MsDUF3700 protein and its encoding gene in regulating plant stress tolerance, growth and yield

Abstract

The application discloses MsDUF3700 protein and an application of a coding gene thereof in regulating plant stress resistance, growth and development and yield. MsDUF3700 The application overexpresses the gene in alfalfa, obtains transgenic alfalfa with overexpressed MsDUF3700, and finds that the gene has a regulating function on aluminum stress, and growth and development (such as plant height and stem diameter) and yield (fresh weight of an aboveground part) of the plant through research on stress resistance, growth and development and yield traits of the transgenic alfalfa with overexpressed MsDUF3700. MsDUF3700 The application fills a blank of no related report of unknown function genes in alfalfa, and provides a theoretical basis and a new method for breeding of a new alfalfa variety with stress resistance and high quality.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the MsDUF3700 protein and its encoding gene in regulating plant stress tolerance, growth and development, and yield. Background Technology

[0002] Aluminum (Al) toxicity is a major factor reducing crop yields in acidic soils. The primary symptom of toxicity is inhibited root growth. 3+ It can disrupt many other functions, including root hair elongation and nutrient absorption, induce oxidative stress, damage the cytoskeleton, and affect intracellular transport, inhibiting root growth and function. This toxicity, in turn, leads to reduced crop yields due to drought and mineral deficiencies.

[0003] alfalfa ( Medicago sativa Alfalfa is the most widely cultivated perennial leguminous forage crop globally, widely used in animal husbandry due to its rich nutritional content, high protein content, and strong adaptability. To meet the growing demand for alfalfa, in addition to increasing alfalfa yield, it is necessary to utilize acidic soils previously considered unsuitable for alfalfa cultivation. In the large red soil regions of southern my country, the strong acidity and high aluminum activity of the soil result in slow alfalfa growth and low yields, hindering large-scale cultivation and severely restricting the development of grassland animal husbandry in the south. Therefore, research on alfalfa aluminum toxicity is of great significance both theoretically and practically. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate plant stress resistance, growth and development and yield.

[0005] To address the aforementioned technical problems, this invention first provides a new use for the MsDUF3700 protein.

[0006] This invention provides the use of MsDUF3700 protein in any of the following 1)-5):

[0007] 1) Regulating plant stress tolerance;

[0008] 2) Regulating plant growth and development;

[0009] 3) Regulating plant yield;

[0010] 4) Cultivate transgenic plants with improved plant height and / or stem diameter and / or yield and / or stress resistance;

[0011] 5) Plant breeding;

[0012] The MsDUF3700 protein is as follows: a1), a2), a3), or a4).

[0013] a1) The amino acid sequence is that of the protein shown in sequence 3;

[0014] a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in sequence 3;

[0015] a3) Proteins related to plant stress resistance and / or growth and / or yield obtained by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence shown in Sequence 3.

[0016] a4) A protein that has 80% amino acid sequence identity with the one shown in sequence 3, is derived from alfalfa, and is associated with plant stress resistance and / or growth and / or yield.

[0017] In the protein described in a2) 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 includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0018] In the protein described in a3) 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.

[0019] In the protein described in a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using an identity search site 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 an identity search on a pair of amino acid sequences, the identity value (%) can be obtained. The identity includes amino acid sequences that have 80% or higher, or 85% or higher, or 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 identity with the amino acid sequence shown in Sequence 3 of this invention.

[0020] The proteins described in a1)-a4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0021] To address the aforementioned technical problems, this invention also provides new uses for biomaterials related to the MsDUF3700 protein.

[0022] This invention provides the use of biomaterials related to the above-mentioned MsDUF3700 protein in any of the following 1)-5):

[0023] 1) Regulating plant stress tolerance;

[0024] 2) Regulating plant growth and development;

[0025] 3) Regulating plant yield;

[0026] 4) Cultivate transgenic plants with improved plant height and / or stem diameter and / or yield and / or stress resistance;

[0027] 5) Plant breeding;

[0028] The biomaterial is any one of the following A1) to A8):

[0029] A1) Nucleic acid molecules encoding the MsDUF3700 protein;

[0030] A2) An expression cassette containing the nucleic acid molecule described in A1);

[0031] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0032] A4) A recombinant vector containing the expression cassette described in A2);

[0033] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0034] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0035] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0036] A8) Recombinant microorganisms containing the recombinant vector described in A4).

[0037] In the above applications, the nucleic acid molecule described in A1) is the gene shown in B1) or B2) below:

[0038] B1) The DNA molecule shown in sequence 1 or sequence 2;

[0039] B2) has 75% or more identity with the nucleotide sequence defined by B1) and is a DNA molecule encoding the MsDUF3700 protein.

[0040] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0041] Those skilled in the art can readily mutate the nucleotide sequence encoding the MsDUF3700 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence encoding the MsDUF3700 protein, provided they encode the MsDUF3700 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0042] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in Sequence 3 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.

[0043] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0044] In the above applications, the expression cassette refers to DNA capable of expressing the MsDUF3700 protein in host cells, and this DNA may include not only the promoter but also... MsDUF3700 The promoter of transcription may also include a terminator. MsDUF3700 Transcription terminators. Further, 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 rbcSE9 terminator, and carmine and octopine synthase terminator.

[0045] In the above applications, the vector refers to a vector that can carry the above nucleic acid molecules into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0046] The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecule with the vector. Existing plant expression vectors can be used to construct recombinant DNA molecules containing the aforementioned recombinant DNA. MsDUF3700 Recombinant vectors for gene expression cassettes. These 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 vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment 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, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). NosThe untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. 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 sources of the translation control signals and start codons are wide-ranging; they 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, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer 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, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0047] In the above applications, the microorganisms can be yeast, bacteria, algae, or fungi. Specifically, the bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0048] The recombinant microorganisms refer to those obtained by manipulating and modifying the genes of a target microorganism, resulting in a functional change. For example, recombinant microorganisms 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 may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0049] In the above applications, the regulation of plant stress tolerance refers to improving plant stress tolerance, that is, when the content and / or activity of MsDUF3700 protein in plants increases, the plant's stress tolerance increases.

[0050] Furthermore, the improvement of plant stress resistance refers to improving the plant's aluminum tolerance.

[0051] Furthermore, the improvement of plant aluminum tolerance refers to improving the aluminum tolerance of the aboveground parts of the plant.

[0052] In one specific embodiment of the present invention, the regulation of plant stress tolerance is manifested as follows: when the plant's... MsDUF3700 When gene expression levels are increased, under aluminum stress, the relative stem elongation of plants increases, the fresh weight of aboveground parts increases, and the stem aluminum content decreases.

[0053] In the above applications, the regulation of plant growth and development refers to promoting plant growth and development, that is, when the content and / or activity of MsDUF3700 protein in plants are increased, the plants grow and develop better.

[0054] Furthermore, the promotion of plant growth and development refers to increasing plant height and / or stem diameter.

[0055] In one specific embodiment of the present invention, the regulation of plant growth and development is manifested as: when the plant's... MsDUF3700 When gene expression levels increase, plant height and stem diameter increase.

[0056] In the above applications, the regulation of plant yield refers to increasing plant yield, that is, when the content and / or activity of MsDUF3700 protein in the plant increases, the plant yield increases.

[0057] Furthermore, the increase in plant yield refers to increasing the fresh weight of the above-ground parts of the plant.

[0058] In one specific embodiment of the present invention, the regulation of plant yield is manifested as: when the plant's... MsDUF3700 When gene expression levels increase, the fresh weight of the aboveground parts of plants increases.

[0059] In the above applications, the purpose of plant breeding is to cultivate aluminum-resistant and / or high-yielding plant varieties.

[0060] To address the aforementioned technical problems, the present invention ultimately provides a method for cultivating transgenic plants with increased plant height and / or stem diameter and / or yield and / or stress tolerance.

[0061] The method for cultivating transgenic plants with improved plant height and / or stem diameter and / or yield and / or stress tolerance provided by the present invention includes the following steps: increasing the content and / or activity of the above-mentioned MsDUF3700 protein in the recipient plant to obtain transgenic plants; wherein the plant height and / or stem diameter and / or yield and / or stress tolerance of the transgenic plants are higher than those of the recipient plants.

[0062] In the above method, the resistance to adverse conditions refers to aluminum resistance.

[0063] Furthermore, the aluminum tolerance refers to the aluminum tolerance of the aboveground parts.

[0064] Furthermore, the stress tolerance of the transgenic plant is higher than that of the target plant, as manifested in any one of the following M1)-M3):

[0065] M1) Under aluminum stress, the relative stem elongation of the transgenic plant was higher than that of the recipient plant;

[0066] M2) Under aluminum stress conditions, the aboveground fresh weight of the transgenic plant was higher than that of the recipient plant;

[0067] M3) Under aluminum stress conditions, the stem aluminum content of the transgenic plant is lower than that of the recipient plant.

[0068] Furthermore, the aluminum stress is AlCl3 stress.

[0069] In a specific embodiment of the present invention, the AlCl3 stress is 200µM AlCl3.

[0070] In the above method, the method for increasing the content and / or activity of the MsDUF3700 protein in the recipient plant is to overexpress the MsDUF3700 protein in the recipient plant.

[0071] Furthermore, the overexpression method involves introducing the gene encoding the MsDUF3700 protein into a recipient plant.

[0072] Furthermore, the nucleotide sequence of the gene encoding the MsDUF3700 protein is shown in Sequence 2.

[0073] In the above method, the transgenic plant is understood to include not only the transgenic plant... MsDUF3700 The first generation of transgenic plants obtained by transforming a recipient plant with a gene, including its progeny. For transgenic plants, the gene can be propagated within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus tissue, whole plants, and cells.

[0074] In any of the above applications or methods, the target plant is a monocotyledonous plant or a dicotyledonous plant, and the dicotyledonous plant may specifically be a legume; the legume may be an alfalfa plant; the alfalfa plant may be alfalfa; the alfalfa may be alfalfa (such as alfalfa variety 1).

[0075] The beneficial effects of this invention are as follows: This invention is the first to... MsDUF3700 The gene was overexpressed in alfalfa, resulting in overexpression. MsDUF3700 Transgenic alfalfa, and through overexpression MsDUF3700 Studies on the stress tolerance, growth and development, and yield traits of transgenic alfalfa revealed the following: MsDUF3700The gene has regulatory functions on aluminum stress, as well as plant growth and development (such as plant height and stem diameter) and yield (fresh weight of aboveground parts). This invention fills the gap of no reports on unknown functional genes in alfalfa, and provides a theoretical basis and new methods for breeding new varieties of alfalfa that combine stress resistance and high quality. Attached Figure Description

[0076] Figure 1 For transgenic plants MsDUF3700 Image showing the results of gene PCR verification.

[0077] Figure 2 For MsDUF3700 alfalfa MsDUF3700 Gene expression level detection results. Among them, WT represents wild-type alfalfa, and 0E1-7 represent different transgenic MsDUF3700 alfalfa.

[0078] Figure 3 The results show the phenotypic, growth, development, and yield traits of wild-type alfalfa and MsDUF3700 trans alfalfa. WT represents wild-type alfalfa, and OE-1, OE-5, and OE-6 represent different trans MsDUF3700 alfalfa varieties.

[0079] Figure 4 Phenotypes of wild-type alfalfa and MsDUF3700 trans alfalfa are shown. WT represents wild-type alfalfa, and OE-1, OE-5, and OE-6 represent different MsDUF3700 trans plants.

[0080] Figure 5 The results show the regeneration rate analysis of wild-type alfalfa and MsDUF3700 transgenic alfalfa. WT represents wild-type alfalfa, and OE-1, OE-5, and OE-6 represent different transgenic MsDUF3700 alfalfa varieties.

[0081] Figure 6 The phenotypes of wild-type alfalfa and MsDUF3700 trans alfalfa under aluminum stress are shown. WT represents wild-type alfalfa, and OE-1, OE-5, and OE-6 represent different trans MsDUF3700 alfalfa varieties.

[0082] Figure 7 The results show the stress tolerance analysis of wild-type alfalfa and MsDUF3700 trans alfalfa under aluminum stress. WT represents wild-type alfalfa, and OE-1, OE-5, and OE-6 represent different trans MsDUF3700 alfalfa varieties. Detailed Implementation

[0083] 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.

[0084] 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.

[0085] The alfalfa variety Zhongmu No. 1 in the following examples is described in the literature “Long RC, Li MN, Kang JM, Zhang TJ, Sun Y, Yang QC. Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs from roots of Medicago sativa and Medicagotruncatula. Physiol Plant. 2015 May;154(1):13-27.”

[0086] The pEarleyGate203 vector used in the following examples is described in the literature “Lu, Y., et al., Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunity. Nature communications, 2020. 11(1): p. 6234-6234.”

[0087] The culture media and their formulations in the following examples are as follows:

[0088] The solvent for YEP liquid culture medium is water, and the solutes and their concentrations are as follows: tryptone 10 g / L, yeast extract 10 g / L, NaCl 5 g / L, pH 7.0.

[0089] The solvent for the S solid medium is water, and the solutes and their concentrations are as follows: MS basal medium (M519, Phyto Technology) 4.43 g / L, sucrose 30 g / L, agar 8 g / L, pH 5.8.

[0090] The solvent for the resuspension was water, and the solutes and their concentrations were as follows: 50 mL / L of 20×N6 bulk mother liquor, 5 mL / L of 200×Ms micro mother liquor, 5 mL / L of 200×iron salt mother liquor, 1 mL / L of 1000×organic components, 3 mg / L of 2,4-D, 0.05 mg / L of KT, 0.6 g / L of MES, 30 g / L of sucrose, and pH 5.4.

[0091] The solvent for the large amount of 20×N6 mother liquor was water, and the solutes and their concentrations were as follows: KNO3 56.6 g / L, (NH4)2SO4 9.2 g / L, CaCl2·2H2O 3.32 g / L, MgSO4·7H2O 3.7 g / L, and KH2PO4 8 g / L.

[0092] The solvent for the 200×Ms trace mother liquor was water, and the solutes and their concentrations were as follows: MnSO4·4H2O 4.46 g / L, ZnSO4·7H2O 1.72 g / L, CuSO4·5H2O 0.005 g / L, Na2MoO4·2H2O 0.05 g / L, CoCl2·6H2O 0.005 g / L, KI 0.166 g / L, and H3BO4 1.24 g / L.

[0093] The solvent for the 200× iron salt mother liquor was water, and the solutes and their concentrations were: FeSO4·7H2O 5.56 g / L and Na2EDTA 7.46 g / L, respectively.

[0094] The solvent for the 1000× organic components is water, and the solutes and their concentrations are: VB1 9.9 g / L, VB6 9.5 g / L, and nicotinic acid 4.5 g / L.

[0095] The solvent for the co-culture medium was water, and the solutes and their concentrations were as follows: 50 mL / L of 20×N6 large volume stock solution, 5 mL / L of 200×Ms micro volume stock solution, 5 mL / L of 200× iron salt stock solution, 1 mL / L of 1000× organic components, 3 mg / L of 2,4-D, 0.05 mg / L of KT, 0.6 g / L of MES, 30 g / L of sucrose, 8 g / L of agar, and pH 5.4.

[0096] The solvent for the callus induction medium was water, and the solutes and their concentrations were as follows: 50 mL / L of 20×N6 large volume stock solution, 5 mL / L of 200×Ms micro volume stock solution, 5 mL / L of 200× iron salt stock solution, 1 mL / L of 1000× organic components, 2 mg / L of 2,4-D, 0.05 mg / L of KT, 0.5 g / L of acid-hydrolyzed casein, 30 g / L of sucrose, 200 mg / L of termethin, 5 mg / L of hygromycin, and pH 5.8.

[0097] The solvent for the differentiation medium was water, and the solutes and their concentrations were as follows: 50 mL / L of 20×N6 large volume stock solution, 5 mL / L of 200×Ms micro volume stock solution, 5 mL / L of 200× iron salt stock solution, 1 mL / L of 1000× organic components, 0.6 mg / L of KT, 0.5 g / L of acid-hydrolyzed casein, 20 g / L of sucrose, 200 mg / L of termethin, 3 mg / L of hygromycin, 8 g / L of agar, and pH 5.8.

[0098] The rooting medium was water as the solvent, and the solutes and their concentrations were as follows: MS basal medium 4.43 g / L, sucrose 20 g / L, agar 8 g / L, termethin 200 mg / L, hygromycin 2 mg / L, pH 5.8.

[0099] Example 1: Preparation of MsDUF3700 alfalfa

[0100] I. Construction of recombinant plasmid pEarleyGate203-DUF3700

[0101] The DNA fragment between the attR1 and attR2 sites in the pEarleyGate203 vector was replaced with the DNA molecule shown in Sequence 2 using a gateway approach, while keeping the other sequences of the pEarleyGate203 vector unchanged, resulting in the recombinant plasmid pEarleyGate203-DUF3700. The recombinant plasmid pEarleyGate203-DUF3700 expresses the MsDUF3700 protein, the amino acid sequence of which is shown in Sequence 3.

[0102] II. Construction of recombinant Agrobacterium pEarleyGate203-DUF3700 / EHA105

[0103] The recombinant plasmid pEarleyGate203-DUF3700 constructed in step one was introduced into Agrobacterium EHA105. After identification, the recombinant Agrobacterium pEarleyGate203-DUF3700 / EHA105 was obtained. The specific steps are as follows:

[0104] 1. Take out the Agrobacterium EHA105 competent cells stored at -80℃, thaw them on ice, gently add the recombinant plasmid pEarleyGate203-DUF3700 to the center of the freshly thawed competent cells, tap them 3-5 times, incubate on ice for 30 minutes, flash freeze in liquid nitrogen for 1 minute, heat shock the competent cells in a water bath at 37℃ for 5 minutes, and then quickly transfer them to ice and let them stand for 2 minutes.

[0105] 2. Add 500µL of YEP liquid medium to the transformed competent cells, invert to mix, and place on a shaker at 28℃ for 2-4 h with shaking at 220rpm.

[0106] 3. Spread 200µL of bacterial culture onto a YEP plate containing 100mg / L kanamycin and 50mg / L rifampin. Incubate at 28℃ in the dark for 2 days. Pick a single colony for colony PCR detection. If the result is positive, it indicates that the recombinant plasmid pEarleyGate203-DUF3700 has been successfully introduced into Agrobacterium. The single clone with the positive result is named recombinant Agrobacterium pEarleyGate203-DUF3700 / EHA105.

[0107] III. Genetic Transformation of Alfalfa

[0108] Using the recombinant Agrobacterium pEarleyGate203-DUF3700 / EHA105 constructed in step two, genetic transformation was performed on alfalfa variety Zhongmu No. 1 as the recipient material to obtain transgenic alfalfa. The specific steps are as follows:

[0109] 1. Take the third healthy compound leaf from the top of alfalfa variety "Zhongmu No. 1" that has grown for 4 to 6 weeks and sterilize it in a 50mL centrifuge tube. Pour in 40mL of 70% ethanol and invert the tube 3-5 times. Pour out the ethanol and add 40mL of 3% NaClO disinfectant solution. Treat the leaf for 10-15 minutes, depending on its condition. Make wounds on the sterilized leaf with a blade and sonicate at 70Hz for 1 minute to enlarge the wounds, thus obtaining the leaf to be infected.

[0110] 2. Inoculate recombinant Agrobacterium pEarleyGate203-DUF3700 / EHA105 into 5 mL of YEP liquid medium containing 50 mg / L Rif and the corresponding vector resistance, and culture in a constant temperature shaker at 28°C and 220 rpm until the bacterial culture reaches OD. 600nm The bacterial culture was prepared by centrifugation at 6000 rpm for 12 min at room temperature, followed by discarding the supernatant and collecting the bacterial cells. 0.1 mM AS was added to the resuspension as the bacterial infection solution. After resuspending the bacterial cells in the infection solution, the cells were placed in the leaf to be infected and incubated for 20 min to obtain the infected leaf.

[0111] 3. Pour off the bacterial solution and use sterile filter paper to absorb the excess bacterial solution on the infected leaves. Then, place the adaxial end of the infected leaves onto the co-culture medium and treat it at 25°C in the dark for 2 days to allow the infected leaves and Agrobacterium to grow together until Agrobacterium grows around the leaves, thus obtaining the co-cultured leaves.

[0112] 4. After co-culturing, the leaves are transferred to callus induction medium after removing the symbiotic Agrobacterium with sterile filter paper. They are subcultured to a new medium every 2-3 weeks, and new callus tissue grows after 6-8 weeks.

[0113] 5. Transfer the newly grown callus tissue to a differentiation medium and expose it to light. Transfer it to a new medium every 2-3 weeks until seedlings differentiate.

[0114] 6. Transfer the seedlings that have differentiated into single compound leaves and stems to a rooting medium until they have rooted and grown into complete seedlings.

[0115] 7. Remove the seedlings from the culture medium, wash the roots, and transplant them into vermiculite. Cover each seedling with a glass cover to maintain humidity and place it in an incubator. Once the seedlings have survived for a week, the glass covers can be removed.

[0116] IV. Identification of Transgenic Plants

[0117] 1. Genomic DNA was extracted from the transgenic plants and verified by PCR using DUF3700-F / DUF3700-R primers, yielding transgenic positive plants. The primer sequences are as follows:

[0118] DUF3700-F: 5'-ATGTTGGGGATATTCAGTAG-3';

[0119] DUF3700-R: 3'-CTACTCTGTGGCTGCAAGAA-5'.

[0120] The results of agarose gel electrophoresis of PCR amplification products are as follows: Figure 1 As shown.

[0121] 2. Approximately 100 mg of leaves from transgenic positive plants were selected, ground in liquid nitrogen, and total RNA was extracted using the Trizol method. Agarose gel electrophoresis confirmed the extraction of high-quality, intact 28S and 18S RNA. RNA concentration and purity were determined using Nanodrop 2000, and OD... 260 / OD 280 All are between 1.8 and 2.0, OD 260 / OD 230 All values ​​>2.0, indicating that the obtained RNA is of good quality and can be used for the synthesis of the first strand of cDNA.

[0122] 3. Using 1µg of the above RNA sample as a template, cDNA was synthesized using the TaKaRa PrimeScript RT reagent Kit for reverse transcription. The cDNA was then used as a template for qRT-PCR analysis, with actin as an internal control gene for detection. MsDUF3700Gene expression levels in different plants. Primer sequences are as follows:

[0123] MsDUF3700-F: 5'-TTGGGACCAGACAATGAAA-3';

[0124] MsDUF3700-R: 3'-GAGATCCTTCAAACAAGCAGA-5';

[0125] actin-F: 5'-CAAAAGATGGCAGATGCTGAGGAT-3';

[0126] actin-R: 3'-CATGACACCAGTATGACGAGGTCG-5'.

[0127] The analysis results of qRT-PCR are as follows: Figure 2 As shown, the results indicate that the MsDUF3700 alfalfa lines OE-1 to OE-7 were transformed... MsDUF3700 Gene expression levels were significantly higher than those of wild-type alfalfa (WT). Transgenic MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6 were selected for the following phenotypic and biological function analyses.

[0128] Example 2: Phenotypic and biological function analysis of MsDUF3700 alfalfa

[0129] I. Phenotypic Analysis of MsDUF3700 Alfalfa Transformation

[0130] Test materials: wild-type alfalfa (WT), and MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6.

[0131] Take stem segments of about 3 cm from the same part of the test material. Each stem segment has a new axillary bud. After pruning the stem segments with a downward oblique cut and an upward flat cut, insert them into the soil. After rooting, select 10 seedlings with the same growth rate and grow for 70 days for phenotypic analysis, including plant height, stem diameter, and fresh weight of the above-ground parts.

[0132] The results are as follows Figure 3As shown, the results indicated that the average plant heights of wild-type alfalfa (WT) and the MsDUF3700 trans alfalfa lines OE-1, OE-5, and OE-6 were 30.4 cm, 46.3 cm, 41.2 cm, and 45.2 cm, respectively; the average stem diameters of wild-type alfalfa (WT) and the MsDUF3700 trans alfalfa lines OE-1, OE-5, and OE-6 were 1.24 mm, 1.46 mm, 1.52 mm, and 1.65 mm, respectively; and the average stem diameters of wild-type alfalfa (WT) and the MsDUF3700 trans alfalfa lines OE-1, OE-5, and OE-6 were 1.24 mm, 1.46 mm, 1.52 mm, and 1.65 mm, respectively. The average aboveground biomass of the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 were 2.9 g, 5.8 g, 4.1 g, and 5.6 g, respectively. Compared with wild-type alfalfa, the plant height of the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 increased by 52.3%, 35.5%, and 48.7%, respectively; the stem diameter increased by 17.7%, 22.6%, and 33.1%, respectively; and the aboveground fresh weight increased by 100%, 41.4%, and 93.1%, respectively. Therefore, overexpression of MsDUF3700 can promote alfalfa growth and development and increase alfalfa yield.

[0133] II. Regeneration Rate Analysis of Alfalfa Converted to MsDUF3700

[0134] Test materials: wild-type alfalfa (WT), and MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6.

[0135] Stem segments of approximately 3 cm in length from the same part of the test material were taken, each segment containing a new axillary bud. After pruning the stem segments with a downward slant and an upward flat cut, they were propagated by cuttings. Once rooted, 10 seedlings with uniform growth were selected and, after 70 days of growth, were harvested for regeneration rate analysis. The specific experimental method is as follows: Cuttings were made at a point 10 cm above the ground at the base of each plant. Plant height was measured daily for 30 consecutive days, and phenotypic images were taken every 5 days.

[0136] The results are as follows Figure 4 and Figure 5 As shown, the results indicated that there were significant differences in plant height between wild-type alfalfa (WT) and the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 after mowing. Furthermore, the regeneration rates of the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 were significantly higher than those of wild-type alfalfa (WT). Therefore, overexpression of MsDUF3700 can promote alfalfa growth and development.

[0137] III. Stress Tolerance Analysis of MsDUF3700 Alfalfa

[0138] Test materials: wild-type alfalfa (WT), and MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6.

[0139] Stem segments approximately 3 cm long from the same part of the test material were taken, each segment containing a new axillary bud. After pruning the stem segments with downward and upward cuts, they were inserted into the soil. Six weeks later, cuttings with uniform growth were selected and subjected to aluminum stress treatment. Phenotypic changes were observed after aluminum stress treatment, and relative root elongation, relative stem elongation, above-ground fresh weight, underground fresh weight, root aluminum content, and stem aluminum content were recorded. The specific steps are as follows:

[0140] 1. Aluminum stress treatment

[0141] Select cuttings with uniform above-ground growth, wash the vermiculite from the roots in clean water, and transfer the cuttings to foam floating boards (each floating board contains 3 wild-type alfalfa plants, 3 MsDUF3700 converted alfalfa plants OE-1, 3 MsDUF3700 converted alfalfa plants OE-5, and 3 MsDUF3700 converted alfalfa plants OE-6). Transfer the floating boards into a plastic bucket containing 2L of 1 / 4 Hoagland's nutrient solution (Beijing Coollab Technology Co., Ltd., product number NS1010). The plants are divided into control and experimental groups depending on whether aluminum stress treatment is performed. The 1 / 4 Hoagland's nutrient solution in the experimental group (pH 4.2) contains 200µM AlCl3, while the 1 / 4 Hoagland's nutrient solution in the control group (pH 4.2) does not contain any other reagents. The nutrient solution was changed every other day for a total of 7 days, with 3 replicates per group.

[0142] 2. Measurement of relative root elongation and relative stem elongation

[0143] Before aluminum stress treatment, the root and stem lengths of all seedlings were measured and recorded. After aluminum stress treatment, the root and stem lengths of all seedlings were measured and recorded. Relative root elongation was then calculated using the following formulas: Relative root elongation = Root length after aluminum stress treatment - Root length before aluminum stress treatment; Relative stem elongation was calculated using the following formulas: Relative stem elongation = Stem length after aluminum stress treatment - Stem length before aluminum stress treatment.

[0144] 3. Determination of aluminum ion content

[0145] Fresh samples were dried in an oven at 65℃ for 48 hours, then ground into powder. 0.1g of the dry powder was weighed and placed in a 50mL Erlenmeyer flask. A funnel was placed on top, and 20mL of concentrated nitric acid was added (operation in a fume hood). The Erlenmeyer flask was placed on a hot plate and heated. The nitric acid condensed and refluxed, evaporating slowly. After evaporation was complete, the Erlenmeyer flask was removed and cooled. Another 10mL of concentrated nitric acid was added to continue nitration until the concentrated nitric acid solution became colorless and transparent. After complete nitration, the nitric acid in the Erlenmeyer flask was evaporated to dryness. The Erlenmeyer flask was removed and cooled to room temperature. Diluted nitric acid solution (0.1M HNO3) was added to rinse the Erlenmeyer flask and funnel. The volumetric flask was then brought to a final volume of 50mL. Standard curve solutions were prepared. The aluminum content in the stems and roots was determined using atomic absorption spectrometry or ICP-MS.

[0146] The results are as follows Figure 6 and Figure 7 As shown, the results indicated that after aluminum stress treatment, the relative stem elongation and aboveground fresh weight of MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6 were significantly higher than those of wild-type alfalfa (WT), while the stem aluminum content was significantly lower than that of wild-type alfalfa (WT). Conversely, after aluminum stress treatment, the underground fresh weight and root aluminum content of MsDUF3700 alfalfa lines OE-1, OE-5, and OE-6 were significantly higher than those of wild-type alfalfa (WT), while the relative root elongation was significantly lower than that of wild-type alfalfa (WT). Among them, the average relative stem elongation of wild-type alfalfa (WT) and the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 were 0.8 cm, 2.6 cm, 2.6 cm, and 3.7 cm, respectively; the average aboveground fresh weight of wild-type alfalfa (WT) and the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 were 0.42 g, 0.85 g, 1.02 g, and 0.73 g, respectively; and the average stem aluminum content of wild-type alfalfa (WT) and the MsDUF3700 transgenic alfalfa lines OE-1, OE-5, and OE-6 were 0.29 mg / g, 0.17 mg / g, 0.16 mg / g, and 0.11 mg / g, respectively. Therefore, overexpression of MsDUF3700 can improve the aluminum tolerance of alfalfa aboveground parts.

[0147] In conclusion, overexpression of MsDUF3700 can promote the growth and development of alfalfa, increase alfalfa yield, and enhance stress resistance.

[0148] 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. Use of a MsDUF3700 protein in any one of 1) to 4) below: 1) increasing aluminum tolerance of a plant; 2) increasing plant height and / or stem diameter; 3) increasing yield of a plant; 4) breeding a transgenic plant with increased plant height and / or stem diameter and / or yield and / or aluminum tolerance; said MsDUF3700 protein is a1) or a2): a1) a protein with an amino acid sequence as set forth in SEQ ID NO: 3; a2) a fusion protein with a tag attached to the N-terminus and / or C-terminus of a protein as set forth in SEQ ID NO: 3; said increasing yield of a plant is increasing fresh weight of above-ground part of a plant; said plant is alfalfa.

2. Use of a biological material related to a MsDUF3700 protein in any one of 1) to 4) below: 1) increasing aluminum tolerance of a plant; 2) increasing plant height and / or stem diameter; 3) increasing yield of a plant; 4) breeding a transgenic plant with increased plant height and / or stem diameter and / or yield and / or aluminum tolerance; said biological material is any one of A1) to A8) below: A1) a nucleic acid molecule encoding a MsDUF3700 protein; A2) an expression cassette comprising the nucleic acid molecule of A1); A3) a recombinant vector comprising the nucleic acid molecule of A1); A4) a recombinant vector comprising the expression cassette of A2); A5) a recombinant microorganism comprising the nucleic acid molecule of A1); A6) a recombinant microorganism comprising the expression cassette of A2); A7) a recombinant microorganism comprising the recombinant vector of A3); A8) a recombinant microorganism comprising the recombinant vector of A4); said MsDUF3700 protein is a1) or a2): a1) a protein with an amino acid sequence as set forth in SEQ ID NO: 3; a2) a fusion protein with a tag attached to the N-terminus and / or C-terminus of a protein as set forth in SEQ ID NO: 3; said increasing yield of a plant is increasing fresh weight of above-ground part of a plant; said plant is alfalfa.

3. Use according to claim 2, characterized in that: A1) the nucleic acid molecule is a DNA molecule as set forth in SEQ ID NO:

2.

4. A method for breeding a transgenic plant with increased plant height and / or stem diameter and / or yield and / or aluminum tolerance, comprising the step of increasing the content and / or activity of a MsDUF3700 protein in a recipient plant to obtain the transgenic plant; the transgenic plant has higher plant height and / or stem diameter and / or yield and / or aluminum tolerance than the recipient plant; said MsDUF3700 protein is a1) or a2): a1) a protein with an amino acid sequence as set forth in SEQ ID NO: 3; a2) a fusion protein with a tag attached to the N-terminus and / or C-terminus of a protein as set forth in SEQ ID NO: 3; said yield is fresh weight of above-ground part; said plant is alfalfa; said method for increasing the content and / or activity of a MsDUF3700 protein in a recipient plant is overexpressing a MsDUF3700 protein in the recipient plant.

5. The method of claim 4, wherein: said transgenic plant has higher aluminum tolerance than the recipient plant as manifested in any one of M1) to M3) below: M1) under aluminum stress, the transgenic plant has higher relative stem elongation than the recipient plant; M2) under aluminum stress, the transgenic plant has higher fresh weight of above-ground part than the recipient plant; M3) the transgenic plant has a lower stem aluminum content than the recipient plant under aluminum stress conditions.

6. The method of claim 4, wherein: The method for overexpression is to introduce the gene sequence encoding the MsDUF3700 protein into the recipient plant.

7. The method of any of claims 4-6, wherein: The gene sequence encoding the MsDUF3700 protein is shown in SEQ ID NO: 2.

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