A method for screening and identifying E2 that works synergistically with plant multi-subunit SCF E3 ligases

Abstract

The present invention discloses a method for screening and identifying an E2 that works in conjunction with a plant multi-subunit SCF E3 ligase. The present invention provides a method for identifying whether a ubiquitin-conjugating enzyme E2 to be tested can work in conjunction with a plant multi-subunit SCF E3 ligase to form a polyubiquitin chain, comprising: preparing a reaction system containing a ubiquitin-conjugating enzyme E2 to be tested, a plant multi-subunit SCF E3 ligase, a ubiquitin activating enzyme E1, and a ubiquitin protein Ubiquitin; placing the reaction system at 22-37°C for a shake reaction for 1-2 hours; and detecting to determine whether the ubiquitin-conjugating enzyme E2 to be tested can work in conjunction with the plant multi-subunit SCF E3 ligase to form a polyubiquitin chain. The present invention is crucial for analyzing the function of plant multi-subunit SCF E3 ligases. The present invention has important theoretical and application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening and identifying E2 that works in cooperation with plant multi-subunit SCF E3 ligase. Background Art

[0002] The balance between protein degradation and synthesis plays an important role in maintaining cellular homeostasis and normal body function. In eukaryotic cells, the ubiquitin-proteasome pathway is one of the major pathways for protein degradation. This pathway consists of ubiquitination of substrate proteins and proteasome-dependent degradation. Protein ubiquitination requires the coordinated efforts of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Among them, SCF E3 ligases are one of the largest ubiquitin ligase subfamily members and are composed of SKP1, Cullin1, RBX1, and a displaceable F-box protein.

[0003] In vitro ubiquitination assays provide an effective platform for studying the molecular mechanisms of E3 ligase activity. To date, research on in vitro reconstitution systems for plant multi-subunit SCF E3 ligase activity remains elusive. Screening and identifying E2s that collaborate with multi-subunit SCF E3 ligases is a bottleneck in establishing such systems. Therefore, establishing a platform for screening and identifying E2s that collaborate with plant multi-subunit SCF E3 ligases is crucial for advancing research into the molecular mechanisms of plant multi-subunit E3 ligase activity. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for screening and identifying E2 that works in conjunction with plant multi-subunit SCF E3 ligase, and to provide some specific ubiquitin conjugating enzymes E2, as well as a method for screening and identifying whether a test protein has ubiquitin conjugating enzyme activity.

[0005] In a first aspect, the present invention claims a method for identifying whether a test ubiquitin-conjugating enzyme E2 can cooperate with a plant multi-subunit SCF E3 ligase to form polyubiquitin chains.

[0006] The method claimed in the present invention for identifying whether a target ubiquitin-conjugating enzyme E2 can cooperate with a plant multi-subunit SCF E3 ligase to form a polyubiquitin chain may comprise the following steps:

[0007] (A1) preparing a reaction system; the reaction system contains the ubiquitin-conjugating enzyme E2 to be tested, plant multi-subunit SCF E3 ligase, ubiquitin activating enzyme E1, and ubiquitin protein Ubiquitin; (A2) placing the reaction system prepared in (A1) at 22-37° C. and shaking for 1-2 hours (e.g., 2 hours);

[0008] (A3) Detecting the system after the reaction in (A2) to determine whether the ubiquitin-conjugating enzyme E2 to be tested can work synergistically with the plant multi-subunit SCF E3 ligase to form a polyubiquitin chain.

[0009] Furthermore, the reaction system further contains a reaction buffer (denoted as reaction buffer 1); the reaction buffer 1 contains Tris (such as pH 7.4), MgCl2, ATP, and DTT.

[0010] In some cases, the reaction system is composed of the following: 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM ATP, 2 mM DTT, 1.67 mg / L ubiquitin activating enzyme E1, 6.67 mg / L of the ubiquitin-conjugating enzyme E2 to be tested, 66.67 mg / L ubiquitin protein, and 6.67 mg / L of the plant multi-subunit SCF E3 ligase (quantified based on F-box protein). The concentrations of the above substances are the final concentrations in the reaction system.

[0011] Furthermore, the order of adding the components in the reaction system is preferably: the ubiquitin-conjugating enzyme E2 to be tested, the plant multi-subunit SCF E3 ligase, the ubiquitin protein Ubiquitin, the ubiquitin activating enzyme E1, and the reaction buffer 1 (e.g., a 20× stock solution, formula: 1M Tris (pH 7.4), 200mM MgCl2, 100mM ATP, 40mM DTT).

[0012] In some cases, in (A2), the reaction temperature is 28°C, the shaking condition is 950 rpm, and the reaction time is 2 hours. The rotation speed of 950 rpm can ensure that the SCF E3 ligase complex can be evenly contacted and mixed with other components in the system and undergo active reaction.

[0013] In step (A2), after the reaction is completed, a step of adding 6×SDS sample loading buffer to terminate the reaction may be further included.

[0014] In step (A3), the detection can be performed by SDS-polyacrylamide gel electrophoresis and Western blotting. The formation of polyubiquitin chains can be used to determine whether the screened E2 can effectively cooperate with the SCF E3 ligase. In some cases, the primary antibody used in Western blotting is an anti-ubiquitin antibody; the secondary antibody used is an HRP-labeled antibody. If the Western blotting results show aggregated ubiquitin signals of varying molecular weights, it indicates that the tested ubiquitin-conjugating enzyme E2 can cooperate with the plant multi-subunit SCF E3 ligase to form polyubiquitin chains. If the Western blotting results do not show aggregated ubiquitin signals of varying molecular weights, it indicates that the tested ubiquitin-conjugating enzyme E2 cannot cooperate with the plant multi-subunit SCF E3 ligase to form polyubiquitin chains.

[0015] In the method, the plant multi-subunit SCF E3 ligase can be prepared according to a method comprising the following steps:

[0016] (B1) fusing the gene encoding the F-box protein in the plant multi-subunit SCF E3 ligase complex with a protein tag coding sequence and introducing the resulting sequence into a recipient plant (e.g., callus tissue of the recipient plant) to obtain a transgenic plant (e.g., transgenic plant callus tissue material);

[0017] The recipient plant material here can be callus tissue, but is not limited to callus tissue. It can also be other plant tissue materials, such as tobacco leaves, Arabidopsis inflorescences, protoplast cells or bioreactors such as wheat embryo in vitro translation systems. The goal is to obtain the expressed F-box protein with a fusion protein tag.

[0018] (B2) taking tissue (such as callus tissue) of the transgenic plant obtained in (B1) and grinding it in liquid nitrogen;

[0019] Furthermore, the method may further include the step of quick-freezing the tissue with liquid nitrogen before grinding in liquid nitrogen.

[0020] (B3) extracting total protein from the ground powder obtained in (B2);

[0021] Furthermore, total protein is extracted as follows: 1-3 mL (e.g., 2 mL) of plant tissue protein lysate is added to 1 g of the ground powder for protein extraction, the plant protein lysate is added to the ground powder, and the mixture is incubated on ice for 25-35 min (e.g., 30 min), shaken every 3-7 min (e.g., 5 min), and centrifuged at 15,000 g at 4°C (e.g., 20 min).

[0022] Furthermore, the plant tissue protein lysate formulation includes: 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM NaF, 2 mM EDTA (pH 8.0), 1 mM sodium orthovanadate, 10% (v / v) Glycerol, 0.5% (v / v) Nonidet P-40, and the following components are added immediately prior to use: 1× complete protease inhibitor cocktail, 1 mM PMSF, and 1 mM DTT. The concentrations of the aforementioned substances are the final concentrations in the plant tissue protein lysate.

[0023] (B4) The protein complex obtained by separating from the total protein obtained in (B3) using a substance that can specifically bind to the protein tag is the plant multi-subunit SCF E3 ligase.

[0024] In some cases, the plant multi-subunit SCF E3 ligase is SCF in the rice strigolactone signaling pathway D3 E3 ligase or SCF in the rice gibberellin signaling pathway GID2 Of course, other SCF E3 ligases are also suitable for use in the methods of the present invention.

[0025] In some cases, when the plant multi-subunit SCF E3 ligase is the SCF D3 When the plant multi-subunit SCF E3 ligase is the SCF GID2 The E3 ligase, wherein the F-box protein is a GID2 protein. Furthermore, the GID2 protein is derived from rice. Furthermore, the amino acid sequence of the GID2 protein is identical to the amino acid sequence encoded by the gene with accession number AB100246.1 in the Genebank database (http: / / www.ncbi.nlm.nih.gov / genbank).

[0026] The protein tags include, but are not limited to, GFP tags, Flag tags, HA tags, or myc tags. The transgenic plants are not limited to transgenic callus expression systems; sources may also include plant bioreactors such as tobacco transient expression systems, protoplast transient expression systems, and wheat germ in vitro translation systems. This method can also be applied to obtaining SCF E3 ligase complexes in eukaryotic organisms other than rice, and can be extended to various species, including Arabidopsis thaliana, tobacco, wheat, soybean, tomato, corn, mice, rats, nematodes, fruit flies, and humans.

[0027] In some embodiments of the present invention, the protein tag is GFP. Accordingly, GFP Trap Agarose is used to obtain active SCF E3 ligase complexes. This product has the advantage that the antibodies bound to the agarose gel undergo a special treatment that eliminates interference from the antibody's heavy and light chains, thereby improving the specificity of subsequent activity analysis experiments.

[0028] In some cases of the present invention, the ubiquitin-conjugating enzyme E2 to be tested is selected from the protein described in (C1) of the third aspect below, or is selected from HsUbcH5C, HsUbcH7 or HsCDC34.

[0029] In a second aspect, the present invention claims a method for preparing a plant multi-subunit SCF E3 ligase.

[0030] The method for preparing the plant multi-subunit SCF E3 ligase claimed in the present invention may include steps (B1) to (B4) described above.

[0031] In a third aspect, the present invention claims protection for any of the following:

[0032] (C1) Use of all or part of the following proteins as ubiquitin-conjugating enzymes:

[0033] OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUB C32 protein, OsUBC44 protein, OsUBC46 protein, OsUBC47 protein; or any of the above proteins with one or more amino acid residues substituted and / or deleted and / or added and derived from rice with the same function; or a protein with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology to any of the above proteins and derived from rice with the same function; or a fusion protein obtained by fusion protein tags at the N-terminus and / or C-terminus of any of the above proteins.

[0034] The amino acid sequences of the above proteins are identical to the amino acid sequences encoded by the genes shown in the corresponding gene accession numbers in Table 1 (the same below).

[0035] In the above-mentioned 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. Such protein tags include, but are not limited to, Flag tags, His tags, MBP tags, HA tags, myc tags, GST tags, and / or SUMO tags.

[0036] 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, 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, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0037] In the above proteins, the 95% or greater homology may be at least 96%, 97%, or 98% identity. The 90% or greater homology may be at least 91%, 92%, 93%, or 94% identity. The 85% or greater homology may be at least 86%, 87%, 88%, or 89% identity. The 80% or greater homology may be at least 81%, 82%, 83%, or 84% identity.

[0038] These proteins all have the function of ubiquitin-conjugating enzyme E2 and form DTT-sensitive OsUBC-Ub conjugates with activated ubiquitin proteins through disulfide bonds.

[0039] (C2) Use of a protein or nucleic acid molecule, or an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the nucleic acid molecule, in the preparation of a product having ubiquitin-conjugating enzyme activity;

[0040] The protein is all or part of the following: OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUBC32 protein, OsUBC44 protein, OsUBC46 protein, OsUBC47 protein.

[0041] The nucleic acid molecule is a nucleic acid molecule encoding the protein; the expression cassette is an expression cassette containing the nucleic acid molecule; the recombinant vector is a recombinant vector containing the nucleic acid molecule or the expression cassette; the recombinant bacteria is a recombinant bacteria containing the nucleic acid molecule or the expression cassette or the recombinant vector; the transgenic cell line is a transgenic cell line containing the nucleic acid molecule or the expression cassette or the recombinant vector.

[0042] The gene accession numbers of the nucleic acid molecules encoding the above-mentioned proteins are shown in Table 1 (the same below). The nucleic acid molecules may also be DNA molecules that hybridize with the above-defined nucleic acid molecules under stringent conditions and encode the corresponding proteins; or DNA molecules that have 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the above-defined nucleic acid molecule sequences and encode the corresponding proteins.

[0043] In the above nucleic acid molecules, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 2×SSC and 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 1×SSC and 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC and 0.1% SDS; and hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC and 0.1% SDS. The membrane can be hybridized in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C and rinsed in 0.1×SSC, 0.1% SDS. The membrane can also be hybridized in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C and rinsed in 0.1×SSC, 0.1% SDS at 65°C. The membrane can also be hybridized in a solution of 6×SSC, 0.5% SDS at 65°C and then washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0044] In the above-mentioned nucleic acid molecules, homology refers to nucleotide sequence identity. Nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of nucleotide sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0045] In the above nucleic acid molecules, the 95% or greater homology may be at least 96%, 97%, or 98% identity. The 90% or greater homology may be at least 91%, 92%, 93%, or 94% identity. The 85% or greater homology may be at least 86%, 87%, 88%, or 89% identity. The 80% or greater homology may be at least 81%, 82%, 83%, or 84% identity.

[0046] (C3) Use of a protein or nucleic acid molecule, or an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the nucleic acid molecule in the preparation of a product for ubiquitination modification analysis;

[0047] The protein is all or part of the following: OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUBC32 protein, OsUBC44 protein, OsUBC46 protein, OsUBC47 protein.

[0048] The nucleic acid molecule is a nucleic acid molecule encoding the protein; the expression cassette is an expression cassette containing the nucleic acid molecule; the recombinant vector is a recombinant vector containing the nucleic acid molecule or the expression cassette; the recombinant bacteria is a recombinant bacteria containing the nucleic acid molecule or the expression cassette or the recombinant vector; the transgenic cell line is a transgenic cell line containing the nucleic acid molecule or the expression cassette or the recombinant vector.

[0049] (C4) A kit for screening for a ubiquitin-conjugating enzyme E2 that works synergistically with a plant multi-subunit SCF E3 ligase to form polyubiquitin chains, comprising the plant multi-subunit SCF E3 ligase described in the first aspect above, a ubiquitin-activating enzyme E1, a ubiquitin protein Ubiquitin, and the reaction buffer described in the first aspect above (i.e., the reaction buffer 1).

[0050] Furthermore, the kit also contains all or part of the following proteins: OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUBC32 protein, OsUBC44 protein, OsUBC46 protein, and OsUBC47 protein.

[0051] (C5) Use of the kit described in (C4) in screening for ubiquitin-conjugating enzymes that can work synergistically with plant multi-subunit SCF E3 ligases to form polyubiquitin chains.

[0052] As used herein, the expression cassette refers to a DNA kit capable of expressing one or more proteins in a host cell. This DNA may include not only a promoter for initiating transcription of the nucleic acid molecule but also a terminator for terminating transcription of the nucleic acid molecule. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0053] In the above text, to facilitate protein purification, all of the above protein expression vectors may be constructed with a protein tag that is fused to the target protein to facilitate expression, detection, tracing, and / or purification of the target protein. Such protein tags include, but are not limited to, GFP tags, Flag tags, HA tags, myc tags, His tags, MBP tags, GST tags, Strep tags, and / or SUMO tags. All constructed vectors for protein expression must be sequenced to confirm that the coding sequence reading frame is correct to ensure proper translation of the entire sequence.

[0054] In order to facilitate the screening and identification of transgenic plant materials (such as transgenic cells, calli or plants), all plant expression vectors can be modified, for example, by adding genes that can be expressed in plants and encode luminescent compounds, enzymes or proteins that can produce color changes (GUS gene, green fluorescent protein GFP gene and luciferase gene, etc.), and antibiotic resistance markers (kanamycin marker, gentamicin marker, hygromycin marker, etc.).

[0055] In this case, a prokaryotic protein expression system was used to express a recombinant plasmid with a His tag fused to the N-terminus. The specific steps are as follows: The expression plasmids of the ubiquitin-binding protein UBCs fused to the His tag were transformed into the Escherichia coli protein expression strain BL21 (DE3); the activated recombinant bacteria were cultured at 37°C until the OD 600 The pH value was set to 0.8, and then a final concentration of 0.3 mM IPTG was added, and protein expression was induced at 16°C and 180 rpm for 16-20 hours; the bacteria were collected by centrifugation, the cells were broken, and the supernatant was purified by Ni column after centrifugation to obtain the target protein.

[0056] In a fourth aspect, the present invention claims a method for detecting whether a protein to be tested has ubiquitin-conjugating enzyme activity.

[0057] The method for detecting whether a protein to be tested has ubiquitin-binding enzyme activity claimed in the present invention may include the following steps:

[0058] (D1) preparing a reaction system; the reaction system contains a protein to be tested, ubiquitin activating enzyme E1 and ubiquitin.

[0059] Furthermore, the reaction system further contains a reaction buffer (denoted as reaction buffer 2); the reaction buffer 2 contains Tris (such as pH 7.4), MgCl2, and ATP; and / or the reaction buffer 2 further contains or does not contain DTT.

[0060] Furthermore, when the reaction buffer 2 does not contain DTT, the reaction system is composed as follows: 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM ATP, 1.67 mg / L ubiquitin activating enzyme E1, and 6.67 mg / L of the protein to be tested; when the reaction buffer contains DTT, the reaction system is composed as follows: 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM ATP, 2 mM DTT, 1.67 mg / L ubiquitin activating enzyme E1, and 6.67 mg / L of the protein to be tested; the concentrations of the above substances are the final concentrations in the reaction system.

[0061] The order of adding samples to the reaction system is preferably: the test protein, ubiquitin, ubiquitin activating enzyme E1, and the reaction buffer 2 (such as 20× mother solution, formula: 1M Tris (pH 7.4), 200mM MgCl2, 100mM ATP, with or without 40mM DTT).

[0062] (D2) The reaction system prepared in (D1) is placed at 28°C for 15-30 minutes (e.g., 30 minutes).

[0063] (D3) Detecting the system after the reaction in (D2) to determine whether the protein to be tested has ubiquitin-binding enzyme activity.

[0064] In step (D2), after the reaction is completed, a step of adding 6×SDS sample loading buffer (with or without 0.6 M DTT) to terminate the reaction may be further included.

[0065] In step (D3), the detection can be performed by Western blotting. In some cases, the primary antibody used is an anti-ubiquitin antibody or an anti-His tag antibody; the secondary antibody used is an HRP-labeled antibody. If a band the size of OsUBC-Ub appears in the Western blotting results of the active reaction sample without the addition of DTT, but the band disappears after the addition of DTT, it indicates that the test protein has ubiquitin conjugating enzyme activity. If no band the size of OsUBC-Ub appears in the Western blotting results, regardless of whether DTT is added, it indicates that the test protein does not have ubiquitin conjugating enzyme activity.

[0066] The in vitro reconstitution system for multi-subunit SCF E3 ligase activity is extremely important for studying the molecular mechanism of action of multi-subunit E3 ligases. However, screening and identifying E2s that specifically and efficiently cooperate with multi-subunit SCF E3 ligases is an extremely important and challenging scientific research field. The present invention helps to solve this key problem and is crucial for the functional analysis of multi-subunit SCF E3 ligases in plants. The present invention has important theoretical and applied value. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Figure 1 shows the purification and quantification of 26 ubiquitin-binding proteins (OsUBCs) from 12 rice subfamilies. A shows the purification and Coomassie staining quantification of OsUBC1 from rice subfamily I, OsUBC4 / 5 / 6 from subfamily II, OsUBC7 / 8 / 9 from subfamily III, OsUBC10 from subfamily IV, and OsUBC25 / 26 from subfamily VII. B shows the purification and Coomassie staining quantification of OsUBC11 from subfamily V and OsUBC14 / 16 / 23 from subfamily VI. C shows the purification and Coomassie staining quantification of OsUBC13 / 18 / 19 / 22 from subfamily VI. D shows the purification and Coomassie blue staining results for OsUBC28 / 29 / 30 / 31 from subfamily VIII, OsUBC32 from subfamily IX, OsUBC44 from subfamily XII, OsUBC46 from subfamily XIV, and OsUBC47 from subfamily XV. Arrows indicate the corresponding OsUBC protein bands after Coomassie blue staining. BSA gradient bands were used as markers to calibrate the target protein content.

[0068] Figure 2 Purification and quantification of rice-derived OsUb and OsE1 proteins. A shows the purification and quantification of His-OsUb protein by Coomassie blue staining. B shows the purification and quantification of GST-OsUb protein by Coomassie blue staining. C shows the purification and quantification of Strep-OsE1 protein by Coomassie blue staining. Arrows indicate the corresponding protein bands of OsUb and OsE1 proteins after Coomassie blue staining. BSA gradient bands were used as markers to calibrate the target protein content.

[0069] Figure 3-9, experimental results of detecting ubiquitin-conjugating enzyme (E2) activity by dithiothreitol (DTT)-sensitive thioester bond formation assay; "+" indicates that the corresponding reaction system contains the substance; "-" indicates that the corresponding reaction system does not contain the substance; arrows point to E2-ubiquitin conjugates and ubiquitin monomers or E2 proteins.

[0070] Figure 3 The E2 binding activity assay results for OsUBC1 (A) from subfamily I and OsUBC4 (B), OsUBC5 (C), and OsUBC6 (D) from subfamily II.

[0071] Figure 4 The E2 binding activity assay results of OsUBC7 (A), OsUBC8 (B), OsUBC9 (C) from subfamily III and OsUBC10 (D) from subfamily IV.

[0072] Figure 5 The E2 binding activity assay results for OsUBC11 (A) from subfamily V and OsUBC13 (B), OsUBC14 (C), and OsUBC16 (D) from subfamily VI.

[0073] Figure 6 The E2 binding activity assay results of OsUBC18 (A), OsUBC19 (B), OsUBC22 (C), and OsUBC23 (D) from subfamily VI.

[0074] Figure 7 The results of the E2 binding activity experiments of OsUBC25 (A) and OsUBC26 (B) from subfamily VII.

[0075] Figure 8 The E2 binding activity assay results of OsUBC28 (A), OsUBC29 (B), OsUBC30 (C), and OsUBC31 (D) from subfamily VIII.

[0076] Figure 9 The results of E2 binding activity experiments of OsUBC32 (A) from subfamily IX, OsUBC44 (B) from subfamily XII, OsUBC46 (C) from subfamily XIV, and OsUBC47 (D) from subfamily XV.

[0077] In summary, Figure 3-9 All OsUBC proteins in the system can form E2-Ub conjugates with Ub proteins in the system, which shows that all OsUBC proteins contained in the present invention are active.

[0078] Figure 10 SCF derived from transgenic callus D3-GFP Purification and quantification of E3 ligase. SCF purified from transgenic rice Act:D3-GFP / d3 callus D3-GFP Detection and quantification of E3 ligase. Arrows point to D3-GFP and GFP proteins, respectively. BSA is used as a standard to calibrate the target protein content.

[0079] Figure 11 HsUbcH5C and HsUbcH7 interact with SCF D3 The experimental results of the specific cooperative working efficiency of E3 ligase. B is the detection of HsUbcH5C and SCF at different temperatures. D3 The efficiency of E3 ligase-specific cooperation.

[0080] Figure 12 Amino acid sequence alignment of human and rice E2 homologous proteins. A shows the amino acid sequence alignment of OsUBC14, OsUBC16, and OsUBC23, which are highly homologous to HsUbcH5C and HsUbcH7 in rice. B shows the amino acid sequence alignment of OsUBC11, which is highly homologous to HsCDC34 in rice.

[0081] Figure 13 To identify SCF in rice D3 E3 ligase specifically works with E2. A is the detection of OsUBC11, OsUBC14 and OsUBC16 with SCF D3 Efficiency of collaborative work of E3 ligases; B is the detection of OsUBC16 and OsUBC23 with SCF D3 The efficiency of E3 ligases working together.

[0082] Figure 14 SCF derived from transgenic callus GID2-GFP Purification and quantification of E3 ligases. SCF purified from transgenic rice UBI:GID2-GFP / NP calli GID2-GFP Quantitative detection of E3 ligase. Arrows point to GID2-GFP and GFP proteins, respectively. BSA is used as a standard to calibrate the target protein content.

[0083] Figure 15 To identify the E2 protein from rice and SCF GID2-GFP Efficiency of E3 ligase cooperation. A is the detection of OsUBC13, OsUBC14 and OsUBC18 respectively with SCF GID2-GFP Efficiency of collaborative work of E3 ligases; B is the detection of OsUBC16, OsUBC19 and OsUBC23 respectively with SCFGID2-GFP Efficiency of collaborative work of E3 ligases; C is the detection of OsUBC16, OsUBC18 and OsUBC22 with SCF GID2-GFP The efficiency of E3 ligase cooperation; the results of experiments A, B and C show that OsUBC18 and SCF GID2-GFP E3 ligases are most efficient when they work together. DETAILED DESCRIPTION

[0084] 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.

[0085] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0086] Example 1, Screening and Identification and SCF D3 E3 ligases specifically and efficiently work with E2

[0087] The present invention relates to 26 ubiquitin-binding proteins from 12 subfamilies of rice, including OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC14 protein, OsUBC16 protein, OsUBC18 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC25 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUBC32 protein, OsUBC44 protein, OsUBC46 protein and OsUBC47 protein. The corresponding encoding gene accession number and protein molecular weight information are shown in Table 1 (ubiquitin protein conjugating enzyme gene information and subtype classification based on bioinformatics are recorded in "Bae, H., and Kim, WT (2014). Classification and interaction modes of 40rice E2 ubiquitin-conjugating enzymes with 17rice ARM-U-box E3ubiquitin ligases. Biochem. Biophys. Res. Commun. 444, 575-580.").

[0088] Table 1. Gene numbers and protein molecular weight information of 26 ubiquitin-binding protein encoding genes of the present invention

[0089]

[0090]

[0091] Note: The gene accession numbers in the table are all from the database "IRGSP (http: / / rice.plantbiology.msu.edu / )".

[0092] 1. Identification of the activity of 26 ubiquitin-binding proteins (OsUBCs) from rice

[0093] 1. Construction of OsUBC protein overexpression vector

[0094] The OsUBC1 gene sequence was amplified using the primer combination GWOSUBC1F and GWOSUBC1R (Table 2) using cDNA from the stem base of Nipponbare rice seedlings as a template. The amplified OsUBC1 gene fragment was then homologously recombined into the intermediate vector pDONR221 using Gateway BP clonase (Invitrogen, Catalog No. P / N 56481). The OsUBC1 gene fragment was then recombined from the intermediate vector pDONR221 into the final protein expression vector pET-61-DEST (Novagen, Catalog No. 71852) using Gateway LR clonase (Invitrogen, Catalog No. P / N 56484). Sequencing confirmed the correctness of the fragment, resulting in the overexpression plasmid pET-61-DEST-OsUBC1. The pET-61-DEST vector contains a His tag at the N-terminus.

[0095] The protein overexpression plasmids pET-61-DEST-OsUBC4, pET-61-DEST-OsUBC5, pET-61-DEST-OsUBC6, pET-61-DEST-OsUBC7, pET-61-DEST-OsUBC8, pET-61-DEST-OsUBC9, pET-61-DEST-OsUBC10, pET-61-DEST-OsUBC11, pET-61-DEST-OsUBC13, pET-61-DEST-OsUBC14, pET-61-DEST-OsUBC16, pET-61-DEST-OsUBC18, and pET- 61-DEST-OsUBC19, pET-61-DEST-OsUBC22, pET-61-DEST-OsUBC23, pET-61-DEST-OsUBC25, pET-61-DEST-OsUBC26, pET-61-DEST-OsUBC28, pET-61-DEST- OsUBC29, pET-61-DEST-OsUBC30, pET-61-DEST-OsUBC31, pET-61-DEST-OsUBC32, pET-61-DEST-OsUBC44, pET-61-DEST-OsUBC46, pET-61-DEST-OsUBC47.

[0096] Protein expression plasmids pET-55-DEST-OsE1, pET-60-DEST-OsUb, and pET-61-DEST-OsUb were cloned using the same method. The pET-55-DEST vector (Novagen, Catalog No. 71846) has a Strep tag at the N-terminus and a His tag at the C-terminus, the pET-60-DEST vector (Novagen, Catalog No. 71851) has a GST tag at the N-terminus, and the pET-61-DEST vector (Novagen, Catalog No. 71852) has a His tag at the N-terminus.

[0097] The accession numbers of the genes encoding the ubiquitin-binding proteins, ubiquitin proteins Ub or E1 involved in each recombinant expression vector are shown in Table 1.

[0098] Table 2. Primers for amplifying protein-coding genes in the present invention

[0099]

[0100]

[0101]

[0102] 2. Expression, purification and activity identification of OsUBC, OsUb and OsE1 proteins

[0103] (A1) The expression plasmids of the ubiquitin-binding protein OsUBCs fused with a His tag, the expression plasmid of the ubiquitin protein OsUb fused with a His tag or a GST tag, and the expression plasmid of the ubiquitin-activating enzyme OsE1 fused with a Strep tag and a His tag obtained in step 1 were transformed into the Escherichia coli protein expression strain BL21(DE3);

[0104] (A2) Single colonies were picked and cultured in 10 mL of LB liquid medium (formula: 10 g / L Tryptone, 5 g / L yeast extract, 10 g / L NaCl) at 37°C overnight for bacterial activation.

[0105] (A3) The activated bacterial culture was expanded at a ratio of 1:50 or 1:100 and cultured at 37°C until the OD600 reached 0.8. Then, IPTG (Sigma, Catalog No. I5502) was added to a final concentration of 0.3 mM and incubated at 16°C and 180 rpm for protein induction. Protein induction typically lasted 16-20 hours.

[0106] (A4) Bacteria were harvested by centrifugation and resuspended in His-tagged protein extraction buffer (formula: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM NaF, 20 mM imidazole (Sigma, Cat. No. 56748), 10% (v / v) glycerol, 0.5% (v / v) Nonidet P-40, plus the following components immediately before use: 1 mM PMSF, 1 mM DTT) or GST-tagged protein extraction buffer (formula: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM NaF, 2 mM EDTA (pH 8.0), 10% (v / v) glycerol, 0.5% (v / v) Nonidet P-40, plus the following components immediately before use: 1 mM PMSF, 1 mM DTT);

[0107] (A5) Cell disruption: Cells were disrupted using a high-pressure cell disruptor (JNBIO, model: JN-3000Plus), followed by centrifugation at 4°C, 15,000 g for 20 min, and the protein supernatant was collected.

[0108] (A6) Protein supernatant was purified using Ni Sepharose 6 Fast Flow medium (GE Health, Catalog No.: 17-5318-01) or glutathione sepharose resin medium (GE Health, Catalog No.: 17-5132-01) according to the manufacturer's instructions;

[0109] (A7) The purified protein was tested for purity and quantity by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, then aliquoted and frozen at -80°C until use.

[0110] The purified OsUBC protein was quantified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Figure 1 As shown in A, B, C and D, from Figure 1 It can be seen that the bands of each OsUBC protein are relatively simple, indicating that the purified OsUBC protein is of high purity. The results of quantification of OsUBC protein based on BSA gradient bands are shown in Table 3. The purified His-OsUb protein, GST-OsUb protein and Strep-OsE1-His protein (hereinafter referred to as Strep-OsE1 protein) were quantified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The results are shown in Table 3. Figure 2 As shown in A, B and C, from Figure 2It can be seen that the His-OsUb protein, GST-OsUb protein and Strep-OsE1-His protein have single bands and high purity, and the results of quantification based on the BSA gradient band are shown in Table 3.

[0111] Table 3. Protein concentration information of OsUBC protein, OsUb protein and OsE1 protein

[0112]

[0113]

[0114] Typically, active ubiquitin-binding proteins form disulfide bonds with ubiquitin molecules to form ubiquitin-binding protein-ubiquitin conjugates (UBC-ubiquitin conjugates) to complete the presentation of ubiquitin during the ubiquitination modification process. Purified OsUBC protein is analyzed for ubiquitin binding activity according to the following steps (B1)-(B4):

[0115] (B1) The activity analysis system of rice ubiquitin-binding proteins (OsUBCs) was 30 μL, and the reaction system components included: 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM ATP, 50 ng OsE1 (i.e., Strep-OsE1 protein), 200 ng of OsUBC1, OsUBC4, OsUBC5, OsUBC6, OsUBC7, OsUBC8, OsUBC9, OsUBC10, OsUBC11, OsUBC13, OsUBC14, OsUBC16, OsUBC18, OsUBC19, OsUBC22, OsUBC23, OsUBC25, OsUBC26, OsUBC28, OsUBC29, OsUBC30, OsUBC31, OsUBC32, OsUBC44, OsUBC46, or OsUBC47, and 2 μg of ubiquitin protein (i.e., His-OsUb or GST-OsUb), with or without 2 mM DTT.

[0116] (B2) To ensure consistent start times for all parallel reactions in the ubiquitin-binding protein activity assay, the order of sample addition for the activity assay was as follows: OsUBC, ubiquitin protein, OsE1, and 20× reaction buffer (formula: 1 M Tris (pH 7.4), 200 mM MgCl2, 100 mM ATP, with or without 40 mM DTT).

[0117] (B3) The reaction conditions for the ubiquitin-binding protein activation reaction were 28°C and the reaction time was 30 min. After the reaction was completed, 6× SDS sample loading buffer (formula: 0.25 M Tris (pH 8.0), 30% (v / v) Glycerol, 10% (w / v) SDS, with or without 0.6 M DTT) was added to terminate the reaction. The 6× SDS sample loading buffer with or without DTT used in this step should be consistent with that used in step (2).

[0118] (B4) Subject the active reaction sample to SDS-PAGE electrophoresis, transfer the membrane to a NC membrane using a semi-dry transfer apparatus, incubate with an anti-ubiquitin antibody (mouse, Cell Signaling, Catalog No. 3936S) and an anti-mouse-HRP antibody (HRP-conjugated secondary antibody, GE Health, Catalog No. NA931V), and develop the membrane. If the experimental results show that an OsUBC-Ub-sized band appears in the active reaction sample without the addition of DTT, but disappears after the addition of DTT, it indicates that the test protein has ubiquitin-conjugating enzyme activity.

[0119] The results showed that OsUBC1( Figure 3 A)、OsUBC4 / 5 / 6( Figure 3 B, C and D), OsUBC7 / 8 / 9( Figure 4 A, B and C), OsUBC10( Figure 4 D), OsUBC11( Figure 5 A)、OsUBC13 / 14 / 16( Figure 5 B, C and D), OsUBC18 / 19 / 22 / 23( Figure 6 A, B, C and D), OsUBC25 / 26( Figure 7 A and B), OsUBC28 / 29 / 30 / 31( Figure 8 A, B, C and D), OsUBC32( Figure 9 A)、OsUBC44( Figure 9 Middle B), OsUBC46( Figure 9 C) and OsUBC47( Figure 9 (D) can form ubiquitin-binding protein-ubiquitin protein conjugates (UBC-Ub conjugates) through disulfide bonds, and these conjugates are all DTT-sensitive.

[0120] In summary, all 26 ubiquitin-binding proteins from rice have ubiquitin-binding enzyme activity.

[0121] 2. Screening and Identification of Rice SCFD3 E3 ligases efficiently work in concert with E2

[0122] 1. Obtaining Rice SCF D3 E3 ligase complex

[0123] The transgenic callus involved in this invention is obtained through Agrobacterium-mediated gene transformation. The experimental material used in this case is transgenic callus overexpressing an F-box protein fused to a GFP tag. However, the source of experimental materials is not limited to transgenic callus expression systems. Other sources include plant bioreactors such as tobacco transient expression systems, protoplast transient expression systems, and wheat germ in vitro translation systems. The fusion protein tag can be, but is not limited to, a GFP tag, a Flag tag, an HA tag, or a myc tag.

[0124] (C1) 10 g of transgenic rice Act:D3-GFP / d3 and Act:GFP callus tissues (described in “Jiang, L., Liu, X., Xiong, G., Liu, H., Chen, F., Wang, L., Meng, X., Liu, G., Yu, H., Yuan, Y., et al. (2013). DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504: 401-405.”, wherein the transgenic rice Act:D3-GFP / d3 material is referred to as “transgenic plants of D3-GFP / d3” in the literature) were collected, the collected callus tissues were quick-frozen in liquid nitrogen, and then the quick-frozen callus tissues were ground in liquid nitrogen.

[0125] (C2) Protein extraction was performed by adding 2 mL of plant tissue protein lysis buffer (formula: 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM NaF, 2 mM EDTA (pH 8.0), 1 mM sodium orthovanadate, 10% (v / v) glycerol, 0.5% (v / v) Nonidet P-40, and the following components added immediately before use: 1× complete protease inhibitor cocktail, 1 mM PMSF, and 1 mM DTT) per 1 g of ground callus. During protein extraction, the protein extract was incubated on ice for 30 min, shaken every 5 min, and centrifuged at 15,000 g for 20 min at 4°C.

[0126] (C3) The supernatant protein solution after centrifugation was transferred to a new 50 mL centrifuge tube, 25 μL of GFP Trap_Agarose (Chromotek, catalog number: gta-20) was added, and the tube was incubated at 4°C and 10 rpm for 2 h.

[0127] (C4) Centrifuge at 4°C and 2,000g for 30 seconds, remove the protein supernatant, add 10 times the volume of plant tissue protein extract as that of GFP Trap Agarose to wash, and after four washes, add a volume of plant tissue protein extract equal to the volume of GFP Trap Agarose to obtain the purified protein complex; the resulting D3-GFP agarose gel and GFP agarose gel are detected and quantified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, and the agarose gel is stored at 4°C and used as soon as possible; here, D3-GFP agarose gel and GFP agarose gel refer to GFP Trap Agarose incubated with Act:D3-GFP / d3 transgenic callus extract and Act:GFP transgenic callus extract, respectively.

[0128] After steps (C1)-(C4), the SCF obtained from the transgenic callus is purified D3-GFP The complex (D3-GFP agarose gel obtained in step (C4)) was subjected to SDS-PAGE electrophoresis for quantitative analysis (quantification based on D3-GFP content), as shown in FIG. Figure 10 As shown, the obtained SCF was analyzed based on the BSA gradient band and D3-GFP protein band. D3-GFP The complex was roughly quantified.

[0129] 2. Preliminary screening can be combined with SCF D3 E3 ligases work efficiently with human E2

[0130] The specific steps are as follows:

[0131] (D1) The E2-E3 specificity screening activity analysis experimental system is 30 μL, and the reaction system components include: 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM ATP, 2 mM DTT, 50 ng HsE1 (Biovision, Catalog No.: 6429), 200 ng of the E2 to be tested, 2 μg of ubiquitin protein (Boston Biochem, Catalog No.: U-110), 200 ng of SCF D3-GFP complex (based on D3-GFP content) or 200 ng GFP (as a negative control).

[0132] (D2) To ensure consistent start times for the E2-E3 specificity screening activity assay, the order of sample loading for the activity assay is as follows: E2 to be tested, SCF complex (or GFP), ubiquitin protein, E1, and 20× reaction buffer (formula: 1 M Tris (pH 7.4), 200 mM MgCl2, 100 mM ATP, 40 mM DTT).

[0133] (D3) E2-E3 specific activity screening reaction was performed at 28°C, shaking at 950 rpm, and reaction time of 2 h. After completion of the reaction, 6× SDS sample loading buffer was added to terminate the reaction.

[0134] (D4) The active reaction sample was subjected to SDS-PAGE electrophoresis and transferred to a NC membrane using a semi-dry transfer apparatus. The membrane was then incubated with an anti-ubiquitin antibody (mouse, Cell Signaling, Catalog No. 3936S) and an anti-mouse-HRP antibody (HRP-labeled secondary antibody, GE Health, Catalog No. NA931V), and developed. If the experimental results show continuous ubiquitin signals of varying molecular weights, i.e., polyubiquitin chain signals, this indicates that the ubiquitin-conjugating enzyme E2 to be tested is capable of synergistically working with the plant multi-subunit SCF E3 ligase to form polyubiquitin chains.

[0135] First, we tested whether the commercial HsUbcH5c protein (Bioton Biochem, Catalog No.: E2-627) and HsUbcH7 protein (Bioton Biochem, Catalog No.: E2-642) could bind to SCF. D3-GFP E3 ligases work together. HsUbcH5C and HsUbcH7 can bind to SCF Fbxl18E3 ligases work together (documented in "Petroski, MD & Deshaies, RJ (2005) In vitro reconstitution of SCF substrate ubiquitination with purified proteins. Method Enzymol. 398, 143-158.", "Skowyra, D., Craig, KL, Tyers, M., Elledge, SJ & Harper, JW (1997) F-box proteins are receptors that recruitphosphorylated substrates to the SCF ubiquitin-ligase complex.Cell91,209-219.","Deffenbaugh,AEet al.(2003)Release of ubiquitin-charged Cdc34-S~ubfrom the RING domain is essential forubiquitination of the SCF Cdc4 -boundsubstrate Sic1.Cell114,611-622." and "Liu,Y.et al.(2015).F-box protein Fbxl18mediates polyubiquitylation and proteasomal degradation of the pro-apoptoticSCF subunit Fbxl7.Cell Death Dis.6,e1630" in four articles).

[0136] The experimental results showed that HsUbcH5C as E2 can efficiently bind to SCF D3-GFP E3 ligases work together to form polyubiquitin chains, but HsUbcH7 cannot bind to SCF D3-GFP E3 ligases work together ( Figure 11 In order to further determine the optimal reaction temperature for the ubiquitination activity system, the present invention simultaneously tested three reaction temperature conditions: 22°C, 28°C, and 37°C (corresponding to the reaction temperature of step (D3)). The experimental results showed that HsUbcH5C could react with SCF at all temperatures, regardless of whether it was 22°C, 28°C, or 37°C. D3 E3 ligases work together efficiently ( Figure 11 B); at 37°C, human HsUbcH5C and SCFD3 The E3 ligase cooperative working efficiency was slightly higher at 22°C and 28°C. Because 28°C is the closest to the optimal growth temperature of rice, we chose 28°C as the reaction temperature for the activity analysis system in all subsequent activity analysis experiments.

[0137] 3. Screening Energy and SCF D3 E3 ligases efficiently work with rice-derived E2

[0138] This case is based on SCF in the rice strigolactone signaling pathway. D3 E3 ligase is used as an example to demonstrate screening and identification with SCF D3 A method system for the ubiquitin-binding enzyme E2 that works in conjunction with E3 ligase; however, the application of this method system is not limited to SCF. D3 E3 ligases can also be used to screen and identify ubiquitin-conjugating enzymes (E2s) that work synergistically with any SCF E3 ligase. For the specific steps, refer to (D1)-(D4) in Step 2, except that OsUBC11, OsUBC14, OsUBC16, and OsUBC23 obtained in Step 1 are used as the E2s to be tested.

[0139] To further identify suitable rice-derived E2s for in vitro recombinant activity, we determined through homologous sequence alignment that the orthologous proteins of HsUbcH5C and HsUbcH7 in rice are OsUBC14, OsUBC16, and OsUBC23. The gene accession numbers encoding the rice-derived OsUBC14, OsUBC16, and OsUBC23 proteins are shown in Table 1. Amino acid sequence homology alignment of human HsUbcH5C and HsUbcH7 with rice OsUBC14, OsUBC16, and OsUBC23 revealed a high degree of amino acid sequence homology ( Figure 12 The homologous protein of human HsCDC34 in rice is OsUBC11. The gene accession number encoding OsUBC11 protein is shown in Table 1. The amino acid sequence homology comparison between human HsCDC34 and rice OsUBC11 showed that they have a high degree of amino acid sequence homology ( Figure 12 Middle B).

[0140] Next, OsUBC11, OsUBC14, OsUBC16 and OsUBC23 ( Figure 1 B) and the SCF obtained in step 2.1 D3-GFP The specific activity analysis of E3 ligases showed that OsUBC14 can bind to SCF with the same efficiency as HsUbcH5C. D3-GFPE3 ligases work together to form polyubiquitin chains ( Figure 13 A), while OsUBC16 and OsUBC23 can interact with SCF D3 E3 ligases work synergistically, but with low efficiency ( Figure 13 A and B); OsUBC11 cannot interact with SCF D3 E3 ligases work together efficiently ( Figure 13 Thus, OsUBC14 can act as E2 and interact with SCF. D3 E3 ligases work together efficiently.

[0141] Example 2, Screening and Identification and SCF GID2 E3 ligases specifically and efficiently work with E2

[0142] 1. Obtaining Rice SCF GID2 E3 ligase complex

[0143] Referring to step 1 of Example 1, primer combination GWGID2F and GWGID2R (Table 2) was used to amplify the OsGID2 gene sequence using cDNA from the stem base of Nipponbare rice seedlings as a template. The GID2 gene fragment was then recombined into the intermediate pDONR221 vector to obtain the pDONR221-OsGID2 plasmid. The GID2 gene fragment was then recombined into the terminal vector pMHb7Fm21GW-UBIL (VIB-UGent Center for Plant Systems Biology, Catalog No.: Vector ID: 3_64; this vector is a binary vector with a C-terminal GFP tag) to obtain the pMHb7Fm21GW-UBIL-GID2 plasmid. The pMHb7Fm21GW-UBIL-GID2 plasmid was transformed into the Agrobacterium EHA109 competent cell strain, and then the Agrobacterium containing the pMHb7Fm21GW-UBIL-GID2 plasmid was used to infect and transform rice callus tissue, thereby obtaining UBI:GID2-GFP transgenic callus tissue material.

[0144] Referring to step 21 of Example 1, after steps (C1) to (C4), the SCF purified from the transgenic rice Ubi:GID2-GFP callus (wherein the gene accession number encoding the GID2 protein is AB100246.1) GID2-GFP The complex was quantitatively analyzed by SDS-PAGE electrophoresis (SCF GID2-GFP The quantification of the complex is mainly based on the content of GID2-GFP protein) Figure 14 ).

[0145] 2. Screening and Identification of Rice SCFGID2 E3 ligases efficiently work in concert with E2

[0146] Because with SCF D3 OsUBC14, an E3 ligase that works efficiently in collaboration with other SCF E3 ligases, is from subfamily VI. When screening for E2s that work efficiently in collaboration with other SCF E3 ligases, we prioritized screening for E2 proteins from subfamily VI. The 26 OsUBC proteins cloned in step 1 of Example 1 of the present invention include 7 OsUBC proteins from subfamily VI: OsUBC13, OsUBC14, OsUBC16, OsUBC18, OsUBC19, OsUBC22, and OsUBC23. The gene accession numbers encoding the OsUBC proteins are shown in Table 1.

[0147] Next, OsUBC13, OsUBC14, OsUBC16, OsUBC18, OsUBC19, OsUBC22 and OsUBC23 ( Figure 1 B and C) were respectively mixed with the SCF obtained in step 1 GID2-GFP The specific activity of E3 ligase was analyzed. The specific steps are shown in (D1)-(D4) in steps 2 and 3 of Example 1. The experimental results showed that OsUBC13, OsUBC14, OsUBC16, OsUBC18, OsUBC19, OsUBC22 and OsUBC23 all have SCF GID2-GFP The activity of E3 ligases working together ( Figure 15 A, B and C), further activity assays revealed that OsUBC18 interacted with SCF GID2 E3 ligases work together to achieve the highest activity ( Figure 15 Therefore, OsUBC18 can act as E2 and interact with SCF GID2 E3 ligases work together efficiently.

[0148] 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 method for identifying whether a target ubiquitin-conjugating enzyme E2 can cooperate with a plant multi-subunit SCF E3 ligase to form polyubiquitin chains, comprising the following steps: (A1) preparing a reaction system; the reaction system contains the ubiquitin-conjugating enzyme E2 to be tested, plant multi-subunit SCF E3 ligase, ubiquitin-activating enzyme E1 and ubiquitin protein; (A2) Place the reaction system prepared in (A1) at 22-37°C and shake for 1-2 hours; (A3) detecting the system after the reaction in (A2) to determine whether the ubiquitin-conjugating enzyme E2 to be tested can cooperate with the plant multi-subunit SCF E3 ligase to form a polyubiquitin chain; The plant multi-subunit SCF E3 ligase is prepared according to a method comprising the following steps: (B1) fusing the gene encoding the F-box protein in the plant multi-subunit SCF E3 ligase complex with a protein tag coding sequence and introducing the resulting sequence into a recipient plant to obtain a transgenic plant; (B2) Grind the tissue of the transgenic plant obtained in (B1) in liquid nitrogen; (B3) extracting total protein from the ground powder obtained in (B2); (B4) using a substance that can specifically bind to the protein tag to separate the protein complex obtained from the total protein obtained in (B3), which is the plant multi-subunit SCF E3 ligase; The plant multi-subunit SCF E3 ligase is the SCF in the rice strigolactone signaling pathway D3 E3 ligase or SCF in the rice gibberellin signaling pathway GID2 E3 ligase; When the plant multi-subunit SCF E3 ligase is the SCF D3 In the case of E3 ligase, the F-box protein is D3 protein; Or, when the plant multi-subunit SCF E3 ligase is the SCF GID2 E3 ligase, the F-box protein is GID2 protein.

2. The method according to claim 1, wherein: The reaction system also contains a reaction buffer; the reaction buffer contains Tris, MgCl2, ATP, and DTT.

3. The method according to claim 2, wherein: The reaction system is composed of: 50 mM Tris pH 7.4, 10 mM MgCl2, 5 mM ATP, 2 mM DTT, 1.67 mg / L ubiquitin activating enzyme E1, 6.67 mg / L of the ubiquitin conjugating enzyme E2 to be tested, 66.67 mg / L of ubiquitin protein Ubiquitin, and 6.67 mg / L of the plant multi-subunit SCF E3 ligase; the content of the plant multi-subunit SCF E3 ligase is calculated based on the amount of F-box protein therein; and / or The order of adding the components in the reaction system is: the ubiquitin-binding enzyme E2 to be tested, the plant multi-subunit SCFE3 ligase, the ubiquitin protein Ubiquitin, the ubiquitin activating enzyme E1, and the reaction buffer.

4. The method according to any one of claims 1 to 3, wherein: In the above (A2), the reaction temperature is 28° C.; and / or the shaking condition is 950 rpm; and / or the reaction time is 2 h.

5. A method for preparing a plant multi-subunit SCF E3 ligase, comprising the following steps: (B1) fusing the gene encoding the F-box protein in the plant multi-subunit SCF E3 ligase complex with a protein tag coding sequence and introducing the resulting sequence into a recipient plant to obtain a transgenic plant; (B2) Grind the tissue of the transgenic plant obtained in (B1) in liquid nitrogen; (B3) extracting total protein from the ground powder obtained in (B2); (B4) using a substance that can specifically bind to the protein tag to separate the protein complex obtained from the total protein obtained in (B3), which is the plant multi-subunit SCF E3 ligase; The plant multi-subunit SCF E3 ligase is the SCF in the rice strigolactone signaling pathway D3 E3 ligase or SCF in the rice gibberellin signaling pathway GID2 E3 ligase; When the plant multi-subunit SCF E3 ligase is the SCF D3 In the case of E3 ligase, the F-box protein is D3 protein; Or, when the plant multi-subunit SCF E3 ligase is the SCF GID2 E3 ligase, the F-box protein is GID2 protein.

6. A kit for screening for a ubiquitin-conjugating enzyme that cooperates with a plant multi-subunit SCF E3 ligase to form polyubiquitin chains, comprising the plant multi-subunit SCF E3 ligase of claim 1, ubiquitin activating enzyme E1, ubiquitin protein, and the reaction buffer of claim 2 or 3.

7. The kit according to claim 6, wherein: The kit also contains the following proteins: OsUBC1 protein, OsUBC4 protein, OsUBC5 protein, OsUBC6 protein, OsUBC7 protein, OsUBC8 protein, OsUBC9 protein, OsUBC10 protein, OsUBC11 protein, OsUBC13 protein, OsUBC19 protein, OsUBC22 protein, OsUBC23 protein, OsUBC26 protein, OsUBC28 protein, OsUBC29 protein, OsUBC30 protein, OsUBC31 protein, OsUBC32 protein, OsUBC44 protein, OsUBC46 protein, and OsUBC47 protein.

8. Use of the kit according to claim 6 in screening for ubiquitin-conjugating enzymes that can cooperate with plant multi-subunit SCF E3 ligases to form polyubiquitin chains.

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