Rice strigolactones receptor OsD14 mutant protein, related biological material and application of rice strigolactones receptor OsD14 mutant protein

Abstract

The invention discloses a rice strigolactone receptor OsD14 mutant protein, a related biological material and application of the rice strigolactone receptor OsD14 mutant protein, and belongs to the technical field of genetic breeding. The mutation type of the rice strigolactone receptor OsD14 mutant protein is any one or three of P1)-P3), and other amino acids in SEQ ID No.1 are kept unchanged. The invention also discloses a preparation method of the rice strigolactone receptor OsD14 mutant protein. P1) the 221 amino acid of the SEQ ID No.1 is mutated from proline to aspartic acid, P2) the 224 amino acid of the SEQ ID No.1 is mutated from glutamic acid to alanine, and P3) the 227 amino acid of the SEQ ID No.1 is mutated from arginine to alanine. Experiments prove that the rice tillering related mutant protein provided by the invention can cause that rice is insensitive to strigolactones signal response, so that the tillering number of the rice is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and in particular relates to a rice strigolactone receptor OsD14 mutant protein, related biomaterials and applications thereof. Background Art

[0002] The three key factors of rice yield include the number of effective panicles, the number of grains per panicle, and the thousand-grain weight. The number of effective panicles is also called the number of effective tillers, which is a specialized plant branch of rice. As a newly discovered hormone that regulates plant branching, strigolactones are also involved in regulating various biological processes such as plant height, branching angle, root morphology, and plant adaptation to drought and phosphorus deficiency. In recent years, it has been discovered that strigolactones also play an important role in the breeding of modern high-yield rice varieties. In the super rice restorer material "Huazhan", the weak allelic mutation HTD1 of the strigolactone synthesis gene HTD1 / D17 is HZ It can effectively increase the number of rice tillers without affecting the fruit setting rate, leading to an increase in biomass and yield, indicating that it is possible to achieve the goal of improving rice plant type by finely regulating the function of strigolactone.

[0003] In rice strigolactone signal transduction, receptor D14 senses strigolactone, forms a complex with F-box protein D3 and inhibitory protein D53, ubiquitinates and degrades transcription inhibitory protein D53 to initiate the strigolactone signaling pathway. At present, structural biologists have proposed a variety of strigolactone signaling perception models based on the crystal structures of D14, D3 and D14-D3 complex. The specific mechanism of how D14 senses strigolactone molecules and activates the signaling pathway is still controversial and has unresolved issues, such as the relationship between strigolactone hydrolysis and signal perception, the genetic function of the key amino acid sites required for the formation of the D14-D3-D53 complex, and the termination method of strigolactone signaling perception.

[0004] OsD14 is a signal receptor component of the rice strigolactone pathway, and its main function is to control rice plant shape, including the number of rice tillers and plant height. Using the OsD14 gene to create a rice strigolactone receptor functional mutant to regulate the number of rice tillers, improve the plant shape, and thus increase rice yield will have broad application prospects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a mutant protein that reduces the number of rice tillers. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The protein provided by the present invention is any one of the following:

[0008] A1) is a protein in which the amino acid at position 221 of SEQ ID No. 1 is mutated from proline to aspartic acid, the amino acid at position 224 of SEQ ID No. 1 is mutated from glutamic acid to alanine, the amino acid at position 227 of SEQ ID No. 1 is mutated from arginine to alanine, and the other amino acids of SEQ ID No. 1 are kept unchanged;

[0009] A2) is a protein in which the amino acid at position 221 of SEQ ID No. 1 is mutated from proline to aspartic acid, while the other amino acids of SEQ ID No. 1 remain unchanged;

[0010] A3) is a protein in which the amino acid at position 224 of SEQ ID No. 1 is mutated from glutamic acid to alanine, while the other amino acids of SEQ ID No. 1 remain unchanged;

[0011] A4) is a protein in which the amino acid at position 227 of SEQ ID No. 1 is mutated from arginine to alanine, while the other amino acids of SEQ ID No. 1 remain unchanged.

[0012] Among the above proteins, the nucleotide sequence of the protein described in A1) is SEQ ID No.10, the nucleotide sequence of the protein described in A2) is SEQ ID No.4, the nucleotide sequence of the protein described in A3) is SEQ ID No.6, and the nucleotide sequence of the protein described in A4) is SEQ ID No.8.

[0013] The present invention also provides a biological material related to the above protein, wherein the biological material is any one of the following:

[0014] B1), a nucleic acid molecule encoding any of the aforementioned proteins;

[0015] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0016] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0017] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0018] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0019] B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0020] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0021] In the above-mentioned biological material, the expression cassette containing nucleic acid molecules described in B2) refers to the DNA capable of expressing the above-mentioned protein in the host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecules of any of the above-mentioned proteins or the DNA of the RNA molecules. The regulatory sequence can guide the coding sequence to express the DNA of any of the above-mentioned proteins or the RNA molecules in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce a specific restriction enzyme site of the vector so as to connect the regulatory sequence with the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the DNA of the protein or the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence can be operably connected to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an mRNA non-translated region that is very important for the translation of the host cell. The leader sequence can be operably connected to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the protein, which can guide the DNA encoding the protein or the RNA molecule into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein or the DNA of the RNA molecule into the secretory pathway of the host cell used can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or the RNA molecule according to the growth of the host cell. Examples of regulatory sequences are systems that can turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification.

[0022] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

[0023] In the above biological materials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0024] Among the above biological materials, the transgenic plant cell lines do not include propagation materials.

[0025] The present invention also provides the use of any of the above-mentioned proteins or a substance for regulating the expression of a gene encoding any of the above-mentioned proteins or a substance for regulating the activity and / or content of any of the above-mentioned proteins in any of the following:

[0026] M1) Regulate the number of plant tillers;

[0027] M2) preparing products for regulating the number of tillers of plants;

[0028] M3) Plant breeding.

[0029] The present invention also provides the use of the aforementioned biomaterial in any of the following:

[0030] M1) Regulate the number of plant tillers;

[0031] M2) preparing products for regulating the number of tillers of plants;

[0032] M3) Plant breeding.

[0033] In the above application, the regulation is to upregulate, enhance or increase the expression of the gene encoding the aforementioned protein or the content or activity of the protein.

[0034] In the above application, the plant is any one of the following:

[0035] G1) Monocots;

[0036] G2) Gramineae;

[0037] G3) Oryza plants;

[0038] G4) Rice plants;

[0039] G5) Rice.

[0040] The present invention also provides a method for regulating the tiller number of rice, comprising the following steps: regulating the expression of the coding gene of the above-mentioned protein in the recipient plant or regulating the activity and / or content of the protein to change the tiller number of the recipient plant.

[0041] The method for regulating plant tillering of the present invention can be implemented by transformation or hybridization, selfing and asexual reproduction, or can be obtained by chemical mutagenesis or gene site-directed mutagenesis, so that the altered plant has the nucleotide sequence of the present invention.

[0042] The present invention also provides a method for increasing the tiller number of rice, comprising the following steps: up-regulating or enhancing or increasing the expression of the coding gene of the above-mentioned protein in the recipient plant or regulating the activity and / or content of the protein, so as to increase the tiller number of the recipient plant.

[0043] The present invention also provides a method for preparing rice with increased tiller number, comprising the following steps: up-regulating or enhancing or increasing the expression of the coding gene of the aforementioned protein in a recipient plant or regulating the activity and / or content of the protein to obtain a plant with increased tiller number, wherein the plant with increased tiller number has a greater number of tillers than the recipient plant.

[0044] In the above method, the plant is any one of the following:

[0045] G1) Monocots;

[0046] G2) Gramineae;

[0047] G3) Oryza plants;

[0048] G4) Rice plants;

[0049] G5) Rice.

[0050] The present invention proves through experiments that the rice tillering-related mutant protein provided by the present invention can cause rice to be insensitive to the signal response of strigolactone, thereby increasing the tillering number of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Systematic analysis of the effects of D14 conserved amino acids on D14-D3 and D14-D53 interactions. (A) Flowchart of yeast screening for key sites of D14 interactions with D3 and D53. (B) Establishment of yeast screening system for key sites of rice D14 interactions with D3 and D53. (C) Screening of D14 conserved amino acids. The amino acids of rice D14, petunia DAD2, pea RMS3 and Arabidopsis D14 were aligned using DNAMAN 8.0 software, and conserved amino acids were marked with red fill. The GenBank amino acid sequence numbers from top to bottom are Q10QA5 (D14), AFR68698 (DAD2), AMB61027.1 (RMS3) and Q9SQR3 (AtD14). (D and E) Overview (D) and detailed information (E) of conserved D14 amino acids with different functions in D14-D3 and D14-D53 interactions.

[0052] Figure 2 The wild-type rice (Nipponbare), the OsD14-deficient d14 mutant, and the transgenic lines Act:D14-GFP / d14, Act:D14E224A -GFP / d14 and Act:D14 R227A -GFP / d14 and overexpression of D14 P221D Rice mutant Act:D14 P221D Phenotype (left) and tiller number (right) of -GFP / d14. Scale bar, 20 cm. Data are mean ± SD.

[0053] Figure 3 The left figure shows the degradation of D53 protein in callus tissue. The left figure shows the treatment of rice d14 mutant, Act:D14-GFP / d14 and Act:D14 with 1 μM rac-GR24. P221D -GFP / d14 callus. The right figure shows the changes in the amount of D53 protein and D14-GFP protein after the rice d14 mutant, Act:D14-GFP / d14, and Act:D14 were treated with 1 μM rac-GR24. E224A -GFP / d14 and Act:D14 R227A -GFP / d14 callus after D53 protein and D14-GFP protein changes.

[0054] Figure 4 The wild-type Nipponbare rice (WT), rice d14 mutant, Act:D14-GFP / d14 and Act:D14 3M -GFP / d14 plant phenotype (left) and tiller number (right). D14 3M ,D14 P221D E224A R227A . Scale bar, 20 cm. Data are mean ± SD.

[0055] Figure 5 The degradation of D53 protein in callus tissue. Rice d14 mutant, Act:D14-GFP / d14 and Act:D14 were treated with 1 μM rac-GR24. 3M -GFP / d14 callus after D53 protein changes. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0058] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0059] rac-GR24, a strigolactone analogue, was purchased from Chiralix with the product number CX2388. MG132 (Proteasome inhibitor) was purchased from Sigma-Aldrich with the product number Cat#474790.

[0060] The preparation methods of AHLG vector, pBI221-GFP vector, rice d14 mutant and D53 rabbit polyclonal antibody are described in the non-patent document "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.". In addition, the 35S:D14-GFP plasmid is recorded in the non-patent document "Hu, Q*., He, Y*., Wang, L., Liu, S., Meng, X., Liu, G., Jing, Y., Chen, M., Song, X., Jiang, L., Yu H, Wang B, Li J. (2017). DWARF14, a receptor covalently linked with the active form of strigolactones, undergoes strigolactone-dependent degradation in rice. Front. Plant Sci. 8, 1935.". The public can obtain this biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0061] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation, and two-tailed Student's t-test was used, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns (not significant).

[0062] Example 1: Identification of amino acid sites in D14 that are critical for OsD14-D3 interaction

[0063] 1. Yeast two-hybrid assay

[0064] Experimental method: The plasmid transformation of yeast was based on the experimental process in the DUAL membrane starter kits and was fine-tuned based on the previous experimental experience of the laboratory. The specific operation process is as follows: (1) Streak the Y2HGold yeast strain on YPDA solid medium and incubate at 30°C for 2-3 days until single colonies appear. (2) Pick 3-4 single colonies and place them in 3 mL YPDA liquid medium and culture them at 250 rpm at 30°C for 12-14 h. (3) Inoculate 3 mL of bacterial solution into 30 mL YPDA liquid medium and continue to culture at 250 rpm at 30°C for 3-5 h until OD 600 Reach 0.6-0.8, room temperature 700 g Centrifuge for 5 min. (4) Discard the supernatant and resuspend the precipitate in 30 mL of sterile water at 700 g Centrifuge again for 5 min. (5) After discarding the supernatant, determine the volume of sterile medium to be added to resuspend the yeast cells according to the number of experimental reactions, place the resuspended yeast competent cells on ice and wait for transformation. (6) When preparing yeast competent cells, denature the salmon sperm single-stranded ssDNA. First boil it at 100°C for 5 min and then place it on ice for 5 min. Repeat 2-3 times. (7) Determine the PEG / LiOAc reaction mixture to be configured according to the number of experimental reactions. Each reaction contains 240 μL 50% PEG, 36 μL LiOAc and 25 μL salmon sperm single-stranded ssDNA. (8) Add the corresponding vector, 300 μL PEG / LiOAc reaction mixture and 100 μL yeast competent cells to a sterilized 1.7mL centrifuge tube, mix well and incubate at 42°C for 45 min. (9) Heat at room temperature at 700 g Centrifuge for 5 min to collect the cells, discard the supernatant and resuspend in 200 μL 0.9% NaCl solution, spread evenly on SD-Leu / -Trp solid yeast defect medium, and invert and culture at 30°C for 3-4 days until colonies appear. (10) Pick about 10 single clones from SD-Leu / -Trp solid yeast defect medium and place them in 100 μL 0.9% NaCl solution. At the same time, prepare 10-fold and 100-fold dilution yeast solutions. (11) Take 6 μL of yeast suspension and spread on SD-Leu / -Trp, SD-Leu / -Trp / -His / -Ade containing acetone and 10 μM rac -GR24 SD-Leu / -Trp / -His / -Ade solid yeast defective medium was incubated at 30°C for 3-7 days before photographing and analysis.

[0065] The results are as follows Figure 1As shown in B, rac-GR24 cannot induce the interaction between AD-D14ΔN and BD-D3, nor can it induce the interaction between AD-D14ΔN and BD-D3-OsSKP1, but it can induce the interaction between AD-D14ΔN and BD-OsSKP1-D3. In addition, rac-GR24 can induce the interaction between AD-D14ΔN and BD-D53. Therefore, the present application uses the combination of AD-D14ΔN and BD-OsSKP1-D3, and AD-D14ΔN and BD-D53 to screen the key amino acids of D14.

[0066] 2. Sequence Alignment

[0067] DNAMAN8.0 software was used to compare the sequences and determine the conserved amino acids in the D14 protein sequences of rice, petunia, pea and Arabidopsis.

[0068] The results are as follows Figure 1 As shown in C, there are 165 highly conserved amino acid residues in D14 among the four species.

[0069] 3. Interaction between D14 protein with conservative amino acid mutations and D3 and D53

[0070] The 165 conservative amino acids of D14 were further mutated into alanine (and alanine was mutated into valine), and the mutant D14 genes were constructed into the pGADT7 vector. The yeast two-hybrid system was then used to analyze the interaction between the conservative amino acid mutated D14 protein and D3 and D53.

[0071] The results are as follows Figure 1As shown in Figures 1D and 1E, among the 165 conserved amino acid sites of D14, mutations in 11% of the amino acid sites significantly inhibit the interaction between D14 and D3 but do not affect the interaction between D14 and D53. These are amino acid sites that specifically affect the interaction between D14 and D3. These amino acid sites are used to construct genetic materials to analyze the biological function of the interaction between D14 and D3 in plants; mutations in 13% of the amino acid sites significantly inhibit the interaction between D14 and D53 but do not affect the interaction between D14 and D3. These are amino acid sites that specifically affect the interaction between D14 and D53. These amino acid sites can be used to The results showed that 26% of the amino acid site mutations significantly inhibited the interaction between D14 and D3 and D14 and D53. These amino acid sites may be the key sites for the interaction between D14-D3 and D14-D53, and may also inhibit the interaction between D14-D3 and D14-D53 by affecting the stability of D14 protein, the binding and hydrolysis of D14 to tricholoma lactone, or the conformational changes of D14. 43% of the amino acid site mutations had no significant effect on the interaction between D14 and D3 and D14 and D53. G66A 、D14 G79A 、D14 S147A 、D14 G153A 、D14 G208A 、D14 R227A 、D14 R233A and D14 H297A The mutation sites all significantly inhibited the interaction between D14 and D3 in yeast, among which D14 G66A 、D14 G79A 、D14 S147A and D14 H297A The mutation also inhibited the interaction between D14 and D53 in yeast. These results indicate that the D14 key site identified in the yeast system plays an important role in the plant strigolactone signaling pathway.

[0072] Example 2: Protein function analysis of OsD14 loss-of-function mutants

[0073] 1. Construction of overexpression vectors of OsD14 and OsD14 loss-of-function mutants

[0074] To construct D14 mutant amino acid forms including D14 P211D 、D14 R224A 、D14 R227A and D14 3M The transgenic expression plasmid is firstly prepared by circularizing and amplifying the corresponding primers for introducing mutations with the 35S:D14-GFP plasmid as a template to obtain a template for site-directed mutation of amino acids.

[0075] D14-P211D-F: 5'-GCGCCCACCGCCAGATCCGCGTACCCCGT-3';

[0076] D14-P211D-R: 5'-ACGGGGTACGCGGATCTGGCGGTGGGCGC-3';

[0077] D14-E224A-F: 5'-CGGTGCAGGCATTCAGCCGCACCCTCTTCAAC-3';

[0078] D14-E224A-R: 5'-CGGCTGAATGCCTGCACCGCCGCCGGCACGTC-3';

[0079] D14-R227A-F: 5'-GGAGTTCAGCGCAACCCTCTTCAACATGCGCCCG-3';

[0080] D14-R227A-R: 5'-GAGGGTGCGCTGAACTCCTGCACCGCCG-3'.

[0081] In the above primers, the red bold ones are the amino acids for site-directed mutagenesis.

[0082] Amplification of D14 using D14-P211D-F and D14-P211D-R P211D Gene, D14 was amplified using D14-E224A-F and D14-E224A-R E224A Gene, D14 was amplified using D14-R227A-F and D14-R227A-R R227A Genes were obtained, and D14 gene (nucleotide sequence is SEQ ID No.2, encoding protein with amino acid sequence is SEQ ID No.1), D14P221D gene (nucleotide sequence is SEQ ID No.4, encoding protein with amino acid sequence is SEQ ID No.3), D14E224A gene (nucleotide sequence is SEQ ID No.6, encoding protein with amino acid sequence is SEQ ID No.5) and D14R227A gene (nucleotide sequence is SEQ ID No.8, encoding protein with amino acid sequence is SEQ ID No.7).

[0083] The gene was then amplified using primers AHLG-D14-F and AHLG-D14-R and homologously recombined into the AHLG plasmid.

[0084] The primers for constructing overexpression mutants are as follows:

[0085] AHLG-D14-F: 5'- ACGATAAGCTTGGGCCC ATGCTGCGATCGACGCATC-3′;

[0086] AHLG-D14-R: 5'- ATGGCGGCCGCTCTAGA GTACCGGGCGAGAGCGC-3′;

[0087] Among the above primers, the bold black sequences are primers for amplifying D14 or D14ΔN, and the underlined sequences are used for homologous recombination with the AH LG vector.

[0088] The D14 overexpression vector AHLG-Act:D14-GFP is a recombinant expression vector obtained by replacing the small fragment between the two enzyme recognition sites ApaI and XbaI of the AHLG vector with SEQ ID No.2 while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the fusion protein D14-GFP.

[0089] D14 P211D Overexpression vector AHLG-Act:D14 P211D -GFP is a recombinant overexpression vector obtained by replacing the small fragment between the two enzyme recognition sites of ApaI and XbaI of the AHLG vector with SEQ ID No.4, while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the protein D14 with the amino acid sequence of SEQ ID No.3 P211D , D14 P211D The difference with D14 is that D14 P211D The 211th amino acid of the gene was mutated from proline to aspartic acid, and the other amino acid sequences remained unchanged.

[0090] D14 E224A Overexpression vector AHLG-Act:D14 E224A -GFP is a recombinant overexpression vector obtained by replacing the small fragment between the two enzyme recognition sites Apal and Xbal of the AHLG vector with SEQ ID No.6, while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the protein D14 with the amino acid sequence of SEQ ID No.5 E224A .D14 E224A The difference with D14 is that D14 E224A The 224th amino acid of the gene was mutated from glutamic acid to alanine, and the other amino acid sequences remained unchanged.

[0091] D14 R227A Overexpression vector AHLG-Act:D14 R227A-GFP is a recombinant overexpression vector obtained by replacing the small fragment between the two enzyme recognition sites of Apal and Xbal of the AHLG vector with SEQ ID No.8, while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the protein D14 with the amino acid sequence of SEQ ID No.7 R227A .D14 R227A The difference with D14 is that D14 R227A The 227th amino acid of the gene was changed from arginine to alanine, and the other amino acid sequences remained unchanged.

[0092] 2. Obtaining overexpression plants

[0093] (1) Conversion

[0094] The d14 mutant was used as the rice receptor. After the seeds of the d14 mutant to be transformed were shelled, the surface was disinfected with 70% (v / v) ethanol for 1 minute, and then the seeds were rotated and washed with sodium hypochlorite with an effective concentration of 2.5% (w / v) for 45 minutes. After washing three times with sterile water, the seeds were sown on NB solid culture medium and cultured at 28°C in the dark for two weeks. After callus tissue grew out of the mature embryo scutellum, the callus tissue was cut off and subcultured on a new NB solid culture medium, and transferred to a new NB solid culture medium every 7 days. After 3-4 times, Agrobacterium infection can be carried out.

[0095] 1L LB solid medium composition: Tryptone 10g, Yeast Extract 5g, NaCl 10g, agar 15g.

[0096] 1L LB liquid culture medium composition: Tryptone 10g, Yeast Extract 5g, NaCl 10g.

[0097] Composition of rice transformation solution: Add the following reagents to NB basic medium: Inositol 2g / L, Glutamine 2g / L, Casein hydrolysate 500mg / L, 10% Synperonic PE 10ml / L, Acetosyringone 100μM.

[0098] When the rice callus tissue is growing well, the plant transgenic expression vector (AHLG-Act:D14-GFP vector, AHL G-Act:D14 P211D -GFP vector, AHLG-Act:D14 E224A -GFP vector or AHLG-Act:D14 R227A-GFP vector) were transformed into Agrobacterium EHA105 strain by high voltage electroporation and spread on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin for 2-3 days. 4-5 single clones were selected for each transformation and inoculated into 7 mL LB liquid medium containing kanamycin and 25 mg / L rifampicin, and then cultured overnight at 250 rpm under the condition of parameter setting at 28°C. The bacteria were centrifuged at 3,000 rpm for 5 min at room temperature and resuspended in rice transformation solution for transformation of rice callus.

[0099] (2) Agrobacterium EHA105 infection of rice callus

[0100] The callus tissue in good condition obtained in step (1) was collected, and an appropriate amount of transformation solution was added until the bacterial solution completely submerged the callus tissue, and the callus tissue was placed at room temperature for 20 minutes, with occasional shaking during the period. Then, the transformed callus tissue was taken out, and the excess bacterial solution was absorbed with sterile filter paper. The callus tissue was placed in a 23°C incubator in the dark for 2-3 days to obtain transgenic callus tissue (AHLG-Act:D14-GFP rice callus tissue, AHLG-Act:D14 P211D -GFP rice callus, AHLG-Act:D14 E224A -GFP rice callus or AHLG-Act:D14 R227A -GFP rice callus).

[0101] (3) Differentiation

[0102] The rice callus obtained in step (2) is transferred to a NB solid screening medium containing hygromycin, cultured in the dark at 28°C for 7-10 days, and then subcultured to a new NB solid screening medium containing hygromycin, and 3-4 rounds of hygromycin screening medium are performed. The callus with good growth status is propagated to a differentiation medium for differentiation and regeneration of seedlings, and after culturing at 28°C for about one month, the grown seedlings are transferred to a rooting medium for about 4 weeks for rooting and strengthening, and then transplanted to a greenhouse for about one month before being transferred to the field.

[0103] 3. Identification of positive plants

[0104] The specific method is as follows: (1) Leaves were taken from individual rice plants, and rice genomic DNA was extracted using the CTAB method. The extracted DNA was used as a template, and PCR amplification was performed using AHLG-F and D14-570-R identification primers, respectively. The PCR products were sent to Beijing Ruiboxing Biotechnology Co., Ltd. for Sanger sequencing. After the sequencing results were obtained, the sequences were compared. The plants with the sequences that matched the expected sequences were T0 generation positive plants. 16 T0 generation positive plants transformed with AHLG-Act:D14-GFP vectors (named Act:D14-GFP / d14 plants) and 13 plants transformed with AHLG-Act:D14 were obtained.P211D -GFP vector T0 generation positive individual (named Act:D14 P211D -GFP / d14 plants), 8 plants transformed with AHLG-Act:D14 E224A -GFP vector T0 generation positive individual (named Act:D14 E224A -GFP / d14 plants), 9 plants transformed with AHLG-Act:D14 R227A -GFP vector T0 generation positive individual (named Act:D14 R227A -GF P / d14 plants). (2) Randomly select 8 Act:D14-GFP / d14 and 8 Act:D14 P211D -GFP / d14, 8 strains Act:D14 E224A -GFP / d14 and 8 strains Act:D14 R227A -GFP / d14 T0 plants are propagated and 8 T1 lines are planted. Six plants are randomly collected from each T1 line and can be used to screen homozygous positive plants. (3) The harvested T1 seeds are germinated and 40 germinated seeds are placed on a 0.5% Agar solid medium containing 50 μg / mL hygromycin (Hyg). After 2-3 days, the root growth of the plants is observed. Plants with normal root growth are positive plants, otherwise they are isolated pseudo-plants. If all seeds of each plant can grow roots normally, it is a homozygous positive line.

[0105] The primers for identifying the homozygous mutant rice transgenic positive seedlings with D14 gene mutation were obtained respectively:

[0106] AHLG-F:5'-TCAGCATTGTTCATCGGTAG-3';

[0107] D14-570-R:5'-CTGCTGTATCCTCCTCCAGCTC-3'.

[0108] PCR reaction system: 1μL DNA template, 2μL 10μM Primer F, 2μL 10μM Primer R and 45μL Gold Mix. Reaction procedure: 98℃ pre-denaturation for 3min; 35 cycles of 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 15s; 72℃ extension for 5min.

[0109] Results A total of 6 Act:D14-GFP / d14 homozygous positive strains and 6 Act:D14 P211D -GFP / d14 homozygous positive single strain, 5 Act:D14 strains E224A -GFP / d14 homozygous positive single strain and 5 Act:D14 strains R227A-GFP / d14 homozygous positive individual strain. The above individual strains used in subsequent experiments were named Act:D14-GFP / d14#1, Act:D14-GFP / d14#2, Act:D14 P211D -GFP / d14#1, Act:D14 P211D -GFP / d14#2, Act:D14 E224A -GFP / d14#1, Act:D14 E224A -GFP / d14#2, Act:D14 R227A -GFP / d14#1 and Act:D14 R227A -GFP / d14#2.

[0110] 4. Count the number of tillers of homozygous positive plants

[0111] The experimental method is as follows: wild-type rice Nipponbare, d14 mutant, the above homozygous plants Act:D14-GFP / d14#1, Act:D14-GFP / d14#2, Act:D14 P211D -GFP / d14#1, Act:D14 P211D -GFP / d14#2, Act:D14 E224A -GFP / d14#1, Act:D14 E224A -GFP / d14#2, Act:D14 R227A -GFP / d14#1 and Act:D14 R227A -GFP / d14#2 was planted in the field of the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing. Each strain was planted with 24 plants with a plant spacing and row spacing of 17 cm. The growth period was from May to November each year. The field phenotypic survey time of rice materials was from September 20 to October 7 each year. The number of tillers counted was the number of effective tillers at maturity (i.e. the number of tillers that grew ears), and the plant height was the distance between the highest ear of rice and the ground. Based on the average number of tillers of each material, representative single-plant transplant pots were selected for photography.

[0112] The results showed that overexpression of D14-GFP could completely restore the tiller number of OsD14 loss-of-function mutant d14 to that of the wild type, that is, the tiller number of d14 was significantly reduced. P211D -GFP, D14 E224A -GFP and D14 R227A -GFP weakly reduced the tiller number of OsD14 loss-of-function mutant d14 rice ( Figure 2 ).

[0113] 5. Detection of D53 and D14-GFP protein levels in positive transgenic plants

[0114] The experimental method is as follows: The degradation of D14-GFP protein and D53 protein is detected by Western Blot experiment. The specific method is as follows: (1) Prepare 10% concentration 12-well or 15-well SDS-PAGE protein gel. The amount of protein sample loaded depends on the experimental requirements. Set the voltage to 80-120V for about 1-2h. (2) Use semi-dry transfer method to transfer the protein to PVDF or NC membrane, the voltage is 15-20V, and the transfer time is 30-60min. (3) Prepare 4% skim milk powder in TBST solution during the transfer. After the transfer is completed, place the PVDF or NC membrane in 4% skim milk powder and incubate it at room temperature for 60min on a shaker at 70rpm. (4) Pour out the skim milk powder, rinse with TBST, add new 4% skim milk powder, add the protein antibody to be detected, and incubate for 1-2h or overnight at 4℃. (5) After incubation with the primary antibody, wash the PVDF or NC membrane three times with TBST, each time at 70 rpm for 5 minutes at room temperature, then add 4% skim milk containing the secondary antibody and incubate at room temperature for 60 minutes on a 60 rpm shaker. (6) After incubation with the secondary antibody, wash the membrane three times with TBST, each time at 70 rpm for 5 minutes at room temperature. Add the color developing solution and use a photosensitive film or a protein developing instrument for protein development. GFP mouse monoclonal antibody (Cat#11814460001) was purchased from Roche. The internal reference protein is Actin, purchased from Abmart, Cat#M20009L.

[0115] The results showed that D53 protein could not be degraded in the OsD14 loss-of-function mutant d14 rice, and overexpression of D14-GFP could restore the degradation of D53 in the OsD14 loss-of-function mutant d14 rice. P211D -GFP, D14 E224A -GFP and D14 R227A -GFP cannot restore D53 degradation in the OsD14 loss-of-function mutant d14 rice. Strigolactone induces its receptor D14 to form a complex with D3 and D53, thereby inducing D53 degradation and initiating signal transduction. P211 、D14 E224A and D14 R227A The mutation inhibits the interaction between D14 and D3 and the subsequent degradation of D53 induced by strigolactone, indicating that D14 P211 、D14 E224A and D14 R227A The site is very important for rice strigolactone signal perception ( Figure 3 ).

[0116] Example 3: Analysis of biological significance of OsD14 loss-of-function mutant protein

[0117] 1. Rice Act: D14 3M Preparation and phenotype of -GFP / d14 strain

[0118] D14 P211D,E224A,R227A Overexpression vector AHLG-Act:D14 P211D,E224A,R227A -GFP is a recombinant overexpression vector obtained by replacing the small fragment between the two enzyme recognition sites of ApaI and XbaI of the AHLG vector with SEQ ID No.10, while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses protein D14 with the amino acid sequence of SEQ ID No.9. D14 3M The difference with D14 is that D14 P211D The 211th amino acid of the gene was mutated from proline to aspartic acid, the 224th amino acid was mutated from glutamic acid to alanine, and the 227th amino acid was mutated from arginine to alanine, while the other amino acid sequences remained unchanged.

[0119] According to the method of Example 2, D14 P211D,E224A,R227A (D14 3M ) overexpression vector instead of AHLG-Act:D14-GFP vector (or AHLG-Act:D14 P211D -GFP vector, or AHLG-Act:D14 E224A -GFP vector or AHLG-Act:D14 R227A -GFP) vector, and obtain transfected D14 P211D,E224A,R227A (D14 3M ) homozygous plants overexpressing the vector. The homozygous plants used in subsequent experiments were named Act:D14 3M -GFP / d14#1 and Act:D14 3M -GFP / d14#2.

[0120] Rice Act: D14 3M The phenotypic results of the -GFP / d14 strain showed that D14-GFP could completely restore the tiller number of the d14 mutant to that of the wild type, while overexpression of D14 3M -GFP cannot restore the tillering phenotype of the d14 mutant ( Figure 4 ). This indicates that the simultaneous mutation of the three amino acid sites makes D14 in rice completely unable to sense the strigolactone signal, and thus cannot restore the tillering phenotype of the d14 mutant.

[0121] 2. Detection of D53 protein levels in positive plants

[0122] The D53 protein level was detected by referring to the method in Example 2. The results showed that the P211D E224A R227A triple mutation of D14 (D14 3M ) was able to completely block the effect of the strigolactone analogs on the interaction between rice D14 and D3 ( Figure 5 ).

[0123] In summary, the three key amino acid sites P211, E224 and R227 are necessary for the interaction between D14 and D3 and play an important role in the strigolactone signaling pathway in rice.

[0124] The sequences involved in the above embodiments are:

[0125] Wild-type amino acid of OsD14 gene (SEQ ID No.1)

[0126] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0127] Wild-type CDS nucleotide sequence of OsD14 gene (SEQ ID No. 2)

[0128] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGT CGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0129] SEQ ID No.3OsD14 P211D :

[0130] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPDLFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYADLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*。

[0131] SEQ ID No.4OsD14 P211D :

[0132] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGT CGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCG GAT CTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0133] SEQ ID No.5 OsD14 E224A :

[0134] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPDLFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQAFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*。

[0135] SEQ ID No.6 OsD14 E224A

[0136] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAG GCA TTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0137] SEQ ID No.7 OsD14 R227A :

[0138] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPDLFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSATLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*。

[0139] SEQ ID No.8 OsD14 R227A :

[0140] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGC GCA ACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0141] SEQ ID No.9 OsD14 P211D,E224A,R227A

[0142] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPDLFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYADLAVGADVPAAVQAFSATLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*。

[0143] SEQ ID No.10 OsD14 P211D,E224A,R227A :

[0144] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCG GAT CTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGCATTCAGCGCAACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0145] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protein, characterized in that The protein is any one of the following: A1) is a protein in which the amino acid at position 221 of SEQ ID No. 1 is mutated from proline to aspartic acid, the amino acid at position 224 of SEQ ID No. 1 is mutated from glutamic acid to alanine, the amino acid at position 227 of SEQ ID No. 1 is mutated from arginine to alanine, and the other amino acids of SEQ ID No. 1 are kept unchanged; A2) is a protein in which the amino acid at position 221 of SEQ ID No. 1 is mutated from proline to aspartic acid, while the other amino acids of SEQ ID No. 1 remain unchanged; A3) is a protein in which the amino acid at position 224 of SEQ ID No. 1 is mutated from glutamic acid to alanine, while the other amino acids of SEQ ID No. 1 remain unchanged; A4) is a protein in which the amino acid at position 227 of SEQ ID No. 1 is mutated from arginine to alanine, while the other amino acids of SEQ ID No. 1 remain unchanged.

2. The protein according to claim 1, characterized in that The nucleotide sequence of the protein A1) is SEQ ID No.10, the nucleotide sequence of the protein A2) is SEQ ID No.4, the nucleotide sequence of the protein A3) is SEQ ID No.6, and the nucleotide sequence of the protein A4) is SEQ ID No.

8.

3. Biomaterial, wherein the biomaterial is any one of the following: B1), a nucleic acid molecule encoding the protein according to any one of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

4. Application, characterized in that, Use of any of the proteins described in claim 1, or a substance that regulates the expression of a gene encoding the protein, or a substance that regulates the activity and / or content of the protein in any of the following: M1) Regulate the number of plant tillers; M2) preparing products for regulating the number of tillers of plants; M3) Plant breeding.

5. Application, characterized in that, Use of the biomaterial according to claim 3 in any of the following: M1) Regulate the number of plant tillers; M2) preparing products for regulating the number of tillers of plants; M3) Plant breeding.

6. The use according to claim 4 or 5, characterized in that: The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein in claim 1 or the content or activity of the protein.

7. A method for regulating the tiller number of rice, comprising the following steps: regulating the expression of the gene encoding the protein described in claim 1 in a recipient plant or regulating the activity and / or content of the protein to change the tiller number of the recipient plant.

8. A method for increasing the tiller number of rice, comprising the steps of upregulating, enhancing or increasing the expression of the gene encoding the protein described in claim 1 in a recipient plant or regulating the activity and / or content of the protein, so as to increase the tiller number of the recipient plant.

9. A method for preparing rice with increased tiller number, comprising the steps of: upregulating or enhancing or increasing the expression of a gene encoding the protein described in claim 1 in a recipient plant or regulating the activity and / or content of the protein to obtain a plant with increased tiller number, wherein the plant with increased tiller number has a greater number of tillers than the recipient plant.

10. The use according to any one of claims 4 to 6 or the method according to any one of claims 7 to 9, characterized in that: The plant is any one of the following: G1) Monocots; G2) Gramineae; G3) Oryza plants; G4) Rice plants; G5) Rice.

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