Trifolium repens MADs-box transcription factor as well as coding gene and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a trifolium repens MADs-box transcription factor as well as a coding gene and application thereof. The length of an open reading frame of the cloned trifolium repens MADs-box transcription factor TrAGL80 gene is 717bp, 238 amino acids are encoded, and after the gene is heterologous overexpressed in a wild type of medicago sativa, ROS accumulation is reduced and malic acid secretion is increased through overexpression of TrAGL80, so that the tolerance of the medicago sativa to Al is improved. By regulating and controlling the expression of the trifolium repens TrAGL80 gene, a strain with high aluminum toxicity stress resistance can be obtained, the tolerance of the plant to aluminum toxicity stress is improved, and the trifolium repens TrAGL80 gene has important significance for cultivating new crop varieties suitable for acid soil cultivation.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a white clover MADs-box transcription factor, a coding gene thereof and an application thereof. Background Art

[0002] In recent years, excessive discharge of industrial acidic waste gas and over-application of nitrogenous fertilizers have led to an increasingly serious soil acidification problem. Aluminum (Al) is the third most abundant element in the earth's crust after oxygen and silicon. As soil acidifies, the active aluminum in the soil will be released. In acidic soils, Al dissolves into trivalent form (Al 3+ ), posing a significant threat to plant growth by impairing root development. It is estimated that approximately 50% of the world's potential arable land is affected by acidification, a problem exacerbated by both natural processes and human activities. The increasing extent of acidified soils directly impacts crop productivity, posing a major challenge to the sustainable development of global agriculture. Solving the problem of growing plants in acid-aluminum soils is a major challenge facing technicians. In the 21st century, the development of modern biotechnology has made it possible to modify plant aluminum tolerance through biotechnology methods such as genetic engineering, thereby improving plant adaptability to acid-aluminum soils. This provides an important and feasible research direction for solving the problem of plant cultivation in acid-aluminum soils.

[0003] MADs-box transcription factors are widely distributed in eukaryotes and play key roles in various stages of plant growth and development, including regulating floral meristem development, seed and root growth and development, fruit ripening, and responses to abiotic stress. A recent study demonstrated that overexpression of GsMAS1 significantly increased the proline content in transgenic Arabidopsis thaliana plants, thereby enhancing their Al tolerance. Similarly, the CaMADS gene in pepper (Capsicum annuum) confers resistance to cold, salt, and osmotic stresses, while OsMADS25 in rice (Oryza sativa) regulates root growth through ABA-mediated signaling and reactive oxygen species (ROS) scavenging mechanisms, thereby enhancing salt tolerance. These findings suggest that MADs-box transcription factors play an important regulatory role in enhancing plant Al tolerance.

[0004] White clover (Trifolium repens L.) is a perennial, cool-season legume forage grass. Compared to other grasses, it exhibits superior tolerance to acidic aluminum. Studies both domestically and internationally have reported that white clover grows well in soils with a pH range of 4.5-6.5, with seed germination and seedling growth only significantly inhibited by 7 mmol / L aluminum stress. Therefore, white clover is renowned for its high tolerance to aluminum toxicity and excellent growth performance in acidic soils. In recent years, significant genomic resources for white clover have been effectively utilized. However, the genetic basis and molecular mechanisms underlying white clover's response to aluminum stress remain largely unknown, and the utilization of white clover genetic resources is limited by high heterozygosity, allopolyploidy, and high levels of genetic heterozygosity. Identifying genes associated with aluminum tolerance in white clover is of great practical significance for alleviating or resolving aluminum toxicity in acidic soils and improving the quality of white clover. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide a MADs-box transcription factor isolated from white clover, which has a positive effect on improving the tolerance of white clover to aluminum toxicity stress and its encoding gene TrAGL80.

[0006] Another object of the present invention is to provide the use of the above-mentioned white clover MADs-box transcription factor and its encoding gene TrAGL80 in improving plant tolerance to aluminum toxicity stress and cultivating aluminum-resistant transgenic plants.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] First, the present invention provides a white clover MADs-box transcription factor, the amino acid sequence of which is shown in (a) or (b) below:

[0009] (a), the amino acid sequence shown in SEQ ID NO. 2; or

[0010] (b) A protein variant derived from the amino acid sequence shown in SEQ ID No. 2 by substitution, deletion or / and insertion of one or more amino acid residues, which still has the function or activity of a MADs-box transcription factor.

[0011] Secondly, the present invention also provides the above-mentioned coding gene TrAGL 80 of the white clover MADs-box transcription factor, the polynucleotide of which is shown in the following (a), (b), (c), (d), and (e):

[0012] (a), the polynucleotide shown in SEQ ID NO.1; or

[0013] (b) a nucleotide sequence encoding the amino acid shown in SEQ ID NO. 2; or

[0014] (c) a polynucleotide that can hybridize with the complementary sequence of the polynucleotide of SEQ ID NO. 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of the white clover MADs-box transcription factor; or

[0015] (d) a polynucleotide having 90% or more homology with the polynucleotide shown in SEQ ID No. 1; or

[0016] (e) A polynucleotide variant having one or more bases deleted, substituted or inserted based on the polynucleotide shown in SEQ ID NO.1, and the protein encoded by the polynucleotide variant still has the function or activity of the white clover MADs-box transcription factor.

[0017] The present invention uses the white clover variety "Latinno" as the material, and by designing the specific primer pair shown in SEQ ID No.3-4, a functional gene TrAGL80 (SEQ ID NO.1) with the ability to improve the tolerance of white clover to aluminum toxicity stress is obtained, and the amino acid sequence encoded by it (SEQ ID NO.2) is deduced. The white clover genome and the TrAGL80 gene are compared using snapgene software, and the comparison shows that the TrAGL80 gene has copies on chromosomes 3O and 3P in the genome. The molecular formula of the TrAGL80 protein is predicted to be C using the ProParam tool. 1182 H 1890 N 334 O 365 S 15The relative molecular mass of TrAGL80 is 27100.93. The secondary structure of the protein, predicted by SOPMA, is composed of 44.96% α-helices, 50.42% random coils, and 4.62% extended chains. NetPhos-3.1 prediction of potential phosphorylation sites for TrAGL80 revealed 10 serine (Ser) sites, 5 threonine (Thr) sites, and 3 tyrosine (Tyr) sites. This protein may be phosphorylated by multiple phosphokinases, including PKC, cdc2, EGFR, CKII, PKG, DNAPK, INSR, and PKA. The three-dimensional structure of the protein was then predicted using the online software SWISS-MODEL. The results showed that TrAGL80 is a monomeric protein containing a MADs-box domain, confirming that the amino acid sequence encoded by the TrAGL80 gene belongs to a MADs-box transcription factor. Through physiological trait experiments and genetic molecular manipulation, the present invention proves that the MADs-box transcription factor TrAGL80 gene encoding white clover is located in the cell nucleus and cytoplasm. It can increase the malic acid content in plant roots and significantly reduce the content of reactive oxygen and aluminum ions, thereby improving the plant's tolerance to aluminum toxicity stress. This is of great significance for cultivating new crop varieties adapted to cultivation in acidic soils.

[0018] Based on the positive effect of the white clover MADs-box transcription factor provided by the present invention on improving the aluminum toxicity stress tolerance of white clover, the present invention provides the use of the transcription factor or its encoding gene in improving the aluminum toxicity stress tolerance of plants and cultivating aluminum-resistant transgenic plants.

[0019] Furthermore, in a specific application of the present invention, the plant is preferably white clover or alfalfa.

[0020] Furthermore, the method for cultivating the aluminum-toxicity-resistant transgenic plant is to use genetic engineering technology to introduce the encoding gene TrAGL80 into plant tissues or cells so that it is overexpressed in the plant tissues or cells, thereby obtaining the aluminum-toxicity-resistant transgenic plant.

[0021] Among them, the genetic engineering technology can adopt the technical means of introducing genes that have been recorded in the art, such as using Agrobacterium-mediated genetic transformation, recombinant plasmids, recombinant bacteria, transgenic cell lines or expression cassettes, etc. to overexpress the introduced genes; in a specific embodiment of the present invention, the genetic engineering technology is Agrobacterium-mediated genetic transformation.

[0022] Furthermore, the method for cultivating the aluminum-toxicity-resistant transgenic plant using the Agrobacterium-mediated genetic transformation method comprises the following steps:

[0023] (1) cloning the coding gene TrAGL80 using white clover as raw material;

[0024] (2) constructing a plant overexpression recombinant vector containing the coding gene of step (1); in a specific embodiment of the present invention, the plant overexpression recombinant vector is obtained by constructing the sequence shown in SEQ ID NO.1 into the pEarleyGate10 plant expression vector;

[0025] (3) The plant overexpression recombinant vector constructed in step (2) is transformed into plant tissues or plant cells using Agrobacterium-mediated genetic transformation. In a specific embodiment of the present invention, the plant overexpression recombinant vector constructed in step (2) can be transformed into Agrobacterium EHA105 to prepare an infection solution to achieve transformation.

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] The white clover MADs-box transcription factor provided by the present invention has the potential to resist aluminum toxicity and peroxidative stress. The expression level of its encoding gene transcripts changes significantly at different time points, at different aluminum concentrations, under different metal stresses, and in different organ and tissue treatments. Overexpression of the white clover MADs-box transcription factor TrAGL80 gene in alfalfa resulted in transgenic plants showing significant aluminum tolerance under Al stress and possessing higher photosynthetic and antioxidant capacities, which is of great significance for cultivating new crop varieties adapted to cultivation in acidic soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0029] Figure 1 The expression characteristics of the TrAGL80 gene shown in Example 2 of the present invention; in the figure, A is the expression profile of the TrAGL80 gene in different varieties of white clover, B is the expression profile of the TrAGL80 gene in the same variety of white clover under different aluminum treatment concentrations, C is the expression profile of the TrAGL80 gene in the same variety of white clover under different metal treatments, and D is the expression profile of the TrAGL80 gene in different parts of the same variety of white clover;

[0030] Figure 2 This is the subcellular localization result of the TrAGL80 gene shown in Example 3 of the present invention;

[0031] Figure 3 The expression characteristics of wild-type alfalfa and alfalfa overexpressing TrAGL80 shown in Example 3 of the present invention;

[0032] Figure 4 The growth conditions of alfalfa overexpressing TrAGL80 and the wild type under normal conditions and aluminum stress in Example 3 of the present invention are shown;

[0033] Figure 5 These are the results of measuring physiological indicators of alfalfa overexpressing TrAGL80 and the wild type after growth under normal conditions and aluminum stress treatment in Example 3 of the present invention; in the figure, -Al represents normal growth, and +Al represents growth under aluminum stress treatment. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0035] The present invention discloses a white clover MADs-box transcription factor and its encoding gene TrAGL80 and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The white clover MADs-box transcription factor and its encoding gene Tr AGL80 and application described in the present invention have been described through examples. Relevant personnel can obviously modify or appropriately change and combine the white clover MADs-box transcription factor and its encoding gene TrAGL 80 and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] In the comparative experiments of the present invention, unless otherwise specified, except for the artificially set differences among the groups (such as the difference in whether the coding sequence of the white clover MADs-box transcription factor TrAGL80 of the present invention was transferred or not), other experimental conditions remained consistent.

[0037] The following further describes a white clover MADs-box transcription factor and its encoding gene TrA GL80 and applications provided by the present invention.

[0038] Example 1: Acquisition of the MADs-box transcription factor TrAGL80 gene from white clover

[0039] 1. Material preparation

[0040] The white clover variety “Latinno” was used as the test material (purchased from Chengdu Green Grass Garden Seed Co., Ltd.). The seeds were disinfected with 75% alcohol and 1% sodium hypochlorite and then hydroponically cultured with Hoagland complete nutrient solution in a light incubator with 12 h of light (23°C) and 12 h of dark (19°C), relative humidity of 75%, and light intensity of 250 μmol·m -2 ·s -1 After 30 days of culture, RNA was extracted and cDNA was synthesized.

[0041] Total RNA was extracted from the leaves of white clover cultivar Ladinno after culturing for 30 days using RNAprep Pure Plant Total RNA Extraction Kit (spin column type) (DP432) from TIANGEN Biotechnology Co., Ltd. The integrity of RNA was detected by 1% ordinary agarose gel electrophoresis, and the OD260 / OD280 ratio was determined using a Nanodrop instrument.

[0042] The complete and uncontaminated RNA was reverse transcribed according to the instructions of the reverse transcription kit (PrimeiScriptTMⅡ1st Strand cDNA Synthesis Kit) to obtain cDNA.

[0043] 2. PCR amplification

[0044] The NCBI database was used to search and compare the white clover genome to obtain homologous gene sequences. Primer Premier 5.0 was used to design primer pairs (SEQ ID NO. 3-4) in the conserved region as the following PCR reaction primers, as follows:

[0045] Forward Primer(5'→3'):GCTCAGATGGCATCATGACGCTGGCC(SE Q ID NO.3);

[0046] Reverse Primer (5'→3'): TGGGAAGAGGAAGAGTTGAATTGAA (SE Q ID NO. 4).

[0047] The target fragment cDNA was amplified by PCR using 2×Phanta Flash Master Mix (Dye Plus). The PCR reaction system is shown in Table 1.

[0048] Table 1. PCR amplification reaction mixture system

[0049] Reagents Dosage <![CDATA[dd H2O]]> 7 μl Phanta Flash Master Mix 10 μl Forward Primer 1 μl Reverse Primer 1 μl cDNA 1 μl Total 20 μl

[0050] The reaction procedure was as follows: (1) 98.0°C, 30.0 s; (2) 98.0°C, 10.0 s; 58.0°C, 5.0 s; 72.0°C, 5.0 s; 35 cycles; (3) 72.0°C, 1.0 min; (4) storage at 4.0°C.

[0051] 3. Obtaining the TrAGL80 gene sequence

[0052] After PCR amplification, the PCR products were detected by 1% agarose gel electrophoresis (electrophoresis conditions: 1×TAE electrophoresis buffer; 120V, 30min) to confirm the correctness of the target band. The electrophoresis product containing a single target band was cut under UV light and purified using a standard agarose gel DNA recovery kit. The purified DNA product was then tailed with an A-Tailing Kit. The A-tailed product was ligated to the pMDTM19-T vector with reference to the pMDTM19-T Vector Cloning Kit, and the resulting ligation product was transformed into DH5α Escherichia coli competent cells according to the instructions. The transformed DH5α Escherichia coli was activated at 200 r / min and 37°C for 60 min, placed in a centrifuge at room temperature and 4000 rpm for 30 s, and the supernatant was removed. The remaining solution was evenly spread on 50 mg / L ampicillin (Amp) and the culture medium was placed upside down for overnight culture. After monoclonal positive colonies grew on LB solid culture medium, the monoclonal positive colonies were picked and the selected colonies were placed in LB liquid culture medium containing Amp for propagation. After shaking for an appropriate time, the propagated culture solution was taken for PCR verification, and then the propagated culture solution was sent to Sangon for sequencing verification. The cDNA sequence of the gene in the propagated culture solution was shown in SEQ ID NO.1, and the gene was named TrAGL80.

[0053] cDNA sequence of TrAGL80 gene (SEQ ID NO.1):

[0054] ATGGCTAGACAAAGGGTGAAACTTGCTTTCATTGTTAATGATGCTGCAAGAAAAGAGACATATAAAAAAAGGAGCAAGAATGTATTGAAAAAAGTTA ATGAACTAAGCACCCTATGCGGAATAGAAGCATGTGCTATAGTCTATGGTCCGTATGAGCCAGAACCTGAGATTTGGCCACCGTCCCCTTCAGATGTCCAAAAGGTGGTATCGAAATTCAGAGCAAAACCTGAATTTGAGCAAAGTAAAAAAAAA TTGAGTCAAGAGGATTACTTGAAGCAGAGCGTTGTGAAGGCTGAAGACCAACTGATGAGGTTGAGGAAAGAAAACAGACAAACTGAGATGCAATTGTTTATGTATGAATATTTTAAAAGAGGTCAAATTGATCTCAACAATATTTCATTGAATGA TTTGAAAGATCTTTCTTGGTTTTATTGACCATAATTTGAAGGACATTGGTAGAAGACTGGAAGTAGGGTATACTGATAATGGTCAAGGTCAAATTATGACTGCTCCAAGCCAACTTCAACTCCAAATGACACCACCGCCTCCATCAGCAGCAACAA CATCTAGCAATGCTGAAATGGCAATGACGATGAATCATGGCCATGTTGGGATGATTAATGATGACATCATGCAAATGGGGTCGTTTATGGATTTGTTGAATGGGAACGGGGATGAGACAATTCCATCTGGCCAGCGTCATGATGCCAATCTTTAA

[0055] The open reading frame (ORF) sequence was predicted at NCBI and protein translation was performed on SNAPGENE to obtain the amino acid sequence encoded by the TrAGL80 gene (shown in SEQ ID NO. 2). Based on the analyzed open reading frame sequence, specific primers TrAGL80 OF (SEQ ID NO. 5): 5'-ATGGCTAGACAAAGGGTGAAA-3' and TrAGL80 OR (SEQ ID NO. 6): 5'-TTAAAGAGGAAGAGTTGAATT-3', covering the entire length of the TrAGL80 gene ORF, were designed for verification.

[0056] Amino acid sequence encoded by TrAGL80 (SEQ ID NO. 2):

[0057] MARQRVKLAFIVNDAARKETYKKRSKNVLKKVNELSTLCGIEACAIVYGPYEPEPEIWPPPSDVQKVVSKFRAKPEFEQSKKKLSQEDYLKQSVVKAEDQLMRLRKENRQTEMQLFMY EYFKRGQIDLNNISLNDLKDLSWFIDHNLKDIGRRLEVGYTDNGQGQIMTAPSQLQLQMTPPPPSAATTSSNAEMAMTMNHGHVGMINDDIMQMGSFMDLLNGNGDETIPSGQRHDANL

[0058] 4. Sequence analysis of the TrAGL80 gene and its encoded protein

[0059] The cDNA sequence of white clover TrAGL80 obtained by snapgene software analysis was 1078bp in length. The open reading frame was predicted using ORFFinder, and the open reading frame obtained by analysis was 717bp long, encoding a total of 238 amino acids (sequence shown in SEQ ID NO.2). NCBI Blast was then used to compare the relationship between this gene and the same gene in other plants online, and the phylogenetic evolutionary tree of TrAGL80 and AGL80 of other species was constructed using the Neighbor-Joining method using MEGA software. According to the phylogenetic evolutionary tree, white clover TrAGL80 and alfalfa (MsAGL80) were clustered on the same evolutionary branch, indicating that the two are closely related and their sequence similarity is very high.

[0060] The snapgene software was used to compare the genome of white clover and the TrAGL80 gene. The comparison revealed that the TrAGL80 gene has copies on chromosomes 3O and 3P in the genome. The ProParam tool predicted that the protein encoded by the TrAGL80 gene has a molecular formula of C 1182 H 1890 N 334 O 365 S 15The protein has a relative molecular mass of 27100.93 and a theoretical isoelectric point of 6.92. It contains 31 negatively charged residues (Asp+Glu) and 31 positively charged amino acid residues (Arg+Lys). Leu is the most abundant peptide, with 20 residues representing 8.4% of the total, followed by Lys with 19 residues representing 8%. The instability index is 44.88, exceeding the threshold of 40, indicating that the protein is relatively unstable. The overall average hydrophobicity index is -0.671, suggesting a hydrophilic nature. The secondary structure of TrAGL80 was predicted using SOPMA, revealing a structure composed of 44.96% α-helices, 50.42% random coils, and 4.62% extended chains. Using NetPhos-3.1 to predict potential phosphorylation sites on TrAGL80, researchers found that the protein contains 10 serine (Ser) sites, 5 threonine (Thr) phosphorylation sites, and 3 tyrosine (Tyr) sites. This protein may be phosphorylated by multiple phosphokinases, including PKC, cdc2, EGFR, CKII, PKG, DNAPK, INSR, and PKA. The online software SWISS-MODEL was then used to predict the protein's three-dimensional structure. The results showed that TrAGL80 is a monomeric protein containing a MADs-box domain.

[0061] Example 2: Analysis of the expression pattern of the TrAGL80 gene

[0062] 1. Method

[0063] Fluorescence quantitative PCR (q-PCR) was used to verify the expression pattern of TrAGL80 gene under different plant treatments.

[0064] Plant treatment methods include the following:

[0065] (1) The Al-tolerant and Al-sensitive varieties of white clover, Haifa and Huia, were used to study the temporal expression pattern of the TrAGL80 gene. One-month-old plants were exposed to 200 μM AlCl 3 (pH = 4.5), and root samples were collected at 0, 1, 4, 12, 24, and 72 h after treatment.

[0066] (2) roots, root tips, stems, young leaves, old leaves, and flowers were sampled from plants grown under normal conditions (pH 5.8);

[0067] (3) For Al concentration studies, plants were treated with 0, 0.1, 0.5, 1, 2, and 4 mM AlCl 3 for 12 h;

[0068] (4) To evaluate the effects of different metals, plants were exposed to 200 μM AlCl 3 , 0.5 μM CuSO 4 , 100 μM ZnSO 4 , 20 μM CdCl 2 , and 100 μM MnSO 4 for 12 h.

[0069] All experiments were performed using three independent biological replicates, and samples were immediately frozen in liquid nitrogen after collection and stored at −80 °C for subsequent analysis.

[0070] The main steps of the q-PCR method are as follows:

[0071] Using TrActin101 screened in the laboratory as the internal reference gene, specific internal reference primer pairs TrActin101-F and TrActin101-R were designed. According to the TrAGL80 gene sequence, q-PCR primer pairs TrAGL80-qPCR F and TrAGL80-qPCRR were designed. The primer sequences are shown in Table 2. The experiment was performed with reference to the fluorescence quantitative kit. The reaction system was prepared according to the instructions of the fluorescence quantitative kit (SYBR Premix Ex Taq II). The two-step method was used for amplification. The amplification program was as follows: 95℃ for 5min; 95℃ for 30s, 58℃ for 30s, for a total of 40 cycles. After completing the above reaction, the data was exported and 2 -ΔΔCt The gene expression levels were calculated and plotted as Figure 1 Expression profiles shown.

[0072] Table 2. Real-time fluorescence quantitative PCR primer sequences

[0073] Primer name Base sequence (5'→3') TrActin101-F (SEQ ID NO. 7) TGCTTGATTCCGGTGATGGTGTG TrActin101-R (SEQ ID NO. 8) TTCTCGGCAGAGGTACTGAAGGAG TrAGL80-qPCR-F (SEQ ID NO. 9) CCATCAGCAGCAACAACATC TrAGL80-qPCR-R (SEQ ID NO. 10) CCCGTTCCCATTCAACAAATC

[0074] 2. Results Analysis

[0075] like Figure 1 As shown: Under Al stress conditions, the expression profiles of TrAGL80 showed significant differences between 'Haifa' and 'Huia' varieties. Quantitative analysis showed that TrAGL80 expression peaked in 'Haifa' 24 h after Al treatment and remained at significantly elevated levels compared to 'Huia' within 12-72 h ( Figure 1 A). Among the different metals, Al induced the highest TrAGL80 expression level ( Figure 1 C). Tissue-specific expression analysis showed that TrAGL80 was highly expressed in flowers ( Figure 1 D).

[0076] Example 3: Verification of the Aluminum Tolerance and Oxidative Stress Resistance of the White Clover TrAGL80 Gene by Heterologous Overexpression in Alfalfa

[0077] 1. Method

[0078] (1) Cultivation of transgenic alfalfa plants using Agrobacterium-mediated genetic transformation

[0079] Based on the pEarleyGate100 vector sequence, primers were designed with homology arm bases containing EcoRV restriction sites at the 5' and 3' ends, respectively: PEG100-TrAGL80-F and PEG100-TrAGL80-R. Their sequences are shown in Table 3 below. TrAGL80 was amplified by PCR using white clover cDNA as a template using a high-fidelity enzyme. The pEarleyGate100 vector was then digested with EcoRV. After seamless cloning and transformation with competent E. coli, single colonies grown in resistance culture medium were selected for PCR verification and sent to Sangon for sequencing. The resulting plant overexpression recombinant vector was designated pEarleyGate100-TrAGL80.

[0080] Table 3. EcoRV restriction primer sequences

[0081]

[0082] Sequencing-verified colonies were expanded and the PEG100-TrAGL80 overexpression recombinant vector was extracted. The recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105 competent cells using the freeze-thaw method. The transformed competent cells were activated and cultured at 28°C, 200 rpm, and shaken for 4 hours. The culture was then centrifuged at 5000 rpm for 1 minute to harvest the bacterial suspension. The suspension was gently pipetted to resuspend the cells and plated onto YEB solid medium supplemented with 50 mg / L Kan and 10 mg / L rifampicin (Rif). The suspension was then inverted and incubated in a 28°C incubator for 2-3 days. Single colonies were picked, shaken, and positive clones were detected using PCR.

[0083] The obtained Agrobacterium containing the recombinant plasmid is cultured and activated to prepare an infiltration solution, which is then inoculated into alfalfa leaves. The infected alfalfa leaves are then inoculated onto a co-culture medium and incubated in the dark for 48-72 hours. The co-cultured leaves are then inoculated onto the corresponding resistance screening medium and incubated for approximately 60 days until callus grows. Normal callus is selected and inoculated onto a differentiation medium corresponding to the resistance and incubated for approximately 60 days. If the callus produces shoots, it is inoculated onto an elongation medium for approximately 60 days. The seedlings are inoculated onto a rooting medium for approximately 30 days before being transferred to soil.

[0084] (2) Phenotypic identification of transgenic alfalfa plants

[0085] For phenotypic analysis of transgenic alfalfa, uniform plants were selected, stem segments were cut into small segments (approximately 3-5 cm), including a leaf node, placed in vermiculite for growth culture, and irrigated with 1 / 2 strength Hoagland solution (pH=5.8) every 4 days. Three weeks later, seedlings were treated with or without 200 μM AlCl 3 in 1 / 2 strength Hoagland nutrient solution (pH=4.5). Alfalfa leaves and roots were collected with four biological replicates for each sample and stored at -80°C.

[0086] (3) Physiological index measurement methods

[0087] The Al content was determined using inductively coupled plasma optical emission spectrometry.

[0088] For hematoxylin staining, roots were immersed in hematoxylin staining solution (0.2% w / v hematoxylin, 0.02% w / v potassium iodide) for 5 min and then washed three times with ddH2O, after which the root tips were excised and imaged under a stereomicroscope (Nikon).

[0089] White clover leaves were dark-adapted for 15 min using a leaf clip connected to a chlorophyll (Chl) fluorometer (Pocket PEA), and the maximum photochemical efficiency of PSII (Fv / Fm) was recorded. To detect O2- and H2O2, white clover leaves were immersed in 1 mM nitro blue tetrazolium (NBT) for 4 h (for O2- staining) or 0.1% 3,3′-diaminobenzidine (DAB) for 12 h (for H2O2 staining). After staining, Chl was removed by decolorizing the leaves in 75% ethanol. Total antioxidant capacity (T-AOC), H2O2, O2-, malondialdehyde (MDA), malic acid, and citric acid contents were measured according to the manufacturer's instructions (Solarbio, Beijing, China). The initial conductivity (C i ) and final conductivity (C f ), and calculate the electrolyte leakage (EL) based on the measured conductivity value.

[0090] 2. Results Analysis

[0091] (1) Subcellular localization of TrAGL80

[0092] The GFP fusion protein expression method was used to generate a GFP fusion construct under the control of the 35S promoter to study the subcellular localization of the TrAGL80 gene. Figure 2 As shown, TrAGL80 is not only located in the nucleus but also in the cytoplasm. These results suggest that the TrAGL80 gene may play a role in organelles in both the nucleus and cytoplasm.

[0093] (2) Identification of alfalfa overexpressing TrAGL80

[0094] The different alfalfa strains obtained were numbered OE1-OE9 and PCR verification was performed. A total of 8 alfalfa strains (OE1-OE7, OE9) had electrophoresis bands consistent with the open reading frame of white clover TrAGL80, and were determined to be alfalfa positive plants. Using MsEF-1α as the internal reference gene, the relative expression level of TrAGL80 was detected by real-time fluorescence quantitative PCR technology, see Figure 3 The results showed that the gene expression levels were relatively high in strains OE2, OE4, and OE5, while slightly lower in strains OE6 and OE8. TrAGL80 was not expressed in the wild type (WT). Based on these results, strains OE2, OE4, and OE5 were selected for subsequent experiments to analyze the aluminum tolerance function of TrAGL80.

[0095] (3) Heterologous expression of TrAGL80 improves Al tolerance in alfalfa

[0096] To further investigate the functional role of TrAGL80, the pEarleyGate100-TrAGL80 recombinant vector was constructed and used to overexpress TrAGL80 in alfalfa. Three transgenic alfalfa seedlings (OE2, OE4, and OE5) were selected based on their relative expression levels for phenotypic and physiological analysis.

[0097] Three independent TrAGL80-OE transgenic lines were grown in Hoagland nutrient solution containing 0 or 200 mM AlCl3 for 2 weeks. Figure 4 、 Figure 5 As shown, alfalfa seedlings overexpressing TrA GL80 showed increased root length and plant height compared with the wild type (WT) ( Figure 4 , Figure 5 B, C); In addition, in the presence of Al, alfalfa seedlings overexpressing TrAGL80 significantly reduced EL, H2O2, and MDA contents ( Figure 5 D, E, F); at the same time, alfalfa seedlings overexpressing TrAGL80 significantly increased Fv / Fm and T-AOC ( Figure 5 G, H), indicating that TrAGL80 can alleviate oxidative damage. Al content measurement showed ( Figure 5 A) Compared with WT, Al content in the root tips of all three transgenic lines was significantly reduced, which was consistent with the results of hematoxylin staining. In addition, compared with WT, malic acid secretion in the root tips of all three transgenic lines was significantly increased, resulting in reduced Al accumulation ( Figure 5These results indicate that overexpression of TrAGL80 reduced ROS accumulation and increased malate secretion, thereby improving the tolerance of alfalfa to Al.

[0098] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A white clover MADs-box transcription factor, characterized in that: Its amino acid sequence is shown in (a) or (b) below: (a), the amino acid sequence shown in SEQ ID NO. 2; or (b) A protein variant derived from the amino acid sequence shown in SEQ ID No. 2 by substitution, deletion or / and insertion of one or more amino acid residues, which still has the function or activity of a MADs-box transcription factor.

2. The gene encoding the white clover MADs-box transcription factor TrAGL80 according to claim 1, characterized in that: The polynucleotides are shown in (a), (b), (c), (d), and (e) below: (a), the polynucleotide shown in SEQ ID NO.1; or (b) a nucleotide sequence encoding the amino acid shown in SEQ ID NO. 2; or (c) a polynucleotide that can hybridize with the complementary sequence of the polynucleotide of SEQ ID NO. 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of the white clover MADs-box transcription factor; or (d) a polynucleotide having 90% or more homology with the polynucleotide shown in SEQ ID No. 1; or (e) A polynucleotide variant having one or more bases deleted, substituted or inserted based on the polynucleotide shown in SEQ ID NO.1, and the protein encoded by the polynucleotide variant still has the function or activity of the white clover MADs-box transcription factor.

3. Use of the transcription factor according to claim 1 or the encoding gene according to claim 2 in improving plant tolerance to aluminum toxicity stress.

4. Use of the transcription factor according to claim 1 or the coding gene according to claim 2 in cultivating transgenic plants resistant to aluminum poisoning.

5. The use according to claim 3 or 4, characterized in that: The plant is white clover or alfalfa.

6. The use according to claim 4, characterized in that: The method for cultivating the aluminum-toxicity-resistant transgenic plant is to use genetic engineering technology to introduce the coding gene of claim 2 into plant tissues or cells so that the gene is overexpressed in the plant tissues or cells.

7. The use according to claim 6, characterized in that: The genetic engineering technology is an Agrobacterium-mediated genetic transformation method.

8. The use according to claim 7, characterized in that: The method for cultivating the aluminum-toxicity-resistant transgenic plant using the Agrobacterium-mediated genetic transformation method comprises the following steps: (1) Using white clover as raw material to clone the coding gene of claim 2; (2) constructing a plant overexpression recombinant vector containing the coding gene of step (1); (3) Using Agrobacterium-mediated genetic transformation, the plant overexpression recombinant vector constructed in step (2) is transformed into plant tissues or plant cells.

9. The use according to claim 8, characterized in that: The plant overexpression recombinant vector in step (2) is obtained by constructing the sequence shown in SEQ ID NO.1 into the pEarleyGate10 plant expression vector.

10. The use according to claim 8, characterized in that: The step (3) includes the step of transforming the plant overexpression recombinant vector constructed in step (2) into Agrobacterium EHA105.

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