACATCTTCACCTCGCTGGGCCTCGAGTACGACGAGAAGGTGCTCCCCTCCATCGGCAACGAGGTGCTCAAGGCCGTCGTCGCGCAGTTCAACGCCGACCAGCTCCTCACCGAGCGCCCCCACGTCTCCGCGCTTGTCCGCGACTCCCTCATCCGCCGCGCCGCCGAGTTCAACATCGTCCTCGACGACGTCGCCATCACGCACCTCGCCTACGGCCCGGAGTTCTCCCAGGCCGTCGAGAAGAAGCAGGTGGCGCAGCAGGAGGCCGAGCGGTCCAGGTTCCTCGTCGCGCGCGCCGAGCAGGAGAGGCGCGCCGCCATCGTCCGCGCCGAGGGGGAGAGCGAGGCCGCGCGCCTCATCTCCGAGGCCACCGCCGCCGCGGGCACCGGCCTGATCGAGCTCAGGAGGATCGAGGCCGCCAAGGAGATCGCCGGGGAGCTTGCGCGCTCCCCCAATGTCTCGTACATCCCGGCCGGCGACAGCAGCCAGATGCTGCTTGGTCTCAGCGGCGCTCGGTGA。

[0023] rice OsmPHBc1 The amino acid sequence of the OsmPHBc1 protein encoded by the gene is shown in SEQ ID NO.2: MAGGGQAAASLLTKLAQAAAGLGIAASAASTALYTVDGGQRAVIFDRFRGVLPETSSEGTHFIVPWLQKPFIFDIRTRPHSFSSTSGTKDLQMVSLTLRVLARPDIDRLPDIFTSLGLEYDEKVLPSIGNEVLKAVVAQFN ADQLLTERPHVSALVRDSLIRRAAEFNIVLDDVAITHLAYGPEFSQAVEKKQVAQQEAERSRFLVARAEQERRAAIVRAEGESEAARLISEATAAAGTGLIELRRIEAAKEIAGELARSPNVSYIPAGDSSQMLLGLSGAR.

[0024] Example 1 OX- OsmPHBc1 Transgenic lines (OX-) OsmPHBc1 Construction of overexpression transgenic lines 1. Extraction of total RNA The Nipponbare japonica rice variety was selected. When the rice seedlings were about two weeks old, leaves were immediately collected, frozen in liquid nitrogen, and stored at -80℃. A portion of the leaves was taken, crushed in a mortar, and transferred into a 1.5 mL EP tube containing Trizol lysis buffer. After thorough shaking, total RNA was extracted, and the quality of the total RNA was determined by electrophoresis.

[0025] 2. Rice mitochondrial-associated protein genes OsmPHBc1 Cloning and construction of plant expression vectors according to OsmPHBc1 Design primers at both ends of the gene sequence: P1: 5'-TCCCCGGGTGAGCTCGGTACCATGGCCGGCGGCGGGCAGGC-3' (SEQ ID NO. 3); P2: 5'-AGCGCCGCACTAGTAAGCTTCCGAGCGCCGCTGAGACCAA-3' (SEQ ID NO. 4).

[0026] The total RNA obtained above was reverse transcribed to synthesize the first strand of cDNA. Using this as a template, PCR amplification was performed using high-fidelity KOD enzyme. The PCR system was as follows (taking a 50 μL system as an example): 25 μL 2×KOD Buffer, 10 μL dNTPs, 1.5 μL upstream primer F, 1.5 μL downstream primer R, 2.0–4.0 μL cDNA template, 0.5 μL KOD high-fidelity polymerase, and ddH2O to 50 μL. The PCR program was as follows: 96℃ pre-denaturation for 3 min, 96℃ denaturation for 30 s, 57℃ annealing for 30 s, 68℃ extension for 1 min, 35 cycles, followed by 68℃ extension for 10 min, and recovery of the target fragment.

[0027] Simultaneously, the pRHVcGFP vector was double-digested with restriction endonucleases SacI and HindIII. Figure 1 The pRHVcGFP vector is disclosed in the literature "A Versatile Vector Toolkit for Functional Analysis of Rice Genes". The enzyme digestion system is as follows: 1 μg of vector plasmid, 5 μL of 10× buffer, 1 μL of restriction endonuclease SacI, 1 μL of restriction endonuclease HindIII, and ddH2O added to 50 μL. The digestion conditions are 37℃ for 60 min. Then, the digested vector fragment is recovered and further processed. OsmPHBc1 Cloning was performed into the pRHVcGFP vector. The ligation system was as follows: Exnase II 2 µL, 5×CE buffer 2 µL, target fragment 2 µL, vector 2 µL, ddH2O 2 µL; 37℃, 30 min. The ligation product was heat-shocked at 42℃ to transform competent E. coli JM109 cells, plated on LB solid medium containing 50 µg / mL kanamycin, and incubated at 37℃ inverted for about 12 h. Successfully transformed single clones were picked and incubated on LB liquid medium containing 50 µg / mL kanamycin for 10-12 h with shaking. Plasmids were extracted, and sequencing was performed to confirm the correct reading frame of the coding region in the expression vector, thus obtaining the recombinant vector pRHV- OsmPHBc1 -GFP sequencing confirms that the reading frame of the coding region in the expression vector is correct.

[0028] 3. Obtaining transgenic plants The recombinant vector pRHV- obtained above OsmPHBc1 -GFP was transferred into Agrobacterium strain EHA105, and further transferred into the japonica rice variety Nipponbare (NPB). The resulting transgenic plants OX- OsmPHBc1 After qRT-PCR verification, transgenic rice was confirmed. The verification steps were as follows: RNA was extracted from young leaves of transgenic rice and reverse transcribed into cDNA. Primers were designed as follows: OsmPHBc1 -F1: 5'-ACGACGAGAAGGTGCTCCC-3' (SEQ ID NO. 5); OsmPHBc1 -F2: 5'-CTCGCTCTCCCCCTCGGCGG-3' (SEQ ID NO. 6).

[0029] qRT-PCR detection OsmPHBc1 Gene expression levels, qRT-PCR reaction system: primers OsmPHBc1 -F1 and OsmPHBc1 1 µL each of -F2, 10 µL of 2×SYBR qPCR Mix, and 8 µL of diluted cDNA; the reaction program was: 95℃ pre-denaturation for 30 s, 95℃ for 10 s, 60℃ for 30 s, 40 cycles, followed by 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s to generate the melting curve. The obtained... OsmPHBc1 Overexpressing plants were subcultured for disease resistance testing in rice. The results showed that the overexpressing plants OX- OsmPHBc1 -GFP-#2 and OX- OsmPHBc1 -GFP-#8 ( Figure 2 ).

[0030] Example 2 OX- OsmPHBc1 Disease resistance identification of transgenic lines Spray inoculation: awaiting OX- OsmPHBc1 When the transgenic rice seedlings reached 3-4 weeks of age and the three-leaf stage, robust, disease-free, and pest-free soil-cultured seedlings were selected as test materials. Simultaneously, the physiological race RB22 of rice blast fungus was cultured in advance, and its colonies were cultured under light conditions for approximately 14 days. Conidia were washed away with a sterile aqueous solution containing 0.1% Tween, and mycelial residues were removed by filtration through Miracloth filter cloth. The spore concentration was then adjusted to 2-2.5 × 10⁻⁶. 6 A homogeneous suspension of *Magnapordica oryzae* spores was obtained by spraying the prepared suspension onto the rice leaves using a spray inoculation method. After inoculation, the inoculation box was sealed with plastic wrap to maintain a high-humidity environment. The seedlings were then cultured at 26°C and a relative humidity of at least 80%. After 7 days, the disease incidence on the rice leaves was observed, the disease severity was assessed, and the number of lesions was counted. Transgenic rice plants exhibiting significant resistance compared to the wild-type control were identified as blast-resistant transgenic plants.

[0031] The results showed that compared with wild-type NPB, OX- OsmPHBc1 Overexpression of transgenic plants enhanced resistance to blast fungus race RB22, specifically manifested as OX- OsmPHBc1 The number of lesions in the overexpressing transgenic plants after inoculation was significantly reduced compared to the control NPB. Figure 3 ).

[0032] Meanwhile, qRT-PCR was used to detect OX- OsmPHBc1 Overexpression of plant defense-related genes OsPBZ1 and OsPAL1 The expression situation. Primer design: OsPBZ1-F1: 5'-CCCTGCCGAATACGCCTAA-3' (SEQ ID NO. 7); OsPBZ1-F2: 5'-CTCAAACGCCACGAGAATTTG-3' (SEQ ID NO. 8); OsPAL1-F1: 5'-ACTCGCTGGGTCTCGTCTCG-3' (SEQ ID NO.9); OsPAL1-F2: 5'-GTGACGCAGTTCTTGACGGA-3' (SEQ ID NO. 10).

[0033] qRT-PCR detection OsmPHBc1 Overexpression of plant defense-related genes OsPBZ1 and OsPAL1 The expression level was determined. The qRT-PCR reaction system consisted of 1 µL each of primers F1 and F2, 10 µL of 2×SYBR qPCR Mix, and 8 µL of diluted cDNA. The reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ for 10 s, 60℃ for 30 s, for 40 cycles, followed by 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s to generate the melting curve.

[0034] qRT-PCR test results are as follows Figure 4 As shown, the results indicate that defense-related genes OsPBZ1 and OsPAL1 The expression level of was significantly increased in overexpressing plants.

[0035] The above results indicate that in rice OsmPHBc1 Overexpression of the gene enhanced rice's resistance to rice blast fungus. Therefore, it holds promise for introducing the gene into plants to improve disease resistance and thus improve plant varieties.

[0036] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of OsmPHBc1 protein in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The amino acid sequence of the OsmPHBc1 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, By increasing the activity or expression level of the OsmPHBc1 protein in rice, the resistance of rice to rice blast can be improved or new rice varieties with high resistance to rice blast can be bred.

3. OsmPHBc1 The application of genes in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, By improving the rice OsmPHBc1 The expression level of genes can be increased to improve the resistance of rice to rice blast or to cultivate new rice varieties with high resistance to rice blast.

5. Includes OsmPHBc1 Application of genetic biomaterials in any of the following: (1) Improve rice blast resistance; (2) Develop new rice varieties with high resistance to rice blast; The OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, The biomaterials include recombinant vectors or recombinant bacteria.

7. A method for improving rice blast resistance, characterized in that, Including overexpression in the rice OsmPHBc1 The steps of gene generation; the described OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The overexpression includes the... OsmPHBc1 The steps include: connecting a gene to an expression vector to construct a recombinant vector; transferring the recombinant vector into Agrobacterium to construct a recombinant engineered bacterium; and then inoculating the recombinant engineered bacterium into rice.

9. A method for cultivating transgenic rice with high resistance to rice blast, characterized in that, Including overexpression in the rice OsmPHBc1 The steps of gene generation; the described OsmPHBc1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that, The overexpression includes the... OsmPHBc1 The steps include: connecting a gene to an expression vector to construct a recombinant vector; transferring the recombinant vector into Agrobacterium to construct a recombinant engineered bacterium; and then inoculating the recombinant engineered bacterium into rice.

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