Application of CDR1 gene in improving rice blast resistance

Abstract

The application of CDR1 gene in improving rice blast resistance belongs to the field of biotechnology, and the mRNA nucleotide sequence of the gene is shown as SEQ ID NO. 1. The results of spore inoculation of rice blast show that the disease spot area of the rice plant overexpressing CDR1 gene is significantly reduced, and the disease spot area of the CRISPR gene editing mutant plant is significantly increased. The quantitative detection of lignin confirms that the lignin content of the overexpression strain is increased, and the lignin content of the mutant is decreased. CDR1 gene can enhance the structural defense ability of rice cell wall by promoting lignin deposition in cell wall, positively regulate the rice blast resistance, and can be applied to rice breeding for resistance to rice blast.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CDR1 gene in improving rice blast resistance. Background Technology

[0002] Rice blast can be spread by air currents and can infect year-round, making it one of the most costly diseases to control in my country's major rice-producing areas. Infected leaves develop typical spindle-shaped lesions; in severe cases, these lesions merge, causing plant death. Infection of the panicle neck and branches directly leads to whiteheads, resulting in significant yield reduction. Current disease-resistant breeding efforts generally face challenges such as a narrow genetic base for resistance and the easy loss of varietal resistance, leading to a heavy reliance on multiple chemical sprays to control the disease.

[0003] Rice's structural defense against rice blast fungus primarily relies on its cell wall. Lignin is deposited in the secondary cell wall through cross-linking, which physically hinders the invasion of pathogenic hyphae and resists the degradation by cell wall-degrading enzymes secreted by the pathogen. Furthermore, the phenylpropanoid metabolic pathway involved in lignin biosynthesis can generate various secondary metabolites with antibacterial activity. Therefore, the level of cell wall lignification in rice plants is closely related to the rice blast resistance of rice varieties.

[0004] The transcriptional expression of lignin synthesis-related genes is precisely regulated by transcription factors. Previous studies have confirmed that the bZIP transcription factor family can participate in the induction of plant defense response genes and simultaneously regulate the phenylpropanone metabolic pathway. In this application, the inventors isolated the bZIP family transcription factor encoding gene CDR1 (Cold and Drought Resistance 1). Pathogen inoculation experiments and lignin content detection confirmed that CDR1 gene overexpression can reduce the area of ​​rice blast lesions and increase the level of lignin accumulation in plants; gene editing and knockout of this gene resulted in the completely opposite phenotype. This demonstrates that CDR1 positively regulates rice blast resistance and promotes lignin accumulation; these functions of the gene have not been previously reported. Summary of the Invention

[0005] This invention provides the application of the CDR1 gene in improving rice blast resistance, wherein the CDR1 gene is a nucleic acid molecule having any of the following nucleotide sequences:

[0006] (1) The mRNA sequence shown in SEQ ID NO.1, with a full length of 498 bp;

[0007] (2) A DNA sequence that can encode the protein corresponding to the sequence in (1);

[0008] (3) A recombinant DNA construct obtained by linking the nucleic acid sequence of (1) or (2) above with a promoter.

[0009] The application of this invention can be divided into two directions:

[0010] Introducing the complete coding sequence of the CDR1 gene into rice plants to achieve gene overexpression can significantly reduce the area of ​​lesions after inoculation with rice blast fungus, increase the lignin content of rice plants, and thus enhance the resistance of rice to rice blast.

[0011] By using gene editing technologies such as CRISPR-Cas9 to inactivate the CDR1 gene or downregulate its expression level, the lesion area of ​​rice infected with rice blast fungus expands and the lignin content of the plant decreases.

[0012] Based on the above characteristics, this invention can both improve the genetic resistance to rice blast by increasing the expression level of the CDR1 gene, and create rice experimental materials with weakened resistance to rice blast by knocking out, inhibiting, or silencing the CDR1 gene. Furthermore, homologous genes of the CDR1 gene in rice can be isolated from rice materials using conventional molecular biology techniques such as probe hybridization and RT-PCR.

[0013] The beneficial effects of this invention are as follows: Spore spray inoculation identification of CDR1 gene overexpression lines (OE-1, OE-2) and gene-edited mutants (cdr1-1, cdr1-2) showed that, compared to the wild type, the lesion area of ​​the CDR1 gene overexpression lines was significantly reduced; the lesion area of ​​the cdr1 knockout mutant was significantly increased (see...). Figure 1 ).

[0014] The lignin content of rice stem and leaf tissues was determined using the acetyl bromide method. The lignin content of the overexpression lines was significantly higher than that of the wild type, while the lignin content of the cdr1 mutant was significantly lower than that of the wild type (see...). Figure 2 The changes in disease resistance phenotype perfectly corresponded to the trends in lignin content, indicating that the CDR1 gene primarily regulates rice resistance to rice blast by promoting lignin deposition in the cell wall. This invention has dual application value in both genetic improvement of rice disease resistance and lignin-related research. Attached Figure Description

[0015] Figure 1 Statistics on lesion phenotype and lesion area after inoculation of WT, CDR1 gene overexpression lines (OE-1, OE-2) and cdr1 knockout mutants (cdr1-1, cdr1-2) with rice blast fungus;

[0016] Figure 2 The results show the lignin content of WT and CDR1 gene overexpression lines (OE-1, OE-2) and cdr1 knockout mutants (cdr1-1, cdr1-2). Detailed Implementation

[0017] The CDR1 gene and transgenic materials were provided by the laboratory of the College of Plant Science, Jilin University, located at No. 5333, Xi'an Road, Luyuan, Changchun, Jilin Province, 130062, China. Contact person: Wu Tao.

[0018] All molecular manipulations were performed using standard methods.

[0019] The test materials consisted of two homozygous overexpression lines (OE-1 and OE-2) and two independent homozygous edited lines (cdr1-1 and cdr1-2). The overexpression lines were obtained by ligating the full-length coding sequence of the CDR1 gene into an expression vector driven by a ubiquitin promoter and then transforming it with Agrobacterium tumefaciens (EHA105). The edited lines were constructed by targeting the coding region of the CDR1 gene using the CRISPR / Cas9 system, and the homozygous mutant genotypes were verified by sequencing after transformation and regeneration of plants.

[0020] Example 1: Identification of rice blast pathogen through inoculation

[0021] The rice blast fungus was activated and cultured on PDA medium, then transferred to rice medium to induce conidia production. Conidia were collected and prepared into a spore suspension. Three-leaf stage rice seedlings were spray-inoculated, followed by 24-hour moistening treatment. Subsequently, they were cultured at 28℃ under 12-hour light / 12-hour dark conditions. Disease phenotypes were photographed and recorded 7-10 days after inoculation, and lesion areas were statistically analyzed. Results showed that the lesion area of ​​OE-1 and OE-2 overexpression lines was significantly smaller than that of the wild type; the cdr1-1 and cdr1-2 knockout mutants not only had significantly larger lesion areas than the wild type, but also showed earlier onset of disease symptoms (e.g., ...). Figure 1 (As shown).

[0022] Example 2: Determination of Lignin Content

[0023] Stem and leaf tissues from various rice materials were collected, dried, and ground into powder. The powder was repeatedly extracted with alcohol solvents to remove pigments and soluble metabolites, yielding cell wall residues. Acetyl bromide-perchloric acid solution was added to the residues, and digestion was performed at 70°C for 30 min. The reaction was terminated with the addition of alkali solution and the volume was adjusted. The absorbance was measured at 280 nm, and the percentage of lignin in the dry weight of the cell wall was calculated using a standard curve. The results showed that the lignin content of the OE-1 and OE-2 overexpression lines was significantly higher than that of the wild type; the lignin content of the cdr1-1 and cdr1-2 knockout mutants was significantly lower than that of the wild type (e.g., ...). Figure 2 (As shown). The test results corroborate the disease resistance phenotype results of Example 1, indicating that CDR1 gene-mediated rice blast resistance may be related to increased lignin deposition levels in plants.

Claims

1. The application of the CDR1 gene in improving rice blast resistance, characterized by: The nucleotide sequence of the CDR1 gene is shown in SEQ ID NO.1; the method of improving rice blast resistance is to increase the expression level of the CDR1 gene in rice, thereby significantly reducing the lesion area and significantly increasing the lignin content of rice plants after inoculation with rice blast fungus.

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