Application of reagent for overexpressing MsGRF3 gene in promoting plant leaf development and method for promoting plant leaf development

Abstract

The invention belongs to the field of plant genetic engineering, and specifically relates to the application of an agent for overexpressing the MsGRF3 gene in promoting plant leaf development and a method for promoting plant leaf development. The invention discloses the MsGRF3 gene and its application, wherein the nucleotide sequence of the MsGRF3 gene is shown in SEQ ID NO.3; and discloses the application of the gene, a plant overexpression vector and a plant chimeric overexpression vector in regulating leaf size and leaf development in alfalfa.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to application of a reagent for overexpressing MsGRF3 gene in promoting plant leaf development and a method for promoting plant leaf development. Background Art

[0002] Alfalfa, Latin name Medicago sativa L., is an excellent leguminous forage grass. It is known as the "king of forage grass" for its high yield, high quality, high efficiency and high nutrition. As the main harvesting organ of alfalfa, the quality of leaves has important economic value. One of the criteria for measuring leaf quality is the size of leaf development. Leaves are an important carrier for photosynthesis in plants, and are also important organs for aerobic respiration and the production of organic matter. Leaves provide plants with the energy and nutrition needed for growth and development. The size of leaves will directly affect the photosynthesis efficiency of plants, thereby improving the economic benefits of crops. Studying the regulatory mechanism of leaf size during leaf development is of great significance for the cultivation and selection of high-quality alfalfa germplasm resources.

[0003] Transcription factors, abbreviated as TFs, are proteins that can recognize and bind to upstream sequences of transcription start sites, regulate transcription activity through DNA-protein interactions, and determine the temporal and spatial specific expression of different genes. Growth regulatory factors are plant-specific transcription factors that regulate plant growth and development. Growth regulatory factors are abbreviated as GRFs, and specific transcription factors are abbreviated as TFs. GRFs transcription factors have two highly conserved domains in the N-terminal region: the QLQ domain and the WRC domain. QLQ is a functional domain that mediates protein-protein interactions and can interact with GRF interacting factors to form transcriptional activators. The English name of the GRF interacting factor is GRF-Interactingfactor 1, abbreviated as GIF1.

[0004] Currently, the functions of the GRF gene family have been discovered and studied in many species, but have not been reported in alfalfa. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of an agent for overexpressing the MsGRF3 gene in promoting plant leaf development and a method for promoting plant leaf development.

[0006] The invention relates to the use of an agent for overexpressing the MsGRF3 gene in promoting plant leaf development, wherein the nucleotide sequence of the MsGRF3 gene is shown in SEQ ID NO.3.

[0007] Preferably, the leaf development is manifested as an increase in plant leaf area.

[0008] Preferably, the reagent includes primer set 1, primer set 2 or primer set 3;

[0009] The sequence of the primer set 1 is shown in SEQ ID NO.5-6;

[0010] The sequences of the primer set 2 are shown in SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10;

[0011] The sequence of the primer set three is shown in SEQ ID NO.11-12.

[0012] Preferably, the reagent further comprises an expression vector pFGC-eYFP, and the nucleotide sequence of pFGC-eYFP is shown as SEQ ID NO.17.

[0013] Preferably, the method for overexpressing the MsGRF3 gene comprises the following steps:

[0014] The amplified product was obtained by PCR amplification using cDNA from plant leaves containing the MsGRF3 gene as a template;

[0015] The amplified product was connected to the expression vector pFGC-eYFP to obtain an overexpression vector;

[0016] The overexpression vector is used to transform Agrobacterium to upregulate the expression level of the MsGRF3 gene.

[0017] Preferably, the pFGC-eYFP expression vector is digested with restriction endonuclease BamHI, and the amplified product and the expression vector are ligated at a molar ratio of 1:2.

[0018] Preferably, the plant is alfalfa.

[0019] Preferably, the variety of alfalfa is Zhongmu No. 1.

[0020] A method for promoting plant leaf development, which comprises obtaining transgenic plants by overexpressing the MsGRF3 to enhance the promotion of plant leaf development.

[0021] Preferably, the leaf development is manifested as an increase in plant leaf area.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention discloses an application of alfalfa GRF transcription factor and MsGRF3 gene, wherein the nucleotide sequence of the MsGRF3 gene is shown as SEQ ID NO.3; and discloses the application of the gene, a plant overexpression vector and a plant chimeric overexpression vector in regulating leaf size and leaf development in alfalfa.

[0024] The present invention cloned a MsGRF3 gene involved in regulating leaf size in alfalfa, and verified its function through genetic transformation of Arabidopsis thaliana and alfalfa. The leaf area of ​​transgenic Arabidopsis thaliana and alfalfa increased. Studying the regulatory mechanism of leaf size during leaf development is of great significance for cultivating and selecting high-quality alfalfa germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a map of the overexpression vector of the gene sequence of the MsGRFs point mutation site;

[0026] Figure 2 is the map of the MsGRF3 and GIF chimeric vector;

[0027] Figure 3 is the leaf size of overexpressed Arabidopsis, where A is the growth graph, B is the phenotype graph, and C is the leaf area statistical graph;

[0028] Figure 4 is a diagram of the transformation process of alfalfa overexpressing MsGRF3;

[0029] Figure 5 The leaf phenotype and leaf area statistics of alfalfa overexpressing MsGRF3, wherein A is the leaf phenotype and B is the leaf area statistics. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified. Example

[0031] Cloning of CDS of MsGRF3 gene

[0032] 1. Extraction of RNA and synthesis of cDNA from alfalfa

[0033] The present invention uses cultivated alfalfa as plant material. The alfalfa seeds are placed in a mixture containing H 2O in a petri dish and then placed in a germination bag. After 7 days, the germinated seedlings were transferred to 1 / 2 Hoagland nutrient solution for growth and culture, and the nutrient solution was changed every 3 days. The plants were placed in an artificial climate incubator with 16 hours of light and 8 hours of darkness, a day and night temperature of 22°C, a relative humidity of 65%, and a light intensity of 150 μmol·m -2 ·s -1 After the materials grew uniformly, they were used for experimental treatment. Mature leaves of alfalfa were taken and total RNA was extracted using a plant RNA extraction kit. RNA was used as a template and reverse transcribed into cDNA using Novozyme HiScriptIII RT SuperMix for qRT-PCR (+gDNA wiper) for subsequent gene cloning.

[0034] Among them, the plant RNA extraction kit was purchased from TakaraBio.

[0035] Design of primers for amplification of MsGRF3 gene

[0036] Based on the cDNA sequence of the MsGRF3 gene of the "Xinjiang Large Leaf" alfalfa variety whose genome has been published, Beijing Qingke Biotechnology Co., Ltd. synthesized and designed primers for amplifying alfalfa MsGRF3.

[0037] The upstream primer for amplifying MsGRF3, MsGRF3-F: 5'-CAACACCGATGACTTCCT-3', is recorded as SEQ ID NO.1;

[0038] The downstream primer for amplifying MsGRF3, MsGRF3-R: 5'-GAGAGCACTACTTCCAACA-3', recorded as SEQ ID NO. 2;

[0039] The coding region of MsGRF3 was amplified and abbreviated as CDS.

[0040] The obtained cDNA and primers for amplifying the MsGRF3 gene were used to amplify the target gene using the Novatomic FastPure Gel DNA Extraction and the Novatomic 2×Phanta Max Master Mix (Dye Plus):

[0041] The total volume of the PCR reaction system is 50 μL: 2×Phanta Max Master Mix 25uL, upstream primer 10μmol·L -1 2 μL, downstream primer 10 μmol·L -1 2 μL, reverse transcription first-strand cDNA 2 μL, ddH 2 O to make up to 50 μL;

[0042] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 2 min, 34 cycles; 72°C for 5 min; the amplified product was detected by 1.2% agarose gel electrophoresis;

[0043] A sequence of about 1920 bp was obtained, and the obtained PCR product was recovered using the FastPure® Gel DNA Extraction Mini Kit gel recovery / DNA purification kit to obtain MsGRF3 CDS.

[0044]

[0045] The amino acid sequence of MsGRF3 is: , recorded as SEQ ID NO.4. Example

[0046] Construction of MsGRF3 overexpression vector

[0047] In this example, three MsGRF3 overexpression vectors were constructed, which were respectively named 35S:MsGRF3, 35S:MsGRIF3, and 35S:rMsGRF3, so as to illustrate in subsequent studies that overexpression vectors constructed in different ways can affect leaf development.

[0048]

[0049] 1. Construction of overexpression vector 35S:MsGRF3

[0050] The plant overexpression vector of MsGRF3 was constructed by homologous recombination technology. According to the nucleotide sequence of MsGRF3 obtained in Example 1, amplification primers with a BamH I single restriction site were designed:

[0051] The upstream primer MsGRF3-Fa for the amplification of the overexpression vector of MsGRF3 was: 5'-TACATTTACAATTACGGATCCATGGATCTTGGTGGAGTGAGTTTAG-3', recorded as SEQ ID NO.5;

[0052] The downstream primers for MsGRF3-Ra amplification of overexpression vector construction were: 5'-CTCGCCCTTGCCCATGGATCCAGGAGTAAAGGAAGTATTATTACCAAGAGT-3', recorded as SEQ ID NO.6;

[0053] The full-length DNA fragment of MsGRF3 was amplified by PCR using MsGRF3-Fa and MsGRF3-Ra. After the amplification, the PCR product was recovered by gel recovery. The recovered product was used as an insert fragment and connected to the plant expression vector pFGC-eYFP using homologous recombinase. During the connection, the pFGC-eYFP expression vector was cut with restriction endonuclease BamHⅠ to form a linearized vector. The insert fragment and the linearized vector were connected at a molar ratio of 1:2. The reaction system was as follows: 2×ClonExpress Mix 5uL, insert fragment 1uL, linearized vector 2uL, ddH2O supplemented to 10μL, 50℃, 5min, and cooled on ice to obtain the connection product;

[0054] Among them, the homologous recombination enzyme was ClonExpress II One Step Cloning Kit, which was purchased from Novozymes Biopharmaceuticals.

[0055] Transformation of Escherichia coli DH5α competent cells: Add the ligation product to 100μL DH5α competent cells and place in ice for 30min; heat shock at 42℃ for 45s, and place in ice for 2min; add the heat-shocked bacteria to 0.9mL LB liquid and culture at 200rpm on a 37℃ constant temperature shaker for 1h. Centrifuge at 5000rpm for 5min, remove 900μL of the supernatant, and leave 100μL of the resuspended bacteria liquid to be spread on LB solid culture medium, and culture inverted in a 37℃ incubator for 12h. It should be noted that similar effects can be achieved by shaking at 200rpm~250rpm on a 37℃ constant temperature shaker and inverting in a 37℃ incubator for 12h~16h.

[0056] Among them, there is kanamycin on the LB solid culture medium.

[0057] Screening, purification and sequencing of overexpression vector 35S: MsGRF3: Single colonies with ligation products were picked for PCR detection. The results of gel electrophoresis showed that there was a single band at about 2150 bp, indicating that the recombinant vector contained the MsGRF3 gene. The positive single clone was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the sequence of the inserted fragment was completely consistent with the sequence of the MsGRF3 coding region, and the restriction site of the inserted fragment was also completely correct, thus proving that the overexpression vector 35S: MsGRF3 was successfully constructed. The expression frame diagram of the recombinant expression vector is shown in the figure below. Figure 1 .

[0058] Construction of GRF3-GIF1 overexpression vector 35S:MsGRIF3

[0059] GIF, as a transcriptional coactivator of GRF, plays an important regulatory role in plant growth, development and stress response. Therefore, we added 4 alanines between MsGRF3 and MsGIF1 to construct a chimeric vector named 35S:MsGRIF3.

[0060] First, the coding sequence of MsGIF1 was amplified from the cDNA of Xinjiang large-leaf alfalfa using primers MsGIF1-Fa and MsGIF1-Ra. In the second step, the amplified nucleotide sequence of MsGRF3 was used as a template and amplified again using primers MsGRF3-Fa and MsGRF3-Rb. At the same time, the amplified coding sequence of MsGIF1 was used as a template and amplified again using primers MsGIF1-Fb and MsGIF1-Ra, so that MsGRF3 produced a 3' end that overlapped with the 5' end of MsGIF1 by 12 bases to obtain the GRF3-GIF1 chimeric gene.

[0061] MsGIF1-Fa: 5'-ATGTTTTGTCACTATCCCTTCCACC-3', recorded as SEQ ID NO. 7;

[0062] MsGIF1-Ra: 5'-CTAGTTCCCATCATCAGAAGATTTGA-3', recorded as SEQ ID NO. 8;

[0063] MsGIF1-Fb: 5'-GCGGCCGCTGCCATGTTTTGTCACTATCCCTTCCACC-3', recorded as SEQ IDNO.9;

[0064] MsGRF3-Rb: 5'-GCGGCCGCTGCCAGGAGTAAAGGAAGTATTATTACCAAGAGT-3', recorded as SEQ IDNO.10;

[0065] After amplification, the PCR product was recovered by gel recovery to obtain the inserted fragment, and the homologous recombinase was used to perform multi-fragment homologous recombination to connect the plant expression vector pFGC-eYFP. During the connection, the pFGC-eYFP expression vector was cut with the restriction endonuclease BamH Ⅰ to form a linearized vector. The inserted fragment and the linearized vector were connected at a molar ratio of 1:2. The reaction system was as follows: 2×ClonExpress Mix 5uL, 1uL of each inserted fragment, 2uL of linearized vector, ddH 2 Add 0 to 10 μL, incubate at 50°C for 5 min, and cool on ice to obtain the ligation product;

[0066] Among them, the homologous recombination enzyme was ClonExpress II One Step Cloning Kit, which was purchased from Novozymes Biopharmaceuticals.

[0067] Transformation of Escherichia coli DH5α competent cells: Add the ligation product to 100μL DH5α competent cells and place in ice for 30min; heat shock at 42℃ for 45s, and place in ice for 2min; add the heat-shocked bacteria to 0.9mL LB liquid and culture at 200rpm on a 37℃ constant temperature shaker for 1h. Centrifuge at 5000rpm for 5min, remove 900μL of the supernatant, and leave 100μL of the resuspended bacteria liquid to be spread on LB solid culture medium, and culture inverted in a 37℃ incubator for 12h. It should be noted that similar effects can be achieved by shaking at 200rpm~250rpm on a 37℃ constant temperature shaker and inverting in a 37℃ incubator for 12h~16h.

[0068] Among them, there is kanamycin on the LB solid culture medium.

[0069] Screening, purification and sequencing of overexpression vector 35S: MsGRIF3: Single colonies with ligation products were picked for PCR detection. The results of gel electrophoresis showed that there was a single band at around 2658 bp, indicating that the recombinant vector contained the GRF3-GIF1 chimeric gene. The positive monoclonal clone was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the sequence of the inserted fragment was completely consistent with the sequence of the GRF3-GIF1 chimeric gene coding region, and the restriction site of the inserted fragment was also completely correct, thus proving that the GRF3-GIF1 overexpression vector 35S: MsGRIF3 was successfully constructed. The expression frame diagram of the recombinant expression vector is shown in the figure. Figure 1 .

[0070] 3. Construction of the overexpression vector 35S:rMsGRF3 with mutation of miR396 binding site

[0071] miR396 negatively regulates the expression of GRF by cleaving GRF mRNA, affecting the signal transduction pathway during plant growth and development. Therefore, we designed an overexpression vector with mutations in the binding sites of MsGRF3 and miR396, named 35S:rMsGRF3.

[0072] PCR amplification was performed using primers MsGRF3-Fc and MsGRF3-Rc and the constructed overexpression vector 35S:MsGRF3 plasmid as a template.

[0073] MsGRF3-Fc: 5'-CGTTCTAGAAAACCAGTAGAGGGC-3', recorded as SEQ ID NO.11;

[0074] MsGRF3-Rc: 5'-TTTCTAGAACGATGGCGTCCT-3', recorded as SEQ ID NO. 12;

[0075] Amplify the linearized DNA fragment of 35S:rMsGRF3 overexpression vector by PCR:

[0076] The total volume of the PCR reaction system is 50 μL: 2×Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 10 μmol·L -1 2 μL, downstream primer 10 μmol·L -1 2 μL, DNA 2 uL, ddHO 2 O to make up to 50 μL;

[0077] PCR reaction program: 95°C 30s; 95°C 15s, 58°C 15s, 72°C 5min, 35 cycles; 72°C 5min; amplification products were detected by 1.2% agarose gel electrophoresis;

[0078] DpnⅠ digestion was performed before recombination cyclization. The reaction system was: DpnⅠ1uL, amplification product 45uL, and the mixture was gently blown and placed at 37℃ for 1h to obtain the digestion product. It should be noted here that the reaction at 37℃ for 1h~2h can obtain similar effects. After the reaction, the digestion product was connected to the plant expression vector pFGC-eYFP with homologous recombinase for recombination cyclization. The reaction system is as follows: 5×CE Ⅱ Buffer 4uL, DpnⅠ digestion product 300ng, Exnase 2uL, ddH2O supplemented to 20μL, 37℃, 30min, and cooled on ice to obtain the connection product;

[0079] Among them, the homologous recombination enzyme was ClonExpress II One Step Cloning Kit, which was purchased from Novozymes Biopharmaceuticals.

[0080] Transformation of Escherichia coli DH5α competent cells: Add the ligation product to 100μL DH5α competent cells and place in ice for 30min; heat shock at 42℃ for 45s, and place in ice for 2min; add the heat-shocked bacteria to 0.9mL LB liquid and culture at 200rpm on a 37℃ constant temperature shaker for 1h. Centrifuge at 5000rpm for 5min, remove 900μL of the supernatant, and leave 100μL of the resuspended bacteria liquid to be spread on LB solid culture medium, and culture inverted in a 37℃ incubator for 12h. It should be noted that similar effects can be achieved by shaking at 200rpm~250rpm on a 37℃ constant temperature shaker and inverting in a 37℃ incubator for 12h~16h.

[0081] Among them, there is kanamycin on the LB solid culture medium.

[0082] Screening, purification and sequencing of overexpression vector 35S:rMsGRF3: Single colonies with ligation products were picked for PCR detection. The results of gel electrophoresis showed that there was a single band at about 2150bp, indicating that the recombinant vector contained the MsGRF3 gene. The positive monoclonal clone was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the sequence of the inserted fragment was completely consistent with the sequence of the MsGRF3 coding region, the corresponding mutation occurred at the miR396 binding site, and the restriction site of the inserted fragment was also completely correct, thus proving that the overexpression vector 35S:rMsGRF3 with a mutation in the miR396 binding site was successfully constructed. The expression frame diagram of the recombinant expression vector is shown in the figure. Figure 2 . Example

[0083] Overexpression screening and phenotypic observation of Arabidopsis

[0084] The pollen infection method was used to infect Arabidopsis thaliana, which was the Columbia wild type:

[0085] 10 mL of LB solution was placed in a 50 mL centrifuge tube, and the corresponding antibiotics and Agrobacterium GV3101 transformed with the overexpression vectors 35S:MsGRF3, 35S:MsGRIF3, and 35S:rMsGRF3 were added, and cultured overnight at 28°C. The overnight culture was transferred to a conical flask containing 200 mL of LB solution containing antibiotics and cultured at 28°C until OD 600 = 1.2. Centrifuge at 8000×g for 8 min and resuspend in 5% sucrose solution to OD 600 = 0.8, add 0.03% silwet1-77 by volume. Put the buds of 4-week-old flowering Arabidopsis into the resuspension solution for 30 seconds, and then transfer them to light for growth after 24 hours in the dark, and harvest them when the seeds are mature.

[0086] The seeds harvested after Arabidopsis thaliana infection by pollen infection were spread on MS medium containing PPT. Plants that can grow normally are considered to be successfully infected, while wild-type plants turn yellow and wilt. The T1 generation strains with phenotypes were functionally verified. The qPCR results showed that the expression level of GRF3 in the three transgenic strains 35S:MsGRF3, 35S:MsGRIF3, and 35S:rMsGRF3 was significantly higher than that in the wild type. The seeds of these three strains were collected to the T3 generation. After growing for 7 days in MS medium, the seeds were moved into plug trays filled with matrix soil and vermiculite. The mass ratio of matrix soil to vermiculite was 1:2. The phenotype was observed after about 3 weeks of growth. The leaves of 35S:MsGRF3, 35S:MsGRIF3, and 35S:rMsGRF3 transgenic Arabidopsis thaliana were significantly larger than those of the wild type, and the number of leaves increased. Figure 3 shown. Example

[0087] Transformation of alfalfa with MsGRF3 gene and identification of transgenic lines

[0088] Preparation of alfalfa, the variety of alfalfa is Zhongmu No. 1: Select fully extended leaves of 2-month-old alfalfa Zhongmu No. 1 plants, clean the leaf surface with 75% alcohol by volume for 1 minute, immerse the leaves in a 15% sodium hypochlorite solution and gently shake for 10 minutes, rinse the leaves with sterile water until there is no foam, and dry the leaves with filter paper for later use. Among them, 1 drop of Tween is added to the 15% sodium hypochlorite solution by mass. It should be noted that selecting fully extended leaves of 1-2 month-old alfalfa Zhongmu No. 1 plants and adding 1-2 drops of Tween to the 15% sodium hypochlorite solution by mass can achieve similar effects.

[0089] Preparation of Agrobacterium: Take out the frozen Agrobacterium containing gene overexpression vectors 35S:MsGRF3, 35S:MsGRIF3, and 35S:rMsGRF3, and pick a small amount of bacterial solution into 2 mL of LB liquid culture medium containing corresponding antibiotics, and shake and culture at 28°C until the bacterial solution becomes turbid. Take 1 mL of bacterial solution and add it to 50 mL of LB liquid culture medium containing corresponding antibiotics, shake and culture at 28°C to make OD600 reach 0.6, centrifuge at 5000 rpm for 10 minutes, remove the supernatant, add SH3a liquid culture medium to OD600 to 0.2 to resuspend the bacterial solution. It should be noted that taking 1-2 mL of bacterial solution and adding it to 50 mL of LB liquid culture medium containing corresponding antibiotics, shaking and culture at 28°C to make OD600 reach 0.6-0.8, and adding SH3a liquid culture medium to OD600 to resuspend the bacterial solution to 0.2-0.4 can achieve similar effects.

[0090] Transformation of alfalfa: Add all the prepared materials into a sterile bottle, vacuum evacuate for 10 minutes, ultrasonicate in an ice bath for 1 minute, and vacuum evacuate for 10 minutes. Spread the dried leaves on SH3a solid culture medium, culture in the dark for 24 hours, subculture to SH3a selective culture medium, continue to culture in the dark, and change to new culture medium every 2 weeks. After callus formation, place the callus on MSBK culture medium and culture under light for no more than one month. When the callus turns green, immediately place the callus on SH9a culture medium, culture under light, and change to new culture medium every 2 weeks. When the callus grows roots, place the callus on MSO culture medium until the transgenic seedlings mature. Figure 4 shown.

[0091] Example 5: Phenotypic analysis of MsGRF3 gene transformed alfalfa

[0092] Using the MtUBC Q gene as an internal reference, quantitative primers for detection were designed based on the MsGRF3 gene cDNA sequence using the online software https: / / blast.ncbi.nlm.nih.gov / Blast.cgi:

[0093] The upstream quantitative primer of MsGRF3 gene, MsGRF3-qF: 5'-CAACACCGATGACTTCCT-3', recorded as SEQ ID NO. 13;

[0094] Downstream quantitative primer of MsGRF3 gene, MsGRF3-qR: 5'-GAGAGCACTACTTCCAACA-3', recorded as SEQ ID NO.14;

[0095] The upstream quantitative primer of the internal reference gene MtUBC Q-qF: 5'-CTGACAGCCCACTGAATTGTGA-3', recorded as SEQ ID NO.15;

[0096] The downstream quantitative primer of the internal reference gene MtUBC Q-qR: 5'-TTTTGGCATTGCTGCAAGC-3', recorded as SEQ ID NO.16;

[0097] MsGRF3 cDNA was diluted 30 times for quantitative PCR template, and the reaction system was 10 μL: 2×ChamQ SYBRqPCR Master Mix (Vazyme) 5 μL, upstream and downstream primers 10 μM each 0.2 μL, cDNA template 4 μL, sterile water 0.6 μL; PCR reaction conditions were set as: 95℃ 30s; 95℃ 10s, 60℃ 30s, 40 cycles; 95℃ 15s, 60℃ 60s, 95℃ 15s. Each sample was set up for 3 replicates, and MtUBC Q was used as the internal reference gene; after the reaction, the melting curve analysis was performed, and the PCR amplification efficiency was above 95%, and the relative expression of the gene was calculated.

[0098] The results of real-time quantitative PCR showed that the expression level of MsGRF3 in the transgenic strain was significantly higher than that in the wild type.

[0099] Figure 5 The leaf phenotype and leaf area statistics of alfalfa overexpressing MsGRF3, wherein A is the leaf phenotype and B is the leaf area statistics.

[0100] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of overexpressing MsGRF3 gene in promoting plant leaf development, characterized in that: The nucleotide sequence of the MsGRF3 gene is shown in SEQ ID NO.3; The leaf development is manifested as an increase in the number of leaves; The plant is Arabidopsis thaliana.

2. The use according to claim 1, characterized in that: The method for overexpressing the MsGRF3 gene comprises the following steps: The amplified product was obtained by PCR amplification using cDNA from plant leaves containing the MsGRF3 gene as a template; The amplified product was connected to the expression vector pFGC-eYFP to obtain an overexpression vector; The overexpression vector is used to transform Agrobacterium to upregulate the expression level of the MsGRF3 gene; The nucleotide sequence of pFGC-eYFP is shown in SEQ ID NO.

17.

3. The use according to claim 2, characterized in that: The pFGC-eYFP expression vector was digested with restriction endonuclease BamHI, and the amplified product and the expression vector were ligated at a molar ratio of 1:

2.

4. A method for promoting plant leaf development, characterized in that: Obtaining a transgenic plant by overexpressing the MsGRF3 described in claim 1 to enhance the development of plant leaves; The leaf development is manifested as an increase in the number of leaves on the plant; The plant is Arabidopsis thaliana.

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