Application of OsUBC11 protein and coding gene thereof in improving plant disease resistance

Abstract

The invention discloses an application of OsUBC11 protein and related biological materials thereof in improving plant disease resistance. The OsUBC11 protein can be specifically the following protein A1), A2) or A3): A1) a protein with an amino acid sequence represented by SEQ ID No.1 in a sequence table; a2) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the protein of A1), has 75% or more of identity with the protein of A1) and has the function of regulating and controlling plant disease resistance; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). An inoculation experiment shows that compared with receptor rice, the resistance of a rice strain for improving expression of the OsUBC11 to rice blast is enhanced, it is indicated that the OsUBC11 is a gene related to plant disease resistance, and the OsUBC11 protein and related biological materials can be used for regulating and controlling the disease resistance of plants. The method disclosed by the invention is simple to operate and low in cost, greatly accelerates the breeding process, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of OsUBC11 protein and its encoding gene in improving plant disease resistance. Background Art

[0002] Rice cultivation has a long and widespread history, feeding over half of the world's population. Therefore, ensuring high and stable yields is crucial for social stability and economic development. Rice blast, caused by the blast fungus Pyricularia oryzae, is a serious fungal disease that severely harms rice in my country. Its prevalence often results in a 10%-35% reduction in rice yield, or even total crop failure. It has been designated a Class I crop disease by my country's Ministry of Agriculture and Rural Affairs as a key national control target. Currently, due to a lack of resistant varieties, rice blast control in my country primarily relies on the application of fungicides. Discovering disease-resistance genes will aid in the development and utilization of disease-resistant rice varieties, reducing the use of chemical pesticides, the resulting environmental pollution, and saving costs.

[0003] The ubiquitin-proteasome pathway plays a crucial role in plant-pathogen interactions. The classic ubiquitin-proteasome system consists of ubiquitin (Ub), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3), and the 26S proteasome. This pathway, catalyzed by a cascade of enzymes, transfers Ub to substrate proteins for ultimate degradation via the 26S proteasome. Studies have shown that the rice U-box ubiquitin ligase SPL11 interacts with the Rho GTPase-activating protein SPIN6 and degrades SPIN6 via the 26S proteasome, significantly increasing ROS levels and the expression of PR-related genes in the plant, enhancing rice resistance to rice blast and bacterial blight. Other studies have shown that the ubiquitin ligases APIP6 and APIP10 can degrade the rice blast effector protein AvrPiz-t through ubiquitination, enhancing rice resistance to the fungus. These results suggest that components of the ubiquitin-proteasome system play a crucial role in rice immune regulation.

[0004] Endoplasmic reticulum-associated protein degradation (ERAD), comprising E2s and E3s located at the ER membrane, plays a crucial role in plant growth and development and in responses to abiotic stresses by promoting the ubiquitination and degradation of misfolded or excess proteins. However, its role in plant disease resistance is rarely investigated. Our previous studies have shown that overexpression of the rice ERAD-associated ubiquitin-conjugating enzyme OsUBC45 not only significantly enhances resistance to rice blast and bacterial blight, but also increases rice yield. Furthermore, it collaborates with the ubiquitin ligase DGS1 to promote the ubiquitination and degradation of two distinct target proteins. However, plants possess numerous E2s and E3s, such as 48 in rice and over 1,500 in rice. Which of these E2s and E3s regulate disease resistance? This is a crucial question that urgently needs to be addressed in ERAD research. Therefore, identifying other ERAD components that play important roles in rice disease resistance will facilitate the development of new disease-resistant rice varieties. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to regulate the disease resistance of plants.

[0006] The present invention provides a protein named OsUBC11, which is the following protein A1), A2) or A3):

[0007] A1) the amino acid sequence is the protein of SEQ ID No. 3 in the sequence listing;

[0008] A2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1) that has 75% or more identity with the protein of A1) and has plant disease resistance regulating activity;

[0009] A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] Among them, SEQ ID No. 1 consists of 169 amino acid residues.

[0011] The above-mentioned protein can be derived from rice.

[0012] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gapexistencecost, Perresiduegapcost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0013] In the above proteins, the 75% or greater identity may be at least 75%, 82%, 83%, 83%, 88%, 95% or 100% identity.

[0014] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc. The amino acid sequences of some of the available tags are shown in Table 1.

[0015] Table 1 Tag sequences

[0016] Label residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0017] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0018] Biological materials related to the protein OsUBC11 also fall within the scope of protection of the present invention.

[0019] The biological material related to the protein OsUBC11 provided by the present invention is any one of the following B1) to B5):

[0020] Any one of the following B1) to B5):

[0021] B1) a nucleic acid molecule encoding the protein OsUBC11;

[0022] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0025] B5) A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette of B2).

[0026] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0027] In the above biological material, the DNA molecule in B1) is any gene shown in b1) to b2) below:

[0028] b1) a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2;

[0029] b2) The nucleotide sequence is the DNA molecule of positions 3243-8805 of SEQ ID No. 1.

[0030] In the above biological materials, the expression cassette (OsUBC11 gene expression cassette) containing the DNA molecule described in B2) refers to a DNA molecule capable of expressing OsUBC11 in a host cell. The DNA molecule may include not only a promoter for initiating transcription of the OsUBC11 gene, but also a terminator for terminating transcription of OsUBC11. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the self-promoter of the gene OsUBC11 (nucleotide sequence as shown in positions 1-3000 of SEQ ID No. 1); the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent No. 200710099169.7)), and promoters specific for seed storage proteins (e.g., the promoters for phaseolin, napin, oleosin, and soybean betaconglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV35S terminator, the tml terminator, the pea rbcSE9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996). 985 ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989)

[0031] Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0032] Existing plant expression vectors can be used to construct a recombinant vector containing the gene encoding the protein OsUBC11 or an expression cassette for the gene encoding the protein OsUBC11. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pCG1301, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pGWB18, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When using OsUBC11 to construct a recombinant vector, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are diverse and can be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or the structural gene.

[0033] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical resistance marker genes (such as herbicide resistance genes).

[0034] In the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi; for example, the bacteria can be Agrobacterium EHA105 strain.

[0035] The application of the above-mentioned protein or the above-mentioned biological material in any of the following C1-C2 also falls within the scope of protection of the present invention:

[0036] C1) Application in regulating plant disease resistance;

[0037] C2) Use in the preparation of products for regulating plant disease resistance.

[0038] In the present invention, the regulation may be upregulation, enhancement or improvement.

[0039] In the above application, the plant may be a grass plant, specifically rice.

[0040] The present invention also provides a method for improving plant disease resistance, comprising step M, wherein step M is to enhance, increase or upregulate the activity and / or content of OsUBC11 protein in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the OsUBC11 protein, so as to improve the plant disease resistance.

[0041] In the above method, the plant may be a grass plant, specifically rice.

[0042] In the above method, the disease resistance can be specifically manifested as the lesion length (lesion area) on the leaves of the target plant after infection with the rice blast pathogen is shorter (smaller) than that of the recipient plant, and the biomass of the rice blast pathogen on the leaves of the target plant is less than that of the recipient plant.

[0043] In order to solve the above technical problems, the present invention also provides a plant agent, which functions to regulate plant disease resistance.

[0044] The plant reagent provided by the present invention contains the protein and / or biological materials related to the protein.

[0045] The active ingredients of the above-mentioned plant reagents may be the above-mentioned proteins and / or biological materials related to the above-mentioned proteins. The active ingredients of the above-mentioned plant reagents may also contain other biological components and / or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned plant reagents based on the plant disease resistance effect.

[0046] The target plant may be a monocot or a dicot. The monocot may be a grass plant, specifically rice.

[0047] Inoculation experiments using rice overexpressing the OsUBC11 gene demonstrated enhanced resistance to rice blast compared to recipient rice, demonstrating that OsUBC11 is a gene associated with plant disease resistance. The method of the present invention is simple to operate, low-cost, and significantly accelerates the breeding process, with broad potential applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The relative expression level of OsUBC11 gene and the test results of rice blast resistance. Figure 1A is the relative expression level of the OsUBC11 gene in the wild-type Nipponbare (WT) and OsUBC11-overexpressing plants (OsUBC11-OE3, OsUBC11-OE4) in Example 2 of the present invention. Figure 1 B-1D is the detection of sensitivity of wild-type Nipponbare (WT), OsUBC11-OE3, and OsUBC11-OE4 to rice blast in Example 2 of the present invention. Figure 1 B shows the phenotypes of wild-type Nipponbare (WT), OsUBC11-OE3, and OsUBC11-OE4 after inoculation with rice blast fungus. Figure 1 C is Figure 1 The statistical results of the lesion size in Figure B. Figure 1 D is Figure 1 The detection results of rice blast fungus biomass in Figure B are shown. In the figure, ** represents the result of significant difference analysis with p < 0.01.

[0049] Figure 2 ROS burst levels and MAPKs activation levels in overexpressing plants. Figure 2 A is the ROS burst level in wild-type Nipponbare (WT) and OsUBC11 overexpressing plants (OsUBC11-OE3, OsUBC11-OE4) at different time points after chitin treatment in Example 3 of the present invention. Figure 2 B is the MAPKs activation level in Nipponbare (WT) and OsUBC11-overexpressing plant OsUBC11-OE3 at different time points after chitin treatment in Example 4 of the present invention. Figure 2 C shows the relative expression levels of disease resistance-related genes in Nipponbare (WT) and OsUBC11-OE3, an OsUBC11-overexpressing plant, after chitin treatment in Example 5 of the present invention. In the figure, ** indicates a significant difference of p < 0.01. The genes associated with disease progression are OsPR1a and OsPR10. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0051] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0052] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0053] The rice variety Nipponbare used in the following examples is a hybrid obtained by crossing "Yamabiko" and "Xingfeng" at the Aichi Prefecture Agricultural Experiment Station in Japan in 1957. It was introduced to China from Japan by the Chinese Academy of Agricultural Sciences in 1967. It was approved by Shandong Province in 1985 and designated Jingyin 153. The public can obtain it from China Agricultural University (the applicant) to replicate the experiments in this application.

[0054] The vector pCG1301 in the following examples is recorded in the non-patent literature "Zhao et al., 2021 (XiaoshengZhao, TianchengQiu, HuijingFeng, ChangfaYin, XunmeiZheng, JunYang, You-LiangPeng, WenshengZhao.2021.A novel glycine-rich domain protein, GRDP1, functions as a critical feedback regulator for controlling cell death and disease resistance in rice, Journal of Experimental Botany, 72(2):608-622.)", which is available to the public from China Agricultural University (i.e., the applicant) to repeat the experiments of this application.

[0055] The Magnaporthe oryzae P131 strain in the following examples is the blast pathogen of rice, which is recorded in the non-patent document "LiuH, LuX, LiM, LunZ, YanX, YinC, YuanG, WangX, LiuN, LiuD, WuM, LuoZ, ZhangY, BhadauriaV, YangJ, TalbotNJ, PengYL. Plant immunity suppression by an exo-β-1,3-glucanase and an elongation factor1αof the rice blast fungus. Nat Commun. 2023Sep7; 14(1): 5491." The public can obtain it from China Agricultural University (i.e., the applicant) to repeat the experiments of this application.

[0056] The components and concentrations of MS medium used in the following examples are shown in Table 2.

[0057] Table 2 MS culture medium components and concentrations used

[0058]

[0059]

[0060] The data in the following examples were processed using GraphPad Prism 9 statistical software. The experimental results were expressed as mean ± standard deviation and tested using t-test. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0061] Example 1. Construction of OsUBC11 overexpression vector

[0062] The genomic sequence of the OsUBC11 gene is shown in SEQ ID No. 1, wherein positions 1-3000 represent its promoter, positions 3243-3294 represent exon 1, positions 3414-3516 represent exon 2, positions 7126-7223 represent exon 3, positions 7308-7408 represent exon 4, positions 8211-8288 represent exon 5, and positions 8728-8805 represent exon 6. The CDS sequence of the OsUBC11 gene is shown in SEQ ID No. 2, and the protein it encodes is named OsUBC11. The amino acid sequence of this protein is shown in SEQ ID No. 3.

[0063] SEQ ID No.1

[0064]

[0065] SEQ ID No.2

[0066] ATGGCGGCTACCATCAGCCAGGCGAGCCTCCTCCTGCAGAAGCAGCTGAAAGATCTCGCGAAGCACCCCGTGGATGGGT

[0067] TCTCGGCTGGGTTAGTTCGACGACAGCAACGTCTTCGAGTGGCAGGTCACAATCATCGGCCCGCCTGACACCCTATATGA

[0068] TGGAGGTTACTTTTAATGCAATAATGAGCTTTCCACAGAATTATCCTAACAGTCCTCCAACTGTCAGATTTACCTCAGAA

[0069] ATGTGGCATCCAAATGTTTATCCGGATGGACGTGTATGCATTTCTATTTCTTCATCCGCCTGGTGATGATCCCAATGGTT

[0070] ATGAGCTTGCGAGTGAGCGTTGGACACCAGTGCATACGGTTGAGAGCATAGTTCTGAGCATCATTTCGATGCTTTCTGG

[0071] TCCAAATGATGAATCACCAGCAAATATTGAAGCAGCTAAGGAATGGAGAGAAGAGGGATGACTTCAAGAAAAAGGTT

[0072] AGGCGCCTTGTAAGGAAATCACAAGAAATGCTCTGA

[0073] SEQ ID No.3

[0074] MAATISQASLLLQKQLKDLAKHPVDGFSAGLVDDSNVFEWQVTIIGPPDTLYDGGYFNAIMSFPQNYPNSPPTVRFTSE

[0075] MWHPNVYPDGRVCISILHPPGDDPNGYELASERWTPVHTVESIVLSIISMLSGPNDESPANIEAAKEWREKRDDFKKKV

[0076] RRLVRKSQEML

[0077] Using the cDNA of the rice variety Nipponbare as a template, the open reading frame sequence of the OsUBC11 gene was amplified by PCR and the PCR product was recovered. The primers used are as follows:

[0078] OsUBC11-GFP-F: 5'-AACACGGGGGACGAGCTC GGTACC ATGGCGGCTACCATCAGC-3' (positions 25-42 are identical to positions 1-18 of SEQ ID No. 2, and the underlined sequence is the KpnI enzyme recognition site sequence);

[0079] OsUBC11-GFP-R: 5'-GCTCACCATGGTGTCGAC TCTAGA GAGCATTTCTTGTGATTTCC-3' (positions 25-44 thereof are reverse complementary to positions 488-507 of SEQ ID No. 2, and the underlined sequence is the XbaI enzyme recognition site sequence).

[0080] The PCR product was recovered to obtain the OsUBC11 gene fragment.

[0081] At the same time, the vector pCG1301 was double-digested with KpnI and XbaI enzymes to obtain the pCG1301 vector backbone.

[0082] The OsUBC11 gene fragment was ligated into the double-enzyme digested vector pCG1301, verified by PCR in the bacterial culture, and then sequenced. The correctly sequenced plasmid is the recombinant expression vector pCG1301-OsUBC11 containing the OsUBC11 gene. The structure of the recombinant expression vector pCG1301-OsUBC11 is described as follows: The small fragment between the recognition sequences for the restriction endonucleases KpnI and XbaI in the vector pCG1301 was replaced with the DNA molecule shown in positions 1-507 of SEQ ID NO. 2, while the other sequences of pCG1301 remained unchanged. In the recombinant expression vector pCG1301-OsUBC11, the 35S promoter drives expression of the OsUBC11 gene.

[0083] Example 2: Establishment, identification, and blast-resistance phenotype analysis of OsUBC11-overexpressing rice lines

[0084] 1. Establishment and identification of OsUBC11 overexpressing rice lines

[0085] To establish an OsUBC11 overexpressing rice line, follow these steps:

[0086] 1. The recombinant expression vector pCG1301-OsUBC11 obtained in Example 1 was introduced into the Agrobacterium strain EHA105 to prepare recombinant Agrobacterium EHA105-OsUBC11.

[0087] 2. Use the recombinant Agrobacterium EHA105-OsUBC11 obtained in step 1 to infect the callus tissue of the rice variety Nipponbare, and obtain T0 generation transformants by hygromycin screening.

[0088] 3. Cut leaves from wild-type Nipponbare and T0-generation transformants and extract DNA from each. Use Hyg-F and Hyg-R primers to amplify the DNA from wild-type Nipponbare and T0-generation transformants as templates. The specific sequences of Hyg-F and Hyg-R are as follows:

[0089] Hyg-F: 5'-TTGGCGACCTCGTATTGGGAA-3';

[0090] Hyg-R: 5'-CAAAGATCGTTATGTTTATCGGCACT-3'.

[0091] The obtained PCR fragments were detected by electrophoresis, and the plants that amplified a hygromycin fragment of about 500 bp were OsUBC11 transgenic positive plants of the T0 generation (ie, OsUBC11 overexpressing plants).

[0092] Transgenic OsUBC11-positive plants from the T0 generation were planted in soil and selfed to obtain T1 seeds. Further planting and selfing were performed to obtain T2 seeds, and the segregation of hygromycin resistance in the T2 generation was analyzed. Several homozygous transgenic lines were selected, two of which were designated OsUBC11-OE3 and OsUBC11-OE4.

[0093] Total RNA was extracted from rice and reverse transcribed to obtain cDNA. qRT-PCR was performed, and the expression levels of the OsUBC11 gene in wild-type Nipponbare (NPB) and each transgenic homozygous line (OsUBC11-OE3 and OsUBC11-OE4) were detected using a fluorescence quantitative PCR instrument (ABI 7500, USA) using the OsUBQ10 gene as an internal reference.

[0094] The primer sequences used are shown in Table 3, and the experiment was repeated three times.

[0095] Table 3 Primer sequences

[0096] Gene name Forward primer 5'-3' Reverse primer 5'-3' OsUBC11 CCCGCCTGACACCCTATATGATG ACATTTGGATGCCACATTTCTGAGG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0097] See the results Figure 1 A, It can be seen that the expression of OsUBC11 gene in transgenic rice lines was significantly upregulated.

[0098] Detection of blast resistance in OsUBC11-overexpressing rice lines

[0099] To detect rice blast resistance, observe the phenotype by scratch inoculation. The specific steps are as follows:

[0100] Seeds of wild-type Nipponbare and two transgenic lines (OsUBC11-OE3 and OsUBC11-OE4) were placed in a 37°C incubator and allowed to germinate for two days by soaking in water. The germinated seeds were then transferred to 9-cm-diameter pots and incubated in a 30°C incubator for approximately 20 days until they reached the four-leaf, one-heart stage. The greenhouse maintained a relative humidity of 70% with 14 hours of light and 10 hours of darkness per day.

[0101] 2. Activate the P131 strain of rice blast fungus (Magnaporthe oryzae) and grow it in an incubator at 28°C for about 7 days.

[0102] 3. Take the penultimate leaf of the rice plant at the four-leaf, one-heart stage in step 1. Take at least 5 leaves from each plant. Cut into 6 to 7 cm lengths and use an insect needle to lightly scratch about 1 mm. 2 Three wounds were made on the main vein of each leaf. The treated scratched leaves were placed in a 10 cm × 10 cm culture dish, and moistened filter paper was placed in the culture dish in advance to maintain humidity.

[0103] 4. Rinse the culture dish in step 2 with 0.025% Tween water, collect the spores of P131 strain, and adjust the spore solution to 1.5×10 5 A P131 spore suspension was prepared at a concentration of spores / mL.

[0104] 5. Spray the leaf surface treated in step 3 evenly with 0.025% Tween water to create a mist to increase adhesion of the spore suspension. Inoculate each wound on the leaf with 10 μL of the P131 spore suspension (see step 4). Keep the inoculated leaves moisturized in a petri dish in the dark for 24 to 36 hours.

[0105] 6. After dark treatment, move the culture dish to light conditions and observe the lesions and take pictures after 4 to 5 days. The phenotypes of wild-type Nipponbare (NPB), OsUBC11-OE3 and OsUBC11-OE4 after inoculation with P131 strain are shown in Figure 1 Figure B.

[0106] 7. Carry out statistics on the length of lesions, as follows:

[0107] The areas of nine lesions in the wild-type and OsUBC11-overexpressing plants infected with the P131 strain were measured, the average values were calculated, and the significant differences were analyzed using the Student's t test. Figure 1 Figure C.

[0108] 8. Quantification of bacterial biomass in rice leaves:

[0109] For specific implementation methods, please refer to "Yoji Kawano, Akira Akamatsu, Keiko Hayashi, Yusuke Housen, Jun Okuda, Ai Yao, Ayako Nakashima, Hiroki Takahashi, Hitoshi Yoshida, HannLing Wong, Tsutomu Kawasaki, Ko Shimamoto. Activation of a Rac GTPase by the NLRfamily disease resistance protein Pit plays a critical role in rice innateimmunity, Cell Host Microbe,2010,7(5):362-75" article.

[0110] After inoculation with the P131 strain, leaves with a size of 2×1 cm, including the lesions, were taken and DNA was extracted using the conventional CTAB method. The DNA concentration was measured and quantified to 3 μg.

[0111] Real-time fluorescence quantitative PCR was used according to the manufacturer's instructions (Genestar). SYBR green I fluorescent dye was added to the PCR system and the expression of the rice blast fungus MoPot2 gene was detected on a fluorescence quantitative PCR instrument (ABI 7500, USA). The rice Ubiquitin gene was used as an internal reference. The primer sequences used are shown in Table 4. The experiment was repeated three times, and the results are shown in Table 4. Figure 1 Figure D.

[0112] Table 4 Primer sequences

[0113] Gene name Forward primer 5'-3' Reverse primer 5'-3' MoPot2 ACGACCCGTCTTTACTTATTTGG AAGTAGCGTTGGTTTTGTTGGAT OsUbiqintin TTCTGGTCCTTCCACTTTCAG ACGATTGATTTAACCAGTCCATGA

[0114] Figure 1 The results showed that compared with the wild type inoculated with the rice blast pathogen, the OsUBC11-overexpressing rice lines inoculated with the rice blast pathogen had enhanced resistance to rice blast, which was manifested in the smaller area of rice blast lesions on rice leaves and the lower biomass of rice blast fungi.

[0115] Example 3. ROS burst levels in OsUBC11-overexpressing rice lines during chitin treatment

[0116] To detect the ROS burst level in OsUBC11-overexpressing rice lines upon chitin treatment, the specific method was as follows:

[0117] Seeds of wild-type Nipponbare (NPB) and OsUBC11-overexpressing plants (OsUBC11-OE3 and OsUBC11-OE4) were placed in a 37°C incubator and allowed to germinate for two days. The germinated seeds were then transferred to 9-cm-diameter pots and incubated in a 30°C room for approximately 20 days until they reached the four-leaf, one-heart stage. The greenhouse maintained a relative humidity of 70% with 14 hours of light and 10 hours of darkness per day.

[0118] 2. Take rice core pieces that are in good growth condition, cut them into small discs using a 4mm diameter punch, and transfer them to sterile water to recover for 12-18 hours.

[0119] 3. Add 50 μL of sterile water to each well of the 96-well plate and use a white pipette tip to pick up a small disc and place it into the well.

[0120] 4. Prepare a reaction solution containing 0.02 mM luminol, 20 μg / ml horseradish peroxidase, and 10 μg / ml chitin or sterile water and add it to eight PCR tubes in a row.

[0121] 5. Use a dispenser to draw up 50 μL of reaction solution and quickly add it to the well with the small disc. Place the test plate in the microplate reader and read the readings at 1 minute / time for 20-40 minutes until the curve gradually decreases. Analyze the results.

[0122] See the results Figure 2 A, The ROS burst level in OsUBC11-overexpressing transgenic rice lines was significantly higher than that in the wild type.

[0123] Example 4: MAPKs activation levels in OsUBC11-overexpressing rice lines during chitin treatment

[0124] To detect the activation level of MAPKs in OsUBC11-overexpressing rice lines under chitin treatment, the specific method is as follows:

[0125] 1. The seeds of wild-type Nipponbare (NPB) and OsUBC11-overexpressing plant OsUBC11-OE3 were treated with 70% alcohol for 3 minutes, washed with 30% laundry detergent for 45 minutes, and then rinsed with plenty of sterile water in a clean bench until there was no foam and dried.

[0126] 2. Spread the seeds on solid 1 / 2MS medium and culture at 28℃ for 5 to 7 days, then transfer to liquid 1 / 2MS medium and culture for 2-3 days.

[0127] 3. Treat the seedlings of the wild type and OsUBC11 overexpressing strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water and stored in a -20°C refrigerator after sonication, and the stock solution concentration is 1 mg / mL), respectively, and sample them at 0, 15, 30, and 60 minutes, respectively.

[0128] 4. To extract total protein from the sample obtained in step 3, add the phosphatase inhibitor PhosSTOP to the protein extraction buffer. TM .

[0129] 5. The protein levels of MAPK3, MAPK6 and the internal reference protein Actin in the above protein samples were detected by western blotting using Phospho-p44 / 42 antibody (used to detect the strain levels of phosphorylated MAPKs) and Actin antibody (both commercial antibodies, Phospho-p44 / 42 antibody catalog number: Cell Signaling Technology 4370; Actin antibody catalog number: EASYBIO BE0027-100).

[0130] Figure 2 B. The results showed that the activation levels of MAPK3 and MAPK6 in OsUBC11-overexpressing transgenic rice lines were significantly higher than those in the wild type.

[0131] Example 5: Identification of Pathogenesis-Related Gene Expression in OsUBC11-Overexpressing Rice Lines

[0132] To detect the expression levels of pathogenesis-related genes in OsUBC11-overexpressing rice lines after chitin treatment, the specific steps are as follows:

[0133] 1. The seeds of wild-type Nipponbare (NPB) and OsUBC11-overexpressing plant OsUBC11-OE3 were treated with 70% alcohol for 3 minutes, washed with 30% laundry detergent for 45 minutes, and then rinsed with plenty of sterile water in a clean bench until there was no foam and dried.

[0134] 2. Spread the seeds on solid 1 / 2MS medium and culture at 28℃ for 5 to 7 days. Then transfer to liquid 1 / 2MS medium and culture for 2-3 days.

[0135] 3. Treat the seedlings of the wild type and OsUBC11 overexpressing strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water, stored in a -20°C refrigerator after sonication, and the stock solution concentration is 1 mg / mL), respectively, and collect samples 6 hours after treatment.

[0136] 4. Extract total RNA from the sample obtained in step 3 and reverse transcribe to obtain cDNA.

[0137] 5. The expression levels of the disease-related genes OsPR1a and OsPR10 in the above cDNA samples were detected by fluorescence quantitative PCR (ABI 7500, USA) (the detection genes and primer sequences are shown in Table 5), and OsUBQ10 was used as the internal reference gene (the sequence is shown in Table 5).

[0138] Table 5

[0139] Gene name Forward primer 5'-3' Reverse primer 5'-3' OsPR1a GTTATCCTGCTGCTTGCTGGTG GCGGGTCCACGAAGTCCTG OsPR10 AGCGTCGTGAAGGTGGAGTC CGGGTTGGCGATGAGGTAGG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0140] See the results Figure 2 C, The induction of pathogenesis-related genes in OsUBC11-overexpressing transgenic rice lines was significantly higher than that in the wild type.

[0141] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A protein, characterized in that The protein is the following protein A1), A2) or A3): A1) the amino acid sequence is the protein of SEQ ID No. 3 in the sequence listing; A2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1) that has 75% or more identity with the protein of A1) and has plant disease resistance regulating activity; A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The protein according to claim 1, characterized in that: The protein is derived from rice.

3. The biomaterial related to the protein according to claim 1 or 2, which is any one of the following B1) to B5): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette of B2).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a gene shown in any one of the following b1)-b2): b1) a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2; b2) A DNA molecule whose nucleotide sequence is SEQ ID No. 1, 3243-8805.

5. Use of the protein according to claim 1 or 2 or the biomaterial according to claim 3 or 4 in the following C1 or C2: C1) Application in regulating plant disease resistance. C2) Use in the preparation of products for regulating plant disease resistance.

6. The use according to claim 5, characterized in that: The regulation is up-regulation, enhancement or improvement.

7. A method for improving plant disease resistance, characterized in that: The method includes step M, which is to enhance, increase or upregulate the activity and / or content of the protein according to claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein according to claim 1 or 2 to improve the plant's disease resistance.

8. The method according to claim 7, wherein: The plant is rice.

9. A plant agent, characterized in that: The reagent contains the protein according to claim 1 or 2 and / or a biological material related to the protein according to claim 3 or 4.

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