Application of LAP1 gene or encoded protein thereof in regulation and control of physical dormancy of plants

Abstract

The invention discloses an application of an LAP1 gene or an encoded protein thereof in regulation and control of physical dormancy of plants. It is found that compared with wild medicago truncatula, transgenic medicago truncatula obtained after the LAP1 gene separated from medicago truncatula is subjected to overexpression in medicago truncatula shows phenotypes such as water swelling or / and seed germination rate improvement and the like, and the transgenic medicago truncatula can be applied to the field of medicago truncatula. The physical dormancy of the transgenic medicago truncatula seeds is obviously broken. The method has application prospects in the aspects of promoting plant seed germination, reducing the production cost during seed germination and the like.

Description

Technical Field

[0001] This invention relates to novel uses of genes isolated from plants in the Leguminosae family, and more particularly to the use of the LAP1 gene or its encoded protein isolated from Medicago truncatula in regulating plant physical dormancy, belonging to the field of novel uses of the LAP1 gene or its encoded protein. Background Technology

[0002] Leguminosae, comprising approximately 700 genera and 18,000 species, is the third largest family after Asteraceae and Orchidaceae. Widely distributed throughout the world, it is a vital source of food, oilseeds, forage, green manure, medicinal herbs, and timber, possessing significant economic, ecological, and biological value. However, the large genome size of most leguminous plants and the immature genetic transformation systems limit research into functional genomics in leguminous plants.

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

[0004] Physical dormancy in seeds is a widespread adaptive characteristic in higher plants, caused by one or more layers of impermeable palisade tissue in the seed or fruit's covering layer. This impermeability of seeds is also known as seed "hardness," and seeds with this characteristic require breaking physical dormancy to germinate.

[0005] In the laboratory, alfalfa seeds require their seed coat to be broken to absorb water and germinate, a time-consuming and labor-intensive process. For field emergence, chemical and physical methods are needed to break the seeds' physical dormancy. Physical methods include sun exposure, hot water soaking, mechanical grinding, and physical milling to accelerate water absorption. Mechanical grinding involves mixing alfalfa seeds with a certain amount of sand and gently rubbing them on a brick surface to create a rough but not broken seed coat, but this often results in some seed loss. Chemical methods include soaking the seeds in concentrated sulfuric acid, or ammonium molybdate and boric acid solutions, followed by immediate rinsing with clean water. These processes not only pose health risks but also pollute the environment with residual chemicals.

[0006] Regulating seed physical dormancy can not only break seed physical dormancy in a timely manner, but also promote seed germination rate; therefore, obtaining genes or proteins that can effectively regulate seed physical dormancy has important application value for promoting seed germination and reducing production costs. Summary of the Invention

[0007] The main objective of this invention is to provide plant-derived genes or their encoded proteins that can regulate plant physical dormancy and to apply them to regulating plant physical dormancy and other related matters.

[0008] This invention discovers that transgenic alfalfa, obtained by overexpressing the LAP1 gene isolated from alfalfa in alfalfa, exhibits significantly disrupted seed dormancy compared to wild-type alfalfa, thus completing this invention.

[0009] This invention primarily provides the use of the LAP1 gene or its encoded protein (LAP1 protein) in regulating plant physical dormancy;

[0010] As a preferred embodiment of the present invention, the regulation of plant physical dormancy is to break the physical dormancy of the plant; more preferably, the breaking of plant physical dormancy includes, but is not limited to, breaking the physical dormancy of plant seeds, wherein breaking the physical dormancy of plant seeds specifically includes: promoting seed water absorption and swelling, promoting seed germination, etc.

[0011] As a possible implementation scheme, overexpression of the gene encoding the LAP1 protein associated with plant physical dormancy can increase the expression level and / or activity of the LAP1 protein, thereby breaking the physical dormancy of seeds and accelerating the process of seed water absorption and swelling.

[0012] As a preferred embodiment, the present invention provides a method for breaking the physical dormancy of plant seeds or promoting seed germination by water absorption, comprising: overexpressing the LAP1 gene in a plant to enhance the expression level of the LAP1 gene or enhance the function or activity of the LAP1 protein; for example, operatively linking the LAP1 gene derived from alfalfa to an expression regulatory element to obtain a recombinant plant expression vector that expresses the gene in a plant; transforming the recombinant plant expression vector into a plant to overexpress the LAP1 gene in the plant, wherein the seeds of the transgenic plant obtained are able to absorb water and swell during germination, thus promoting seed germination.

[0013] The nucleotide sequence of the CDS of the LAP1 gene described in this invention is selected from any one of the nucleotide sequences described in (a)-(e) below:

[0014] (a) The polynucleotide sequence shown in SEQ ID No. 2;

[0015] (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1;

[0016] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy;

[0017] (d) A polynucleotide sequence having at least 75% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; preferably, a polynucleotide sequence having at least 85% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; more preferably, a polynucleotide sequence having at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; most preferably, a polynucleotide sequence having at least 95% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy;

[0018] (e) A polynucleotide sequence that is complementary to any of the polynucleotide sequences described in (a)-(d), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy.

[0019] The nucleotide sequence of the LAP1 gene described in this invention is shown in SEQ ID No. 3.

[0020] The percentage of sequence identity described in this invention can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0021] In addition, those skilled in the art can optimize the nucleotides shown in SEQ ID NO.2 to enhance their expression efficiency in plants.

[0022] The present invention can also produce mutants by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to the 5′ and / or 3′ end of the amino acid sequence of the protein encoded by its gene.

[0023] Those skilled in the art can readily mutate the nucleotide sequence of the LAP1 gene using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence of the LAP1 gene, provided that the encoded protein has the function of regulating plant physical dormancy, are derived from and are equivalent to the nucleotide sequence of this invention.

[0024] In addition, the nucleotide sequence described in this invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; or it can be RNA, such as mRNA or hnRNA.

[0025] The amino acid sequence of the LAP1 protein described in this invention is selected from any of the following amino acid sequences (a)-(d):

[0026] (a) The amino acid sequence shown in SEQ ID NO.1;

[0027] (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, which still has the function or activity of regulating plant physical dormancy;

[0028] (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, which still has the function or activity of regulating plant physical dormancy;

[0029] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, which is derived from alfalfa and is associated with plant physical dormancy.

[0030] To facilitate the purification or detection of the LAP1 protein, a tag can be attached to the amino terminus and / or carboxyl terminus of the protein. As an example, the present invention provides some specific tags that can be attached to the amino terminus or carboxyl terminus of the protein. The specific amino acid sequences of these tags can be as shown in Table 1, such as Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc, etc.

[0031] Table 1 Label Sequence

[0032] Label residues 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

[0033] The LAP1 protein described in this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0034] Another aspect of the present invention further provides an expression cassette, recombinant vector, or recombinant host cell containing the LAP1 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.

[0035] For reference, this invention provides a plant recombinant expression vector for the LAP1 gene, comprising: operably linking the LAP1 gene derived from *Alfalfa truncatum* to an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector may consist of a 5′ untranslated region, the nucleotides shown in SEQ ID NO. 2, and a 3′ untranslated region; wherein, the 5′ untranslated region may include a promoter sequence, an enhancer sequence, and / or a translational enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, or a tissue or organ-specific promoter; the 3′ untranslated region may include a terminator sequence, an mRNA cleavage sequence, etc. A suitable terminator sequence may be obtained from the Ti-plasmid of *Agrobacterium tumefaciens*, such as the terminator regions of octopine synthase and carmine synthase.

[0036] The recombinant plant expression vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicides. Furthermore, the marker genes may also include phenotypic markers, such as β-galactosidase and fluorescent proteins.

[0037] The transformation scheme and the scheme for introducing the polynucleotide or polypeptide into the plant can vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods can be used to deliver the MtMET1 gene from alfalfa to the plant. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986.5:81-84).

[0038] Another aspect of the invention provides the use of the LAP1 gene or its encoded protein (LAP1 protein) in prolonging the physical dormancy of plant seeds, including: mutating the LAP1 gene in plants to reduce the expression level of the LAP1 gene or to defect the normal function of the LAP1 protein.

[0039] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the nucleotide sequence of the LAP1 gene or its promoter. Specifically, the mutations can be obtained through physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis and space breeding; chemical mutagenesis methods include mutagenesis induced by treatment with mutagens such as EMS; and gene editing methods include, but are not limited to, ZFN, TALE, and / or CRISPR / Cas.

[0040] Those skilled in the art can use conventional methods such as gene knockout or gene editing techniques to knock out or mutate the LAP1 gene in plants, such as constructing a LAP1 gene knockout vector or using gene editing technology to construct a CRISPR / Cas9 gene editing vector for the LAP1 gene, to knock out or mutate the LAP1 gene in plants. These methods are well known to those skilled in the art.

[0041] Those skilled in the art will understand that the main principle of the CRISPR / Cas gene editing system or method is to use a nucleic acid fragment called guide RNA (gRNA) to locate the site for gene editing in the host genome, i.e., the target DNA sequence, and then use Cas proteins to cut the DNA. In this application, the Cas proteins include, but are not limited to, Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14 proteins.

[0042] Interference with the normal expression or function of the LAP1 gene or promoter can be achieved using RNA interference (RNAi) technology. This involves interfering with the normal expression of the LAP1 protein-coding gene or its promoter, or causing defects in its normal function. RNA interference is a conventional technique in this field. It involves the specific binding of a 21-23 bp short double-stranded RNA (siRNA) or a long double-stranded RNA (dsRNA) to the homologous region of the mRNA expressing the target gene, thereby degrading the mRNA and inhibiting gene expression.

[0043] The plants described in this invention include dicotyledonous plants or monocotyledonous plants, preferably legumes; wherein, the legume is preferably alfalfa.

[0044] Definitions of terms involved in this invention

[0045] 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 pertains.

[0046] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, specific nucleic acid sequences implicitly encompass variants of their conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0047] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0048] In this invention, "rigorous hybridization conditions" refers to conditions of low ionic strength and high temperature known in the art. Typically, under rigorous conditions, the detectability of a probe hybridizing with its target sequence is significantly higher than that with other sequences (e.g., at least twice the background level). Rigorous hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the rigor of hybridization or washing conditions. Detailed 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 acidassays," 1993). More specifically, the rigorous conditions are typically chosen to be below the melting point (T0) of the specific sequence at a specified ionic strength pH. m Approximately 5-10℃. mThe temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence under equilibrium conditions (at specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is in excess, therefore at T...). m (Under equilibrium conditions, 50% of the probe is occupied). Strict conditions may include: a salt concentration of less than approximately 1.0 M sodium ions at pH 7.0 to 8.3, typically approximately 0.01 to 1.0 M sodium ions (or other salts), and a temperature of at least approximately 30 °C for short probes (including (but not limited to) 10 to 50 nucleotides) and at least approximately 60 °C for long probes (including (but not limited to) more than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal may be at least twice the background hybridization, and, where appropriate, 10 times the background hybridization. Exemplary strict hybridization conditions may 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 performed for 5, 15, 30, 60, 120 minutes or longer.

[0049] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may be maintained as, for example, a non-integrating vector of a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell, and may also be a monocotyledonous or dicotyledonous plant cell.

[0050] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.

[0051] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, an engineered selection marker for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0052] The term "transformation" refers to the 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 transgene in plant cells.

[0053] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells. Attached Figure Description

[0054] Figure 1 Phenotypic diagrams of wild-type (WT) alfalfa R108 and transgenic LAP1 alfalfa, and graphs showing the expression levels of the MtLAP1 gene.

[0055] Figure 2 Images of seed germination of wild-type (WT) alfalfa R108 and transgenic LAP1 alfalfa.

[0056] Figure 3 Statistical graphs showing the water absorption and germination rate of wild-type (WT) alfalfa R108 and transgenic LAP1 alfalfa seeds. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0058] Experiment 1: Obtaining and Phenotypic Analysis of Alfalfa LAP1 Transgenic Plants

[0059] 1. Construction of the plant high-efficiency expression vector pEarlyGate202-MtLAP1

[0060] A complete open reading frame containing the attB site was amplified from the CDS of young leaves of *Alfalfa truncatum* using primers MtLAP1-attB(F) and MtLAP1-attB(R). The amplified product and the entry vector pDNOR207 were subjected to a backpropagation (BP) reaction. The CCDB gene in the pDNOR207 vector was replaced with the MtLAP1 gene (SEQ ID No. 2) to construct an entry vector containing the target gene MtLAP1. This vector was transformed into *E. coli* DH5α, and recombinant plasmids were screened by PCR amplification and sequenced for identification. The correctly sequenced entry vector pDNOR207-MtLAP1 was then subjected to an LR reaction with the expression vector pEarlyGate202. The CCDB gene in the expression vector was replaced with the target gene MtLAP1 to construct the plant expression vector pEarlyGate202-MtLAP1. This vector was transformed into *E. coli* DH5α, and recombinant plasmids were identified by PCR amplification.

[0061] 2. Genetic transformation of alfalfa

[0062] Agrobacterium containing the plant expression vector pEarlyGate202-MtLAP1 was cultured to OD. 600Centrifuge at 3800 rpm for 15 min with a concentration of 0.8-2.0, discard the supernatant, resuspend the bacterial precipitate in CO2 permeate, and use it to infect young leaves of alfalfa.

[0063] Young leaves were taken from sterile alfalfa seedlings that were about 5 weeks old. The leaves were then punctured with a knife to create wounds. Agrobacterium resuspension was added to the leaves, and the mixture was sonicated for 1-2 minutes, followed by standing for 30 minutes. The leaves were then laid flat and cultured in the dark for 2 days before being transferred to a plant resistance induction medium. Callus tissue was selected for further culture, transferred to a light-treated medium for differentiation until green shoots appeared, and then transferred to a rooting medium until new seedlings emerged. Phenotypic data were statistically analyzed during plant growth. Figure 1 A, B).

[0064] Compared to wild-type alfalfa R108, plants overexpressing LAP1 are entirely purple, and anthocyanins accumulate in the leaves, stems, petals, pods, and seeds.

[0065] 3. PCR detection at the RNA level

[0066] Leaves and flowers from two randomly selected positive *Alfalfa* plants identified at the DNA level were used as materials to extract total RNA. Using cDNA obtained through reverse transcription as a template, the LAP1 gene was amplified using primers consisting of LAP1-qF / LAP1-qR. The *Alfalfa* internal control gene MtActin was amplified using primers consisting of MtActin-qF / MtActin-qR. Quantitative real-time PCR (qRT-PCR) was performed, showing that the expression levels of the LAP1 gene in the leaves and flowers of both transgenic plants were significantly higher than those in the wild type. Figure 1 C and D) further verified that the overexpression of the LAP1 gene led to the breaking of physical dormancy in alfalfa, indicating that the LAP1 gene plays an important role in the regulation of physical dormancy in alfalfa.

[0067] Table 2 Primer sequences used in this experiment

[0068]

[0069]

[0070] Experiment 2: Verification Experiment on the Breaking of Physical Dormancy in Seeds of Alfalfa LAP1 Transgenic Plants

[0071] Wild-type and transgenic MtLAP1 alfalfa seeds were placed separately in sterile water. Compared to wild-type seeds, the transgenic MtLAP1 alfalfa seeds began to absorb water, and the seed coat swelled and ruptured, indicating that the physical dormancy of the transgenic alfalfa seeds was broken, while the wild-type seeds remained unchanged. Ultimately, the transgenic alfalfa seeds were able to germinate, while the wild-type seeds remained unchanged. Figure 2 ).

[0072] Approximately 100 mg of mature wild-type and transgenic LAP1 seeds were soaked in sterile water at room temperature, and the weight was measured every 30 minutes in three independent experiments. The water absorbed was equal to the total weight of the swollen seed minus the original weight of the dry seed, and this was used to evaluate the water absorption of the seed.

[0073] Experimental results showed that the water absorption of transgenic LAP1 seeds gradually increased over time, while the wild type showed no significant change. This indicates that the LAP1 gene can improve seed germination rate by breaking the physical dormancy of seeds. Figure 3 A).

[0074] Further analysis of the water absorption rate and germination rate of approximately 100 mg of mature wild-type and transgenic LAP1 seeds revealed that the mature transgenic LAP1 seeds had a water absorption rate of 0.47% and a germination rate of 0.3%. Figure 3 B). This further demonstrates that the LAP1 gene can break the physical dormancy of alfalfa seeds and improve seed germination rate.

Claims

1. The use of the LAP1 gene, LAP1 protein, or expression cassettes containing the LAP1 gene, recombinant plant expression vectors, or recombinant host cells in regulating plant physical dormancy.

2. The use according to claim 1, characterized in that, The regulation of plant physical dormancy mentioned above refers to breaking the physical dormancy of plant seeds.

3. The use according to claim 2, characterized in that, Breaking the physical dormancy of plant seeds includes: promoting seed water absorption and swelling and / or promoting seed germination.

4. The use according to claim 1 or 2, characterized in that, include: Overexpression of the LAP1 gene in plants can increase the expression level or enhance the function or activity of the LAP1 protein, thereby breaking the physical dormancy of plant seeds.

5. The use according to claim 1, characterized in that, The nucleotide sequence of the CDS of the LAP1 gene is selected from any of the following nucleotide sequences (a)-(e): (a) The polynucleotide sequence shown in SEQ ID No. 2; (b) A 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 strict hybridization conditions, the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; (d) A polynucleotide sequence having at least 75% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; preferably, a polynucleotide sequence having at least 85% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; more preferably, a polynucleotide sequence having at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; most preferably, a polynucleotide sequence having at least 95% or more identity with any of the polynucleotide sequences shown in (a)-(c), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy; (e) A polynucleotide sequence that is complementary to any of the polynucleotide sequences described in (a)-(d), the polynucleotide sequence being derived from alfalfa and having the function of regulating plant physical dormancy.

6. The use according to claim 1, characterized in that, The nucleotide sequence of the LAP1 gene genome is shown in SEQ ID No.

3.

7. The use according to claim 1, characterized in that, The amino acid sequence of the LAP1 protein is selected from any of the following amino acid sequences (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, which still has the function or activity of regulating plant physical dormancy; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, which still has the function or activity of regulating plant physical dormancy; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, which is derived from alfalfa and is associated with plant physical dormancy.

8. The use according to claim 1, characterized in that, The regulation of plant physical dormancy refers to prolonging the physical dormancy of plant seeds.

9. The use according to claim 8, characterized in that, include: Mutating the LAP1 gene in plants can reduce the expression level of the LAP1 gene or impair the normal function of the LAP1 protein.

10. The use according to claim 1, characterized in that, The plants mentioned include dicotyledonous or monocotyledonous plants, preferably legumes, and more preferably, alfalfa.

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