OsCARD1 gene, application of OsCARD1 gene in improvement of resistance of rice to weeds, and protein

Abstract

The invention relates to the technical field of gene engineering, in particular to an OsCARD1 gene, application of the OsCARD1 gene to improvement of resistance of rice to weeds and protein. The nucleotide sequence of the OsCARD1 gene is as shown in SEQ ID No.1 (sequence identifier number 1). Through knockout of the OSCARD1 gene, the resistance of rice to weeds is remarkably improved, a new germplasm with weed resistance is cultivated, and a good growth state can be kept under the weed symbiosis condition. The result not only embodies an important role of the OSCARD1 gene in response to weed allelopathy signals of rice, but also provides a new thought and resource for molecular breeding of weed resistance of rice.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an OsCARD1 gene and its application in improving rice resistance to weeds, and a protein. Background Art

[0002] Weed damage is a major biotic stress in rice production, leading to severe rice yield reductions. The competition between rice and weeds is not limited to the competition for light, water, and nutrients. Weeds also inhibit rice growth and development by secreting allelopathic substances. Currently, chemical control remains the main means of weed control in rice fields. However, the long-term use of herbicides has led to increasing weed resistance, pesticide residues, and environmental pollution, highlighting the importance of developing green herbicides. Allelopathy, as a natural plant-to-plant interaction mechanism, shows great potential in the green and safe control of weeds. Therefore, elucidating the molecular mechanism of rice's response to weed allelopathic signals is of great significance for the breeding of weed-tolerant rice varieties. Summary of the Invention

[0003] To address the above problems, the present invention provides an OsCARD1 gene and its application in improving rice resistance to weeds, as well as a protein. The present invention clarifies the function of the OsCARD1 gene in rice responding to weed allelopathic signals, providing a theoretical basis for its in-depth application in the subsequent breeding of weed-resistant rice.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides an OsCARD1 gene, the nucleotide sequence of the OsCARD1 gene is shown as SEQ ID No.1.

[0006] The present invention also provides the use of the OsCARD1 gene described in the above technical solution in improving the resistance of rice to weeds.

[0007] Preferably, knocking out the OsCARD1 gene in rice improves the weed resistance of rice.

[0008] Preferably, knocking out the OsCARD1 gene in rice improves the resistance of rice to quinone allelopathic substances secreted by weeds.

[0009] Preferably, the quinone allelochemical includes 2,6-dimethoxy-1,4-benzoquinone.

[0010] Preferably, the weeds include Leptochloa chinensis.

[0011] The present invention also provides a protein encoded by the OsCARD1 gene in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No.2.

[0012] Beneficial effects of the present invention:

[0013] 1. Revealing the function of OsCARD1 gene at the molecular level

[0014] The present invention cloned the OsCARD1 gene and verified its function using genetic methods, elucidating the mechanism of action of the OsCARD1 gene in rice weed resistance and providing valuable theoretical basis and gene resources for breeding weed-resistant rice.

[0015] 2. Improve rice's weed resistance and develop new weed-resistant varieties

[0016] Knockout of the OsCARD1 gene significantly improved rice resistance to weeds, enabling the development of new weed-resistant germplasm that can maintain optimal growth under weed-infested conditions. This result not only demonstrates the important role of the OsCARD1 gene in rice's response to weed allelopathic signals but also provides new insights and resources for molecular breeding of rice for weed resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 Identification of OsCARD1 gene loss-of-function mutant materials; (a), Schematic diagram of the primary structure of OsCARD1 and OsCARD1-CD proteins; LRR: leucine-rich repeat sequence; TM: transmembrane domain; JM: membrane-proximal region; KD: kinase domain; CT: C-terminal region; (b), qRT-PCR analysis of OsCARD1 expression after DMBQ treatment; (c), OsCARD1-CD protein purification diagram; S: supernatant after centrifugation; R: column protein; E: eluted OsCARD1-CD protein; (d), qRT-PCR analysis of Oscard1 mutant materials; (e), West-Blot analysis of Oscard1 mutant materials;

[0019] Figure 2 Bioassay analysis of OsCARD1 gene functional deficiency materials; (ac), determination of growth and development indicators of different rice varieties treated with DMBQ; (df), determination of growth and development indicators of different rice varieties interacting with weeds. DETAILED DESCRIPTION

[0020] The present invention provides an OsCARD1 gene, the nucleotide sequence of which is shown in SEQ ID No. 1, and is as follows:

[0021]

[0022] The present invention also provides the use of the OsCARD1 gene described in the above technical solution in improving rice resistance to weeds. In the present invention, knocking out the OsCARD1 gene in rice improves rice resistance to weeds. Knocking out the OsCARD1 gene in rice improves rice resistance to weeds, which inhibit the growth and development of rice. In the present invention, knocking out the OsCARD1 gene in rice improves rice resistance to quinone allelopathic substances secreted by weeds. In the present invention, the quinone allelopathic substances preferably include 2,6-dimethoxy-1,4-benzoquinone. In the present invention, the weeds preferably include Leptochloa chinensis.

[0023] The present invention also provides a protein encoded by the OsCARD1 gene described in the above technical solution, the amino acid sequence of which is shown in SEQ ID No. 2, as follows:

[0024] MGVSPWIIIFLLIVLVQAFVASADTNAQDTSGLNGLAGSWGSAPSNWAGNDPCGDKWIGIICTGNRVTSIRLSSFGLSGTLSGDIQSLSELQYLDSYNKNLNGPLPSTIGTLSKLQNLILVGCGFTGEIPKEIGQLSNLIFLSLNSNKFTGSIPPSLGGLSKLYWFDLADNQLTGGLPISNATSPGLDNLTSTKHFHFGINQLSGSIPSQIFNS.

[0025] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] Basic information of OsCARD1 gene and identification of genetic materials.

[0028] Based on the genome sequence of Leptochloa crusgalli, specific primers were designed to amplify the OsCARD1 gene. The forward primer sequences are as follows: OsCARD1-F (SEQ ID No. 3): 5'-ATGGGGGTTTCTCCATGGATTATC-3'; OsCARD1-R (SEQ ID No. 4): 5'-CTTGGGCTCCACCCTCATCGA-3'. The complete coding sequence (CDS) of the OsCARD1 gene (SEQ ID No. 1) was amplified from rice cDNA. PCR amplification conditions included 35 cycles of initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes. The amplified product was confirmed by agarose gel electrophoresis and then purified.

[0029] Bioinformatics analysis of the structure of OsCARD1 revealed that it has the ability to transmit extracellular signals into cells, which may mediate the response of rice to external stress ( Figure 1 In addition, qRT-PCR experiments showed that the expression of OsCARD1 was induced in wild-type rice (Nip) after DMBQ treatment ( Figure 1 b). The OsCARD1-CD (intracellular domain) DNA fragment obtained by PCR amplification was ligated into the prokaryotic expression vector pET32a using the homologous recombination method. The ligation product was transformed into Escherichia coli Top10 by the heat shock method. By screening the LB culture medium containing ampicillin, the positive colonies were selected, and then PCR detection and sequencing were performed to confirm the correct recombinant vector. The constructed recombinant vector was transformed into Escherichia coli BL21 (DE3), and the LB culture medium containing ampicillin was selected for culture and induced expression. Under appropriate conditions, such as 37°C and after the addition of IPTG, protein induced expression was performed. The bacteria were broken by the cell disruption method, and the supernatant was separated by centrifugation. The target protein in the supernatant was affinity purified using Ni beads (containing agarose and Ni ions). After rinsing and elution steps, the pure target protein was finally obtained, and its authenticity was detected by SDS-PAGE ( Figure 1Four-week-old female ICR mice (Slack Jingda Animal Laboratory, 20-22g) were selected and fed for one week in the animal room. Subsequently, OsCARD1 protein (50μg) was mixed with complete Freund's adjuvant in a 1:1 ratio and shaken on a vortex shaker to ensure that the protein and adjuvant were completely mixed without stratification, forming a stable oil-in-water structure. Then, each mouse was intraperitoneally injected with 200μL of the mixed protein. Two weeks after the first injection, OsCARD1 protein (50μg) was mixed with incomplete Freund's adjuvant in a 1:1 ratio and shaken on a vortex shaker to ensure that the protein and adjuvant were completely mixed without stratification. Then, each mouse was intraperitoneally injected with 200μL of the mixed protein again to enhance the immune effect. After another week, the mice were stunned with ether, and their eyeballs were removed with forceps to collect blood from their orbits. Sodium citrate anticoagulant was added, and the blood was centrifuged at 12,000 g for 20 minutes at 4°C. The supernatant was used as the primary polyclonal antibody for OsCARD1. Simultaneously, Oscard1 mutants were generated using CRISPR-Cas9 gene editing technology, and subsequently identified at the transcriptional and protein levels using qRT-PCR and Western Blot experiments. Figure 1 The results showed that the mutant material could be used for subsequent experiments.

[0030] Example 2

[0031] The OsCARD1 gene is involved in the growth and development of rice regulated by the allelopathic signal DMBQ.

[0032] Different rice varieties cultured in hydroponics were treated with exogenous 10 μM DMBQ to analyze its effects on rice growth. The results showed that DMBQ significantly inhibited the growth of wild-type rice, while having little effect on the Oscard1 mutant ( Figure 2 The field experiment of Leptochloa crus-rice in the same hole further showed that Leptochloa crus-ferns significantly inhibited the plant height of wild-type and OsCARD1 overexpressing materials, but had no significant inhibition on OsCARD1 functional loss materials; in terms of tiller number, all materials were inhibited, but the inhibition rate of Oscard1-22 material (CARD1 mutant material) was significantly lower than that of wild-type ( Figure 2 The results indicate that OsCARD1 plays a key role in regulating rice growth and development through weed allelopathic signals.

[0033] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An OsCARD1 gene, characterized in that: The nucleotide sequence of the OsCARD1 gene is shown in SEQ ID No.

1.

2. Use of the OsCARD1 gene according to claim 1 in improving the weed resistance of rice.

3. The use according to claim 2, characterized in that Knocking out the OsCARD1 gene in rice improves rice resistance to weeds.

4. The use according to claim 3, characterized in that Knocking out the OsCARD1 gene in rice improves rice's resistance to quinone allelopathic chemicals secreted by weeds.

5. The use according to claim 4, characterized in that The quinone allelopathic substances include 2,6-dimethoxy-1,4-benzoquinone.

6. The use according to claim 2, characterized in that The weeds include Leptochloa chinensis.

7. A protein encoded by the OsCARD1 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No. 2.

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