Use of RH1 gene or its encoded protein in regulating plant physical dormancy

Abstract

The present invention discloses RH1 The invention relates to a gene or a protein encoded by the gene for regulating plant physical dormancy. RH1 Medicago truncatula RH1 Gene was used to obtain transgenic Medicago truncatula. Compared with wild-type Medicago truncatula, transgenic Medicago truncatula seeds showed phenotypes such as water absorption and swelling and increased seed germination rate, indicating that the physical dormancy of transgenic Medicago truncatula seeds was significantly broken, confirming RH1 The gene or its encoded protein has the function of regulating plant physical dormancy. The invention has application prospects in promoting plant seed germination, reducing production costs during seed germination, etc.

Description

Technical Field

[0001] The present invention relates to Leguminosae ) plants, particularly Medicago truncatula ( Medicago truncatula ) RH1 Use of a gene or its encoded protein in regulating plant physical dormancy, belonging to RH1 New areas of use for genes or their encoded proteins. Background Art

[0002] Leguminosae Leguminosae ) Plants have about 700 genera and 18,000 species, making them the third largest family after Asteraceae and Orchidaceae. They are widely distributed throughout the world and are important sources of food and oil for human beings, as well as forage, green manure, medicinal materials and timber, with important economic, ecological and biological values. However, the research on functional genomics of leguminous plants is limited by the large genome size of most leguminous crops and the immature genetic transformation system.

[0003] Medicago truncatula Medicago truncatula ) is an annual herb and grazing forage. It is an ideal model plant for genetics and genomics research in legumes due to its short growth cycle, small ploidy, small genome, self-pollination and nitrogen fixation.

[0004] Seed physical dormancy is an adaptive trait widely found in higher plants. It is a phenomenon caused by one or more layers of impermeable palisade tissue in the seed or fruit coating. This impermeability of seeds is also called the "hardness" of seeds. Seeds with this characteristic need to break physical dormancy to germinate.

[0005] In the laboratory, the seed coat of Medicago truncatula seeds needs to be broken before they can absorb water and germinate, and the process is time-consuming and laborious. Field emergence requires chemical and physical methods to break the physical dormancy of plant seeds. Physical methods include: sun exposure, hot water immersion, mechanical grinding, physical grinding and other methods to accelerate water absorption. Mechanical grinding refers to mixing a certain amount of sand and gravel into alfalfa seeds and gently rubbing them on the brick floor to make the seed coat rough but not broken, but some seeds are often lost during the actual operation. Chemical methods include: soaking the seeds in concentrated sulfuric acid, or ammonium molybdate and boric acid solution, and immediately washing them with clean water after soaking. The processing process will not only cause harm to the human body, but the residual chemical reagents will also pollute the environment.

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

[0007] The main purpose of the present invention is to RH1 Gene, RH1 protein, containing RH1 Gene expression cassette or containing RH1 The recombinant plant expression vector of the gene is used to regulate plant physical dormancy;

[0008] To achieve the above purpose, the main technical solutions adopted by the present invention include:

[0009] One aspect of the present invention is to RH1 Gene, RH1 protein, containing RH1 Gene expression cassette or containing RH1 The recombinant plant expression vector of the gene is used to regulate plant physical dormancy and other aspects.

[0010] In a preferred specific embodiment of the present invention, the regulating plant physical dormancy is breaking the physical dormancy of the plant; more preferably, the breaking the physical dormancy of the plant includes but is not limited to breaking the physical dormancy of plant seeds; wherein, the breaking the physical dormancy of plant seeds specifically includes: promoting water absorption and swelling of plant seeds or promoting germination of plant seeds.

[0011] For reference, the present invention provides an implementation scheme, that is, by overexpressing the coding gene of the RH1 protein related to plant physical dormancy in plants, the expression amount or activity of the RH1 protein related to plant physical dormancy is increased, thereby breaking the physical dormancy of seeds, and then promoting the water absorption and swelling of plant seeds or promoting the germination of plant seeds.

[0012] A preferred specific embodiment of the present invention is a method for breaking the physical dormancy of plant seeds, promoting the swelling of plant seeds by water absorption, or promoting the germination of plant seeds by water absorption, comprising: overexpressing in a plant RH1 Gene, RH1 The expression of the gene is increased or the function or activity of the RH1 protein is enhanced; for example, RH1 The gene is connected to the expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into plants, so that RH1 The gene is overexpressed in the plant, and the seeds of the resulting transgenic plants can absorb water and swell during germination, thus promoting seed germination.

[0013] For reference, the present invention provides a RH1 Gene plant recombinant expression vector, comprising: a gene derived from Medicago truncatula RH1The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.2 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0014] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0015] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell to be transformed. 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 may be used to transform Medicago truncatula. RH1 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0016] A preferred embodiment of the present invention is a method for extending the physical dormancy of plant seeds, comprising: RH1 Gene mutation RH1 Reduced gene expression may cause defects in the normal function of RH1 protein.

[0017] The mutations include RH1 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; chemical mutagenesis includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes, but is not limited to, ZFN, TALE and / or CRISPR / Cas, etc.

[0018] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. RH1Gene knockout mutation, such as constructing RH1 Gene knockout vectors or constructed using gene editing technology RH1 CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants RH1 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0019] 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 location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the targeted DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0020] The interference RH1 The normal expression or normal function of the gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the RH1 protein or its promoter or to cause defects in its normal function. RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short-chain double-stranded RNA (siRNA) or a long-chain double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0021] The plants described in the present invention include dicotyledonous plants or monocotyledonous plants, preferably leguminous plants; wherein the leguminous plants are preferably Medicago truncatula.

[0022] Another aspect of the present invention is to provide a Medicago truncatula-derived compound capable of regulating plant physical dormancy. RH1 gene or its encoded protein.

[0023] The present invention RH1 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) to (e) below:

[0024] (a) the polynucleotide sequence shown in SEQ ID No. 2;

[0025] (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1;

[0026] (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the polynucleotide sequence still has the function of regulating plant physical dormancy;

[0027] (d) a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in any one of (a) to (c), and the polynucleotide sequence still has the function of regulating plant physical dormancy;

[0028] (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), and the polynucleotide sequence still has the function of regulating plant physical dormancy.

[0029] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0030] In addition, those skilled in the art can also optimize the nucleotide sequence shown in SEQ ID NO. 2 to enhance the expression efficiency in plants.

[0031] The present invention can also be used to obtain mutants by deleting one or several codons for 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 connecting the coding sequence of the tag shown in Table 1 to the 5' end and / or 3' end of the amino acid sequence of the protein encoded by its gene.

[0032] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to RH1 The nucleotide sequence of the gene is mutated. RH1 Nucleotides with 75% or higher identity to the nucleotide sequence of a gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating plant physical dormancy.

[0033] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0034] The amino acid sequence of the RH1 protein described in the present invention is selected from the amino acid sequence shown in any one of the following (a)-(d):

[0035] (a) the amino acid sequence shown in SEQ ID NO.1;

[0036] (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, wherein the protein variant still has the function or activity of regulating plant physical dormancy;

[0037] (c) a protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of regulating plant physical dormancy;

[0038] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, wherein the protein still has the function or activity of regulating plant physical dormancy.

[0039] In order to facilitate the purification or detection of the RH1 protein, a tag can be connected to the amino terminus or and / or carboxyl terminus of the protein. For example, the present invention provides some specific tags that can be connected to the amino terminus or carboxyl terminus of the protein. The specific amino acid sequences of these tags can be shown in Table 1, for example, they can be Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc, etc.

[0040] Table 1 Tag sequence

[0041]

[0042] The RH1 protein of the present invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0043] Definitions of terms used in this invention

[0044] Unless defined otherwise, 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.

[0045] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof 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 reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0046] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, 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 encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0047] The "stringent hybridization conditions" described herein refer to conditions of low ionic strength and high temperature as known in the art. Generally, under stringent conditions, the detectable degree of hybridization of the probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times above background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to 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 usually selected to be lower than the thermal melting point (T) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. T m The temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T mStringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, 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 may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent 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 may be performed for 5, 15, 30, 60, 120 minutes or longer.

[0048] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0049] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0050] The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0051] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0052] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0053] 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

[0054] Figure 1Medicago truncatula R108 wild type (WT) and transgenic RH1 Phenotype of Medicago truncatula RH1 Gene expression detection diagram; among them, Figure 1 -A is the phenotype diagram of wild type (WT) of Medicago truncatula R108; Figure 1 -B is transgenic RH1 Phenotype diagram of Medicago truncatula; Figure 1 -C for Medicago truncatula R108 wild type (WT) and transgenic RH1 Medicago truncatula RH1 Gene expression detection graph;

[0055] Figure 2 Medicago truncatula R108 wild type (WT) and transgenic RH1 Medicago truncatula seed germination diagram;

[0056] Figure 3 Medicago truncatula R108 wild type (WT) and transgenic RH1 Statistical chart of water absorption rate, swelling rate and germination rate of Medicago truncatula seeds; among them, Figure 3 -A is Medicago truncatula R108 wild type (WT) and transgenic RH1 Diagram of water absorption rate of Medicago truncatula seeds; Figure 3 -B is Medicago truncatula R108 wild type (WT) and transgenic RH1 Statistical chart of imbibition rate and germination rate of Medicago truncatula seeds. DETAILED DESCRIPTION

[0057] 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, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0058] Test Example 1 Medicago truncatula RH1 Obtaining transgenic plants and phenotypic analysis

[0059] 1 Expression vector pEarlyGate203- RH1 Construction

[0060] Using primers cacc-RH1-CDS-F and RH1-CDS-R A complete open reading frame containing the cacc site was amplified from the CDS of young leaves of Medicago truncatula to generate a blunt-end PCR product. The PCR product was cloned into the pENTR vector through a TOPO cloning reaction to construct a vector containing the target gene. RH1The entry vector was transformed into E. coli DH5α, and the recombinant plasmid was screened by PCR amplification and sequenced for identification. pENTR - RH1 LR reaction was carried out with the plant overexpression vector pEarlyGate203 to CCDB Gene replacement with target gene RH1 , construct plant expression vector pEarlyGate203 - RH1 , transformed into Escherichia coli DH5α, and the recombinant plasmid was amplified and identified by PCR.

[0061] The amino acid sequence of RH1 protein is as follows: MANTSGVRKGAWTYEEDNCLKAYILKHGVGKWHLIPERTGLNRCRKSCRLRWVNYLNPYINRENFSKDEVDLILRLHNLLGNRWSLIAGRLPGRTANDVKNYWNTYLRKKVES EAKEKEKSNETMKAHEVIKPRPITLSSRSHFVHDSNKDRYVPIYQDDSSETMVPSQIGGDYASAVQPSLGNNVQTPCAMWSDSLWDMGSSEKIGSCSSLQEVNNFNMDFPDDSFWDFNFSDFEFLRDL (SEQ ID NO. 1).

[0062] RH1The nucleotide sequence of the gene is as follows: (SEQ ID NO.2).

[0063] 2 Genetic transformation of Medicago truncatula

[0064] Plant expression vector pEarlyGate203 - RH1 Agrobacterium was cultured to OD 600 The concentration of the culture medium was 0.8-2.0, and the mixture was centrifuged at 3800 rpm for 15 min. The supernatant was discarded and the bacterial precipitate was resuspended in CO2 infiltration solution for infecting young leaves of Medicago truncatula.

[0065] Take the young leaves of sterile Medicago truncatula seedlings that have grown for about 5 weeks, cut the leaves with a knife to make wounds, add the resuspended Agrobacterium solution to the leaves, ultrasonicate for 1-2 minutes and let it stand for 30 minutes, finally spread the leaves flat and culture in the dark for 2 days, then transfer them to an induction medium with plant resistance, select callus tissue for further culture, transfer to a light medium for differentiation until green shoots grow, and transfer to a rooting medium until new seedlings grow, and count the phenotypes of the plants during their growth.

[0066] The phenotype of the plant during growth is as follows Figure 1 -A. Figure 1 -B, compared with the wild-type Medicago truncatula R108, overexpression RH1 The entire Medicago truncatula plant is purple, and anthocyanins are accumulated in the leaves, stems, petals, pods, and seeds of the plant.

[0067] 3 PCR detection of RNA level

[0068] The leaves and flowers of two positive Medicago truncatula plants identified at the DNA level were randomly selected as materials, and total RNA was extracted. The cDNA obtained by reverse transcription was used as a template. RH1 Gene specific quantitative primers RH1 -qF / RH1 -qR primer pair amplification RH1 Gene, and primers MtActin -qF / MtActin -qR primer pair to amplify the internal reference gene of Medicago truncatula MtActin , and Quantitative Real-time PCR (qRT-PCR) was performed.

[0069] The results of qRT-PCR are as follows Figure 1 -C, leaves and flowers of two transgenic plants RH1 The expression levels of the genes were significantly higher than those of the wild type, further verifying that RH1 Overexpression of the gene causes the physical dormancy of Medicago truncatula to be released, indicating that RH1 Genes play an important role in the regulation of physical dormancy in Medicago truncatula.

[0070] Table 2 Primer sequences used in this experiment

[0071]

[0072] Test Example 2 Medicago truncatula RH1 A test to verify that the physical dormancy of transgenic plant seeds has been released

[0073] Wild-type and transgenic RH1Medicago truncatula seeds were placed in sterile water and the wild type and transgenic RH1 The test results are as follows: Figure 2 As shown, compared with wild-type seeds, transgenic RH1 The Medicago truncatula seeds began to absorb water, and the seed coat swelled and burst, indicating that the physical dormancy of the transgenic Medicago truncatula seeds was broken, while the wild-type seeds remained intact. Eventually, the transgenic Medicago truncatula seeds were able to germinate, while the wild-type seeds remained intact without change.

[0074] About 100 mg of wild-type and transgenic RH1 Mature seeds were soaked in sterile water at room temperature, and their weight was measured every 30 min in three independent experiments. The absorbed water was equal to the total weight of the swollen seeds minus the original weight of the dry seeds, which was used to evaluate the water absorption of the seeds.

[0075] Wild type and transgenic RH1 Changes in the water absorption rate of mature seeds Figure 3 -A, transgenic RH1 The water absorption of seeds gradually increased over time, while there was no obvious change in the wild type, indicating that RH1 Genes can improve seed germination rates by breaking the physical dormancy of seeds.

[0076] About 100 mg of wild-type and transgenic RH1 The imbibition rate and germination rate of mature seeds. The results of imbibition rate and germination rate are as follows Figure 3 -B, transgenic RH1 The imbibition rate of mature seeds reached 0.44 and the germination rate reached 0.43. RH1 The gene can break the physical dormancy of Medicago truncatula seeds and increase the seed germination rate.

Claims

1. RH1 Gene, RH1 protein, containing RH1 Gene expression cassette or containing RH1 Use of a recombinant plant expression vector of a gene in regulating the physical dormancy of Medicago truncatula; RH1 The nucleotide sequence of the CDS of the gene is selected from the nucleotide sequence described in (a) or (b) below: (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; The amino acid sequence of the RH1 protein is shown in SEQ ID NO.1: The method of regulating the physical dormancy of Medicago truncatula is to break the physical dormancy of Medicago truncatula seeds.

2. The use according to claim 1, characterized in that The method of breaking the physical dormancy of the Medicago truncatula seeds includes: promoting the Medicago truncatula seeds to absorb water and swell or promoting the Medicago truncatula seeds to germinate.

3. The use according to claim 1 or 2, characterized in that include: improve RH1 The expression amount or level of the gene, or the function or activity of the RH1 protein is enhanced.

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