Gene osslm and its use

Abstract

The application discloses a gene OsSLM and application thereof. The application obtains a rice lesion mimic mutant slm through EMS mutagenesis, detects the ROS content level after treating the leaves of a wild type Zhongheng 8015 and the mutant with fungi chitin and bacterial flagellin, artificially inoculates Magnaporthe grisea and identifies the resistance level, compares and analyzes the expression level of defense-related genes of the mutant and the wild type by using fluorescent quantitative PCR technology, further clones a gene for controlling the rice lesion, and then verifies the gene function through genetic transformation, and finally successfully separates an immune negative regulation gene OsSLM of rice. The experimental results show that in the OsSLM function loss mutant obtained through EMS mutagenesis, the resistance to rice fungal disease Magnaporthe grisea of the mutant is extremely significantly improved compared to the wild type, the mutant can be effectively applied to the molecular improvement work of the disease resistance of rice, and has great development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the gene OsSLM and its applications. Background Technology

[0002] To cope with the damage caused by various pathogens (viruses, bacteria, and fungi), plants have evolved two innate immune systems: pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI relies on pattern recognition receptors (PRRs) on the cell membrane surface and is generally considered to have weak but broad-spectrum resistance. ETI, on the other hand, is triggered by plant-specific resistance proteins (R proteins) recognizing effector proteins secreted by pathogens, and has strong species-specific resistance. Upon sensing pathogen infection, the plant's immune system is rapidly activated, blocking further infection and spread of the pathogen by quickly initiating programmed cell death (PCD) at the infection site, while simultaneously transmitting signals to neighboring tissues to induce acquired resistance. Within the plant, these two pathways interact and work together to resist pathogen infection.

[0003] Rice is one of the world's most important food crops, with over 50% of the global population relying on it as their staple food, and this proportion is increasing annually. With global warming and frequent natural disasters, maintaining stable and high rice yields is crucial for ensuring global food security. Utilizing rice's own immune system to resist diseases and reduce yield losses is one of the more reasonable and effective measures. Rice lesion mutants are essentially a manifestation of uncontrolled plant disease progression (PCD) caused by phytosensitive response (HR). Simultaneously, most plant lesion mutants exhibit activated defense responses, significantly enhancing resistance to some plant pathogens. Therefore, plant lesion mutants have become ideal materials for studying plant PCD and defense responses, and are of significant research value in revealing plant defense pathways and signal regulatory networks. With the rise of modern molecular biology, using molecular methods to discover rice disease-resistant genes, analyze rice's autoimmune response mechanisms, cultivate superior disease-resistant new varieties, and enhance rice's own disease resistance are of great importance for ensuring safe rice production.

[0004] Plant defense responses are triggered by the recognition of PAMPs by certain PPRs on the cell membrane, known as the PTI response. Upon PTI activation, some PPRs may possess endocytic motifs, transmitting immune signals downstream via endocytosis when the defense response is activated. After plant PAMP receptors recognize signaling molecules, ubiquitination and phosphorylation occur. Following a sorting process, the PAMPs return to the plasma membrane via vesicle transport or are transported to vacuoles for degradation. This transport process, mediated by the ESCRT complex, is crucial for establishing a normal immune response in plants.

[0005] Isolating and cloning disease resistance-related genes is a prerequisite for studying the disease resistance mechanism of rice. Furthermore, compared to the application of disease resistance genes, the application of disease resistance-related genes can provide plants with broader and longer-lasting resistance. Improving the disease resistance of rice varieties by inhibiting the function of genes related to negative disease resistance regulators will further enhance plant disease resistance and broaden the spectrum of plant resistance. Summary of the Invention

[0006] The purpose of this invention is to provide the gene OsSLM and its applications.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides the application of the gene OsSLM in regulating disease resistance in rice.

[0008] Furthermore, the regulation is negative regulation.

[0009] Among them, the gene OsSLM is:

[0010] i) The nucleotide sequence shown in SEQ ID NO: 1; or

[0011] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 that has been substituted, deleted, and / or had one or more nucleotides added and expresses a protein with the same function; or

[0012] iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO: 1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or

[0013] iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0014] The disease resistance described in this invention is resistance to rice blast. For example, the pathogen is Magnaporthe grisea Barr.

[0015] Secondly, the present invention provides a method for improving the disease resistance of rice by using genetic engineering techniques to perform site-directed mutations on the rice gene OsSLM, thereby causing the gene to lose its function and thus improving the disease resistance of rice.

[0016] Furthermore, site-directed mutagenesis was performed on the rice gene OsSLM, resulting in a 4bp deletion (GGGG) in the fifth exon of the gene, and ultimately a frameshift mutation caused premature termination of amino acid translation at codon 192 (SEQ ID NO: 4).

[0017] Thirdly, the present invention provides an OsSLM protein mutant, which is:

[0018] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4; or

[0019] (b) Proteins derived from (a) with the sequence shown in SEQ ID NO: 4 substituted, deleted or added with one or more amino acids and having the same function.

[0020] Fourthly, the present invention provides a gene encoding the mutant of the said protein.

[0021] Fifthly, the present invention provides biological materials containing the said gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0022] In a sixth aspect, the present invention provides the application of the gene or biological material containing the gene in improving the disease resistance of rice.

[0023] Furthermore, the application includes:

[0024] 1) To make the plant contain the said gene; or

[0025] 2) Cause the plant to express the protein mutant.

[0026] In a seventh aspect, the present invention provides the use of transgenic rice obtained according to the method or application in plant breeding.

[0027] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0028] This invention obtained a rice lesion mutant, slm, through EMS mutagenesis. ROS levels were measured after treating wild-type rice blast fungus (SLM) and mutant leaves with fungal chitin and bacterial flagellin. The rice blast fungus was artificially inoculated, and resistance levels were identified. Quantitative real-time PCR was used to compare and analyze the expression levels of defense-related genes in the mutant and wild type. Genes controlling rice lesions were then cloned, and gene function was verified through genetic transformation. Finally, a negative regulatory gene for rice immunity—OsSLM—was successfully isolated. Experimental results show that the OsSLM loss-of-function mutant obtained through EMS mutagenesis exhibits significantly enhanced resistance to rice blast fungus compared to the wild type. This mutant has significant potential for molecular improvement of rice disease resistance and can be effectively applied to this field, demonstrating great promise for future development and application. Attached Figure Description

[0029] Figure 1 This is a comparison of the programmed cell death phenotypes of wild-type Zhonghui 8015 and mutant slm in a preferred embodiment of the present invention. ab shows the lesion-like phenotypes of plants (a) and leaves (b) at the tillering stage (60 days after sowing). cd shows the lesion-like phenotypes of plants (c) and leaves (d) at the heading stage.

[0030] Figure 2 The following are comparative diagrams showing the results of resistance identification for artificial inoculation with *Magnapordica oryzae* in a preferred embodiment of the present invention. (a) is a comparative diagram of leaf lesion phenotypes inoculated with *Magnapordica oryzae*, (b) is a comparative diagram of the relative lesion area of ​​leaves inoculated with *Magnapordica oryzae*, and (c) is a comparative diagram of the relative fungal biomass of leaves inoculated with *Magnapordica oryzae*.

[0031] Figure 3 The figure shows the relative expression levels of eight defense-related genes (OsNPR5, OsNPR1, OsPR1a, OsPR1b, OsJAZ1, OsPR10, OsWRKY45, and OsPAL1) in a preferred embodiment of the present invention. (a) is the tillering stage, and (b) is the heading stage.

[0032] Figure 4 The following are dynamic analysis diagrams of ROS accumulation in a preferred embodiment of the present invention. (a) is a dynamic analysis diagram of ROS after chitin treatment, and (b) is a dynamic analysis diagram of ROS accumulation after Flg22 treatment.

[0033] Figure 5 The following are the mutation modes of OsSLM in a preferred embodiment of the present invention. (a) Structure and mutation mode of the OsSLM gene. (b) Expression level analysis of OsSLM in WT and slm. (c) Amino acid sequence analysis of OsSLM and osslm.

[0034] Figure 6The following are phenotypic comparisons of transgenic complementary materials in a preferred embodiment of the present invention. (a) Comparison of the entire plant, (b) Comparison of a single leaf, (c) Transcriptional levels of OsSLM in WT, slm and complementary plants, (d) DAB staining of leaves of WT, slm and complementary plants, (e) Identification results of rice blast resistance in wild-type, mutant and complementary plants. Detailed Implementation

[0035] The present invention aims to provide a rice immune negative regulatory protein OsSLM related to rice disease resistance, its encoding gene and its application.

[0036] The present invention adopts the following technical solution:

[0037] This invention provides a rice immune negative regulatory protein OsSLM, which is:

[0038] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4; or

[0039] (b) Proteins derived from (a) with the sequence shown in SEQ ID NO: 4 substituted, deleted or added with one or more amino acids and having the same function.

[0040] This invention provides a gene encoding the above-mentioned protein.

[0041] Furthermore, the genomic gene sequence encoding the rice immune negative regulatory protein OsSLM is shown in (A) or (B):

[0042] (A) The nucleotide sequence shown in SEQ ID NO: 1;

[0043] (B) A mutant gene, allele, or derivative that encodes a protein with the same function by adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence defined in (A).

[0044] Furthermore, the cDNA sequence encoding the rice immune negative regulatory protein OsSLM is shown in (C) or (D):

[0045] (C) The nucleotide sequence shown in SEQ ID NO: 2;

[0046] (D) cDNA that encodes a protein with the same function by adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence defined in (C).

[0047] The aforementioned proteins and genes can be applied to the molecular improvement of disease resistance in rice.

[0048] This invention also provides a mutant protein of rice immune negative regulatory protein OsSLM that enhances rice disease resistance, its encoding gene, expression vector, transformant, and applications.

[0049] Furthermore, the mutant protein of the rice immune negative regulatory protein OsSLM has the amino acid sequence shown in SEQ ID NO: 4.

[0050] The present invention also provides a gene encoding the above-mentioned protein.

[0051] The present invention also provides an expression vector or transformant containing the above-mentioned genes.

[0052] The present invention also provides the application of the above-mentioned proteins, genes or expression vectors, and transformants in improving the disease resistance of rice.

[0053] Furthermore, the disease resistance is resistance to rice blast.

[0054] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0055] The fungal chitin and bacterial flagellin Flg22 used in the following examples were purchased from Santa Cruz and Guangzhou Qiyun Biotechnology Co., Ltd., respectively.

[0056] The term PAMP stands for pathogen-associated molecular patterns.

[0057] Example 1: Obtaining SLM mutants and identifying programmed cell death phenotypes

[0058] A lesion-like mutant was obtained from the indica rice restorer line Zhonghui 8015 (wild type, WT) through EMS mutagenesis screening. Under natural field conditions, approximately 60 days after sowing and transplanting, reddish-brown lesions appeared at the base of the midrib of the leaf. This mutant was named slm (spotted leaf midrib). As the rice grew and developed, the lesions on the midrib gradually progressed upwards and expanded to both sides of the midrib, eventually covering the entire leaf by the heading stage. Figure 1 ).

[0059] Example 2: Identification of rice blast resistance

[0060] Rice blast inoculation was performed using the perforation method. Wild-type Zhonghui 8015 and mutant SLM seeds were soaked and germinated before being sown in cylindrical culture pots (15cm in diameter and 15cm in height) filled with sterilized nutrient soil, with 5 plants per pot. The pots were then cultured at a constant temperature of 26℃ in an artificial greenhouse for 12 hours of light followed by 12 hours of darkness until the peak tillering stage. Once the mutant-like lesion phenotype appeared, circular wounds (1.5mm in diameter) were artificially created on the surface of the third leaf from the bottom using a perforator, and 10 μL of pre-prepared spores (approximately 10 μg / L) were extracted. 5 Rice blast fungus physiological race 12-144-1-1 (see Zhang Y, Liu Q, Zhang Y, Chen Y, Yu N, Cao Y, Zhan X, Cheng S, Cao L. LMM24 Encodes Receptor-Like Cytoplasmic Kinase 109, Which Regulates Cell Death and Defense Responses in Rice. Int J Mol Sci. 2019 Jul 2; 20(13): 3243. doi: 10.3390 / ijms20133243. PMID: 31269643; PMCID: PMC6651581.) was dropped onto the wound and fixed with transparent tape. The bacteria were cultured in an artificial climate chamber with constant light and temperature and humidity adjusted to 80% to promote disease development. The size of the lesions was investigated and samples were taken after 14 days. DNA was extracted from rice leaves and *Magnaporum oryzae* from the inoculation site. Real-time PCR was used to relatively quantify the DNA of the *Magnaporum oryzae* gene Pot2 (NCBI accession number: CP050928) using rice Actin as an internal reference gene, representing the ratio of their biomass. The amplification primers are as follows (5′-3′):

[0061] OsActin-F: CAGGCCGTCCTCTCTCTGTA

[0062] OsActin-R:AAGGATAGCATGGGGGAGAG

[0063] MoPot2-F: ACGACCCGTCTTTACTTATTTGG

[0064] MoPot2-R: AAGTAGCGTTGGTTTTGTTGGAT

[0065] The results showed that, compared with the wild type Zhonghui 8015, the mutant slm exhibited significantly improved resistance to blast fungus pathotype 12-144-1-1, with a significantly smaller lesion area than the wild type. The proliferation rate of blast fungus in the mutant leaves was also significantly and effectively inhibited. Figure 2).

[0066] Example 3: Analysis of the expression levels of defense-related genes

[0067] Wild-type Zhonghui 8015 and the mutant slm were planted in experimental fields of the China National Rice Research Institute under normal field management. At the peak tillering and heading stages, three vigorous individual plants were selected, and the leaf tips (approximately 1 / 3 of the total leaf length) of the third leaf from the top were harvested. Total RNA was extracted using the RNAprep Pure plant total RNA extraction kit from Beijing Tiangen Biotech Co., Ltd., and the first strand cDNA was synthesized using the Toyobo First strand cDNA Synthesis Kit Rever Tra Ace kit. Quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit from Novizan, following the manufacturer's instructions. The primers used are as follows (5′-3′):

[0068] qOsPR5CTTCTGCCCATAATGCATCATCTGATTATCGATCAAGGTGTCGT

[0069] qOsNPR1 CACTGCACTACGCCGTCGAACTCTCTTCGCCTCGCAGCAA

[0070] qOsPOXGCTCCAAGGTGAACTCCTAATTATATGGGTATATGTGGTGTGGC

[0071] qOsPR1aCGTGTCGGCGTGGGTGTGGCGAGTAGTTGCAGGTGATG

[0072] qOsPR1b CATTGCTTTGGCCATGGTAGGAACCCCAGAAGAGGTTCTC

[0073] qOsJAZ1 GCGCTCCCGGAGArGCCGATTTCGCTCGTTGTCGTGATCCTGT

[0074] qOsPR10CTCAAGATGATCGAGGACTACCAGAAAGGCACATAAACACAACC

[0075] qOsWRKY4 GCCGACGACCAGCACGATCACC

[0076] 5 ACGAGCCGACGCCGCCCTC

[0077] qOsAOS2 AAGCTGCTGCAATACGTGTACTGGCGACGAGCAACAGCCTTCCG

[0078] qOsPAL1 GACCCTGTATTTTCTTCGTTCGAGTAGCAATACTTTCACCCCAA

[0079] The results showed that all eight defense-related genes were upregulated in the mutant, with upregulation folds ranging from 3 to over 400 times. This further indicates that the mutant gene plays a negative regulatory role in the immune stress response. Figure 3 ).

[0080] Example 4: Dynamic Analysis of ROS Accumulation After PAMP Treatment

[0081] The planting method was the same as in the rice blast inoculation section of Example 2. Leaves of WT and slm rice, approximately 60 days after sowing and transplanting, were sampled using a 0.5cm perforator, avoiding the midrib. The samples were then placed in double-distilled water overnight in the dark to eliminate ion penetration and defense reactions caused by physical damage. Three leaves were randomly selected with tweezers, blotted dry on clean filter paper, and then carefully placed into 1.5mL centrifuge tubes. 1μL of peroxidase-streptavidin, 100μL of Mmunstar-HRP substrate, 1μL of Chitin (800nM) or 1μL of Flg22 (10μM), and 1μL of double-distilled water were added sequentially as a control (Mock). The centrifuge tubes were placed in a Glomax (Promega, E5311) instrument to measure luminescence at 10s intervals for a total of 20 minutes. The results are as follows: Figure 4 As shown, the mutant slm is more sensitive to both Chitin and Flg22, and the RO5S (reactive oxygen species) accumulation rate and peak value are significantly higher than those of the wild-type Zhonghui 8015.

[0082] Example 5 Cloning of the OsSLM gene

[0083] F1 was obtained by crossing slm as the female parent and WT as the male parent, and the F2 segregating population was obtained by self-pollination after bagging. DNA was extracted from 30 F2 plants with lesion-like phenotypes and mixed in the same proportion to construct a mutant pool. The WT pool was constructed in the same way, and library construction and sequencing were performed. Gene annotation of candidate sites was performed using ANNOVAR, and genes containing sites that cause frameshift mutations, nonsynonymous mutations, or alternative splicing were preferentially selected as candidate genes. Based on the distribution of SNPs and InDels, and referring to the functional annotation of the rice variety 9311 genome, it was found that only the Indel deletion (-4bp, GGGG) of the gene BGIOSGA018999 (LOC_Os05g01250) on chromosome 5 caused a frameshift mutation. Therefore, LOC_Os05g01250 was listed as a candidate gene for analysis.

[0084] To verify whether the candidate gene LOC_OsO5g01250 was mutated in slm, the full-length genome sequences of LOC_Os05g01250 in WT and slm were amplified by PCR using the gene sequence annotated by RGAP (http: / / rice.uga.edu / ) as a template, and then sequenced and aligned. The results showed that a 4bp (GGGG) deletion mutation occurred at the junction of the fifth exon and intron of the LOC_Os05g01250 gene in slm. To verify whether the mutation caused a frameshift in the coding sequence, RNA was extracted from WT and slm, reverse transcribed, and the CDS sequence of LOC_Os05g01250 was amplified by PCR. Sequencing and alignment revealed that the mutation caused a 4bp (GGGG) deletion in the fifth exon, and the frameshift mutation ultimately led to premature termination of amino acid translation at codon 192 (SEQ ID NO: 4). The transcriptional level of this gene after mutation was verified using qRT-PCR. The results showed that the transcriptional level of this gene was significantly reduced in slm. Figure 5 ).

[0085] Example 6: Transgenic Complementation Verification of OsSLM Gene Function

[0086] To verify whether the lesion-like phenotype was caused by the OsSLM mutation, a 6.8Kb sequence from WT, including the complete coding region of the OsSLM gene (3.7Kb), 2.5Kb before the start codon, and 1.6Kb after the stop codon), was inserted into the complementary vector pCAMBIA1300. The recombinant plasmid was then transformed into slm callus tissue via Agrobacterium infection. Under natural field conditions, all T1-positive transgenic plants exhibited growth and development consistent with WT, with no lesion-like phenotype appearing throughout the growth cycle. Subsequently, the OsSLM transcription level in the complementary transgenic plants was detected, and the results showed that the OsSLM transcription level was restored in the complementary transgenic plants. DAB staining was then performed on the leaves of the plants at the heading stage, as shown... Figure 6 As shown, the leaves of the T1 generation complementary plants did not exhibit the brown precipitate found in slm, and the H2O2 level returned to the normal level of WT. Correspondingly, the results of rice blast resistance identification showed that the rice blast resistance of the complementary plants also returned to a level comparable to that of the wild type. These results indicate that the lesion-like phenotype of slm and the significantly enhanced rice blast resistance are both caused by the loss of function of OsSLM.

[0087] The above results fully demonstrate that OsSLM is a resistance-related protein that mainly plays a negative regulatory role in the immune stress response of rice. With the development of CRISP genome-directed editing technology and the increasing maturity of other molecular biology techniques, this gene can be effectively applied to the molecular improvement of rice resistance in the future, and has great development and utilization value and significance.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Mutant gene OsSLM Application in improving rice disease resistance The mutation causes the gene to... OsSLM Functionality missing; in, Gene OsSLM for: The nucleotide sequence shown in SEQ ID NO:1 The disease resistance mentioned refers to resistance to rice blast.

2. A method for improving the disease resistance of rice, characterized in that, Using genetic engineering techniques to modify rice genes OsSLM Perform site-directed mutations to make this gene OsSLM Functional deficiency, thereby improving the disease resistance of rice; Among them, genes OsSLM for: The nucleotide sequence shown in SEQ ID NO:1 The disease resistance mentioned refers to resistance to rice blast.

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