Method for preparing rice with changed tiller number

Abstract

The invention discloses a method for preparing rice with changed tiller number, and belongs to the technical field of genetic breeding. The method comprises the following steps: introducing a coding gene of an OsD14 mutant into target rice to obtain rice with changed tiller number, the tiller number of the rice with changed tiller number is higher or lower than that of the target rice, and the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to another amino acid and keeping the other amino acids of SEQ ID NO: 1 unchanged. Experiments prove that whether the OsD14 is subjected to ubiquitination degradation or not is related to the phosphorylation state of the OsD14, and the phosphorylation state of the OsD14 is used for adjusting the tillering number of the rice and improving the plant type of the plant, so that the application prospect is wide when the yield 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 method for preparing rice with changed tillering number. Background Art

[0002] Rice is one of the most important food crops in the world. It feeds more than half of the world's population. Rice yield is of great significance to world food security. Rice tillering is an important agronomic trait and one of the important factors determining rice yield. Therefore, in-depth research on the molecular mechanism of regulating rice tillering formation and applying it to agricultural production will be of great practical significance for increasing rice yield.

[0003] Rice has unique advantages as a model plant for monocotyledon research. As a special branch, rice tillering has advantages in morphogenesis and genetics research, providing a very important research model for the study of the mechanism of branching in higher plants. Therefore, in-depth research on the molecular mechanism of rice tillering has important guiding significance for revealing the regulatory mechanism of plant branching.

[0004] Recently, strigolactones were discovered as a new plant hormone that regulates plant branching, which greatly promoted the study of the branching development mechanism of higher plants. Rice has a special branching form, tillering, which plays a very key role in the study of strigolactones regulating plant branching development. In recent years, research on the anabolism, transport and signal transduction of strigolactones has made great progress, and a series of key genes have been identified. However, there are still many unresolved issues in the study of strigolactone anabolism and signal transduction.

[0005] OsD14 The gene comes from rice and belongs to the strigolactone receptor protein. Its main function is to control the rice plant type, including the number of tillers and plant height. In the strigolactone signaling pathway, OsD14 forms a complex with OsD3 and OsD53, inducing OsD53 to undergo ubiquitination and degradation to relieve the inhibition of downstream corresponding genes. OsD14 itself can also undergo ubiquitination and degradation. Whether it undergoes ubiquitination and degradation and the regulation it is subject to can affect the signal transduction of strigolactones to varying degrees, thereby affecting the number of rice tillers. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing rice with changed tiller number. The technical problem to be solved is not limited to the technical subject described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a method for preparing rice with altered tiller number. The method comprises the steps of introducing a gene encoding an OsD14 mutant into target rice to obtain rice with altered tiller number, wherein the tiller number of the rice with altered tiller number is higher or lower than that of the target rice, and the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO:1 from serine to another amino acid, while keeping other amino acids of SEQ ID NO:1 unchanged.

[0008] In the above method, the other amino acid is alanine or aspartic acid.

[0009] In the above method, the nucleotide sequence of the gene encoding the OsD14 mutant can be obtained by mutating the nucleotides at positions 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO: 2 into codons for alanine, or by mutating the nucleotides at positions 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO: 2 into codons for aspartic acid.

[0010] The present invention also provides a method for changing the tiller number of rice, the method comprising introducing a gene encoding an OsD14 mutant into the target rice to change the tiller number of the recipient rice, wherein the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to another amino acid, while keeping the other amino acids of SEQ ID NO: 1 unchanged.

[0011] In the above method, the nucleotide sequence of the gene encoding the OsD14 mutant can be obtained by mutating the nucleotides at positions 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO: 2 into codons for alanine, or by mutating the nucleotides at positions 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO: 2 into codons for aspartic acid.

[0012] In the above method, the other amino acid is alanine or aspartic acid.

[0013] In the above method, when the codons for the amino acids at positions 10, 11, 13, and 15 of OsD14 are mutated from serine to alanine, OsD14 is in a non-phosphorylated state, and the tiller number of the recipient rice increases relative to before the mutation. When the codons for the amino acids at positions 10, 11, 13, and 15 of OsD14 are mutated from serine to aspartic acid, OsD14 is in a phosphorylated state, and the tiller number of the recipient rice decreases relative to before the mutation.

[0014] The present invention also provides a method for increasing the tiller number of rice under low nitrogen conditions, the method comprising introducing a gene encoding an OsD14 mutant into the target rice to change the tiller number of the recipient rice, wherein the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to alanine, while keeping the other amino acids of SEQ ID NO: 1 unchanged.

[0015] The low nitrogen condition in the above method refers to the application of nitrogen fertilizer at a weight of 50 kg per hectare per plant line in the field of the transgenic base of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing.

[0016] The present invention also provides a method for reducing the tiller number of rice under high nitrogen conditions, the method comprising introducing a gene encoding an OsD14 mutant into the target rice to change the tiller number of the recipient rice, wherein the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to aspartic acid, while keeping the other amino acids of SEQ ID NO: 1 unchanged.

[0017] The high nitrogen condition in the above method refers to the application of nitrogen fertilizer at a weight of 300 kg per hectare per plant line in the field of the transgenic base of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing.

[0018] The present invention also provides a protein, wherein the protein is any one of the following: A1) is a protein in which the amino acids 10, 11, 13 and 15 of SEQ ID NO: 1 are mutated from serine to alanine, while the other amino acids of SEQ ID NO: 1 remain unchanged; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

[0019] The present invention also provides a biomaterial, wherein the biomaterial is any one of the following: B1), a nucleic acid molecule encoding the aforementioned protein; 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).

[0020] The present invention also provides another protein, which is any one of the following: A1) is a protein in which the amino acids 10, 11, 13 and 15 of SEQ ID NO: 1 are mutated from serine to aspartic acid, while the other amino acids of SEQ ID NO: 1 remain unchanged; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

[0021] The present invention also provides a biological material related to the aforementioned another protein, wherein the biological material is any one of the following: B1), a nucleic acid molecule encoding the aforementioned protein; 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).

[0022] The codons described in this application are triplet codons composed of four bases of DNA.

[0023] In the above biological materials, the expression cassette containing nucleic acid molecules described in B3) refers to DNA that can express the above-mentioned proteins in host cells. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing the nucleic acid molecules of any of the above-mentioned proteins. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins 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 specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker can be provided. The regulatory sequence may be a suitable promoter sequence, i.e., 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 protein expression. 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. 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. 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 encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used can be used in the present invention. It may also be necessary to add a regulatory sequence that can regulate protein expression according to the growth of the host cell. Examples of regulatory sequences are those systems that turn gene expression on or off in response to a chemical or physical stimulus, including in the presence of a regulatory compound. Other examples of regulatory sequences are those that allow gene amplification.

[0024] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

[0025] In the above biological materials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0026] Among the above biological materials, the transgenic plant cell lines do not include propagation materials.

[0027] The present invention proves through experiments that whether OsD14 undergoes ubiquitination degradation is related to its phosphorylation state. The phosphorylation state of OsD14 is used to regulate the number of rice tillers, improve the plant type, and thus increase the yield of rice, which will have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A in the figure is a schematic diagram of the phosphorylation site of D14 in the N-terminal domain, and B is the mass spectrometry detection result. 4SA Refers to D14 with S10A, S11A, S13A, S15A mutations, D14 4SD It refers to D14 with S10D, S11D, S13D, and S15D mutations.

[0029] Figure 2 Phosphorylation analysis of D14-GFP in transgenic calli was performed by SDS-PAGE after treatment with 50 μM λ protein phosphatase (λPP).

[0030] Figure 3 To stabilize D14-GFP and D14 in transgenic lines 4SA -GFP and D14 4SD -Ubiquitination of GFP in induced callus.

[0031] Figure 4 For 20 μM rac -GR24 treatment-induced stable transgenic lines in callus D14-GFP, D14 4SA -GFP and D14 4SD -GFP degradation rate.

[0032] Figure 5 The wild type (NP), d14 , Act:D14-GFP / d14 , Act:D14 4SA -GFP / d14 and Act:D14 4SD -GFP / d14 Morphology and tiller number of . Scale bar, 20 cm. Data are mean ± SD.

[0033] Figure 6 strigolactone downstream tillering inhibitory gene OsTB1 (left) and strigolactone biosynthesis genes (right) D10 Transcript levels in the bud base of 2-week-old seedlings.

[0034] Figure 7 Shown is the phosphorylation status of D14-GFP under high nitrogen (HN) and low nitrogen (LN) conditions.

[0035] Figure 8 is the relative protein abundance of D14-GFP. The D14-GFP signal of seedlings grown under HN conditions was set to 1.00.

[0036] Figure 9 Wild type (NP), d14, Act: D14-GFP / d14, Act: D14 4SA -GFP / d14, Act: D14 4SD -Phenotype of GFP / d14 at heading stage in HN, MN, or LN fields. HN, high nitrogen (300 kg ha-1); MN, medium nitrogen (150 kg ha-1); LN, low nitrogen (50 kg ha-1). Scale bar, 20 cm. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0040] Protein phosphatase (λPP) was purchased from NEB, with the catalog number P0753S. GFP-trap beads were purchased from ChromoTek, with the catalog number Gta-200. Phosphatase inhibitors were purchased from CoWin Biosciences, with the catalog number CW2383S. rac-GR24, a strigolactone analog, was purchased from Chiralix, with the catalog number CX2388. MG132 (Proteasome inhibitor) was purchased from Sigma-Aldrich, with the catalog number Cat# 474790.

[0041] The AHLG vector is 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). DWARF53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401-405.". The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0042] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. Two-tailed Student's t-test was used, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns (not significant) or One-way ANOVA test was used, P < 0.05 (*) indicated a significant difference.

[0043] Example 1: Analysis of phosphorylation sites of OsD14 protein in rice In the hormone signal transduction pathway, signal termination is the key to regulating the duration and intensity of signal transduction. In rice, D14-sensing strigolactone molecules interact with D3 to form a D14-D3-D53 complex, promote the degradation of D53, and activate the strigolactone signaling pathway, but the inactivation mechanism of rice strigolactone signaling is still unclear. Previous work in this study found that strigolactone induced D14-GFP to undergo ubiquitination and degradation dependent on 26S ubiquitination-proteasome. However, the regulatory mechanism of D14 ubiquitination and degradation is still unclear.

[0044] Experimental method: To identify the phosphorylation site of D14, immunoprecipitation was performed from Act:D14-GFP / D14 D14-GFP protein was enriched in callus induced by transgenic seeds. Act:D14-GFP / D14The transgenic seeds 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.". The public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes. Total protein was extracted from callus tissue using IP protein extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.5% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF, 50 μM MG132, and phosphatase inhibitors). Then, 25 μL of GFP-Trap beads were added to 1 mL of the supernatant and incubated at 4°C with gentle rotation for 2 h. The beads were washed 3-5 times with IP wash buffer (50 mMTris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.1% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF, 50 μM MG132, and phosphatase inhibitors) and then washed with 50 μL Elution with SDS-PAGE buffer was performed and SDS-PAGE was performed. One-third of the protein gel was used for subsequent experiments, and the protein was digested with trypsin and analyzed by LC-MS / MS using a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientific).

[0045] Experimental results: The post-transcriptional modification of D14 was analyzed, and IP-MS detection revealed that S10, S11, S13 and S15 residues were phosphorylated ( Figure 1 ).

[0046] Example 2: Analysis of the phosphorylation function of OsD14 protein in rice and its regulatory effect on rice tillering 1. OsD14, OsD14 4SA 、OsD14 4SD Construction of mutant overexpression vector Build OsD14, OsD14 4SA and OsD14 4SD The specific method is as follows: First, a pair of forward and reverse primers containing the mutation site were designed (OsD14 4SA -F and OsD14 4SA -R is a group, OsD14 4SD -F and OsD14 4SD -R as a group), the primers and template plasmid 35S:D14-Flag After annealing the vector, the PCR product was obtained by cyclic extension, which contained a circularized plasmid containing the D14 mutation and a template plasmid. The PCR product was digested with DpnⅠ to remove the normal 35S:D14-Flag The enzyme digestion product was transformed into Escherichia coli to obtain a vector containing the D14 mutation. 35S:OsD14 4SA -Flag and 35S:OsD14 4SD -Flag Circularized plasmid. 35S:D14-Flag The vector 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. et al. (2017). DWARF14, a receptor covalently linked with the active form of strigolactones, undergoes strigolactone-dependentdegradation in rice. Front. Plant Sci. 8, 1935.". The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0047] The primers for introducing different D14 amino acid site mutations are as follows: OsD14 4SA -F: 5'-CATCCGCCGCCCgcTgcCCCGgcCAGCgcCAGCAGCGGCGGCGGCGGGGGCGGGGGTCG-3'; OsD14 4SA -R: 5'-GCCGCCGCCGCTGCTGgcGCTGgcCGGGgcAgcGGGCGGCGGATGCGTCGATCGCAG-3'; OsD14 4SD -F: 5'-CATCCGCCGCCCgaTgaCCCGgaCAGCgaCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCG-3'; OsD14 4SD -R: 5'-GCCGCCGCCGCTGCTGtcGCTGtcCGGGtcAtcGGGCGGCGGATGCGTCGATCGCAG-3'; Then the above D14 plasmids containing different amino acid mutations ( 35S:OsD14 4SA -Flag or 35S:OsD14 4SD - Flag The circularized plasmid was used as a template and primers AHLG-D14-F and AHLG-D14-R were used to amplify OsD14 and OsD14 4SA and OsD14 4SD Different fragments, different DNA fragments are recombined into the AHLG vector to obtain a transgenic expression plasmid for construction.

[0048] AHLG-D14-F: 5'- ACGATAAGCTTGGGCCC ATGCTGCGATCGACGCATC-3′; AHLG-D14-R: 5'- ATGGCGGCCGCTCTAGA GTACCGGGCGAGAGCGC-3'.

[0049] Among the above primers, the ununderlined sequences are amplification D14 or primers with different mutation forms, the underlined sequences are used for homologous recombination with the AHLG vector.

[0050] D14 overexpression vector AHLG- Act:D14-GFP The recombinant expression vector is obtained by replacing a small fragment between two enzyme recognition sites ApaI and XbaI of the AHLG vector with SEQ ID NO: 2, while keeping other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the fusion protein D14-GFP.

[0051] D14-4SA overexpression vector AHLG- Act:D14 4SA -GFP So D14-4SAA recombinant expression vector is obtained by gene replacing a small fragment between the two enzyme recognition sites ApaI and XbaI of the AHLG vector, while keeping other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses D14-4SA protein, and the D14-4SA protein is obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO:1 from serine to alanine, while keeping other amino acid sequences unchanged.

[0052] D14-4SD overexpression vector AHLG- Dct:D14 4SD -GFP So D14-4SD A recombinant expression vector is obtained by gene replacing a small fragment between the two enzyme recognition sites ApaI and XbaI of the AHLG vector, while keeping other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses D14-4SD protein, and the D14-4SD protein is obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO:1 from serine to aspartic acid, while keeping other amino acid sequences unchanged.

[0053] 2. Obtaining overexpression plants (1) Conversion use d14 The mutant is used as the rice recipient, and the d14 After the mutant seeds were shelled, they were surface disinfected with 70% (v / v) ethanol for 1 min, then washed with 2.5% (w / v) sodium hypochlorite for 45 min, washed three times with sterile water, and then sown on NB solid medium and cultured at 28°C in the dark for two weeks. After callus tissue grew on the mature embryo scutellum, the callus tissue was cut and subcultured on a new NB solid medium, and transferred to a new NB solid medium every 7 days. After 3-4 times, Agrobacterium infection can be carried out.

[0054] 1 L LB solid medium composition: Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g, agar 15 g.

[0055] 1 L LB liquid culture medium composition: Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g.

[0056] Composition of rice transformation solution: Add the following reagents to NB basic medium: Inositol 2 g / L, Glutamine 2 g / L, Casein hydrolysate 500 mg / L, 10% Synperonic PE 10 ml / L, Acetosyringone 100 μM.

[0057] When the rice callus tissue is growing well, the plant transgenic expression vector (AHLG- Act:D14-GFP Carrier, AHLG- Act:D14 4SA -GFP、 AHLG- Act:D14 4SD -GFP ) 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 from each transformation were inoculated into 7 mL LB liquid medium containing kanamycin and 25 mg / L rifampicin, and then cultured overnight at 250 rpm at 28ºC. 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.

[0058] (2) Agrobacterium EHA105 infects rice callus Collect the callus tissue in good condition obtained in step (1), add an appropriate amount of transformation solution until the bacterial solution completely submerges the callus tissue, and place it at room temperature for 20 minutes, shaking it from time to time. Then take out the transformed callus tissue, absorb the excess bacterial solution with sterile filter paper, and culture it in a 23ºC incubator away from light for 2-3 days to obtain transgenic callus tissue.

[0059] (3) Differentiation The rice callus obtained in step (2) is transferred to 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 differentiation medium for differentiation and regeneration of seedlings. After about one month of light culture at 28°C, the grown seedlings are transferred to rooting medium and cultured for about 4 weeks for rooting and seedling strengthening. They are transplanted to a greenhouse for about one month and then transferred to the field.

[0060] 3. Identification of positive plants 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. The PCR products were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for Sanger sequencing. After the sequencing results were obtained, the sequences were compared. The plants with the expected sequences were T0 positive plants, and 16 AHLG-transformed plants were obtained. Act:D14-GFP The T0 positive individual of the vector (named Act:D14-GFP / d14 plants), 10 plants were transformed with AHLG- Act:D14 4SA -GFP The T0 positive individual of the vector (named Act: D14 4SA -GFP / d14 plants), 15 plants were transformed with AHLG- Act:D14 4SD -GFP The T0 positive individual of the vector (named Act: D14 4SD -GFP / d14 (2) Randomly select 8 plants Act:D14-GFP / d14 Plant 、 8 strains Act:D14 4SA -GFP / d14 Plants and 8 plants Act:D14 4SD -GFP / d14 The T0 generation plants of the plant were propagated and 8 T1 generation strains were planted. Six plants were randomly collected from each T1 generation strain and used to screen homozygous positive plants. (3) The harvested T1 generation seeds were germinated and 40 germinated seeds were placed on a 0.5% Agar solid medium containing 50 μg / mL hygromycin (Hyg). After 2-3 days, the root growth of the plants was observed. Plants with normal root growth were positive plants, otherwise they were isolated pseudo-singles. If all seeds of each plant can grow roots normally, it is a homozygous positive strain.

[0061] Get D14 Primers for identifying positive transgenic rice seedlings with homozygous mutations: AHLG-F: 5'-TCAGCATTGTTCATCGGTAG-3'; D14-570-R: 5'-CTGCTGTATCCTCCTCCAGCTC-3'.

[0062] PCR reaction system: 1 μL DNA template, 2 μL 10 μM Primer F, 2 μL 10 μM Primer R and 45 μL Gold Mix. Reaction program: 98℃ pre-denaturation for 3 min; 35 cycles of 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 15 s; 72℃ extension for 5 min.

[0063] As a result, a total of 6 strains were obtained Act:D14-GFP / d14 Homozygous positive individual 、 5 strains Act:D14 4SA -GFP / d14 Homozygous positive single plant and 5 plants Act:D14 4SD -GFP / d14 The above-mentioned single plants used in subsequent experiments were named Act:D14-GFP / d14#1 , Act:D14-GFP / d14#2 , Act:D14 4SA -GFP / d14#1 , Act:D14 4SA -GFP / d14#2 , Act:D14 4SD -GFP / d14#1 and Act:D14 4SD -GFP / d14#2 .

[0064] 4. SDS-PAGE analysis of D14-GFP phosphorylation in transgenic callus Protein phosphatase (λPP) storage conditions: 100 mM NaCl, 50 mM HEPES (pH 7.5), 0.1 mM MnCl2, 0.1 mM EGTA, 2 mM DTT, 0.01% BSA and 50% glycerol. Store at -70 ℃.

[0065] (1) Treatment with protein phosphatase (λPP) working solution Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1The total protein of callus tissue was extracted by IP protein extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.5%Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF and50 μM MG132). Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1 Each callus total protein was divided into two samples, for a total of 6 samples. After adding 20 µL GFP-Trapbeads to each sample, it was placed at 4ºC and incubated for 3 h, washed 3 times with IP protein extraction solution, and finally added 50 microliters of IP protein extraction solution. Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1 One of the samples of callus IP protein was added with 20 μL protein phosphatase (λPP), and the other was used as a control. After reacting at 30℃ for 2 h, protein loading buffer was added and boiled. Then SDS-PAGE and phos-tag gel electrophoresis were performed.

[0066] (2) Detect the phosphorylation of D14-GFP protein in the callus tissue treated in step (1) by Western Blot experiment. The specific method is as follows: (1) Prepare 10% concentration 12-well SDS-PAGE protein gel or phos-tag gel electrophoresis. The amount of protein sample loaded depends on the experimental requirements. Set the voltage to 80-120 V for about 1-2 h. (2) Transfer the protein to PVDF or NC membrane using semi-dry transfer method. The voltage is 15-20 V and the transfer time is 30-60 min. (3) During the transfer, prepare 4% skim milk powder in TBST solution. After the transfer, place the PVDF or NC membrane in 4% skim milk powder and incubate it at room temperature at 70 rpm on a shaker for 60 min. (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 1-2 h or overnight at 4ºC. (5) After incubation with the primary antibody, wash the PVDF or NC membrane three times with TBST, each time at 70 rpm for 5 min at room temperature, then add 4% skim milk containing the secondary antibody and incubate at room temperature for 60 min 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 min at room temperature. Add the color developing solution and use a photographic film or protein developing instrument to develop the protein. Use GFP antibody (1:3000, Roche, Cat#11814460001) to detect GFP fusion protein.

[0067] The results showed that phosphorylation of D14-GFP could be inhibited by protein phosphatase (λPP) treatment or by mutation of S10, S11, S13 and S15 to alanine (abbreviated as D14 4SA ) is suppressed by the four mutations ( Figure 2 ), indicating that S10, S11, S13, and S15 are the main phosphorylation sites of OsD14.

[0068] 5. Ubiquitination and degradation of D14 20 μM rac -GR24 working fluid: Dissolve in acetone rac -GR24 is obtained, where rac The content of -GR24 was 20 μM.

[0069] Experimental method: Select Act:D14-GFP / d14 rice callus and Act:D14∆N-GFP / d14 rice callus with vigorous growth, i.e., pale yellow, smooth appearance, and compact morphology, and transfer them to new NB solid selection medium containing antibiotics and culture them at 28℃ in the dark for 4-6 days. Then transfer the callus to NB liquid medium and culture them at 28℃ and 50 rpm in the dark for 1-2 h. For the ubiquitination experiment of D14, transfer the above-mentioned callus to NB liquid medium. Act:D14-GFP / d14#1, Act:D14 4SA -GFP / d14#1 and Act: D14 4SD -GFP / d14#1 The calli were pretreated with 50 μM MG132 for 1 hour. The calli were treated with or without 20 μM rac-GR24 for 1 hour and then frozen in liquid nitrogen. Total protein was extracted in IP protein extraction buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.5% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF and 50 μM MG132). Then, 25 μL GFP-Trap beads were added to 1 mL of supernatant and incubated at 4°C with gentle rotation for 2 h. GFP-Trap beads were washed 3-5 times with IP wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.1% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail). Then, GFP-Trap beads were eluted with 50 μL SDS-PAGE buffer and subjected to SDS-PAGE and Western blot. Blot detection.

[0070] For the degradation experiment of D14, 20 μM rac-GR24 working solution was added for 1 h or 3 h. After treatment, callus tissue was collected and frozen in liquid nitrogen. Callus tissue was collected and added with 2 volumes of RIPA protein extraction solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 1.0% (v / v) Nonidet P-40, 10 mMNaF, 2 mM EDTA, 0.1% SDS sodium orthovanadate, 1 x Roche cOmplete™ ProteaseInhibitor Cocktail, 1 mM PMSF and 50 μM MG132). Vortex thoroughly to mix and place on ice. Vortex thoroughly for 10 s every 8 min, vortex 3-4 times and centrifuge. Incubate at 4°C for 18,000 min. gCentrifuge for 20 min, pipette 200 μL of supernatant into a new 1.7 mL centrifuge tube, add 50 μL of protein loading buffer, denature the sample at 100°C for 5 min, and perform Western Blot.

[0071] The results showed that D14 4SA Enhanced ubiquitination and degradation of D14 4SD Significantly reduced the ubiquitination and degradation of D14 ( Figure 3 and 4 ), indicating that phosphorylation can inhibit the ubiquitination and degradation of rice D14.

[0072] 6. Count the number of tillers of homozygous positive plants The experimental method is as follows: wild-type rice Nipponbare, d14 Mutants, homozygous plants Act:D14-GFP / d14#1 , Act:D14-GFP / d14#2 , Act:D14 4SA -GFP / d14#1 , Act:D14 4SA -GFP / d14#2 , Act:D14 4SD - GFP / d14#1 and Act:D14 4SD -GFP / d14#2 Planted in the field at the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing, 24 plants were planted in each line with a plant spacing and row spacing of 17 cm, and 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 for each material, representative single-plant transplant pots were selected for photography.

[0073] The results showed that overexpression of D14 4SA -GFP partially rescued the high tillering phenotype of D14, while overexpression of D14 4SD -GFP resulted in even more tillers than the wild type ( Figure 5 ), indicating that D14 phosphorylation inhibits rice tillering development ( Figure 5 ).

[0074] 7. Detection of expression levels of SL-responsive genes (1) Detection of strigolactone biosynthesis genes D10 Relative expression of genes The experimental method is as follows: Act:D14-GFP / d14#1 ,Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1 Rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28°C for 16 h of light and 25°C for 8 h of darkness. The light intensity (150–200 μM m -2 s -1 ), humidity was 70%. After culturing in Kimura B nutrient solution for 2 weeks, root tissues of seedlings of different materials were collected.

[0075] RNA was extracted from rice seedlings using the Trizol method (Life Technologies: 15596-026). The reagents and experimental consumables used in the RNA extraction process had been free of RNase. Rice tissue samples were thoroughly ground in liquid nitrogen. The samples were kept frozen during the entire grinding process. About 100-150 mg of powdered sample was placed in a 1.7 mL centrifuge tube, 1 mL of Trizol was added, and the mixture was thoroughly vortexed and allowed to stand at room temperature for 5 min. After that, 200 μL of chloroform was added and immediately inverted for 15 seconds, allowed to stand at room temperature for 5 min, and then incubated at 4°C for 12,000 min. g Centrifuge for 15 min; take 500 μL of the supernatant into a 1.7 mL centrifuge tube, add an equal volume of isopropanol, invert 20 times to mix, let stand at room temperature for 10 min, and then incubate at 4°C at 12,000 g Centrifuge for 10 min; discard the supernatant, add 1 mL 70% (v / v) ethanol to the precipitated RNA, invert 30 times, and g Centrifuge for 5 min; discard the supernatant, add 50 μL of RNase-free water after the precipitate is dried, and place on ice for more than 3 h to fully dissolve the RNA; use NanoDrop 2000 UV-Vis spectrophotometer to measure the OD values ​​at 230 nm, 260 nm, and 280 nm to evaluate the overall quality of RNA preparation. RNA samples can be directly used for RNA reverse transcription experiments or placed at -20°C for use.

[0076] Using TURBO DNA-free TM The kit was used to remove residual DNA from total RNA. For detailed steps, see the instructions. Each reaction system was set to 20 μL, total RNA was 12.5 μg, incubated at 37°C for 30 min, 2 μL DNase inactivation reagent was added and mixed thoroughly at room temperature for 5 min, and then heated at room temperature for 12,000 gCentrifuge for 5 min, and 4 μL of supernatant is used for reverse transcription experiment. The first strand of cDNA is synthesized with SuperScript®III First-Strand Synthesis System Reverse Transcription Kit. For detailed steps, see the instructions. Finally, add 180 μL of nuclease-free double distilled water to dissolve the reverse transcription product, and mix thoroughly before use.

[0077] Detection of SL-biosynthetic genes in roots of transgenic rice seedlings using quantitative PCR D10 The transcription level of each reaction system in fluorescence quantitative PCR was 10 μL, including 5 μL SsoFast EvaGreen supermix, 0.5 μL sense primer (5 μM), 0.5 μL antisense primer (5 μM), 2.0 μL diluted cDNA and 2 μL ddH2O. The instrument used for fluorescence quantitative PCR was a real-time fluorescence quantitative PCR instrument (model: CFX96) from BIO-RAD. The PCR running program parameter setting information was: 98ºC, 30 s; (98ºC, 5 s → 60ºC, 5 s → information collection), 40 cycles; 60-95ºC, 0.5ºC / 5 s, information collection / 5 s. After the program was completed, the data was processed by BIO-RAD CFXManager software.

[0078] D10 Fluorescence quantitative PCR primers for gene expression detection: qD10-F: 5'-CGTGGCGATATCGATGGT-3'; qD10-R: 5'-CGACCTCCTCGAACGTCTT-3'.

[0079] qUbiquitin-F: 5'-AACCAGCTGAGGCCCAAGA-3'; qUbiquitin-R: 5'-ACGATTGATTTAACCAGTCCATGA-3'.

[0080] (2) Detection OsTB1 Relative expression of genes The experimental method is as follows: Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1Rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28°C for 16 h of light and 25°C for 8 h of darkness. The light intensity (150–200 μM m -2 s -1 ), humidity was 70%. After culturing in Kimura culture medium for 2 weeks, stem base tissues (1 cm) of seedlings of different materials were collected. Detection of rice tillering inhibition gene in transgenic rice using fluorescent quantitative PCR OsTB1 The specific method is as described above, and the internal reference is qUbiquitin.

[0081] OsTB1 Fluorescence quantitative PCR primers for gene expression detection: OsTB1-F: 5'-CGACAGCGGCAGCTACTAC-3'; OsTB1-R: 5'-GCGAATTGGCGTAGACGA-3'.

[0082] The results showed that compared with the wild type, the strigolactone-responsive genes that inhibit rice tillering function OsTB1 exist Act:D14 4SA – GFP / d14 The expression level was reduced in the transgenic lines. Act:D14 4SD – GFP / d14 The expression level was increased in the transgenic lines ( Figure 6 ). SL biosynthesis genes regulated by feedback D10 The expression level in Act:D14 4SA – GFP / d14 In the rise, Act:D14 4SD – GFP / d14 The expression level decreased ( Figure 6 ). In summary, D14 phosphorylation enhances SL signaling and inhibits rice tillering.

[0083] Example 3: Application of regulating rice by changing the phosphorylation state of D14 In order to analyze the regulatory role of OsD14 phosphorylation in low nitrogen response, the effect of different nitrogen content conditions on the number of rice tillers was explored. The experiment was repeated three times, and each repetition was as follows: 1. Experimental Grouping High nitrogen group (HN): Transgenic homozygous plants Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1Planted in the field of the transgenic base of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing, the weight of nitrogen fertilizer applied was 300 kg per hectare per plant line, and 24 plants were planted with a plant and row spacing of 17 cm. The number of rice tillers was counted according to the above method.

[0084] Medium nitrogen group (MN): The difference between this group and the high nitrogen group (HN) is that the weight of nitrogen fertilizer applied is 150 kg per hectare, and the rest of the operations are the same as the high nitrogen group (HN).

[0085] Low nitrogen group (LN): The difference between this group and the high nitrogen group (HN) is that the weight of nitrogen fertilizer applied is 50 kg per hectare, and the rest of the operations are the same as the high nitrogen group (HN).

[0086] 2. Phosphorylation and protein levels of OsD14 under different nitrogen levels Experimental methods: To identify the phosphorylation level and protein level of D14 phosphorylation sites under different nitrogen conditions, the transgenic rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28ºC for 16 h of light and 25ºC for 8 h of darkness, with a light intensity of 150-200 μM m-2 s-1 and a humidity of 70%. After culturing in Kimura B nutrient solution for 1 week, the seedlings were further cultured in different nitrogen concentrations for 7-10 days.

[0087] Table 1 Formula of Kimura B nutrient solution

[0088] Note: To prepare 20 mM FeSO4-EDTA solution, add 5.57 g FeSO4·7H2O and 7.45 g Na2-EDTA to 800 mL deionized water and heat in a 70°C oven (or water bath) for 1-2 days, mixing manually until completely dissolved. (NH4)6Mo7O 24 ·4H2O can be replaced by Na2MoO4·2H2O (0.128 mg / L). The pH of the rice nutrient solution should be between 5.5-6.0, and the nutrient solution needs to be replaced every 2 days. When preparing culture solutions with different nitrogen concentrations, first prepare Kimura nutrient solution without nitrogen source. Then replace ammonium ((NH4)2SO4) and nitrate (KNO3, Ca(NO3)2) ions with 1 M NH4NO3, and add the corresponding volume of NH4NO3 as needed to adjust the nitrogen concentration. Finally, add 0.365 mMCaCl2 to make up the calcium ions. The final concentration of 0.15 mM NH4NO3 is a low-nitrogen hydroponic solution, and the final concentration of 2.5 mM NH4NO3 is a high-nitrogen culture solution.

[0089] The stem base tissues (1 cm) of seedlings of different materials were collected and D14-GFP protein was enriched by immunoprecipitation. Total protein was extracted in IP protein extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.5% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF, 50 μM MG132, and phosphatase inhibitors). Then, 25 μL GFP-Trap beads were added to 1 mL of supernatant and incubated with gentle rotation at 4°C for 2 h. The beads were washed 3-5 times with IP wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 0.1% Nonidet P-40, 1x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF, 50 μM MG132, and phosphatase inhibitors). Elution was performed with 50 μL SDS-PAGE buffer and SDS-PAGE and Western Blot were performed as described above.

[0090] The experimental results showed that the phosphorylation level of OsD14 was enhanced under low nitrogen conditions ( Figure 7 ), while the protein abundance of D14-GFP increased significantly ( Figure 8 ). This indicates that low nitrogen triggers the phosphorylation of OsD14 and inhibits its ubiquitination degradation.

[0091] 3. Count the number of tillers The number of tillers of rice was counted according to the method described in "6. Counting the number of tillers of homozygous positive plants" in Example 2.

[0092] The results showed that the number of rice tillers decreased significantly under low nitrogen conditions ( Fig. 9 ), indicating that low nitrogen conditions trigger D14 phosphorylation and stabilize D14 protein, thereby enhancing SL perception and inhibiting tillering development. Therefore, the response to low nitrogen can be regulated by regulating the phosphorylation level of OsD14. 4SA -GFP overexpression led to an increase in the number of tillers under low nitrogen conditions, indicating that the number of rice tillers under low nitrogen conditions can be regulated by changing the phosphorylation state of D14. Fig. 9 It also shows that D14 4SD -GFP led to a decrease in tiller number under high nitrogen conditions, further demonstrating the feasibility of improving rice tiller number by changing the phosphorylation state of the strigolactone receptor OsD14.

[0093] The sequences involved in the above embodiments SEQ ID NO:1 OsD14 Wild-type amino acid sequence of the gene MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0094] SEQ ID NO:2 OsD14 Wild-type CDS nucleotide coding sequence of the gene 5'-ATGCTGCGATCGACGcatccgccgccCAGTAGCCCGAGCAGCAGcagcagcggcggcggcgggggcggggggtcgTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTA0CTAA-3'.

[0095] Mutant D144SA The amino acid sequence MLRSTHPPPAAPASASSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0096] Mutant D14 4SD The amino acid sequence MLRSTHPPPDDPDSDSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0097] 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 method for preparing rice with altered tillering number, characterized in that: The method comprises the steps of introducing a gene encoding an OsD14 mutant into target rice to obtain rice with altered tiller number, wherein the tiller number of the rice with altered tiller number is higher or lower than that of the target rice, and the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to another amino acid, while keeping other amino acids of SEQ ID NO: 1 unchanged.

2. The method according to claim 1, characterized in that: The other amino acid is alanine or aspartic acid.

3. A method for changing the number of rice tillers, characterized in that: The method comprises introducing a gene encoding an OsD14 mutant into target rice to change the tiller number of the recipient rice, wherein the OsD14 mutant is a protein obtained by mutating the 10th, 11th, 13th and 15th amino acids of SEQ ID NO: 1 from serine to another amino acid, while keeping the other amino acids of SEQ ID NO: 1 unchanged.

4. The method according to claim 3, characterized in that The other amino acid is alanine or aspartic acid.

5. A protein, characterized in that The protein is any of the following: A1) is a protein in which the amino acids 10, 11, 13 and 15 of SEQ ID NO: 1 are mutated from serine to alanine, while the other amino acids of SEQ ID NO: 1 remain unchanged; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

6. Biomaterial, wherein the biomaterial is any one of the following: B1), a nucleic acid molecule encoding the protein according to any one of claim 5; 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).

7. Use of the protein according to claim 5 or the biomaterial according to claim 6 in any of the following: K1) Increase the number of tillers of rice; K2) preparing products for regulating the number of rice tillers; K3) Rice breeding.

8. A protein, characterized in that The protein is any of the following: A1) is a protein in which the amino acids 10, 11, 13 and 15 of SEQ ID NO: 1 are mutated from serine to aspartic acid, while the other amino acids of SEQ ID NO: 1 remain unchanged; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

9. Biomaterial, wherein the biomaterial is any one of the following: B1), a nucleic acid molecule encoding the protein according to any one of claim 8; 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).

10. Use of the protein according to claim 8 or the biomaterial according to claim 9 in any of the following: K1) Increase the number of tillers of rice; K2) preparing products for regulating the number of rice tillers; K3) Rice breeding.

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