Plant disease resistance and yield increase related OsUBC46 protein as well as related biological materials and application thereof

Abstract

The invention discloses an OsUBC46 protein related to plant disease resistance and yield increase as well as a related biological material and application of the OsUBC46 protein. The OsUBC46 protein can be specifically a protein of A1), A2) or A3) as follows: A1) a protein of which the amino acid sequence is SEQ ID No.2 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 activity of regulating and controlling plant disease resistance and yield increase; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The OsUBC46 protein and related biological materials thereof can be used for regulating and controlling the disease resistance and yield increase of plants.

Description

Technical Field

[0001] The present invention relates to OsUBC46 protein related to plant disease resistance and yield increase in the field of biotechnology and related biological materials and applications thereof. Background Art

[0002] Rice blast, caused by Magnaporthe oryzae, is an important fungal disease in rice production, which seriously affects rice yield and quality and is one of the constraints on food security. Breeding new disease-resistant rice varieties is the most economical and environmentally friendly measure to prevent and control rice blast. Therefore, it is crucial to study the rice disease resistance regulatory network and explore excellent genes that are disease-resistant without affecting rice yield.

[0003] The ubiquitin proteasome pathway plays an important role in plant growth and development and in responding to various environmental stresses. This process is catalyzed by a cascade of three enzymes: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3. After multiple rounds of reactions, ubiquitin molecules are added in series to the lysine of the substrate protein and eventually degraded by the proteasome in the cytoplasm. Studies have shown that multiple ubiquitin ligases participate in the rice resistance to rice blast by regulating the stability of substrate proteins. For example, the U-box type E3 ligase OsPIE3 is a negative regulator of rice resistance to rice blast. It interacts with the plasma membrane-localized receptor-like kinase PID2. When OsPIE3 is co-expressed with PID2, it promotes the translocation of the latter from the plasma membrane to the nucleus and is degraded by OsPIE3 in the nucleus. Although some other E3 ligases have also been reported to be involved in the regulation of rice blast, their targets in rice are still unclear. For example, the RING-type E3 ligase ORGLG5 is a positive regulator of rice resistance to rice blast. OsRGLG5 knockout plants are more susceptible to the disease, while overexpression plants are more resistant. Studies have shown that ORGLG5 can promote the degradation of the rice blast effector protein AvrPi9, and AvrPi9 also interferes with the stability of RGLG5. However, ORGLG5 does not participate in the resistance mediated by the rice resistance protein Pi9, and its target in rice is unclear. Although multiple ubiquitin ligase E3s have been reported to regulate rice resistance to rice blast, there are relatively few reports on E2.

[0004] Endoplasmic reticulum-associated protein degradation is a special proteasome reaction, which is catalyzed by E2 or E3 located in the endoplasmic reticulum. It mainly mediates the degradation of misfolded proteins or partially normally folded signaling pathway proteins, helping plants adapt to various environmental stresses. Our previous studies have shown that both rice blast infection and chitin treatment can induce the expression of genes related to rice unfolded protein response, indicating that rice blast infection causes endoplasmic reticulum stress. Through further research, we found that the ubiquitin-binding enzyme OsUBC45 positively regulates rice disease resistance and yield at the same time. It forms an E2-E3 pair with the E3 ligase OsDGS1, and participates in rice disease resistance and yield regulation by regulating two different target proteins. This directly proves the important role of endoplasmic reticulum-associated protein degradation pathway components in rice disease resistance and yield. Studying the functions of other key components of the endoplasmic reticulum-associated degradation pathway in rice resistance to rice blast is expected to provide us with more excellent gene resources and lay the foundation for breeding new disease-resistant rice varieties. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the resistance of rice to rice blast and increase the rice yield.

[0006] The present invention provides a protein named OsUBC46, which is the following protein A1), A2) or A3):

[0007] A1) the amino acid sequence is the protein of SEQ ID No. 2 in the sequence list;

[0008] A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having 75% or more identity with the protein of A1) and having the activity of regulating plant disease resistance and increasing yield;

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

[0010] Among them, SEQ ID No. 2 consists of 240 amino acid residues.

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

[0012] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0013] In the above proteins, the above 75% or more identity may be at least 75%, 80%, 85%, 90%, 95%, 98%, 99% 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 DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a 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 WSHQ c-myc 10 EQKLISEEDL

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

[0018] Biological materials related to protein OsUBC46 also fall within the protection scope of the present invention.

[0019] The biological material related to the protein OsUBC46 provided by the present invention is any one of the following B1) to B5):

[0020] B1) a nucleic acid molecule encoding the protein OsUBC46;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] 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);

[0024] B5) A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B2).

[0025] The nucleic acid molecule may be a DNA molecule, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be an RNA molecule, such as mRNA or hnRNA.

[0026] In the above biological material, the nucleic acid molecule described in B1) is a gene shown in any one of the following b1)-b3):

[0027] b1) the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1;

[0028] b2) The nucleotide sequence is the DNA molecule of positions 3001-7583 of SEQ ID No.3.

[0029] In the above biological materials, the expression cassette (OsUBC46 gene expression cassette) containing the DNA molecule described in B2) refers to a DNA molecule capable of expressing OsUBC46 in a host cell, and the DNA molecule may include not only a promoter for initiating transcription of the OsUBC46 gene, but also a terminator for terminating transcription of OsUBC46. Further, 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 OsUBC46 (the nucleotide sequence is shown in positions 1-3000 of SEQID No.3); the constitutive promoter 35S of the cauliflower mosaic virus; the wound-inducible promoter from tomato, the leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 10099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolus, napin, oleosin and soybean beta conglycin (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 CaMV 35S terminator, the tml terminator, the pea rbcS E9 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) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0030] Existing plant expression vectors can be used to construct a recombinant vector containing the protein OsUBC46 encoding gene or the protein OsUBC46 encoding gene expression cassette. 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 OsUBC46 is used 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., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are wide-ranging and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0031] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as 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), etc.

[0032] In the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi; for example, the bacteria can be Agrobacterium EHA105 strain.

[0033] The application of the above-mentioned protein or the above-mentioned biological material in any of the following C1-C2 also falls within the protection scope of the present invention:

[0034] C1) Application in regulating plant disease resistance and / or plant yield;

[0035] C2) Use in the preparation of products for regulating plant disease resistance and / or plant yield.

[0036] In the present invention, the regulation may be up-regulation, enhancement or increase.

[0037] In the above application, the plant may be a grass plant, specifically rice.

[0038] The present invention also provides a method for improving plant disease resistance, the method comprising step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of OsUBC46 protein in a recipient plant, or / and, enhance, increase or up-regulate the expression level of a gene encoding the OsUBC46 protein in the recipient plant, so as to obtain a target plant having stronger disease resistance than the recipient plant.

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

[0040] In the above method, the target plant that obtains stronger disease resistance than the recipient plant can be specifically manifested in that the length of the lesions (lesion area) on the leaves of the target plant after being infected 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.

[0041] The present invention also provides a method for increasing plant yield, which comprises step N, wherein step N is to enhance, increase or up-regulate the activity and / or content of OsUBC46 protein in a recipient plant, or / and, enhance, increase or up-regulate the expression level of a gene encoding OsUBC46 protein in the recipient plant, so as to obtain a target plant having a higher yield than that of the recipient plant.

[0042] In the above method, the plant may be a grass plant, specifically rice.

[0043] In the above method, the target plant with higher yield than that of the recipient plant can be specifically manifested in that the ear length of the target plant is longer than that of the recipient plant, the seed length of the target plant is longer than that of the recipient plant, the thousand-grain weight of the seeds of the target plant is heavier than that of the recipient plant, and the single-plant yield of the target plant is higher than that of the recipient plant.

[0044] In order to solve the above technical problems, the present invention also provides a plant reagent, which functions to regulate the disease resistance and yield increase of plants.

[0045] The plant reagent provided by the present invention contains the protein and / or biological materials related to the protein.

[0046] 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 may determine the other active ingredients of the above-mentioned plant reagents based on the plant disease resistance and yield-increasing effects.

[0047] The target plant may be a monocot or a dicot. The monocot may be a grass plant, specifically rice.

[0048] Experiments on overexpressing the OsUBC46 gene in rice have shown that, compared with the corresponding wild-type rice, the transgenic rice overexpressing the OsUBC46 protein has enhanced resistance to rice blast, and the yield has not decreased but increased, indicating that the OsUBC46 protein is a gene related to plant disease resistance and yield increase, and overexpressing the OsUBC46 protein improves the disease resistance of plants and increases yield. The method of the present invention is simple to operate, low in cost, greatly accelerates the breeding process, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The relative expression levels of the OsUBC46 gene in the wild-type Nipponbare (NPB) and the OsUBC46 overexpressing plant OsUBC46-OE in Example 2 of the present invention, wherein OsUBC46-OE2 in the OsUBC46 overexpressing plant OsUBC46-OE is marked as 2#, OsUBC46-OE7 is marked as 7#, and OsUBC46-OE15 is marked as 15#. In the figure, ** represents the result of the significant difference analysis as P<0.01.

[0050] Figure 2 These are the test results of the sensitivity of the wild-type Nipponbare and the OsUBC46 overexpressing plants to rice blast in Example 2 of the present invention, wherein OsUBC46-OE2 in the OsUBC46 overexpressing plant OsUBC46-OE is marked as 2#, OsUBC46-OE7 is marked as 7#, and OsUBC46-OE15 is marked as 15#. Figure 2 Figure A shows the phenotypes of wild-type Nipponbare and OsUBC46 overexpressing plants after inoculation with strain P131. Figure 2 Figure B is Figure 2 Statistical results of the lesion area in Figure A. Figure 2 The C diagram is Figure 2The detection results of rice blast fungus biomass in Figure A. In the figure, * represents the significant difference analysis result of P<0.05, and ** represents the significant difference analysis result of P<0.01.

[0051] Figure 3 The ROS burst levels in the wild-type Nipponbare (NPB) and OsUBC46 overexpressing plants (OsUBC46-OE2, OsUBC46-OE7, OsUBC46-OE15) at different time points after chitin treatment in Example 3 of the present invention.

[0052] Figure 4 The MAPKs activation levels in the wild-type Nipponbare (NPB) and the OsUBC46-overexpressing plant OsUBC46-OE2 at different time points after chitin treatment in Example 4 of the present invention.

[0053] Figure 5 The relative expression levels of disease resistance-related genes in wild-type Nipponbare (NPB) and OsUBC46-overexpressing plant OsUBC46-OE2 after chitin treatment in Example 5 of the present invention. In the figure, * represents the result of significant difference analysis as P<0.05, and ** represents the result of significant difference analysis as P<0.01. The genes related to the course of disease are OsPR1b, OsPR10 and OsAOS2.

[0054] Figure 6 The yield statistics of the wild-type Nipponbare (NPB) and the OsUBC46 overexpression plants in Example 6 of the present invention are shown, wherein OsUBC46-OE2 in the OsUBC46 overexpression plant OsUBC46-OE is marked as 2#, OsUBC46-OE7 is marked as 7#, and OsUBC46-OE15 is marked as 15#. Figure 6 Figure A is a photo of rice ears. Figure 6 Figure B shows the statistical results of ear length. Figure 6 Figure C shows the statistical results of single plant yield. Figure 6 Figure D is a photo of seeds. Figure 6 Figure E shows the statistical results of seed thousand-grain weight. In the figure, * represents the statistical results of significant difference analysis of P < 0.05, and ** represents the statistical results of significant difference analysis of P < 0.01. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0057] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0058] The rice variety Nipponbare in the following examples is a hybrid variety obtained by crossing "Yamabiko" and "Xingfeng" at the Aichi Prefecture Agricultural Experiment Station in Japan in 1957, and was introduced to China from Japan by the Chinese Academy of Agricultural Sciences in 1967. It was approved by Shandong in 1985 and numbered: Jingyin 153. The public can obtain it from China Agricultural University (i.e., the applicant) to repeat the experiment of this application.

[0059] The vector pCG1301 in the following embodiments is recorded in the non-patent literature "Zhao et al., 2021 (XiaoshengZhao, Tiancheng Qiu, Huijing Feng, Changfa Yin, Xunmei Zheng, Jun Yang, You-LiangPeng, Wensheng Zhao. 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 can be obtained by the public from China Agricultural University (ie, the applicant) to repeat the experiments of this application.

[0060] The Magnaporthe oryzae P131 strain in the following embodiment is a rice blast pathogen, which is recorded in the non-patent document "Liu H, Lu X, Li M, Lun Z, Yan X, Yin C, Yuan G, Wang X, Liu N, Liu D, Wu M, Luo Z, Zhang Y, Bhadauria V, Yang J, Talbot NJ, Peng YL. Plant immunity suppression by an exo-β-1,3-glucanase and an elongation factor 1α of the rice blast funus. Nat Commun. 2023 Sep 7; 14 (1): 5491. doi: 10.1038 / s41467-023-41175-z.", which is available to the public from China Agricultural University (i.e., the applicant) to repeat the experiment of this application.

[0061] The components and concentrations of MS medium used in the following examples are shown in Table 2.

[0062] Table 2 MS medium composition and concentration used

[0063]

[0064] The data in the following examples were processed using GraphPad Prism 9 statistical software, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the analysis, and P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0065] Example 1. Construction of OsUBC46 overexpression vector

[0066] The genomic sequence of the OsUBC46 gene is shown in SEQ ID No.3, wherein positions 1-3000 are its own promoter, positions 3348-3404 are the first exon, positions 3509-3563 are the second exon, positions 5053-5091 are the third exon, positions 5207-5270 are the fourth exon, positions 5462-5523 ​​are the fifth exon, positions 5655-5694 are the sixth exon, positions 6085-6112 are the seventh exon, positions 6530-6621 are the eighth exon, and positions 6910-7195 are the ninth exon. The CDS sequence of the OsUBC46 gene is shown in SEQ ID No.1, and the protein encoded by it is named OsUBC46, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0067] SEQ ID No.1

[0068] ATGGCGGAGAAGGGATGCCTCAAGCGCCTCCAGAAGGAGTACCACTCGCTCTGCAAGGAGCCGCCGCCGCAGATCGTGGCTCGCCCGCTGCCCAACGACATATTGGAGTGGCATTTTGTACTTGAAGGTAGTGCTGGCACACCATTTGAAGGTGGATATTATTACGGGAAACTCAAATTTCCACCTGATTACCCTTTTAAGCCTCCAAGCATCAGCATGACAACTCCTAGCGGAAGGTTTGCCCCTCACAAAAGAATATGCCTATCAATGAGTGACTTTCATCCGGAATCTTGGAATCCTATGTGGTCTGTGGCAAGCATTCTCACGGGCCTCCTTTCATTCATGATGGATGATGCTCTGACAACTGGAAGCATCAGGAGTACAGAGGGGGAAAAGAGACGTTTAGCAAAGGCTTCCCTTGCCTACAACTGTGAGAGCAAAAATTGCCCTCACTTCAGGAAAATGTTTCCAGAGTACGTCGAGAAATACAACCAACAGAAACAGATGGAGCAAACCGTAGCGGAACCAGAAACTCAAGAGAACCCTGCCCCAGCTCCATCTCCTGCGGTCCAACAACAGGCTGCAGTAGTAGCCAATAAGGCGAAACCTGCGGCGGAGGCCGCAGGCGAGCAGAAGCAGAAGAAGCGAGTGCCCTTCTGGATGATGCTGGTTATGTTTTCTGTTTTTGGTGCGGTGATGGCCTTGCCCCTGATGCAACTCTGA

[0069] SEQ ID No.2

[0070] MAEKGCLKRLQKEYHSLCKEPPPQIVARPLPNDILEWHFVLEGSAGTPFEGGYYYGKLKFPPDYPFKPPSISMTTPSGRFAPHKRICLSMSDFHPESWNPMWSVASILTGLLSFMMDDALTTGSIRSTEGEKRRLAKASLAYNCESKNCPHFRKMFPEYVEKYNQQKQMEQTVAEPETQENPAPAPSPAVQQQAAVVANKAKPAAEAAGEQKQKKRVPFWMMLVMFSVFGAVMALPLMQL

[0071] SEQ ID No.3

[0072]

[0073] Using the cDNA of the rice variety Nipponbare as a template, the open reading frame sequence of the OsUBC46 gene was obtained by PCR amplification and the PCR product was recovered. The primers used are as follows:

[0074] OsUBC46-GFP-F: 5'-AACACGGGGGACGAGCTC GGTACC ATGGCGGAGAAGGGATGC-3' (positions 25-42 are identical to positions 1-18 of SEQ ID No. 1, and the underlined sequence is the Kpn I enzyme recognition site sequence);

[0075] OsUBC46-GFP-R: 5'-GCTCACCATGGTGTCGAC TCTAGA GAGTTGCATCAGGGGCAA-3' (positions 25-42 thereof are reverse complementary to positions 703-720 of SEQ ID No. 1, and the underlined sequence is the Xba I enzyme recognition site sequence).

[0076] The PCR product was recovered to obtain the OsUBC46 gene fragment.

[0077] At the same time, the vector pCG1301 was double-digested with Kpn I enzyme and Xba I enzyme to obtain the pCG1301 vector backbone.

[0078] The OsUBC46 gene fragment was connected to the vector pCG1301 after double restriction digestion, and sequenced after verification by bacterial liquid PCR. The plasmid with correct sequencing is the recombinant expression vector pCG1301-OsUBC46 containing the OsUBC46 gene. The structure of the recombinant expression vector pCG1301-OsUBC46 is described as follows: the small fragment between the recognition sequences of the restriction endonucleases Kpn I and Xba I of the vector pCG1301 is replaced with the DNA molecule shown in positions 1-720 of SEQ ID NO.1, and the other sequences of pCG1301 are kept unchanged to obtain a recombinant expression vector. In the recombinant expression vector pCG1301-OsUBC46, the 35S promoter drives the expression of the OsUBC46 gene.

[0079] Example 2: Establishment, identification and analysis of rice blast resistance phenotype of OsUBC46 overexpressing rice lines

[0080] 1. Establishment and identification of OsUBC46 overexpressing rice lines

[0081] To establish OsUBC46 overexpressing rice lines, the steps are as follows:

[0082] 1. The recombinant expression vector pCG1301-OsUBC46 obtained in Example 1 was introduced into the Agrobacterium strain EHA105 to prepare the recombinant Agrobacterium EHA105-OsUBC46.

[0083] 2. Use the recombinant Agrobacterium EHA105-OsUBC46 obtained in step 1 to infect the callus tissue of the rice variety Nipponbare, and obtain T0 generation transformants by hygromycin screening.

[0084] 3. Cut the leaves of wild-type Nipponbare and T0-generation transformants and extract DNA respectively. Use Hyg-F and Hyg-R primers to amplify the DNA of wild-type Nipponbare and T0-generation transformants as templates. The specific sequences of Hyg-F and Hyg-R are as follows:

[0085] Hyg-F: 5'-TTGGCGACCTCGTATTGGGAA-3';

[0086] Hyg-R: 5'-CAAAGATCGTTATGTTTATCGGCACT-3'.

[0087] The obtained PCR fragments were subjected to electrophoresis detection, and the plants that amplified a hygromycin fragment of about 500 bp were OsUBC46 transgenic positive plants of the T0 generation (ie, OsUBC46 overexpressing plants).

[0088] The OsUBC46 transgenic positive plants of the T0 generation were planted in the soil, self-pollinated to obtain T1 generation seeds, and then self-pollinated to obtain T2 generation seeds after further planting, and the hygromycin resistance segregation of the T2 generation was analyzed. Several transgenic homozygous lines were selected, of which three transgenic homozygous lines were numbered OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15.

[0089] Total RNA was extracted from rice and reverse transcribed to obtain cDNA. qRT-PCR was performed, and the OsUBQ10 gene was used as an internal reference. The expression level of the OsUBC46 gene in the wild-type Nipponbare (NPB) and each transgenic homozygous strain (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) was detected by fluorescent quantitative PCR instrument (ABI 7500, USA).

[0090] The primer sequences used are shown in Table 3, and the experiment was repeated three times.

[0091] Table 3 Primer sequences

[0092] Gene name Forward primer 5'-3' Reverse primer 5'-3' OsUBC46 AAGCCTCCAAGCATCAGCAT AAAGGAGGCCCGTGAGAATG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0093] Results Figure 1It can be seen that the expression of OsUBC46 gene in transgenic rice lines was significantly upregulated.

[0094] Detection of resistance to rice blast in OsUBC46-overexpressing rice lines

[0095] In order to detect rice blast resistance, the phenotype was observed by scratch inoculation. The specific steps are as follows:

[0096] 1. The seeds of wild-type Nipponbare and three overexpressing transgenic lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were placed in a 37°C incubator and soaked in water for two days for germination. The germinated seeds were then transferred into small pots with a diameter of 9 cm and cultured in a 30°C culture room for about 20 days until the four-leaf one-heart stage. The greenhouse maintained a relative humidity of 70%, 14 hours of light and 10 hours of darkness per day.

[0097] 2. Activate the Magnaporthe oryzae P131 strain and grow it in an incubator at 28°C for about 7 days.

[0098] 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 strain. Cut into 6 to 7 cm lengths and use an insect pin to lightly scratch about 1 mm. 2 Three wounds were made on the main vein of each leaf. The scratched leaves were placed in a 10 cm × 10 cm culture dish, and moist filter paper was placed in the culture dish in advance to maintain humidity.

[0099] 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.

[0100] 5. Spray the surface of the leaves treated in step 3 evenly with 0.025% Tween water to form a mist to increase the adhesion of the spore suspension. Inoculate 10 μL of P131 spore suspension (see step 4) at each wound on the leaves. Keep the inoculated leaves moisturized in a petri dish and place them in a dark environment for 24 to 36 hours.

[0101] 6. After the dark treatment, move the culture dish to the light condition, observe the lesions and take pictures after 4 to 5 days. The phenotypes of wild-type Nipponbare (NPB), OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15 after inoculation with P131 strain are shown in Figure 2 Figure A.

[0102] 7. Statistical analysis of the length of the lesions is as follows:

[0103] The 9 lesion areas of the wild-type and OsUBC46 overexpressing plants infected with the P131 strain were measured, and the average values ​​were calculated. The Student's t test was used to calculate the significant differences. The results are shown in Figure 2 Figure B.

[0104] 8. Quantification of bacterial biomass in rice leaves:

[0105] 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.

[0106] After inoculation with the P131 strain, leaves with a size of 2×1 cm including lesions were taken, and DNA was extracted using the conventional CTAB method. The DNA concentration was measured and quantified to 3 μg.

[0107] Using real-time fluorescence quantitative PCR technology, according to the instructions provided by the manufacturer (Genestar), SYBR green I fluorescent dye was added to the PCR system, and the expression of the rice blast fungus MoPot2 gene was detected in a fluorescence quantitative PCR instrument (ABI 7500, USA), with the rice Ubiquitin gene 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 Figure 2 Figure C.

[0108] Table 4 Primer sequences

[0109] Gene name Forward primer 5'-3' Reverse primer 5'-3' MoPot2 ACGACCCGTCTTTACTTATTTGG AAGTAGCGTTGGTTTTGTTGGAT OsUbiqintin TTCTGGTCCTTCCACTTTCAG ACGATTGATTTAACCAGTCCATGA

[0110] Figure 2 The results showed that compared with the wild type inoculated with the rice blast pathogen, the OsUBC46 overexpressing rice lines inoculated with the rice blast pathogen had enhanced resistance to rice blast, which was specifically manifested in the reduction of the area of ​​rice blast lesions on rice leaves and the reduction of the biomass of the rice blast pathogen.

[0111] Example 3. ROS burst levels in OsUBC46 overexpressing rice lines during chitin treatment

[0112] To detect the level of ROS burst in OsUBC46 overexpressing rice lines under chitin treatment, the specific method is as follows:

[0113] 1. The seeds of wild-type Nipponbare (NPB) and OsUBC46 overexpressing plants (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were placed in a 37°C incubator and soaked in water for two days for germination. The germinated seeds were then transferred into small pots with a diameter of 9 cm and cultured in a 30°C culture room for about 20 days until the four-leaf one-heart stage. The greenhouse maintained a relative humidity of 70%, 14 hours of light and 10 hours of darkness per day.

[0114] 2. Take rice heart slices with good growth condition, make small discs with a 4mm diameter puncher, and transfer them to sterile water to recover for 12-18 hours.

[0115] 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.

[0116] 4. Prepare the reaction solution, including 0.02mM luminol, 20μg / ml horseradish peroxidase, 10μg / ml chitin or sterile water, and add it to eight rows of PCR tubes.

[0117] 5. Use a spray gun to draw 50 μL of reaction solution and quickly add it to the hole with the small disc. Place the test plate in the ELISA instrument and read the readings at 1 minute / time for 20-40 minutes until the curve drops gently and analyze the results.

[0118] Results Figure 3 The ROS burst level in OsUBC46 overexpressing transgenic rice lines was significantly higher than that in the wild type.

[0119] Example 4: MAPKs activation levels in OsUBC46 overexpressing rice lines during chitin treatment

[0120] To detect the activation level of MAPKs in OsUBC46 overexpressing rice lines under chitin treatment, the specific method is as follows:

[0121] 1. Treat the seeds of wild-type Nipponbare (NPB) and OsUBC46-overexpressing plant OsUBC46-OE2 with 70% alcohol for 3 minutes, wash with 30% laundry detergent for 45 minutes, and then rinse the seeds with plenty of sterile water in a clean bench until there is no foam and dry them.

[0122] 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.

[0123] 3. Treat the seedlings of the wild type and OsUBC46 overexpression strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water and stored in a -20°C refrigerator after ultrasound, and the mother solution concentration is 1 mg / mL), and take samples at 0, 15, 30, and 60 minutes, respectively.

[0124] 4. To extract total protein from the sample obtained in step 3, add the phosphatase inhibitor PhosSTOP to the protein extraction buffer. TM .

[0125] 5. The protein content of MAPK3 and MAPK6 and the internal reference protein Actin in the above protein samples was detected by western blotting, using Phospho-p44 / 42 antibody (used to detect the strain level of phosphorylated MAPKs) and Actin antibody (both are commercial antibodies, Phospho-p44 / 42 antibody catalog number: Cell Signaling Technology 4370; Actin antibody catalog number: EASYBIO BE0027-100).

[0126] Figure 4 The results showed that the activation levels of MAPK3 and MAPK6 in OsUBC46 overexpressing transgenic rice lines were significantly higher than those in the wild type.

[0127] Example 5: Identification of the expression levels of genes related to disease progression in OsUBC46 overexpressing rice lines

[0128] In order to detect the expression levels of pathogenesis-related genes in OsUBC46 overexpressing rice lines after chitin treatment, the specific steps are as follows:

[0129] 1. Treat the seeds of wild-type Nipponbare (NPB) and OsUBC46-overexpressing plant OsUBC46-OE2 with 70% alcohol for 3 minutes, wash with 30% laundry detergent for 45 minutes, and then rinse the seeds with plenty of sterile water in a clean bench until there is no foam and dry them.

[0130] 2. Spread the seeds on solid 1 / 2MS medium, culture at 28℃ for 5 to 7 days, and then transfer to liquid 1 / 2MS medium and culture for 2-3 days.

[0131] 3. Treat the seedlings of the wild type and OsUBC46 overexpression strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water and stored in a -20°C refrigerator after ultrasound, and the mother solution concentration is 1 mg / mL), respectively, and take samples 6 hours after treatment.

[0132] 4. Extract total RNA from the sample obtained in step 3 and reverse transcribe it to obtain cDNA.

[0133] 5. The expression levels of the disease-related genes OsPR1b, OsPR10, and OsAOS2 in the above cDNA samples were detected by fluorescent quantitative PCR instrument (ABI 7500, USA) (the detection genes and primer sequences are shown in Table 5), and OsUBQ10 was used as the internal reference gene, and the sequence is shown in Table 5.

[0134] Table 5

[0135] Gene name Forward primer 5'-3' Reverse primer 5'-3' OsPR1b TACGCCAGCCAGAGGAGC GCCGAACCCCAGAAGAGG OsPR10 GTCCGGGCACCATCTACACC CAAGCTTCGTCTCCGTCGAGT OsAOS2 AAGCTGCTGCAATACGTGTACTGG CGACGAGCAACAGCCTTCCG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0136] Results Figure 5 It can be seen that the induction of pathogenesis-related genes in OsUBC46-overexpressing transgenic rice lines was significantly higher than that in the wild type.

[0137] Example 6. Yield determination of OsUBC46 overexpressing rice lines

[0138] Count the length of the spike:

[0139] The rice panicles of wild-type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7, and OsUBC46-OE15) were photographed. Figure 6 Figure A. The length of the rice panicle of the wild type and three overexpression transgenic lines was measured. Eight rice panicles were measured and the average value was calculated. The results showed that the panicle length of the three overexpression transgenic lines was significantly greater than that of the wild type line. Figure 6 Figure B.

[0140] Statistics of single plant yield:

[0141] Rice panicles of wild-type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7, and OsUBC46-OE15) were taken, all seeds on the rice panicles were threshed, and the seed weight of each plant was weighed. The seed weights of ten rice panicles were weighed separately, and the average value was calculated. The results showed that the yield per plant of the three overexpression transgenic lines was significantly greater than that of the wild-type line, as shown in Figure 6 Figure C.

[0142] Compare seed sizes:

[0143] Take 10 seeds of each of the wild-type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15), place them head to tail and take photos. The seed length of the three overexpression transgenic lines is longer than that of the wild type, and the seed width is wider than that of the wild type. For details, see Figure 6 Figure D.

[0144] Measuring thousand-grain weight of seeds:

[0145] 1000 seeds of each of the wild-type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were weighed, and the thousand-grain weights of ten rice plants were weighed and the average value was calculated. The results showed that the thousand-grain weights of the seeds of the three overexpression transgenic lines were significantly greater than the thousand-grain weights of the wild-type seeds. Figure 6 Figure E.

[0146] Based on the above results, overexpression of OsUBC46 gene can significantly improve rice resistance to rice blast disease and increase rice yield.

[0147] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended 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. 2 in the sequence list; A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having 75% or more identity with the protein of A1) and having the activity of regulating plant disease resistance and increasing yield; A3) A fusion protein obtained by connecting 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 biological material 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 described in B1), or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B2).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a gene shown in any of the following b1)-b3): b1) the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1; b2) The nucleotide sequence is the DNA molecule of positions 3001-7583 of SEQ ID No.

3.

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 and / or plant yield; C2) Use in the preparation of products for regulating plant disease resistance and / or plant yield.

6. The use according to claim 4, characterized in that: The regulation is up-regulation, enhancement or increase.

7. A method for improving plant disease resistance and / or plant yield, characterized in that: The method comprises step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein according to claim 1 or 2 in the recipient plant, or / and, enhance, increase or up-regulate the expression level of the gene encoding the protein according to claim 1 or 2 in the recipient plant, so as to obtain a target plant with stronger disease resistance than the recipient plant.

8. A method for increasing plant yield, characterized in that: The method comprises step N, wherein step N is to enhance, increase or up-regulate the activity and / or content of the protein according to claim 1 or 2 in the recipient plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein according to claim 1 or 2 in the recipient plant, so as to obtain a target plant with a higher yield than that of the recipient plant.

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

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