Method for promoting flowering of plants and application

Abstract

The invention provides a method for promoting flowering of plants and application. The method comprises the following steps: editing a wild-type florigen FT gene of a target plant so as to promote flowering of the target plant; the editing comprises the following steps: editing a wild-type florigenic FT gene of a target plant, so that a wild-type florigenic FT protein expressed by the target plant is mutated into a mutant FT protein, and the target plant is promoted to bloom; the editing comprises the following steps: replacing at least one nucleotide in the wild-type florigen FT gene to mutate the protein coded by the wild-type florigen FT gene to obtain a mutant FT protein; the method can solve the problem of lack of a method for rapidly and effectively promoting plant flowering in the prior art, and is suitable for the field of plant breeding.

Description

Technical Field

[0001] The invention relates to the field of plant breeding, and in particular to a method and application for promoting plant flowering. Background Art

[0002] Flowering of plants is a critical stage in their life cycle, and precise regulation of its timing is crucial to crop production and ecological adaptability. In the agricultural field, especially for gramineous crops such as wheat, corn, and rice, the regulation of heading / flowering period is directly related to the yield, quality, and adaptability of crops to environmental changes. If the flowering time of crops is inappropriate, it will seriously affect their growth cycle, resulting in a decrease in yield, or losses due to the inability to adapt to the environmental conditions of a specific season.

[0003] Florigin FT (flowering locus T), as the core integrator of flowering regulation by various environmental signals such as photoperiod, temperature, and hormones, is widely and conservatively present in flowering plants and plays a vital role in flowering time. FT protein, belonging to the phosphatidylethanolamine binding protein (PEBP) family, forms heterodimers with the bZIP transcription factor FLOWERING LOCUSD (FD) in apical meristem (SAM) cells through long-distance transport in the phloem, activates downstream flowering gene expression, promotes flower primordium differentiation, and thus promotes plant flowering. FT homologs, such as Hd3a in rice, SbFT1, SbFT8, and SbFT10 in sorghum, ZCN8 in maize, SFT in tomato, and GmFT1a and GmFT2a / 5a in soybean, all control flowering time in a photoperiod-dependent manner in different plants. However, the functions of FT proteins vary in different species, and these differences often originate from subtle changes in their amino acid sequences, which poses a challenge to regulating flowering time through precise molecular design.

[0004] At present, the field of plant breeding and genetic improvement mainly uses traditional hybridization, mutagenesis screening and gene knockout techniques to regulate the flowering time of plants. However, these methods are often inefficient, time-consuming, and difficult to accurately control the flowering time. Especially for complex crops such as wheat and corn, whose flowering regulation mechanism is more complicated, traditional genetic improvement methods are difficult to achieve the ideal regulation effect. Summary of the invention

[0005] The main purpose of the present invention is to provide a method and application for promoting plant flowering, so as to solve the problem that there is a lack of a method for promoting plant flowering quickly and effectively in the prior art.

[0006] In order to achieve the above object, according to a first aspect of the present invention, a method for promoting plant flowering is provided, the method comprising:

[0007] The wild-type florigen FT gene of the target plant is edited so that the wild-type florigen FT protein expressed by the target plant is mutated into a mutant FT protein, thereby promoting flowering of the target plant; the editing includes: replacing at least one nucleotide in the wild-type florigen FT gene so that the protein encoded by the wild-type florigen FT gene is mutated to obtain a mutant FT protein; the wild-type florigen FT protein includes: the protein shown in SEQ ID NO: 7, or a homologous protein having at least 75% sequence homology and the same functional conserved sites as the protein shown in SEQ ID NO: 7; the mutation includes: in the wild-type florigen FT protein, corresponding to the protein shown in SEQ ID NO: 7, any one or more of the following mutations are performed: mutation of valine at position 70 to isoleucine; mutation of cysteine ​​at position 164 to serine; mutation of tyrosine at position 162 to phenylalanine; mutation of threonine at position 31 to isoleucine. Homology.

[0008] Further, the wild-type florigen FT protein includes any one of the following proteins a1)-a2): a1) Arabidopsis FT protein shown in SEQ ID NO: 7, the PmFTL1 protein shown in SEQ ID NO: 3, the PmFTL2 protein shown in SEQ ID NO: 4, or the TaVRN3-7B protein shown in SEQ ID NO: 10; or a2) a protein having the same function obtained by connecting a protein tag to the N-terminus or / and C-terminus of the FT protein in a1); the mutation includes any one or more of the following: mutating the valine at position 70 in SEQ ID NO: 7 to isoleucine; mutating the cysteine ​​at position 164 in SEQ ID NO: 7 to serine; mutating the tyrosine at position 162 in SEQ ID NO: 7 to phenylalanine; mutating the threonine at position 31 in SEQ ID NO: 7 to isoleucine; mutating the asparagine at position 65 in SEQ ID NO: 4 to isoleucine; mutating the valine at position 70 in SEQ ID NO: 10 to isoleucine.

[0009] Further, the wild-type florigen FT gene includes: b1) a gene encoding Arabidopsis FT protein, PmFTL1 protein, PmFTL2 protein or TaVRN3-7B protein; the gene encoding Arabidopsis FT protein is an FT gene having a nucleotide sequence shown in SEQ ID NO: 6; the gene encoding PmFTL1 protein is an FT gene having a nucleotide sequence shown in SEQ ID NO: 1; the gene encoding PmFTL2 protein is an FT gene having a nucleotide sequence shown in SEQ ID NO: 2; the gene encoding TaVRN3-7B protein is an FT gene having a nucleotide sequence shown in SEQ ID NO: 9; b2) an FT gene that has at least 75% homology with the coding gene in b1) and encodes any one of the FT proteins in a1)-a2); or b3) an FT gene that hybridizes with the coding gene in b1) under strict conditions and encodes any one of the FT proteins in a1)-a2).

[0010] Further, the method comprises: introducing genetic material into the target plant to obtain a plant with an early flowering time; preferably, introducing genetic material into the tissue or cell of the target plant, and culturing the introduced cell or tissue into a complete plant to obtain a plant with an early flowering time; preferably, the genetic material comprises a DNA circular plasmid, a DNA linear fragment, or in vitro transcribed RNA; preferably, the genetic material comprises a recombinant vector containing a gene mutation site; preferably, the editing method comprises seamless cloning, error-prone PCR, CRISPR / Cas9, CRISPR / Cas12 or RED recombination; preferably, introducing The method includes gene gun method, Agrobacterium infection method, PEG-induced protoplast method, electrode method, silicon carbide fiber-mediated method or vacuum infiltration method, more preferably Agrobacterium infection method; preferably, the cells of the target plant include protoplast cells or suspension cells; preferably, the tissues of the target plant include callus, immature embryos, mature embryos, leaves, stem tips, young ears or hypocotyls; preferably, the target plant includes monocotyledonous plants or dicotyledonous plants; preferably, the monocotyledonous plants include millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; preferably, the dicotyledonous plants include Arabidopsis, rapeseed, mustard, soybean, cotton, sweet orange or pecan.

[0011] In order to achieve the above-mentioned object, according to a second aspect of the present invention, a protein for promoting plant flowering is provided, the protein comprising: the amino acid sequence of a wild-type florigen FT protein is as shown in SEQ ID NO: 7; a homologous protein of the wild-type florigen FT protein has at least 75% sequence homology with the wild-type florigen FT protein, and a mutation site in the homologous protein of the wild-type florigen FT protein corresponds to a functionally conserved site in the wild-type florigen FT protein in the homologous protein; the mutation comprises: in the wild-type florigen FT protein or a homologous protein of the wild-type florigen FT protein, corresponding to the wild-type florigen FT protein, any one or more of the following mutations are performed: a mutation of valine at position 70 to isoleucine; or a mutation of cysteine ​​at position 164 to serine; or a mutation of tyrosine at position 162 to phenylalanine; or a mutation of threonine at position 31 to isoleucine.

[0012] Further, the wild-type florigen FT protein includes any one of the following proteins a1)-a2): a1) PmFTL1 protein shown in SEQ ID NO: 3, PmFTL2 protein shown in SEQ ID NO: 4, TaVRN3-7B protein shown in SEQ ID NO: 10 or Arabidopsis FT protein shown in SEQ ID NO: 7; or a2) a protein having the same florigen function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the protein in a1); or a3) a protein having at least 75% homology with the protein in a1) and having the same function; or a4) a protein having the same function obtained by connecting a protein tag to the N-terminus or / and C-terminus of the FT protein in a1), a2) or a3);

[0013] The mutations include: mutating the asparagine at position 65 in SEQ ID NO: 4 to isoleucine; or mutating the valine at position 70 in SEQ ID NO: 10 to isoleucine; or mutating the valine at position 70 in SEQ ID NO: 7 to isoleucine; or mutating the cysteine ​​at position 164 in SEQ ID NO: 7 to serine; or mutating the tyrosine at position 162 in SEQ ID NO: 7 to phenylalanine; or mutating the threonine at position 31 in SEQ ID NO: 7 to isoleucine.

[0014] In order to achieve the above object, according to the third aspect of the present invention, a nucleic acid molecule for promoting plant flowering is provided, wherein the nucleic acid molecule is a nucleic acid molecule encoding the protein nucleic acid molecule for promoting plant flowering.

[0015] In order to achieve the above object, according to a fourth aspect of the present invention, a biological material for promoting plant flowering is provided, the biological material comprising: a biological material for editing the FT gene of a target plant in the above method for promoting plant flowering.

[0016] Furthermore, the biological material includes: one or more of DNA, RNA, protein or recombinant vector; preferably, the biological material also includes a host cell transformed with a recombinant vector; preferably, the host cell includes Escherichia coli or Agrobacterium; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium includes GV3101; preferably, the host cell is a non-plant host cell; preferably, the target plant includes monocotyledonous plants and dicotyledonous plants; preferably, the monocotyledonous plants include millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; preferably, the dicotyledonous plants include Arabidopsis thaliana, rapeseed, mustard, soybean, cotton, sweet orange or pecan.

[0017] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided a method for promoting plant flowering, or the protein for promoting plant flowering, or the nucleic acid molecule for promoting plant flowering, or the biological material for promoting plant flowering for use in regulating the flowering time of plants; preferably, regulating the flowering time of plants includes shortening the flowering time of plants or prolonging the flowering time of plants.

[0018] By applying the technical solution of the present invention, after editing the FT gene of the target plant, one or more of the following mutations occur in the amino acid of the FT protein encoded by the FT gene: V70I, C164S, Y162F or T31I, which can change the florigen function of the FT protein, promote plant flowering, and shorten the time required for the plant from the seedling stage to flowering / heading, thereby optimizing the planting cycle, avoiding adverse environmental influences, reducing planting costs, and facilitating the development of crop production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 A schematic diagram of single nucleotide differences in the coding sequences (CDS) of millet PmFTL1 and PmFTL2 according to Example 1 of the specification of the present invention is shown.

[0021] Figure 2 A schematic diagram of the amino acid sequence alignment of PmFTL1 and PmFTL2 according to Example 1 of the specification of the present invention is shown.

[0022] Figure 3A difference diagram of the total leaf number (flowering period) of overexpressing four PmFTL-CDS in the Arabidopsis thaliana Col-0 background under long-day conditions according to Example 1 of the present specification is shown. The total leaf number is the sum of the number of rosette true leaves and stem leaves. Figure 3 A in the middle is the plants in the Mock control (Col-0) group. Figure 3 B in the figure is PmFTL1 I65F102 -OE plants, Figure 3 C in the figure is PmFTL2 N65L102 -OE plants, Figure 3 D in the figure is PmFTL2M I65L102 -OE plants, Figure 3 E in the middle is PmFTL1M N65F102 -OE plants, Figure 3 The scale bars in A, B, C, D, and E are all 1 cm; Figure 3 F in the middle is Mock control (Col-0) and PmFTL1 I65F102 -OE、PmFTL2 N65L102 -OE、PmFTL2M I65L102 -OE and PmFTL1M N65F102 -OE transgenic Arabidopsis thaliana overexpression lines at the time of bud initiation. The M in 1M and 2M is the abbreviation of mutant.

[0023] Figure 4 A schematic diagram showing the amino acid sequence alignment of millet PmFTLs and Arabidopsis FT and FT homologous proteins of some monocotyledonous plants according to Example 1 of the present specification is shown.

[0024] Figure 5 A statistical graph showing the leaf number (experimental replication 1) after the site-directed mutation of amino acid V at position 70 of wheat VRN3-7B to I in Arabidopsis thaliana according to Example 2 of the present specification is shown.

[0025] Figure 6 A statistical graph showing the leaf number (experimental replication 2) after the site-directed mutation of amino acid V at position 70 of wheat VRN3-7B to I in Arabidopsis thaliana according to Example 2 of the present specification.

[0026] Figure 7 The figure shows the statistical graph of leaf numbers (experimental replication 1) of random mutation of FT-CDS in Arabidopsis thaliana and verification of amino acid mutation types C164S, Y162F, and T31I according to Example 3 of the present specification.

[0027] Figure 8The figure shows a statistical graph of leaf numbers (experimental replication 2) of random mutation of FT-CDS in Arabidopsis thaliana and verification of amino acid mutation types C164S, Y162F, and T31I according to Example 3 of the present specification.

[0028] Fig. 9 A schematic diagram showing the amino acid sequence alignment of Arabidopsis FT and FT homologous proteins of some dicotyledonous plants according to Example 3 of the present specification is shown. DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0030] Directed evolution: It simulates Darwin's evolutionary mechanism at the molecular level. It uses molecular biological methods to create a gene mutation library in vitro, applies selection pressure according to needs and purposes, and screens out proteins with expected characteristics, simulating mutation, recombination and selection at the molecular level.

[0031] Error-prone PCR: It is a commonly used method for constructing mutant libraries. It uses the fact that Taq DNA polymerase does not have a 3'→5' proofreading function to introduce mismatched bases while amplifying the target gene, thereby obtaining various mutation types of the gene and constructing a random mutation library.

[0032] Seamless cloning is a DNA cloning technology based on homologous recombination. By designing homologous sequences of specific length (usually 15-25bp) and GC content (40-60%), the target DNA fragment and the linearized vector are precisely matched at the junction, and the synergistic action of exonucleases, polymerases and ligases is used to achieve traceless connection (without introducing additional bases or restriction enzyme sites). This technology is widely used in gene editing, vector construction, and complex DNA assembly in synthetic biology.

[0033] As mentioned in the background technology, the prior art mainly uses traditional hybridization, mutagenesis screening and gene knockout technology to regulate the flowering time of plants. However, these methods are often inefficient, time-consuming, and difficult to accurately control the flowering time and promote plant flowering. Based on this, in this application, the inventor attempts to provide a new method for promoting plant flowering through precise mutation of amino acid sites to optimize the planting cycle of the target plant, and thus proposes a series of protection schemes of this application.

[0034] In a first typical embodiment of the present application, a method for promoting plant flowering is provided, the method comprising: editing a wild-type florigen FT gene of a target plant so that the wild-type florigen FT protein expressed by the target plant mutates into a mutant FT protein, thereby promoting flowering of the target plant; the editing comprises: replacing at least one nucleotide in the wild-type florigen FT gene so that the protein encoded by the wild-type florigen FT gene mutates to obtain a mutant FT protein; the wild-type florigen FT protein comprises: a protein as shown in SEQ ID NO: 7, or a homologous protein having at least 75% (including but not limited to 75%, 80%, 85%, 90%, 95%, 98%, 99.5%) sequence homology and the same functional conserved sites as the protein as shown in SEQ ID NO: 7; the mutation comprises: in the wild-type florigen FT protein, a nucleotide corresponding to SEQ ID The protein shown in NO:7 undergoes any one or more of the following mutations: the valine at position 70 mutates to isoleucine; the cysteine ​​at position 164 mutates to serine; the tyrosine at position 162 mutates to phenylalanine; the threonine at position 31 mutates to isoleucine.

[0035] The above-mentioned functional conserved sites refer to amino acid positions in homologous proteins that have the same or similar functions as the mutation sites of wild-type proteins, and the same or similar functions include but are not limited to maintaining protein structural stability, participating in the formation of protein active sites, or interacting with other molecules. For example, the 70th valine in the wild-type florigen FT protein SEQ ID NO: 7 of Arabidopsis thaliana corresponds to the 65th isoleucine in the wild-type florigen FT protein SEQ ID NO: 3 of millet, and the 65th asparagine in SEQ ID NO: 4, and the wild-type florigen FT protein in wheat corresponds to the 70th valine in SEQ ID NO: 10, i.e., the above-mentioned functional conserved sites in homologous proteins.

[0036] In the prior art, attempts have been made to regulate the flowering time of plants through various methods, such as hybridization, mutagenesis screening, and gene knockout technology, in order to cope with various challenges in agricultural production, including environmental changes, improving crop yield and quality, etc., but these methods have problems such as low efficiency and ineffective regulation of flowering time. At the molecular biology level, it is difficult for the prior art to provide an effective and rapid method for promoting plant flowering, especially to improve the flowering promotion function of FT protein through precise amino acid sequence modification, and to achieve directional genetic improvement of crop heading / flowering time. For example, in monocotyledonous and dicotyledonous crops, the understanding of the conservation and variability of FT homologous proteins is still insufficient. How to improve or change the activity of FT through slight changes in amino acid sequences, thereby effectively regulating the heading / flowering time of crops, is a difficulty and blank point in current research. After a lot of research, the inventors in this application found that by editing the nucleotides of the FT gene and causing mutations at specific amino acid sites, the flowering of the target plant can be promoted, providing new strategies and technical support for regulating the flowering time of plants.

[0037] The present application provides a precise editing strategy for the wild-type florigen FT gene, which can effectively and quickly promote plant flowering by targetedly changing the amino acids of the wild-type florigen FT protein. The present application can not only accelerate the flowering process of plants, but also provide a strong theoretical basis and technical support for the directional genetic improvement of the heading / flowering period of monocotyledonous and dicotyledonous crops. Compared with traditional genetic improvement methods, the method of the present application has higher accuracy and efficiency, and provides the possibility for rapid, directional, and optimized growth cycle regulation of crops. It has important scientific research value and broad application prospects, and has opened up new ways for agricultural production and crop breeding. It is expected to play a key role in optimizing the crop growth cycle, increasing crop yields and adaptability.

[0038] In a preferred embodiment, the wild-type florigen FT protein includes any one of the following proteins a1)-a2): a1) Arabidopsis FT protein shown in SEQ ID NO: 7, the PmFTL1 protein shown in SEQ ID NO: 3, the PmFTL2 protein shown in SEQ ID NO: 4, or the TaVRN3-7B protein shown in SEQ ID NO: 10; or a2) a protein having the same function obtained by connecting a protein tag to the N-terminus or / and C-terminus of the FT protein in a1); the mutation includes any one or more of the following: mutating the valine at position 70 in SEQ ID NO: 7 to isoleucine; mutating the cysteine ​​at position 164 in SEQ ID NO: 7 to serine; mutating the tyrosine at position 162 in SEQ ID NO: 7 to phenylalanine; mutating the threonine at position 31 in SEQ ID NO: 7 to isoleucine; mutating the asparagine at position 65 in SEQ ID NO: 4 to isoleucine; mutating the valine at position 70 in SEQ ID NO: 10 to isoleucine.

[0039] SEQ ID NO: 3: MDPLVMAHVIQDVLDPFTPTVPLRITYNNRLLLAGAELKPSAVLSKPRVDLGGNDMRAFYTLVLIDPDAPSHPSLREYLHWMVIDIPETTSVSFGKELVFYERPEPRSGIHRMVFVLFRQLGRGTVFAPEMRHNFNCRSFARQYHLNIATATYFNCQREAGSGGRRFRDE.

[0040] SEQ ID NO: 4: MDPLVMAHVIQDVLDPFTPTVPLRITYNNRLLLAGAELKPSAVLSKPRVDLGGNDMRAFYTLVLNDPDAPSPSHPSLREYLHWMVIDIPETTSVSFGKELVLYERPEPRSGIHRMVFVLFRQLGRGTVFAPEMRHNFNCRSFARQYHLNIATATYFNCQREAGSGGRRFRDE.

[0041] SEQ ID NO: 10: MAGRDRDPLVVGRVVGDVLDPFVRTTNLRVTFGNRTVSNGCELKPSMVAQQPRVEVGGNEMRTFYTLVMVDPDAPSPSDPNLREYLHWLVTDIPGTTGASFGQEVMCYESPRPTMGIHRFVLVLFQQLGRQTVYAPGWRQNFNTRDFAELYNLGPPVAAVYFNCQREAGSGGRRMYN.

[0042] SEQ ID NO: 7: MSINIRDPLIVSRVVGDVLDPFNRSITLKVTYGQREVTNGLDLRPSQVQNKPRVEIGGEDLRNFYTLVMVDPDVPSPSNPHLREYLHWLVTDIPATTGTTFGNEIVCYENPSPTAGIHRVVFILFRQLGRQTVYAPGWRQNFNTREFAEIYNLGLPVAAVFYNCQRESGCGGRRL.

[0043] In the early stage of this application, two wild-type florigen FT homologous genes PmFTL1 and PmFTL2 (PmFT-like-1 SEQ ID NO: 1, PmFT-like-2 SEQ ID NO: 2) were cloned from a broomcorn germplasm resource with an extremely early heading period. The PmFTL1 and PmFTL2 (PmFT-like-1 SEQ ID NO: 3, PmFT-like-2 SEQ ID NO: 4) proteins encoded by them respectively have two differences (65th and 102nd positions) in the amino acid sequence. Through overexpression experiments in Arabidopsis, it was proved that the 65th amino acid 65I (isoleucine, IIe) of broomcorn PmFTL1 has a stronger function of promoting flowering. 65I of PmFTL1 corresponds to 70V of wheat florigen FT homologous protein VRN3-7B (SEQ ID NO: 10), and also corresponds to 70V of Arabidopsis FT (SEQ ID NO: 7). Through directed mutagenesis of the 70V site on the amino acid sequence of wheat VRN3-7B, it was confirmed that the mutation from V to I can advance the heading date of wheat.

[0044] Furthermore, the present application introduces mismatched bases into the coding sequence of the wild-type florigen FT gene (SEQ ID NO: 6) of Arabidopsis thaliana, establishes a random mutation library and transfers the FT functional loss mutant to complement its late flowering phenotype, thereby screening out plants with early flowering; further, through site-directed mutagenesis of the coding sequence of the Arabidopsis FT gene, the early flowering phenotype of the screened functional amino acid sites is verified, and the mutation types C164S, Y162F, and T31I that can advance the flowering period of Arabidopsis thaliana are identified. These functional amino acid sites are expected to be applied to the modification of florigens in dicotyledonous crops.

[0045] SEQ ID NO: 1: atggatcctttggttatggctcatgtcatacaggatgtgttggatccctttacaccaactgttccactcagaataacgtacaacaacaggctacttctggcaggtgctgagctaaagccatctgcg gttttaagtaaaccacgagttgatcttggtggcaatgacatgagggctttctacaccctggtactgattgacccggatgccccaagtccgagccatccctctctaagggagtacttgcactggatggtgat agatattcctgaaacaactagcgtcagctttggcaaagagctagtattttatgagagaccagagccaagatctggcatacacaggatggtatttgtgctgttccggcaacttggcaggggcacagtttttg caccagaaatgcgccacaacttcaactgcagaagctttgcacggcaatatcacctcaatattgccactgccacatatttcaactgtcaaagggaagctgggtcgggtggaagaaggtttagggatgagtag.

[0046] SEQ ID NO:2:atggatcctttggtcatggctcatgtcatacaggatgtgttggatccctttacaccaaccgttccactcagaataacatacaacaacaggctacttctggcaggtgctgagctaaagccatctgcggttttaagtaaaccacgagttgatcttggtggcaatgacatgagggctttctacaccctggtactgaatgacccggatgccccaagtccgagccatccctctctaagggagtacttgcactggatggtgatagatattcctgaaacaactagcgtcagctttggcaaagagctagtactttatgagagaccagagccaagatctggcatccacaggatggtatttgtgctgttccggcaacttggcaggggtacagtttttgcaccagaaatgcgccacaacttcaactgcagaagctttgcacggcaatatcacctcaatattgccactgccacatatttcaactgtcaaagggaagctgggtcgggcggaagaaggtttagggatgagtag。

[0047] SEQ ID NO: 6:.

[0048] The above-mentioned FT proteins include proteins in millet, wheat and Arabidopsis, and also include endogenous homologous proteins in other plants; and also include exogenous amino acid mutated FT protein-related biological materials transformed into millet, wheat and Arabidopsis that do not contain such homologous proteins, so as to express the above-mentioned exogenous FT proteins in plants. The above-mentioned applications can regulate the function or transport ability of the FT proteins of millet, wheat and Arabidopsis, and can achieve the regulation of the flowering time of plants, and further regulate the growth cycle of plants. The above-mentioned same functions include the ability to regulate the flowering time and growth cycle of plants containing FT proteins.

[0049] In a preferred embodiment, the wild-type florigen FT gene includes: b1) a gene encoding Arabidopsis FT protein, PmFTL1 protein, PmFTL2 protein or TaVRN3-7B protein; the gene encoding Arabidopsis FT protein is a FT gene having a nucleotide sequence shown in SEQ ID NO: 6; the gene encoding PmFTL1 protein is a FT gene having a nucleotide sequence shown in SEQ ID NO: 1; the gene encoding PmFTL2 protein is a FT gene having a nucleotide sequence shown in SEQ ID NO: 2; the gene encoding TaVRN3-7B protein is a FT gene having a nucleotide sequence shown in SEQ ID NO: 9; b2) a FT gene that has at least 75% homology with the coding gene in b1) and encodes any one of the FT proteins in a1)-a2); or b3) a FT gene that hybridizes with the coding gene in b1) under strict conditions and encodes any one of the FT proteins in a1)-a2).

[0050] SEQ ID NO:9:.

[0051] Preferably, the editing includes: preferably, replacing the A at position 194 of the wild-type florigen FT gene containing the nucleotide sequence shown in SEQ ID NO: 2 with T; preferably, replacing the G at position 208 of the wild-type florigen FT gene in the nucleotide sequence shown in SEQ ID NO: 9 with A; preferably, replacing the G at position 491 of the wild-type florigen FT gene in the nucleotide sequence shown in SEQ ID NO: 6 with C; preferably, replacing the A at position 485 of the wild-type florigen FT gene in the nucleotide sequence shown in SEQ ID NO: 6 with T; preferably, replacing the C at position 92 of the wild-type florigen FT gene in the nucleotide sequence shown in SEQ ID NO: 6 with T.

[0052] "Homology" in this specification refers to similarity or identity, and particularly refers to homology. "Homology of amino acid sequences" refers to homology relative to the entire amino acid sequence. "Homology" between amino acid sequences refers to the total ratio of amino acid residues of the same type in these amino acid sequences. "Homology" between nucleotide sequences is the total ratio of nucleotides of the same type in the nucleotide sequence. "Similarity" between amino acids refers to the total ratio of amino acid residues of the same type in these amino acid sequences and the ratio of amino acid residues with similar properties of the side chains. The homology of amino acid sequences and nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool).

[0053] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0054] Substitution and replacement rules. Generally speaking, the effects of replacing amino acids with similar properties are similar. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0055] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0056] The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

[0057] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0058] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0059] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0060] As used herein, the term "DNA hybridization under stringent conditions" means that the nucleotide sequence specifically hybridizes to the target sequence in an amount that is detectably stronger than non-specific hybridization. Stringent conditions can include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalent at a temperature of about 50°C to 70°C.

[0061] The wild-type florigen FT gene can encode FT protein with florigen function. Based on the sequence (a), the nucleotides are mutated, hybridized with the DNA molecule specified in (a) under strict conditions, and no frameshift mutation occurs. If the mutation occurs in the nucleotide encoding the active site of the protein, it may cause the key amino acid binding site of the encoded protein to change, affecting the function of the protein encoded by the gene, resulting in an increase or decrease in its activity or even loss of activity; if the mutation occurs in the nucleotide encoding the inactive site of the protein, it may affect the folding mode, three-dimensional structure and other properties of the encoded protein, thereby affecting the physicochemical properties and activity of the protein. Homology of more than 75% means that the FT gene with more than 75%, 85%, 95% or 99% homology and encoding proteins with the same function, the active site, active pocket, active mechanism, etc. of the encoded protein are most likely the same as the gene provided by the sequence (a), and is a homologous gene obtained by nucleotide mutation.

[0062] In a preferred embodiment, the method comprises: introducing genetic material into the target plant to obtain a plant with early flowering time; preferably, introducing genetic material into the tissue or cell of the target plant, and culturing the introduced cell or tissue into a complete plant to obtain a plant with early flowering time; preferably, the genetic material comprises a DNA circular plasmid, a DNA linear fragment, or an in vitro transcribed RNA; preferably, the genetic material comprises a recombinant vector containing a gene mutation site; preferably, the editing method comprises seamless cloning, error-prone PCR, CRISPR / Cas9, CRISPR / Cas12 or RED recombination; preferably, the introduction method comprises a gene gun method, Agrobacterium infection method, PEG-induced protoplast method, electrode method, silicon carbide fiber-mediated method or vacuum infiltration method, more preferably Agrobacterium infection method; preferably, the cells of the target plant comprise protoplast cells or suspension cells; preferably, the tissue of the target plant comprises callus, immature embryos, mature embryos, leaves, stem tips, young spikes or hypocotyls.

[0063] In the above method, the above recombinant vector is introduced into the target plant by plant virus vector, gene gun or Agrobacterium infection. The above method for preparing transgenic plants uses a variety of methods such as plant virus vector, gene gun or Agrobacterium infection to introduce the expression cassette, recombinant vector or host cell into the target plant to obtain a transgenic plant with shortened flowering time. The above method can affect the expression of the FT gene of the target plant, the activity and protein function of the FT protein by means of establishing a mutant library, obtaining mutant families, and causing mutations at the nucleotide sites of the FT gene, thereby obtaining a transgenic plant with shortened flowering time. CRISPR-Cas9-mediated gene editing technology can also be used to achieve knockout, knock-in, and single base substitution of the FT gene, thereby changing the expression of the FT gene of the target plant, the activity and protein function of the FT protein, and obtaining a transgenic plant with shortened flowering time.

[0064] Precise gene editing can accelerate plant breeding and genetic improvement. Gene editing tools represented by the CRISPR system can achieve base knockout, insertion or replacement. For example, the Primeediting system (PE) developed based on the CRISPR / CAS9 system can accurately replace a single base.

[0065] In a preferred embodiment, the target plant includes a monocotyledon or a dicotyledon; preferably, the monocotyledon includes millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; more preferably, the monocotyledon includes millet or wheat; preferably, the dicotyledon includes Arabidopsis, rapeseed, mustard, soybean, cotton, sweet orange or pecan; more preferably, the dicotyledon includes Arabidopsis.

[0066] According to a second typical embodiment of the present application, a protein for promoting plant flowering is provided, the protein comprising: an amino acid sequence of a wild-type florigen FT protein as shown in SEQ ID NO: 7; a homologous protein of the wild-type florigen FT protein has at least 75% sequence homology with the wild-type florigen FT protein, and a mutation site in the homologous protein of the wild-type florigen FT protein corresponds in the homologous protein to a functionally conserved site in the wild-type florigen FT protein; the mutation comprises: in the wild-type florigen FT protein or a homologous protein of the wild-type florigen FT protein, corresponding to the wild-type florigen FT protein, any one or more of the following mutations are performed: a mutation of valine at position 70 to isoleucine; or a mutation of cysteine ​​at position 164 to serine; or a mutation of tyrosine at position 162 to phenylalanine; or a mutation of threonine at position 31 to isoleucine.

[0067] In a preferred embodiment, the wild-type florigen FT protein includes the following: a1) the Arabidopsis FT protein shown in SEQ ID NO: 7, the PmFTL1 protein shown in SEQ ID NO: 3, the PmFTL2 protein shown in SEQ ID NO: 4, or the TaVRN3-7B protein shown in SEQ ID NO: 10; or a2) a protein having the same function obtained by connecting a protein tag to the N-terminus or / and C-terminus of the FT protein in a1); the mutation includes any one or more of the following: mutating the valine at position 70 in SEQ ID NO: 7 to isoleucine; mutating the cysteine ​​at position 164 in SEQ ID NO: 7 to serine; mutating the tyrosine at position 162 in SEQ ID NO: 7 to phenylalanine; mutating the threonine at position 31 in SEQ ID NO: 7 to isoleucine; mutating the asparagine at position 65 in SEQ ID NO: 4 to isoleucine; mutating the valine at position 70 in SEQ ID NO: 10 to isoleucine.

[0068] According to a third typical embodiment of the present application, a nucleic acid molecule for promoting plant flowering is provided, wherein the nucleic acid molecule is a nucleic acid molecule encoding the above-mentioned protein for promoting plant flowering.

[0069] The coding nucleotides corresponding to the key amino acid residues affecting the function of florigen in the present application (i.e. the above-mentioned nucleic acid molecules) can also be used as SNP (single nucleotide polymorphism) molecular markers for the identification of heading / flowering period of target plants.

[0070] According to a fourth typical embodiment of the present application, a biological material for promoting plant flowering is provided, the biological material comprising a biological material for editing the FT gene of a target plant in the above-mentioned method for promoting plant flowering.

[0071] In a preferred embodiment, the biological material includes: one or more of DNA, RNA, protein or recombinant vector; preferably, the biological material also includes a host cell transformed with a recombinant vector; preferably, the host cell includes Escherichia coli or Agrobacterium; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium includes GV3101; preferably, the host cell is a non-plant host cell; preferably, the target plant includes monocotyledonous plants and dicotyledonous plants; preferably, the monocotyledonous plants include millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; preferably, the dicotyledonous plants include Arabidopsis thaliana, rapeseed, mustard, soybean, cotton, sweet orange or pecan.

[0072] The above-mentioned recombinant vector includes a translation control signal; preferably, the translation control signal includes an enhancer; preferably, the enhancer includes a translation enhancer and / or a transcription enhancer; preferably, the translation control signal is derived from a natural sequence or an artificially synthesized sequence; preferably, the recombinant vector includes a plant expression vector; preferably, the plant expression vector includes a binary vector for Agrobacterium transformation and a vector for gene gun bombardment; preferably, the plant expression vector includes pPZP221B or pB2GW7; preferably, the recombinant vector includes a reporter gene; preferably, the reporter gene includes a resistance gene or a gene expressing an enzyme that produces a color change or a luminescent compound; preferably, the resistance gene includes an antibiotic resistance gene or a chemical agent resistance gene.

[0073] The above-mentioned recombinant vector, comprising the wheat leaf rust resistance gene or the above-mentioned expression cassette, may also comprise other nucleic acid fragments such as a replication initiation site, a multiple cloning site, a translation control signal, etc. The translation control signal derived from a natural sequence or an artificially synthesized sequence includes an enhancer, a molecular chaperone, and other nucleotide sequences that can affect protein translation. The above-mentioned enhancer includes a translation enhancer and / or a transcription enhancer, which can be used alone or in combination to regulate protein transcription and translation. The above-mentioned recombinant vector can be a plant expression vector, which can be transformed into a plant, express the target gene in the plant, produce the target protein, and thus play a role; the plant expression vector includes but is not limited to a binary vector for Agrobacterium transformation and a vector for gene gun bombardment, which can be introduced into plant cells by different transformation methods to improve the transformation efficiency, and the above-mentioned plant expression vector includes but is not limited to pPZP221B or pB2GW7 used in the examples.

[0074] The above-mentioned recombinant vector may also include a reporter gene; preferably, the reporter gene includes but is not limited to a resistance gene or a gene that expresses an enzyme or luminescent compound that produces a color change, so as to judge whether the recombinant vector is successfully transformed and expressed by various methods such as resistance screening, color screening, and fluorescence screening; wherein the resistance gene includes but is not limited to an antibiotic resistance gene or a chemical agent resistance gene, and antibiotics, chemical agents and other drugs can be used to efficiently screen the transformed mother to judge whether the recombinant vector is successfully transformed and expressed. Considering the safety of transgenics, it is also possible not to add any reporter gene, and directly screen whether the transformation is successful by phenotype.

[0075] The host cells are transformed with recombinant vectors, and can carry recombinant vectors to perform various functions such as recombinant vector copying, gene expression, gene integration into chromosomes, etc. The host cells can be various strains such as Escherichia coli and Agrobacterium, among which Escherichia coli can be the commonly used DH5α, and Agrobacterium can be the commonly used GV3101 or EHA105.

[0076] In a fifth typical embodiment of the present application, a method for promoting plant flowering or the use of the above-mentioned biological material for promoting plant flowering in regulating the flowering time of plants is provided; regulating the flowering time of plants includes shortening the flowering time of plants or extending the flowering time of plants.

[0077] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0078] The experimental methods not specifically noted in the examples of this application (such as DNA extraction, preparation of culture medium, connection of DNA fragments / vectors, transformation) are all conventional methods, and reference can be made to the fourth edition of the original book Molecular Cloning Experiment Guide (translated by He Fuchu, Science Press, 2017). The Escherichia coli strains used in the present invention are all DH5α, the Agrobacterium strains used to transfect Arabidopsis thaliana are all GV3101, the restriction endonucleases are all purchased from NEB, T4 DNA ligase is selected from T4 DNA Ligase (NEB, M0202M), and the DNA fragments used for cloning into the vector are all 2×Phanta Max Master Mix (Vazyme, C515) unless otherwise specified, and the gel recovery kit is selected from Gel Extraction Kit (Omega, D2500-02), used for E. coli plasmid extraction Plasmid Mini Kit I (Omega, D6943-02), RNA extraction kit Super total RNA extraction kit (Promega, LS1040), the reagent used for reverse transcription was M-MLV Reverse Transcriptase (Promega, M1701), Recombinant Ribonuclease Inhibitor (Promega, N2515), dNTPMix (Promega, U151B), seamless cloning Ultra One Step Cloning Kit (Vazyme, C115-02), sequencing and primer synthesis services were provided by Qingdao Branch of Beijing Qingke Biotechnology Co., Ltd.

[0079] The growth conditions of Arabidopsis thaliana in the examples of the present application are: 22°C, 16h light / 8h dark. The present invention uses the Agrobacterium inflorescence infection method to transfer the target gene into Arabidopsis thaliana, and the steps are as follows:

[0080] 1. Prepare 200 mL of Arabidopsis thaliana in full flowering stage and culture the Agrobacterium solution until the OD value reaches 1.0;

[0081] 2. Preparation of the dye solution: 200 mL ddH2O, 10 g sucrose (Shanghai Test, 10021418), 0.1 g MES (Sigma, M3671-50G), mix thoroughly, adjust the pH to 5.7 with KOH solution, and add 100 μL Silwet (BIOBYING, BY-S9430);

[0082] 3. Centrifuge the bacterial solution at 4000 rpm for 20 min, remove the supernatant, and resuspend in the staining solution;

[0083] 4. Cut off the fruit pods of the plants before dipping, immerse the above-ground parts of the plants in the bacterial solution for 30-40 seconds, cover the infected plants with a tray to maintain humidity and darkness, and place the plants under normal conditions for growth after 16-24 hours;

[0084] 5. Collect T1 seeds after Arabidopsis siliques mature.

[0085] The steps of screening Arabidopsis transgenic positive seedlings using selective medium are as follows:

[0086] 1. Place the collected T1 seeds in a solution containing 75% ethanol (Shanghai Test, 10009218) and 1% Triton TM Immerse in a solution of X-100 (Sigma-Aldrich, 9002-93-1) for 15 min;

[0087] 2. Pour off the solution in a clean bench, suspend the seeds with anhydrous ethanol and pour them onto filter paper to dry;

[0088] 3. Sprinkle the seeds evenly on the selective medium containing antibiotics;

[0089] 4. Place the plate in a dark place at 4°C for three days to homogenize. After growing in an incubator (22°C, 16h light, 8h dark) for three days, the positive seedlings can be transplanted into the soil.

[0090] Example 1

[0091] In the previous study of this application, the heading / flowering period of multiple germplasm resources of the grass crop Panicum miliaceum L. was identified (culture conditions: short day, 10 hours of light / 14 hours of darkness, 22°C), and a line with the shortest heading / flowering period (25 days) was screened. Combined with the genome sequencing data of foxtail millet, and homologous cloning, the coding sequences of two FT homologous genes (CDS, SEQ ID NO: 1, SEQ ID NO: 2) were cloned from the allotetraploid grass crop foxtail millet. The comparison results of the two genes are as follows: Figure 1 As shown, namely PmFTL1-CDS and PmFTL2-CDS (the corresponding encoded proteins are named PmFTL1 and PmFTL2, respectively). Figure 1 In the figure, the nucleotide differences are highlighted in different colors, and the red arrows indicate that PmFTL1-CDS and PmFTL2-CDS can be cut by restriction endonuclease EcoN1.

[0092] The amino acid sequences of PmFTL1 and PmFTL2 differ at positions 65 and 102, respectively. The comparison of their amino acid sequences is shown in Figure 2 As shown, (SEQ ID NO: 3 and SEQ ID NO: 4), Figure 2 In the figure, the red rectangular box is the common characteristic sequence of PEBP family proteins; in PmFTL1-CDS and PmFTL2-CDS, the nucleotide sites that cause differences in the encoded amino acid residues are at positions 194 and 304.

[0093] PmFT-like-1-CDS and PmFT-like-2-CDS contain 8 single nucleotide polymorphisms (SNPs), of which only nucleotides T194→A or T304→C lead to missense amino acid mutations I65→N65 or F102→L102 in PmFTL1 or PmFTL2 protein, respectively. 60 TLVLI 65 DPDAPSPSHPSLREYLH 82 (SEQ ID NO: 95), which is the common characteristic sequence of PEBP (phosphatidylethanolamine binding protein) family proteins.

[0094] Use restriction endonuclease EcoN1 to cut PmFTL1-CDS and PmFTL2-CDS into two fragments at the 5' and 3' ends between the 228 / 229 nucleotides of PmFTL1-CDS and PmFTL2-CDS, respectively. Then use T4 ligase to connect the 5' end fragment of PmFTL1-CDS and the 3' end fragment of PmFTL2-CDS, and the 5' end fragment of PmFTL2-CDS and the 3' end fragment of PmFTL1-CDS, respectively. The new sequences after connection were named PmFTL2M-CDS (M is the abbreviation of mutant) and PmFTL1M-CDS, respectively.

[0095] The above four PmFTL-CDS sequences were further inserted into the downstream of the 35S promoter of the overexpression vector pB2GW7 using the Gateway cloning method (Invitrogen, Carlsbad, CA, USA) to construct overexpression vectors, 35S::PmFTL1-CDS(65I, 102F)-OE; 35S::PmFTL2-CDS(65N, 102L)-OE; 35S::PmFTL2M-CDS(65I, 102L)-OE; 35S::PmFTL1M-CDS(65N, 102F)-OE. The above overexpression vectors were transformed into Agrobacterium strain GV3101, and the obtained Agrobacterium bacterial solution was transformed into wild-type Arabidopsis thaliana (Col-0 ecotype) by the floral dipping method.

[0096] The mature seeds of the transformed plants were further harvested, and the positive seedlings that were successfully transformed were screened on the glufosinate-ammonium herbicide screening medium to obtain PmFTL1 I65F102 -OE、PmFTL2 N65L102 -OE、PmFTL2M I65L102 -OE and PmFTL1M N65F102 -OE transgenic Arabidopsis thaliana overexpression lines were transplanted into culture medium and grown under long-day conditions (16 h light / 8 h dark, 22°C).

[0097] When the bolt height of the transgenic Arabidopsis line was about 2 cm after flowering, the total number of leaves (the sum of the number of true leaves of the rosette and the number of stem leaves) was counted. The earlier the flowering time, the fewer the total number of leaves. The difference in the effect of 65I / N and 102F / L in promoting flowering in the PmFTL protein of millet was analyzed. 65 F 102 -OE(B) and PmFTL2M-I 65 L 102 -OE (D) overexpression lines appeared flower buds, Mock control (Col-0) (A), PmFTL2-N 65 L102 -OE(C),PmFTL1M-N 65 F 102 -OE(E) plant phenotypes Figure 3 As shown, Figure 3 A in the middle is the plants in the Mock control (Col-0) group. Figure 3 B in the figure is PmFTL1 I65F102 -OE plants, Figure 3 C in the figure is PmFTL2 N65L102 -OE plants, Figure 3 D in the figure is PmFTL2M I65L102 -OE plants, Figure 3 E in the middle is PmFTL1M N65F102 -OE plants, Figure 3 The scale bars in A, B, C, D, and E are all 1 cm; Figure 3 F in the middle is Mock control (Col-0) and PmFTL1 I65F102 -OE、PmFTL2 N65L102 -OE、PmFTL2M I65L102 -OE and PmFTL1M N65F102 - Total leaf statistics of the OE transgenic Arabidopsis overexpression line at the beginning of flower bud appearance, Figure 3 The least significant difference (LSD) multiple comparison test was used to indicate significant differences (p < 0.05).

[0098] The amino acid sequences of PmFTL1-CDS and PmFTL2-CDS were compared with those of FT from other plants. Figure 4 As shown in Table 1, the corresponding amino acid types of 65I / N and 102F / L in the amino acid sequence of millet PmFTL in other plants were found, and their physical and chemical properties were preliminarily analyzed. The CDS sequences of florigens from other plants were compared to obtain nucleotide sites for modification, and the results are shown in Table 1.

[0099] Figure 4 Included are FT homologs from other plants:

[0100] Protein sequence number: PmFT-chr5-longmi024158 (SEQ ID NO: 80), PmFT-like-1-chr3 (SEQ ID NO: 3), PmFT-like-2-chr10 (SEQ ID NO: 4), derived from Panicum miliaceum;

[0101] PhHd3a-like (XP_025817314.1), from Panicum hallii (SEQ ID NO: 81);

[0102] SiHd3a (XP_004972176.1), from Setaria italica (SEQ ID NO: 78);

[0103] PvFT1 (XP_039807977.1), PvHd3a (XP_039837531.1), derived from Panicum virgatum (SEQ ID NO: 75, SEQ ID NO: 76);

[0104] ZCN8 (NP_001106247.1), from Zea mays (SEQ ID NO: 77);

[0105] TuRFT1-like (XP_048546114.1), from Triticum urartu (SEQ ID NO: 82);

[0106] TdRFT1-like (XP_037472921.1), from emmer wheat Triticum dicoccoides (SEQ ID NO: 83);

[0107] Hd3a (BAB61030.1), Heading date 3A (FT-like), from Oryza sativa Japonica Group (SEQ ID NO: 79);

[0108] BmFT-like (AQQ11817.1), derived from Bambusa multiplex (SEQ ID NO: 84);

[0109] VRN3-7B (ABK32208.1), derived from wheat Triticum aestivum (SEQ ID NO: 10);

[0110] FT (AAF03936.1), FLOWERING LOCUS T, is derived from Arabidopsis thaliana (SEQ ID NO: 7).

[0111] Table 1

[0112] Gene Protein number CDS number or accession number (NCBI) Codon Amino Acids PmFTL1 SEQ ID NO:3 SEQ ID NO:1 193-195nt: ATT <![CDATA[65 th ,I(IIe)]]> PmFTL2 SEQ ID NO:4 SEQ ID NO:2 193-195nt: AAT <![CDATA[65 th ,N(Asn)]]> PvRFT1 SEQ ID NO:75 XM_039952043.1 389-391nt: ATT <![CDATA[65 th ,I(IIe)]]> PvHd SEQ ID NO:76 XM_039981597.1 279-281nt: ATC <![CDATA[65 th ,I(IIe)]]> ZCN8 SEQ ID NO:77 NM_001112776.1 202-204nt: ATT <![CDATA[68 th ,I(IIe)]]> HkDJ SEQ ID NO:78 XM_004972119.2 202-204nt: GTG <![CDATA[68 th ,V(Val)]]> H3A SEQ ID NO:79 AB052944.1 214-216nt: GTA <![CDATA[72 nd ,V(Val)]]> VRN3-7B SEQ ID NO:10 DQ890165.1 208-210nt: GTA <![CDATA[70 ty ,V(Val)]]>

[0113] The amino acid sequences of florigen proteins from plants such as millet PmFTL1 (SEQ ID NO: 3), millet PmFTL2 (SEQ ID NO: 4), Arabidopsis FT (SEQ ID NO: 7), and wheat VRN3-7B (SEQ ID NO: 10) were compared using the CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw) online analysis tool, and it was found that the 65I site of PmFT1 corresponds to the 70V of wheat VRN3-7B, as shown in Figure 2 . Figure 4 shown.

[0114] In addition, the above research results can also provide potential nucleotide sites for gene editing and modification of FT homologs in sorghum, millet, switchgrass and other plants, namely the sites shown in Table 1. For example, if the nucleotide corresponding to the 65th amino acid of switchgrass PvFT1 (XP_039807977.1) or PvHd3a (XP_039837531.1) (SEQ ID NO: 75, SEQ ID NO: 76) (ATT or ATC) is edited to AAT or AAC, the 65th amino acid can be changed from I (IIe) to N (Asn), which may delay the heading date and extend the vegetative period and biomass of switchgrass as a bioenergy crop. If the nucleotides 202 to 204 of the maize ZCN8 gene coding sequence (NM_001112776.1) are changed from ATT to AAT, the 68th amino acid I (IIe) of ZCN8 (SEQ ID NO: 77) becomes N (Asn), which may lead to a delay in the maize heading date and an increase in the yield of forage stems and leaves.

[0115] Example 2

[0116] A site-directed mutagenesis experiment was performed on the amino acid position (70V) of the wheat florigen VRN3-7B gene CDS (SEQ ID NO: 9) corresponding to the amino acid position 65I of PmFTL1 to confirm the function of the mutation from V to I.

[0117] The steps of site-directed mutagenesis experiment are as follows:

[0118] The Arabidopsis thaliana genomic DNA extracted by the CTAB method was used as a template, and pFT-R (for pPZP221B): 5'-CGAGCTCGGTACCCGGGATTCGGGAATCTATAAACCCAGG-3' (SEQ ID NO: 11) and pFT-R (for pPZP221B): 5'-AGCTTGCATGCCTGCAGCTTTGATCTTGAACAAACAGGTG-3' (SEQ ID NO: 12) were used as primers to amplify the Arabidopsis thaliana FT gene promoter sequence (488-8095 bp in SEQ ID NO: 5), and a PCR product of about 7642 bp was recovered. The pPZP221B vector was double-digested with restriction endonucleases XmaI and PstI, the digestion product was recovered, and seamlessly cloned and ligated with the above-recovered product to construct the pPZP221B-pFT vector.

[0119] Using Arabidopsis genomic DNA extracted by the CTAB method as a template, and using FT Ter-F: 5'-GGAGGAAGAAGACTTTAGATGGCTTCTTCCTTTATAACC-3' (SEQ ID NO: 13) and FT Ter-R: 5'-CAGTGCCAAGCTTGCATGCCTAACGTCGAAGTCCATTAG-3' (SEQ ID NO: 14) as primers, the FT gene terminator sequence, i.e., SEQ ID NO: 8, was amplified, and a PCR product of about 617 bp was recovered and named fragment A.

[0120] RNA was extracted from the wheat (Triticum aestivum) variety "Chinese Spring" and reverse transcribed to synthesize cDNA. Using cDNA as a template and VRN3-7B CDS-F: 5'-CTGTTTGTTCAAGATCAAAGATGGCCGGTAGGGATAGGGACC-3' (SEQ ID NO: 15) and VRN3-7B CDS-R: 5'-ATAAAGGAAGAAGCCATTCAATTGTACATCCTCCTGCCG-3' (SEQ ID NO: 16) as primers, the VRN3-7B-CDS sequence, i.e., SEQ ID NO: 9, was amplified and a PCR product of about 571 bp was recovered and named fragment B.

[0121] Using the PCR product fragment B diluted 1 / 50 and the above fragment A as templates, VRN3-7B-CDS-F and FT Ter -R is a primer, and the VRN3-7B-CDS sequence (SEQ ID NO: 9) and the terminator sequence FT of the FT gene are overlapped by PCR. Ter(SEQ ID NO: 8) and the PCR product of about 1153 bp was recovered and seamlessly cloned into the pPZP221B-pFT vector digested with restriction endonuclease PstI to construct pPZP221B-pFT-VRN3-7B-FT TER carrier.

[0122] SEQ ID NO: 8:.

[0123] pPZP221B-pFT-VRN3-7B-FT TERThe vector was used as a template, VRN3-7B V70I-F: 5'-ACACACTCGTGATGATTGACCCAGATGCTCC-3' (SEQ ID NO: 17) and VRN3-7B V70I-R: 5'-GGAGCATCTGGGTCAATCATCACGAGTGTGT-3' (SEQ ID NO: 18) were used as intermediate primers, VRN3-7B CDS-F and FT Ter-R primers were used to amplify the final fragment, and the 70th amino acid residue V of the VRN3-7B gene was mutated to I by overlapping extension PCR, and the amplified fragment was seamlessly cloned into the pPZP221B-pFT vector digested with the restriction endonuclease PstI to construct pPZP221B-pFT-VRN3-7B (V70I)-FT TER carrier.

[0124] The vector pPZP221B-pFT-VRN3-7B-FT TER ,pPZP221B-pFT-VRN3-7B(V70I)-FT TER The vectors were transferred into Agrobacterium and then into Arabidopsis thaliana ft-1 mutants by Agrobacterium inflorescence infection. When the bolting height of the transgenic T1 plants was about 2 cm, the total number of leaves was counted. The leaf statistics of replicate experimental group 1 and replicate experimental group 2 were as follows: Figure 5 and Figure 6 As shown, the numbers represent the average of the total number of leaves in the test group, and "n" represents the number of samples (the figures of the present application specification Figure 7 , Figure 8 The numbers and letters have the same meanings as herein), and the results showed that in wheat, the mutation of the 70th amino acid residue of florigen from V to I enhanced its function of promoting flowering.

[0125] Example 3

[0126] Random mutations were performed on the FT-CDS of Arabidopsis, and the mutation types Y162F, C164S, and T31I that could advance the flowering period of Arabidopsis were initially screened out. Site-directed mutagenesis experiments were then used to verify the effects of Y162F, C164S, and T31I on the flowering period of Arabidopsis.

[0127] Here are the steps:

[0128] 1. pENTR TM 4-pFT-FT vector construction

[0129] The genomic DNA of Arabidopsis thaliana was extracted by CTAB method and used as template to clone the DNA with pFT-F (for pENTR TM4): 5'-CATGCCATGGGATTACCTCCCAGCACCAAAGACA-3' (SEQ ID NO: 19) and pFT-R (for pENTR TM 4): 5'-ATTTGCGGCCGCCTTTGATCTTGAACAAACAGGTGG-3' (SEQ ID NO: 20) was used as primer to PCR amplify the FT gene promoter sequence, i.e., SEQ ID NO: 5, and recover a PCR product of about 8123 bp. The gel-recovered product of the amplified FT gene promoter sequence (SEQ ID NO: 5) and pENTR TM The 4 vectors were double-digested with restriction endonucleases NcoI and NotI, and the two digestion products were recovered and ligated with T4 DNA ligase to construct pENTR TM 4-pFT vector.

[0130]

[0131] Arabidopsis RNA was extracted and reverse transcribed to synthesize cDNA. The cDNA was used as a template and FT CDS-F (for pENTR TM 4): 5'-ATTTGCGGCCGCATGTCTATAAATATAAGAGACCCTC-3' (SEQ ID NO: 21) and FT CDS-R (forpENTR TM 4): 5'-CCGCTCGAGCTAAAGTCTTCTTCCTCCGCAGC-3' (SEQ ID NO: 22) was used as primer to amplify the FT gene CDS sequence, i.e., SEQ ID NO: 6, and a PCR product of about 549 bp was recovered. The gel-recovered product of the amplified FT gene CDS (SEQ ID NO: 6) was mixed with pENTR TM The 4-pFT vector was double-digested with restriction endonucleases NotI and XhoI, and the digestion products were recovered and ligated with T4 DNA ligase to construct pENTR TM 4-pFT-FT vector.

[0132] 2. Random Mutation of FT Gene

[0133] Using 100-fold diluted pENTR TM Using 4-pFT-FT vector as template, StarMut Random Mutation Kit (GenStar, T115-01) was used to amplify the FT-CDS sequence (SEQ ID NO: 6) by error-prone PCR to generate a randomly mutated FT gene sequence. Primer sequences: FT MUT-F: 5'-GTTCAAGATCAAAGGCGGCCGCATG-3' (SEQ ID NO: 23); FTMUT-R: 5'-GCTGGGTCTAGATATCTCGAGCTA-3' (SEQ ID NO: 24).

[0134] The PCR reaction system was: template 2μL, FT MUT-F 1μL, FT MUT-R 1μL, 2xStarMut RandomSystem 25μL, StarMut Enhancer 10μL, ddH2O 11μL. The PCR reaction program was: 95℃2min; 94℃30sec, 60℃1min, 72℃1min, 25cycles; 72℃7min. A PCR product of about 571bp was recovered. pENTR TMThe 4-pFT vector was double-digested with restriction endonucleases NotI and XhoI, and the digestion product was recovered and seamlessly cloned with the gel-recovered product of the randomly mutated FT-CDS sequence, transformed into competent E. coli, and spread on LB agar plates containing 50 mg / L kanamycin. All colonies grown on the plates were collected, and the plasmid was extracted and named pENTR TM 4-pFT-FT(RM) vector.

[0135] use LR Clonase TM II Enzyme mix (Invitrogen, 11791020) catalyzes pENTR TM 4-pFT-FT(RM) was recombined with pBGW vector to construct pBGW-pFT-FT(RM) vector.

[0136] The pBGW-pFT-FT(RM) vector was transformed into Agrobacterium competent cells and spread on LB agar plates containing 100 mg / L spectinomycin and 25 mg / L rifampicin. The colonies were resuspended in LB liquid culture medium containing 100 mg / L spectinomycin and 25 mg / L rifampicin. 50% (v / v) sterilized glycerol was added to the resuspended liquid at a ratio of 1:1 to preserve the strain. The mutant library of the FT gene was obtained and stored in a -80°C refrigerator.

[0137] 3. Screening of early-flowering FT mutant plants

[0138] 100 μL of the FT gene mutation library strain stored in a -80℃ refrigerator was spread on an LB agar plate containing 100 mg / L spectinomycin and 25 mg / L rifampicin for activation. All colonies on the plate were collected and cultured in 200 mL of LB liquid medium containing 100 mg / L spectinomycin and 25 mg / L rifampicin until the OD value reached 1.0. The pBGW-pFT-FT(RM) vector carrying the randomly mutated FT gene was transferred into the FT gene mutated ft-10 late-flowering mutant of Arabidopsis thaliana by the Agrobacterium inflorescence infection method. Transgenic plants were screened using a selective medium containing 22 mg / L glufosinate, and a total of 1764 positive seedlings were transplanted.

[0139] The genomic DNA of the early flowering plants among the positive marker plants was extracted by SDS method. The genomic DNA was used as a template, and 2×Es Taq MasterMix (Dye) (CW0690L) was used to amplify the FT-CDS sequence carried by the pBGW-pFT-FT (RM) vector inserted into the genome of these plants with FT seq-F: 5'-GTGTAGAGGGTTCATGCCTATGATAC-3' (SEQ ID NO: 25) and FT seq-R: 5'-CTCCCATATGGTCGACCTGCAG-3' (SEQ ID NO: 26) as primers. The PCR product was sequenced using FT seq-F as a sequencing primer. If the sequencing result was a double peak, the PCR product needed to be recovered from the gel and connected to the pCE2 TA / Blunt-Zero (Vazyme, C601) vector, transformed into competent E. coli, and 3-5 single colonies were selected for sequencing. The sequencing results were compared with the FT-CDS (SEQ ID NO: 6) and the amino acid sequence (SEQ ID NO: 7). The nucleotide and amino acid mutation types containing three or less amino acid mutation sites are shown in Table 2.

[0140] Table 2

[0141]

[0142]

[0143] 4. Site-directed mutagenesis experiments verified the early flowering phenotype of FT mutant plants

[0144] The Arabidopsis genomic DNA extracted by the CTAB method was used as a template, and pFT-R (for pPZP221B): 5'-CGAGCTCGGTACCCGGGATTCGGGAATCTATAAACCCAGG-3' (SEQ ID NO: 27) and pFT-R (for pPZP221B): 5'-AGCTTGCATGCCTGCAGCTTTGATCTTGAACAAACAGGTG-3' (SEQ ID NO: 28) were used as primers to amplify the FT gene promoter sequence (488-8095 bp in SEQ ID NO: 5), and a PCR product of about 7642 bp was recovered. The pPZP221B vector was double-digested with restriction endonucleases XmaI and PstI, the digestion product was recovered, and seamlessly cloned and ligated with the above-mentioned recovered product to construct the pPZP221B-pFT vector.

[0145] Using Arabidopsis cDNA as a template, FT CDS-F (for pPZP221B): 5'-CTGTTTGTTCAAGATCAAAGATGTCTATAAATATAAGAGACC-3' (SEQ ID NO: 29) and FT CDS-R (for pPZP221B): 5'-ATAAAGGAAGAAGCCATCTAAAGTCTTCTTCCTCCGC-3' (SEQ ID NO: 30) as primers, FT-CDS, i.e., SEQ ID NO: 6, was amplified and a PCR product of about 565 bp was recovered and named fragment C.

[0146] The Arabidopsis genomic DNA extracted by CTAB was used as template and FT Ter -F: 5'-GGAGGAAGAAGACTTTAGATGGCTTCTTCCTTTATAACC-3' (SEQ ID NO: 31) and FT Ter -R: 5'-CAGTGCCAAGCTTGCATGCCTAACGTCGAAGTCCATTAG-3' (SEQ ID NO: 32) was used as a primer to amplify the FT gene terminator sequence, namely SEQ ID NO: 8, and a PCR product of about 617 bp was recovered and named fragment A.

[0147] Using 1 / 50 diluted PCR products fragment C and fragment A as templates, FT CDS-F (for pPZP221B) and FT Ter -R was used as primer, and FT-CDS (SEQ ID NO: 6) and terminator sequence (SEQ ID NO: 8) were spliced ​​by overlap extension PCR, and the PCR product of about 1147 bp was recovered and seamlessly cloned into the pPZP221B-pFT vector digested with restriction endonuclease PstI to construct pPZP221B-pFT-FT-FT TER carrier.

[0148] pPZP221B-pFT-FT-FT TER The vector was used as the template, and the primer sequences with the mutated bases in Table 3 were the intermediate primers, FT-CDS-F (for pPZP221B) and FT Ter -R primer was used to amplify the final fragment, and the FT gene was site-directed mutated by overlap extension PCR. The amplified fragment was seamlessly cloned into the pPZP221B-pFT vector digested with restriction endonuclease PstI to construct a site-directed mutagenesis verification vector as shown in Table 4.

[0149] Table 3

[0150]

[0151]

[0152] Table 4

[0153]

[0154]

[0155] The constructed pPZP221B-pFT-FT-FTTER and the site-directed mutagenesis verification vectors in Table 4 were transferred into Agrobacterium, and these vectors were transferred into the ft-1 late-flowering mutant with a mutation in the Arabidopsis FT gene by the inflorescence infection method. The T1 transgenic positive plants were screened using a selective medium containing 22 mg / L glufosinate ammonium. When the bolting height of Arabidopsis thaliana was about 2 cm after flowering, the total number of leaves (the sum of the number of true leaves of the rosette leaves and the number of stem leaves) was counted. The earlier the flowering time, the fewer the total number of leaves. It was found that mutations in the three sites of Y162F, C164S, and T31I can enhance the function of florigen to promote flowering and make the plant bloom earlier. The results of repeated experiments 1 and 2 are shown as follows: Figure 7 and Figure 8 shown.

[0156] The amino acid sequences of the florigen proteins of some dicotyledonous plants were compared using the online analysis tool CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw), and the mutation types Y162F, C164S, and T31I that can advance the flowering period of Arabidopsis were found. The Y162 amino acid site is consistent in the florigen homologous proteins in the cruciferous plants Brassicanapus, Brassica juncea, and Raphanus sativus, while the C164 and T31 amino acid sites are consistent in a variety of dicotyledonous plants, such as soybean (Glycine max), Raymond's cotton (Gossypium raimondii), sweet orange (Citrus sinensis), and American pecan (Carya illinoinensis) (Compare with Fig. 9 The results show that the functional mutations of Y162F, C164S and T31I are expected to be applied to the modification of florigen in dicotyledonous crops.

[0157] Fig. 9 The protein sequence number in is:

[0158] CiHd3a-like[Carya illinoinensis],XP_042987078.1 (SEQ ID NO: 85);

[0159] PnVRN3[Populus nigra],XP_061985178.1 (SEQ ID NO: 86);

[0160] PpFT[Prunus persica],ACH73165.1(SEQ ID NO:87);

[0161] PmFT[Prunus mume],BAH82787.1 (SEQ ID NO: 88);

[0162] GrHd3a[Gossypium raimondii],XP_012476940.1 (SEQ ID NO: 89);

[0163] GmFT[Glycine max],NP_001240185.1 (SEQ ID NO: 90);

[0164] CsHd3a-like[Citrus sinensis],XP_006480698.1 (SEQ ID NO: 91);

[0165] BnFT[Brassica napus],XP_022571536.1 (SEQ ID NO: 92);

[0166] BjFT[Brassica juncea],AFQ39836.1(SEQ ID NO:93);

[0167] PsFT[Raphanus sativus],XP_018470278.1 (SEQ ID NO: 94);

[0168] FT [Arabidopsis thaliana], AAF03936.1. (SEQ ID NO: 7).

[0169] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present application has found that after editing the FT gene of the target plant, the FT protein amino acid encoded by the FT gene undergoes one or more of the following mutations: millet N65I, wheat V70I, Arabidopsis Y162F, C164S or T31I. By directing mutations to these sites, the florigen function of the FT protein can be changed, plant flowering can be promoted, and the planting cycle can be further optimized. It also provides a theoretical basis for regulating the flowering cycle of other plants containing homologous FT proteins, which is beneficial to the development of crop breeding.

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for promoting plant flowering, characterized in that: The method comprises: Editing the wild-type florigen FT gene of the target plant so that the wild-type florigen FT protein expressed by the target plant is mutated into a mutant FT protein, thereby promoting flowering of the target plant; The editing comprises: replacing at least one nucleotide in the wild-type florigen FT gene, causing the protein encoded by the wild-type florigen FT gene to mutate, thereby obtaining the mutant FT protein; The wild-type florigen FT protein includes: the protein shown in SEQ ID NO: 7, or a homologous protein having at least 75% sequence homology and the same functional conserved sites as the protein shown in SEQ ID NO: 7; The mutation includes: in the wild-type florigen FT protein, corresponding to the protein shown in SEQ ID NO: 7, performing any one or more of the following mutations: The valine at position 70 mutated to isoleucine; The cysteine ​​at position 164 was mutated to serine; There is a mutation at position 162, where the tyrosine is mutated to phenylalanine; The threonine at position 31 is mutated to isoleucine.

2. The method according to claim 1, characterized in that: The wild-type florigen FT protein includes any one of the following proteins a1)-a2): a1) the Arabidopsis FT protein shown in SEQ ID NO: 7, the PmFTL1 protein shown in SEQ ID NO: 3, the PmFTL2 protein shown in SEQ ID NO: 4, or the TaVRN3-7B protein shown in SEQ ID NO: 10; or a2) a protein having the same function as the FT protein in a1) obtained by connecting a protein tag to the N-terminus or / and the C-terminus; The mutations include any one or more of the following: The valine at position 70 in SEQ ID NO: 7 was mutated to isoleucine; The cysteine ​​at position 164 in SEQ ID NO: 7 was mutated to serine; The tyrosine at position 162 in SEQ ID NO: 7 was mutated to phenylalanine; The threonine at position 31 in SEQ ID NO: 7 is mutated to isoleucine; The asparagine at position 65 in SEQ ID NO: 4 was mutated to isoleucine; The valine at position 70 in SEQ ID NO: 10 was mutated to isoleucine.

3. The method according to claim 2, characterized in that The wild-type florigen FT gene comprises: b1) a gene encoding the Arabidopsis FT protein, the PmFTL1 protein, the PmFTL2 protein or the TaVRN3-7B protein; The Arabidopsis FT protein encoding gene is the FT gene having the nucleotide sequence shown in SEQ ID NO: 6; The gene encoding the PmFTL1 protein is the FT gene having the nucleotide sequence shown in SEQ ID NO: 1; The gene encoding the PmFTL2 protein is the FT gene having the nucleotide sequence shown in SEQ ID NO: 2; The gene encoding the TaVRN3-7B protein is the FT gene having the nucleotide sequence shown in SEQ ID NO: 9; b2) having at least 75% homology with the gene encoding b1) and encoding the FT gene of any one of a1) to a2); or b3) A FT gene that hybridizes with the coding gene in b1) under stringent conditions and encodes the FT protein described in any one of a1) to a2).

4. The method according to claim 1, characterized in that: The method comprises: introducing genetic material into the target plant to obtain a plant with an early flowering time; Preferably, genetic material is introduced into tissues or cells of the target plant, and the introduced cells or tissues are cultured into complete plants to obtain plants with early flowering time; Preferably, the genetic material comprises a circular DNA plasmid, a linear DNA fragment, or an in vitro transcribed RNA; Preferably, the genetic material includes a recombinant vector containing a gene mutation site; Preferably, the editing method comprises seamless cloning, error-prone PCR, CRISPR / Cas9, CRISPR / Cas12 or RED recombination; Preferably, the introduction method includes gene gun method, Agrobacterium infection method, PEG-induced protoplast method, electrode method, silicon carbide fiber-mediated method or vacuum infiltration method, more preferably Agrobacterium infection method; Preferably, the target plant cells include protoplast cells or suspension cells; Preferably, the target plant tissue includes callus, immature embryo, mature embryo, leaf, stem tip, young panicle or hypocotyl; Preferably, the target plant includes a monocotyledon or a dicotyledon; Preferably, the monocotyledonous plant comprises millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; Preferably, the dicotyledonous plant comprises Arabidopsis thaliana, rapeseed, mustard, soybean, cotton, sweet orange or pecan.

5. A protein for promoting plant flowering, characterized in that: The protein includes: a protein obtained by mutating a wild-type florigen FT protein or a homologous protein of the wild-type florigen FT protein; The amino acid sequence of the wild-type florigen FT protein is shown in SEQ ID NO: 7; The homologous protein of the wild-type florigen FT protein has at least 75% sequence homology with the wild-type florigen FT protein, and the mutation site in the homologous protein of the wild-type florigen FT protein corresponds to the functional conserved site in the wild-type florigen FT protein in the homologous protein; The mutation includes: in the wild-type florigen FT protein or a homologous protein of the wild-type florigen FT protein, corresponding to the wild-type florigen FT protein, any one or more of the following mutations are performed: The valine at position 70 mutated to isoleucine; The cysteine ​​at position 164 was mutated to serine; There is a mutation at position 162, where the tyrosine is mutated to phenylalanine; The threonine at position 31 is mutated to isoleucine.

6. The protein according to claim 5, characterized in that The wild-type florigen FT protein includes any one of the following proteins a1)-a2): a1) the Arabidopsis FT protein shown in SEQ ID NO: 7, the PmFTL1 protein shown in SEQ ID NO: 3, the PmFTL2 protein shown in SEQ ID NO: 4, or the TaVRN3-7B protein shown in SEQ ID NO: 10; or a2) a protein having the same function as the FT protein in a1) obtained by connecting a protein tag to the N-terminus or / and the C-terminus; The mutations include any one or more of the following: The valine at position 70 in SEQ ID NO: 7 was mutated to isoleucine; The cysteine ​​at position 164 in SEQ ID NO: 7 was mutated to serine; The tyrosine at position 162 in SEQ ID NO: 7 was mutated to phenylalanine; The threonine at position 31 in SEQ ID NO: 7 is mutated to isoleucine; The asparagine at position 65 in SEQ ID NO: 4 was mutated to isoleucine; The valine at position 70 in SEQ ID NO: 10 was mutated to isoleucine.

7. A nucleic acid molecule for promoting plant flowering, characterized in that: The nucleic acid molecule is a nucleic acid molecule encoding the plant flowering promoting protein according to any one of claims 5 to 6.

8. A biological material for promoting plant flowering, characterized in that: The biological material includes a biological material for editing the FT gene of a target plant in the method for promoting flowering of a plant according to any one of claims 1 to 4.

9. The biomaterial according to claim 8, characterized in that The biological material includes: one or more of DNA, RNA, protein or recombinant vector; Preferably, the biological material further comprises a host cell transformed with the recombinant vector; Preferably, the host cell comprises Escherichia coli or Agrobacterium; Preferably, the Escherichia coli comprises DH5α; Preferably, the Agrobacterium comprises GV3101; Preferably, the host cell is a non-plant host cell; Preferably, the target plants include monocots and dicots; Preferably, the monocotyledonous plant comprises millet, wheat, rice, sorghum, corn, foxtail millet or switchgrass; Preferably, the dicotyledonous plant comprises Arabidopsis thaliana, rapeseed, mustard, soybean, cotton, sweet orange or pecan.

10. Use of the method for promoting plant flowering according to any one of claims 1 to 4, or the protein for promoting plant flowering according to any one of claims 5 or 6, or the nucleic acid molecule for promoting plant flowering according to claim 7, or the biological material for promoting plant flowering according to any one of claims 7 to 9 in regulating the flowering time of plants; Preferably, regulating the flowering time of a plant comprises shortening the flowering time of a plant or extending the flowering time of a plant.

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