MsPIF8 protein and its coding gene in regulating the growth of alfalfa under short-day conditions

Abstract

The application discloses application of MsPIF8 protein and a coding gene thereof in regulating growth of alfalfa under short-day conditions. The MsPIF8 protein is any one of the following proteins: a) a protein with an amino acid sequence shown in SEQ ID NO. 2; b) a fusion protein obtained by connecting a label to an N terminal and / or a C terminal of the protein shown in SEQ ID NO. 2; c) a protein with the same function obtained by substitution, deletion and / or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO. 2; and d) a protein with 75% or above identity with the amino acid sequence shown in SEQ ID NO. 2 and with the same function. Experiments prove that overexpression of the MsPIF8 gene in alfalfa can increase plant height and stem node number of the alfalfa under short-day conditions. The application has important significance for improving yield of the alfalfa.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of the MsPIF8 protein and its encoding gene in regulating the growth of alfalfa under short-day conditions. Background Technology

[0002] Alfalfa (Medicago sativa L.) is an excellent perennial leguminous forage grass, high in crude protein, rich in nutrients, and highly palatable, earning it the title of "King of Forages." It plays a vital role in agricultural and livestock production. With the continuous improvement of living standards, the demand for livestock products is also constantly increasing. Therefore, cultivating superior alfalfa varieties and continuously increasing alfalfa production is of practical significance for promoting the development of animal husbandry.

[0003] Light provides energy to plants and also acts as an important environmental signal, regulating plant growth and development, such as photomorphogenesis, dark morphogenesis, and shading response. Changes in light quality and photoperiod can affect plant growth processes. Alfalfa is a long-day plant; when the photoperiod changes in autumn, alfalfa undergoes dormancy, leading to alterations in its growth and morphology. This affects its cold resistance and overwintering ability, ultimately reducing alfalfa yield and quality, which is detrimental to livestock development. Summary of the Invention

[0004] The purpose of this invention is to provide the MsPIF8 protein, its encoding gene, and its applications.

[0005] Firstly, this invention claims protection for a protein.

[0006] The protein claimed in this invention is derived from alfalfa and is named MsPIF8. The MsPIF8 protein is as shown in a) or b) or c) or d) below:

[0007] a) The amino acid sequence is that of the protein shown in SEQ ID NO.2;

[0008] b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2;

[0009] c) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0010] d) Proteins that have 75% or more of the same amino acid sequence as shown in SEQ ID NO.2 and have the same function.

[0011] In the protein described in b) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] In the protein described in c) above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0013] In the protein described in d) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the identity of a pair of amino acid sequences, the identity value (%) can be obtained. The identity includes amino acid sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO.2 of this invention.

[0014] The proteins described in a), b), c), or d) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0015] Secondly, the present invention claims protection for biological materials related to the aforementioned MsPIF8 protein.

[0016] The biological material related to the aforementioned MsPIF8 protein claimed in this invention is any one of the following A1) to A8):

[0017] A1) The nucleic acid molecule encoding the MsPIF8 protein described above;

[0018] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0019] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0020] A4) A recombinant vector containing the expression cassette described in A2);

[0021] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0022] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0023] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0024] A8) Recombinant microorganisms containing the recombinant vector described in A4).

[0025] Furthermore, the nucleic acid molecule described in A1) is a gene as shown in 1) or 2) below:

[0026] 1) Its coding sequence is the DNA molecule shown in SEQ ID NO.1;

[0027] 2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the MsPIF8 protein described above.

[0028] Those skilled in the art can readily mutate the nucleotide sequence encoding the MsPIF8 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the MsPIF8 nucleotide sequence isolated according to this invention, as long as they encode the MsPIF8 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0029] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0030] The expression cassette refers to DNA capable of expressing the MsPIF8 protein in host cells. This DNA may include not only promoters that initiate MsPIF8 transcription but also terminators that terminate it. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters; and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0031] The vector can be a plasmid, granule, bacteriophage, or viral vector. The recombinant vector can be a vector containing the MsPIF8 gene expression cassette constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3′ untranslated region of the foreign gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3′ end of the mRNA precursor. Similar functions exist in the untranslated regions transcribed at the 3′ end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nosine synthase gene) and plant genes (such as the soybean storage protein gene). When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signal and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed. This can involve adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic marker genes (such as the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methotrexate, and the EPSPS gene for resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0032] In the above applications, the microorganism can be yeast, bacteria, algae, or fungi, such as Agrobacterium. The recombinant microorganism refers to a recombinant microorganism whose function has been altered by manipulating and modifying the genes of the target microorganism. For example, the recombinant microorganism obtained after introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0033] Thirdly, the present invention claims protection for new uses of the aforementioned MsPIF8 protein or the aforementioned biological material.

[0034] This invention claims protection for the use of the above-described MsPIF8 protein or the above-described biological material in any of the following M1)-M10):

[0035] M1) regulates plant growth;

[0036] M2) to prepare products that regulate plant growth;

[0037] M3) regulates plant height;

[0038] M4) to prepare products that regulate plant height;

[0039] M5) regulates the number of stem nodes in plants;

[0040] M6) is used to prepare products that regulate the number of stem nodes in plants;

[0041] M7) Develop transgenic plants with increased plant height and / or increased number of stem nodes;

[0042] M8) Products prepared by cultivating transgenic plants with increased plant height and / or increased number of stem nodes;

[0043] M9) Plant breeding;

[0044] M10) is used to prepare plant breeding products.

[0045] In the above applications, the regulation of plant growth refers to promoting plant growth under short-day conditions.

[0046] The regulation of plant height refers to increasing plant height under short-day conditions.

[0047] The regulation of plant stem node number refers to increasing the number of plant stem nodes under short-day conditions.

[0048] The indicators for plant breeding are plant height or number of stem nodes (plant height or number of stem nodes under short-day conditions).

[0049] The purpose of the plant breeding is to cultivate plant varieties with increased plant height and / or increased number of stem nodes (cultivating plant varieties with increased plant height and / or increased number of stem nodes under short-day conditions).

[0050] Fourthly, the present invention claims protection for a method of cultivating transgenic plants with increased plant height and / or increased number of stem nodes, or a method of increasing plant height and / or number of stem nodes.

[0051] The method for cultivating transgenic plants with increased plant height and / or increased number of stem nodes claimed in this invention includes the following steps: increasing the expression level and / or activity of the above-mentioned MsPIF8 protein in the target plant to obtain a transgenic plant; wherein the plant height and / or number of stem nodes of the transgenic plant is higher than that of the target plant.

[0052] The method for increasing plant height and / or number of stem nodes claimed in this invention includes the following steps: increasing the expression level and / or activity of MsPIF8 protein in the target plant to increase plant height and / or number of stem nodes.

[0053] The above-mentioned method for cultivating transgenic plants with increased plant height and / or increased number of stem nodes is a method for cultivating transgenic plants with increased plant height and / or increased number of stem nodes under short-day conditions.

[0054] The methods described above for increasing plant height and / or number of stem nodes are methods for increasing plant height and / or number of stem nodes under short-day conditions.

[0055] In the above method, the method for increasing the expression level and / or activity of the MsPIF8 protein in the target plant is to overexpress the MsPIF8 protein in the target plant.

[0056] Furthermore, the overexpression method involves introducing the gene encoding the MsPIF8 protein into the target plant.

[0057] Furthermore, the gene encoding the MsPIF8 protein is the DNA molecule shown in SEQ ID NO.1.

[0058] In any of the above applications or methods, short daylight refers to sunshine duration of less than (excluding equal to) 12 hours.

[0059] In some implementations, the short daylight period is defined as 8 hours of sunshine and 16 hours of darkness.

[0060] In any of the above-described applications or methods, the transgenic plant includes not only the first-generation transgenic plant obtained by transforming the MsPIF8 gene into the target plant, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plant includes seeds, callus tissue, intact plants, and cells.

[0061] In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0062] Furthermore, the dicotyledonous plant is a leguminous plant.

[0063] Furthermore, the legume in question is a plant of the genus *Alfalfa*.

[0064] Furthermore, the clover species mentioned are alfalfa.

[0065] In some implementations, the alfalfa is clover ('Zhongmu No. 1').

[0066] This invention constructs a transgenic alfalfa with enhanced MsPIF8 gene expression. Experiments demonstrate that, compared to wild-type alfalfa, the transgenic alfalfa with enhanced MsPIF8 gene expression exhibits significantly increased plant height and number of stem nodes under short-day conditions. This invention is of significant importance for improving alfalfa yield. Attached Figure Description

[0067] Figure 1 Identification and phenotype of MsPIF8 transgenic alfalfa. a: Schematic diagram of the pMDC83-MsPIF8 vector. b: RT-qPCR detection of MsPIF8 gene expression in MsPIF8 transgenic alfalfa. MsActin was used as an internal reference gene for alfalfa. c: Phenotypes of alfalfa before and after long-day and short-day treatments. First row: Phenotypes of alfalfa before long-day and short-day treatments; Second row: Phenotypes of alfalfa after 4 weeks of long-day and short-day treatments. Scale bar: 5cm. d: Plant height and number of stem nodes of alfalfa after 4 weeks of long-day and short-day treatments. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0070] The alfalfa 'Zhongmu No. 1' in the following examples is described in the literature "Shi K, Liu J, Liang H, Dong H, Zhang J, Wei Y, Zhou L, Wang S, Zhu J, Cao M, Jones CS, Ma D, Wang Z. An alfalfaMYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation. J Integr PlantBiol. 2024 Apr; 66(4):683-699."

[0071] Example 1: Cloning and Obtaining the MsPIF8 Gene

[0072] 1. Extraction of RNA from alfalfa and synthesis of cDNA

[0073] Total RNA was extracted from 'Zhongmu No. 1' alfalfa seedlings using the TRIzoL (Invitrogen, USA) method, and the RNA was analyzed using a reverse transcription kit from TransGen Biotech. The One-Step gDNA Removal and cDNA Synthesis SuperMix (catalog number: AT311) method involves reverse transcription of extracted RNA to obtain cDNA.

[0074] 2. Using the cDNA obtained in step 1 as a template, amplification was performed using primers T-MsPIF8-F / T-MsPIF8-R to obtain the PCR amplification product, which is the coding region sequence of the MsPIF8 gene. The primer sequences are as follows:

[0075] T-MsPIF8-F: 5'-ATGAGTCAGTGTGTTCCTCGTTGGGA-3';

[0076] T-MsPIF8-R: 5'-TCAATTCTTTGAACCTGGTGCTGGAG-3'.

[0077] The PCR amplification system was as follows: 10 μL of 2*KOD one, 0.6 μL of T-MsPIF8-F (10 μmol / L), 0.6 μL of T-MsPIF8-R (10 μmol / L), 1 μL of alfalfa cDNA, and ddH2O was added to bring the final volume to 20 μL.

[0078] The PCR reaction program was as follows: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 68℃ extension for 20 s, 40 cycles; 12℃ for 5 min.

[0079] 3. The PCR amplification product obtained in step 2 was ligated into the T vector and sequenced. The plasmid with correct sequencing was named T-MsPIF8. Sequencing results showed that the coding region sequence of the MsPIF8 gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2, consisting of the MsPIF8 protein, which is composed of 492 amino acid residues.

[0080] Example 2: Preparation of MsPIF8 transgenic alfalfa

[0081] I. Construction of the MsPIF8 overexpression vector pMDC83-MsPIF8

[0082] A plant overexpression vector for MsPIF8 was constructed using Gateway Technology. The specific steps are as follows:

[0083] 1. Based on the CDS sequence of MsPIF8, design primer pairs pDONR207-MsPIF8-F / pDONR207-MsPIF8-R. The primer sequences are as follows:

[0084] pDONR207-MsPIF8-F:

[0085] 5'-ggggacaagtttgtacaaaaaagcaggcttcATGAGTCAGTGTGTTCCTCGTTG-3';

[0086] pDONR207-MsPIF8-R:

[0087] 5'-ggggaccactttgtacaagaaagctgggtcATTCTTTGAACCTGGTGCTGG-3'.

[0088] 2. Using the T-MsPIF8 plasmid constructed in Example 1 as a template, gene amplification was performed using the primer pair in step 1 to obtain the PCR product, which was named PCR-pDONR207-MsPIF8.

[0089] The PCR amplification system consisted of: 10 μL of 2*KOD one, 0.6 μL of p207-MsPIF8-F (10 μmol / L), 0.6 μL of p207-MsPIF8-R (10 μmol / L), and 0.5 μL of T-MsPIF8 plasmid, with ddH2O added to a final volume of 20 μL. The PCR reaction program was as follows: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 56℃ annealing for 5 s, 68℃ extension for 20 s, for 40 cycles; and storage at 12℃ for 5 min.

[0090] 3. After the PCR-pDONR207-MsPIF8 in step 2 is recovered from the gel, it is ligated to the pDONR207 vector using Gateway BP Clonase II (Thermo Fisher). The plasmid with correct sequencing results is named pDONR207-MsPIF8. The Gateway BP Clonase II ligation system is as follows: 2 μL PCR-pDONR207-MsPIF8, 0.5 μL pDONR207 vector, and 0.5 μL BP enzyme.

[0091] 4. Using Gateway LR Clonase II (Thermo Fisher), the pDONR207-MsPIF8 obtained in step 3 was ligated to the pMDC83 vector (NovoPro, V013531). The correctly sequenced plasmid was named pMDC83-MsPIF8. A schematic diagram of the pMDC83-MsPIF8 plasmid is shown below. Figure 1 As shown in a.

[0092] The Gateway LP Clonase II ligation system is as follows: 0.5 μL of pDONR207-MsPIF8 plasmid, 2 μL of pMDC83 vector, and 0.5 μL of BP enzyme.

[0093] II. Obtaining and Identifying MsPIF8 Transgenic Alfalfa

[0094] 1. The recombinant plasmid pMDC83-MsPIF8 constructed in step one was transformed into competent Agrobacterium tumefaciens cells EHA105. After identification, Agrobacterium tumefaciens cells carrying the pMDC83-MsPIF8 recombinant plasmid were obtained. Then, the recombinant plasmid pMDC83-MsPIF8 was transformed into leaves of alfalfa 'Zhongmu No. 1' using Agrobacterium-mediated transformation. Transgenic seedlings were obtained through tissue culture.

[0095] 2. Genomic DNA was extracted from transgenic seedlings using the CTAB method. Primer pair 35S and pMDC83-MsPIF8-R were used to detect whether the pMDC83-MsPIF8 vector was inserted into the plant. Seedlings containing the correct target band (about 1500bp) were considered positive.

[0096] 3. Leaves from the same parts of positive seedlings and alfalfa 'Zhongmu No. 1' were collected, and RNA was extracted using the Trizol method, followed by reverse transcription to obtain cDNA. Using primer pairs qMsPIF8-F / qMsPIF8-R and MsActin-F / q-MsActin-R, real-time quantitative PCR was performed with the extracted cDNA as a template to detect MsPIF8 expression levels and screen for transgenic alfalfa with elevated MsPIF8 expression. Primer sequences are as follows:

[0097] 35S: 5'-CGCAAGACCTCCTCTATATAAG-3';

[0098] qMsPIF8-F: 5'-GGCGGTGGATGCGTTGGTTC-3';

[0099] qMsPIF8-R: 5'-CCTCCTTCGTCCTGCGTTGC-3';

[0100] q-MsActin-F: 5'-CAAAAGATGGCAGATGCTGAGGAT-3';

[0101] q-MsActin-R: 5'-CATGACACCAGTATGACGAGGTCG-3'.

[0102] The results are as follows Figure 1 As shown in b, the results indicated that the expression level of MsPIF8 in the MsPIF8 transgenic alfalfa lines OE-MsPIF8-1, OE-MsPIF8-3, and OE-MsPIF8-10 was significantly higher than that in wild-type alfalfa. The MsPIF8 transgenic alfalfa lines OE-MsPIF8-1, OE-MsPIF8-3, and OE-MsPIF8-10 were selected for the following biological function analysis.

[0103] Example 3: Phenotypic Analysis of MsPIF8 Transgenic Alfalfa

[0104] Test materials: wild-type alfalfa 'Zhongmu No. 1' (CK), MsPIF8 transgenic alfalfa lines OE-MsPIF8-1, OE-MsPIF8-3, and OE-MsPIF8-10.

[0105] The experimental method is as follows:

[0106] (1) Propagation of test materials by cuttings: After 4 weeks of growth, select seedlings with uniform growth and transfer them to nutrient soil for further cultivation. After 4 weeks of growth, cut the seedlings to a stubble length of 6cm to obtain alfalfa plants.

[0107] (2) The alfalfa plants from step (1) were placed in long-day (light:dark = 16h:8h) and short-day (light:dark = 8h:16h) incubators, respectively, at a temperature of 25℃ and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 After 4 weeks of growth, the phenotype was observed, and the plant height and number of stem nodes were counted.

[0108] The results are as follows Figure 1 c and Figure 1 As shown in d, the results showed that after 4 weeks of treatment under long-day conditions, MsPIF8 transgenic alfalfa did not differ from wild-type alfalfa (CK) in plant height and number of stem nodes; however, after 4 weeks of treatment under short-day conditions, MsPIF8 transgenic alfalfa showed a significant increase in both plant height and number of stem nodes compared to wild-type alfalfa (CK). Under short-day conditions, the plant heights of wild-type alfalfa (CK), MsPIF8 transgenic alfalfa lines OE-MsPIF8-1, OE-MsPIF8-3, and OE-MsPIF8-10 were 20.3 cm, 24.2 cm, 24.7 cm, and 25.2 cm, respectively. The number of stem nodes in these lines were 6.4, 8, 9, and 8.3, respectively. This indicates that the MsPIF8 gene can regulate plant growth under short-day conditions.

[0109] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a protein or a biological material associated with said protein in any of the following M1)-M6): M1) Increases plant height under short-day conditions; M2) Prepare products that increase plant height under short-day conditions; M3) Increases the number of stem nodes in plants under short-day conditions; M4) Prepare products that increase the number of stem nodes in plants under short-day conditions; M5) Transgenic plants with increased plant height and / or increased number of stem nodes were cultivated under short-day conditions; M6) Products prepared by cultivating transgenic plants with increased plant height and / or increased number of stem nodes under short-day conditions; The plant in question is alfalfa; The protein is the protein shown in either a) or b) below: a) The amino acid sequence is that of the protein shown in SEQ ID NO.2; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2; The biomaterial is any one of the following A1) to A8): A1) The nucleic acid molecule that encodes the protein; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganisms containing the nucleic acid molecules described in A1); A6) Recombinant microorganisms containing the expression cassette described in A2); A7) Recombinant microorganisms containing the recombinant vector described in A3); A8) Recombinant microorganisms containing the recombinant vector described in A4).

2. The application according to claim 1, characterized in that: A1) The nucleic acid molecule is a gene as shown in 1) or 2) below: 1) Its coding sequence is the DNA molecule shown in SEQ ID NO.1; 2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the protein.

3. A method for cultivating transgenic plants with increased plant height and / or increased number of stem nodes under short-day conditions, comprising the following steps: increasing the expression level of a protein in a target plant to obtain a transgenic plant; wherein the transgenic plant has a higher plant height and / or number of stem nodes under short-day conditions than the target plant; The plant in question is alfalfa; The protein is the protein shown in either a) or b) below: a) The amino acid sequence is that of the protein shown in SEQ ID NO.2; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.

2.

4. A method for increasing plant height and / or number of stem nodes under short-day conditions, comprising the following steps: increasing the expression level of a protein in the target plant to increase plant height and / or number of stem nodes under short-day conditions; The plant in question is alfalfa; The protein is the protein shown in either a) or b) below: a) The amino acid sequence is that of the protein shown in SEQ ID NO.2; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.

2.

5. The method according to claim 3 or 4, characterized in that: The method for increasing the expression level of the protein in the target plant is to overexpress the protein in the target plant.

6. The method according to claim 5, characterized in that: The overexpression method involves introducing the gene encoding the protein into the target plant.

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