A method for making rice plants with altered tiller number

Abstract

The application 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, wherein 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 in SEQ ID NO: 1 into other amino acids and keeping other amino acids in SEQ ID NO: 1 unchanged. It is proved by experiments that whether ubiquitination degradation of the OsD14 occurs is related to a phosphorylation state of the OsD14, the number of rice tillers is adjusted by using the phosphorylation state of the OsD14, and the plant type is improved, so that the yield of the rice is improved, and the method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology, specifically relating to a method for preparing rice with altered tiller number. Background Technology

[0002] Rice is one of the world's most important food crops, feeding more than half the world's population. Rice yield is of great significance to global food security. Rice tillering is an important agronomic trait and one of the key factors determining rice yield. Therefore, in-depth research into the molecular mechanisms regulating rice tillering and applying them to agricultural production will have significant practical implications for increasing rice yield.

[0003] Rice, as a model plant for monocotyledonous plant research, possesses unique advantages. Rice tillering, as a special form of branching, offers significant advantages in morphological and genetic studies, providing a crucial research model for understanding the mechanisms of branching in higher plants. Therefore, in-depth research into the molecular mechanisms of rice tillering is of great guiding significance for elucidating the regulatory mechanisms of plant branching.

[0004] Recently, strigolactones have been discovered as a novel plant hormone regulating branching, greatly advancing research into the mechanisms of branching development in higher plants. Rice, with its unique tillering branching system, plays a crucial role in studies of strigolactone regulation of branching development. In recent years, significant progress has been made in the research of strigolactone synthesis, metabolism, transport, and signal transduction, with the identification of a series of key genes. However, many unresolved issues remain regarding strigolactone synthesis, metabolism, and signal transduction.

[0005] OsD14 The gene originates from rice and belongs to the strigolactone receptor protein family. Its main function is to control rice plant architecture, 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, thus relieving the inhibition of downstream corresponding genes. OsD14 itself can also undergo ubiquitination and degradation. Whether it undergoes ubiquitination and degradation, as well as the regulation it receives, can affect strigolactone signal transduction to varying degrees, thereby affecting the number of tillers in rice. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for preparing rice with altered tiller number. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] This invention provides a method for preparing rice with altered tiller number. The method includes introducing the coding gene of an OsD14 mutant into a target rice variety to obtain rice with altered tiller number. The tiller number of the altered rice variety is higher or lower than that of the target rice variety. The OsD14 mutant is a protein obtained by mutating serine to another amino acid at positions 10, 11, 13, and 15 of SEQ ID NO:1, while keeping the other amino acids of SEQ ID NO:1 unchanged.

[0009] In the above method, the other amino acid is alanine or aspartic acid.

[0010] In the above method, the nucleotide sequence of the encoding gene of the OsD14 mutant can be obtained by mutating nucleotides 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO:2 to alanine codons, or by mutating nucleotides 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO:2 to aspartic acid codons.

[0011] The present invention also provides a method for changing the number of tillers in rice, the method comprising introducing the coding gene of an OsD14 mutant into a target rice to change the number of tillers in the recipient rice, wherein the OsD14 mutant is a protein obtained by mutating serine to another amino acid at positions 10, 11, 13 and 15 of SEQ ID NO:1, while keeping the other amino acids of SEQ ID NO:1 unchanged.

[0012] In the above method, the nucleotide sequence of the encoding gene of the OsD14 mutant can be obtained by mutating nucleotides 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO:2 to alanine codons, or by mutating nucleotides 28-30, 31-33, 37-39, and 43-45 of SEQ ID NO:2 to aspartic acid codons.

[0013] In the above method, the other amino acid is alanine or aspartic acid.

[0014] In the above method, when the codons for amino acids 10, 11, 13, and 15 of OsD14 are all mutated from serine to alanine, OsD14 exhibits a non-phosphorylated state, and the number of tillers in the recipient rice increases compared to before the mutation. When the codons for amino acids 10, 11, 13, and 15 of OsD14 are all mutated from serine to aspartic acid, OsD14 exhibits a phosphorylated state, and the number of tillers in the recipient rice decreases compared to before the mutation.

[0015] The present invention also provides a method for increasing the number of tillers in rice under low nitrogen conditions, the method comprising introducing the coding gene of an OsD14 mutant into a target rice to change the number of tillers in 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.

[0016] The low-nitrogen conditions mentioned above refer to the application of nitrogen fertilizer at a rate of 50 kg per hectare per line in the field of the transgenic base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing.

[0017] The present invention also provides a method for reducing the number of tillers in rice under high nitrogen conditions, the method comprising introducing the coding gene of an OsD14 mutant into a target rice to change the number of tillers in 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 amino acids, while keeping the other amino acids of SEQ ID NO:1 unchanged.

[0018] The high-nitrogen conditions mentioned above refer to the application of nitrogen fertilizer at a rate of 300 kg per hectare per line in the field of the transgenic base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing.

[0019] The present invention also provides a protein, said protein being any of the following:

[0020] A1) is a protein in which the amino acids at positions 10, 11, 13, and 15 of SEQ ID NO:1 are all mutated from serine to alanine, while keeping the other amino acids of SEQ ID NO:1 unchanged.

[0021] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1).

[0022] A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0023] The present invention also provides a biomaterial, wherein the biomaterial is any one of the following:

[0024] B1) Nucleic acid molecules that encode the aforementioned proteins;

[0025] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0026] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0027] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0028] 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);

[0029] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0030] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2).

[0031] The present invention also provides another protein, said protein being any of the following:

[0032] A1) is a protein in which the amino acids at positions 10, 11, 13, and 15 of SEQ ID NO:1 are all mutated from serine to aspartic alanine, while keeping the other amino acids of SEQ ID NO:1 unchanged.

[0033] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1).

[0034] A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0035] The present invention also provides a biomaterial related to another protein mentioned above, wherein the biomaterial is any one of the following:

[0036] B1) Nucleic acid molecules that encode the aforementioned proteins;

[0037] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0038] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0040] 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);

[0041] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0042] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2).

[0043] The codons described in this application are triplet codons composed of the four bases of DNA.

[0044] In the aforementioned biological materials, the expression cassette containing nucleic acid molecules described in B3) refers to DNA capable of expressing the proteins described above in host cells. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for expressing any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in suitable host cells. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may 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 transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of 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 and thus terminate transcription. The termination sequence is operatively attached 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 this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached 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 this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein into the secretion pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those systems that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification.

[0045] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0046] In the above-mentioned biological materials, the microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0047] Among the aforementioned biological materials, the transgenic plant cell lines do not include propagation materials.

[0048] This invention demonstrates through experiments that whether OsD14 undergoes ubiquitination degradation is related to its phosphorylation state. Utilizing the phosphorylation state of OsD14 to regulate the number of rice tillers and improve plant architecture, thereby increasing rice yield, has broad application prospects. Attached Figure Description

[0049] Figure 1 In the diagram, A represents the phosphorylation site of D14 in the N-terminal domain, and B represents the mass spectrometry detection result. (D14) 4SA This refers to D14 cells with mutations in S10A, S11A, S13A, and S15A. 4SD This refers to D14 mutated by S10D, S11D, S13D, and S15D.

[0050] Figure 2 Phosphorylation of D14-GFP in transgenic callus was analyzed by labeled SDS-PAGE after treatment with 50 μM λ protein phosphatase (λPP).

[0051] Figure 3 To stabilize D14-GFP and D14 in transgenic lines 4SA -GFP and D14 4SD -GFP ubiquitination in induced callus.

[0052] Figure 4 For use of 20 μM rac -GR24 treatment induced D14-GFP, D14 4SA -GFP and D14 4SD -GFP degradation rate.

[0053] Figure 5 Wild type (NP) d14 , Act:D14-GFP / d14 , Act:D14 4SA -GFP / d14 and Act:D14 4SD -GFP / d14 Morphology and tiller number. Scale bar, 20 cm. Data are mean ± standard deviation.

[0054] Figure 6 For strigolactone downstream tillering repressor gene OsTB1 (Left) and strigolactone biosynthesis gene (Right) D10 Transcriptional levels in 2-week-old seedling buds.

[0055] Figure 7 The phosphorylation of D14-GFP under high nitrogen (HN) and low nitrogen (LN) conditions is shown.

[0056] Figure 8 The relative protein abundance of D14-GFP is given. The D14-GFP signal of seedlings grown under HN conditions is set to 1.00.

[0057] Figure 9 Wild type (NP), d14, Act: D14-GFP / d14, Act: D14 4SA -GFP / d14, Act: D14 4SD -GFP / d14 phenotype 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 Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0061] Protein phosphatase (λPP) was purchased from NEB, catalog number P0753S. GFP-trap beads were purchased from ChromoTek, catalog number Gta-200. The phosphatase inhibitor was purchased from CoWin Biosciences, catalog number CW2383S. rac-GR24, a strigolactone analog, was purchased from Chiralix, catalog number CX2388. MG132 (a proteasome inhibitor) was purchased from Sigma-Aldrich, catalog number Cat# 474790.

[0062] The AHLG vector is described in the non-patent literature “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.” It is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biomaterial is intended solely for replicating experiments related to this invention and may not be used for any other purpose.

[0063] The following examples use GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Two-tailed Student's t-tests were used, with P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and ns (not significant) as values. Alternatively, one-way ANOVA tests were used, with P < 0.05 (*) indicating a significant difference.

[0064] Example 1: Analysis of phosphorylation sites of OsD14 protein in rice

[0065] In hormone signal transduction pathways, signal termination is crucial for regulating the duration and intensity of signal transduction. In rice, D14 senses the interaction between strigolactone molecules and D3, forming the D14-D3-D53 complex, which promotes the degradation of D53 and activates the strigolactone signaling pathway. However, the inactivation mechanism of strigolactone signal sensing in rice remains unclear. Previous work in this study found that strigolactone-induced D14-GFP formation depends on 26S ubiquitination-proteasome ubiquitination and degradation. However, the regulatory mechanisms of D14 ubiquitination and degradation are still unclear.

[0066] Experimental methods: To identify the D14 phosphorylation site, immunoprecipitation was performed on the site. Act:D14-GFP / D14 D14-GFP protein was enriched in callus induced by transgenic seeds. Act:D14-GFP / D14The transgenic seeds are documented in non-patent literature: “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.” They are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is intended solely for replicating experiments related to this invention and may not be used for any other purpose. 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 a phosphatase inhibitor). Then, 25 μL of GFP-Trap beads were added to 1 mL of supernatant and incubated gently at 4°C for 2 hours. The beads were washed 3-5 times with IP washing 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 a phosphatase inhibitor), followed by 50 μL of... After elution with SDS-PAGE buffer, SDS-PAGE was performed. One-third of the protein gel was then used for subsequent experiments. Proteins were digested with trypsin and analyzed by LC-MS / MS using a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientific).

[0067] Experimental results: Analysis of post-transcriptional modifications of D14 revealed phosphorylation of S10, S11, S13, and S15 residues using IP-MS. Figure 1 ).

[0068] Example 2: Analysis of the phosphorylation function of OsD14 protein in rice and its regulatory effect on rice tillering.

[0069] 1. OsD14, OsD14 4SA OsD14 4SDConstruction of overexpression vectors for mutants

[0070] Construct OsD14, OsD14 4SA and OsD14 4SD The transgenic expression plasmid was prepared using the following method:

[0071] First, design a pair of forward and reverse primers containing the mutation site (OsD14). 4SA -F and OsD14 4SA -R is a group, OsD14 4SD -F and OsD14 4SD -R is a group), primers and template plasmid 35S:D14-Flag After vector annealing, cyclic extension yielded PCR products containing a circularized plasmid with the D14 mutation and the template plasmid. The PCR product was then digested with DpnI to remove the normal plasmid. 35S:D14-Flag Vector. The enzyme digestion product was transformed into E. coli to obtain vectors containing the D14 mutation. 35S:OsD14 4SA -Flag and 35S:OsD14 4SD -Flag Circulated plasmids. Among them... 35S:D14-Flag The vector is described in the non-patent literature “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-dependent degradation in rice. Front. Plant Sci. 8, 1935.” It is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biomaterial is intended solely for replicating experiments related to this invention and may not be used for any other purpose.

[0072] The primers for introducing mutations at different D14 amino acid sites are as follows:

[0073] OsD14 4SA -F: 5'-CATCCGCCGCCCgcTgcCCCGgcCAGCgcCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCG-3';

[0074] OsD14 4SA-R: 5'-GCCGCCGCCGCTGCTGgcGCTGgcCGGGgcAgcGGGCGGCGGATGCGTCGATCGCAG-3';

[0075] OsD14 4SD -F: 5'-CATCCGCCGCCCgaTgaCCCGgaCAGCgaCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCG-3';

[0076] OsD14 4SD -R: 5'-GCCGCCGCCGCTGCTGtcGCTGtcCGGGtcAtcGGGCGGCGGATGCGTCGATCGCAG-3';

[0077] Then, the D14 plasmid containing different amino acid mutations was used. 35S:OsD14 4SA -Flag or 35S:OsD14 4SD - Flag Using a circularized plasmid as a template, OsD14 and OsD14 were amplified using primers AHLG-D14-F and AHLG-D14-R. 4SA and OsD14 4SD Different DNA fragments are recombined into the AHLG vector to obtain plasmids for constructing transgenic expression.

[0078] AHLG-D14-F: 5'- ACGATAAGCTTGGGCCC ATGCTGCGATCGACGCATC-3';

[0079] AHLG-D14-R: 5'- ATGGCGGCCGCTCTAGA GTACCGGGCGAGAGCGC-3'.

[0080] Among the primers mentioned above, those without underlined sequences are for amplification. D14 Primers with different mutation forms or underlined sequences are used for homologous recombination with the AHLG vector.

[0081] D14 overexpression vector AHLG- Act:D14-GFP The recombinant expression vector is obtained by replacing the small fragment between the two restriction 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. This recombinant expression vector expresses the fusion protein D14-GFP.

[0082] D14-4SA overexpression vector AHLG- Act:D144SA -GFP Therefore D14-4SA A recombinant expression vector was obtained by replacing a small fragment between the ApaI and XbaI restriction sites of the AHLG vector with a gene, while keeping the other nucleotide sequences of the AHLG vector unchanged. This recombinant expression vector expresses the D14-4SA protein, which is 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 acid sequences unchanged.

[0083] D14-4SD overexpression vector AHLG- Dct:D14 4SD -GFP Therefore D14-4SD A recombinant expression vector was obtained by replacing a small fragment between the ApaI and XbaI restriction sites of the AHLG vector with a gene, while keeping the other nucleotide sequences of the AHLG vector unchanged. This recombinant expression vector expresses the D14-4SD protein, which is 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 acid sequences unchanged.

[0084] 2. Obtaining overexpression plants

[0085] (1) Transformation

[0086] use d14 The mutant serves as a rice acceptor, transforming the rice seedlings... d14 After removing the husk from the mutant seeds, they were surface-sterilized with 70% (v / v) ethanol for 1 min, followed by vortex washing with 2.5% (w / v) sodium hypochlorite for 45 min, and then rinsed three times with sterile water. The seeds were then sown on NB solid medium and incubated at 28ºC in the dark for two weeks. Once callus tissue grew from the mature embryo scutellaria, it was excised and subcultured on fresh NB solid medium, transferred every 7 days. After 3-4 subcultures, Agrobacterium infection could be performed.

[0087] The composition of 1 L LB solid culture medium is: Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g, and agar 15 g.

[0088] The composition of 1 L LB liquid culture medium is: Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g.

[0089] Composition of rice conversion solution: The following reagents were added to NB basic medium: Inositol 2 g / L, Glutamine 2 g / L, Casein hydrolysate 500 mg / L, 10% Synperonic PE 10 ml / L, and Acetosyringone 100 μM.

[0090] When the rice callus tissue is in good condition, the plant transgenic expression vector (AHLG-) is introduced. Act:D14-GFP Vector, AHLG- Act:D14 4SA -GFP、 AHLG- Act:D14 4SD -GFP The cells were transformed into Agrobacterium EHA105 strain by high voltage electroporation and plated on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin for 2-3 days. For each transformation, 4-5 single colonies were picked and inoculated into 7 mL of LB liquid medium containing kanamycin and 25 mg / L rifampin. The cells were then incubated overnight at 28ºC and 250 rpm. The cells were centrifuged at 3,000 rpm for 5 min at room temperature and resuspended in rice transformation solution for transformation of rice callus tissue.

[0091] (2) Agrobacterium EHA105 infects rice callus tissue

[0092] 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, place at room temperature for 20 min, shaking occasionally during the period, then take out the transformed callus tissue, absorb the excess bacterial solution with sterile filter paper, and incubate in a 23ºC incubator in the dark for 2-3 days to obtain transgenic callus tissue.

[0093] (3) Differentiation

[0094] The rice callus obtained in step (2) was transferred to NB solid selection medium containing hygromycin and cultured at 28ºC in the dark for 7-10 days. The culture was then subcultured into a new NB solid selection medium containing hygromycin for 3-4 rounds of hygromycin selection. The callus in good condition was propagated to differentiation medium for differentiation and regeneration, and seedlings were cultured at 28ºC under light for about one month. The resulting seedlings were then transferred to rooting medium and cultured for about 4 weeks to promote rooting and seedling growth. After about one month of growth in a greenhouse, the seedlings were transplanted to the field.

[0095] 3. Identification of positive plants

[0096] The specific methods are as follows: (1) Leaves were taken from individual rice plants, and rice genomic DNA was extracted using the CTAB method. Using the extracted DNA as a template, PCR amplification was performed using AHLG-F and D14-570-R identification primers. The PCR products were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger sequencing. After obtaining the sequencing results, the sequences were compared. Those sequences that matched the expected sequences were T0 generation positive plants, and 16 plants were obtained that were transformed with AHLG-F. Act:D14-GFP T0 generation positive single plant of the vector (named Act:D14-GFP / d14 10 plants were converted to AHLG- Act:D14 4SA -GFP T0 generation positive single plant of the vector (named Act: D14 4SA -GFP / d14 15 plants were converted to AHLG- Act:D14 4SD -GFP T0 generation positive single plant 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 were propagated, and 8 T1 generation lines were planted. Six individual plants were randomly collected from each T1 generation line for screening homozygous positive individual plants. (3) The harvested T1 generation seeds were germinated, and 40 germinated seeds were placed on 0.5% Agar solid medium containing 50 μg / mL hygromycin (Hyg). The root growth of the plants was observed after 2-3 days. Plants with normal root growth were positive, while those without were pseudo-individual plants. If all seeds of each plant could grow roots normally, it was a homozygous positive line.

[0097] Obtained respectively D14 Primers for identifying homozygous mutant transgenic rice seedlings with gene mutations:

[0098] AHLG-F: 5'-TCAGCATTGTTCATCGGTAG-3';

[0099] D14-570-R: 5'-CTGCTGTATCCTCCTCCAGCTC-3'.

[0100] PCR reaction mixture: 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, 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 15 s; 72℃ extension for 5 min.

[0101] A total of 6 plants were obtained. Act:D14-GFP / d14 homozygous positive single plant 、 5 strains Act:D14 4SA -GFP / d14 homozygous positive single plant and 5 plants Act:D14 4SD -GFP / d14 Homozygous positive single plants. The single plants mentioned above 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 .

[0102] 4. SDS-PAGE analysis of D14-GFP phosphorylation in transgenic callus

[0103] 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 °C.

[0104] (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#1Total protein from callus tissue was extracted using the following method: 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 and 50 μM MG132) Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1 Total protein from callus tissue was collected, with each type of callus tissue divided into two aliquots, resulting in a total of six samples. Each sample was added with 20 µL of GFP-Trapbeads and incubated at 4ºC for 3 h with inversion. The samples were then washed three times with IP protein extraction buffer, and finally, 50 µL of IP protein extraction buffer was added. Act:D14-GFP / d14#? , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1 One sample of callus IP protein was added with 20 μL of protein phosphatase (λPP), while the other served as a control. After reacting at 30°C for 2 h, protein loading buffer was added and the samples were boiled. SDS-PAGE and phos-tag gel electrophoresis were then performed.

[0105] (2) The phosphorylation of D14-GFP protein in the callus tissue treated in step (1) was detected 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 and the time to 1-2 h. (2) Use semi-dry transfer method to transfer the protein to PVDF or NC membrane. 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 is completed, put the PVDF or NC membrane into 4% skim milk powder and incubate at room temperature for 60 min at 70 rpm on a shaker. (4) Discard 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 primary antibody incubation, wash the PVDF or NC membrane three times with TBST, each time at 70 rpm on a shaker at room temperature for 5 min. Then add 4% skim milk containing secondary antibody and incubate at 60 rpm on a shaker at room temperature for 60 min. (6) After secondary antibody incubation, wash the membrane three times with TBST, each time at 70 rpm on a shaker at room temperature for 5 min. Add chromogenic solution and develop the protein using photographic film or a protein developing instrument. Detect the GFP fusion protein using GFP antibody (1:3000, Roche, Cat#11814460001).

[0106] The results showed that phosphorylation of D14-GFP could be achieved by treatment with protein phosphatase (λPP) or by mutation of the S10, S11, S13, and S15 sites to alanine (abbreviated as D14). 4SA The four mutations that suppress () Figure 2 This indicates that S10, S11, S13, and S15 are the major phosphorylation sites of OsD14.

[0107] 5. Ubiquitination and degradation of D14

[0108] 20 μM rac -GR24 working solution: dissolved in acetone rac -GR24 is obtained, where rac The concentration of -GR24 was 20 μM.

[0109] Experimental Methods: Vigorous growth, characterized by pale yellow color, smooth appearance, and compact morphology, was selected from Act:D14-GFP / d14 and Act:D14∆N-GFP / d14 rice calluses and transferred to new NB solid selection medium containing antibiotics. The calluses were cultured at 28°C in the dark for 4-6 days. Then, the calluses were transferred to NB liquid medium and cultured at 28°C for 50 rpm in the dark for 1-2 hours. For the ubiquitination experiment of D14, the above-mentioned calluses were... Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act: D14 4SD -GFP / d14#1 The callus tissues were pretreated with 50 μM MG132 for 1 hour. The callus tissues were treated with or without 20 μM rac-GR24 for 1 hour and then frozen in liquid nitrogen. Total protein was extracted 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 and 50 μM MG132). Then, 25 μL of GFP-Trap beads were added to 1 mL of supernatant and incubated gently at 4°C for 2 hours. The GFP-Trap beads were washed 3-5 times with IP washing 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), followed by elution with 50 μL SDS-PAGE buffer. SDS-PAGE and Western spectroscopy were then performed. Blot analysis.

[0110] For the degradation experiment of D14, 20 μM rac-GR24 working solution was added and treated for 1 h or 3 h. After treatment, callus tissue was collected and frozen with liquid nitrogen. The collected callus tissue was then mixed with 2 volumes of RIPA protein extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 1.0% (v / v) Nonidet P-40, 10 mM NaF, 2 mM EDTA, 0.1% SDS sodium orthovanadate, 1 x Roche cOmplete™ Protease Inhibitor Cocktail, 1 mM PMSF and 50 μM MG132), vortexed thoroughly, and placed on ice. The mixture was vortexed for 10 s every 8 min, 3-4 times, and then centrifuged. The mixture was then incubated at 4ºC for 18,000 seconds. g Centrifuge for 20 min, transfer 200 μL of supernatant to a new 1.7 mL centrifuge tube, and add 50 μL of protein loading buffer. Denature the sample at 100ºC for 5 min, and then perform Western blotting.

[0111] The results showed that D14 4SA It enhanced the ubiquitination and degradation of D14, while D14 4SD Significantly reduced the ubiquitination and degradation of D14 ( Figure 3 and 4 This indicates that phosphorylation can inhibit ubiquitination and degradation of rice D14.

[0112] 6. Count the number of tillers in homozygous positive plants.

[0113] The experimental method is as follows: Wild-type rice Nipponbare, d14 Mutant, the above homozygous plant 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#2In the field at the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing, each rice line was planted with 24 plants at a spacing of 17 cm between plants and rows, and the growing season was from May to November each year. The field phenotypic survey of the rice materials was conducted from September 20th to October 7th each year. The number of tillers was the number of effective tillers at maturity (i.e., the number of tillers that produced panicles), and the plant height was the distance from the tallest panicle to the ground. Representative individual plants were transplanted into pots and photographed using the average number of tillers for each material as the standard.

[0114] 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 a higher number of tillers than the wild type ( Figure 5 This indicates that D14 phosphorylation inhibits rice tiller development. Figure 5 ).

[0115] 7. Detect the expression level of SL response genes.

[0116] (1) Detection of strigolactone synthesis gene D10 Relative expression level of genes

[0117] 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) under 28ºC light for 16 h followed by 25ºC in darkness for 8 h, with a light intensity of 150-200 μMm. -2 s -1 The humidity was 70%. After culturing in Kimura B nutrient solution for 2 weeks, root tissues of seedlings from different materials were collected.

[0118] RNA extraction from rice seedlings was performed using the Trizol method (Life Technologies: 15596-026). All reagents and consumables used in the RNA extraction process were RNase-free. Rice tissue samples were thoroughly ground in liquid nitrogen, ensuring the samples remained frozen throughout the grinding process. Approximately 100-150 mg of powder 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. Then, 200 μL of chloroform was added, and the tube was immediately inverted for 15 s, allowed to stand at room temperature for 5 min, and then centrifuged at 4ºC for 12,000 seconds. gCentrifuge for 15 min; transfer 500 μL of supernatant to 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, then centrifuge at 4ºC for 12,000 minutes. g Centrifuge for 10 min; discard the supernatant, add 1 mL of 70% (v / v) ethanol to the RNA precipitate, invert 30 times, and centrifuge at 7,500 rpm. g Centrifuge for 5 min; discard the supernatant, and after the precipitate has dried, add 50 μL of RNase-free water and place on ice for at least 3 h to allow the RNA to fully dissolve; measure the OD values ​​at 230 nm, 260 nm, and 280 nm using a NanoDrop 2000 UV-Vis spectrophotometer to assess the overall quality of RNA preparation. RNA samples can be directly used for RNA reverse transcription experiments or placed at -20°C for later use.

[0119] Use TURBO DNA-free TM The kit removes residual DNA from total RNA. Detailed steps are described in the instructions. Each reaction volume is 20 μL, with 12.5 μg of total RNA. Incubate at 37ºC for 30 min, then add 2 μL of DNase inactivation reagent and mix thoroughly at room temperature for 5 min. Continue incubation at room temperature for 12,000 minutes. g Centrifuge for 5 min, and use 4 μL of the supernatant for reverse transcription experiments. Synthesize the first strand of cDNA using the SuperScript® III First-Strand Synthesis System reverse transcription kit. See the instructions for detailed steps. Finally, add 180 μL of nuclease-free double-distilled water to dissolve the reverse transcription product, mix thoroughly, and it is ready for use.

[0120] The SL-biosynthesis gene in the roots of transgenic rice seedlings was detected using quantitative real-time PCR. D10The transcription level was measured. Each reaction volume in the 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 quantitative PCR was a BIO-RAD real-time quantitative PCR instrument (model: CFX96). The PCR program parameters were set as follows: 98ºC, 30 s; (98ºC, 5 s → 60ºC, 5 s → data acquisition), 40 cycles; 60-95ºC, 0.5ºC / 5 s, data acquisition / 5 s. After the program was completed, the data was processed using BIO-RAD CFXManager software.

[0121] D10 Primers for quantitative real-time PCR to detect gene expression levels:

[0122] qD10-F: 5'-CGTGGCGATATCGATGGT-3';

[0123] qD10-R: 5'-CGACCTCCTCGAACGTCTT-3'.

[0124] qUbiquitin-F: 5'-AACCAGCTGAGGCCCAAGA-3';

[0125] qUbiquitin-R: 5'-ACGATTGATTTAACCAGTCCATGA-3'.

[0126] (2) Detection OsTB1 Relative expression level of genes

[0127] 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) under 28ºC light for 16 h followed by 25ºC in darkness for 8 h, with a light intensity of 150-200 μMm. -2 s -1The humidity was 70%. After culturing in Kimura medium for 2 weeks, stem base tissue (1 cm) of seedlings from different materials was collected. The rice tillering inhibitor gene in transgenic rice was detected using quantitative real-time PCR. OsTB1 The transcriptional level was determined using the method described above, with qUbiquitin as the internal reference.

[0128] OsTB1 Primers for quantitative real-time PCR to detect gene expression levels:

[0129] OsTB1-F: 5'-CGACAGCGGCAGCTACTAC-3';

[0130] OsTB1-R: 5'-GCGAATTGGCGTAGACGA-3'.

[0131] The results showed that, compared with the wild type, the strigolactone-responsive gene with the function of inhibiting rice tillering... OsTB1 exist Act:D14 4SA –GFP / d14 The expression level was reduced in the transgenic lines. Act:D14 4SD –GFP / d14 Increased expression levels in transgenic lines ( Figure 6 SL biosynthesis genes under feedback regulation D10 The level of expression at Act:D14 4SA –GFP / d14 The middle rises, while Act:D14 4SD –GFP / d14 The expression level in the middle decreased ( Figure 6 In summary, D14 phosphorylation enhances SL signaling and inhibits rice tillering.

[0132] Example 3: Regulating the application of rice by changing the phosphorylation state of D14

[0133] To analyze the regulatory role of OsD14 phosphorylation in the low-nitrogen response, the effects of different nitrogen contents on the number of rice tillers were investigated. The experiment was repeated three times, with each repetition as follows:

[0134] 1. Experimental Grouping

[0135] High-nitrogen group (HN): transgenic homozygous plants Act:D14-GFP / d14#1 , Act:D14 4SA -GFP / d14#1 and Act:D14 4SD -GFP / d14#1In a field planted at the transgenic base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Shunyi District, Beijing, 300 kg of nitrogen fertilizer was applied per hectare per line, with 24 plants planted at a spacing of 17 cm between plants and rows. The number of tillers was counted according to the aforementioned method.

[0136] Medium nitrogen group (MN): The difference between this group and the high nitrogen group (HN) is that the amount of nitrogen fertilizer applied is 150 kg per hectare, and the rest of the operation is the same as the high nitrogen group (HN).

[0137] Low nitrogen group (LN): The difference between this group and the high nitrogen group (HN) is that the amount of nitrogen fertilizer applied is 50 kg per hectare, and the rest of the operation is the same as the high nitrogen group (HN).

[0138] 2. Phosphorylation and protein levels of OsD14 under different nitrogen levels

[0139] Experimental Methods: To identify the phosphorylation and protein levels of the D14 phosphorylation site under different nitrogen conditions, the transgenic rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) under 28ºC light for 16 h followed by 25ºC in darkness for 8 h, with a light intensity of 150-200 μM m⁻² s⁻¹ and a humidity of 70%. After one week of cultivation in Kimura B nutrient solution, the seedlings were further cultured for 7-10 days in culture solutions with different nitrogen concentrations.

[0140] Table 1. Formula for Kimura B Nutrient Solution

[0141]

[0142] Note: When preparing a 20 mM FeSO4-EDTA solution, add 5.57 g FeSO4·7H2O and 7.45 g Na2-EDTA to 800 mL of deionized water, and heat in a 70°C oven (or water bath) for 1-2 days, stirring manually until completely dissolved. (NH4)6Mo7O 24 • 4H₂O can be replaced with Na₂MoO₄·2H₂O (0.128 mg / L). The pH of the rice nutrient solution should be between 5.5 and 6.0, and the nutrient solution needs to be changed every 2 days. When preparing culture solutions with different nitrogen concentrations, first prepare Kimura nutrient solution without nitrogen source. Then, replace ammonium ions ((NH₄)₂SO₄) and nitrate ions (KNO₃, Ca(NO₃)₂) with 1 M NH₄NO₃, and add the appropriate volume of NH₄NO₃ as needed to adjust the nitrogen concentration. Finally, add 0.365 mM CaCl₂ to replenish calcium ions. A final concentration of 0.15 mM NH₄NO₃ is a low-nitrogen hydroponic solution, and a final concentration of 2.5 mM NH₄NO₃ is a high-nitrogen culture solution.

[0143] Stem base tissue (1 cm) of seedlings from different materials was collected, and D14-GFP protein was enriched by immunoprecipitation. Total protein was extracted 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 a phosphatase inhibitor). Then, 25 μL of GFP-Trap beads were added to 1 mL of supernatant and incubated gently by rotation at 4°C for 2 hours. The beads were then washed 3–5 times with IP washing 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 a phosphatase inhibitor). Elute with 50 μL SDS-PAGE buffer, then perform SDS-PAGE and Western Blot as described above.

[0144] Experimental results show that under low nitrogen conditions, the phosphorylation level of OsD14 is enhanced. Figure 7 Meanwhile, the protein abundance of D14-GFP was significantly increased. Figure 8 This indicates that low nitrogen levels trigger the phosphorylation of OsD14, inhibiting its ubiquitination and degradation.

[0145] 3. Count the number of tillers

[0146] The number of rice tillers was counted according to the method described in Example 2, "6. Counting the number of tillers of homozygous positive plants".

[0147] The results showed that under low nitrogen conditions, the number of tillers in rice was significantly reduced ( Figure 9 This indicates that low-nitrogen conditions trigger D14 phosphorylation, stabilize the D14 protein, thereby enhancing SL sensing and inhibiting tiller development. Therefore, the response to low-nitrogen conditions can be regulated by modulating the phosphorylation level of OsD14. Simultaneously, D14... 4SA -GFP overexpression led to an increase in tiller number under low nitrogen conditions, indicating that the tiller number in rice under low nitrogen conditions can be regulated by altering the phosphorylation status of D14. Meanwhile, Figure 9 It also indicates that D14 4SD-GFP leads to a decrease in tiller number under high nitrogen conditions, further demonstrating the feasibility of improving rice tiller number by altering the phosphorylation status of the strigolactone receptor OsD14.

[0148] The sequences involved in the foregoing embodiments

[0149] SEQ ID NO:1 OsD14 Wild-type amino acid sequence of the gene

[0150] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0151] SEQ ID NO:2 OsD14 Wild-type CDS nucleotide coding sequence of the gene

[0152] 5'-ATGCTGCGATCGACGcatccgccgccCAGTAGCCCGAGCAGCAGcagcagcggcggcggcgggggcggggggtcgTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTA0CTAA-3'.

[0153] Mutant D144SA amino acid sequence

[0154] MLRSTHPPPAAPASASSGGGGGGGSSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0155] Mutant D14 4SD amino acid sequence

[0156] MLRSTHPPPDDPDSDSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSHGFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILASIRRPD LFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDISLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0157] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, characterized in that, The protein is a protein encoded by the nucleotide sequence of SEQ ID NO:2, in which the amino acids at positions 10, 11, 13, and 15 are all mutated from serine to alanine, while keeping the other amino acids of SEQ ID NO:1 unchanged.

2. Biomaterials, wherein the biomaterials are any one of the following: B1) A nucleic acid molecule encoding the protein 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

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