Plant disease-resistant yield-increasing osubc46 protein and related biological materials and applications thereof

Abstract

The application discloses plant disease-resistant yield-increasing related OsUBC46 protein and related biological materials and application thereof. The OsUBC46 protein can be specifically the protein of A1), A2) or A3) as follows: A1) the protein with the amino acid sequence of SEQ ID No. 2 in the sequence listing; A2) the protein obtained by substitution, deletion and / or addition of one or more amino acid residues of the protein of A1) and having more than 75% identity with the protein of A1) and having the activity of regulating plant disease resistance and yield increase; and A3) the fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of the protein of A1) or A2). The OsUBC46 protein and related biological materials can be used for regulating plant disease resistance and yield increase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and relates to an OsUBC46 protein related to plant disease resistance and yield increase, and a biological material and application thereof. BACKGROUND

[0002] Magnaporthe oryzae causes rice blast, which is an important fungal disease in rice production, seriously affecting the yield and quality of rice, and is one of the factors restricting food security. Cultivating new rice varieties with disease resistance is the most economical and environmentally friendly measure to prevent rice blast, so it is crucial to study the rice disease resistance regulatory network and to mine excellent genes that are resistant to disease while not affecting the yield of rice.

[0003] The ubiquitin proteasome pathway plays an important role in plant growth and development and response to various environmental stresses. The process is catalyzed by three enzymes, ubiquitin activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, in a cascade. After multiple rounds of reactions, ubiquitin molecules are linked to the lysine of the substrate protein, and are finally degraded by the cytoplasmic proteasome. Studies have shown that multiple ubiquitin ligases regulate the stability of substrate proteins and are involved in rice resistance to rice blast. 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 in the nucleus. Some E3 ligases have also been reported to be involved in rice blast regulation, but their targets in rice are not clear. 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 disease, while overexpression plants are more resistant to disease. Studies have shown that ORGLG5 can promote the degradation of the rice blast effector protein AvrPi9, while AvrPi9 also interferes with the stability of RGLG5. However, ORGLG5 is not involved in the resistance mediated by the rice resistance protein Pi9, and its target in rice is not clear. 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 catalyzed by E2 or E3 located in the endoplasmic reticulum, which mainly helps plants adapt to various environmental stresses by mediating the degradation of misfolded proteins or partially normal signaling pathway proteins. Our previous studies showed that rice blast infection and chitin treatment could induce the expression of rice unfolded protein response-related genes, indicating that rice blast infection caused endoplasmic reticulum stress. Through further research, we found that ubiquitin-conjugating enzyme OsUBC45 positively regulates rice disease resistance and yield, and it forms an E2-E3 pair with E3 ligase OsDGS1, which regulates two different target proteins and is involved in rice disease resistance and yield regulation, respectively. Directly proving 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 may provide us with more excellent gene resources and lay the foundation for breeding new varieties of disease-resistant rice. SUMMARY

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

[0006] The present application provides a protein named OsUBC46, which is the protein of A1), A2) or A3) as follows:

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

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

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

[0010] SEQ ID No. 2 is composed of 240 amino acid residues.

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

[0012] In the above protein, the 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 of the NCBI homepage. For example, the identity (%) of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and then obtaining the value of the identity (%).

[0013] In the above protein, the identity of more than 75% can be at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%.

[0014] In the above protein, the protein tag refers to a polypeptide or protein that is expressed in fusion with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking 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, and the amino acid sequences of some of the tags are shown in Table 1.

[0015] Table 1 Sequence of tags

[0016] Tag 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 protein can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0018] The biological material related to the protein OsUBC46 also falls within the protection scope of the present application.

[0019] The biological material related to the protein OsUBC46 provided by the present application 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 of B1);

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

[0023] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

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

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

[0026] The above biological material, the nucleic acid molecule of B1) is as shown in any one of the following b1) to 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 a DNA molecule of SEQ ID No. 3, 3001-7583.

[0029] In the above-mentioned 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 host cells. This DNA molecule may include not only a promoter to initiate OsUBC46 gene transcription but also a terminator to terminate OsUBC46 transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this 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 OsUBC46 gene (nucleotide sequence as shown in SEQ ID No. 3, positions 1-3000); the constitutive promoter 35S of cauliflower mosaic virus; and the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology). 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline inducible promoter (US Patent 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 bean globule protein, 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 full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, for example, Odell et al. (I)). 985Nature 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] The recombinant vector containing the protein OsUBC46-encoding gene or the protein OsUBC46-encoding gene expression cassette can be constructed using an existing plant expression vector. The plant expression vector can be a Gateway system vector or an Agrobacterium binary vector, such as pCG1301, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pGWB18, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing the recombinant vector using OsUBC46, any one of the enhancer, constitutive, tissue-specific, or inducible promoters can be added before the transcription initiation nucleotide, such as the Cauliflower Mosaic Virus (CAMV) 35S promoter, the ubiquitin gene (Ubiqutin) promoter (pUbi), and the like, which can be used alone or in combination with other plant promoters; in addition, when constructing the plant expression vector using the gene of the present application, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be the ATG initiation codon or the adjacent region initiation codon, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the initiation codon is broad, and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or a 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 a gene that can express an enzyme or a luminescent compound that can produce a color change (GUS gene, luciferase gene, etc.), a resistant antibiotic marker (gentamicin marker, kanamycin marker, etc.), or an anti-chemical reagent marker gene (such as an anti-herbicide gene).

[0032] The recombinant microorganism can be yeast, bacteria, algae and fungi, and the bacteria can be Agrobacterium tumefaciens EHA105 strain.

[0033] The application of the protein or any one of C1-C2 of the biological material also belongs to the protection scope of the present application.

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

[0035] C2) application in preparing a product for regulating plant disease resistance and / or plant yield.

[0036] In the present application, the regulation can be up-regulation or enhancement or increase.

[0037] In the above application, the plant can be a plant of the family Poaceae, and specifically can be rice.

[0038] The present application also provides a method for increasing plant disease resistance, which comprises the step M of enhancing, increasing or up-regulating the activity and / or content of OsUBC46 protein in a receptor plant, or / and, enhancing, increasing or up-regulating the expression amount of the gene encoding OsUBC46 protein in the receptor plant, to obtain a target plant with stronger disease resistance than the receptor plant.

[0039] In the above method, the plant can be a plant of the family Poaceae, and specifically can be rice.

[0040] In the above method, the target plant with stronger disease resistance than the receptor plant can specifically exhibit shorter (smaller) lesion length (lesion area) on the leaves of the target plant after infection with Magnaporthe oryzae pathogen, and less biomass of Magnaporthe oryzae pathogen on the leaves of the target plant than the receptor plant.

[0041] The present application also provides a method for increasing plant yield, which comprises the step N of enhancing, increasing or up-regulating the activity and / or content of OsUBC46 protein in a receptor plant, or / and, enhancing, increasing or up-regulating the expression amount of the gene encoding OsUBC46 protein in the receptor plant, to obtain a target plant with higher yield than the receptor plant.

[0042] In the above method, the plant can be a plant of the family Poaceae, and specifically can be rice.

[0043] In the above method, the target plant with higher yield than the receptor plant can specifically exhibit longer panicle length, longer seed length, heavier thousand seed weight, and higher yield per plant of the target plant than the receptor plant.

[0044] In order to solve the above technical problems, the present application also provides a plant agent for regulating plant disease resistance and yield increase.

[0045] The plant agent provided by the present application contains the protein or the biological material related to the protein.

[0046] The active ingredient of the plant agent can be the protein or the biological material related to the protein, and the active ingredient of the plant agent can also contain other biological components or / and non-biological components, and the other active ingredients of the plant agent can be determined by those skilled in the art according to the plant disease resistance and yield increase effect.

[0047] The target plant can be a monocotyledon or a dicotyledon. The monocotyledon can be a plant of the family Poaceae, and specifically can be rice.

[0048] The experiment of overexpressing the OsUBC46 gene in rice proves that the transgenic rice overexpressing the OsUBC46 protein has enhanced resistance to rice blast compared with the corresponding wild-type rice, and the yield is not only not reduced, but also increased, which indicates that the OsUBC46 protein is a gene related to plant disease resistance and yield increase, and overexpression of the OsUBC46 protein enhances the disease resistance of plants while increasing the yield. The method of the present application is simple in operation and low in cost, greatly accelerates the breeding process, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0050] Figure 2 The detection results of the sensitivity of wild-type Nipponbare and OsUBC46 overexpression plants to rice blast in Example 2 of the present application, wherein the OsUBC46-OE2 in the OsUBC46 overexpression plant OsUBC46-OE is marked as 2#, the OsUBC46-OE7 is marked as 7#, and the OsUBC46-OE15 is marked as 15#. Figure 2 The A figure of FIG. 1 is the phenotype of wild-type Nipponbare and OsUBC46 overexpression plants inoculated with P131 strain. Figure 2 The B figure of FIG. 1 is Figure 2 The statistical results of the lesion area in the A figure of FIG. 1. Figure 2 The C figure of FIG. 1 is Figure 2Figure 1A shows the detection results of Magnaporthe grisea biomass in the A figure. In the figure, * represents the significant difference analysis result P < 0.05, and ** represents the significant difference analysis result P < 0.01.

[0051] Figure 3 Figure 2 shows the ROS burst level in wild type Nipponbare (NPB) and OsUBC46 overexpression plants (OsUBC46-OE2, OsUBC46-OE7, OsUBC46-OE15) at different time points after chitin treatment in Example 3 of the present application.

[0052] Figure 4 Figure 3 shows the MAPKs activation level in wild type Nipponbare (NPB) and OsUBC46 overexpression plant OsUBC46-OE2 at different time points after chitin treatment in Example 4 of the present application.

[0053] Figure 5 Figure 4 shows the relative expression level of disease resistance related genes in wild type Nipponbare (NPB) and OsUBC46 overexpression plant OsUBC46-OE2 after chitin treatment in Example 5 of the present application. In the figure, * represents the significant difference analysis result P < 0.05, and ** represents the significant difference analysis result P < 0.01. The disease course related genes are OsPR1b, OsPR10 and OsAOS2.

[0054] Figure 6 Figure 5 shows the yield statistics of wild type Nipponbare (NPB) and OsUBC46 overexpression plants in Example 6 of the present application, wherein OsUBC46 overexpression plant OsUBC46-OE2 is marked as 2#, OsUBC46-OE7 is marked as 7#, and OsUBC46-OE15 is marked as 15#. Figure 6 Figure 5A is a photo of rice panicles. Figure 6 Figure 5B is the statistics result of panicle length. Figure 6 Figure 5C is the statistics result of single plant yield. Figure 6 Figure 5D is a photo of seeds. GGTACC Figure 5E is the statistics result of thousand seed weight. In the figure, * represents the significant difference analysis result P < 0.05, and ** represents the significant difference analysis result P < 0.01. DETAILED DESCRIPTION

[0055] The present application will be further described in conjunction with the specific embodiments, and the embodiments provided below serve only to illustrate the present application, rather than to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0056] In the quantitative test in the following examples, three repeated experiments were set, and the average value was taken.

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

[0058] The rice variety Nipponbare in the following examples is a hybrid variety obtained by crossing “Yamahiko” and “Kachikaze” in Aichi Agricultural Experiment Station, Japan in 1957, which was introduced into China by Chinese Academy of Agricultural Sciences in 1967. It was approved in Shandong in 1985 with the number of Jingyin 153. The public can obtain it from China Agricultural University (i.e. the applicant) to repeat the experiments of the present application.

[0059] The vector pCG1301 in the following examples is all described in the non-patent literature “Zhao et al., 2021 (Xiaosheng Zhao, Tiancheng Qiu, Huijing Feng, Changfa Yin, Xunmei Zheng, Jun Yang, You-Liang Peng, 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.)”. The public can obtain it from China Agricultural University (i.e. the applicant) to repeat the experiments of the present application.

[0060] The Magnaporthe oryzae P131 strain in the following examples is a rice blast pathogen, which is described in the non-patent literature "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-beta-1, 3-glucanase and an elongation factor 1 alpha of the rice blast fungus. 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 experiments of the present application.

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

[0062] Table 2 Components and use concentrations of MS medium

[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, t-test was used for inspection, P<0.05 (*) indicated significant difference, P<0.01 (**) indicated extremely 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 the positions 1-3000 are its own promoter, the positions 3348-3404 are the first exon, the positions 3509-3563 are the second exon, the positions 5053-5091 are the third exon, the positions 5207-5270 are the fourth exon, the positions 5462-5523 are the fifth exon, the positions 5655-5694 are the sixth exon, the positions 6085-6112 are the seventh exon, the positions 6530-6621 are the eighth exon, and the positions 6910-7195 are the ninth exon. The CDS sequence of the OsUBC46 gene is shown in SEQ ID No. 1, which encodes a protein 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] The open reading frame sequence of OsUBC46 gene was obtained by PCR amplification using the cDNA of rice variety Nipponbare as template, and the PCR product was recovered. The primers used were as follows:

[0074] OsUBC46-GFP-F: 5'-AACACGGGGGACGAGCTC TCTAGA ATGGCGGAGAAGGGATGC-3' (the 25th-42nd positions are the same as the sequence of the 1st-18th positions of SEQ ID No. 1, and the sequence indicated by underlining is the Kpn I enzyme recognition site sequence);

[0075] OsUBC46-GFP-R: 5'-GCTCACCATGGTGTCGAC Gene name GAGTTGCATCAGGGGCAA-3' (the 25th-42nd positions are the reverse complement of the sequence of the 703rd-720th positions of SEQ ID No. 1, and the sequence indicated by underlining is the Xba I enzyme recognition site sequence).

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

[0077] Meanwhile, 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 ligated to the double-digested vector pCG1301, and after verification by bacterial liquid PCR, sequencing was performed. The plasmid with correct sequencing was 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 enzymes Kpn I and Xba I of the vector pCG1301 was replaced with the DNA molecule shown in SEQ ID No. 1, positions 1-720, while the other sequences of pCG1301 were kept unchanged, to obtain the 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 Anti-rice blast Phenotype of OsUBC46 Overexpression Rice Strains

[0080] I. Establishment and identification of OsUBC46 overexpression rice strains

[0081] The steps for establishing OsUBC46 overexpression rice strains are as follows:

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

[0083] 2. The recombinant Agrobacterium EHA105-OsUBC46 obtained in step 1 was used to infect callus of rice variety Nipponbare, and T0 generation transformants were obtained by hygromycin selection.

[0084] 3. Leaf blades of wild-type Nipponbare and T0 generation transformants were cut, and DNA was extracted from each. The DNA of wild-type Nipponbare and T0 generation transformants was used as a template for amplification using primers Hyg-F and Hyg-R, and 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 PCR fragments obtained were detected by electrophoresis, and plants in which a hygromycin fragment of about 500 bp was amplified were T0 generation OsUBC46 transgenic positive plants (i.e. OsUBC46 overexpression plants).

[0088] The T0 generation OsUBC46 transgenic positive plants were planted in soil, selfed to obtain T1 generation seeds, and the T2 generation seeds were obtained by continued selfing and planting, and the hygromycin resistance segregation of the T2 generation was analyzed. Several transgenic homozygous lines were selected, and three of them were numbered as OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15.

[0089] Total RNA was extracted from rice, and cDNA was obtained by reverse transcription, qRT-PCR was performed, and the OsUBQ10 gene was used as an internal reference. The expression amount of the OsUBC46 gene in wild-type Nipponbare (NPB) and each transgenic homozygous line (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) was detected by a fluorescence 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] Forward primer 5'-3' Reverse primer 5'-3' OsUBC46 AAGCCTCCAAGCATCAGCAT AAAGGAGGCCCGTGAGAATG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG Figure 1

[0093] The results are shown in Figure 2The expression of OsUBC46 gene in transgenic lines of rice was significantly up-regulated.

[0094] II. Detection of rice blast resistance in OsUBC46 overexpression lines of rice

[0095] To detect the rice blast resistance, the phenotype was observed by wound inoculation, and the specific steps were as follows:

[0096] 1. The seeds of wild type Nipponbare and three overexpression transgenic lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were soaked in water for germination for two days in a 37°C incubator, and then the germinated seeds were moved 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 stage. The greenhouse maintained a relative humidity of 70%, with 14 hours of light and 10 hours of darkness per day.

[0097] 2. The Magnaporthe oryzae P131 strain was activated and grown in a 28°C incubator for about 7 days.

[0098] 3. The penultimate leaf of the four-leaf stage rice in step 1 was taken, and at least 5 leaves were taken for each line. The leaves were cut to a length of 6 to 7 cm, and about 1 mm 2 wounds were lightly scratched on each leaf vein with an insect needle. The treated wounded leaves were placed in 10 cm x 10 cm culture dishes, and the culture dishes were placed with wet filter paper in advance to maintain humidity.

[0099] 4. The culture dishes in step 2 were rinsed with 0.025% Tween water, and the P131 strain spores were collected, and the spore solution was adjusted to a concentration of 1.5 x 10 5 / mL to prepare a P131 spore suspension.

[0100] 5. The surface of the leaves treated in step 3 was evenly sprayed with 0.025% Tween water to form a mist to increase the adhesion of the spore suspension. 10 μL of P131 spore suspension (see step 4) was inoculated at each wound site on the leaves. The inoculated leaves were kept moist in the culture dishes and placed in a dark environment for 24 to 36 hours.

[0101] 6. After dark treatment, the culture dishes were moved to light conditions, and after 4 to 5 days, the lesions were observed and photographed. The phenotypes of wild type Nipponbare (NPB), OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15 after inoculation with P131 strain are shown in Figure 2 A.

[0102] 7. The lesion length was counted as follows:

[0103] The average value was calculated and the significant difference was calculated by Student's t test. The results are shown in Figure 2B. Figure 2

[0104] 8. Quantification of the biomass of the fungus in the rice leaf:

[0105] The specific implementation method refers to the article "Yoji Kawano, Akira Akamatsu, Keiko Hayashi, Yusuke Housen, Jun Okuda, Ai Yao, Ayako Nakashima, Hiroki Takahashi, Hitoshi Yoshida, Hann Ling Wong, Tsutomu Kawasaki, Ko Shimamoto. Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity, Cell Host Microbe, 2010, 7(5): 362-75".

[0106] The 2x1 cm size leaf containing the lesion after inoculation with P131 strain was taken, and the DNA was extracted by the conventional CTAB method. After measuring the DNA concentration, 3 μg was quantified.

[0107] According to the use method provided by the manufacturer (Genestar), SYBR green I fluorescent dye was added in the PCR system, and the expression of the Magnaporthe oryzae MoPot2 gene was detected by the fluorescence quantitative PCR instrument (ABI 7500, USA) with the rice Ubiquitin gene as the internal reference. The primer sequences used are shown in Table 4. The experiment was set in triplicate, and the results are shown in Figure 2C. Gene name

[0108] Table 4 Primer sequences

[0109] Forward primer 5'-3' Reverse primer 5'-3' MoPot2 ACGACCCGTCTTTACTTATTTGG AAGTAGCGTTGGTTTTGTTGGAT OsUbiqintin TTCTGGTCCTTCCACTTTCAG ACGATTGATTTAACCAGTCCATGA Figure 2

[0110] Figure 3 The results show that compared with the wild type inoculated with the Magnaporthe oryzae pathogen, the OsUBC46 overexpression rice strain inoculated with the Magnaporthe oryzae pathogen has enhanced resistance to rice blast, which is specifically manifested in that the rice blast lesion area on the rice leaf is smaller, and the biomass of the Magnaporthe oryzae fungus is reduced.

[0111] ​​Example 3, ROS burst level in OsUBC46 overexpressed rice lines upon chitin treatment

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

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

[0114] 2. The well-grown rice heart pieces were made into small round pieces with a 4 mm diameter puncher, and were transferred to sterile water for 12-18 hours of recovery.

[0115] 3. 50 μL of sterile water was added to each well of a 96-well plate, and the small round pieces were placed in the wells with a white gun head.

[0116] 4. The reaction solution was prepared, containing 0.02 mM luminol, 20 μg / ml horseradish peroxidase, 10 μg / ml chitin or sterile water, and was added to an eight-row PCR tube.

[0117] 5. 50 μL of the reaction solution was quickly added to the well containing the small round pieces using a row gun, and the detection plate was placed in a microplate reader, reading at 1 minute per time, measuring for 20-40 minutes until the curve gently declined, and analyzing the results.

[0118] The results are shown in Figure 4 The ROS burst level in OsUBC46 overexpressed transgenic lines of rice was significantly higher than that in the wild type.

[0119] Example 4, MAPKs activation level in OsUBC46 overexpressed rice lines upon chitin treatment

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

[0121] 1. The seeds of wild type Nipponbare (NPB) and OsUBC46 overexpressed plants OsUBC46-OE2 were treated with 70% alcohol for 3 minutes, then washed with 30% laundry detergent for 45 minutes, and then rinsed with a large amount of sterile water in a clean bench until there was no foam and dried.

[0122] 2. The seeds are spread on solid 1 / 2MS medium and cultured at 28°C for 5 to 7 days, and then transferred to liquid 1 / 2MS medium and cultured for 2 to 3 days.

[0123] 3. The seedlings of wild type and OsUBC46 overexpression lines are treated with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water, ultrasonic after stored in -20°C refrigerator, mother liquor concentration is 1 mg / mL) respectively, and samples are taken at 0, 15, 30, 60 minutes respectively.

[0124] 4. The total protein of the sample obtained in step 3 is extracted, and phosphatase inhibitor PhosSTOP needs to be added in the protein extraction buffer. TM .

[0125] 5. The protein content of MAPK3 and MAPK6 and the internal reference protein Actin in the above protein sample is detected by Western blotting, and Phospho-p44 / 42 antibody (for detecting the strain level of phosphorylated MAPKs) and Actin antibody (both are commercial antibodies, Phospho-p44 / 42 antibody item number: Cell Signaling Technology 4370; Actin antibody item number: EASYBIO BE0027-100) are used.

[0126] Gene name The results show that the activation level of MAPK3 and MAPK6 in OsUBC46 overexpression transgenic lines of rice is significantly higher than that of MAPK3 and MAPK6 in wild type.

[0127] Example 5, Identification of the expression amount of pathogenesis-related genes in OsUBC46 overexpression rice lines

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

[0129] 1. The seeds of wild type Nipponbare (NPB) and OsUBC46 overexpression plant OsUBC46-OE2 are treated with 70% alcohol for 3 minutes, then washed with 30% laundry detergent for 45 minutes, and then washed with a large amount of sterile water in a clean bench until there is no foam and dried.

[0130] 2. The seeds are spread on solid 1 / 2MS medium and cultured at 28°C for 5 to 7 days, and then transferred to liquid 1 / 2MS medium and cultured for 2 to 3 days.

[0131] 3. Wild type and OsUBC46 overexpression lines were treated with ultrapure water and 10 μg / mL chitin suspension (chitin suspension was prepared with water, stored in -20 °C refrigerator after ultrasonic, mother liquor concentration was 1 mg / mL) respectively, and sampled after 6 hours of treatment.

[0132] 4. Total RNA was extracted from the samples obtained in step 3, and cDNA was obtained by reverse transcription.

[0133] 5. The expression levels of pathogenesis-related genes OsPRlb, OsPR10 and OsAOS2 in the above cDNA samples were detected by a fluorescence quantitative PCR instrument (ABI 7500, USA) (see Table 5 for the sequences of the detected genes and primers), with OsUBQ10 as the internal reference gene, and the sequence is shown in Table 5.

[0134] Table 5

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

[0136] The results are shown in Figure 6 It can be seen that the induction of pathogenesis-related genes in the OsUBC46 overexpression transgenic lines of rice is significantly higher than that of pathogenesis-related genes in the wild type.

[0137] Example 6, yield determination of OsUBC46 overexpression rice lines

[0138] The length of the ear was counted:

[0139] The ears of wild type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were taken for photography, as shown in Figure 6 Figure A. The length of the ears of wild type and three overexpression transgenic lines of rice was measured, and the average value was calculated by measuring eight ears of rice respectively. The results showed that the ear length of the three overexpression transgenic lines was significantly longer than that of the wild type, and the results are shown in Figure 6 Figure B.

[0140] The yield per plant was counted:

[0141] The ears of wild type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were taken, and all the seeds on the ears of rice were threshed, and the weight of the seeds per plant was measured. The weight of the seeds of ten ears of rice was measured respectively, and the average value was calculated. The results showed that the yield per plant of the three overexpression transgenic lines was significantly higher than that of the wild type, and the details are shown in Figure 6 Figure C.

[0142] The size of the seeds was compared:

[0143] Ten seeds of wild type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were taken, arranged head to tail and photographed. The length of seeds of three overexpression transgenic lines was longer than that of wild type, and the width of seeds of three overexpression transgenic lines was wider than that of wild type, as shown in the D graph of FIG. 4. Figure 6

[0144] The thousand seed weight was measured:

[0145] 1000 seeds of wild type Nipponbare (NPB) and OsUBC46 overexpression lines (OsUBC46-OE2, OsUBC46-OE7 and OsUBC46-OE15) were taken and weighed, and the thousand seed weight of ten rice plants was measured and the average was calculated. The results showed that the thousand seed weight of seeds of three overexpression transgenic lines was significantly greater than that of wild type seeds, as shown in the E graph of FIG. 5. ​

[0146] In summary, overexpression of OsUBC46 gene can significantly improve the blast resistance of rice, and the yield of rice is increased.

[0147] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In general, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.​​

Claims

1. Use of a protein or a biological material related to the protein in C1 or C2 below: C1) in modulating plant disease resistance and / or plant yield; C2) in preparing a product for modulating plant disease resistance and / or plant yield; the modulation is up-regulation or enhancement or improvement of plant disease resistance and / or plant yield by overexpression of the protein; the plant is rice; the disease resistance is disease resistance against rice blast; the protein is a protein of A1) or A2) below: A1) a protein with an amino acid sequence of SEQ ID No. 2 in the sequence listing; A2) a fusion protein obtained by linking a protein tag to the N terminus or / and C terminus of A1); the biological material related to the protein is any one of B1) to B4) below: B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3).

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

3. Use according to claim 1, characterized in that: B1) the nucleic acid molecule is a gene of any one of b1) to b2) below: b1) a DNA molecule with a coding sequence of SEQ ID No. 1 encoding a chain; b2) a DNA molecule with a nucleotide sequence of SEQ ID No. 3 from position 3001 to 7583.

4. A method for increasing disease resistance and / or yield of a plant, characterized by: The method comprises step M of enhancing, improving or up-regulating the content of the protein of claim 1 or 2 in a recipient plant, or / and, enhancing, improving or up-regulating the expression amount of the gene encoding the protein of claim 1 or 2 in a recipient plant, to obtain a target plant with stronger disease resistance than the recipient plant; the plant is rice; the disease resistance is disease resistance against rice blast.

5. A method of increasing yield in a plant, comprising: The method comprises step N of enhancing, improving or up-regulating the content of the protein of claim 1 or 2 in a recipient plant, or / and, enhancing, improving or up-regulating the expression amount of the gene encoding the protein of claim 1 or 2 in a recipient plant, to obtain a target plant with higher yield than the recipient plant; the plant is rice; the disease resistance is disease resistance against rice blast.

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