Medicago truncatula leaf development protein as well as coding gene and application thereof

Abstract

The invention discloses medicago truncatula leaf development protein as well as a coding gene and application thereof. The method comprises the following steps: firstly, cloning an MtPIF4 gene related to plant leaf development from wild medicago truncatula R108 through sequence analysis, then overexpressing the MtPIF4 gene in medicago truncatula through a transgenic technology, and identifying and screening a transgenic MtPIF4 medicago truncatula strain with stable inheritance. And finally, through phenotype analysis on the transfected MtPIF4 medicago truncatula strain, it is found that compared with a wild type R108, the transfected MtPIF4 medicago truncatula strain has obvious phenotypes of leaf enlargement, main stem thickening, branch number increase and plant height increase. The invention lays a theoretical foundation for researching the MtPIF4 gene in regulating and controlling leaf development in plants.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Medicago truncatula leaf development protein and a coding gene and application thereof. Background Art

[0002] Plant biomass is an important production trait. Leaf size is an important indicator for evaluating plant biomass, which directly affects plant yield, and high biomass yield is a key target trait in crop breeding. Leaves are the main organs for photosynthesis in plants. The size of leaf area directly affects the light interception ability of plants and plays an important role in plant growth and development as well as the establishment of yield traits. Therefore, studying the leaf development process is of great significance for high-yield and high-quality crop breeding.

[0003] Leguminosae is not only the main source of protein in human food, but also an important feed and oil crop. Medicago truncatula ) is an annual plant of the genus Medicago in the Leguminosae family. It is another plant with a large-scale genome sequencing after the completion of the genome sequencing of Arabidopsis thaliana and rice. After the cotyledons of Medicago truncatula emerge from the soil, the first true leaf is a simple leaf, which continues to grow and unfold into three-pinnate compound leaves until the entire life cycle is completed. This process requires a balance between cell proliferation and cell expansion, and is also regulated by light conditions, plant hormones and small molecule RNA. In past studies, many genes have been identified to form a complex regulatory network that is involved in leaf growth and development and plant morphology. Since the regulation of plant leaf growth and development requires the coordination of different types of genes, the functional study of key regulatory genes for leaf development in leguminous plants is crucial to effectively increase plant yields. Therefore, it is necessary to provide a plant leaf development control gene and the protein it encodes. Summary of the invention

[0004] The purpose of the present invention is to provide a Medicago truncatula leaf development protein and its encoding gene and application.

[0005] In order to achieve the above object, the present invention first provides a protein derived from Medicago truncatula ( Medicago truncatula ), named MtPIF4, the MtPIF4 protein is any one of a1) or a2) or a3) or a4): a1) the amino acid sequence is the protein shown in SEQ ID No. 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID No. 2; a3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; a4) A protein having 90% identity with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

[0006] In the protein described in a2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0007] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0008] In the protein described in a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively and searching 1, the identity of the amino acid sequence is calculated, and then the value of the identity (%) can be obtained. The identity includes an amino acid sequence having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in SEQ ID No. 2 of the present invention.

[0009] The protein described in a1) or a2) or a3) or a4) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0010] In order to achieve the above object, the present invention further provides biological materials related to the above MtPIF4 protein.

[0011] The biological material related to the MtPIF4 protein provided by the present invention is a nucleic acid molecule encoding the MtPIF4 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule.

[0012] In the above biological material, the nucleic acid molecule is the gene shown in B1) or B2) below: B1) a DNA molecule represented by SEQ ID No.1 or SEQ ID No.3; B2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in B1) and encodes the above-mentioned MtPIF4 protein.

[0013] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0014] Those skilled in the art can easily mutate the nucleotide sequence encoding the MtPIF4 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding the MtPIF4 protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the MtPIF4 protein and have the same function.

[0015] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.2. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0016] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0017] In the above biological material, the expression cassette refers to a DNA capable of expressing MtPIF4 protein in a host cell, which may include a promoter MtPIF4 The promoter of transcription may also include the terminator MtPIF4The expression cassette may further include an enhancer sequence. The promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminators.

[0018] In the above biological materials, the vector can be a plasmid, a cosmid, a phage or a virus vector. The recombinant vector can be constructed by using an existing plant expression vector containing MtPIF4 Vectors of gene expression cassettes. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos ), the non-translated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes ( GUS genes, luciferase genes, etc.), antibiotic marker genes (such as those that confer resistance to kanamycin and related antibiotics nptII Gene that confers resistance to the herbicide phosphinothricin bar Gene that confers resistance to the antibiotic hygromycin hph genes, and those conferring resistance to methotrexate dhfrGenes, such as EPSPS genes that confer resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0019] In some embodiments, the recombinant vector may be a recombinant plasmid 35S: MtPIF4 CDS -GFP. The recombinant plasmid 35S: MtPIF4 CDS -GFP is the pCAMBIA1300-35S:GFP vector Kpn I and Sal The plasmid was obtained by replacing the DNA fragment between the restriction endonuclease I sites with the DNA molecule shown in SEQ ID No. 1 in the sequence list, and keeping the other sequences of the pCAMBIA1300-35S:GFP vector unchanged.

[0020] In the above-mentioned biological materials, the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium. The recombinant microorganism refers to a recombinant microorganism with a changed function obtained by operating and modifying the genes of the target microorganism. For example, a recombinant microorganism obtained by introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood to refer not only to a specific recombinant microorganism, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0021] In some embodiments, the recombinant microorganism is a recombinant plasmid containing the above-mentioned 35S: MtPIF4 CDS -GFP-expressing Agrobacterium EHA105.

[0022] In order to achieve the above objectives, the present invention also provides new uses of the above MtPIF4 protein or biomaterial.

[0023] The present invention provides the use of the above-mentioned MtPIF4 protein or biological material in the following 1)-6): 1) Regulate plant leaf development; 2) Regulate plant height; 3) Regulate the number of plant branches; 4) Regulate plant stem thickness; 5) Cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems; 6) Plant breeding.

[0024] In the above application, the regulation of plant leaf development includes regulating the size of plant leaves and / or the size of individual cells in the leaves and / or the number of cells contained in a single leaf. The manner of regulating plant leaf development is positive regulation, that is, when the protein content and / or activity of MtPIF4 in the plant increases, the plant leaves become larger, the individual cells in the leaves become larger, and the number of cells contained in a single leaf increases; when the protein content and / or activity of MtPIF4 in the plant decreases or is absent, the plant leaves become smaller, the individual cells in the leaves become smaller, and the number of cells contained in a single leaf decreases. In some embodiments, regulating plant leaf development is promoting plant leaf development, which is specifically embodied in that when MtPIF4 When the gene expression level increases, the plant leaf area increases, the area of ​​a single cell in the leaf increases, and the number of cells contained in a single leaf increases.

[0025] In the above application, the method of regulating plant height is positive regulation, that is: when the MtPIF4 protein content and / or activity in the plant increases, the plant height increases; when the MtPIF4 protein content and / or activity in the plant decreases or is absent, the plant height decreases.

[0026] In some embodiments, the regulating plant height is to increase the plant height, which is specifically embodied in that when the plant MtPIF4 When gene expression is increased, plant height increases.

[0027] In the above application, the method of regulating the number of plant branches is positive regulation, that is: when the MtPIF4 protein content and / or activity in the plant increases, the number of plant branches increases; when the MtPIF4 protein content and / or activity in the plant decreases or is absent, the number of plant branches decreases.

[0028] In some embodiments, the regulating the number of plant branches is to increase the number of plant branches, which is specifically embodied by MtPIF4 When the gene expression level increases, the number of branches on the main stem of the plant increases.

[0029] In the above application, the method of regulating plant stem thickness is positive regulation, that is: when the MtPIF4 protein content and / or activity in the plant increases, the plant stem thickness increases; when the MtPIF4 protein content and / or activity in the plant decreases or is absent, the plant stem thickness decreases.

[0030] In some embodiments, the regulating plant stem thickness is to increase the plant stem thickness, which is specifically embodied in that when the plant MtPIF4 When the gene expression level increases, the thickness of the plant's main stem increases.

[0031] In the above application, the purpose of plant breeding is to cultivate plant varieties with larger leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

[0032] In order to achieve the above objectives, the present invention finally provides a method for cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

[0033] The method for cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems provided by the present invention comprises the following steps: increasing the content and / or activity of MtPIF4 protein in the recipient plant to obtain transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

[0034] In the above method, the enlargement of the leaves is reflected in that the leaf area and / or the area of ​​a single cell in the leaves and / or the number of cells contained in a single leaf of the transgenic plant are larger than those of the recipient plant.

[0035] In the above method, the method for increasing the content and / or activity of the MtPIF4 protein in the recipient plant is to overexpress the MtPIF4 protein in the recipient plant.

[0036] Furthermore, the overexpression method is to introduce the coding gene of the MtPIF4 protein into the recipient plant.

[0037] Furthermore, the coding gene sequence of the protein is shown as SEQ ID No. 1 in the sequence listing.

[0038] In any of the above-mentioned uses or methods, the transgenic plant is understood to include not only MtPIF4 The first generation of transgenic plants obtained by gene transformation of the recipient plant also includes its progeny. For transgenic plants, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, complete plants and cells.

[0039] In any of the above uses or methods, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0040] Furthermore, the dicotyledonous plant is a leguminous plant.

[0041] Furthermore, the leguminous plant is a plant of the genus Medicago.

[0042] Furthermore, the Medicago plant is alfalfa (such as Medicago truncatula ecotype R108).

[0043] Compared with the prior art, the present invention can achieve the following technical effects: 1. The present invention cloned a gene in Medicago truncatula that can promote cell growth, increase leaf area, promote branching, and increase plant height and stem thickness. This gene was first discovered in Medicago truncatula and can regulate leaf development, branching, plant height and stem thickness, providing a research basis for innovative research on forage germplasm resources.

[0044] 2. The cloned genes in the present invention can also provide evidence for the study of genes related to leaf development in leguminous crops such as Medicago truncatula and other dicotyledonous plants.

[0045] The present invention first cloned a gene related to plant leaf development from Medicago truncatula R108 after sequence analysis. MtPIF4 Genes, and then through genetic modification technology MtPIF4 The gene was overexpressed in Medicago truncatula, and the stable transgenic strains were identified and screened. MtPIF4 Medicago truncatula strains, finally through the transformation MtPIF4 Phenotypic analysis of Medicago truncatula strains revealed that compared with wild type R108, MtPIF4 The Medicago truncatula strain showed obvious phenotypes of enlarged leaves, thickened main stems, increased number of branches and increased plant height. MtPIF4 This laid the theoretical foundation for how genes regulate leaf development in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 yes MtPIF4 Gene structure and transfer MtPIF4 Medicago truncatula strain MtPIF4 Detection of gene expression. Where A is MtPIF4 Gene structure diagram; B is the recombinant plasmid 35S: MtPIF4 CDS -GFP structure diagram; C is DNA level identification of transfection MtPIF4 Compared with wild type R108, the Medicago truncatula strains 35S Specific primers and MtPIF4 The gene primers amplified a fragment of 488 bp; D is the wild-type R108 and the transgenic MtPIF4 Medicago truncatula strain MtPIF4 Compared with wild-type R108, the expression of MtPIF4 Medicago truncatula strain MtPIF4 The expression of the gene increased tenfold.

[0047] Figure 2 The wild type R108 and the transgenic MtPIF4 Phenotypic diagram of Medicago truncatula strains. A is the phenotype diagram of wild-type R108 leaves; B is the phenotype diagram of wild-type R108 leaf cells; C is the phenotype diagram of transgenic MtPIF4 Phenotypic diagram of leaves of Medicago truncatula strains; D is the MtPIF4Phenotypic diagram of leaf cells of Medicago truncatula strains; E is the whole plant phenotype of wild type R108; F is the transgenic MtPIF4 Figure 2 shows the whole plant phenotype of Medicago truncatula strains; Figure G shows the stem thickness phenotype of wild type R108; Figure H shows the stem thickness phenotype of transgenic MtPIF4 Stem thickness phenotype of Medicago truncatula strains; I is the branch number phenotype of wild type R108; J is the transgenic MtPIF4 Branch number phenotype of Medicago truncatula lines.

[0048] Figure 3 For wild-type R108 and transgenic MtPIF4 Statistical results of leaf phenotype and leaf area size of Medicago truncatula strains.

[0049] Figure 4 For wild-type R108 and transgenic MtPIF4 Statistical results of the number of cells contained in a single leaf of Medicago truncatula strains.

[0050] Figure 5 For wild-type R108 and transgenic MtPIF4 Statistical results of plant height of Medicago truncatula strains.

[0051] Figure 6 For wild-type R108 and transgenic MtPIF4 Statistical results of main stem diameter of Medicago truncatula strains.

[0052] Figure 7 For wild-type R108 and transgenic MtPIF4 Statistical results of the number of main stem branches of Medicago truncatula strains. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0055] The Medicago truncatula ecotype R108 in the following example is recorded in the document “Comparison of whole genome sequence structural variations of two Medicago truncatula ecotypes: Jemalong A17 and R108 [J], BMC Plant Biology, 2022”.

[0056] The pCAMBIA1300-35S:GFP vector in the following examples is a product of Glaucomannan Biotechnology, with the product number being HZB813118.

[0057] Embodiment 1, MtPIF4 Gene cloning and acquisition 1. Extract RNA from Medicago truncatula ecotype R108 and reverse transcribe it into cDNA.

[0058] 2. Using the cDNA obtained in step 1 as a template, use primer MtPIF4 CDS -F and primer MtPIF4 CDS -R primer pair to obtain PCR amplification products.

[0059] MtPIF4 CDS -F:5'-ATGAACAACACCATTCCTGATTG-3'; MtPIF4 CDS -R:5'-TTAACCCATTTTGCCACTCACAG-3'.

[0060] 3. Connect the PCR amplification product obtained in step 2 to the T vector and sequence it to obtain MtPIF4 The coding region sequence (CDS sequence) of the gene, whose nucleotide sequence is shown in SEQ ID No.1 in the sequence listing. MtPIF4 The gene encodes a protein shown in SEQ ID No. 2 in the sequence list, which is composed of 511 amino acid residues. The protein shown in SEQ ID No. 2 in the sequence list is named MtPIF4. MtPIF4 The CDS sequence of the gene was compared on the Medicago truncatula website (http: / / blast.jcvi.org / Medicago-Blast / ) and the MtPIF4 The genomic sequence of the gene, its nucleotide sequence is shown as SEQID No.3 in the sequence table. MtPIF4 The structure of the gene Figure 1 As shown in A.

[0061] Embodiment 2, transfer MtPIF4 Preparation of Medicago truncatula 1. Recombinant plasmid 35S: MtPIF4 CDS -GFP construction The pCAMBIA1300-35S:GFP vector was inserted into Kpn I and SalThe DNA fragment between the restriction sites I was replaced with the DNA molecule shown in SEQ ID No.1 in the sequence list, and the other sequences of the pCAMBIA1300-35S:GFP vector were kept unchanged to obtain the recombinant plasmid 35S: MtPIF4 CDS -GFP. Recombinant plasmid 35S: MtPIF4 CDS -GFP structure diagram Figure 1 As shown in B.

[0062] 2. Recombinant Agrobacterium 35S: MtPIF4 CDS -GFP / EHA105 construction The recombinant plasmid 35S constructed in step 1: MtPIF4 CDS -GFP was introduced into Agrobacterium EHA105, and recombinant Agrobacterium 35S was obtained after identification: MtPIF4 CDS -GFP / EHA105, the specific steps are as follows: 1. Take out the competent cells of Agrobacterium tumefaciens EHA105 stored in -80℃ refrigerator, thaw them on ice, and transfer the recombinant plasmid 35S: MtPIF4 CDS -GFP was gently added to the center of the freshly thawed competent cells, flicked 3-5 times, placed in an ice bath for 30 minutes, and quickly frozen in liquid nitrogen for 1 minute. The competent cells were heat-shocked at 37°C in a water bath for 5 minutes, and quickly placed on ice for 2 minutes.

[0063] 2. Add 500µL YEP liquid medium to the competent cells after transformation prepared in step 1, mix by inversion, and place on a shaker at 28°C and shake at 220rpm for 2-4 hours. The solvent of YEP liquid medium is water, and the solutes and their concentrations are 10g / L tryptone, 10g / L yeast extract, and 5g / L NaCl, respectively. Adjust the pH to 7.0.

[0064] 3. Take 200 µL of the bacterial solution prepared in step 2 and spread it on a YEP plate. Incubate it upside down at 28°C in the dark for 2 days. Pick a single colony for colony PCR detection. The test result is positive, indicating that the recombinant plasmid 35S: MtPIF4 CDS -GFP has been successfully introduced into Agrobacterium, and the single clone with positive test results was named recombinant Agrobacterium 35S: MtPIF4 CDS -GFP / EHA105, preservation of recombinant Agrobacterium 35S: MtPIF4 CDS -GFP / EHA105 bacterial solution.

[0065] 3. Genetic transformation of Medicago truncatula Using the recombinant Agrobacterium 35S constructed in step 2: MtPIF4 CDS -GFP / EHA105 bacterial solution, using Medicago truncatula ecotype R108 as the recipient material for genetic transformation to obtain transgenic Medicago truncatula, the specific steps are as follows: 1. Select full R108 seeds of Medicago truncatula ecotype in a 50mL centrifuge tube, add an appropriate amount of 75% ethanol, rinse for 1min; discard the ethanol, add 10mL 50% H2SO4, shake on a shaker at room temperature for 5min; discard the concentrated H2SO4, wash 3-5 times with sterile water, add 20mL 5% sodium hypochlorite, shake on a shaker at room temperature for 40min; discard the sodium hypochlorite, wash 5-6 times with sterile water to obtain the washed seeds. The washed seeds were placed on MS solid culture medium for germination, and sterile seedlings grew after 7 days. The solvent of MS solid culture medium is water, and the solutes and their concentrations are MS basic culture medium (M519, Phyto Technology) 4.43g / L, sucrose 30g / L, agar 8g / L, pH 5.8.

[0066] 2. Take 100 µL of the stored recombinant Agrobacterium tumefaciens 35S: MtPIF4 CDS -GFP / EHA105 bacterial solution was added to 20 mL YEP liquid medium, and cultured at 28°C and 220 rpm for 14 h. Then As was added to 150 µM and shaken for another 2 h until the bacterial solution OD 600 When the value reaches 0.8-1.0, centrifuge at 3000rpm for 10min to collect the bacteria, and then resuspend them in 20mL of resuspension solution to the OD value of the bacterial solution. 600The value reached 0.5-0.6, and the suspension was vibrated at 28°C and 80rpm for 2h to obtain the prepared Agrobacterium bacterial solution. The solvent of the resuspension was water, and the solutes and their concentrations were 20×N6 bulk mother solution 50mL / L, 200×Ms micro-mother solution 5mL / L, 200×iron salt mother solution 5mL / L, 1000×organic component 1mL / L, 2,4-D 3mg / L, KT 0.05mg / L, MES 0.6g / L, sucrose 30g / L, pH 5.4. Among them, the solvent of 20×N6 bulk mother solution was water, and the solutes and their concentrations were KNO3 56.6g / L, (NH4)2SO4 9.2g / L, CaCl2·2H2O 3.32g / L, MgSO4·7H2O 3.7g / L, KH2PO4 8g / L. The solvent of 200×Ms trace mother solution is water, and the solutes and their concentrations are MnSO4·4H2O 4.46g / L, ZnSO4·7H2O 1.72g / L, CuSO4·5H2O 0.005g / L, Na2MoO4·2H2O0.05g / L, CoCl2·6H2O 0.005g / L, KI 0.166g / L, H3BO41.24g / L. The solvent of 200×ferric salt mother solution is water, and the solutes and their concentrations are FeSO4·7H2O 5.56g / L, Na2EDTA 7.46g / L. The solvent of 1000×organic component is water, and the solutes and their concentrations are VB19.9g / L, VB69.5g / L, and niacin 4.5g / L.

[0067] 3. Cut the cotyledons of sterile seedlings grown for 7-8 days into 3-4 mm 2 Cut the hypocotyl into small pieces of 2-3 mm, pour the prepared explants into the prepared Agrobacterium solution, bathe on ice for 20 minutes, vacuum at -0.8 MPa for 10 minutes, and slow-infect at 28℃ and 80rpm for 30 minutes.

[0068] 4. Discard the Agrobacterium bacterial solution, transfer the explants to sterile filter paper, dry them slightly (more than 30 minutes), and spread the explants on the co-culture medium. A layer of filter paper is laid on the medium in advance, and it is placed in the dark. Culture for 2-3 days until the bacterial plaques begin to appear. The solvent of the co-culture medium is water, and the solutes and their concentrations are 20×N6 macromolecular solution 50mL / L, 200×Ms micromolecular solution 5mL / L, 200×iron salt mother solution 5mL / L, 1000×organic component 1mL / L, 2,4-D 3mg / L, KT 0.05mg / L, MES 0.6g / L, sucrose 30g / L, agar 8g / L, pH 5.4.

[0069] 5. After the co-cultivation, the explants were washed 3-5 times with sterile water, dried on sterile filter paper, and transferred to callus induction medium. After two weeks, the hygromycin concentration in the medium was increased to 10 mg / L. The solvent of the callus induction medium was water, and the solutes and their concentrations were 20×N6 macromolecular solution 50 mL / L, 200×Ms micromolecular solution 5 mL / L, 200×iron salt solution 5 mL / L, 1000×organic component 1 mL / L, 2,4-D 2 mg / L, KT 0.05 mg / L, acid hydrolyzed casein 0.5 g / L, sucrose 30 g / L, timentin 200 mg / L, hygromycin 5 mg / L, pH 5.8.

[0070] 6. After another 2 weeks, the green, sticky callus was inoculated on the differentiation medium, subcultured every 2 weeks, and the continuously formed torpedo-shaped embryos were inoculated on the rooting medium. The solvent of the differentiation medium is water, and the solutes and their concentrations are 20×N6 macromolecular solution 50mL / L, 200×Ms micromolecular solution 5mL / L, 200×iron salt solution 5mL / L, 1000×organic component 1mL / L, KT 0.6mg / L, acid hydrolyzed casein 0.5g / L, sucrose 20g / L, timentin 200mg / L, hygromycin 3mg / L, agar 8g / L, pH 5.8. The solvent of the rooting medium is water, and the solutes and their concentrations are MS basic medium 4.43g / L, sucrose 20g / L, agar 8g / L, timentin 200mg / L, hygromycin 2mg / L, pH 5.8.

[0071] 7. When the seedlings on the rooting medium grow to a height of about 10 cm and the root system is healthy, use tweezers to remove the sterile seedlings, wash the root medium, and move them into nutrient soil for further cultivation to obtain transgenic plants.

[0072] 4. Identification of transgenic plants 1. Extract genomic DNA of transgenic plants using primers ID-35S-F and ID-MtPIF4 CDS -R primer pair was used for PCR identification to obtain transgenic positive plants. The primer sequences are as follows: ID-35S-F: 5'-CGACTCTAGAGGATCCCCGGGTACC-3'; ID-MtPIF4 CDS -R: 5'-CAAGGTGATGAAACCTCGGAGCCG-3'.

[0073] The results of agarose gel electrophoresis of PCR amplification products were as follows Figure 1 As shown in C.

[0074] After two generations of identification, three transgenic positive lines with stably inherited phenotypes were obtained.

[0075] 2. About 100 mg of leaves from transgenic positive strains and Medicago truncatula ecotype R108 were selected and ground in liquid nitrogen. Total RNA was extracted according to the Trizol method and reversely transcribed into cDNA. Then, qRT-PCR analysis was performed using cDNA as a template and actin as an internal reference gene. MtPIF4 The expression levels of genes in different plants. The primer sequences are as follows: MtPIF4-F: 5'-TGGGATCTGGCATGACACCA-3'; MtPIF4-R: 5'-AACGGACAGATCCAGAGGCA-3'; actin-F: 5'-CAAAAGATGGCAGATGCTGAGGAT-3'; actin-R: 5'-CATGACA CCAGTATGACGAGGTCG-3'.

[0076] The results of qRT-PCR analysis are shown in Figure 1 D shows that: MtPIF4 Medicago truncatula strain OE-MtPIF4#1 、 OE-MtPIF4#2, OE-MtPIF4#3 MtPIF4 The expression levels were significantly higher than those of Medicago truncatula ecotype R108. MtPIF4 Medicago truncatula lines OE-MtPIF4#1, OE-MtPIF4#2, and OE-MtPIF4#3 were used for the following phenotypic and biological function analyses.

[0077] Embodiment 3, transfer MtPIF4 Phenotypic Analysis of Medicago truncatula Test materials: Medicago truncatula ecotype R108 (referred to as wild type R108), MtPIF4 Medicago truncatula strains OE-MtPIF4#1, OE-MtPIF4#2, and OE-MtPIF4#3.

[0078] 1. Plant the test materials according to the conventional method. During the branching period, select the leaves from the same part of the test materials (the third true leaf from the top), cut them gently with scissors, take pictures, and count the leaf area.

[0079] 2. Plant the test materials according to the conventional method, cut the terminal leaflets at the same position of the test materials during the branching period, decolorize them with 70% ethanol, observe the cell size at the same magnification under a stereomicroscope, then take pictures, and count the single cell area of ​​the leaf and the number of cells contained in a single leaf.

[0080] 3. Plant the test materials according to conventional methods, count the plant height, main stem thickness and number of main stem branches of the test materials during the branching period, and take photos.

[0081] Phenotypic observations Figure 2 As shown, A is the leaf phenotype of wild-type R108R108, B is the leaf cell size phenotype of wild-type R108R108; C is the transformed MtPIF4 Leaf phenotype of Medicago truncatula OE#1; D is the transformed MtPIF4 Leaf cell size phenotype of Medicago truncatula OE#1; E is the whole plant phenotype of wild type R108; F is the transgenic MtPIF4 The whole plant phenotype of Medicago truncatula strain OE#1; G is the stem thickness phenotype of wild type R108; H is the transgenic MtPIF4 The stem thickness phenotype of Medicago truncatula strain OE#1; I is the branch number phenotype of wild type R108; J is the transgenic MtPIF4 Branch number phenotype of Medicago truncatula line OE#1.

[0082] As can be seen from the figure, compared with the wild-type R108, MtPIF4 The leaf area and single cell area of ​​Medicago truncatula strains increased significantly ( Figure 2 AD, Figure 3 ), and the total number of cells contained in a single leaf also increased significantly ( Figure 4 ), while plant height also increased significantly ( Figure 2 E, F, Figure 5 ). In addition, MtPIF4 The main stem diameter and number of main stem branches of Medicago truncatula strains also changed. Compared with the wild type R108, the stem diameter was significantly thicker ( Figure 2 G, H, Figure 6 ), and the number of branches has increased significantly ( Figure 2 I, J, Figure 7 ).

[0083] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Protein, the protein being any one of a1), a2), a3), or a4): a1) the amino acid sequence is the protein shown in SEQ ID No. 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID No. 2; a3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; a4) A protein having 90% identity with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

2. A biological material related to the protein according to claim 1, wherein the biological material is a nucleic acid molecule encoding the protein according to claim 1 or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule.

3. The biomaterial according to claim 2, Features: The nucleic acid molecule is the gene shown in B1) or B2) below: B1) a DNA molecule represented by SEQ ID No.1 or SEQ ID No.3; B2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in B1) and encodes the protein of claim 1.

4. Use of the protein according to claim 1 or the biomaterial according to claim 2 or 3 in the following 1)-6): 1) Regulate plant leaf development; 2) Regulate plant height; 3) Regulate the number of plant branches; 4) Regulate plant stem thickness; 5) Cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems; 6) Plant breeding.

5. The use according to claim 4, Features: The regulating plant leaf development includes regulating the plant leaf size and / or the size of a single cell in the leaf and / or the number of cells contained in a single leaf.

6. A method for cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems, comprising the following steps: increasing the content and / or activity of the protein according to claim 1 in a recipient plant to obtain transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

7. The method according to claim 6, Features: The method for increasing the content and / or activity of the protein of claim 1 in a recipient plant is to overexpress the protein of claim 1 in the recipient plant.

8. The method according to claim 7, Features: The overexpression method is to introduce the gene encoding the protein of claim 1 into a recipient plant.

9. The method according to any one of claims 6 to 8, Features: The coding gene sequence of the protein is shown in SEQ ID No. 1 in the sequence listing.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, Features: The plant is a dicotyledon or a monocotyledon; And / or, the dicotyledonous plant is a leguminous plant; And / or, the leguminous plant is a plant of the genus Medicago; And / or, the alfalfa plant is alfalfa; And / or, the alfalfa is Medicago truncatula.

Images