Plant lateral branch development related protein as well as coding gene and application thereof

Abstract

The invention discloses a plant lateral branch development related protein as well as a coding gene and application thereof. The invention provides a DSI1 protein derived from medicago truncatula and related to plant lateral branch development, the amino acid sequence of the DSI1 protein is as shown in SEQ ID NO.1, the nucleotide sequence of CDS of the coding gene of the DSI1 protein is as shown in SEQ ID NO.2, and the nucleotide sequence of the genome sequence of the coding gene of the DSI1 protein is as shown in SEQ ID NO.3. The invention further provides application of the DSI1 protein related to plant lateral branch development and the coding gene or the genome sequence of the DSI1 protein in regulation and control of plant lateral branch development, which comprises the following steps: mutating the coding gene of the DSI1 protein in a plant to cause defects in normal functions of the DSI1 protein, or interfering normal expression of the coding gene of the DSI1 protein, so that the DSI1 protein related to plant lateral branch development is obtained. The number of lateral branches of plants is reduced, and the number of inflorescences is increased.

Description

Technical Field

[0001] The present invention relates to genes isolated from plants of the Leguminosae family and their applications, and particularly to genes related to plant lateral branch development isolated from Medicago truncatula and their applications, belonging to the field of genes related to plant lateral branch development and their applications. Background Art

[0002] There are approximately 7,000 genera and 18,000 species of plants in the Leguminosae family, which is the third largest family after the Asteraceae and Orchidaceae families. It is widely distributed throughout the world and is an important source of human food, oil, forage, green manure, medicinal materials, and wood, having important economic, ecological, and biological values. However, due to factors such as the relatively large genomes of most leguminous crops and the immature genetic transformation systems, the research on the functional genomics of leguminous plants has been restricted.

[0003] Medicago truncatula is an annual herbaceous plant and a grazing forage. Due to its short growth cycle, low ploidy, small genome, self-pollination, and nitrogen fixation characteristics, it is an ideal model plant for the genetics and genomics research of leguminous plants.

[0004] The shoot system of higher plants is an important part of the plant morphological structure. Shoots include main branches and lateral branches. For gramineous plants such as rice and wheat, it is necessary to shape a suitable plant type structure to resist lodging. In solanaceous vegetables such as tomatoes and cucumbers, excessive growth of lateral branches will cover the lower leaves, inhibit reproductive growth, be unfavorable for field ventilation and management, and reduce land utilization rate. Therefore, to ensure high-quality and high-yield fruits and avoid excessive growth of lateral branches, artificial pruning is usually required, consuming a large amount of labor. In addition, for ornamental plants, it is also necessary to artificially regulate the branching to meet the preferences of consumers. Therefore, regulating the growth of plant lateral branches can not only increase crop yields but also meet market demands. Summary of the Invention

[0005] One object of the present invention is to provide a protein related to plant lateral branch development;

[0006] Another object of the present invention is to provide a coding gene for the protein related to plant lateral branch development;

[0007] A further object of the present invention is to provide an expression vector containing the coding gene for the protein related to plant lateral branch development;

[0008] Yet another object of the present invention is to apply the protein related to plant lateral branch development or its coding gene to regulate plant lateral branch development.

[0009] To achieve the above objects, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide a DSI1 protein related to plant lateral branch development, and the amino acid sequence of the DSI1 protein related to plant lateral branch development is selected from any one of the amino acid sequences shown in the following (a)-(d):

[0011] (a) The amino acid sequence shown in SEQ ID NO.1;

[0012] (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating plant lateral branch development;

[0013] (c) A protein variant obtained by inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating plant lateral branch development;

[0014] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, which is derived from Medicago truncatula and is related to plant lateral branch development.

[0015] In order to facilitate the purification or detection of the DSI1 protein provided by the present invention, a tag can be connected to the amino terminus or carboxyl terminus of this protein. Those skilled in the art can connect the corresponding tag to the amino terminus or carboxyl terminus of this protein according to the needs of purification or detection, and these are all well-known technical means in the art.

[0016] As an example, the present invention provides some specific tags that can be connected to the amino terminus or carboxyl terminus of this protein. The specific amino acid sequences of these tags can be as shown in Table 1. For example, they can be Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc, etc.

[0017] Table 1 Sequences of tags

[0018] Label Residue Sequence Poly-Arg 5 - 6 (usually 5) RRRRR Poly-His 2 - 10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0019] The DSI1 protein described in the present invention can be artificially synthesized, or can be obtained by first synthesizing its coding gene and then performing biological expression.

[0020] Another aspect of the present invention is to provide a coding gene of the DSI1 protein related to plant lateral branch development, and the nucleotide sequence of the CDS of the coding gene is selected from any one of the nucleotide sequences described in the following (a)-(e):

[0021] (a) The polynucleotide sequence shown in SEQ ID No.2;

[0022] (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1;

[0023] (c) A polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches;

[0024] (d) A polynucleotide sequence having at least 75% or more identity with any one of the polynucleotide sequences shown in (a)-(c), which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; preferably, a polynucleotide sequence having at least 85% or more identity with any one of the polynucleotide sequences shown in (a)-(c), which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; more preferably, a polynucleotide sequence having at least 90% or more identity with any one of the polynucleotide sequences shown in (a)-(c), which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; most preferably, a polynucleotide sequence having at least 95% or more identity with any one of the polynucleotide sequences shown in (a)-(c), which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches;

[0025] (e) A polynucleotide sequence capable of complementary pairing with any one of the polynucleotide sequences described in (a)-(d), which polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches.

[0026] The present invention also provides the genomic sequence of the coding gene of the DSI1 protein related to the development of plant lateral branches, and its nucleotide sequence is shown in SEQ ID No.3.

[0027] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including the Myers and Miller algorithms, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the algorithm of Karlin and Altschul, which are well-known to those skilled in the art.

[0028] In addition, those skilled in the art can also optimize the nucleotides shown in SEQ ID NO.2 to enhance the expression efficiency in plants.

[0029] The coding gene of the DSI1 protein in the present invention can be a mutant obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutations of one or several base pairs, and / or linking the coding sequences of the tags shown in Table 1 at its 5′ end and / or 3′ end.

[0030] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the DSI1 protein of the present invention by using known methods, such as directed evolution or point mutation methods. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the DSI1 protein isolated from the present invention, as long as they encode the DSI1 protein, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0031] In addition, the nucleotides in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; or they can be RNA, such as mRNA or hnRNA, etc.

[0032] Another aspect of the present invention further provides an expression cassette, recombinant vector or recombinant host cell containing the coding gene; preferably, the expression cassette, recombinant vector or recombinant host cell is a recombinant eukaryotic expression cassette, recombinant eukaryotic expression vector or recombinant plant cell; more preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or recombinant plant expression vector.

[0033] Another aspect of the present invention is to apply the DSI1 protein related to plant lateral branch development, its coding gene, and the expression vector containing the coding gene to regulate plant lateral branch development; wherein, the regulation of plant lateral branch development can be to promote the generation of plant lateral branches or inhibit the generation of plant lateral branches, preferably to inhibit the generation of plant lateral branches, including reducing the number of lateral branch branches, increasing the number of inflorescences, etc.

[0034] As a reference implementation, by mutating or interfering with the coding gene of the DSI1 protein related to plant lateral branch development in plants, the expression level and / or activity of the DSI1 protein related to plant lateral branch development can be reduced, thereby reducing the number of lateral branch branches, increasing the number of inflorescences, etc.

[0035] The mutations include substitution, deletion, and / or addition of one or more nucleotides in the nucleotide sequence of the coding gene of the DSI1 protein or its promoter. Specifically, the mutations can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; the methods of chemical mutagenesis include mutagenesis caused by treating with mutagens such as EMS, etc.; the methods of gene editing include, but are not limited to, ZFN, TALEN, and / or CRISPR / Cas, etc.

[0036] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing techniques to knockout the coding gene of the DSI1 protein in plants. For example, construct a knockout vector of the coding gene of the DSI1 protein or use gene editing techniques to construct a CRISPR / Cas9 gene editing vector of the coding gene of the DSI1 protein, etc., to knockout or mutate the coding gene of the DSI1 protein in plants. These methods are all proficiently mastered by those skilled in the art.

[0037] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the position to be gene-edited in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the target DNA sequence, and then cut the DNA by the Cas protein. In this application, the Cas protein includes, but is not limited to, proteins such as Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14, etc.

[0038] To interfere with the normal expression or normal function of the coding gene or promoter of the DSI1 protein, RNA interference technology (RNAi) can be used to interfere with the normal expression of the coding gene of the DSI1 protein or its promoter or cause defects in its normal function. RNA interference technology is a conventional technology in this field. It specifically binds to the homologous region of the mRNA expressed by the target gene through short double-stranded RNA (siRNA) of 21 - 23bp or long double-stranded RNA (dsRNA; double-strand RNA), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0039] Another aspect of the present invention is to provide a method for inhibiting the development of lateral branches and increasing the number of inflorescences in plants, including: causing mutations in the coding gene of the DSI1 protein in plants to cause defects in the normal function of the coding gene of the DSI1 protein; or interfering with the normal expression or normal function of the coding gene of the DSI1 protein.

[0040] Another aspect of the present invention is to provide a method for promoting the development of plant lateral branches, including: overexpressing the coding gene of the DSI1 protein derived from Medicago truncatula in a plant, and the resulting transgenic plant has enhanced lateral branch development; for example, the coding gene of the DSI1 protein derived from Medicago truncatula is operably linked to an expression regulatory element to obtain a recombinant plant expression vector for expressing the coding gene in a plant; transforming the recombinant plant expression vector into a plant to overexpress the coding gene of the DSI1 protein derived from Medicago truncatula in the plant, and the resulting transgenic plant has enhanced lateral branch development.

[0041] The present invention also discloses a recombinant expression vector containing the coding gene of the DSI1 protein derived from Medicago truncatula and a recombinant host cell containing the recombinant expression vector. The coding gene of the DSI1 protein derived from Medicago truncatula is operably linked to an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector can consist of a 5'-untranslated region, the nucleotide shown in SEQ ID NO.2, and a 3'-untranslated region; wherein, the 5'-untranslated region may include a promoter sequence, an enhancer sequence or / and a translation enhancer sequence; the promoter can be a constitutive promoter, an inducible promoter, a tissue- or organ-specific promoter; the 3'-untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. A suitable terminator sequence can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

[0042] The recombinant plant expression vector may further contain a selectable marker gene for selecting transformed cells to select the transformed cells or tissues. The marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds, etc. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins, etc.

[0043] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell to be transformed. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment, etc. In a specific embodiment, various transient transformation methods can be used to provide the Medicago truncatula MtMET1 gene to a plant. Stable transformed plants can be regenerated from the transformed cells using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0044] The plant described in the present invention is selected from any one of the following (c1) to (c6): (c1) dicotyledonous plants; (c2) monocotyledonous plants; (c3) leguminous plants; (c4) Medicago truncatula; (c5) Medicago truncatula R108; (c6) mutant dsi1.

[0045] Term definitions involved in the present invention

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form, and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically specified, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0048] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

[0049] As used herein, "stringent hybridization conditions" refers to conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence with a detectable degree that is higher than that of hybridization to other sequences (e.g., at least 2-fold above background). Stringent hybridization conditions are sequence-dependent and will vary in different environmental conditions. Longer sequences hybridize specifically at higher temperatures. By controlling the stringency of hybridization or the washing conditions, target sequences that are 100% complementary to the probe can be identified. Exhaustive guidance on nucleic acid hybridization can be found in the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays, 1993). More specifically, the stringent conditions are typically selected to be about 5 - 10 °C below the thermal melting point (T m ) of the specific sequence at a specified ionic strength and pH. T m is the temperature at which 50% of the probe that is complementary to the target hybridizes to the target sequence at equilibrium (at a specified ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, 50% of the probe is occupied at equilibrium at T m ). Stringent conditions can be the following: where the salt concentration is less than about 1.0 M sodium ion concentration at pH 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30 °C for short probes (including (but not limited to) 10 to 50 nucleotides), and at least about 60 °C for long probes (including (but not limited to) greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice the background hybridization, optionally 10-fold background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C; or 5×SSC, 1% SDS, incubated at 65 °C, washed in 0.2×SSC, and washed in 0.1% SDS at 65 °C. The washing can be carried out for 5, 15, 30, 60, 120 minutes or longer.

[0050] The term "recombinant host cell line" or "host cell" means a cell containing the polynucleotide of the present invention, regardless of the method used for insertion to generate the recombinant host cell, such as direct uptake, transduction, f-mating or other methods known in the art. The exogenous polynucleotide can be maintained as a non-integrating vector such as a plasmid or can be integrated into the host genome. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

[0051] The term "operably linked" refers to a functional linkage between two or more elements, and the elements that are operably linked can be adjacent or non-adjacent.

[0052] The term "recombinant plant expression vector" means one or more DNA vectors for realizing plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors together with vectors having helper plasmids are mostly commonly used for Agrobacterium tumefaciens-mediated transformation. Binary vectors generally include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be able to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0053] The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism. The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0054] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a phenotypic diagram of the lateral branches / secondary inflorescences of Medicago truncatula R108 wild type (WT) and Medicago truncatula dsi mutant lateral branches / secondary inflorescences.

[0056] Figure 2 It is a schematic diagram of the structure of the DSI gene and the insertion positions of Tnt1 in the gene in three mutants, dsi-1, dis-2, and dsi-3.

[0057] Figure 3 It is a phenotypic diagram of three dsi mutants and allele verification. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0059] Example 1: Obtaining of Medicago truncatula dsi mutant and phenotypic analysis

[0060] By screening the Medicago truncatula Tnt1 insertion mutant library, a mutant NF9910 with increased secondary inflorescences at the leaf axils was obtained and named double secondery inflorescence (dsi). Thirty seeds of mutant dsi or Medicago truncatula R108 were respectively taken, vernalized at 4 °C for 7 d, planted in nutrient soil, and cultured in an artificial climate chamber (16 h light / 8 h dark), and the phenotypes were observed in detail. The phenotypes of the lateral branches and inflorescences of the wild type and dsi mutant after flowering are as Figure 1 shown. Compared with the wild type, after phase transition, the lateral branches at the leaf axils of the dsi mutant were transformed into inflorescences( Figure 1 A, B). With the development of the plant, the number of inflorescences at the leaf axils of the internodes in the dsi mutant became two or three( Figure 1 C, D).

[0061] Example 2: Confirmation of the linkage relationship between DSI1 and the phenotypes of multiple inflorescences and reduced lateral branches and obtaining of its coding gene DSI1

[0062] 1. According to the flanking sequences of NF9910 published on the Medicago truncatula Tnt1 website (https: / / medicago-mutant.noble.org / mutant / ), the corresponding primers DSI1-Insertion-6-F and DSI1-Insertion-6-R were designed and combined with the primers of Tnt1 (LTR6 or LTR31) respectively to screen the Tnt1 insertion sites co-segregating with the dsi1 mutant phenotype, and the target gene was initially locked.

[0063] LRT31-F: 5’-CTCCTCTCGGGGTCGTGGTT-3’

[0064] LTR6-R: 5’-GCTACCAACCAAACCAAGTCAA-3’

[0065] DSI1-Insertion-F: 5’-ATGAGTATTGTAACAGATCCTCTTGCT-3’

[0066] DSI1-Insertion-R: 5’-CAGGTACGGATCACTAGGGCCAGGCACA-3’

[0067] Finally, a gene DSI1 linked to the mutant was discovered. In the NF9910 mutant, Tnt1 was inserted into the first exon of this gene. To further prove that the phenotypes of multiple inflorescences and reduced lateral branches of this mutant were caused by the mutation of the DSI gene, two other mutants of this gene were ordered: NF18411 (dsi-2, Tnt1 inserted into the first exon) and NF20902 (dsi-3, Tnt1 inserted between the +8 and +9 bases before the start codon), and the previous NF9910 was named dsi-1 (see Figure 2 A). Phenotypic analysis showed that dsi-2 and dsi-3 exhibited exactly the same phenotypes of multiple inflorescences and reduced lateral branches as dsi-1. The linkage type between DSI and the above phenotypes was further clarified.

[0068] 2. Total RNA was extracted from the apical meristems (Apex) of Medicago truncatula R108 (wild type) after phase conversion and reverse transcribed into cDNA as a template. The primer pair DSI1-F / DSI1-R was used for amplification.

[0069] DSI-F: 5’-TCTTGTTCACCTGCTTCACC-3’;

[0070] DSI-R: 5’-GCATTAGCACGTACAATCACTTT-3’.

[0071] The obtained PCR amplification products were sequenced to obtain the coding region sequence of the target gene. The nucleotide sequence of its CDS is shown as SEQ ID No.1. The deduced amino acid sequence of the protein encoded by this CDS is shown as SEQ ID No.2. This protein consists of 173 amino acid residues. The CDS sequence of this gene was aligned on the Medicago website (http: / / blast.jcvi.org / Medicago-Blast / ) to obtain the genomic sequence of the DSI gene. The nucleotide sequence of this genomic sequence is shown as SEQ ID No.3. The protein with the amino acid sequence shown as SEQ ID No.2 was named DSI, and the coding gene of DSI was named DSI gene.

[0072] 3. Total RNA was extracted from wild type R108 and the three dsi1 mutants respectively, and the reverse transcribed cDNA was used as a template. The primer pair composed of the DSI1 specific primers DSI1-F / DSI1-R was used to amplify the WF1 gene, and the primer pair RT-MtActin-F / RT-MtActin-R was used to amplify the Medicago truncatula internal reference gene MtActin for Real-time PCR (RT-PCR), showing that the expression levels of the DSI1 gene in the three mutants were significantly lower than those in the wild type ( Figure 2B), further verified that the deletion of the DSI1 gene led to abnormal development of lateral branches in Medicago truncatula and an increase in the number of secondary inflorescences at the axils of leaves, indicating that the DSI1 gene plays an important role in the regulation of lateral branch development in Medicago truncatula.

[0073] DSI1-F: 5’-ATGAGTATTGTAACAGATCCTCTTGCT-3’

[0074] DSI1-R: 5’-TCAACGTCTTCTGGCAGCTGT-3’

[0075] MtActin-F: 5’-TCTTACTCTCAAGTACCCCATTGAGC-3’

[0076] MtActin-R: 5’-GTGGGAGTGCATAACCCTCATAGATT-3’

[0077] 4. To determine that the abnormal phenotype of dsi1 lateral branch development was caused by DSI1 mutation, this experiment was further verified by allelic verification.

[0078] F1 generation plants were constructed pairwise for dsi-1, dis-2, and dsi-3 through hybridization. Phenotypic analysis showed that the above F1 generation plants all exhibited the same phenotypes as the dsi-1, dis-2, and dsi-3 homozygous mutants, namely abnormal lateral branch development and increased inflorescence number ( Figure 3 ).

Claims

1. The DSI1 protein related to plant lateral branch development, characterized in that, The amino acid sequence of the DSI1 protein is selected from any one of the amino acid sequences shown in the following (a)-(d): (a) The amino acid sequence shown in SEQ ID NO.1; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating the development of plant lateral branches; (c) A protein variant obtained by inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating the development of plant lateral branches; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, which is derived from Medicago truncatula and is related to the development of plant lateral branches.

2. The DSI1 protein related to plant lateral branch development according to claim 1, wherein A tag is connected to the amino terminus or carboxyl terminus of the DSI1 protein; preferably, the tag is selected from Poly-Arg, Poly-His, FLAG, Strep-tagII or c-myc.

3. The coding gene of the DSI1 protein related to plant lateral branch development according to claim 1, characterized in that, The nucleotide sequence of the CDS of the encoding gene is selected from any one of the nucleotide sequences described in the following (a)-(e): (a) The polynucleotide sequence shown in SEQ ID No.2; (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1; (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; (d) A polynucleotide sequence having at least 75% or more identity with any one of the polynucleotide sequences shown in (a)-(c), and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; preferably, a polynucleotide sequence having at least 85% or more identity with any one of the polynucleotide sequences shown in (a)-(c), and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; more preferably, a polynucleotide sequence having at least 90% or more identity with any one of the polynucleotide sequences shown in (a)-(c), and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; most preferably, a polynucleotide sequence having at least 95% or more identity with any one of the polynucleotide sequences shown in (a)-(c), and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches; (e) A polynucleotide sequence that can be complementary to any one of the polynucleotide sequences described in (a)-(d), and this polynucleotide sequence is derived from Medicago truncatula and has the function of regulating the development of plant lateral branches.

4. The genomic sequence of the coding gene of the DSI1 protein related to plant lateral branch development according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID No.

3.

5. A chimeric gene or expression cassette containing the encoding gene according to claim 3 or the genomic sequence according to claim 4.

6. A recombinant expression vector containing the chimeric gene or expression cassette according to claim 5.

7. A recombinant host cell containing the recombinant expression vector according to claim 6. Use of the DSI1 protein related to plant lateral branch development according to claim 1 or 2, the coding gene according to claim 2, and the genomic sequence according to claim 3 in regulating plant lateral branch development; preferably, the regulation of plant lateral branch development is to inhibit the generation of plant lateral branches, including reducing the number of lateral branch branches and increasing the number of inflorescences.

9. A method for inhibiting the development of plant lateral branches and increasing the number of inflorescences, comprising: Mutating the coding gene of the DSI1 protein in a plant to cause a defect in the normal function of the coding gene of the DSI1 protein; Or interfering with the normal expression or normal function of the coding gene of the DSI1 protein.

10. The method according to claim 9, characterized in that The plant is selected from any one of the following (c1) to (c6): (c1) dicotyledonous plants; (c2) monocotyledonous plants; (c3) leguminous plants; (c4) Medicago truncatula; (c5) Medicago truncatula R108; (c6) mutant dsi1.

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