Application of PiK1 gene in regulating rice blast resistance

Abstract

The application of the PiK1 gene in regulating rice blast resistance belongs to the field of biotechnology, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The phenotype identification results by inoculation of Magnaporthe oryzae show that, compared with wild type rice, the disease spot area of the PiK1 gene overexpression strain is significantly reduced, and the disease spot area of the gene editing knockout mutant is significantly increased, which confirms that the PiK1 gene positively regulates the rice blast resistance. The application provides a new gene resource for rice blast resistance molecular breeding, and has important application value for genetic improvement of rice disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the PiK1 gene in regulating rice blast resistance. Background Technology

[0002] Rice blast, caused by the fungus *Magnaporthe oryzae*, is the most widespread fungal disease affecting rice production. In the field, leaf blast lesions spread rapidly; neck blast causes whiteheads, and in years of severe outbreaks, yield losses can exceed 30%. Current production relies primarily on chemical pesticides for disease control. However, rice varieties are susceptible to resistance variations due to physiological races of *Magnaporthe oryzae*, losing resistance after several years of promotion. The scarcity of resistance genes has become a key bottleneck restricting the development of rice disease-resistant breeding.

[0003] Upon recognizing pathogenic microorganisms, plants initiate the MAPK cascade signaling pathway (MAPKKK-MAPKK-MAPK). This pathway amplifies the signal through stepwise phosphorylation, thereby activating the expression of downstream transcription factors and disease-related genes. The activation intensity and response speed of the plant immune response are largely determined by the components of this cascade pathway. While there are reports on the involvement of rice MAPK family members in regulating rice blast resistance, most studies remain at the stage of basic functional analysis.

[0004] The inventors of this application cloned the MPK-like kinase gene PiK1 (Pyricularia-resistance Kinase 1) from rice, with a full-length coding sequence of 1836 bp. Functional identification through pathogen inoculation confirmed that overexpression of the PiK1 gene significantly reduced the area of ​​rice lesions; gene editing that inactivated the gene significantly increased the area of ​​rice lesions. These results demonstrate that PiK1 positively regulates rice resistance to rice blast, and there are currently no publicly available reports on the disease resistance function of this gene. Summary of the Invention

[0005] This invention provides the application of the PiK1 gene in regulating rice blast resistance, wherein the PiK1 gene has any one of the following nucleotide sequences:

[0006] (1) The mRNA sequence shown in SEQ ID NO.1 has a length of 1836 bp;

[0007] (2) A DNA sequence encoding the same protein as the sequence described in (1), containing a codon degenerate variant;

[0008] (3) Recombinant DNA molecules obtained by linking the nucleic acid sequence of (1) or (2) with a promoter.

[0009] The application of this invention is as follows: The full-length coding sequence of the PiK1 gene is introduced into an expression vector, and then introduced into rice plants through genetic transformation. This enhances the rice's resistance to rice blast fungus, and the area of ​​rice lesions is significantly reduced after inoculation with the pathogen. Using gene editing technologies such as CRISPR to inactivate the PiK1 gene or downregulate its expression level significantly reduces rice blast resistance.

[0010] Therefore, on the one hand, rice blast resistance can be improved by increasing the expression level of the PiK1 gene; on the other hand, rice materials with enhanced susceptibility to rice blast can be obtained through gene knockout, gene suppression, and gene silencing. Simultaneously, nucleic acid probes can be prepared using the PiK1 gene sequence, or homologous genes of this gene can be isolated and identified from rice using PCR technology.

[0011] Beneficial effects:

[0012] This invention created PiK1 overexpression lines (OE-1, OE-2) and gene-edited mutants (pik1-1, pik1-2) (see...). Figure 1 Rice seedlings at the three-leaf stage were sprayed with a suspension of rice blast fungus spores. Post-inoculation phenotypic results showed that PiK1 gene overexpression lines had fewer and smaller lesions; gene-edited mutants had larger lesions that expanded rapidly; and wild-type plants exhibited disease severity between the two (see...). Figure 2 The expression level of the PiK1 gene is positively correlated with the strength of rice blast resistance in rice plants, confirming that the PiK1 gene positively regulates rice blast resistance. This gene can be used as a candidate gene for molecular breeding of rice disease resistance. Attached Figure Description

[0013] Figure 1 Identification of PiK1 gene overexpression lines (A) and gene-edited mutants (B);

[0014] Figure 2 Statistics on lesion phenotype and lesion area after inoculating WT, PiK1 gene overexpression lines (OE-1, OE-2) and knockout mutants (pik1-1, pik1-2) with rice blast fungus. Detailed Implementation

[0015] The PiK1 gene material used in this invention was provided by the laboratory of the College of Plant Science, Jilin University; contact address: College of Plant Science, No. 5333, Xi'an Road, Lvyuan District, Changchun City, Jilin Province, 130062, China; contact person: Wu Tao.

[0016] The molecular manipulations and rice genetic transformations involved in this embodiment are all conventional experimental techniques in this field.

[0017] Example 1: Obtaining PiK1 gene overexpression lines and pik1 mutants

[0018] Using rice seedling cDNA as a template, the full-length 1836 bp PiK1 coding region sequence was amplified. An overexpression vector was constructed, and rice callus tissue was transformed using Agrobacterium tumefaciens EHA105. After plant regeneration, resistance selection, and self-pollination, genetically homozygous overexpression lines OE-1 and OE-2 were obtained. qRT-PCR and Western blot results confirmed that the target gene-encoded protein achieved high-level accumulation in both lines (e.g., ...). Figure 1 (As shown in A).

[0019] Gene-editing mutants were created using CRISPR / Cas9 technology: sgRNAs were designed targeting the coding region of the PiK1 gene, gene-editing vectors were constructed, and rice callus tissue was transformed into regenerated plants. PCR amplification and sequencing verification of the editing target region were performed, resulting in two independent homozygous edited lines, pik1-1 and pik1-2. Sequencing results showed that base insertions or deletions occurred at the editing target site in both lines, inducing frameshift mutations that caused the loss of function of the kinase domain of the encoded protein (e.g., ...). Figure 1 (as shown in B).

[0020] Example 2: Identification of rice blast resistance

[0021] The rice blast pathogen was activated on PDA plates and transferred to rice culture medium to induce sporulation. Spores were collected and a spore suspension was prepared using sterile water. Three-leaf stage rice seedlings were spray-inoculated and cultured under moist conditions for 24 h. Subsequent culture conditions were set at 25–28 °C with alternating 12 h light / 12 h dark cycles. Disease surveys were conducted 7 days after inoculation, and the area of ​​individual lesions was counted and measured. Results showed that compared to the wild type, the lesion area was significantly reduced in OE-1 and OE-2 overexpression lines, and lesion expansion was significantly inhibited; the lesion area of ​​pik1-1 and pik1-2 knockout mutants was significantly larger than that of the wild type (e.g., [example missing]). Figure 2 (As shown in the figure). This demonstrates that the expression level of the PiK1 gene is positively correlated with rice blast resistance, and that upregulating PiK1 gene expression can be used to improve rice blast resistance.

Claims

1. The application of the PiK1 gene in improving rice blast resistance, characterized by: The nucleotide sequence of the PiK1 gene is shown in SEQ ID NO.1; The method to improve rice blast resistance is to increase the expression level of the PiK1 gene in rice, thereby significantly reducing the lesion area of ​​rice plants after inoculation with rice blast fungus.

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