CN119161439A - Method for preparing rice with increased tillering number - Google Patents

Abstract

The invention discloses a method for preparing rice with increased tiller number, belonging to the technical field of genetic breeding. The method comprises the steps of carrying out mutation on a OsD coding gene in target rice containing OsD coding genes to obtain rice with increased tillering number, wherein the tillering number of the rice with increased tillering number is larger than that of the target rice, and the mutation is to mutate a 644 th amino acid codon of coding Os D3 in the coding genes in a target rice genome into a codon of coding other amino acids. Experiments prove that the mutant protein has reduced response to strigolactone signal due to reduced D3 function, so that the mutant protein has a dwarf multi-tillering phenotype, and the mutant creating method can be used for improving genetic characters.

Description

Method for preparing rice with increased tiller number

Technical Field

The invention belongs to the technical field of genetic breeding, and particularly relates to a method for preparing rice with increased tiller number.

Background

Strigolactone is a plant hormone synthesized in roots, enters overground parts of plants in an upward transportation mode, and inhibits the occurrence of plant branches. The strigolactone inhibits the discovery of plant branching, so that people fully realize that the occurrence of plant branching and the growth environment (nutrition condition) of plant roots are in close connection, and a direct research clue is provided for 'field fertilizer seedling strengthening' summarized in agricultural cultivation for thousands of years. Besides transporting to the overground part, strigolactone can be secreted from the root to the periphery of the rhizosphere and used as an important information molecule for communication between plants and the external environment, so that the symbiotic growth of plants and symbiotic arbuscular fungi can be promoted, and bad growth conditions can be overcome by a symbiotic method. Under the condition that the nutrition condition of the soil is unfavorable, the strigolactone is released to the periphery of the rhizosphere in a large amount, symbiotic arbuscular fungi and plants are attracted, the absorption and utilization capacity of the plants to the nutrition in the soil is improved, meanwhile, strigolactone is transported upwards to the stem, excessive nutrition growth of the plants is inhibited, the main stem is ensured to fully utilize limited nutrition, and the life cycle is completed.

In the traditional crop improvement, the yield is often increased by introducing a few genetic loci, such as a famous green revolution, and the introduction of genetic loci such as sd1, rht and the like changes plant height factors in plant types through regulating signal paths of gibberellin, so that lodging resistance of plants is realized. The strigolactone brings our hint, can coordinate plant type and its ecological environment of root synthetically, achieve the improvement of crop yield, namely we can regulate and control the function of branching through strigolactone and improve the overground plant type of the crops, make it suitable for the production practice on the one hand, on the other hand we can also initiate the symbiotic of plant and symbiotic arbuscular fungi through strigolactone, realize the optimization to the ecological microenvironment of plant growth, compare with traditional crop improvement, improvement to plant type and optimization to ecological environment at the same time, have apparent advantage. Meanwhile, the effect of strigolactone in plants is revealed continuously, and at present, strigolactone has been found to change root morphology, stimulate root nodule generation and the like, which shows that strigolactone has great potential and can comprehensively regulate the overall structure (above ground and underground) of plants. The strigolactone is very common in plants, and the symbiotic of the plants and symbiotic arbuscular fungi widely occurs in the plant kingdom, so that the strigolactone can realize the synergistic optimization of plant types and ecological environments in many crops. Compared with the traditional plant hormones of several major classes, strigolactone is found later, but the strigolactone is not prevented from becoming a plant hormone with wide application prospect.

However, strigolactone, which is an important hormone for controlling rice tillering, has extremely high tillering and dwarf phenotype with serious decrease in fruiting rate and yield, and is difficult to be directly applied to rice breeding practice. However, by finely regulating the function of strigolactone, it is possible to achieve the object of plant type improvement, for example, in the "Huazhan" restorer material of super rice variety, the weak allele mutation HTD1 ^HZ of strigolactone synthesis gene HTD1/D17 can effectively increase the tillering number of rice without affecting the setting rate, and the increase of biomass and yield is brought about, and HTD1 ^HZ and green revolution gene SD1 ^DGWG are commonly selected and widely used by breeders in high-yield rice breeding. OsD3 genes are derived from rice, belong to signal components of strigolactone pathway, and have the main functions of controlling rice plant types, including the tiller number and plant height of the rice. The method utilizes site-directed editing to create OsD functional reduction allelic mutants to accurately regulate and control strigolactone signal paths, regulate the tiller number and plant height of rice, and further improve the plant type of the rice.

Disclosure of Invention

The invention aims to solve the technical problem of a method for preparing rice with increased tiller number. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

In order to solve the technical problems, the invention provides the following technical scheme:

The invention provides a method for preparing rice with increased tillering number, which comprises the steps of mutating a coding gene OsD3 in target rice containing a coding gene OsD to obtain rice with increased tillering number, wherein the tillering number of the rice with increased tillering number is larger than that of the target rice, osD is protein with an amino acid sequence of SEQ ID No.1, 644 th amino acid of OsD is 644 amino acid of SEQ ID No.1, 644 th amino acid is leucine, and mutating a codon encoding the 644 th amino acid of OsD3 in the coding gene in a genome of the target rice to codons encoding other amino acids.

In the above method, the other amino acid may specifically be serine.

The invention also provides a method for increasing the tiller number of rice, which comprises the steps of mutating a coding gene in acceptor rice containing OsD coding genes to increase the tiller number of the acceptor rice, wherein OsD is protein with an amino acid sequence of SEQ ID No.1, the 644 th amino acid of OsD3 is the 644 th amino acid of SEQ ID No.1, the 644 th amino acid is leucine, and mutating codons of the coding genes in target rice genome, which code for the 644 th amino acid of Os D3, into codons of coding other amino acids.

In the above method, the other amino acid may specifically be serine.

The invention also provides an application, wherein the application is the application of a protein or a substance for regulating the expression of a coding gene of the protein or a substance for regulating the activity and/or the content of the protein in any one of the following:

m1) regulating and controlling the tillering number of plants;

M2) preparing a product for regulating and controlling the tillering number of plants;

M3) plant breeding;

the protein is OsD, and OsD is a protein with an amino acid sequence of SEQ ID No. 1.

In the above application, the modulation is inhibition or reduction or downregulation of the expression of the OsD gene encoding said OsD or the content or activity of said OsD.

In the above application, the substance is any one of the following:

C1 A nucleic acid molecule that inhibits or reduces or down-regulates the expression of the gene encoding OsD;

C2 Expression of the gene encoding the nucleic acid molecule of C1);

C3 An expression cassette containing the gene of C2);

c4 A recombinant vector comprising the gene of C2) or a recombinant vector comprising the expression cassette of C3);

C5 A recombinant microorganism comprising the gene of C2), or a recombinant microorganism comprising the expression cassette of C3), or a recombinant microorganism comprising the recombinant vector of C4);

c6 A transgenic plant cell line containing the gene of C2), or a transgenic plant cell line containing the expression cassette of C3), or a transgenic plant cell line containing the recombinant vector of C4);

C7 A transgenic plant tissue containing the gene of C2), or a transgenic plant tissue containing the expression cassette of C3), or a transgenic plant tissue containing the recombinant vector of C4);

C8 A transgenic plant organ containing the gene of C2), or a transgenic plant organ containing the expression cassette of C3), or a transgenic plant organ containing the recombinant vector of C4).

Among the above, the expression cassette containing a nucleic acid molecule as described in C2) refers to a DNA capable of expressing the above-described protein in a host cell. The expression cassette may also include single-or double-stranded nucleic acid molecules expressing all the regulatory sequences necessary for the nucleic acid molecules of any of the proteins described above or the DNA of the RNA molecule. The regulatory sequences are capable of directing the expression of the DNA of any of the above proteins or the RNA molecules in a suitable host cell under conditions compatible with the regulatory sequences. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include promoters and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector in order to ligate the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a ligated regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e.a nucleic acid sequence which is recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein or the DNA of the RNA molecule. The promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The control sequence may also be a suitable transcription termination sequence, a sequence that is recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequences may also be suitable leader sequences, i.e., untranslated regions of mRNA which are important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that is functional in the host cell of choice may be used in the present invention. The regulatory sequence may also be a signal peptide coding region which codes for an amino acid sequence attached to the amino terminus of the protein and which directs DNA encoding the protein or the RNA molecule into the cell's secretory pathway. Signal peptide coding regions that direct the expressed protein or the DNA of the RNA molecule into the secretory pathway of host cells used may be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or the DNA of the RNA molecule depending on the growth of the host cell. Examples of regulatory sequences are those systems which are capable of opening or closing gene expression in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those which enable the amplification of a gene.

Among the above, the vector may be a plasmid, cosmid, phage or viral vector.

Among the above, the microorganism may be yeast, bacteria, algae or fungi, such as agrobacterium.

In the above, none of the transgenic plant cell lines include propagation material.

The invention also provides an application, wherein the application is the application of a protein or a substance for regulating the expression of a coding gene of the protein or a substance for regulating the activity and/or the content of the protein in any one of the following:

m1) regulating and controlling the tillering number of plants;

M2) preparing a product for regulating and controlling the tillering number of plants;

M3) plant breeding;

The protein is OsD mutant, and the OsD mutant is a protein which mutates 644 th amino acid of SEQ ID No.1 from leucine to serine and keeps other amino acids of SEQ ID No.1 unchanged.

In the above application, the modulation is up-regulation or enhancement or increase of the expression of the OsD mutant encoding gene or the content or activity of the OsD mutant.

In the above application, the nucleic acid molecule of B1) above is a gRNA targeting the coding gene of OsD.

The target sequence of the gRNA may be 5'-CCGGAGCCTGACATTGCCAG-3'. The target sequence of the gRNA targets nucleotides 2000-2019 of the wild-type genomic sequence of the OsD gene.

The aforementioned proteins, nucleic acid molecules and substances, i.e. biological materials, are also within the scope of the present invention.

The nucleic acid molecule may specifically be a DNA molecule in which the coding sequence of the coding strand is obtained by mutating the 1931 st thymine deoxyribonucleotide of SEQ ID No.2 to cytosine deoxyribonucleotide while keeping the other nucleotides of SEQ ID No.2 unchanged.

The allelic mutation creating method provided by the invention can accurately obtain the mutant with reduced functions of the rice tillering control gene OsD, and the mutant shows reduced response to strigolactone signals due to reduced functions of D3, so that the mutant presents a dwarf multi-tillering phenotype, and the mutant creating method can be used for genetic character improvement.

Drawings

FIG. 1 shows the effect of a yeast assay to detect the D3 key amino acid site mutation on D14 and D3 interactions.

FIG. 2 shows the effect of rice protoplast assay to detect D3-critical amino acid site mutation on D14 and D3 interactions.

FIG. 3 is genomic editing information for rice CRI-d3 ^L644S. And constructing the CRI-d3 ^L644S genome site-directed editing material by adopting an adenine base editing technology. Grey highlighted TT is the predicted edited base when the target was designed.

The rice CRI-d3 ^L644S phenotype is shown on the left of FIG. 4, scale 20 cm. On the right are statistical analyses of the tillers of wild type and mutant CRI-d3 ^L644S, the values represent mean ± standard deviation (n=15).

FIG. 5 shows D53 protein levels of wild type (NP) and mutant CRI-D3 ^L644S.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The following examples were run using GRAPHPAD PRISM statistical software and the experimental results were expressed as mean ± standard deviation using a two-tailed student t-test, P <0.05 (, P <0.01 (, P <0.001 (, ns (not significant)).

Example 1 analysis of the Critical loci of the D3 Gene affecting the interaction of D3 with D14

1. Yeast two-hybrid experiments

Corresponding vectors include ：pGADT7、pGADT7-D14ΔN、pGBKT7、pGBKT7-OsSKP1-D3、pGBKT7-OsSKP1-D3^T599A、pGBKT7-OsSKP1-D3^D606A、pGBKT7-OsSKP1-D3^L644A、pGBKT7-OsSKP1-D3^H668A、pGBKT7-OsSKP1-D3^R702A and pGBKT7-OsSKP1-D3 ^E704A. pGADT7 (AD) and pGBKT7 (BK) are described in non-patent literature "Song,X.,Lu,Z.,Yu,H.,Shao,G.,Xiong,J.,Meng,X.,Jing,Y.,Liu,G.,Xiong,G.,Duan,J.,et al.(2017).IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice.Cell Res.27,1128-1141."., which is a biological material available from research on genetics and developmental biology of national academy of sciences, and which is used only for repeated experiments related to the present invention and not for other uses.

The construction primers for pGADT7-D14ΔN (AD-D14ΔN) were:

AD-NdeI-D14ΔN-F:

AD-EcoRI-D14ΔN-R:

Among the above primers, the black bolded sequence was the primer for amplification of d14Δn, and the underlined sequence was used for homologous recombination with pGADT7 vector. The AD-D14 delta N vector is a recombinant expression vector obtained by replacing small fragments between NdeI and EcoRI cleavage recognition sites of the pGADT7 vector with a OsD delta N gene CDS nucleotide sequence and keeping other nucleotide sequences of the pGADT7 vector unchanged.

The construction primers for pGBKT7-OsSKP1-D3 (BK-OsSKP 1-D3) were:

BK-NdeI-OsSKP1-D3-F:

BK-EcoRI-OsSKP1-D3-R:

Among the above primers, the black bolded sequence was the primer for amplifying OsSKP a 1-D3, and the underlined sequence was used for homologous recombination with pGBKT7 vector. The BK-OsSKP1-D3 vector is a recombinant expression vector obtained by replacing a small fragment between NdeI and EcoRI cleavage recognition sites of the pGBKT7 vector with a CDS nucleotide sequence encoded by OsSKP1-D3 fusion protein and keeping other nucleotide sequences of the pGBKT7 vector unchanged.

To construct D3 mutant amino acid forms including pGBKT7-OsSKP1-D3^T599A、pGBKT7-OsSKP1-D3^D606A、pGBKT7-OsSKP1-D3^L644A、pGBKT7-OsSKP1-D3^H668A、pGBKT7-OsSKP1-D3^R702A and pGBKT7-OsSKP1-D3 ^E704A, final vectors of amino acid site-directed mutations were obtained by cyclic amplification using BK-OsSKP1-D3 vectors as templates, respectively, with corresponding mutation-introducing primers. In the following primers, the base sequence of the site-directed mutant amino acid site is bolded in black.

D3-T599A-F:

D3T599A-R:

D3-D606A-F:

D3-D606A-R:

D3-L644A-F:

D3-L644A-R:

D3-H668A-F:

D3-H668A-R:

D3-R702A-F:

D3-R702A-R:

D3-E704A-F:

D3-E704A-R:

Experimental methods Yeast plasmid transformation was based on the experimental procedure in DUAL membrane STARTER KITS and fine-tuned according to laboratory foreman experimental experience, the specific procedures were (1) streaking Y2HGold Yeast strain in YPDA solid medium, culturing for 2-3 days at 30℃with inversion to single colony appearance. (2) 3-4 single colonies were picked and cultured in 3mL YPDA liquid medium at 30℃for 12-14h at 250 rpm. (3) 3mL of the bacterial liquid is inoculated into 30mL of YPDA liquid culture medium, the culture is continued for 3-5h at 250rpm under the condition of 30 ℃, and the culture is centrifuged for 5min at 700g at room temperature until OD ₆₀₀ reaches 0.6-0.8. (4) After discarding the supernatant, the pellet was resuspended in 30mL of sterile water and centrifuged once more at 700g for 5min. (5) After discarding the supernatant, the added sterile medium volume of resuspended yeast cells was determined based on the number of experimental reactions, and the resuspended yeast competent cells were placed on ice and waited for transformation. (6) In preparation of yeast competent cells, salmon sperm single-stranded ssDNA was denatured, boiled at 100℃for 5min, and then placed on ice for 5min, and repeated 2-3 times. (7) The configured PEG/LiOAc reaction mixtures were determined based on the number of experimental reactions, each containing 240. Mu.L of 50% PEG, 36. Mu.L of LiOAc and 25. Mu.L of salmon sperm single stranded ssDNA. (8) To the sterilized 1.7mL centrifuge tube, the corresponding carrier (see above), 300. Mu.L of PEG/LiOAc reaction mixture and 100. Mu.L of yeast competent cells were added, and after mixing well, incubated at 42℃for 45min. (9) The thalli are collected by centrifugation at 700g for 5min at room temperature, the supernatant is discarded and then resuspended in 200 mu L of 0.9% NaCl solution, and all the supernatant is uniformly coated on SD-Leu/-Trp solid yeast defect culture medium, and the culture is inverted and carried out at the constant temperature of 30 ℃ for 3-4 days until colonies appear. (10) About 10 single clones were picked from SD-Leu/-Trp solid yeast defect medium and placed in 100. Mu.L of 0.9% NaCl solution, while 10-fold and 100-dilution yeast solutions were prepared. (11) 6. Mu.L of yeast suspension was spotted on SD-Leu/-Trp, SD-Leu/-Trp/-His/-Ade containing acetone and SD-Leu/-Trp/-His/-Ade containing 10. Mu.M rac-GR24 solid yeast defect medium, and after inversion culture at 30℃for 3-7 days, photographic analysis was performed.

The formula of the yeast culture medium comprises:

1L of YPAD broth included 10g of Bacto-eye extract, 20 g of Bacto-peptone.

20 Grams of Dextrose and 100 milligrams Adenine sulfate.

1L of YPAD solid medium was prepared by adding 15 g of Agar to the above YPAD liquid medium.

1L of yeast amino acid deficiency medium comprises 6.7g of yeast nitrogen source basal medium without amino acid, 0.6-0.7g of Dropout mix (added according to the specification of specific deficiency SD), 20g of Glucose and 20g of Agar, and the formulation of the yeast medium needs to be autoclaved at 121 ℃ for 15min and stored at 4 ℃.

The results indicate that after D606, L644 and E704 sites of D3 on the interaction interface were mutated, the interaction of D14 with D3 in yeast was lost, and after T599, H668 and R702 sites of D3 were mutated, the interaction of D14 with D3 in yeast was significantly reduced (fig. 1 a).

2. Yeast three-hybrid experiments

The corresponding vectors include ：pGADT7-D53、pBridge、pBridge-MCS1-OsSKP1-D3-MCS2-Blank、pBridge-MCS1-OsSKP1-D3-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^T599-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^D606A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^L644A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^H668A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^R702A-MCS2-D14ΔN and pBridge-MCS1-OsSKP1-D3 ^E704A -MCS 2-D14. DELTA.N. Wherein pBridge is an empty vector, and is described in non-patent literature "Yu,H.,Yang,L.,Long,H.,Su,X.,Wang,Y.,Xing,Q.,Yao,R.,Zhang,M.,and Chen,L.(2022).Strigolactone signaling complex formation in yeast:A paradigm for studying hormone-induced receptor interaction with multipledownstream proteins.Methods Enzymol.674,519-541."., the biological material is obtained from research on genetics and developmental biology of national academy of sciences, and is only used for repeated experiments related to the invention, and can not be used for other purposes. pGADT7-D53, abbreviated as AD-D53, is described in non-patent literature "Song,X.,Lu,Z.,Yu,H.,Shao,G.,Xiong,J.,Meng,X.,Jing,Y.,Liu,G.,Xiong,G.,Duan,J.,et al.(2017).IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice.Cell Res.27,1128-1141."., and is a biological material available from research on genetics and developmental biology of national academy of sciences, which is used only for repeated experiments related to the present invention and is not used for other purposes.

The pBridge-MCS1-OsSKP1-D3-MCS2-Blank vector is a recombinant expression vector obtained by adding a CDS nucleotide sequence encoded by OsSKP1-D3 fusion protein to the BamH1 single cleavage recognition site of the pBridge vector and keeping other nucleotide sequences of the pBridge vector unchanged.

The construction primers for pBridge-MCS1-OsSKP1-D3-MCS2-Blank are:

BamHI-OsSKP1-D3-F:

BamHI-OsSKP1-D3-R:

Among the above primers, the black bolded sequence was the primer for amplifying OsSKP a 1-D3, and the underlined sequence was used for homologous recombination with pGBKT7 vector.

The pBridge-MCS1-OsSKP1-D3-MCS2-D14 delta N vector is a recombinant expression vector which is obtained by adding the CDS nucleotide sequence of the OsD delta N gene between the NotI and BglII cleavage recognition sites of the pBridge-MCS1-OsSKP1-D3-MCS2-Blank vector and keeping the other nucleotide sequences of the pBridge-MCS1-OsSKP1-D3-MCS2-Blank vector unchanged.

Not I-MCSI I-D14ΔN-F:

BglII-MCSII-D14ΔN-R:

Among the above primers, the black bolded sequence is the primer for amplifying D14ΔN, and the underlined sequence is used for homologous recombination with pBridge-MCS1-OsSKP1-D3-MCS2-Blank vector.

To construct the D3 mutant amino acid forms comprising pBridge-MCS1-OsSKP1-D3^T599A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^D606A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^L644A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^H668A-MCS2-D14ΔN、pBridge-MCS1-OsSKP1-D3^R702A-MCS2-D14ΔN and pGBKT7-OsSKP1-D3 ^E704A, respectively, the corresponding introduced mutations were used the primer of (2) takes BK-OsSKP1-D3 carrier as template for cyclizing amplification to obtain final carrier of amino acid site-directed mutation. Among the primers described below, the following primers, the black bolded is the base sequence of the site-directed mutant amino acid site.

D3-T599A-F:

D3-T599A-R:

D3-D606A-F:

D3-D606A-R:

D3-L644A-F:

D3-L644A-R:

D3-H668A-F:

D3-H668A-R:

D3-R702A-F:

D3-R702A-R:

D3-E704A-F:

D3-E704A-R:

Experimental methods Yeast triple hybridization methods refer to the steps (1) to (10) in the "1 st, yeast double hybridization experiment" experimental methods in example 1. The final step was adjusted by spotting 6. Mu.L of yeast suspension onto SD-Leu/-Trp, SD-Leu/-Trp/-His/-Ade/-Met containing acetone and SD-Leu/-Trp/-His/-Ade-Met containing 10. Mu.M rac-GR24 solid yeast defect medium and performing photographic analysis after culturing for 3-7 days at 30℃in an inverted state.

The results indicate that the D3T 599A, D606A, L644A, H668A, R a and E704A mutations impair or block the formation of the D3-D14-D53 complex (B in fig. 1).

3. D3 mutant inhibits rac-GR24 induced D14-D3 interaction

The experimental method comprises the following steps:

The 35S: D14-FLAG plasmid is a recombinant expression plasmid obtained by inserting the OsD gene CDS nucleotide sequence into 35S: FLAG. Wherein the primer sequences are:

OsD14-SpeI-SCC-3FLAG-F:

OsD14-XhoI-SCC-3FLAG-R:

among the above primers, the black bolded sequence is the primer for amplifying OsD14, and the underlined sequence is used for homologous recombination with 35S: FLAG vector.

The 35S: D3-GFP plasmid is a recombinant expression plasmid obtained by inserting the OsD gene CDS nucleotide sequence into 35S: GFP. Wherein the D3 mutant amino acid site-directed mutagenesis primer is used for double hybridization and triple hybridization experiments of yeast.

The 35S: D3 ^T599A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^T599A into 35S: GFP. D3 ^T599A is obtained by mutating the amino acid position A at position 599 of the amino acid sequence of D3.

The 35S: D3 ^H668A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^H668A into 35S: GFP. D3 ^H668A is obtained by mutating the amino acid position A at position 668 of the amino acid sequence of D3.

The 35S: D3 ^E704A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^E704A into 35S: GFP. D3 ^E704A is obtained by mutating the amino acid position 704 of the amino acid sequence of D3 from the E mutation position A.

The 35S: D3 ^D606A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^D606A into 35S: GFP. D3 ^D606A is obtained by mutating the amino acid position A at position 606 of the amino acid sequence of D3.

The 35S: D3 ^L644A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^L644A into 35S: GFP. D3 ^L644A is obtained by mutating the amino acid position A at position 644 of the amino acid sequence of D3.

The 35S: D3 ^R702A -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^R702A into 35S: GFP. D3 ^R702A is obtained by mutating the amino acid position 702 of the amino acid sequence of D3 from the position A of R.

The 35S: D3 ^3M -GFP plasmid is a recombinant expression plasmid obtained by inserting the sequence of D3 ^3MA into 35S: GFP. D3 ^3M is obtained by mutating amino acid position 606 from D mutation position A, amino acid position 644 from L mutation position A and amino acid position 702 from R mutation position A of the amino acid sequence of D3.

Protoplasts of rice seedlings were prepared according to reference "Bart,R.,Chern,M.,Park,C.J.,Bartley,L.,and Ronald,P.C.(2006).A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.Plant Methods 2,13." and plasmids 35S: D3-GFP plasmid, 35S: D3 ^T599A -GFP plasmid, 35S: D3 ^H668A -GFP plasmid, 35S: D3 ^E704A -GFP plasmid, 35S: D3 ^D606A -GFP plasmid, 35S: D3 ^L644A -GFP plasmid, 35S: D3 ^R702A -GFP plasmid or 35S: D3 ^3M -GFP plasmid were co-transformed with the 35S: D14-FLAG plasmid, respectively, in the aforementioned rice protoplasts.

Rice protoplast co-immunoprecipitation was performed by combining 10 reactions (10 2mL centrifuge tubes) each, culturing at 28℃for 14h in the absence of light, followed by centrifugation at 150g for 3min to collect protoplasts, adding 0.2mL of W5 solution (154mM NaCl,125mM CaCl ₂, 5mM KCl,2mM MES,pH 5.7) to each 2mL centrifuge tube to resuspend the transformed 35S: D3-GFP plasmid+35S: D14-GFP plasmid, 35S: D3 ^T599A -GFP plasmid+35S: D14-GFP plasmid, 35S: D3 ^H668A -GFP plasmid+35S: D14-GFP plasmid or 35S: D3 ^E704A -GFP plasmid+35S: D14-GFP plasmid, 35S: D3 ^D606A -GFP plasmid +35S: D14-GFP plasmid, 35S: D3 ^L644A -GFP plasmid +35S: D14-GFP plasmid, 35S: D3 ^R702A -GFP plasmid +35S: D14-GFP plasmid or 35S: D3 ^3M -GFP plasmid +35S: D14-FLAG plasmid, 10 reactions in each combination were combined into one 5mL centrifuge tube and aliquoted into two new 2mL centrifuge tubes, 1. Mu.L of 50mM rac-GR24 or 1. Mu.L of acetone was added to the protoplasts, after incubation at 28℃for 1h in the dark, protoplasts were collected by centrifugation at 150g for 3min, the protoplasts were lysed with 1mL of IP protein extract (50mM Tris-HCl(pH 7.5),150mM NaCl,10％(v/v)glycerol,0.5％ Nonidet P-40,1x Roche cOmplete^TMProtease Inhibitor Cocktail,1mM PMSF and 50μM MG132), centrifuged at 4℃for 10min at 18,000g, the supernatants were collected into new 1.7mL centrifuge tubes, 1. Mu.L of 50mMrac-GR24 or 1. Mu.L of acetone were added, 25. Mu.L of GFP-Trap (ChromoTek, cat# Gta-200) were simultaneously added, and after incubation for 3h in 4℃with a flip-over incubation with IP protein wash (50mM Tris-HCl(pH 7.5),150mM NaCl,10％(v/v)glycerol,0.1％ Nonidet P-40,1x Roche cOmplete^TMProtease Inhibitor Cocktail), 25. Mu.L of protein extract was added, vortexing was vortexed. Samples were denatured at 100℃for 5min, after which Western Blot experiments were performed in which FLAG antibody was murine monoclonal antibody (Sigma-Aldrich, cat#F1804) diluted at a ratio of 1:3,000 and GFP antibody was murine monoclonal antibody (Roche, cat# 11814460001) diluted at a ratio of 1:3,000.

The results indicate that the D14-D3 interactions induced by rac-GR24 are also significantly inhibited by the D3T 599A, D606A, L644A, H668A, R A and E704A mutations in rice (FIGS. 2A and B).

The triple mutation of D606A, L644A and R702A completely blocked strigolactone-induced D14 and D3 interactions (B in fig. 2).

From this, it can be seen that the D3 amino acid in the AtD14-D3-ASK1 complex interaction interface is very important for direct interaction of D14 and D3.

EXAMPLE 2D 3 Gene L644 Multi-tillering phenotype of fixed-point editing mutant

The pH-PABE-7 (pH-PABE-7-sgRNA) vector source is described in non-patent literature "Li,C.,Zong,Y.,Wang,Y.,Jin,S.,Zhang,D.,Song,Q.,Zhang,R.,and Gao,C.(2018).Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion.Genome Biol.19,59.",, the biological material is obtained from research on genetic and developmental biology of the national academy of sciences, and is only used for repeated experiments related to the invention, and can not be used for other purposes.

1. Vector construction

The recombinant vector D3L644-pH-PABE-7 is obtained by inserting nucleotide CCGGAGCCTGACATTGCCAG at 1917-1936 (namely nucleotide 2000-2019 of the wild-type genome sequence of OsD gene) in SEQ ID No.2 into Bsa I cleavage recognition site of the pH-PABE-7 vector, and keeping other nucleotide sequences of the pH-PABE-7 vector unchanged.

D3L644 genome site-specific oligo linker primer:

D3L644-ABE-F:

D3L644-ABE-R:

Wherein the black bolded line is the editing target sequence of D3.

1) The adaptor primers D3L644-ABE-F and D3L644-ABE-R were dissolved in 100. Mu.m mother liquor, 5. Mu.L each was taken and mixed to obtain a primer mixture, and further diluted to 1. Mu.M with water. The mixture was left at 95℃for 30 seconds, and then the PCR instrument was turned off, and the annealing was completed to obtain an oligo dimer linker.

2) Cleavage of the pH-PABE-7 vector 2. Mu.g of the pH-PABE-7 vector was cleaved with 25U of Bsa I in a 50. Mu.L reaction system for 5h, followed by purification to give the cleaved pH-PABE-7 vector.

The adaptor was ligated with the digested vector in 1. Mu.L of 10 XT 4 DNA LIGASE buffer, 1. Mu. L T4 DNA LIGASE, 0.5. Mu.L of diluted sgRNA mixture (oligo-dimer adaptor) and 1. Mu.L of digested linear vector (pH-PABE-7 vector) and finally ddH ₂ O was added to a total volume of 10. Mu.L and ligated overnight at 16℃to give D3L644-pH-PABE-7 recombinant vector.

2. Obtaining transgenic plants

(1) Transformation

Seeds of japonica rice Nippon-P to be transformed were dehulled using japonica rice Nippon-P as a rice receptor, surface-sterilized with 70% (v/v) ethanol for 1min, then washed with 2.5% (w/v) sodium hypochlorite by rotation for 45min, washed three times with sterile water, and then sown on NB solid medium, and left to stand at 28℃for two weeks in a dark culture. After the callus grows out from the mature embryo scutellum, cutting off the callus, subculturing the callus on a new NB solid medium, transferring the callus onto the new NB solid medium every 7 days, and carrying out infection of agrobacterium after 3-4 times.

1L LB solid medium consists of 10g of Tryptone, 5g of Yeast Extract, 10g of NaCl and 15g of agar.

1L LB liquid culture medium comprises 10g of Tryptone, 5g of Yeast Extract and 10g of NaCl.

The rice transformation liquid comprises adding the following reagents into NB basic culture medium ,Inositol 2g/L、Glutamine 2g/L、Casein hydrolysate 500mg/L、10％ Synperonic PE 10ml/L、Acetosyringone 100μM.

When the growth state of the rice callus is good, the recombinant vector D3L644-pH-PABE-7 is transformed into an agrobacterium EHA105 strain through high-voltage excitation, and is coated on LB solid medium containing 50mg/L kanamycin and 25mg/L rifampicin for 2-3 days, 4-5 monoclonals of each transformation are inoculated into 7mL LB liquid medium containing kanamycin and 25mg/L rifampicin, then the culture is carried out for overnight under the condition that the parameters are set to 28 ℃, the cells are centrifuged for 5min at 3,000rpm at room temperature, and the rice transformation liquid is used for transformation of the rice callus.

(2) Infection of rice callus by agrobacterium EHA105

Collecting the callus in good state obtained in the step (1), adding a proper amount of transformation liquid until the callus is completely submerged by the bacterial liquid, standing at room temperature for 20min, shaking at intervals, taking out the transformed callus, sucking the excessive bacterial liquid by using sterile filter paper, and carrying out light-proof static culture in a 23 ℃ incubator for 2-3 days to obtain the transgenic callus.

(3) Differentiation

Transferring the rice callus obtained in the step (2) to a hygromycin-containing NB solid screening medium, culturing at 28 ℃ in a dark place for 7-10 days, and then subsequently transferring to a new hygromycin-containing NB solid screening medium, wherein the hygromycin-containing NB solid screening medium is 3-4 rounds of hygromycin screening medium. And (3) propagating the callus with good growth state to a differentiation culture medium for differentiation regeneration and seedling emergence, transferring the grown seedlings to a rooting culture medium for culturing for about 4 weeks after culturing for about one month at 28 ℃, transplanting to a greenhouse for growing for about one month, and transferring to a field.

3. Positive plant identification

The specific method is that leaves are respectively taken from rice according to single plants, and the genome DNA of the rice is extracted by a CTAB method. The extracted DNA is used as a template, the PCR amplification is respectively carried out by using D3-detect (1563) -transgenic-F and D3-detect (2533) -transgenic-R identification primers, and the PCR products are sent to the Beijing Rui Boxing family biotechnology Co., ltd for Sanger sequencing, thus obtaining the sequencing result. The sequencing result is compared with the Japanese sunny sequence of the wild type plant, and if only one PCR amplification product of the regenerated plant is identical with the nucleotide sequence of the Japanese sunny PCR amplification product of the wild type plant, the regenerated plant is wild type. If the regenerated plant has two PCR amplification products, one of which is identical to the nucleotide sequence of the PCR amplification product of Japanese sunny of the wild-type plant and the other of which is mutated (mutation includes deletion, insertion or substitution of one or more nucleotides) as compared with the nucleotide sequence of the PCR amplification product of Japanese sunny of the wild-type plant, the regenerated plant is heterozygous. If the regenerated plant has two PCR amplification products, the regenerated plant is double allelic mutant, in which the PCR amplification products are mutated (the mutation includes deletion, insertion or substitution of one or more nucleotides) as compared with the nucleotide sequence of the PCR amplification product of Japanese sunny wild type plant. If the PCR amplification product of the regenerated plant is one and is mutated (the mutation includes deletion, insertion or substitution of one or more nucleotides) as compared to the nucleotide sequence of the PCR amplification product of Japanese sunny of the wild-type plant, the regenerated plant is homozygously mutated. If the PCR amplification products of the regenerated plant are more than three, the regenerated plant is chimeric. Plants of heterozygous, bi-allelic mutant, homozygous mutant, and chimeric type are collectively referred to as editing plants.

Homozygous mutant rice transgenic positive seedling identification primer for D3 gene mutation:

D3-detect(1563)-transgenic-F:5'-GCTCAGGTCACTCTCCCTATG-3';

D3-detect(2533)-transgenic-R:5'-GCCAATTTGCCAATTACTCTTG-3'。

The PCR reaction system was 1. Mu.L of DNA template, 2. Mu.L of 10. Mu.M Primer F, 2. Mu.L of 10. Mu.M Primer R and 45. Mu.L of gold plate Mix. The reaction procedure was 98℃pre-denaturation for 3min, 35 cycles, 98℃denaturation for 10s,55℃annealing for 15s,72℃extension for 15s, and 72℃extension for 5min.

Plants with non-synonymous mutations at leucine 644 were the subject of subsequent stress study. As a result, 4 homozygous individual strains of D3L644-pH-PABE-7 were obtained in total. The homozygous individual used in the subsequent experiments was designated CRI-d3 ^L644S plant. In comparison with wild-type rice, the D3 gene in both homologous chromosomes of CRI-D3 ^L644S has been mutated in such a way that nucleotide 2014 of the wild-type genomic sequence of the OsD gene is mutated from T mutation C, corresponding to nucleotide 1931 of CDS (SEQ ID No. 2), and further in such a way that amino acid 644 is mutated from leucine Leu (L) to serine Ser (FIG. 3).

4. Counting tillering number of homozygous cationic plants

The experimental method comprises the steps of planting japonica rice Nippon NP, wherein the homozygous plant CRI-d3 ^L644S is planted in a field of an experimental base of the institute of genetic and developmental biology of China academy of sciences in Changping area of Beijing, 24 plants are planted in each plant line in a mode of 17cm in plant spacing and line spacing, and the growth time is 5 months to 11 months per year. The field phenotype investigation time of the rice material is 9 months 20 days to 10 months 7 per year, the counted tillering number is the effective tillering number of the mature period (i.e. the tillering number of the growing spike), and the plant height is the distance between the highest spike of the rice and the ground. Taking average tillering number of each material as standard, and taking photos from representative single plant transplanted flowerpots.

The results indicate that the D3 site-directed editing homozygous mutant CRI-D3 ^L644S exhibits a dwarf multi-tiller phenotype (FIG. 4).

5. D53 protein level detection of CRI-D3 ^L644S

The degradation of D53 protein in CRI-D3 ^L644S is detected by Western Blot experiment, and the method comprises (1) preparing 10% concentration 12-hole or 15-hole SDS-PAGE protein gel, and setting voltage at 80-120V for about 1-2 hr according to the requirement of experiment. (2) Transferring the protein onto PVDF or NC membrane by semi-dry transfer membrane method, and transferring the membrane for 30-60min at voltage of 15-20V. (3) 4% of skimmed milk powder is prepared in TBST solution during film transfer, after film transfer, PVDF or NC film is put into 4% of skimmed milk powder, and the film is placed in a 70rpm shaker to be incubated for 60min at room temperature. (4) The skimmed milk powder was poured off, rinsed with TBST, fresh 4% skimmed milk powder was added, the protein antibody to be detected was added, and incubation time was 1-2h or 4℃overnight. (5) After incubation of the primary antibody, PVDF or NC membrane was washed 3 times with TBST, 5min at room temperature on a 70rpm shaker, then 4% skim milk containing the secondary antibody was added and incubated on a 60rpm shaker for 60min at room temperature. (6) After the secondary antibody incubation, the membranes were washed 3 times with TBST, 5min at room temperature on a 70rpm shaker. Adding color developing solution, and developing protein with photosensitive film or protein developing instrument. GFP murine monoclonal antibody (Cat# 11814460001) was purchased from Roche. The reference protein is Actin, purchased from Abmart, and the product number is Cat#M20009L.

D53 is used as a core inhibition protein of the strigolactone signal pathway, and the amount of the protein is directly related to the strength of strigolactone signals. The results showed that the D53 protein levels in the stem base of seedlings were significantly higher for wild type and CRI-D3 ^L644S mutants than for wild type (FIG. 5), consistent with typical strigolactone synthesis or signal mutant D53 accumulation, indicating CRI-D3 ^L644S is a typical strigolactone pathway mutant.

Wild type amino acid sequence encoded by SEQ ID No.1D3 gene

MAEEEEVEEGRSSSSAILDLPEPLLLHILSFLTDVRSRHRAALACGRMRAAERATRSELSLRGDPRSPGFLFLSHAFRFPALEHLDLSLVSPWGHPLLSSVPPCGGGGGGAPSASSSSGMNVYHPEAISEQNAFIAARLAGCFPAVTSLAVYCRDPTTLANLTPHWQASLRRVKLVRWHQRPPTLPDGADLEPLLETCAALRELDLSEFYCWTEDVVRALTTHPSATAALTHLDLGLAAATDGFKSSELGPIAASCPNLRKLVAPCLFNPRFSDCVGDDALLSLATSCPRLTVLRLSEPFEAAANIQREEAAITVAGLVAFFAALPALEDFTMDLQHNVLEAAPAMEALARRCPRIKFLTLGSFQGLCKASWLHLDGVAVCGGLESLYMKNCQDLTDASLAAIGRGCRRLAKFGIHGCDLVTSAGIRRLAFTLRPTLKEVTVLHCRLLHTAECLTALSPIRDRIESLEINCVWNTTEQPCSVANGTTTECDPEDDELGEVYESAAKKCRYMEFDDLGSWEMLRSLSLWFSAGQLLSPLISAGLDSCPVLEEISIKVEGDCRTCPRPAPRTIFGLSDLAGFPVLAKMKLDLSEAVGYALTAPTGQMDLSLWERFYLHGIESLQTLYELDYWPPQDKDVHHRSLTLPAVGLIQRCVGLRKLFIHGTTHEHFMTFFLSIPNLRDMQLREDYYPAPENDLMFTEMRAESWLRFEVQLNSRQIDD*.

SEQ ID No.2D3 gene coding CDS nucleotide sequence

5'-ATGGCGGAAGAGGAGGAGGTGGAGGAGGGGAGGTCCTCGTCGTCGGCGATACTGGACCTGCCGGAGCCGCTGCTGCTGCACATCCTGAGCTTCCTGACGGACGTGAGGTCTCGGCACAGGGCGGCGCTGGCGTGCGGGAGGATGCGGGCGGCGGAGCGGGCGACGAGGTCGGAGCTCTCGCTGAGGGGCGACCCGAGGTCGCCGGGGTTCCTGTTCCTCTCGCACGCGTTCCGCTTCCCGGCGCTGGAACACCTCGACCTCTCGCTCGTCTCGCCGTGGGGGCATCCGCTTCTCTCCTCCGTGCCGCCCTGCGGCGGCGGCGGCGGCGGCGCGCCCTCGGCGTCGTCGTCGTCGGGGATGAACGTGTACCACCCCGAGGCGATCTCCGAGCAGAACGCCTTCATCGCCGCCCGCCTCGCGGGCTGCTTCCCGGCGGTGACCTCGCTCGCCGTCTACTGCCGCGACCCCACCACGCTCGCCAACCTCACCCCGCACTGGCAGGCCTCCCTCCGCCGCGTCAAGCTCGTGCGCTGGCACCAGCGCCCGCCCACCCTCCCCGACGGCGCGGATCTCGAGCCGCTGCTGGAGACCTGCGCCGCGCTCCGGGAGCTCGACCTGTCGGAGTTCTACTGCTGGACCGAGGACGTCGTGAGGGCGCTCACCACGCACCCTTCCGCCACCGCGGCGCTCACCCACCTCGACCTCGGCCTCGCCGCCGCCACCGACGGCTTCAAATCCTCCGAGCTTGGGCCAATCGCGGCCTCCTGCCCCAACCTCCGCAAGCTCGTGGCGCCATGCTTGTTCAACCCACGGTTCAGCGATTGCGTCGGCGACGACGCGCTGCTCTCGCTGGCCACCAGCTGCCCGCGGCTGACCGTCTTGCGGCTCAGCGAGCCGTTCGAGGCTGCGGCCAACATCCAGAGGGAGGAGGCGGCCATCACCGTTGCGGGGCTAGTCGCCTTCTTCGCGGCGCTCCCCGCGCTGGAGGATTTCACCATGGATCTCCAGCACAATGTGCTGGAGGCCGCGCCCGCGATGGAGGCGCTTGCCCGAAGGTGCCCGCGGATCAAGTTCTTGACCCTGGGTTCCTTCCAGGGGCTGTGTAAGGCCTCTTGGTTGCATCTTGATGGTGTTGCGGTGTGCGGTGGGCTGGAGTCACTTTACATGAAGAATTGCCAGGATCTCACGGATGCCAGCCTTGCGGCAATTGGCCGTGGGTGCCGGAGGCTTGCTAAGTTCGGCATCCATGGCTGTGACCTTGTCACTTCGGCTGGGATCAGGAGGCTTGCATTCACGCTTCGGCCTACTCTCAAGGAAGTCACTGTCTTGCACTGCCGGCTTCTGCACACTGCAGAATGTCTCACTGCTCTAAGTCCGATCCGTGATCGCATTGAAAGTCTTGAGATCAACTGTGTCTGGAACACAACCGAACAACCCTGCAGTGTTGCAAATGGCACCACCACCGAATGCGATCCTGAGGATGATGAGCTTGGTGAAGTGTACGAGTCTGCAGCCAAGAAATGTAGGTACATGGAATTTGATGATCTTGGAAGCTGGGAGATGCTCAGGTCACTCTCCCTATGGTTCTCTGCTGGCCAGCTTCTCTCTCCGCTCATTTCTGCTGGTCTCGATAGCTGTCCCGTGCTTGAGGAGATCTCAATTAAGGTGGAGGGTGATTGCCGGACATGCCCACGACCTGCTCCAAGAACAATTTTTGGCTTAAGTGATCTTGCAGGCTTCCCAGTATTAGCCAAGATGAAATTGGACCTCAGTGAAGCTGTGGGTTATGCACTTACTGCACCAACAGGGCAGATGGATCTTTCACTATGGGAGCGATTTTATTTGCATGGTATCGAATCACTGCAGACTTTGTATGAATTGGACTACTGGCCGCCCCAAGACAAGGATGTGCACCACCGGAGCCTGACATTGCCAGCCGTGGGATTGATCCAACGCTGCGTTGGACTCAGGAAGCTTTTCATCCATGGCACCACACATGAGCACTTCATGACCTTCTTCCTTTCAATTCCAAACTTGCGGGACATGCAGTTGCGGGAGGACTATTATCCAGCCCCAGAGAATGATCTGATGTTCACAGAGATGCGGGCTGAATCTTGGCTTAGGTTTGAGGTGCAACTGAACAGCCGGCAAATTGATGATTAG-3'.

Wild type genomic sequence of OsD gene

5'-GCTTCACCCCAAATCCCTCAACGGCAGCAAGAGAGAGAAAGAAGAGAGAGAGAGAGAGAGAGAGAGAGGAGTAGACGTCGCCCATGGCGGAAGAGGAGGAGGTGGAGGAGGGGAGGTCCTCGTCGTCGGCGATACTGGACCTGCCGGAGCCGCTGCTGCTGCACATCCTGAGCTTCCTGACGGACGTGAGGTCTCGGCACAGGGCGGCGCTGGCGTGCGGGAGGATGCGGGCGGCGGAGCGGGCGACGAGGTCGGAGCTCTCGCTGAGGGGCGACCCGAGGTCGCCGGGGTTCCTGTTCCTCTCGCACGCGTTCCGCTTCCCGGCGCTGGAACACCTCGACCTCTCGCTCGTCTCGCCGTGGGGGCATCCGCTTCTCTCCTCCGTGCCGCCCTGCGGCGGCGGCGGCGGCGGCGCGCCCTCGGCGTCGTCGTCGTCGGGGATGAACGTGTACCACCCCGAGGCGATCTCCGAGCAGAACGCCTTCATCGCCGCCCGCCTCGCGGGCTGCTTCCCGGCGGTGACCTCGCTCGCCGTCTACTGCCGCGACCCCACCACGCTCGCCAACCTCACCCCGCACTGGCAGGCCTCCCTCCGCCGCGTCAAGCTCGTGCGCTGGCACCAGCGCCCGCCCACCCTCCCCGACGGCGCGGATCTCGAGCCGCTGCTGGAGACCTGCGCCGCGCTCCGGGAGCTCGACCTGTCGGAGTTCTACTGCTGGACCGAGGACGTCGTGAGGGCGCTCACCACGCACCCTTCCGCCACCGCGGCGCTCACCCACCTCGACCTCGGCCTCGCCGCCGCCACCGACGGCTTCAAATCCTCCGAGCTTGGGCCAATCGCGGCCTCCTGCCCCAACCTCCGCAAGCTCGTGGCGCCATGCTTGTTCAACCCACGGTTCAGCGATTGCGTCGGCGACGACGCGCTGCTCTCGCTGGCCACCAGCTGCCCGCGGCTGACCGTCTTGCGGCTCAGCGAGCCGTTCGAGGCTGCGGCCAACATCCAGAGGGAGGAGGCGGCCATCACCGTTGCGGGGCTAGTCGCCTTCTTCGCGGCGCTCCCCGCGCTGGAGGATTTCACCATGGATCTCCAGCACAATGTGCTGGAGGCCGCGCCCGCGATGGAGGCGCTTGCCCGAAGGTGCCCGCGGATCAAGTTCTTGACCCTGGGTTCCTTCCAGGGGCTGTGTAAGGCCTCTTGGTTGCATCTTGATGGTGTTGCGGTGTGCGGTGGGCTGGAGTCACTTTACATGAAGAATTGCCAGGATCTCACGGATGCCAGCCTTGCGGCAATTGGCCGTGGGTGCCGGAGGCTTGCTAAGTTCGGCATCCATGGCTGTGACCTTGTCACTTCGGCTGGGATCAGGAGGCTTGCATTCACGCTTCGGCCTACTCTCAAGGAAGTCACTGTCTTGCACTGCCGGCTTCTGCACACTGCAGAATGTCTCACTGCTCTAAGTCCGATCCGTGATCGCATTGAAAGTCTTGAGATCAACTGTGTCTGGAACACAACCGAACAACCCTGCAGTGTTGCAAATGGCACCACCACCGAATGCGATCCTGAGGATGATGAGCTTGGTGAAGTGTACGAGTCTGCAGCCAAGAAATGTAGGTACATGGAATTTGATGATCTTGGAAGCTGGGAGATGCTCAGGTCACTCTCCCTATGGTTCTCTGCTGGCCAGCTTCTCTCTCCGCTCATTTCTGCTGGTCTCGATAGCTGTCCCGTGCTTGAGGAGATCTCAATTAAGGTGGAGGGTGATTGCCGGACATGCCCACGACCTGCTCCAAGAACAATTTTTGGCTTAAGTGATCTTGCAGGCTTCCCAGTATTAGCCAAGATGAAATTGGACCTCAGTGAAGCTGTGGGTTATGCACTTACTGCACCAACAGGGCAGATGGATCTTTCACTATGGGAGCGATTTTATTTGCATGGTATCGAATCACTGCAGACTTTGTATGAATTGGACTACTGGCCGCCCCAAGACAAGGATGTGCACCACCGGAGCCTGACATTGCCAGCCGTGGGATTGATCCAACGCTGCGTTGGACTCAGGAAGCTTTTCATCCATGGCACCACACATGAGCACTTCATGACCTTCTTCCTTTCAATTCCAAACTTGCGGGACATGCAGTTGCGGGAGGACTATTATCCAGCCCCAGAGAATGATCTGATGTTCACAGAGATGCGGGCTGAATCTTGGCTTAGGTTTGAGGTGCAACTGAACAGCCGGCAAATTGATGATTAGTTATGTGGGCACAAAATGGTTTGAAGCTGAATACAGAGATTTATCTGGATGGTGCCATTGCTCCACTGTGCAATGGCAGGGGATTCCTGGTGAGTTGGTTATGATTATGGGTGGAGTCGTGTGTATTGCTGCAGTGCCATTGAGGAGAGTAGTATACTGGCAGCACTTGGATCTGTCAGCAAAGTAACCTTCTCCAGTTGCTTTTTTACCCCCTTTTTGATGTAATAAGAGAGTTGGGTCGGAAATGAGATATTTGCAGGAGATAAGATTATAAATTAGGCTTCATGGAAAATTTTCCAAGAAAAAAAAACATTTTGTTTTTAAGATGGTCTGAGTTGTGAACACCGGCAAGAGTAATTGGCAAATTGGCATGGTTCTAGCGGTTTGTAACATTTGAACTCTGTAAACAAAAGAAAAACGCCACTCGCTTTTCTTATGCCCTTTGCTTCATGGGTGAAAGTGGCTCATCTAATATTGGTCAGTGTTTTACTGTTTTCAATGGATGGGCAACGGAGTTCAGTACTACTGCGATAGGAAACTATTTTGATGTGTACATAACAGCTCTATTAATCCAAAATTATGTGCCTTTGCTCTTTGAGTTTATTTCTGTCCCTTTCCTTTTCCATTTCATGCACAGGCTTGGTGACAAATGGGATGGCGTGTGCAGATGTGCAAGCCAGTTTTGCATGTCATTATCGGGCATGTTGAGTTGCAACACCGGCTACAACAAGGTTTACTTTAATACACAGCAGTAAGGATTTAATCTGATAGAATGTTGAAAGGTTTTCTCTTTTTTATGCAGGATTGAAAGCTGCTTTAGTTTCCAAGGAACAACCAATCAGTTCCATCAGAACTGACTACCACCATTTGCTGATTCGTTCTCTGCAGTGATCCCCAATGGAGACCTGGCCTTTGCTTCATCTCATCCTCAAACTGTAGCATGACAAGAAAGACGTGCGGAACAAGATCAGCATCAGAACATGACAAAAAGATTTTCGCACTAGAAGCCGGTTTATCCAATTCCTCTGCTTGCCTCTTCCATTCAGCTAGTTAAGATAATGCGAGATCACTGGTTTTGTCAAAGCAAATCCAGACGATTCTTGGTGCCATGGAAAAAGAAGTCGTCCATGAGTGAGAGAGCTCTCTTGTCCTAATCCATCAGCAAGGGCAATAATGCTTTTTTGATTAAGCAGCTGGAGGCCTCACAAAATAATGCTTAAGCAAGTACTACTACAGGATTAAAAGCTGGCCTCTAGAAGGAGTAGATTAGGTAGAGGAGAAGCTTCTCCTTTCCTCTTTTGCCACGTTGAGGCTTATTGCTCACATGATTGTCAATCAACCACGTCACACAAGCACATACACACACTTATTATTTGCTCATAGTTTTAGTTATTTATCAACATTTGGCAGATATGTGTTGGAAATTCAGATGCTCCTTGATGCCATTTTATCTTGCTGGTACATCTGCATAACTCGCTCATTACTATCTGTTGCAATATTATTATACATTTGGATATTTAATGTGCTGATCGCTTCTTGCTACCTATCTAATTTCAGAATGTCTTGAAAGCAAAGCTGGTGACAGGAGAGATGTCATATGAGCAGGCAGTCAGTTGCAGCCTTGTGGTTGCACCTCCCTTTCCTGCTGTCCTCTTTTTTTTGTTTTCCCTGGTTTCTCTTCATCAGAATTAAAGCAGATATTTTTGTGGCGGCCGTGGATTTCCATTCATCTGATGATCAGCTAGTAATATTCTGCAAGTGCCTTCCGATAGAATTGAGCGATGATTCTGAGGTCAGATACTCATCAGATGCTCGAAGGATAAGTGGGTTTCAGACGATGCTGCAAAAGATATTAGCCGGCGAGTGAGGTGAGGCTACTAGCCTACTAGGCTTATCGCCTTGTATAAAACTAAAGGAGTCAGTACAGAGACATCAGGTAAAGTCAGCATATGCAGCTATCCATCTATGCATGCATGCACCAACTGCAAAGTAAAGGTAAAAGTGAAAGCTTTGTCTTCCAGGTGTATGGAAGAAAATTATCCTTGTGATGGTTATGCAACTCACTCTTTTGTTTGCACCCAGATATGTCATATTGGCTTTTTCACCATGGTGTCAAGGTTGATGTGAATGTGAATGTGATTCTACTAATTTGAGGTCATGTAGAGTAATTAAGTAAGCAAATGGAGCCCGTCATGTCGTCATGCACTGACAAGGATATAGTCCTATGGACCTCTATTTTGATTAATTGGTGGAGAAAATTCAGAGGAAAAATATGTGTGTGCTGGAATATGTTGGTATTTTTTTTTTACAA-3'.

OsSKP 1A 1-D3 fusion protein coding CDS nucleotide sequence

5'-ATGGCGGCTGAGGGAGAGAAGAAGATGATCACCCTGAAGAGCTCCGACGGGGAGGAGTTCGAGGTGGAGGAGGCGGTGGCGATGGAGTCGCAGACGATCCGCCACATGATCGAGGACGACTGCGCCGACAACGGCATCCCGCTCCCCAACGTCAACTCCAAGATCCTCTCCAAGGTCATCGAGTACTGCAACAAGCACGTGCACGCCGCGGCCGCCGCGGCCTCCAAGGCCGCCGACGACGCCGCGTCCGCCGCCGCAGCCGTGCCGCCGCCCTCCGGCGAGGACCTCAAGAACTGGGACGCCGACTTCGTCAAGGTCGACCAGGCCACCCTCTTCGACCTCATCCTGGCCGCGAACTACCTCAACATCAAGGGGTTGCTGGACCTTACTTGCCAGACTGTTGCTGACATGATCAAGGGGAAGACTCCTGAGGAGATCCGCAAGACCTTCAACATCAAGAACGACTTCACCCCTGAGGAGGAAGAGGAGATCCGCAGGGAGAACCAGTGGGCTTTTGAGCATATGGCCATGGAGGCCGAATTCATGGCGGAAGAGGAGGAGGTGGAGGAGGGGAGGTCCTCGTCGTCGGCGATACTGGACCTGCCGGAGCCGCTGCTGCTGCACATCCTGAGCTTCCTGACGGACGTGAGGTCTCGGCACAGGGCGGCGCTGGCGTGCGGGAGGATGCGGGCGGCGGAGCGGGCGACGAGGTCGGAGCTCTCGCTGAGGGGCGACCCGAGGTCGCCGGGGTTCCTGTTCCTCTCGCACGCGTTCCGCTTCCCGGCGCTGGAACACCTCGACCTCTCGCTCGTCTCGCCGTGGGGGCATCCGCTTCTCTCCTCCGTGCCGCCCTGCGGCGGCGGCGGCGGCGGCGCGCCCTCGGCGTCGTCGTCGTCGGGGATGAACGTGTACCACCCCGAGGCGATCTCCGAGCAGAACGCCTTCATCGCCGCCCGCCTCGCGGGCTGCTTCCCGGCGGTGACCTCGCTCGCCGTCTACTGCCGCGACCCCACCACGCTCGCCAACCTCACCCCGCACTGGCAGGCCTCCCTCCGCCGCGTCAAGCTCGTGCGCTGGCACCAGCGCCCGCCCACCCTCCCCGACGGCGCGGATCTCGAGCCGCTGCTGGAGACCTGCGCCGCGCTCCGGGAGCTCGACCTGTCGGAGTTCTACTGCTGGACCGAGGACGTCGTGAGGGCGCTCACCACGCACCCTTCCGCCACCGCGGCGCTCACCCACCTCGACCTCGGCCTCGCCGCCGCCACCGACGGCTTCAAATCCTCCGAGCTTGGGCCAATCGCGGCCTCCTGCCCCAACCTCCGCAAGCTCGTGGCGCCATGCTTGTTCAACCCACGGTTCAGCGATTGCGTCGGCGACGACGCGCTGCTCTCGCTGGCCACCAGCTGCCCGCGGCTGACCGTCTTGCGGCTCAGCGAGCCGTTCGAGGCTGCGGCCAACATCCAGAGGGAGGAGGCGGCCATCACCGTTGCGGGGCTAGTCGCCTTCTTCGCGGCGCTCCCCGCGCTGGAGGATTTCACCATGGATCTCCAGCACAATGTGCTGGAGGCCGCGCCCGCGATGGAGGCGCTTGCCCGAAGGTGCCCGCGGATCAAGTTCTTGACCCTGGGTTCCTTCCAGGGGCTGTGTAAGGCCTCTTGGTTGCATCTTGATGGTGTTGCGGTGTGCGGTGGGCTGGAGTCACTTTACATGAAGAATTGCCAGGATCTCACGGATGCCAGCCTTGCGGCAATTGGCCGTGGGTGCCGGAGGCTTGCTAAGTTCGGCATCCATGGCTGTGACCTTGTCACTTCGGCTGGGATCAGGAGGCTTGCATTCACGCTTCGGCCTACTCTCAAGGAAGTCACTGTCTTGCACTGCCGGCTTCTGCACACTGCAGAATGTCTCACTGCTCTAAGTCCGATCCGTGATCGCATTGAAAGTCTTGAGATCAACTGTGTCTGGAACACAACCGAACAACCCTGCAGTGTTGCAAATGGCACCACCACCGAATGCGATCCTGAGGATGATGAGCTTGGTGAAGTGTACGAGTCTGCAGCCAAGAAATGTAGGTACATGGAATTTGATGATCTTGGAAGCTGGGAGATGCTCAGGTCACTCTCCCTATGGTTCTCTGCTGGCCAGCTTCTCTCTCCGCTCATTTCTGCTGGTCTCGATAGCTGTCCCGTGCTTGAGGAGATCTCAATTAAGGTGGAGGGTGATTGCCGGACATGCCCACGACCTGCTCCAAGAACAATTTTTGGCTTAAGTGATCTTGCAGGCTTCCCAGTATTAGCCAAGATGAAATTGGACCTCAGTGAAGCTGTGGGTTATGCACTTACTGCACCAACAGGGCAGATGGATCTTTCACTATGGGAGCGATTTTATTTGCATGGTATCGAATCACTGCAGACTTTGTATGAATTGGACTACTGGCCGCCCCAAGACAAGGATGTGCACCACCGGAGCCTGACATTGCCAGCCGTGGGATTGATCCAACGCTGCGTTGGACTCAGGAAGCTTTTCATCCATGGCACCACACATGAGCACTTCATGACCTTCTTCCTTTCAATTCCAAACTTGCGGGACATGCAGTTGCGGGAGGACTATTATCCAGCCCCAGAGAATGATCTGATGTTCACAGAGATGCGGGCTGAATCTTGGCTTAGGTTTGAGGTGCAACTGAACAGCCGGCAAATTGATGATTAG-3'.

OsD14 gene CDS nucleotide sequence

5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'.

OsD 14A 14 DeltaN Gene CDS nucleotide sequence

5'-ATGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGT CGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1. A method for preparing rice with increased tillering number is characterized by comprising the steps of mutating a coding gene OsD3 in target rice containing a coding gene OsD to obtain rice with increased tillering number, wherein the tillering number of the rice with increased tillering number is larger than that of the target rice, osD is protein with an amino acid sequence of SEQ ID No.1, 644 th amino acid of OsD is 644 th amino acid of SEQ ID No.1, 644 th amino acid is leucine, and the mutation is to mutate a codon encoding the 644 th amino acid of OsD3 in the coding gene in a genome of the target rice into a codon encoding other amino acids.

2. A method for increasing the tiller number of rice is characterized by comprising the steps of mutating a coding gene in acceptor rice containing OsD coding genes to increase the tiller number of the acceptor rice, wherein OsD is protein with an amino acid sequence of SEQ ID No.1, 644 th amino acid of OsD3 is 644 th amino acid of SEQ ID No.1, 644 th amino acid is leucine, and mutating a codon encoding 644 th amino acid of Os D3 in the coding gene in a target rice genome into a codon encoding other amino acids.

3. Use, characterized in that it is a protein or a substance regulating the expression of a gene encoding said protein or a substance regulating the activity and/or the content of said protein, in any of the following:

m1) regulating and controlling the tillering number of plants;

M2) preparing a product for regulating and controlling the tillering number of plants;

M3) plant breeding;

the protein is OsD, and OsD is a protein with an amino acid sequence of SEQ ID No. 1.

4. The use according to claim 3, wherein the modulation is inhibition or reduction or downregulation of the expression of the OsD gene encoding or the OsD3 content or activity.

5. A use according to claim 3, wherein the substance is any one of the following:

c1 A nucleic acid molecule which inhibits or reduces or down-regulates the expression of the gene encoding OsD,

C2 Expression of the gene encoding the nucleic acid molecule according to C1),

C3 An expression cassette containing the gene of C2),

C4 A recombinant vector containing the gene of C2) or a recombinant vector containing the expression cassette of C3),

C5 A recombinant microorganism containing the gene of C2), a recombinant microorganism containing the expression cassette of C3), or a recombinant microorganism containing the recombinant vector of C4),

C6 A transgenic plant cell line containing the gene of C2), or a transgenic plant cell line containing the expression cassette of C3), or a transgenic plant cell line containing the recombinant vector of C4),

C7 A transgenic plant tissue containing the gene of C2), or a transgenic plant tissue containing the expression cassette of C3), or a transgenic plant tissue containing the recombinant vector of C4),

C8 A transgenic plant organ containing the gene of C2), or a transgenic plant organ containing the expression cassette of C3), or a transgenic plant organ containing the recombinant vector of C4).

6. Use, characterized in that it is a protein or a substance regulating the expression of a gene encoding said protein or a substance regulating the activity and/or the content of said protein, in any of the following:

m1) regulating and controlling the tillering number of plants;

M2) preparing a product for regulating and controlling the tillering number of plants;

M3) plant breeding;

The protein is OsD mutant, and the OsD mutant is a protein which mutates 644 th amino acid of SEQ ID No.1 from leucine to serine and keeps other amino acids of SEQ ID No.1 unchanged.

7. The use according to claim 6, wherein the modulation is up-regulation or enhancement or increase of the expression of the gene encoding the OsD mutant or the content or activity of the OsD mutant.

8. A protein characterized in that it is a protein obtained by mutating amino acid 644 of SEQ ID No.1 from leucine to serine while keeping the other amino acids of SEQ ID No.1 unchanged.

9. A nucleic acid molecule encoding the protein of claim 1.

10. A biomaterial, characterized in that it is any of the substances according to claim 5.