Application of rice blast resistance related gene OsCBP60B1

Abstract

The invention discloses application of a rice blast resistance related gene OsCBP60B1, and belongs to the technical field of plant genetic engineering. The expression quantity of the OsCBP60B1 is increased after the OsCBP60B1 is induced by pathogenic bacteria of rice blast in susceptible and disease-resistant materials. After the gene is over-expressed, the rice blast resistance can be improved. The CDS sequence of the OsCBP60B1 is connected to an overexpression vector pOX to transform rice, and the result shows that the gene can positively regulate and control the rice blast resistance. The overexpression vector constructed by the invention can be applied to rice blast-resistant breeding of rice. According to the invention, the action mechanism of the OsCBP60B1 can be better understood, the cloning of the OsCBP60B1 lays a foundation for further understanding of rice-rice blast pathogen interaction and a disease-resistant signal transduction pathway, and the OsCBP60B1 has a great application value in breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to the application of a rice disease resistance-related gene, specifically a rice blast resistance-related gene. OsCBP60B1 Applications. Background Technology

[0002] Rice blast can be classified into several types based on the time and location of infection, including seedling blast, leaf blast, and panicle blast. Among these, leaf blast and panicle blast are the most common and cause the most severe damage. Leaf blast can cause the entire field of leaves to appear as if burned, resulting in stunted new leaf growth, stunted plant growth, and difficulty in heading. Panicle blast causes numerous white panicles or shriveled grains, significantly reducing yield. The incubation period of the rice blast pathogen is related to the age of the rice plant: 4-7 days for leaf blast and 10-14 days for panicle blast, with even shorter incubation periods for young plants. The pathogen attaches to the leaf cuticle by secreting a sticky substance from the spore tip, forming appressoria and generating significant turgor pressure. This mechanical pressure penetrates the epidermis and cuticle to form infection spikes, which then expand into infective hyphae. Necrotic lesions appear on the leaves after about 5-7 days, and new spores spread by wind and other means, initiating a new round of infection. Under suitable conditions, the rice blast pathogen infects rice plants extremely rapidly, easily causing outbreaks and severe yield and economic losses. In the long-term evolution of plants and pathogens, plants recognize pathogen PAMPs through PRRs on their cell membranes, activating the PTI response of their immune system and limiting pathogen invasion and replication. Pathogens, in turn, invade plant tissues through secretion or exocytosis, allowing virulence effectors to invade and alter signal transduction processes to suppress the PTI response. Plants, on the other hand, specifically recognize effectors through NBS-LRR proteins, triggering a higher-level ETI response. Pathogens can escape ETI recognition by losing or altering effectors, while plants evolve new R genes to recognize new effectors, triggering the ETI response again, forming a complex zigzag plant immune system mechanism.

[0003] Studies have shown that transcription factors are ubiquitous in all organisms, occupying an important part of the organism's genomic information, and can specifically bind to gene promoter regions to initiate the transcription of downstream target genes. Among them, CBP60s, as one of the plant-specific calmodulin-binding protein families, have been successfully isolated from plants such as maize, tobacco, Arabidopsis thaliana, and common bean. These proteins are responsible for transducing signals after plant cells sense external signals or stimuli to trigger appropriate physiological responses.

[0004] Calmodulin-like protein (CaM) and calmodulin-like protein (CML) are the main sources of calcium ions (Ca). 2+ ) Receptors. Although they themselves do not possess catalytic activity, they can regulate various physiological and biochemical activities of cells by modulating the activity of downstream target proteins.2+ As an important second messenger, calmodulin can transmit signals to downstream effector proteins, such as enzymes, transcription factors, transport proteins, and cytoskeletal proteins, participating in the regulation of cellular processes such as metabolism, gene expression, transmembrane transport, and development, thereby affecting plant growth, development, and environmental adaptability. Although 23 calmodulin-binding proteins have been identified in rice, OsCBP60B1 Its specific role in rice blast immunity remains unclear. Further research on its function will help improve rice disease resistance and promote crop breeding.

[0005] Identifying disease resistance-related genes helps to elucidate the disease resistance mechanisms in rice and the specific interaction mechanisms between rice and pathogens. This will further enable the breeding of related disease-resistant varieties, allowing for better control and reduction of the damage caused by rice blast fungus to rice, and enhancing the plant's disease resistance. These studies have significant application value for rice gene function research and disease-resistant rice breeding. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a rice blast resistance-related gene. OsCBP60B1 New applications.

[0007] OsCBP60B1 (LOC_Os11g44680) is located on chromosome 11, with a full-length genome sequence of 5990 bp (SEQ ID NO:1) and a full-length cDNA sequence of 1857 bp (SEQ ID NO:2). It contains a total of 12 exons and 11 introns, encoding a protein of 618 amino acids (SEQ ID NO:3). The OsCBP60B1 protein structure includes a conserved calmodulin-binding protein central domain (176-467 amino acids).

[0008] The OsCBP60B1 protein encoded by this gene belongs to the CBP60 family of transcription factors, and its expression is significantly upregulated inducible by rice blast fungus infection. Functional validation revealed that... OsCBP60B1 Overexpression of the gene resulted in significantly enhanced resistance to rice blast, while gene knockout lines exhibited a susceptible phenotype. In conclusion, overexpression of the gene can help control the disease. OsCBP60B1 This invention aims to improve the resistance of rice to rice blast fungus, thereby controlling rice blast disease. The objective is achieved through the following technical solution: Overexpression of rice blast resistance-related genes OsCBP60B1 The application is any one of the following: I. Application in improving rice blast resistance; II. Application in the breeding of high-yield rice blast-resistant rice III. Application in the prevention and control of rice blast.

[0009] Furthermore, the aforementioned rice blast resistance-related gene OsCBP60B1 The encoded protein sequence is shown in SEQ ID NO: 3.

[0010] Furthermore, the aforementioned rice blast resistance-related gene OsCBP60B1 The full-length genome sequence is shown in SEQ ID NO: 1.

[0011] Furthermore, the rice blast resistance-related gene OsCBP60B1 The full-length cDNA sequence is shown in SEQ ID NO: 2.

[0012] Furthermore, the application I is: by overexpressing the rice blast resistance-related gene. OsCBP60B1 This improves the resistance of rice to rice blast fungus.

[0013] Furthermore, the application II is: overexpressing the rice blast resistance-related gene in rice. OsCBP60B1, To cultivate and obtain transgenic rice with enhanced resistance to rice blast fungus.

[0014] Furthermore, application III involves overexpressing the rice blast resistance-related gene in rice. OsCBP60B1 This reduces the occurrence of rice blast.

[0015] Furthermore, the overexpression is achieved through target gene overexpression mediated by an expression vector.

[0016] Furthermore, the overexpression is achieved by constructing a recombinant vector for overexpressing the target gene.

[0017] Furthermore, the overexpression is achieved through a host bacterium containing a "strong promoter-ORF" expression vector without altering the genome.

[0018] Used to amplify the above-mentioned rice blast resistance-related genes. OsCBP60B1 Application of primers in the comparison, identification and / or evaluation of rice blast resistance.

[0019] Furthermore, the application steps are as follows: detecting the presence of [a certain substance] in the rice plants under test using real-time quantitative PCR. OsCBP60B1 Gene expression levels, if OsCBP60B1 If the expression level is significantly higher than that of the control rice plant, then the rice plant exhibits stronger resistance to rice blast than the control rice plant; if OsCBP60B1 If the expression level is significantly lower than that of the control rice plant, then the rice plant has weaker resistance to rice blast than the control rice plant.

[0020] This invention relates to genes related to rice blast resistance. OsCBP60B1 Application in plant disease resistance breeding. This invention... OsCBP60B1 The expression pattern analysis revealed OsCBP60B1 Induced by rice blast fungus. Overexpression OsCBP60B1 This can significantly enhance rice's resistance to rice blast. (This reveals...) OsCBP60B1 It can positively regulate the resistance response of rice to rice blast. The overexpression vector pOX- constructed in this invention... OsCBP60B1 It can be used for research on the function of rice disease resistance-related genes and for molecular breeding improvement.

[0021] OsCBP60B1 Positive regulation of rice blast resistance. By constructing corresponding vectors to transform rice, the disease resistance pathways involved were studied, and these pathways were integrated into a complex disease resistance regulatory network, confirming... OsCBP60B1 This gene participates in the defense response of rice against rice blast pathogen and is an important positive regulator of rice disease resistance. Researching the disease resistance pathways it participates in and integrating it into a complex disease resistance regulatory network will provide theoretical guidance for breeding rice varieties resistant to rice blast.

[0022] Compared with the prior art, the present invention has the following advantages and effects: The method of this invention uses reverse transcription PCR technology to clone a gene of a CBP60 family protein from rice. OsCBP60B1 Confirmed OsCBP60B1 This gene participates in the defense response of rice against rice blast fungus and is an important positive regulatory gene involved in rice disease resistance. This invention contributes to a better understanding of... OsCBP60B1 Mechanism of action, OsCBP60B1 The identification of this data lays the foundation for further understanding of rice-pathogen interactions and has significant application value in breeding. Attached Figure Description

[0023] Figure 1 yes OsCBP60B1 A schematic diagram of the full-length gene structure; where the black areas are exons, the blank rectangular areas are the 5' untranslated region and the 3' untranslated region, and the red areas are the conserved domains of the calmodulin family. Figure 2 yes OsCBP60B1 The results of expression in disease-resistant materials being induced by rice blast fungus are shown in the figure. Figure 3 yes OsCBP60B1 Graph showing the results of transcription activation property detection; Figure 4 yes OsCBP60B1 Figure showing the identification of transgenic plants and the results of quantitative detection of gene overexpression; Figure 5 After inoculation with rice blast fungus OsCBP60B1 Phenotypic diagram of overexpressing transgenic plants; WT represents Zhonghua 11 (ZH11); among them, OsCBP60B1- OX is OsCBP60B1 Overexpression transformants. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0025] Unless otherwise specified, all raw materials and equipment used in this invention are commercially available products that can be directly purchased from the market.

[0026] The primer sequences used in the embodiments of this invention were all synthesized by Beijing Qingke Biotechnology Co., Ltd. and Shanghai Sangon Biotech Co., Ltd.

[0027] The Lijiang Xintuan Black Rice (LTH) used in this embodiment of the invention has been disclosed in the literature “Study and utilization of the universal sensitivity characteristics of rice variety Lijiang Xintuan Black Rice [J]. Chinese Agricultural Science, 2001, 01:121-121.”

[0028] The rice strains with high resistance to rice blast used in the embodiments of the invention Pilk-H4 NIL has been described in the literature "Rice Blast Resistance Proteins" Spade 2 -H4 The cloning of genes and screening of interacting proteins [J]. Guangdong Agricultural Sciences, 2014, (04): 156-160.

[0029] The rice blast fungus used in the embodiments of this invention ( M. oryzae GDYJ7 has been published in the literature “Wu Pengzhi, Identification and preliminary functional study of rice microRNAs responding to rice blast fungus infection (D), 2019.”

[0030] The rice blast fungus used in the embodiments of this invention ( M. oryzae Zhong1 has already published in the paper "Yu, S.; Li, S.; Wang, W.; Tang, D. OsCAMTA3 Negatively Regulates Disease Resistance to Magnaporthe oryzae by Associating with OsCAMTAPL in Rice. Int. J. Mol. Sci. 2024, 25 , 5049. ” is published.

[0031] The overexpression vector pOX used in this invention embodiment is disclosed in the literature "Cloning and Functional Analysis of Rice SDG711 and SDG723 [D]. South China Agricultural University, 2012".

[0032] The plasmid pGBKT7 used in the embodiments of this invention is a commercially available plasmid.

[0033] Example 1: OsCBP60B1 Sequence analysis and cloning 1) Extraction of total RNA from rice leaves and OsCBP60B1 Cloning Four-leaf stage rice seedlings were ground into powder using liquid nitrogen in a mortar and transferred to a 1.5 mL centrifuge tube. Trizol reagent (Invitrogen) was added at a ratio of 1 mL per 100 mg of material, and the mixture was thoroughly mixed. Chloroform was added at a ratio of 200 μL per 100 mg of material, and the mixture was thoroughly mixed. The mixture was centrifuged at 10,000 g at 4°C for 15 min, and the lower organic phase was discarded. The aqueous phase was collected and transferred to a centrifuge tube. 600 μL of isopropanol was added, and the mixture was thoroughly mixed. The mixture was allowed to stand at room temperature for 20 min, then centrifuged at 10,000 g at 4°C for 15 min. The precipitate was collected, and after the isopropanol evaporated, it was dissolved in RNase-free ultrapure water. Total RNA from rice leaves stored at -80°C was extracted and 2 μL of Oligo(dT) was added. 16 (10 mM), mix well and incubate at 70℃ for 5 min; after 5 min incubation, add 2 μL of dNTP Mixture (10 mM), 4 μL of 5×RT Buffer, 1 μL of RNase inhibitor (10 U / μL), 8 μL of RNase-free ddH2O, and 1 μL of ReverTraAce to the EP tube on ice in sequence; place the EP tube in a PCR instrument and react at 30℃ for 10 min, 42℃ for 60 min, 99℃ for 5 min, and 40℃ for 5 min to obtain the first-stranded single-stranded cDNA, using primers: OsCBP60B1 -F、 OsCBP60B1 PCR was performed using the R-R method. The PCR reaction system consisted of: 2 μL cDNA template, 2 μL each of forward and reverse primers (10 μM), 25 μL 2×PhantaMax Buffer, 1 μL dNTP mix (10 mM each), 1 μL PhantaMax Super-Fidelity DNA Polymerase, and ddH2O added to a final volume of 50 μL. The amplification conditions were: 35 cycles of denaturation at 95℃ for 3 min, followed by denaturation at 57℃ for 15 s and 72℃ for 2 min, and extension at 72℃ for 5 min to obtain the full-length cDNA. OsCBP60B1 The cDNA was then sent to Beijing Qingke Company for sequencing analysis. OsCBP60B1 -F、 OsCBP60B1 The sequence of -R is as follows: OsCBP60B1 -F: 5'-ATGCACACGAAGCGTCCG-3'; OsCBP60B1 -R: '-AACTGGTCCTGGATATGCAGAGC-3'.

[0034] 2) OsCBP60B1 Sequence analysis and homology alignment analysis OsCBP60B1 The full-length genome sequence is 5990 bp, and the full-length CDS is 1857 bp, containing 12 exons and 11 introns, encoding 618 amino acids, including a conserved calmodulin family domain. Figure 1 The OsCBP60B1 protein structure was detected using NCBI BlastProtein, which showed a conserved CBP60 domain. This domain was then analyzed using CLC sequencing. OsCBP60B1 Comparison of the gene sequence with homologous family gene sequences revealed that the OsCBP60B1 protein contains a conserved calmodulin family domain sequence. Figure 2 ).

[0035] Example 2: OsCBP60B1 Expression pattern analysis Disease-resistant varieties Pilk-H4 NIL and the susceptible variety Lijiang Xintuan Black Rice (LTH) were inoculated with rice blast virus (ddH2O as control), and samples were taken at different time points after inoculation. Real-time fluorescence quantitative PCR was used to detect the virus. OsCBP60B1 The expression changes were observed. The primer sequences used are as follows: Actin-F: 5'-TGTATGCCAGGTGGTCGTACCA-3'; Actin-R: 5'-CCAGCAAGGTCGAGACGAA-3'; OsCBP60B1 -qPCR-F: 5'-AAGAGTCCTGCTGCCATCACT-3'; OsCBP60B1 -qPCR-R: 5'-ATAGCTGTACCATCCTCCGC-3'.

[0036] The results showed that after infection by rice blast fungus, OsCBP60B1 The expression patterns of rice blast fungal infection differed significantly between the two varieties. OsCBP60B1 The level at Pilk-H4 In NIL, OsCBP60B1 The accumulation of [a certain substance] shows a trend of "first rising and then falling," increasing sharply 12 hours after vaccination and reaching its peak at 24 hours after vaccination. Figure 3 This discovery further confirms... OsCBP60B1 The response to rice blast fungus infection indicates OsCBP60B1It participates in the immune-related processes of rice.

[0037] Example 3: OsCBP60B1 Transcription activation activity analysis OsCBP60B1 The sequence 176-467 aa is a calcium ion-binding family domain, divided into three segments: the N-terminus (176-320 aa), the calcium ion-binding family domain (332-399 aa), and the C-terminus (406-464 aa). These segments and the full-length sequence were ligated to the pGBKT7 plasmid to construct recombinant plasmids. These plasmids were then transformed into the yeast strain Y2HGold, and the specific transcriptional activation region of the transcription factor OsCBP60B1 was detected in the yeast expression system. The primer sequences used are as follows: BD- OsCBP60B1 -NF: 5'-tcagaggaggacctgcatatgGTTGAGTTGAGGTTCCTCAATAAGTTG-3'; BD- OsCBP60B1 -NR:5'-tcgacggatccccgggaattcGTAAACCTGCACCACAACAGGA-3'; BD- OsCBP60B1 -Domain-F:5'-tcagaggaggacctgcatatgAAGCTAAAAGATGAAGTACATCGTTTAAA-3'; BD- OsCBP60B1 -Domain-R:5'-tcgacggatccccgggaattcACACTCTTTAGCATGTTTAATTGTATCTTT-3'; BD- OsCBP60B1 -CF: 5'-tcagaggaggacctgcatatgAAGCTGAAATTATATAGAGCTGAAGAGC-3'; BD- OsCBP60B1 -CR:5'-tcgacggatccccgggaattcAATGCTATCCAGCTCATCATATGC-3'.

[0038] The results showed that only the full-length OsCBP60B1 protein and its fragmented form... OsCBP60B1 -Domain has transcriptional activation properties ( Figure 4 This indicates that the conserved domain of the OsCBP60B1 protein is only responsible for the activation properties of the transcription factor itself.

[0039] Example 4: OsCBP60B1 Identification of rice blast resistance in transgenic plants 1) OsCBP60B1 Construction of transgenic rice with overexpression Using pOX- OsCBP60B1 -F and pOX- OsCBP60B1 -R amplification OsCBP60B1 The cDNA fragment was ligated into the overexpression vector pOX using homologous recombination. This yielded pOX-OsCBP60B1 (…). OsCBP60B1 The -OX recombinant vector was used to transform *E. coli* DH5α, and positive transformants were identified by sequencing for subsequent *Agrobacterium* transformation experiments. The transformation material was *Zhonghua 11*. The primer sequences used are as follows: OsCBP60B1 -OX-F: 5'-tccccgggtgagctcggtaccATGCACACGAAGCGTCCG-3'; OsCBP60B1 -OX-R: 5'-agcggccgcactagtaagcttAACTGGTCCTGGATATGCAGAGC-3'.

[0040] 3) Identification of rice blast resistance in transgenic plants After screening and identification by hygromycin and quantitative real-time PCR, we selected three homozygous overexpressing lines for the next stage of experiments. (Correctly identified lines...) OsCBP60B1 -OX transgenic lines were inoculated with rice blast fungus (Zhong1) at the three-leaf stage, with WT as a control. The length and area of ​​leaf lesions were observed 5 days after inoculation.

[0041] The results showed that the size of the lesions varied significantly compared to wild-type plants. ,OsCBP60B1 Overexpression resulted in significant changes in plant resistance. Figure 5 ), but OsCBP60B1 Quantitative fluorescence detection of disease-related genes in overexpressed plants PR1b、 PR1a, PR10 The expression level of [a certain substance] was significantly higher than that of the wild type, suggesting that... OsCBP60B1 Positive regulation of rice blast resistance can be achieved by overexpressing [a specific gene] in rice germplasm. OsCBP60B1 To improve resistance to rice blast.

[0042] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of overexpression of a blast resistance related gene OsCBP60B1 characterized in that The application is any one of the following applications: I. Application in improving rice blast resistance; II. Application in cultivating rice with high blast resistance; III. Application in preventing rice blast.

2. The overexpression of rice blast resistance-related genes according to claim 1 OsCBP60B1 The application of which is characterized by: The rice blast resistance-related gene OsCBP60B1 The encoded protein sequence is shown as SEQ ID NO:

3.

3. The overexpression of rice blast resistance-related genes according to claim 1 OsCBP60B1 The application of which is characterized by: The rice blast resistance-related gene OsCBP60B1 The full-length genomic sequence of the rice blast resistance-related gene is shown as SEQ ID NO:

1.

4. The overexpression of rice blast resistance-related genes according to claim 1 OsCBP60B1 The application of which is characterized by: The rice blast resistance-related gene OsCBP60B1 The full-length cDNA sequence of the rice blast resistance-related gene is shown as SEQ ID NO:

2.

5. The overexpression of rice blast resistance-related genes according to claim 1 OsCBP60B1 The application of which is characterized by: The application I is: improving the resistance of rice to Magnaporthe grisea by overexpressing the rice blast resistance related gene OsCBP60B1 . The application II is: overexpressing the rice blast resistance related gene in rice OsCBP60B1 Transgenic rice with enhanced resistance to Magnaporthe grisea is obtained by breeding The application III is: overexpressing the rice blast resistance related gene in rice to reduce the occurrence of rice blast. OsCBP60B1 , reduce the occurrence of rice blast.

6. The overexpression of the rice blast resistance-related gene according to any one of claims 1-5 OsCBP60B1 for use, characterized in that: The overexpression is achieved by overexpression of the target gene mediated by an expression vector.

7. The overexpression of rice blast resistance-related genes according to claim 6 OsCBP60B1 The application of which is characterized by: The overexpression is achieved by constructing a recombinant vector for overexpression of the target gene.

8. The overexpression of rice blast resistance-related genes according to claim 6 OsCBP60B1 The application of which is characterized by: The overexpression is achieved by a host bacterium containing an "strong promoter-ORF" expression vector without modifying the genome.

9. Use of primers for amplifying the rice blast resistance related gene according to any one of claims 1 to 5 in rice blast resistance comparison, identification and / or evaluation. OsCBP60B1 of the rice blast resistance related gene according to any one of claims 1 to 5 in rice blast resistance comparison, identification and / or evaluation.

10. The application of claim 9, wherein: The application steps are as follows: Detection of [certain substances] in the rice plants under test using real-time quantitative PCR. OsCBP60B1 Gene expression levels, if OsCBP60B1 If the expression level is significantly higher than that of the control rice plant, then the rice plant has stronger resistance to rice blast than the control rice plant. If OsCBP60B1 If the expression level is significantly lower than that of the control rice plant, then the rice plant has weaker blast resistance than the control rice plant.

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