Application of truncated alfalfa transcription factor MtERF009 and / or alfalfa transcription factor MfERF009 in regulation and control of seed development

Abstract

The invention provides application of a truncated alfalfa transcription factor MtERF009 and / or a medicago falcata transcription factor MfERF009 in regulation and control of seed development, and belongs to the technical field of plant genetic engineering. The invention provides application of a truncated alfalfa transcription factor MtERF009 and / or a medicago falcata transcription factor MfERF009 in regulation and control of seed development. The result of the embodiment shows that the single-pod seed number, the whole-plant seed number, the whole-plant seed weight, the lateral branch number and the pod number can be remarkably increased by overexpressing the alfalfa transcription factor MfERF009 in the truncated alfalfa; the transcription factor MtERF009 expression is inhibited in the cut-type alfalfa, the grain length and the grain width of the cut-type alfalfa seeds can be increased, and the thousand grain weight of the cut-type alfalfa seeds can be increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of truncate Medicago truncatula transcription factor MtERF009 and / or yellow Medicago truncatula transcription factor MfERF009 in regulating seed development. Background Art

[0002] Medicago truncatula L. is a model legume crop, characterized by a short growth cycle and a small genome. Transcription factors (TFs), also known as trans-acting factors, are DNA-binding proteins that specifically interact with cis-acting elements in the promoter region of eukaryotic genes. Through interactions between these elements and other related proteins, they activate or repress the transcription of their target genes. In recent years, a series of TFs have been isolated from higher plants that regulate the expression of genes related to drought, low temperature, high salt levels, hormones, pathogen responses, and development. The AP2 / EREBP ethylene-responsive element-binding protein transcription factor family is a large family of transcription factors found widely in plants. Family members play important regulatory roles in plant growth and development, organ organization, adverse stresses, and hormone signaling responses. Currently, there is a lack of technology to improve new varieties of yellow alfalfa. If molecular genetics and gene editing techniques can be used to improve the growth rate of truncatula, it will greatly promote its application in production and have important practical significance for molecular breeding research on forages.

[0003] Currently, there are many reports on the effects of different transcription factors on the growth and development of alfalfa plants, but relatively few reports on the direct effects of related transcription factors on alfalfa seed development. Summary of the Invention

[0004] In view of the defects in the prior art, the present invention aims to provide the use of truncate alfalfa transcription factor MtERF009 and / or yellow alfalfa transcription factor MfERF009 in regulating seed development. The MtERF009 and MfERF009 can regulate the development of alfalfa seeds.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides an application of a truncate Medicago truncatula transcription factor MtERF009 and / or a Medicago flavescentis transcription factor MfERF009 in regulating seed development; the regulating seed development comprises any one or more of the following (1) to (8): (1) regulating the number of seeds in a single pod; (2) regulating the number of seeds in a whole plant; (3) regulating the weight of seeds in a whole plant; (4) regulating the length of seeds; (5) regulating the width of seeds; (6) regulating the thousand-grain weight of seeds; (7) regulating the number of side branches; and (8) regulating the number of pods.

[0007] Preferably, the amino acid sequence of MtERF009 is shown as SEQ ID NO.27; the amino acid sequence of MfERF009 is shown as SEQ ID NO.30.

[0008] Preferably, overexpression of the MfERF009 gene increases any one or more of the number of seeds in a single pod, the number of seeds in a whole plant, and the weight of seeds in a whole plant; inhibition of the expression of the MtERF009 gene reduces any one or more of the number of seeds in a single pod, the number of seeds in a whole plant, the weight of seeds in a whole plant, the number of side branches, and the number of pods.

[0009] Preferably, inhibiting the expression of the MtERF009 gene can increase any one or more of seed length, seed width and seed 1000-grain weight; overexpressing the MfERF009 gene can reduce any one or more of seed length, seed width and seed 1000-grain weight.

[0010] Preferably, the method for overexpressing the MfERF009 gene comprises: transferring a preparation for overexpressing the MfERF009 gene into alfalfa; the preparation for overexpressing the MfERF009 gene comprises one or more of an expression cassette, vector or transgenic cell containing the MfERF009 gene.

[0011] Preferably, the method for inhibiting MtERF009 gene expression comprises: introducing an agent for inhibiting MtERF009 gene expression into alfalfa; the agent for inhibiting MtERF009 gene expression comprises one or more of an expression cassette, vector or transgenic cell containing the sgDNA gene of the MtERF009 gene.

[0012] Preferably, the seeds comprise truncate alfalfa seeds.

[0013] The present invention provides a primer for preparing a MtERF009 gene editing fragment, including a MtERF009DT1DT2-F0 / R0 primer pair and a MtERF009DT1DT2-BSF / BSR primer pair;

[0014] The MtERF009 DT1DT2-BSF / BSR primer pair includes MtERF009 DT1-BSF and MtERF009 DT2-BSR; the nucleotide sequence of the MtERF009 DT1-BSF is shown in SEQ ID NO.7; the nucleotide sequence of the MtERF009 DT2-BSR is shown in SEQ ID NO.10;

[0015] The MtERF009 DT1DT2-F0 / R0 primer pair includes MtERF009 DT1-F0 and MtERF009 DT2-R0; the nucleotide sequence of the MtERF009 DT1-F0 is shown in SEQ ID NO.8; the nucleotide sequence of the MtERF009 DT2-R0 is shown in SEQ ID NO.9.

[0016] The present invention provides the use of the primers described in the above technical solution in preparing a preparation for inhibiting the expression of the MtERF009 gene.

[0017] The present invention provides a method for regulating alfalfa seed development, comprising:

[0018] introducing an agent that inhibits the expression of the MtERF009 gene into alfalfa;

[0019] Alternatively, a preparation overexpressing the MfERF009 gene is introduced into alfalfa.

[0020] Beneficial effects of the present invention:

[0021] The present invention provides an application of a truncate Medicago truncatula transcription factor MtERF009 and / or a Medicago flavescentis transcription factor MfERF009 in regulating seed development; the regulating seed development comprises any one or more of the following (1) to (8): (1) regulating the number of seeds in a single pod; (2) regulating the number of seeds in a whole plant; (3) regulating the weight of seeds in a whole plant; (4) regulating the length of seeds; (5) regulating the width of seeds; (6) regulating the thousand-grain weight of seeds; (7) regulating the number of side branches; and (8) regulating the number of pods. The results of the embodiments of the present invention show that overexpressing the transcription factor MfERF009 of yellow alfalfa in truncation alfalfa can significantly increase the number of seeds in a single pod of truncation alfalfa seeds, increase the number of seeds in a whole plant of truncation alfalfa, and increase the seed weight of the whole plant of truncation alfalfa; inhibiting the expression of the transcription factor MtERF009 in truncation alfalfa can increase the grain length and width of truncation alfalfa seeds, increase the thousand-grain weight of truncation alfalfa seeds, reduce the number of seeds in a single pod of truncation alfalfa seeds, reduce the number of seeds in a whole plant of truncation alfalfa, reduce the seed weight of the whole plant of truncation alfalfa, reduce the number of side branches of truncation alfalfa, and reduce the number of pods of truncation alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the expression of the MtERF009 gene in the roots, stems, leaves, flowers and pods of Medicago truncatula;

[0023] Figure 2 This is the result of MtERF009 protein subcellular localization;

[0024] Figure 3 This is the target mutation detection diagram of the MtERF009 mutant strain;

[0025] Figure 4 This is the peak diagram of the target mutation sequencing of the MtERF009 mutant strain;

[0026] Figure 5 This is the expression level detection result of the gene-edited mutant MtERF009 strain;

[0027] Figure 6 This is the result of the expression level detection of the MfERF009 overexpressing strain;

[0028] Figure 7 This is the result of the change in the number of side branches of truncate alfalfa after editing the MtERF009 gene;

[0029] Figure 8 This is a graph showing the change in the number of pods per plant of truncate alfalfa after editing the MtERF009 gene;

[0030] Figure 9 The following are photos of the seed phenotypes of Medicago truncatula after editing the MtERF009 gene and overexpressing the MfERF009 gene in Medicago truncatula;

[0031] Figure 10 This is a graph showing the statistical results of various phenotypic indicators of Medicago truncatula seeds after editing the MtERF009 gene and overexpressing the MfERF009 gene in Medicago truncatula;

[0032] Figure 11 The diagrams show the results of the binding experiment of the MtERF009 transcription factor to the TTGTTG element of the promoter of the MtSPL15 gene and the TCGCGC element of the promoter of the MtFTa gene;

[0033] Figure 12 This is a graph showing the detection results of the fluorescence signal between the MtERF009 transcription factor and the MtSPL15 gene promoter in the dual luciferase assay. DETAILED DESCRIPTION

[0034] The present invention provides an application of a truncate Medicago truncatula transcription factor MtERF009 and / or a Medicago flavescentis transcription factor MfERF009 in regulating seed development; the regulating seed development comprises any one or more of the following (1) to (8): (1) regulating the number of seeds in a single pod; (2) regulating the number of seeds in a whole plant; (3) regulating the weight of seeds in a whole plant; (4) regulating the length of seeds; (5) regulating the width of seeds; (6) regulating the thousand-grain weight of seeds; (7) regulating the number of side branches; and (8) regulating the number of pods.

[0035] As an optional embodiment of the present invention, the amino acid sequence of MtERF009 is shown in SEQ ID NO. 27; the nucleotide sequence of the MtERF009 gene is shown in SEQ ID NO. 26; the amino acid sequence of MfERF009 is shown in SEQ ID NO. 30; and the nucleotide sequence of the MfERF009 gene is shown in SEQ ID NO. 29. As an optional embodiment of the present invention, the seeds include truncate alfalfa seeds.

[0036] The present invention shows through the subcellular localization results of the Medicago truncatula transcription factor MtERF009 that the Medicago truncatula transcription factor MtERF009 protein is localized in the cell nucleus and can increase the number of lateral branches of Medicago truncatula by regulating the downstream gene MtSPL15.

[0037] The results of the embodiments of the present invention show that overexpressing the MfERF009 gene in truncated alfalfa can increase any one or more of the following: the number of alfalfa seeds per pod, the number of alfalfa seeds per plant, and the weight of alfalfa seeds per plant; and inhibiting the expression of the MtERF009 gene in truncated alfalfa can reduce any one or more of the following: the number of alfalfa seeds per pod, the number of alfalfa seeds per plant, the weight of alfalfa seeds per plant, the number of alfalfa side branches, and the number of alfalfa pods.

[0038] The results of the examples of the present invention show that inhibiting the expression of the MtERF009 gene in truncated alfalfa can increase any one or more of alfalfa grain length, alfalfa grain width and alfalfa 1000-grain weight; overexpressing the MfERF009 gene in truncated alfalfa can reduce any one or more of alfalfa grain length, alfalfa grain width and alfalfa 1000-grain weight.

[0039] As an optional embodiment of the present invention, a method for overexpressing the MfERF009 gene includes: introducing a preparation that overexpresses the MfERF009 gene into alfalfa; the preparation that overexpresses the MfERF009 gene includes one or more of an expression cassette, vector, or transgenic cell containing the MfERF009 gene. The present invention does not specifically limit the preparation method of the MfERF009 gene expression cassette, vector, or transgenic cell; any preparation method conventional in the art that can overexpress the MfERF009 gene can be used. As an optional embodiment of the present invention, the preparation that overexpresses the MfERF009 gene includes a plasmid that overexpresses the MfERF009 gene, Agrobacterium containing the plasmid that overexpresses the MfERF009 gene, or plant cells containing the Agrobacterium. In the present invention, the method for preparing the plasmid that overexpresses the MfERF009 gene includes: inserting the MfERF009 gene into a backbone vector to obtain a plasmid that overexpresses the MfERF009 gene. As an optional embodiment of the present invention, the backbone vector includes pCAMBIA1307. The present invention has no particular limitation on the preparation method of the Agrobacterium containing the plasmid overexpressing the MfERF009 gene or the plant cell containing the Agrobacterium, and any conventional method in the art can be used.

[0040] As an optional embodiment of the present invention, the method for inhibiting the expression of the MtERF009 gene includes: transferring an agent that inhibits the expression of the MtERF009 gene into alfalfa; the agent that inhibits the expression of the MtERF009 gene includes one or more of an expression cassette, vector, or transgenic cell containing the sgDNA gene of the MtERF009 gene. The present invention does not specifically limit the preparation method of the expression cassette, vector, or transgenic cell containing the sgDNA gene of the MtERF009 gene, and any conventional preparation method in the art that can inhibit the expression of the MtERF009 gene can be used. As an optional embodiment of the present invention, the sgDNA gene can be used as a primer for preparing the MtERF009 gene editing fragment, and the intermediate vector p5CBC plasmid can be used as a template to perform PCR amplification to obtain the target fragment. In the present invention, the primers used to prepare the MtERF009 gene editing fragment include using the online software http: / / crispor.tetor.net / crispor.py to screen potential targets of the MtERF009 gene, selecting targets with a GC content between 40% and 60% and an off-target rate of less than 50%, and designing gene editing primers based on the targets. As an optional embodiment of the present invention, the primers for preparing the MtERF009 gene editing fragment include a MtERF009 DT1DT2-F0 / R0 primer pair and a MtERF009 DT 1DT2-BSF / BSR primer pair; the MtERF009DT1DT2-BSF / BSR primer pair includes MtERF009 DT1-BSF and MtERF009 DT2-BSR; the nucleotide sequence of the MtERF009 DT1-BSF is shown in SEQ ID NO.7; the nucleotide sequence of the MtERF009 DT2-BSR is shown in SEQ ID NO.10; the MtERF009 DT1DT2-F0 / R0 primer pair includes MtERF009 DT1-F0 and MtERF009 DT2-R0; the nucleotide sequence of the MtERF009 DT1-F0 is shown in SEQ ID NO.8; the nucleotide sequence of the MtERF009 DT2-R0 is shown in SEQ ID NO.9.

[0041] As an optional embodiment of the present invention, the agent for inhibiting MtERF009 gene expression includes a plasmid containing the sgDNA gene of the MtERF009 gene, Agrobacterium containing the plasmid, or plant cells containing the Agrobacterium. In the present invention, the method for preparing the plasmid containing the sgDNA gene of the MtERF009 gene includes inserting the sgDNA gene into a backbone vector to obtain a plasmid containing the sgDNA gene of the MtERF009 gene. In the present invention, the backbone vector includes the pHSE6401 plasmid. In the present invention, the sgDNA gene preparation method includes: using the online software http: / / crispor.tetor.net / crispor.py to screen potential targets of the MtERF009 gene, selecting targets with a GC content between 40% and 60% and an off-target rate of less than 50%, designing a gene editing vector based on the target, constructing primers, and using the intermediate vector p5CBC containing the sgRNA sequence as a template. The constructed primers are used for PCR amplification using a high-fidelity enzyme. The amplified product is an sgDNA gene containing the potential target sequence of MtERF009. In the present embodiment, the sgDNA gene is referred to as MtERF009 Target-5CBC. After obtaining the sgDNA gene, the present invention recovers it and uses Bsa I and T4 DNA Ligase to cut and ligate it to the plasmid vector pHSE401. Screening obtains a positive plasmid pHSE401-MtERF009, thus obtaining a plasmid containing the sgDNA gene of the MtERF009 gene. The present invention has no particular limitation on the preparation method of the Agrobacterium containing the plasmid or the plant cell containing the Agrobacterium, and any conventional preparation method in the art may be used for preparation.

[0042] The present invention provides primers for preparing a MtERF009 gene editing fragment, comprising a MtERF009DT1DT2-F0 / R0 primer pair and a MtERF009 DT1DT2-BSF / BSR primer pair; the MtERF009 DT1DT2-BSF / BSR primer pair comprises MtERF009 DT1-BSF and MtERF009 DT2-BSR; the nucleotide sequence of the MtERF009 DT1-BSF is shown in SEQ ID NO.7; the nucleotide sequence of the MtERF009 DT2-BSR is shown in SEQ ID NO.10; the MtERF009 DT1DT2-F0 / R0 primer pair comprises MtERF009 DT1-F0 and MtERF009 DT2-R0; the nucleotide sequence of the MtERF009DT1-F0 is shown in SEQ ID NO. NO.8; the nucleotide sequence of the MtERF009 DT2-R0 is shown in SEQ ID NO.9.

[0043] The present invention provides the use of the primers described in the above technical solution in preparing a formulation for inhibiting the expression of the MtERF009 gene. The primers provided by the present invention are capable of amplifying and obtaining an sgDNA gene for inhibiting the MtERF009 gene. The present invention inserts the sgDNA gene into a backbone vector to obtain a plasmid containing the sgDNA gene of the MtERF009 gene. The plasmid is then transformed into Agrobacterium, which is then further transformed into plant cells, thereby inhibiting the expression of the MtERF009 gene in the plant cells.

[0044] The present invention provides a method for regulating alfalfa seed development, comprising:

[0045] introducing an agent that inhibits the expression of the MtERF009 gene into alfalfa;

[0046] Alternatively, a preparation overexpressing the MfERF009 gene is introduced into alfalfa.

[0047] The present invention provides a method for regulating alfalfa seed development, comprising: introducing an agent that inhibits the expression of the MtERF009 gene into alfalfa. As an optional embodiment of the present invention, the alfalfa includes truncated alfalfa. The present invention does not particularly limit the method of introduction, and any conventional introduction method in the art can be used. As an optional embodiment of the present invention, the introduction method includes the leaf disc transformation method mediated by Agrobacterium EHA105. After the present invention introduces the agent that inhibits the expression of the MtERF009 gene into alfalfa leaves, the obtained leaves are preferably cultured using a plant tissue culture method to obtain plants, and then the plants are cultured to obtain transgenic alfalfa. In the transgenic alfalfa obtained by the method described in the above technical solution of the present invention, the expression level of the MtERF009 gene is significantly reduced, and at the same time, the seed width and length of the transgenic alfalfa are increased, and the thousand-grain weight of the seeds is significantly increased.

[0048] The present invention provides a method for regulating alfalfa seed development, comprising: introducing a preparation that overexpresses the MfERF009 gene into alfalfa. As an optional embodiment of the present invention, the alfalfa includes truncated alfalfa. The present invention does not particularly limit the method of introduction, and any conventional introduction method in the art can be used. As an optional embodiment of the present invention, the introduction method includes the leaf disc transformation method mediated by Agrobacterium EHA105. After the present invention introduces the preparation that overexpresses the MfERF009 gene into alfalfa leaves, the obtained leaves are preferably cultured using a plant tissue culture method to obtain plants, and then the plants are cultured to obtain transgenic alfalfa. The transgenic alfalfa obtained by the method described in the above technical solution of the present invention has a significantly increased expression level of the MfERF009 gene, and at the same time, the number of seeds per pod, the number of seeds per plant, the weight of seeds per plant, the number of side branches, and the number of pods of the transgenic alfalfa are significantly improved.

[0049] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Medicago truncatula is a truncate Medicago truncatula. The primer information used in the following technical scheme is shown in Table 1.

[0051] Table 1 Primer information

[0052]

[0053] Example 1 Acquisition of Medicago truncatula MtERF009 transcription factor

[0054] Total RNA was extracted from leaf tissue of wild-type Medicago truncatula R108 plants using liquid nitrogen grinding and Trizol RNA extraction reagent from Takara Biotechnology. Reverse transcription and cDNA synthesis were performed according to the instructions for the Takara Biotechnology Reverse Transcription Kit (6210A). RT-PCR was performed using primers MtERF009-F (sequence shown in SEQ ID NO. 1) and MtERF009-R (sequence shown in SEQ ID NO. 2). PCR conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 98°C for 10 seconds; annealing at 55°C for 30 seconds; extension at 68°C for 1 minute, followed by 30 cycles of denaturation and extension; post-extension at 68°C for 10 minutes; and storage at 4°C. After the PCR reaction, the target fragment (approximately 500 bp) was recovered using the SanPrep column-based DNA gel recovery kit from Sangon Biotechnology Co., Ltd., following the instructions in the kit. The CDS sequence of the Medicago truncatula MtERF009 gene was obtained using the above method. The nucleotide sequence of the MtERF009 gene is shown in SEQ ID NO. 26, and the amino acid sequence is shown in SEQ ID NO. 27.

[0055] SEQ ID NO.26:

[0056]

[0057] SEQ ID NO.27:

[0058]

[0059] Example 2 Analysis of the expression pattern of the Medicago truncatula MtERF009 gene

[0060] Plant material: wild-type truncate alfalfa that had grown normally for 45 days was selected. The root, stem, leaf, and flower tissues were separated and wrapped in tin foil, quickly frozen in liquid nitrogen, and stored at -80°C. RNA was extracted separately, and cDNA was obtained by reverse transcription. This cDNA was used as a template, MtERF009DL-F (sequence shown in SEQ ID NO.3) and MtERF009DL-R (sequence shown in SEQ ID NO.4) were used as primers, and the truncate alfalfa actin MtActin gene was used as an internal reference gene. The primer sequences were shown in SEQ ID NO.24 and SEQ ID NO.25. The operation was performed according to the instructions of the fluorescence quantitative kit (RR047Q) of TAKARA Biological Company. The expression of the MtERF009 gene in roots, stems, leaves, flowers and pods is shown in Tables 2 and 3. Figure 1 As shown: MtERF009 was found to have the highest relative expression level in leaves.

[0061] Table 2 Analysis of the expression patterns of the MtERF009 gene in roots, stems, leaves, flowers and pods

[0062] Group root stem leaf flower pods Relative expression level 1 0.905167239 3.914790348 9.05339853 5.321779419 4.803048521 Relative expression level 2 1.110208216 3.432891039 8.6680587 5.566045906 4.23445596 Relative expression level 3 0.984624545 3.643955279 9.924977292 5.373755153 4.220817095 Average relative expression 1 3.663878889 9.215478174 5.420526826 4.419440525

[0063] Example 3 Subcellular localization analysis of the Medicago truncatula MtERF009 gene

[0064] 1. Construction of recombinant plasmid pBE-GFP-MtERF009. The cDNA of MtERF009 cloned in Example 1 was used as a template. PCR was performed using MtERF009 PBE-F (sequence shown in SEQ ID NO. 5) and MtERF009 PBE-R (sequence shown in SEQ ID NO. 6) to obtain the full-length cDNA of MtERF009 with BamH I and Sal I restriction sites. The PCR conditions were as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 sec; annealing at 60°C for 30 sec; extension at 68°C for 1 min, 30 cycles of denaturation and extension; post-extension at 68°C for 10 min; and storage at 4°C. The pBE empty plasmid (Wang, ZW, Li, JW and Min, Y., et al.. The ERF072 Transcription Factor Directly Regulates MtSOC1-Like Expression and Mediates Drought-Accelerated Flowering in Medicago truncatula [J]. Plant, Cell & Environment, 2025.) was double-digested with BamH I and Sal I. The system is shown in Table 3.

[0065] Table 3 pBE vector restriction enzyme digestion system

[0066] Reagents Dosage pBE plasmid (100 ng / μL) 10 μL 10×T Buffer 3μL BamH I 1 μL Sal I 1 μL <![CDATA[ddH2O]]> Up to 20μL

[0067] After mixing the system, enzyme digestion was carried out at 37℃ for 1h. Use the HD Cloning Kit to perform homologous recombination ligation of the target fragment and the pBE vector. Transform the ligation product into E. coli DH5α, test the colony by PCR, and send it for sequencing. Positive clones verified by sequencing are expanded and the plasmid is extracted for later use.

[0068] 2. Transformation of Agrobacterium GV3101 with the recombinant plasmid pBE-GFP-MtERF009: Pipette 5 μL of the recombinant plasmid pBE-GFP-MtERF009 and the pBE empty vector into competent Agrobacterium GV3101 cells. Gently mix, incubate on ice for 5 minutes, add liquid nitrogen for 5 minutes, transfer to a 37°C water bath for a heat shock of 5 minutes, and then in an ice-water bath for 5 minutes. Add 700 μL of LB liquid medium equilibrated to room temperature in a laminar flow hood and incubate at 28°C at 200 rpm for 2 hours. Centrifuge at 6000 rpm for 1 minute, discard 500 μL of the supernatant, and spread 80 μL of the remaining liquid onto LB solid medium containing 50 mg / L Kan+, 50 mg / L genta, and 25 mg / L Rif. Incubate inverted at 28°C for 36-48 hours until single colonies emerge.

[0069] 3. Injection infection of the lower epidermis of tobacco leaves: A single colony of positive Agrobacterium GV3101 was picked and inoculated in YEB liquid medium containing 50 mg / L Kan+, 50 mg / L genta, and 25 mg / L Rif. After shaking culture at 28°C for 24 hours, 500 μL of the bacterial solution was added to 40 mL YEB triple antibody (containing 50 mg / L Kan+, 50 mg / L genta, and 25 mg / L Rif) containing 500 μL 1 M MES and 5 μL 200 mM AS. + , 50 mg / L genta, 25 mg / L Rif) liquid culture medium, shake culture at 28 ° C until OD 600 After collecting the bacteria, add invasion dye solution (1 mL 1M MES, 1 mL 1M MgCl2 and 100 μL 200mM MAS per 100 mL ddH2O) and resuspend until the OD 600 The value was 1.0. After standing in the dark for 4 hours, the lower epidermis of 4-week-old tobacco leaves was injected and cultured in a plant tissue culture room for 36 hours.

[0070] 4. Observe the fluorescence of leaves under laser confocal microscope. Cut the infected tobacco leaves and place them in a culture dish. DAPI is dropped on the leaves. After staining for 20 minutes, wash them twice with PBS buffer, each time for 20 minutes. Place the stained leaves on a slide and observe the lower epidermis of the leaves under a laser confocal microscope. The results are as follows. Figure 2 As shown (the scale bar in the figure is 25 μm), among which 35S:GFP is the observation result after Agrobacterium GV3101 was transformed with empty pBE-GFP and then injected to infect the lower epidermis of tobacco leaves; 35S:MtERF009-GFP is the observation result after Agrobacterium GV3101 was transformed with recombinant plasmid pBE-GFP-MtERF009 and then injected to infect the lower epidermis of tobacco leaves.

[0071] Depend on Figure 2 It was found that MtERF009 protein was localized in the cell nucleus in subcellular localization experiments.

[0072] Example 4 Obtaining the mutant MtERF009 truncate Medicago

[0073] 1. Construction of the editing vector pHSE401-ED-MtERF009: The MtERF009 gene sequence was obtained from the Medicago truncatula database (https: / / modms.lzu.edu.cn). The MtERF009 gene was cloned using primers MtERF009 F (sequence shown in SEQ ID NO. 1) and MtERF009 R (sequence shown in SEQ ID NO. 2). Potential target sites of the MtERF009 gene were screened using the online software http: / / crispor.tetor.net / crispor.py. Target sites with a GC content between 40% and 60% and an off-target rate less than 50% were selected. Gene editing primers were designed according to the target site: MtERF009 DT1-BSF (sequence shown in SEQ ID NO.7), MtERF009 DT1-F0 (sequence shown in SEQ ID NO.8), MtERF009 DT2-R0 (sequence shown in SEQ ID NO.9) and MtERF009 DT2-BSR (sequence shown in SEQ ID NO.10). The pHSE401 editing vector was constructed (Zhu F, Ye Q, Chen H, Dong J, Wang T. Multigene editing reveals that MtCEP1 / 2 / 12 redundantly control lateral root and nodule number in Medicago truncatula. J Exp Bot. 2021: 72(10): 3661-3676.).

[0074] Using the PCR method, the intermediate vector p5CBC plasmid was used as a template and four gene editing primers were used to amplify the sgDNA module MtERF009 Target-5CBC (such as SEQ ID NO. 28) containing the potential target sequence of MtERF009. The reaction system is shown in Table 4, and the reaction degree is shown in Table 5.

[0075] SEQ ID NO.28:

[0076]

[0077] Table 4 PCR reaction system

[0078]

[0079]

[0080] Table 5 PCR reaction procedure

[0081]

[0082] After the reaction was completed, 1% agarose gel was used for electrophoresis detection, and the PCR product of about 840 bp was recovered from the gel fragment.

[0083] The MtERF009 Target-5CBC fragment and the pHSE401 gene editing vector were used to construct the pHSE401-MtERF009 recombinant binary expression vector by cutting and ligating. The system is shown in Table 6, and the reaction procedure is shown in Table 7.

[0084] Table 6 PCR reaction system

[0085] Element volume MtERF009 Target-5CBC recovered fragment 400ng pHSE401 (400 ng / μL) 400ng <![CDATA[10×T4Ligase Buffer]]> 1.5 μL <![CDATA[T4 DNA Ligase]]> 1 μL 10×BSA 1.5 μL Bsa I 1 μL Total volume 15 μL

[0086] Table 7 Reaction procedure

[0087] temperature time 37℃ 5h 50℃ 5min 80℃ 10min

[0088] 2. Transformation of Agrobacterium EHA105 with the gene editing vector pHSE401-MtERF009

[0089] Take 500ng of gene editing vector pHSE401-MtERF009 and add it to 50μL EHA105 competent medium. Place on ice for 5min, quick freeze in liquid nitrogen for 5min, heat shock at 37℃ for 5min, place on ice for 5min, add 600μL YEB liquid medium, and culture at 28℃ and 200rpm for 2h. Take 100μL of cultured bacterial liquid and spread it on YEB solid medium containing kanamycin (50mg / L) and rifampicin (75mg / L), invert and culture at 28℃ for 2 days, and then screen for positive colonies. Select positive single colonies and expand the culture to OD 600 The Agrobacterium liquid was centrifuged at 2400 r / min for 15 min at room temperature, the supernatant was discarded, and the bacteria were suspended in MES buffer (buffer composition see Table 16) to an OD of 0.6 to 0.8. 600 When it reaches 0.2-0.3, prepare the infection solution for use.

[0090] 3. Leaf disc transformation of Medicago truncatula mediated by Agrobacterium tumefaciens EHA105

[0091] The plant material selected was Medicago truncatula R108, and the infection material was selected from leaves that had been cultivated for four weeks in good growth condition. The Medicago truncatula was transformed using the EHA105-mediated leaf disc method. The infection material was washed with ultrapure water, then sterilized for 10 minutes with 20 mL of 5% hypochlorous acid rinse solution and 20 μL of Tween-20. The prepared infection solution was mixed with the leaves, and the leaves were vacuum-infiltrated at 0.08-0.09 MPa for 10 minutes. The leaves were then ultrasonically treated at 40 kHz at room temperature for 5 minutes, and then vacuum-infiltrated again at 0.08-0.09 MPa for 10 minutes. The Agrobacterium suspension was discarded, and the Agrobacterium on the leaf surface was removed by blotting on sterile filter paper. The leaves were then plated on a co-culture medium (SH3α medium without TMT and Hyg) and incubated in the dark for 24-30 hours. Explants were transferred to selective medium (SH3α medium) supplemented with antibiotics (hygromycin HYG and timentin TMT). Callus culture was subcultured every two weeks for 5-6 weeks. Well-growing calli were transferred to MSBK medium and cultured for 15 days until green embryoids appeared. Calli with green embryoids were transferred to SH9 medium for shoot differentiation. Subculture was performed every three to four weeks until shoots developed, which took six to eight weeks on this medium. When shoots developed two to three fully expanded leaves, they were transferred to MSO medium for rooting. (If robust seedlings failed to root within one month, 1 mg / L IAA was added.) Rooted plants were transplanted to a mixture of vermiculite and perlite (1:1 by volume) for hardening. Surviving plants were transferred to a mixture of nutrient soil and vermiculite (1:1 by volume). The composition of the culture medium is shown in Table 8.

[0092] Table 8 Culture medium composition

[0093]

[0094]

[0095] 4. Detection of transgenic Medicago truncatula: Gene-edited lines were verified using PCR, target site detection, and RT-qPCR. PCR amplification was performed using DNA from regenerated Medicago truncatula plants as template, the pHSE401-MtERF009 recombinant plasmid as a positive control, and wild-type Medicago truncatula genomic DNA as a negative control. The primers MtERF009-DT1-BSF and MtERF009-DT2-BsR were used. The reaction system was as follows: 10 μL of 2× Taq mix, 1 μL of MtERF009-DT1-BSF, 1 μL of MtERF009-DT2-BsR, 1 μL of template DNA, and 7 μL of ddH2O, in a total reaction volume of 20 μL. The reaction procedure was: 95°C for 3 min; 95°C for 30 sec; 55°C for 30 sec; 72°C for 1 min; 35 cycles; and 72°C for 5 min. The size of PCR product fragments was detected by agarose gel electrophoresis, and positive plants were confirmed to be transgenic plants.

[0096] The positive plants were numbered, and the DNA sequences were amplified using primers MtERF009-F and MtERF009-R. After ligation into the pMD19T vector, the competent E. coli cells were transformed and plated on LBA solid medium. The cells were cultured at 37°C for 12 h. Twelve monoclonal strains were randomly selected for streaking culture. The PCR-positive colonies were sequenced and the target site sequences were compared ( Figure 3 ) and compared the sequencing peaks ( Figure 4 ). Leaves of mutant-positive strains were selected to extract RNA and reverse transcribed into cDNA, and then fluorescence quantitative RT-qPCR was used to calculate the relative expression level of MtERF009 in each strain. MtERF009 DL-F (sequence as shown in SEQ ID NO.3) and MtERF009 DL-R (sequence as shown in SEQ ID NO.4) were used as primers, MtActin gene was used as internal reference gene, and the operation was carried out according to the instructions of TAKARA Bio's fluorescence quantitative kit (RR047Q). The expression detection results of mutant MtERF009 strains are shown in the figure. Figure 5 As shown, wT is the wild-type plant, and ED1, ED5, ED8, ED15, and ED17 are different mutant MtERF009 lines. Figure 5 The results showed that the expression levels of the mutant MtERF009 strains were significantly reduced compared to the wild type (WT). These results prove that the MtERF009 gene has been successfully edited in the Medicago truncatula genome.

[0097] 5. Obtaining Medicago truncatula overexpressing MfERF009 Our laboratory previously overexpressed the MfERF009 gene in Medicago truncatula through genetic transformation technology, and used the obtained transgenic Medicago truncatula (OE) overexpressing the MfERF009 gene as a control to observe the function of the ERF009 gene in Medicago truncatula.

[0098] (1) Construction of overexpression vector pCAM1307-OE-MfERF009

[0099] The empty plasmid pCAMBIA1307 (hereinafter referred to as p1307) was double-digested with BamH I and Xba I. The enzyme digestion system is shown in Table 9.

[0100] Table 9 Enzyme Digestion System

[0101] Reagents Dosage p1307 plasmid (100 ng / μL) 10 μL 10×K Buffer 1 μL BamH I 1 μL Xba I 1 μL <![CDATA[ddH2O]]> Up to 20μL

[0102] After the system is mixed, enzyme digestion is performed at 37°C for 1 hour to recover the linearized plasmid vector.

[0103] Using Medicago fruticosa cDNA as a template, MfERF009 OE-F (sequence shown in SEQ ID NO. 21) and MfERF009 OE-R (sequence shown in SEQ ID NO. 22) were used to obtain the full-length OE-MfERF009 cDNA with BamHI and XbaI restriction sites. PCR conditions were the same as in step 1 of Example 3. The nucleotide sequence of the MfERF009 gene is shown in SEQ ID NO. 29. The amino acid sequence is shown in SEQ ID NO. 30.

[0104] SEQ ID NO.29:

[0105]

[0106]

[0107] SEQ ID NO.30:

[0108]

[0109] Recover the target fragment OE-MfERF009 and ligate each fragment into the linearized p1307 vector at a fragment:vector molar ratio of 3:1. Transform the fragment into competent E. coli DH5α cells, plate the cells, and select positive clones for PCR analysis. Sequencing-verified positive clones were expanded and the plasmids were extracted for later use.

[0110] (2) Transformation of Agrobacterium EHA105 with overexpression vector pCAM1307-OE-MfERF009 The transformation method was the same as step 2 in Example 4.

[0111] (3) Leaf disc transformation of truncate alfalfa mediated by Agrobacterium EHA105 The transformation method was the same as step 3 in Example 4.

[0112] (4) Detection of transgenic Medicago truncatula Transgenic lines were verified by PCR and RT-qPCR. Transgenic plants were selected after survival (about 30 days) and leaf DNA was extracted for PCR identification. Primers MfERF009 OE-F and 3×Flag-R (sequence shown in SEQ ID NO. 23) were used. The PCR conditions were as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 sec; annealing at 57°C for 30 sec; extension at 68°C for 1 min, denaturation to extension for 30 cycles; post-extension at 68°C for 10 min; and storage at 4°C.

[0113] The leaves of the lines that were initially detected as positive were selected to extract RNA and reverse transcribe into cDNA. Then, the relative expression level of MfERF009 in each line was calculated by fluorescence quantitative RT-qPCR. MfERF009 DL-F and MfERF009 DL-R were used as primers and the operation was carried out according to the instructions of the fluorescence quantitative kit (RR047Q) of TAKARA Bio. Figure 6 As shown in the figure, compared with the wild type (WT), the expression levels of the transgenic lines overexpressing the MfERF009 gene were significantly increased. The above results prove that the MfERF009 gene has been integrated into the Medicago truncatula genome and has been overexpressed.

[0114] Example 5 Developmental phenotype experiment of mutant strains

[0115] 1. Planting of mutant truncation alfalfa Seeds of T1 mutant truncation alfalfa (T1 is the first generation of transgenic plants, i.e., genetically transformed regenerated seedlings that have been successfully gene-edited) were selected, treated with concentrated sulfuric acid for 8 minutes, washed twice with sterile water, disinfected with HgCl2 for 5 minutes, and washed five times with sterile water. The seeds were placed in 1 / 2MS solid culture medium, vernalized in the dark at 4°C for 3 days, cultured in an incubator, and transplanted into a matrix of vermiculite: nutrient soil = 3:2 (v / v) after 2 weeks. Wild-type truncation alfalfa was planted in the same batch using the same method.

[0116] 2. Developmental phenotypes of mutant strains The wild-type plants and mutant plants at the end of the entire growth cycle in vermiculite: nutrient soil = 3:2 (v / v) were selected for measurement and statistics of lateral branch and seed phenotypes. The results are as follows: Figures 7-10 As shown in Tables 10 to 14. Figure 7 The figure shows the changes in the number of lateral branches of truncate alfalfa after editing the MtERF009 gene, where WT is the wild-type truncate alfalfa, and ED-1, ED-2, and ED-3 are truncate alfalfa mutant lines after editing the MtERF009 gene, the same below; Figure 8 This is a graph showing the changes in the number of pods per plant of truncate alfalfa after editing the MtERF009 gene. Figure 9 The following are photos of the seed phenotype of truncate alfalfa after editing the MtERF009 gene and overexpressing the MfERF009 gene in truncate alfalfa. Among them, OE1 is a mutant strain of truncate alfalfa after overexpressing the MfERF009 gene, and the same below. Figure 10 This is a graph showing the statistical results of various seed phenotypic indicators of truncation alfalfa after editing the MtERF009 gene and overexpressing the MfERF009 gene in truncation alfalfa.

[0117] Table 10 Number of seeds per pod (unit: grain)

[0118]

[0119]

[0120] Table 11 Number of seeds per plant

[0121]

[0122] Table 12 Whole plant seed weight

[0123]

[0124]

[0125] Table 13 Grain length and width (unit: mm)

[0126]

[0127] Table 14 Thousand-grain weight (unit: g)

[0128]

[0129] like Figure 7 and Figure 8 As shown in Figure 2, compared with the wild type, the number of lateral branches and pods of the mutant plants of truncate alfalfa after editing the MtERF009 gene were reduced. Figures 9-10 The results show that compared with the control (WT) plants and plants overexpressing the MfERF009 gene, the number of seeds per plant and weight of seeds decreased, the grain width and length increased, and the 1000-grain weight increased in the truncate alfalfa mutant plants after editing the MtERF009 gene. This indicates that after editing the MtERF009 gene, the grain length, width, and 1000-grain weight of the truncate alfalfa mutant plants were increased. Compared with the control plants, the number of seeds per pod, the number of seeds per plant, and the weight of seeds per plant were significantly increased in the plants overexpressing the MfERF009 gene.

[0130] Example 6 Yeast One-Hybrid Transcription Factor of Medicago truncatula MtERF009

[0131] 1. Vector Construction The DNA of the MtERF009 gene cloned in Example 1 was used as a template, and the primer sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12 were used for PCR amplification to obtain the full-length sequence encoding MtERF009. The full-length sequence encoding MtERF009 was inserted into the pGADT7 vector (Wang, Z. W., Li, J. Wan and Min, Y., et al.. The ERF072 Transcription Factor Directly Regulates MtSOC1-Like Expression and Mediates Drought-Accelerated Flowering in Medicago truncatula [J]. Plant, Cell & Environment, 2025.) to construct a fusion expression vector.

[0132] Single-stranded primers such as primers shown in SEQ ID NO.13 and SEQ ID NO.14 are subjected to DNA annealing reaction to synthesize double strands to obtain double-stranded DNA of the promoter TTGTTG element of the MtSPL15 gene; single-stranded primers such as primers shown in SEQ ID NO.15 and SEQ ID NO.16 are subjected to DNA annealing reaction to synthesize double strands to obtain double-stranded DNA of the promoter TCGCGC element of the MtFTa gene.

[0133] The promoter elements TTGTTG of the MtSPL15 gene and TCGCGC of the MtFTa gene were constructed into the pABAi vector, respectively, and designated pABAi-SPL15 and pABAi-FTa. Primers were designed with Hind III and Kpn I restriction enzyme sites at both ends to facilitate ligation to the pAbAi vector. Three bases were removed from the 5' and 3' ends immediately adjacent to the cis-acting elements (TTGTTG and TCGCGC), and this fragment was repeated three times to design primers specific for the promoter cis-acting elements. The pABAi vector was double-digested with Hind III and Kpn I, and then T4 DNA ligase was used to ligate the promoter elements TTGTTG of the MtSPL15 gene and TCGCGC of the MtFTa gene to the pABAi vector, respectively. The resulting primers were designated pABAi-SPL15 and pABAi-FTa. (Wang, ZW, Li, JWand Min, Y., et al..The ERF072 Transcription Factor Directly Regulates MtSOC1-Like Expression and Mediates Drought-AcceleratedFlowering in Medicago truncatula[J].Plant, Cell&Environment, 2025.)

[0134] 2. Preparation of Competent Yeast Strain Y1H

[0135] The Y1H yeast strain was inoculated into 5 mL YPDA liquid medium and cultured at 30°C, 200 rpm for 24 h for activation. The activated bacterial solution was inoculated into 50 mL YPDA liquid medium and cultured at 30°C, 200 rpm for 2-3 h until the OD 600 =0.5-0.8. Transfer the bacterial suspension to a 50 mL centrifuge tube and centrifuge at 3000 rpm for 5 minutes. Discard the supernatant and collect the cells. Wash the cells with sterile ddH2O at 3000 rpm for 5 minutes, discard the supernatant, and repeat the wash. Resuspend the pellet in 1× LiAc, aliquot the suspension into 100 μL aliquots, centrifuge at 3000 rpm for 5 minutes, and discard the supernatant. Competent cells are now ready.

[0136] 3. Obtaining pAbAi-SPL15 and pAbAi-FTa yeast strains

[0137] The linearized pAbAi-SPL15 and pAbAi-FTa plasmids were transformed into Y1H Gold yeast strain competent cells and coated with SD-Ura medium.

[0138] Prepare a premix as shown in Table 15. Add 360 μL of the premix to the Y1H competent yeast cells and pipette to mix thoroughly to suspend the yeast cells in the premix. Incubate in a 30°C waterbath for 30 minutes (invert and mix every 10 minutes); incubate in a 42°C waterbath for 30 minutes (invert and mix every 10 minutes); centrifuge at 12,000 rpm for 15 seconds, discard the supernatant, resuspend the pellet in YPDA, and incubate at 30°C with shaking for 1 hour; centrifuge at 12,000 rpm for 15 seconds, discard the supernatant, resuspend the cells in ddH2O, spread 100 μL of the suspension evenly on SD-Ura solid medium, and incubate at 30°C with shaking for 72 hours; pick a single clone from the plate, resuspend the cells in 100 μL of ddH2O, boil in boiling water for 10 minutes, and snap freeze in liquid nitrogen for 10 minutes. Repeat twice, then boil in boiling water for 10 minutes, ice-bath for 10 minutes, centrifuge at 12,000 rpm for 5 minutes, and use the supernatant as a template. Perform PCR verification on the culture.

[0139] Table 15 Premix

[0140] Ingredients volume Linearized pAbAi-SPL15 or pAbAi-FTa plasmid 1000ng PEG solution 240 μL LiAc solution 36μL Salmon sperm DNA (10 μg / μL) 10 μL <![CDATA[ddH2O]]> Up to 360μL

[0141] 4. Screening for AbA concentration that inhibits background expression of pAbAi

[0142] The Y1H strain containing pAbAi-SPL15 and the Y1H strain containing pAbAi-FTa were resuspended separately with 0.9% NaCl solution, and 100 μL of the resuspension was spread on SD / -Ura solid culture medium containing different concentrations of AbA (0 ng / mL, 100 ng / mL, 200 ng / mL) for culture. The yeast was grown at 30°C in a constant temperature incubator for 3 to 5 days, and the growth of the yeast was observed to obtain the lowest AbA concentration that completely inhibited the growth of the yeast strain. It was determined that 200 ng / mL of ABA can inhibit the growth of the Y1H strain of pAbAi-SPL15 and the Y1H strain of pAbAi-FTa, and subsequent experiments can be carried out.

[0143] 5. Transform pGADT7-ERF009 into the Y1H strain containing pAbAi-SPL15 and the Y1H strain containing pAbAi-FTa using the same transformation method as in step 3 above.

[0144] 6. Interaction experiment between pGADT7-ERF009 protein and pBait-AbAi element

[0145] The Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-SPL15 and the Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-FTa were inoculated into YPDA medium, shaken at 30°C and 200 rpm, and the bacterial solution OD 600When the concentration of the aliquot was 1.0, serial dilutions were performed. At dilution factors of 0, 10, and 100, 10 μL was taken and cultured on SD / -Ura / -Leu solid medium containing 200 ng / mL AbA, and cultured in an inverted manner at 30°C for 3 days. The results showed that on SD / -Ura / -Leu plates, the Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-SPL15, the Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-FTa, the negative control strain and the positive control strain grew well, indicating that the plasmid was successfully transformed into the strain (Wang, ZW, Li, JWand Min, Y., et al..The ERF072 Transcription Factor Directly Regulates MtSOC1-Like Expressionand Mediates Drought-Accelerated Flowering in Medicago truncatula[J].Plant, Cell & Environment, 2025.); the Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-SPL15, the Y1H strain co-transformed with pGADT7-ERF009 and pAbAi-FTa, the negative control strain and the positive control strain grew well, indicating that the plasmid was successfully transformed into the strain (Wang, ZW, Li, JWand Min, Y., et al..The ERF072 Transcription Factor Directly Regulates MtSOC1-Like Expressionand Mediates Drought-Accelerated Flowering in Medicago truncatula[J].Plant, Cell & Environment, 2025.); The H strain and the positive control strain grew well on SD / -Ura / -Leu plates containing 200 ng / ml AbA, while the negative control was inhibited, indicating that the ERF009 transcription factor interacted directly with the TTGTTG element in the promoter of the MtSPL15 gene and the TCGCGC element in the promoter of the MtFTa gene ( Figure 11 ).

[0146] Example 7

[0147] Dual luciferase activity assay

[0148] 1. Vector Construction: Medicago truncatula genomic DNA was used as a template and primer sequences such as SEQ ID NO. 17 and SEQ ID NO. 18 were used as primers to perform PCR to clone the MtSPL15 gene promoter. The MtSPL15 gene promoter fragment was approximately 2000 bp in length. It was double-digested with Kpn I and BamH I and then ligated to the pGreenII 0800 vector using T4 DNA ligase.

[0149] Using Medicago truncatula genomic DNA as a template and primer sequences such as SEQ ID NO.19 and SEQ ID NO.20 as primers, PCR was performed to obtain the full-length CDS sequence of the ERF009 gene. The full-length CDS sequence of the ERF009 gene was double-digested with BamHI and Kpn I and then ligated into the pGreenII 62SK vector using T4 DNA ligase.

[0150] 2. Transformation of Recombinant Plasmid into Agrobacterium

[0151] (1) Take 500 ng of pGreenII 0800-ProSPL15 recombinant plasmid and pGreenII 62SK-ERF009 recombinant plasmid and add them into 50 μL GV3101 competent cells respectively, put them in ice bath for 5 min, freeze them in liquid nitrogen for 5 min, incubate them at 30℃ for 5 min, and put them in ice water bath for 5 min.

[0152] (2) Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C, 200 rpm for 3 h.

[0153] (3) Centrifuge at 5000 rpm for 1 min, discard 600 μL of supernatant, and suspend the bacteria with the remaining supernatant. Spread the suspension on LB solid dual-antibody culture medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and culture inverted at 28°C for 2 days.

[0154] (4) Pick a single clone and streak it for expansion, and verify the positive strain by bacterial PCR.

[0155] 3. Preparation of infection medium: A single colony of GV3101 containing the pGreenII 0800-ProSPL15 recombinant plasmid and a single colony of GV3101 containing the pGreenII 62SK-ERF009 recombinant plasmid were placed in 5 mL of LB liquid medium (containing 50 mg / L kanamycin and 25 mg / L rifampicin) and cultured at 28°C with shaking at 210 rpm for 24 hours. 100 μL of the bacterial solution was inoculated into 30 mL of double-antibody LB liquid medium, and 500 μL of MES (1 M) and 5 μL of AS (200 mM) were added. The culture was shaken at 200 rpm at 28°C until the OD reached 0. 600 = 1.0. Centrifuge at 8000 rpm for 10 min to collect the cells. Use MES-MgCl2 buffer (Table 16) to suspend the cells and adjust the suspension to OD 600 =1.0, prepare the infection solution and let it stand in the dark for 3h.

[0156] Table 16 MES-MgCl2 buffer

[0157] Element volume MES (1 mol / L) 1mL AS (200mmol / L) 100 μL <![CDATA[MgCl2(1mol / L)]]> 1mL

[0158] 4. Injecting tobacco

[0159] One-month-old Nicotiana benthamiana plants were selected and the impregnation solution was injected into the lower epidermis of the tobacco leaves. The tobacco leaves were incubated in the dark for 8 hours and then under normal light for 36 hours. Four injection combinations were used: the upper left side of the leaf was co-injected with Agrobacterium carrying the pGreenII 62SK empty vector and Agrobacterium carrying the pGreenII 0800 empty vector; the upper right side of the leaf was co-injected with Agrobacterium carrying the pGreenII 62SK empty vector and Agrobacterium carrying the pGreenII 0800-ProMtSPL15 empty vector; and the lower right side of the leaf was co-injected with Agrobacterium carrying the pGreenII 62SK empty vector and Agrobacterium carrying the pGreenII 0800-ProMtSPL15 empty vector.

[0160] 5. In vivo observation

[0161] The color-developing substrate was added to the injection area, and the reaction was carried out in the dark for 10 minutes before being placed in the plant living imaging system. Figure 12 The results showed that a significant fluorescence signal was detected in tobacco leaves injected with ERF009+proMtSPL15, indicating an interaction between the MtERF009 gene and the MtSPL gene promoter in a dual-luciferase assay. Furthermore, the fluorescence signal was stronger in the experimental group (pGreenII62SK-ERF009+pGreenII 0800-ProMtSPL15) than in the control group (pGreenII 62SK+pGreenII0800-ProMtSPL15), indicating that the ERF009 transcription factor can positively regulate the expression of the MtSPL15 gene, thereby regulating the development of lateral branches in the plant.

[0162] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of the Medicago truncatula transcription factor MtERF009 and / or the Medicago flavescentis transcription factor MfERF009 in regulating seed development; the regulating seed development comprises any one or more of the following (1) to (8): (1) Regulate the number of seeds per pod; (2) regulating the number of seeds per plant; (3) regulating the weight of the whole seed plant; (4) regulating seed length; (5) regulating seed width; (6) regulating seed thousand-grain weight; (7) Regulate the number of side branches; (8) Regulate the number of pods.

2. The application according to claim 1, characterized in that The amino acid sequence of MtERF009 is shown in SEQ ID NO.27; the amino acid sequence of MfERF009 is shown in SEQ ID NO.

30.

3. The application according to claim 1, characterized in that Overexpression of the MfERF009 gene increases any one or more of the number of seeds in a single pod, the number of seeds in a whole plant, and the weight of seeds in a whole plant; inhibition of the expression of the MtERF009 gene reduces any one or more of the number of seeds in a single pod, the number of seeds in a whole plant, the weight of seeds in a whole plant, the number of side branches, and the number of pods.

4. The application according to claim 1, characterized in that Inhibiting the expression of the MtERF009 gene can increase any one or more of seed length, seed width and seed 1000-grain weight; overexpressing the MfERF009 gene can reduce any one or more of seed length, seed width and seed 1000-grain weight.

5. The use according to claim 3 or 4, characterized in that: The method for overexpressing the MfERF009 gene comprises: transferring a preparation for overexpressing the MfERF009 gene into alfalfa; the preparation for overexpressing the MfERF009 gene comprises one or more of an expression cassette, a vector or a transgenic cell containing the MfERF009 gene.

6. The use according to claim 3 or 4, characterized in that: The method for inhibiting the expression of the MtERF009 gene comprises: introducing a preparation for inhibiting the expression of the MtERF009 gene into alfalfa; the preparation for inhibiting the expression of the MtERF009 gene comprises one or more of an expression cassette, a vector or a transgenic cell containing the sgDNA gene of the MtERF009 gene.

7. The use according to claim 1, characterized in that The seeds include truncate alfalfa seeds.

8. A primer for preparing a MtERF009 gene editing fragment, characterized in that: Includes the MtERF009 DT1DT2-F0 / R0 primer pair and the MtERF009 DT1DT2-BSF / BSR primer pair; The MtERF009 DT1DT2-BSF / BSR primer pair includes MtERF009 DT1-BSF and MtERF009 DT2-BSR; the nucleotide sequence of the MtERF009 DT1-BSF is shown in SEQ ID NO.7; the nucleotide sequence of the MtERF009 DT2-BSR is shown in SEQ ID NO.10; The MtERF009DT1DT2-F0 / R0 primer pair includes MtERF009 DT1-F0 and MtERF009 DT2-R0; the nucleotide sequence of the MtERF009DT1-F0 is shown in SEQ ID NO.8; the nucleotide sequence of the MtERF009 DT2-R0 is shown in SEQ ID NO.

9.

9. Use of the primer according to claim 8 in preparing a preparation for inhibiting the expression of the MtERF009 gene.

10. A method for regulating alfalfa seed development, characterized in that: include: introducing an agent that inhibits the expression of the MtERF009 gene into alfalfa; Alternatively, a preparation overexpressing the MfERF009 gene is introduced into alfalfa.

Images