N-terminal motif deletion protein of rice strigolactone receptor OsD14 and application of N-terminal motif deletion protein

Abstract

The invention discloses an N-terminal motif deletion protein of a rice strigolactone receptor OsD14 and application of the N-terminal motif deletion protein, and belongs to the technical field of genetic breeding. The amino acid sequence of the N-terminal motif deletion protein of the rice strigolactone receptor OsD14 is the 51st-322nd site of SEQ ID No.1, and the N-terminal motif deletion protein of the rice strigolactone receptor OsD14 can be used for regulating and controlling the tiller number of plants. Experiments show that the N-terminal motif deleted protein stability of the rice strigolactone receptor OsD14 is improved, the rice strigolactone receptor OsD14 has higher susceptibility to strigolactone, and a plant shows a strigolactone signal enhanced phenotype and shows a less tillering phenotype.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and in particular relates to an N-terminal motif-deficient protein of a rice strigolactone receptor OsD14 and an application thereof. Background Art

[0002] Rice is one of the most important crops. The increasing world population has put forward higher and higher requirements for the improvement of rice yield. After decades of efforts by breeders, rice yield has been greatly improved, but it still cannot meet people's demand for total food. Rice plant type is a key factor in determining yield, which is mainly determined by plant height, tiller number, angle and panicle type.

[0003] The formation of rice plant type mainly depends on factors such as plant height, number of tillers, tillering angle and panicle morphology. Appropriate tillering force can increase the biological yield of the group. At the same time, tillering is also the basis for yield formation. A large number of experiments have shown that when the tillering force is too weak, the automatic adjustment ability is often poor, and it is difficult to form a group with high biological yield in the early stage, resulting in low biological yield and limiting the increase in economic yield; while when the tillering force is too strong, it is easy to cause the stems to be thin and weak, and the lodging resistance is reduced, which is also unfavorable to production. This requires moderate tillering force. Therefore, the control of rice tillering is an important prerequisite for achieving the "ideal plant type".

[0004] Strigolactone is a hormone synthesized in the roots that inhibits plant branch formation. Strigolactone has a wide range of effects in plants. With the physiological and genetic studies of a series of dwarf multi-tillering mutants, some important members of the strigolactone synthesis and signaling pathways have been cloned in various model plants. The common characteristics of these member mutants are the loss of apical dominance and the release of lateral bud growth and development, resulting in a dwarf multi-branching phenotype. However, to date, people's understanding of the biosynthesis process and signal transduction pathway of strigolactone is still very limited, and its function in the growth and development of plants needs to be further explored.

[0005] As an important hormone regulating rice tillering, mutants with complete loss of its synthesis and signaling show extreme multi-tillering and dwarf phenotypes, accompanied by severe reductions in fruit set rate and yield, making it difficult to directly apply it to rice breeding practice. However, by finely regulating the function of strigolactones, it is possible to achieve the goal of plant type improvement. For example, in the restorer line material "Huazhan" of the super rice variety, the weak allele HTD1 of the strigolactone synthesis gene HTD1 / D17 is HZ It can effectively increase the number of rice tillers without affecting the fruiting rate, resulting in an increase in biomass and yield. HZ Green Revolution Gene SD1 DGWGIt has been selected and widely used by breeders in high-yield rice breeding. The OsD14 gene comes from rice. As a receptor component of the strigolactone pathway, its main function is to control the rice plant type, including the number of rice tillers and plant height. Regulating the number of rice tillers and improving the plant type by regulating the OsD14-mediated strigolactone signal perception will have broad application prospects in increasing rice yield. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a mutant protein with a missing N-terminal motif of a rice strigolactone receptor. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides the use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity and / or content of the protein in any of the following:

[0009] M1) Regulate the number of plant tillers;

[0010] M2) preparing products for regulating the number of tillers of plants;

[0011] M3) Plant breeding;

[0012] The protein is any of the following:

[0013] A1) a protein whose amino acid sequence is the 51st to 322nd position of SEQ ID No. 1 or a protein whose amino acid sequence is the SEQ ID No. 1;

[0014] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues;

[0015] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

[0016] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (H is-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0017] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0018] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0019] In the above application, the regulation is to upregulate, enhance or increase the expression of the gene encoding the aforementioned protein or the content or activity of the protein.

[0020] In the above application, the regulation is to down-regulate or inhibit or reduce the expression of the gene encoding the aforementioned protein or the content or activity of the protein.

[0021] In the above application, the substance is a biological material, and the biological material is any one of the following:

[0022] B1), RNA molecules that inhibit, reduce or down-regulate the expression of the gene encoding the aforementioned protein or RNA molecules that inhibit, reduce or down-regulate the activity or content of the protein;

[0023] B2), expressing the coding gene of the RNA molecule described in B1);

[0024] B3), an expression cassette containing the gene described in B2);

[0025] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0026] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

[0027] B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4);

[0028] B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4);

[0029] B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4);

[0030] B9), a nucleic acid molecule encoding the aforementioned protein;

[0031] B10), an expression cassette containing the nucleic acid molecule described in B9);

[0032] B11), a recombinant vector containing the nucleic acid molecule described in B9), or a recombinant vector containing the expression cassette described in B10); B12), a recombinant microorganism containing the nucleic acid molecule described in B9), or a recombinant microorganism containing the expression cassette described in B10), or a recombinant microorganism containing the recombinant vector described in B3);

[0033] B13), a transgenic plant cell line containing the nucleic acid molecule described in B9), or a transgenic plant cell line containing the expression cassette described in B10);

[0034] B14), transgenic plant tissue containing the nucleic acid molecule described in B9), or transgenic plant tissue containing the expression cassette described in B10);

[0035] B15), a transgenic plant organ containing the nucleic acid molecule described in B9), or a transgenic plant organ containing the expression cassette described in B10).

[0036] In the above application, B1) the RNA molecule targets the gene encoding the protein.

[0037] In the above application, the protein encoding gene is the wild-type genomic sequence of the OsD14 gene, and B1) the RNA molecule targets nucleotides 232-251 of the wild-type genomic sequence of the OsD14 gene, targets nucleotides 280-299 of the wild-type genomic sequence of the OsD14 gene, targets nucleotides 342-361 of the wild-type genomic sequence of the OsD14 gene, or targets nucleotides 384-403 of SEQ ID No.2.

[0038] In the above application, the coding sequence of the nucleic acid molecule described in B9) is SEQ ID No.2.

[0039] The present invention also provides a method for regulating the tiller number of rice, comprising the following steps: regulating the expression of the coding gene of the above-mentioned protein in the recipient plant or regulating the activity and / or content of the protein to change the tiller number of the recipient plant.

[0040] In the above method, the recipient plant may or may not contain the gene encoding the protein.

[0041] The present invention also provides the use of a polypeptide or a substance for regulating the expression of a gene encoding the polypeptide or a substance for regulating the activity and / or content of the polypeptide in any of the following:

[0042] M1) Regulate the number of plant tillers;

[0043] M2) preparing products for regulating the number of tillers of plants;

[0044] M3) Plant breeding;

[0045] The protein is any of the following:

[0046] A1) the amino acid sequence is amino acids 1 to 50 of SEQ ID No. 1;

[0047] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues;

[0048] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

[0049] The substance is a biological material, and the biological material is any one of the following:

[0050] K1), an RNA molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or an RNA molecule that inhibits, reduces or down-regulates the activity or content of the protein;

[0051] K2), expressing the coding gene of the RNA molecule described in K1);

[0052] K3), an expression cassette containing the gene described in K2);

[0053] K4), a recombinant vector containing the gene described in K2), or a recombinant vector containing the expression cassette described in K3);

[0054] K5), a recombinant microorganism containing the gene described in K2), or a recombinant microorganism containing the expression cassette described in K3), or a recombinant microorganism containing the recombinant vector described in K4);

[0055] K6), a transgenic plant cell line containing the gene described in K2), or a transgenic plant cell line containing the expression cassette described in K3), or a transgenic plant cell line containing the recombinant vector described in K4);

[0056] K7), transgenic plant tissue containing the gene described in K2), or transgenic plant tissue containing the expression cassette described in K3), or transgenic plant tissue containing the recombinant vector described in K4);

[0057] K8), a transgenic plant organ containing the gene described in K2), or a transgenic plant organ containing the expression cassette described in K3), or a transgenic plant organ containing the recombinant vector described in K4);

[0058] K9), a nucleic acid molecule encoding the aforementioned protein;

[0059] K10), an expression cassette containing the nucleic acid molecule described in K9);

[0060] K11), a recombinant vector containing the nucleic acid molecule described in K9), or a recombinant vector containing the expression cassette described in K10); K12), a recombinant microorganism containing the nucleic acid molecule described in K9), or a recombinant microorganism containing the expression cassette described in K10), or a recombinant microorganism containing the recombinant vector described in K3);

[0061] K13), a transgenic plant cell line containing the nucleic acid molecule described in K9), or a transgenic plant cell line containing the expression cassette described in K10);

[0062] K14), transgenic plant tissue containing the nucleic acid molecule described in K9), or transgenic plant tissue containing the expression cassette described in K10);

[0063] K15), a transgenic plant organ containing the nucleic acid molecule described in K9), or a transgenic plant organ containing the expression cassette described in K10).

[0064] In the above application or the above substance, the expression cassette containing nucleic acid molecules described in B3) refers to the DNA capable of expressing the RNA molecules described above in the host cell. The expression cassette containing nucleic acid molecules described in B10) refers to the DNA capable of expressing the protein described above in the host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecules of any of the above proteins or the DNA of the RNA molecules. The regulatory sequence can guide the coding sequence to express any of the above proteins or the DNA of the RNA molecules in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce a specific restriction enzyme site into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the DNA of the protein or the RNA molecule. The promoter can be any nucleic acid sequence that has transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence can be operably connected to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an mRNA non-translated region that is very important for the translation of the host cell. The leader sequence can be operably connected to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the protein, which can guide the DNA encoding the protein or the RNA molecule into the cell secretion pathway. Signal peptide coding regions that can direct the expressed protein or the DNA of the RNA molecule into the secretory pathway of the host cell used can be used in the present invention. It may also be necessary to add regulatory sequences that can regulate the expression of the DNA of the protein or RNA molecule according to the growth of the host cell. Examples of regulatory sequences are those systems that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can enable gene amplification.

[0065] In the above substances, the vector can be a plasmid, a cosmid, a phage or a virus vector.

[0066] In the above substances, the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0067] In the above substances, the transgenic plant cell lines do not include propagation materials.

[0068] The plant mentioned above is any one of the following:

[0069] G1) Monocots;

[0070] G2) Gramineae;

[0071] G3) Oryza plants;

[0072] G4) Rice plants;

[0073] G5) Rice.

[0074] The aforementioned protein or a substance that regulates the expression of the gene encoding the protein or a substance that regulates the activity and / or content of the protein also falls within the scope of protection of the present invention.

[0075] The present invention shows through experiments that the stability of the N-terminal motif-deleted protein of the rice strigolactone receptor OsD14 is improved, the sensitivity to strigolactone is stronger, the plant shows a phenotype of enhanced strigolactone signal, and presents a phenotype of less tillering. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Functional analysis of the NTD motif of OsD14 protein in rice. (A) Protein degradation of D14-GFP and D14ΔN-GFP in callus. Act:D14–GFP / d14 and Act:D14–GFPΔN / d14 callus were treated with 20 μM rac-GR24 for 0, 1, and 3 h, respectively. (B) Subcellular localization of D14-GFP and D14ΔN-GFP in rice protoplasts. (C) GST Pull-Down to detect the interaction between D14ΔN-GFP and GST-D3. (D) GST Pull-Down to detect the interaction between D14ΔN-GFP and GST-D53.

[0077] Figure 2The regulatory role of the NTD motif of OsD14 protein in rice on tiller development. (A) Comparison of plant morphology and tiller number of wild-type Nipponbare rice (NP), d14 mutant, Act:D14-GFP / d14 overexpression line, and Act:D14ΔN-GFP / d14 overexpression line. Scale bar, 20 cm. Data are mean ± SD. (B) D53 and D14-GFP protein levels in the aboveground substrate of seedlings of wild-type (NP), d14 mutant, Act:D14-GFP / d14, and Act:D14ΔN-GFP / d14. Data are mean ± SD, n = 3. (C) Transcript levels of strigolactone biosynthesis gene D10 in seedling roots of wild-type (NP), d14 mutant, Act:D14-GFP / d14, and Act:D14ΔN-GFP / d14. Data are mean ± SD, n = 3. (D) 4DO levels in root exudates and root tissues of wild-type (NP), d14, Act:D14-GFP / d14, and Act:d14ΔN-GFP / d14 seedlings. (E) Transcript levels of the tillering suppressor gene OsTB1 gene at the stem base of wild-type (NP), d14, Act:D14-GFP / d14, and Act:d14ΔN-GFP / d14 seedlings. Data are mean ± SD, n = 3.

[0078] Figure 3 Creation of NTD motif knockout mutants of OsD14 and their effects on tillering. (A) Information of rice D14 genome editing d14ΔN-1 and d14ΔN-2 mutants. The left figure shows the genomic DNA sequence of d14ΔN-1 and d14ΔN-2 mutants, the blue line indicates the N-terminal domain of D14, the gray font indicates the deleted sequence, and the red font indicates the inserted base pairs; the right figure shows the amino acid sequence of d14ΔN-1 and d14ΔN-2 proteins, the blue line indicates the N-terminal domain of D14, the green font indicates the truncated N-terminal sequence, and the orange font indicates the mutated N-terminal sequence. (B) The left figure shows the phenotypes of wild type (NP), d14ΔN-1 and d14ΔN-2 mutants; the right figure shows the tillering number statistics of wild type (NP), d14ΔN-1 and d14ΔN-2 mutants. Scale bar, 20 cm. Data are mean ± SD. (C) D53 protein level in the stem base of 2-week-old seedlings of wild type (NP), d14ΔN-1, and d14ΔN-2. Data are mean ± SD. n = 3. (D) Transcript level of SL-biosynthesis gene D10 in roots of wild type (NP), d14ΔN-1, and d14ΔN-2 seedlings. Data are mean ± SD, n = 3. (E) Transcript level of rice tillering inhibitor gene OsTB1 in the stem base of wild type (NP), d14ΔN-1, and d14ΔN-2 seedlings. Data are mean ± SD, n = 3. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0080] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0081] rac-GR24, a strigolactone analogue, was purchased from Chiralix with the catalog number CX2388.

[0082] MG132 (Proteasome inhibitor) was purchased from Sigma-Aldrich, Cat#474790.

[0083] The quantitative tests in the following examples were all repeated at least three times, and the results were averaged.

[0084] The preparation of AHLG vector, pBI221-GFP vector, rice d14 mutant and D53 rabbit polyclonal antibody 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). DWARF 53 acts as a repressor of strigolacto nesignalling in rice. Nature 504, 401-405.". The public can obtain the biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0085] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation, and two-tailed Student's t-test was used, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns (not significant).

[0086] Example 1: Functional analysis of the NTD motif of OsD14 protein in rice

[0087] 1. Fragment amplification and vector construction of D14 and D14ΔN

[0088] Total RNA of Nipponbare wild-type rice was extracted and reverse transcribed into cDNA. Using the above cDNA as a template, the D14 gene was amplified using AHLG-D14-F and AHLG-D14-R, and the D14ΔN gene was amplified using AHLG-D14ΔN-F and AHLG-D14ΔN- to obtain the D14 gene (the nucleotide sequence is SEQ ID No. 2, encoding the protein with the amino acid sequence of SEQ ID No. 1) and the D14ΔN gene (the nucleotide sequence is the 151-957th positions of SEQ ID No. 2, encoding the protein with the amino acid sequence of SEQ ID No. 1 from the 51st to the 322nd positions).

[0089] AHLG-D14-F:

[0090] AHLG-D14-R:

[0091] AhLG-D14ΔN-F:

[0092] AHLG-D14ΔN-R:

[0093] Among the above primers, the bold black sequences are primers for amplifying D14 or D14ΔN, and the underlined sequences are used for homologous recombination with the AH LG vector.

[0094] The D14 overexpression vector AHLG-Act:D14-GFP is a recombinant expression vector obtained by replacing the small fragment between the two enzyme recognition sites ApaI and XbaI of the AHLG vector with SEQ ID No.2 while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the fusion protein D14-GFP.

[0095] The D14ΔN overexpression vector AHLG-Act:D14ΔN-GFP is a recombinant expression vector obtained by replacing the small fragment between the two enzyme recognition sites of ApaI and XbaI of the AHLG vector with 51-322 of SEQ ID No.1, while keeping the other nucleotide sequences of the AHLG vector unchanged. The recombinant expression vector expresses the fusion protein D14ΔN-GFP.

[0096] 2. Callus induction containing recombinant vectors Act:D14-GFP or Act:D14ΔN-GFP

[0097] The specific operations are as follows:

[0098] (1) Induction and subculture of rice callus

[0099] NB basic medium composition: KNO3, 2830mg / L, (NH4)2SO4 463mg / L, KH2PO4 400mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 166mg / L, FeSO4·7H2O 27.8mg / L, Na2EDTA 37.5mg / L, MnSO4·4H2O 10mg / L, H3BO3 3mg / L, ZnSO4·7H2O 2mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O0.025mg / L, CoCl2·6H2O 0.025mg / L, KI 0.75mg / L, Vitamin B1 10mg / L, Vitamin B6 1mg / L, Nicotinic acid 1mg / L, Myo-inositol 100mg / L, Casein hydrolysate 300mg / L, Glutamine 500mg / L, Glycine 2mg / L, Proline 1000mg / L, 2,4-D 2mg / L, the rest is water. Use the d14 mutant as the rice receptor, remove the seeds of the d14 mutant to be transformed, disinfect them with 70% (v / v) ethanol for 1min, then use 2.5% (w / v) sodium hypochlorite for 45min, wash them three times with sterile water, sow the seeds on NB solid medium, and culture them at 28℃ in the dark for two weeks. After the mature embryo scutellum grows callus tissue, cut off the callus tissue and subculture it on a new NB solid medium, transfer it to a new NB solid medium every 7 days, and after 3-4 times, Agrobacterium infection can be carried out.

[0100] (2) Cultivation of Agrobacterium tumefaciens EHA105

[0101] 1L LB solid culture medium composition: Tryptone 10g, Yeast Extract 5g, NaCl 10g, agarose 15g.

[0102] 1L LB liquid culture medium composition: Tryptone 10g, Yeast Extract 5g, NaCl 10g.

[0103] Rice transformation solution composition: Add the following reagents to NB basic medium: Inositol 2g / L, Glutamine 2g / L, Casein hydrolysate 500mg / L, 10% Synperonic PE 10ml / L, Acetosyringone 100

[0104] μM.

[0105] When the growth state of rice callus is good, the plant transgenic expression vector (AHLG-Act:D14-GFP vector or AHLG-Act:D14ΔN-GFP vector) is transformed into Agrobacterium EHA105 strain by high-voltage electroporation, and spread on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin for 2-3 days. 4-5 single clones of each transformation are inoculated into 7 mL LB liquid medium containing kanamycin and 25 mg / L rifampicin, and then cultured overnight at 250 rpm under the condition of parameter setting at 28°C. The bacteria are centrifuged at 3,000 rpm for 5 min at room temperature and resuspended with rice transformation solution for transformation of rice callus.

[0106] (3) Agrobacterium EHA105 infection of rice callus

[0107] The callus tissue in good condition obtained in step (2) was collected, and an appropriate amount of transformation solution was added until the bacterial solution completely submerged the callus tissue, and the callus tissue was placed at room temperature for 20 minutes, during which time it was shaken from time to time. Then, the transformed callus tissue was taken out, and the excess bacterial solution was absorbed with sterile filter paper. The callus tissue was cultured in a 23°C incubator in the dark for 2-3 days to obtain transgenic callus tissue (Act:D14-GFP / d14 rice callus tissue, Act:D14ΔN-GFP / d14 rice callus tissue).

[0108] 3. rac-GR24 treatment of callus containing recombinant vectors Act:D14-GFPΔN / d14 or Act:D14-GFP / d14

[0109] 10 μM rac-GR24 working solution: obtained by dissolving rac-GR24 in acetone, wherein the content of rac-GR24 is 10 μM.

[0110] 20 μM rac-GR24 working solution: obtained by dissolving rac-GR24 in acetone, wherein the content of rac-GR24 is 20 μM.

[0111] The specific operation is as follows: select Act:D14-GFP / d14 rice callus and Act:D14ΔN-GFP / d14 rice callus with vigorous growth, i.e., light yellow color, smooth appearance and compact morphology, and transfer them to new NB solid screening medium containing antibiotics, and culture them at 28°C in the dark for 4-6 days. Transfer the callus to NB liquid medium, and culture them at 28°C and 50rpm in the dark for 1-2 hours. Then, add 10μM or 20μM rac-GR24 working solution and treat for 1 hour or 3 hours. After treatment, collect the callus and freeze it with liquid nitrogen.

[0112] 4. Total protein extraction of Act:D14-GFPΔN / d14 and Act:D14-GFP / d14 from callus

[0113] Collect the callus tissue in step 3 and extract the total protein. The specific process is as follows: (1) Grind the callus tissue thoroughly in liquid nitrogen. Make sure the sample is frozen during the entire grinding process. (2) Weigh the same weight of powder sample of each sample into a 1.7mL centrifuge tube that has been frozen in advance, add 2 volumes of RIPA protein extraction solution, vortex and shake thoroughly to mix, place on ice, shake thoroughly for 10 seconds every 8 minutes, shake 3-4 times and then centrifuge. (3) Centrifuge at 18,000g for 20 minutes at 4°C, aspirate 200μL of supernatant into a new 1.7mL centrifuge tube, and add 50μL of protein loading buffer. Denature the sample at 100°C for 5 minutes and perform Western Blot.

[0114] 5. Western Blot detection of the degradation of D14-GFP protein and D14ΔN-GFP protein

[0115] The degradation of D14-GFP protein and D14ΔN-GFP protein was detected by Western Blot experiment. The specific method is as follows: (1) Prepare 10% concentration 12-well or 15-well SDS-PAGE protein gel. The amount of protein sample loaded depends on the experimental requirements. Set the voltage to 80-120V for about 1-2h. (2) Use semi-dry transfer method to transfer the protein to PVDF or NC membrane, the voltage is 15-20V, and the transfer time is 30-60min. (3) Prepare 4% skim milk powder in TBST solution during the transfer. After the transfer, put the PVDF or NC membrane in 4% skim milk powder and incubate it at room temperature for 60min on a shaker at 70rpm. (4) Pour out the skim milk powder, rinse with TBST, add new 4% skim milk powder, add the protein antibody to be detected, and incubate for 1-2h or overnight at 4℃. (5) After incubation with the primary antibody, wash the PVDF or NC membrane three times with TBST, each time at 70 rpm for 5 minutes at room temperature, then add 4% skim milk containing the secondary antibody and incubate at room temperature for 60 minutes on a 60 rpm shaker. (6) After incubation with the secondary antibody, wash the membrane three times with TBST, each time at 70 rpm for 5 minutes at room temperature. Add the color developing solution and use a photosensitive film or a protein developing instrument for protein development. GFP mouse monoclonal antibody (Cat#11814460001) was purchased from Roche. The internal reference protein is Actin, purchased from Abmart, Cat#M20009L.

[0116] The results are as follows Figure 1As shown in A, after rac-GR24 treatment, the D14-GFP protein in Act:D14-GFP / d14 rice callus decreased 1 hour after treatment, and D14-GFP was significantly degraded 3 hours after treatment of transgenic callus; however, the degradation of D14ΔN-GFP in Act:D14ΔN-GFP / d14 rice callus was significantly inhibited ( Figure 1 These results suggest that the NTD motif promotes the degradation of OsD14.

[0117] 6. Subcellular localization detection of D14-GFP and D14ΔN-GFP

[0118] The specific operation is as follows: To construct the 35S:D14-GFP plasmid, the AHLG-Act:D14-GFP plasmid was used as a template, and the D14 fragment was amplified with primers PBI221-D14-F and PBI221-D14-R and homologously recombined into the pBI221-GFP plasmid. To construct the 35S:D14ΔN-GFP plasmid, the D14ΔN fragment was amplified with primers PBI221-D14ΔN-F and PBI221-D14ΔN-R and homologously recombined into the pBI221-GFP vector. The plasmids 35S:D14-GFP plasmid and 35S:D14ΔN-GFP plasmid were transformed into rice protoplasts separately, and cultured in a 28°C incubator in the dark for 14-16 hours. The GFP fluorescence signal was observed using a fluorescence confocal microscope (FluoView 1000; Olympus) with the fluorescence excitation light of 488 nm.

[0119] PBI221-D14-F:

[0120] PBI221-D14-R:

[0121] PBI221-D14ΔN-F:

[0122] PBI221-D14ΔN-R:

[0123] Among the above primers, the bold black sequences are primers for amplifying D14 or D14ΔN, and the underlined sequences are used for homologous recombination with the AH LG vector.

[0124] The results are as follows Figure 1 As shown in B, D14-GFP protein and D14ΔN-GFP protein are expressed in the nucleus and cytoplasm, showing similar expression patterns, indicating that D14ΔN does not affect its own subcellular localization ( Figure 1 (middle B).

[0125] 7. Prokaryotic expression and purification of GST fusion protein

[0126] The specific operation is as follows: To express Glutathone S-transferase (GST)-D3 and GST-D53 fusion proteins, the plasmid was transformed into the expression strain BL21 (DE3), 3-4 single clones were selected and placed in 4 mL LB liquid medium, and shaken at 37°C overnight; the next morning, 3 mL of the bacterial solution was transferred to 300 mL LB liquid medium, and cultured at 37°C for 3-4 hours, and then the OD 600nm The concentration of fusion protein was between 0.6 and 1.0, 90 μL of 1 M IPTG was added, and cultured at 16°C for about 16-20 h to induce the expression of fusion protein.

[0127] Purification of GST fusion protein was performed according to the instructions of Glutathione Sepharose 4Fast Flow. Collect the cells by centrifugation at 4,000 rpm for 10 min at 4°C, and resuspend with 20 mL of GST-tagged protein extract (Appendix H) for every 300 mL of bacterial liquid; use a high-pressure cell disruptor to disrupt the cells 2-3 times until the cells are clear, centrifuge at 15,000 rpm for 30 min at 4°C, and collect the supernatant; add 200-300 μL of GST column filler for every 20 mL of protein supernatant, where the column filler needs to be washed 2-3 times with GST-tagged protein extract in advance, centrifuged at 500g for 5 min at 4°C each time; after incubation at 4°C for 2-3 h, wash 3 times with GST-tagged protein extract, and after the last wash, transfer the beads to a 2 or 5 mL centrifuge tube, centrifuge at 500g for 5 min at 4°C, remove the supernatant, and add 1.6 mL of PBS buffer for use.

[0128] 8. GST-pull down detection of the interaction between D14ΔN-GFP and GST-D3 and GST-D53

[0129] The specific operation is as follows: first, use IP protein extraction solution containing a final concentration of 50μM MG132 (Proteasome inhibitor) to extract plant total protein from Act:D14ΔN-GFP rice callus at a ratio of w / v = 1:2; add 30μL of GST-D3 or GST-D53 column filler (Beads with coupled protein obtained in the previous step) and 1μL 20mM rac-GR24 or 1μL acetone to each 1mL of total protein of plant callus; incubate at 4℃ with rotation for 2-3h, then wash 3-4 times with IP protein extraction solution, boil at 100℃ for 8min with 60μL SDS loading, and detect protein-protein interactions by Western-blot. GFP fusion protein was detected using GFP antibody (1:3000, Roche, Cat#11814460001), and GST fusion protein was detected by staining with Ponceau S or GST mouse monoclonal antibody (1:5000, Abmart, Cat#M20007) at a dilution ratio of 1:5,000.

[0130] The results showed that compared with the full-length form of D14-GFP, D14ΔN-GFP interacted more strongly with GST-D3 and GST-D53 in the absence of exogenous application of rac-GR24. After exogenous application of rac-GR24, the interaction of D14-GFP and D14ΔN-GFP with GST-D3 was enhanced, showing no significant difference. At the same time, after exogenous application of rac-GR24, the interaction of D14-GFP and D14ΔN-GFP with GST-D53 was also significantly enhanced, showing no significant difference ( Figure 1 These results indicate that D14ΔN has the ability to sense strigolactone signals and can significantly inhibit the degradation of D14.

[0131] Example 2: Regulatory effect of the NTD motif of OsD14 protein on tillering development in rice

[0132] 1. Obtaining transgenic rice overexpressing D14 or D14ΔN

[0133] The specific method is as follows: the Act:D14-GFP / d14 rice callus and Act:D14ΔN-GFP / d14 rice callus obtained in Example 1 "2. Callus induction containing recombinant vector Act:D14-GFP or Act:D14ΔN-GFP" are transferred to NB solid screening medium containing hygromycin, cultured in the dark at 28°C for 7-10 days, and then subcultured to a new NB solid screening medium containing hygromycin, and 3-4 rounds of hygromycin screening medium are performed. The callus with good growth status is propagated to differentiation medium for differentiation and regeneration, and after about one month of light culture at 28°C, the grown seedlings are transferred to rooting medium for about 4 weeks for rooting and seedling strengthening, and transplanted to a greenhouse for about one month before being transferred to the field.

[0134] 2. Identification of positive plants

[0135] The specific method is as follows: (1) Leaves were taken from individual rice plants, and rice genomic DNA was extracted using the CTAB method. The extracted DNA was used as a template and PCR amplification was performed using AHLG-F and D14-570-R identification primers. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. After the sequencing results were obtained, the sequences were compared. The plants with the sequences consistent with the expected sequences were T0 generation positive plants. 16 T0 generation positive plants transformed with AHLG-Act:D14-GFP vector (named Act:D14-GFP / d14 plants) and 20 T0 generation positive plants transformed with AHLG-Act:D14ΔN-GFP vector (named Act:D14ΔN-GFP / d14 plants) were obtained. (2) Eight Act:D14-GFP / d14 and eight Act:d14ΔN-GFP / d14 positive T0 generation plants were randomly selected for breeding, and eight T1 generation lines were planted respectively. Six plants were randomly collected from each T1 generation line, which can be used to screen homozygous positive plants. (3) The harvested T1 generation seeds were germinated, and 40 germinated seeds were placed on a 0.5% Agar solid culture 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 plants, and those with abnormal root growth were isolated pseudo-plants. If all seeds of each plant can grow roots normally, they are homozygous positive lines. (4) Six Act:D14-GFP / d14 homozygous positive plants were obtained, and five Act:d14ΔN-GFP / d14 homozygous positive plants were obtained. The Act:D14-GFP / d14 homozygous positive individual plants (T2 generation) and Act:d14ΔN-GFP / d14 homozygous positive individual plants (T2 generation) used in subsequent experiments were named Act:D14-GFP / d14#1, Act:D14-GFP / d14#2, Act:d14ΔN-GFP / d14#1 and Act:d14ΔN-GFP / d14#2, respectively.

[0136] Primers for identification of transgenic positive seedlings:

[0137] AHLG-F:5'-TCAGCATTGTTCATCGGTAG-3';

[0138] D14-570-R:5'-CTGCTGTATCCTCCTCCAGCTC-3'.

[0139] PCR reaction system: 1μL DNA template, 2μL 10μM Primer F, 2μL 10μM Primer R and 45μL Gold Mix. Reaction procedure: 98℃ pre-denaturation for 3min; 35 cycles of 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 15s; 72℃ extension for 5min.

[0140] 3. Count the number of tillers of homozygous positive plants

[0141] The experimental method is as follows: wild-type rice Nipponbare, d14 mutant, the above homozygous plants Act:D14-GFP / d14#1, Act:D14-GFP / d14#2, Act:d14ΔN-GFP / d14#1 and Act:d14ΔN-GFP / d14#2 were planted in the field of the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing. Each strain was planted with 24 plants with a plant spacing and row spacing of 17 cm. The growth period was from May to November each year. The field phenotypic survey time of rice materials was from September 20 to October 7 each year. The number of tillers counted was the number of effective tillers at maturity (i.e., the number of tillers that grew ears), and the plant height was the distance between the highest ear of rice and the ground. The average number of tillers for each material was used as the standard, and representative single-plant transplant pots were selected for photography.

[0142] The results showed that both transgenic materials were able to restore the dwarf multi-tillering phenotype of the d14 mutant to the wild-type level when the overexpression levels were basically the same, and Act:D14ΔN-GFP / d14 plants showed a phenotype with fewer tillers than Act:D14-GFP / d14 plants ( Figure 2 (A).

[0143] 4. Detection of D53, D14 / D14ΔN-GFP protein levels in positive plants

[0144] The experimental method is as follows: Referring to the method shown in Example 1 "5. Western Blot detection of degradation of D14-GFP protein and D14ΔN-GFP protein", the levels of D53 protein, D14-GFP protein and D14ΔN-GFP protein in positive plants were detected by Western Blot experiment.

[0145] The results showed that the protein level of D14ΔN-GFP in Act:D14ΔN-GFP / d14 was significantly higher than that of D14-GFP in Act:D14-GFP / d14, and the protein level of D53 in Act:D14ΔN-GFP / d14 was significantly lower than that in wild-type and Act:D14-GFP / d14 plants ( Figure 2 (B) This indicates that overexpression of D14ΔN-GFP will cause the plant to show a phenotype of enhanced strigolactone signaling.

[0146] 5. Detection of the transcription level of SL-biosynthesis gene D10 in the roots of transgenic rice seedlings

[0147] The experimental method is as follows: rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28°C for 16 h of light and 25°C for 8 h of darkness. The light intensity (150–200 μM m -2 s -1 ), and the humidity was 70%. After culturing in Kimura B nutrient solution for 2 weeks, the root tissues of seedlings of different materials were collected.

[0148] The Trizol method (Life Technologies: 15596-026) was used to extract RNA from rice seedlings. The reagents and experimental consumables used in the RNA extraction process had been free of RNase. Rice tissue samples were thoroughly ground in liquid nitrogen. During the whole grinding process, the samples were kept frozen. About 100-150 mg of powdered samples were placed in a 1.7 mL centrifuge tube, 1 mL of Trizol was added, and the mixture was thoroughly vortexed and allowed to stand at room temperature for 5 min. After that, 200 μL of chloroform was added and immediately inverted for 15 s, allowed to stand at room temperature for 5 min, and then centrifuged at 12,000 g for 15 min at 4 °C. 500 μL of supernatant was placed in a 1.7 mL centrifuge tube, an equal volume of isopropanol was added, the tube was inverted 20 times to mix, and allowed to stand at room temperature for 10 min, and then centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was discarded, 1 mL of 70% (v / v) ethanol was added to the precipitated RNA, the tube was inverted 30 times, and centrifuged at 7,500 g for 5 min. The supernatant was discarded, and after the precipitate was dried, 50 μL of RNase-free water was added, and the tube was placed on ice for more than 3 h to fully dissolve the RNA. The supernatant was then removed and the tube was precipitated. The OD values ​​at 230 nm, 260 nm, and 280 nm were measured using a 2000 UV-Vis spectrophotometer to evaluate the overall quality of RNA preparation. RNA samples were directly used for RNA reverse transcription experiments or placed at -20 °C for later use.

[0149] Using TURBO DNA-free TM The kit removes residual DNA from total RNA. For detailed steps, see the instructions. Each reaction system is set to 20 μL, total RNA is 12.5 μg, incubate at 37°C for 30 min, add 2 μL DNase inactivation reagent and mix thoroughly at room temperature for 5 min, centrifuge at 12,000g for 5 min, and use 4 μL supernatant for reverse transcription experiments. III First-Strand Synthesis System reverse transcription kit to synthesize the first strand of cDNA. For detailed steps, see the instructions. Finally, add 180 μL of nuclease-free double-distilled water to dissolve the reverse transcription product and mix thoroughly before use.

[0150] Fluorescence quantitative PCR was used to detect the transcription level of SL-biosynthesis gene D10 in the roots of transgenic rice seedlings. Each reaction system in fluorescence quantitative PCR was 10μL, including 5μL SsoFast EvaGreen supermix, 0.5μL sense primer (5μM), 0.5μL antisense primer (5μM), 2.0μL diluted cDNA and 2μL dd H2O. The instrument used for fluorescence quantitative PCR was BIO-RAD's real-time fluorescence quantitative PCR instrument (model: CFX96). The PCR running program parameter setting information was: 98℃, 30s; (98℃, 5s→60℃, 5s→information collection), 40cycles; 60-95℃, 0.5℃ / 5s, information collection / 5s. After the program was completed, the data was processed by BIO-RAD CFX Manager software.

[0151] Fluorescence quantitative PCR primers for detecting D10 gene expression:

[0152] qD10-F:5'-CGTGGCGATATCGATGGT-3';

[0153] qD10-R:5'-CGACCTCCTCGAACGTCTT-3'.

[0154] qUbiquitin-F:5'-AACCAGCTGAGGCCCAAGA-3';

[0155] qUbiquitin-R:5'-ACGATTGATTTAACCAGTCCATGA-3'.

[0156] The results showed that compared with the wild type and Act:D14-GFP / d14, the expression of the strigolactone biosynthesis gene D10 was decreased in the D14ΔN-GFP overexpression line ( Figure 2 Middle C).

[0157] 6. Detection of 4DO levels in root exudates and root tissues of transgenic rice seedlings

[0158] The experimental method is as follows: rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28°C for 16 h of light and 25°C for 8 h of darkness. The light intensity (150–200 μM m -2 s -1), humidity was 70%. After 1 week of culture in normal Kimura culture medium, culture in phosphorus-deficient culture medium was continued for 2 weeks, and new culture medium was replaced every 3 days. The aqueous solution was replaced on the day of sampling, and 5 hours later, the hydroponic solution was collected, secretion into the hydroponic solution was detected, and rice roots were collected at the same time. The samples were sent to the hormone platform of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for determination of the content of 4DO in rice root secretion and hydroponic solution.

[0159] The results showed that compared with the wild type and Act:D14-GFP / d14, the endogenous SL content in the D14ΔN-GFP overexpression line was reduced ( Figure 2 Combined with the changes in D10 expression, it is suggested that the enhanced signal of strigolactones in the D14ΔN-GFP overexpression line is not due to the synthesis of more strigolactones in the plant.

[0160] 7. Detection of the transcription level of rice tillering inhibitory gene OsTB1 at the stem base of transgenic rice seedlings

[0161] The experimental method is as follows: rice seedlings were grown in a rice incubator (MLR-351H, SANYO, Japan) at 28°C for 16 h of light and 25°C for 8 h of darkness. The light intensity (150–200 μM m -2 s -1 ), and the humidity was 70%. After culturing in Kimura culture medium for 2 weeks, the stem base tissues (1 cm) of seedlings of different materials were collected. Fluorescence quantitative PCR was used to detect the transcription level of the rice tillering inhibitory gene OsTB1 in transgenic rice. The specific method was as described above, and the internal reference was qUbiquitin.

[0162] Fluorescence quantitative PCR primers for detecting OsTB1 gene expression:

[0163] OsTB1-F:5'-CGACAGCGGCAGCTACTAC-3'

[0164] OsTB1-R:5'-GCGAATTGGCGTAGACGA-3'

[0165] The results showed that compared with the wild type and Act:D14-GFP / d14, the expression of OsTB1, a tillering inhibitory gene downstream of strigolactone, was increased in the D14ΔN-GFP overexpression line ( Figure 2 (E). This result further demonstrates that Act:D14ΔN-GFP / d14 has an enhanced strigolactone signaling pathway.

[0166] In summary, the protein stability of D14 was improved after the NTD end was deleted, and the sensitivity to strigolactone was stronger. The plants showed a phenotype of enhanced strigolactone signaling and less tillering phenotype.

[0167] Example 3: Creation of NTD motif knockout mutant of OsD14 and its effect on tillering

[0168] 1. Construction of knockout vector

[0169] The pYLCRISPR / Cas9Pubi-H, pYLgRNA-OsU3 and pYLgRNA-OsU6a vectors in the above embodiments have been recorded in: Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y., et al. (2015). A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274-1284. The public can obtain the biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0170] The CRIPSR / Cas9 system was used to edit the N-terminal genomic region of rice OsD14. The specific steps are as follows:

[0171] (1) Selection of target sequences

[0172] Target site T1: 5′-GGCTTGCCAATCCGCCGCGC-3′, located at nucleotides 232-251 of the wild-type genomic sequence of the OsD14 gene.

[0173] Target site T2: 5′-GTCGAGCTCGGAGAAGACGA-3′, located at nucleotides 342-361 of the wild-type genomic sequence of the OsD14 gene.

[0174] Target site T3: 5′-CCGCCCAGTAGCCCGAGCAG-3′, located at nucleotides 280-299 of the wild-type genomic sequence of the OsD14 gene.

[0175] Target site T4: 5′-GGGAGGAGGGAGCGGGTCGG-3′, located at nucleotides 384-403 of the wild-type genomic sequence of the OsD14 gene.

[0176] (2) Use Overlapping PCR to construct the gene target sgRNA expression cassette.

[0177] Take 2-5 ng pYLgRNA-OsU3 plasmid as template, and use 4 primers in one reaction: 0.2μM each of UF and D14-N-CRI-U3-R1, 0.1μM each of D14-N-CRI-U3-F1 and gR-R. 25-28 cycles: 94℃10s, 58℃15s, 68℃20s, and the PCR product of OsU3 promoter-driven sgRNA expression cassette containing gene target site T1 is obtained, named OsU3-D14-T1. Similarly, take 2-5 ng pYLgRNA-OsU6a plasmid as template, and use 4 primers in one reaction: 0.2μM each of UF and D14-N-CRI-U6a-R1, 0.1μM each of D14-N-CRI-U6a-F1 and gR-R. 25-28 cycles: 94°C for 10 s, 58°C for 15 s, and 68°C for 20 s, to obtain the PCR product of the OsU6a promoter-driven sgRNA expression cassette containing the gene target site T2, named OsU6a-D14-T2.

[0178] OsD14 NTD motif genome editing primers:

[0179] D14-N-CRI-U3-F1:

[0180] D14-N-CRI-U3-R1:

[0181] D14-N-CRI-U6a-F1:

[0182] D14-N-CRI-U6a-R1:

[0183] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3';

[0184] gR-R: 5'-CGGAGGAAAATTCCATCCAC-3'.

[0185] (3) The first-round PCR products OsU3-D14-T1 and OsU6a-D14-T2 were diluted 10 times with H2O, and new PCR fragments OsU3-D14-N1 and OsU6a-D14-N1 were amplified using primers B1' and B2 and B1' / B2 primers, respectively.

[0186] Uctcg-B1′: 5′-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG--3′ (U3 upstream primer);

[0187] gRctga-B2: 5′-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3′ (RNA downstream primer);

[0188] Uctga-B2′: 5′-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3′ (U6a upstream primer);

[0189] gRcggt-BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3' (complementary to the right side of the vector (BL')).

[0190] (4) Assemble the sgRNA expression cassette into the pYLCRISPR / Cas9 vector using the cutting and ligation method. The system is as follows:

[0191] Table 1 10μL 1x BsaI digestion ligation reaction solution

[0192]

[0193] The PCR instrument was set up with the following program: 37°C for 5 min; 10°C for 5 min, 20°C for 5 min, 10-15 cycles, and finally 37°C for 5 min.

[0194] (5) Chemical heat shock transformation can also obtain positive clones, extract plasmids, and detect the s gRNA expression box junction fragment by Mlu I or Asc I restriction electrophoresis. Send it to the company for sequencing. This CRISPR plasmid containing T1 and T2 dual targets is named pYLCRISPR / Cas9Pubi-H-D14-N1 plasmid.

[0195] A CRISPR plasmid containing T3 and T4 dual targets was constructed according to the above method and named pYLCRISPR / Cas9Pubi-H-D14-N2 plasmid. The primers used are as follows. The rest of the operations are the same as the construction of pYLCRISPR / Cas9Pubi-H-D14-N1 plasmid.

[0196] D14-N-CRI-U3-F2: 5'-CTGCTCGGGCTACTGGGCGG gttttagagctagaaat-3';

[0197] D14-N-CRI-U3-R2: 5'-CCGCCCAGTAGCCCGAGCAG Tgccacggatcatctgc-3';

[0198] D14-N-CRI-U6a-F2: 5'-GGGAGGAGGGAGCGGGTCGG gttttagagctagaaat-3';

[0199] D14-N-CRI-U6a-R2: 5'-CCGACCCGCTCCCTCCTCCC Cggcagccaagccagca-3'.

[0200] 2. Obtaining transgenic rice with knockout of D14 NTD motif

[0201] The wild-type Nipponbare was used to replace the d14 mutant in Example 1 “2. Callus induction containing recombinant vectors Act:D14-GFP or Act:D14ΔN-GFP”, and the pYLCRISPR / Cas9Pubi-H-D14-N1 plasmid and the pYLCRISPR / Cas9Pubi-H-D14-N2 plasmid were used to replace the AHLG-Act:D14-GFP vector or the AHLG-Act:D14ΔN-GFP vector in Example 1 “2. Callus induction containing recombinant vectors Act:D14-GFP or Act:D14ΔN-GFP”. The remaining operations were the same as those in Example 1 “2. Callus induction containing recombinant vectors Act:D14-GFP or Act:D14ΔN-GFP”.

[0202] 3. Identification of positive plants

[0203] The specific method is as follows: Select normally growing plants with hygromycin resistance for identification of mutation sites. The specific methods of extracting total plant DNA and PCR identification are as described above. The amplified PCR product is purified and sent to the company for sequencing. The sequencing results are compared with the sequence of the wild-type plant Nipponbare. If there is only one PCR amplification product of the regenerated plant, and it is consistent with the nucleotide sequence of the PCR amplification product of the wild-type plant Nipponbare, the regenerated plant is wild type. If there are two PCR amplification products of the regenerated plant, one is consistent with the nucleotide sequence of the PCR amplification product of the wild-type plant Nipponbare, and the other is mutated compared with the nucleotide sequence of the PCR amplification product of the wild-type plant Nipponbare (mutation includes one or more nucleotide deletions, insertions or substitutions), the regenerated plant is heterozygous. If there are two PCR amplification products of the regenerated plant, both of which are mutated compared with the nucleotide sequence of the PCR amplification product of the wild-type plant Nipponbare (mutation includes one or more nucleotide deletions, insertions or substitutions), the regenerated plant is a biallelic mutant. If the PCR amplification product of the regenerated plant is one, and a mutation occurs compared to the nucleotide sequence of the PCR amplification product of the wild-type plant Nipponbare (the mutation includes the deletion, insertion or substitution of one or more nucleotides), the regenerated plant is a homozygous mutant. If the PCR amplification product of the regenerated plant is three or more, the regenerated plant is a mosaic. Heterozygous, biallelic mutant, homozygous mutant, and mosaic plants are collectively referred to as edited plants.

[0204] Among the 88 gene-edited candidate plants, two homozygous plants were obtained and named d14ΔN-1 and d14ΔN-2. The mutant forms are shown in Figure 3 Middle A. d14ΔN-1 is a homozygous plant obtained by transformation with pYLCRISPR / Cas9Pubi-H-D14-N1 plasmid. d14ΔN-2 is a homozygous plant obtained by transformation with pYLCRISPR / Cas9Pubi-H-D14-N2 plasmid. Both mutants truncate most of the NTD region of D14, which can be used to deeply analyze the function of NTD on D14 and the regulatory mechanism of rice tillering development.

[0205] Compared with wild-type rice, the D14 genes in the two homologous chromosomes of the homozygous mutant d14ΔN-1 rice underwent the following mutations: nucleotides 249-358 of the wild-type genomic sequence of the OsD14 gene were deleted, a total of 110 nucleotides were deleted, and no frameshift mutation occurred in the protein sequence outside the N-terminus of the OsD14 protein.

[0206] Compared with wild-type rice, the homozygous mutant d14ΔN-2 rice showed the following mutations in the D14 genes in the two homologous chromosomes of the homozygous mutant d14ΔN-1 rice: nucleotides 249-358 of the wild-type genomic sequence of the OsD14 gene were deleted and inserted, nucleotides 241-368 were deleted, a total of 128 nucleotides were deleted, and a T base was inserted between nucleotides 399 and 400 of the wild-type genomic sequence of the OsD14 gene. No frameshift mutation occurred in the protein sequence outside the N-terminus of the OsD14 protein.

[0207] 4. Count the tillering number of positive plants

[0208] The experimental method is as follows: the wild-type rice Nipponbare and the d14 mutant in "4. Counting the number of tillers of homozygous positive plants" in Example 2 are replaced by the wild-type rice Nipponbare and the above-mentioned homozygous plants d14ΔN-1 and d14ΔN-2, and the above-mentioned homozygous plants Act:D14-GFP / d14#1, Act:D14-GFP / d14#2, Act:d14ΔN-GFP / d14#1 and Act:d14ΔN-GFP / d14#2. Other operations are the same as those in Example 2 "4. Counting the number of tillers of homozygous positive plants".

[0209] The results showed that compared with the wild type, the tiller numbers of d14ΔN-1 and d14ΔN-2 were less than that of the wild type ( Figure 3 (middle B).

[0210] 5. Detection of D53 protein levels in positive plants

[0211] The experimental method is as follows: The level of D53 protein in positive plants was detected by Western Blot experiment, and the specific method is as described in Example 1 "5. Western Blot detection of degradation of D14-GFP protein and D14ΔN-GFP protein". The internal reference protein is Actin, purchased from Abmart, Cat#M20009L. The specific method is as described above.

[0212] The results showed that D53 protein level was significantly decreased compared with the wild type ( Figure 3 Middle C).

[0213] 6. Detection of the transcription level of SL-biosynthesis gene D10 in the roots of transgenic rice seedlings

[0214] The experimental method is as follows: Fluorescence quantitative PCR is used to detect the transcription level of SL-biosynthesis gene D10 in transgenic rice seedling roots. The specific method is as described in Example 2, "5. Detection of transcription level of SL-biosynthesis gene D10 in transgenic rice seedling roots".

[0215] The results showed that the expression of the strigolactone biosynthesis gene D10 was decreased in the d14ΔN strain compared with the wild type ( Figure 3 (middle D).

[0216] 7. Detection of the transcription level of rice tillering inhibitory gene OsTB1 at the stem base of transgenic rice seedlings

[0217] The experimental method is as follows: Fluorescence quantitative PCR is used to detect the transcription level of the rice tillering inhibitory gene OsTB1 in transgenic rice. The specific method is as described in Example 2, "7. Detecting the transcription level of the rice tillering inhibitory gene OsTB1 at the base of the stem of transgenic rice seedlings."

[0218] The results showed that compared with the wild type, the expression of OsTB1, a tillering inhibitory gene downstream of strigolactone, was increased ( Figure 3 Middle E).

[0219] In summary, the NTD motif of D14 promotes rice tillering by inhibiting strigolactone perception.

[0220] The above embodiment involves the following sequence:

[0221] Wild-type amino acid sequence of OsD14 gene SEQ ID No.1

[0222] MLRSTHPPPSSPSSSSSGGGGGGGSSASSSSEKTMVGGGGGGGGGSGSAAPSGAKLLQILNVRVVGSGERVVVLSH

[0223] GFGTDQSAWSRVLPYLTRDHRVVLYDLVCAGSVNPDHFDFRRYDNLDAYVDDLLAILDALRIPRCAFVGHSVSAMIGILA

[0224] SIRRPDLFAKLVLIGASPRFLNDSDYHGGFELEEIQQVFDAMGANYSAWATGYAPLAVGADVPAAVQEFSRTLFNMRPDI

[0225] SLHVCQTVFKTDLRGVLGMVRAPCVVVQTTRDVSVPASVAAYLKAHLGGRTTVEFLQTEGHLPHLSAPSLLAQVLRRALARRCGQS*.

[0226] Wild-type CDS sequence of OsD14 gene SEQ ID No.2

[0227] 5'-ATGCTGCGATCGACGCATCCGCCGCCCAGTAGCCCGAGCAGCAGCAGCAGCGGCGGCGGCGGGGGCGGGGGGTCGTCGGCGTCGTCGAGCTCGGAGAAGACGATGGTGGGCGGCGGGGGAGGAGGGGGAGGAGGGAGCGGGTCGGCGGCGCCGAGCGGGGCGAAGCTGCTGCAGATCCTGAACGTGCGGGTGGTGGGGAGCGGCGAGCGGGTGGTGGTGCTGTCGCATGGCTTCGGGACGGACCAGTCGGCGTGGAGCCGCGTGCTGCCGTACCTCACCCGCGACCACCGCGTCGTGCTCTACGACCTCGTCTGCGCCGGCAGCGTCAACCCGGACCACTTCGACTTCCGCCGCTACGACAACCTCGACGCCTACGTCGACGACCTGCTCGCCATCCTCGACGCGCTCCGCATCCCGCGCTGCGCCTTCGTCGGCCACTCCGTCTCCGCCATGATCGGCATCCTCGCCTCCATCCGACGACCTGACCTCTTCGCCAAGCTTGTCCTCATCGGCGCCTCTCCCCGGTTCTTGAACGACAGCGACTACCACGGCGGGTTCGAGCTGGAGGAGATACAGCAGGTGTTCGACGCGATGGGGGCGAACTACTCGGCGTGGGCGACGGGGTACGCGCCTCTGGCGGTGGGCGCCGACGTGCCGGCGGCGGTGCAGGAGTTCAGCCGCACCCTCTTCAACATGCGCCCGGACATCTCCCTCCACGTCTGCCAGACCGTCTTCAAGACCGACCTCCGCGGCGTGCTCGGCATGGTCCGCGCCCCCTGCGTCGTCGTCCAGACCACCCGCGACGTCTCCGTCCCGGCCTCCGTCGCCGCCTACCTCAAGGCCCACCTCGGCGGCCGCACCACCGTCGAGTTCCTCCAGACCGAGGGTCACCTCCCCCACCTCAGCGCCCCCAGCCTCCTCGCCCAGGTGCTCCGCCGCGCTCTCGCCCGGTACTAA-3'。

[0228] Wild-type genomic sequence of the OsD14 gene

[0229]

[0230]

[0231] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that, Use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity and / or content of the protein in any of the following: M1) Regulate the number of plant tillers; M2) preparing products for regulating the number of tillers of plants; M3) Plant breeding; The protein is any of the following: A1) a protein whose amino acid sequence is the 51st to 322nd position of SEQ ID No. 1 or a protein whose amino acid sequence is the SEQ ID No. 1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

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

3. The use according to claim 1, characterized in that: The regulation is to down-regulate, inhibit or reduce the expression of the gene encoding the protein in claim 1 or the content or activity of the protein.

4. The use according to any one of claims 1 to 3, characterized in that: The substance is a biological material, and the biological material is any one of the following: B1), an RNA molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein of claim 1 or an RNA molecule that inhibits, reduces or down-regulates the activity or content of the protein; B2), expressing the coding gene of the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4); B9), a nucleic acid molecule encoding the protein according to claim 1; B10), an expression cassette containing the nucleic acid molecule described in B9); B11), a recombinant vector containing the nucleic acid molecule described in B9), or a recombinant vector containing the expression cassette described in B10); B12), a recombinant microorganism containing the nucleic acid molecule described in B9), or a recombinant microorganism containing the expression cassette described in B10), or a recombinant microorganism containing the recombinant vector described in B3); B13), a transgenic plant cell line containing the nucleic acid molecule described in B9), or a transgenic plant cell line containing the expression cassette described in B10); B14), transgenic plant tissue containing the nucleic acid molecule described in B9), or transgenic plant tissue containing the expression cassette described in B10); B15), a transgenic plant organ containing the nucleic acid molecule described in B9), or a transgenic plant organ containing the expression cassette described in B10).

5. The use according to claim 4, characterized in that: B1) The RNA molecule targets the gene encoding the protein.

6. The use according to claim 4, characterized in that: B9) The coding sequence of the nucleic acid molecule is SEQ ID No.

2.

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

8. Application, characterized in that, Use of a polypeptide or a substance for regulating the expression of a gene encoding the polypeptide or a substance for regulating the activity and / or content of the polypeptide in any of the following: M1) Regulate the number of plant tillers; M2) preparing products for regulating the number of tillers of plants; M3) Plant breeding; The protein is any of the following: A1) the amino acid sequence is amino acids 1 to 50 of SEQ ID No. 1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of A1) or A2).

9. The use according to claims 1 to 6 or claim 8 or the method according to claim 7, characterized in that: The plant is any one of the following: G1) Monocots; G2) Gramineae; G3) Oryza plants; G4) Rice plants; G5) Rice.

10. The protein according to any one of claims 1 to 5, or a substance that regulates the expression of a gene encoding the protein, or a substance that regulates the activity and / or content of the protein.

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