A Medicago truncatula leaf development protein, its encoding gene and applications

Abstract

The present invention discloses a Medicago truncatula leaf development protein, its coding gene and applications. First, through sequence analysis, the present invention clones the gene related to plant leaf development from wild-type Medicago truncatula R108, MtPIF4 then overexpresses the gene in Medicago truncatula through transgenic technology, and MtPIF4 identifies and screens out the stably inherited transgenic MtPIF4 Medicago truncatula lines. Finally, through phenotypic analysis of the transgenic MtPIF4 Medicago truncatula lines, it is found that compared with the wild-type R108, the transgenic MtPIF4 Medicago truncatula lines show obvious phenotypes of larger leaves, thicker main stems, increased number of branches and increased plant height. The present invention lays a theoretical foundation for studying the MtPIF4 gene regulating leaf development in plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and more specifically, relates to a Medicago truncatula leaf development protein, its encoding gene, and applications thereof. Background Art

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

[0003] Leguminous plants are not only the main source of protein in human food but also important feed and oil crops. Medicago truncatula ( Medicago truncatula ) is an annual plant belonging to the genus Medicago in the legume family and is another plant with a large-scale genome sequencing completed after Arabidopsis thaliana and rice. The first true leaf of Medicago truncatula after cotyledon emergence is a simple leaf, and it continues to grow and develop into a trifoliate compound leaf 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 RNAs. In past studies, many genes have been identified to form a complex regulatory network involved in leaf growth and development and plant morphogenesis. Since the regulation of plant leaf growth and development requires the coordination of different types of genes, the functional study of key regulatory genes in leguminous plant leaf development is crucial for effectively improving plant yield. Therefore, it is necessary to provide a plant leaf development control gene and the protein encoded by it. Summary of the Invention

[0004] The object of the present invention is to provide a Medicago truncatula leaf development protein, its encoding gene, and applications thereof.

[0005] To achieve the above object, the present invention first provides a protein, which is derived from Medicago truncatula ( Medicago truncatula ) and is named MtPIF4. The MtPIF4 protein is any one of a1), a2), a3), or a4):

[0006] a1) a protein with the amino acid sequence shown in SEQ ID No.2;

[0007] a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No.2;

[0008] a3) A protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No.2;

[0009] a4) A protein with 90% identity to the amino acid sequence shown in SEQ ID No.2 and having the same function.

[0010] In the protein described in a2) above, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing, and / or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues or no more than 9 amino acid residues or no more than 8 amino acid residues or no more than 7 amino acid residues or no more than 6 amino acid residues or no more than 5 amino acid residues or no more than 4 amino acid residues or no more than 3 amino acid residues or no more than 2 amino acid residues or no more than 1 amino acid residue.

[0012] 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 by using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of the amino acid sequence pair, and then the identity value (%) can be obtained. The identity includes amino acid sequences having 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology to the amino acid sequence shown in SEQ ID No.2 of the present invention.

[0013] The protein described in a1), a2), a3), or a4) above can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0014] To achieve the above object, the present invention also provides a biological material related to the above-mentioned MtPIF4 protein.

[0015] The biological material related to the above-mentioned MtPIF4 protein provided by the present invention is a nucleic acid molecule encoding the above-mentioned MtPIF4 protein, or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.

[0016] Among the above biological materials, the nucleic acid molecule is a gene shown in B1) or B2) below:

[0017] B1) A DNA molecule shown in SEQ ID No.1 or SEQ ID No.3;

[0018] B2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in B1) and encoding the above-mentioned MtPIF4 protein.

[0019] Among them, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0020] Those of ordinary skill 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 methods. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence encoding the MtPIF4 protein, as long as they encode the MtPIF4 protein and have the same function, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0021] As used herein, the term "identity" refers to the sequence similarity with a natural nucleic acid sequence. "Identity" includes a nucleotide sequence having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the protein composed of the amino acid sequence shown in SEQ ID No.2 of the present invention. 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.

[0022] The above 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0023] Among the above-mentioned biological materials, the expression cassette refers to DNA capable of expressing the MtPIF4 protein in a host cell, and this DNA may not only include a promoter for MtPIF4 transcription, but may also include a terminator for MtPIF4 transcription. Further, the expression cassette may also include an enhancer sequence. 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 terminator.

[0024] Among the above-mentioned biological materials, the vector can be a plasmid, cosmid, phage or viral vector. The recombinant vector can be a vector constructed using an existing plant expression vector and containing MtPIF4 a gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also contain the 3´ untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3´ end of the mRNA precursor. For example, the genes of Agrobacterium crown gall-inducing (Ti) plasmids (such as the nopaline synthase gene Nos ), and the 3´ untranslated regions transcribed from plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants ( GUS gene, luciferase gene, etc.), a marker gene for antibiotics (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hphgenes, and those that confer resistance to methotrexate dhfr Genes such as the EPSPS gene that confers glyphosate resistance, chemical resistance marker genes (such as herbicide resistance genes), and the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose can be added. For the safety of transgenic plants, it is possible to directly screen transformed plants using stress without adding any selectable marker genes.

[0025] 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, while keeping the other sequences of the pCAMBIA1300-35S:GFP vector unchanged.

[0026] In the aforementioned biological materials, the microorganisms may be yeast, bacteria, algae, or fungi, such as Agrobacterium. Recombinant microorganisms are those obtained by genetically manipulating and modifying the target microorganism, thereby obtaining a functionally altered recombinant microorganism. For example, a recombinant microorganism is obtained by introducing the aforementioned recombinant vector into the target microorganism. Recombinant microorganisms may be understood to refer not only to the specific recombinant microorganism, but also to the progeny of such cells. Due to natural, accidental, or intentional mutations and / or alterations, such progeny may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0027] In some embodiments, the recombinant microorganism comprises the recombinant plasmid 35S: MtPIF4 CDS -GFP-expressing Agrobacterium EHA105.

[0028] In order to achieve the above objectives, the present invention also provides new uses of the above MtPIF4 protein or biomaterial.

[0029] The present invention provides the use of the above-mentioned MtPIF4 protein or biomaterial in the following 1)-6):

[0030] 1) Regulate plant leaf development;

[0031] 2) Regulating plant height;

[0032] 3) Regulate the number of plant branches;

[0033] 4) Regulate plant stem thickness;

[0034] 5) Cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thickened stem diameter;

[0035] 6) Plant breeding.

[0036] In the above applications, the regulation of plant leaf development includes regulating the size of plant leaves and / or the size of individual leaf cells and / or the number of cells contained in a single leaf. The way of regulating plant leaf development is positive regulation, that is: when the content and / or activity of MtPIF4 protein in the plant increases, the plant leaves become larger, the individual leaf cells become larger, and the number of cells contained in a single leaf increases; when the content and / or activity of MtPIF4 protein in the plant decreases or is absent, the plant leaves become smaller, the individual leaf cells become smaller, and the number of cells contained in a single leaf decreases.

[0037] In some embodiments, the regulation of plant leaf development is to promote plant leaf development, specifically manifested as when MtPIF4 the gene expression level increases, the leaf area of the plant becomes larger, the area of individual leaf cells becomes larger, and the number of cells contained in a single leaf increases.

[0038] In the above applications, the way of regulating plant height is positive regulation, that is: when the content and / or activity of MtPIF4 protein in the plant increases, the plant height increases; when the content and / or activity of MtPIF4 protein in the plant decreases or is absent, the plant height decreases.

[0039] In some embodiments, the regulation of plant height is to increase plant height, specifically manifested as when MtPIF4 the gene expression level increases, the plant height increases.

[0040] In the above applications, the way of regulating the number of branches of plants is positive regulation, that is: when the content and / or activity of MtPIF4 protein in the plant increases, the number of branches of the plant increases; when the content and / or activity of MtPIF4 protein in the plant decreases or is absent, the number of branches of the plant decreases.

[0041] In some embodiments, the regulation of the number of branches of plants is to increase the number of branches of plants, specifically manifested as when MtPIF4 the gene expression level increases, the number of branches of the main stem of the plant increases.

[0042] In the above applications, the way of regulating the stem diameter of plants is positive regulation, that is: when the content and / or activity of MtPIF4 protein in the plant increases, the stem diameter increases; when the content and / or activity of MtPIF4 protein in the plant decreases or is absent, the stem diameter decreases.

[0043] In some embodiments, the regulation of the stem diameter of plants is to increase the stem diameter of plants, specifically manifested as when MtPIF4When the gene expression level increases, the main stem diameter of the plant increases.

[0044] In the above application, the purpose of the 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.

[0045] To achieve the above object, the present invention finally provides a method for cultivating transgenic plants with larger leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

[0046] The method for cultivating transgenic plants with larger leaves and / or increased plant height and / or increased number of branches and / or thicker stems provided by the present invention includes the following steps: increasing the content and / or activity of MtPIF4 protein in the recipient plant to obtain transgenic plants with larger leaves and / or increased plant height and / or increased number of branches and / or thicker stems.

[0047] In the above method, the larger leaves are manifested as the leaf area and / or the area of a single cell in the leaf and / or the number of cells contained in a single leaf of the transgenic plant being greater than that of the recipient plant.

[0048] In the above method, the method for increasing the content and / or activity of MtPIF4 protein in the recipient plant is to overexpress the above MtPIF4 protein in the recipient plant.

[0049] Further, the method of overexpression is to introduce the coding gene of MtPIF4 protein into the recipient plant.

[0050] Furthermore, the coding gene sequence of the protein is as shown in SEQ ID No.1 in the sequence listing.

[0051] In any of the above applications or methods, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the recipient plant with the MtPIF4 gene, but also its offspring. For transgenic plants, the gene can be propagated in this species, or transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

[0052] In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0053] Further, the dicotyledonous plant is a leguminous plant.

[0054] Still further, the leguminous plant is a Medicago plant.

[0055] Furthermore, the Medicago plant is Medicago truncatula (such as the R108 ecotype of Medicago truncatula).

[0056] Compared with the prior art, the present invention can achieve the following technical effects:

[0057] 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 diameter. This gene was discovered for the first time in Medicago truncatula and can regulate leaf development, branching, plant height and stem diameter, providing a research basis for innovative research on forage germplasm resources.

[0058] 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 dicotyledons.

[0059] The present invention first cloned a gene related to plant leaf development from Medicago truncatula R108 after sequence analysis. MtPIF4 gene, and then through genetic modification technology MtPIF4 The gene was overexpressed in Medicago truncatula, and the stably inherited transgenic strain was 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, the transgenic 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

[0060] Figure 1 yes MtPIF4 Gene structure and transgene MtPIF4 Medicago truncatula strains 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 strains MtPIF4 The detection of gene expression showed that compared with wild type R108, MtPIF4 Medicago truncatula strains MtPIF4 Gene expression increased tenfold.

[0061] Figure 2 The wild type R108 and the transgenic MtPIF4Phenotype diagrams of Medicago truncatula lines. Among them, A is the phenotype diagram of the leaves of wild-type R108; B is the phenotype diagram of the leaf cells of wild-type R108; C is the MtPIF4 phenotype diagram of the leaves of the Medicago truncatula line; D is the MtPIF4 phenotype diagram of the leaf cells of the Medicago truncatula line; E is the whole-plant phenotype diagram of wild-type R108; F is the MtPIF4 whole-plant phenotype diagram of the Medicago truncatula line; Figure G is the phenotype diagram of the stem diameter of wild-type R108; H is the MtPIF4 phenotype diagram of the stem diameter of the Medicago truncatula line; I is the phenotype diagram of the number of branches of wild-type R108; J is the MtPIF4 phenotype diagram of the number of branches of the Medicago truncatula line.

[0062] Figure 3 Statistics of the leaf phenotypes and leaf area sizes of wild-type R108 and the Medicago truncatula line. MtPIF4 Statistics of the number of cells contained in a single leaf of wild-type R108 and the Medicago truncatula line.

[0063] Figure 4 Statistics of the plant heights of wild-type R108 and the Medicago truncatula line. MtPIF4 Statistics of the main stem diameters of wild-type R108 and the Medicago truncatula line.

[0064] Figure 5 Statistics of the number of main stem branches of wild-type R108 and the Medicago truncatula line. MtPIF4 Specific embodiments

[0065] Figure 6 The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way. MtPIF4

[0066] Figure 7 MtPIF4

[0067]

[0068]

[0069] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0068]

[0069] ​The Medicago truncatula ecotype R108 in the following examples is described in the literature "Comparison of Genome-wide Structural Variations between Two Medicago truncatula Ecotypes: Jemalong A17 and R108[J], BMC Plant Biology, 2022".

[0070] The pCAMBIA1300-35S:GFP vector in the following examples is a product of Cyanidioschyzon merolae, with the product number HZB813118.

[0071] Example 1 MtPIF4 Cloning and Obtaining of Genes

[0072] 1. Extract the RNA of Medicago truncatula ecotype R108 and reverse transcribe it into cDNA.

[0073] 2. Using the cDNA obtained in step 1 as a template, perform amplification with the primer pair composed of primer MtPIF4 CDS -F and primer MtPIF4 CDS -R to obtain a PCR amplification product.

[0074] MtPIF4 CDS -F: 5’-ATGAACAACACCATTCCTGATTG-3’;

[0075] MtPIF4 CDS -R: 5’-TTAACCCATTTTGCCACTCACAG-3’.

[0076] 3. After ligating the PCR amplification product obtained in step 2 to the T vector, perform sequencing to obtain MtPIF4 the coding region sequence (CDS sequence) of the gene, and its nucleotide sequence is as shown in SEQ ID No.1 in the sequence listing. MtPIF4 The gene encodes the protein shown in SEQ ID No.2 in the sequence listing. This protein is composed of 511 amino acid residues. Name the protein shown in SEQ ID No.2 in the sequence listing as MtPIF4. Compare the CDS sequence of the MtPIF4 gene on the Medicago truncatula website (http: / / blast.jcvi.org / Medicago-Blast / ) to obtain MtPIF4 the genomic sequence of the gene, and its nucleotide sequence is as shown in SEQ ID No.3 in the sequence listing. MtPIF4 The structural diagram of the gene is as shown in Figure 1 A.

[0077] Example 2. Preparation of Transgenic Medicago truncatula MtPIF4 I. Recombinant plasmid 35S:

[0078] I. Recombinant plasmid 35S:MtPIF4 CDS -GFP construction

[0079] The pCAMBIA1300-35S:GFP vector was inserted into Kpn I and Sal The DNA fragment between the restriction endonuclease I and II enzyme cutting sites 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.

[0080] 2. Recombinant Agrobacterium 35S: MtPIF4 CDS Construction of -GFP / EHA105

[0081] 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:

[0082] 1. Take out the Agrobacterium EHA105 competent cells stored in -80℃ refrigerator, thaw them on ice, and recombinant plasmid 35S: MtPIF4 CDS Gently add -GFP to the center of the freshly thawed competent cells, flick 3-5 times, place on ice for 30 minutes, quick-freeze in liquid nitrogen for 1 minute, heat shock the competent cells in a water bath at 37°C for 5 minutes, and quickly transfer to ice and let stand for 2 minutes.

[0083] 2. Add 500 µL of YEP liquid medium to the transformed competent cells prepared in step 1, mix thoroughly by inversion, and incubate on a shaker at 220 rpm at 28°C for 2-4 hours. YEP liquid medium contains water as the solvent, 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl as the solutes, and their concentrations, respectively. Adjust the pH to 7.0.

[0084] 3. Take 200µL of the bacterial solution prepared in step 2 and spread it on a YEP plate. Incubate it upside down at 28℃ in the dark for 2 days. Pick a single colony for colony PCR test. 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, storing the recombinant Agrobacterium tumefaciens 35S: MtPIF4 CDS -GFP / EHA105 bacterial liquid.

[0085] III. Genetic transformation of Medicago truncatula

[0086] Using the recombinant Agrobacterium tumefaciens 35S: MtPIF4 CDS -GFP / EHA105 bacterial liquid constructed in Step II, Medicago truncatula ecotype R108 was used as the receptor material for genetic transformation to obtain transgenic Medicago truncatula. The specific steps are as follows:

[0087] 1. Select plump Medicago truncatula ecotype R108 seeds into a 50 mL centrifuge tube, add an appropriate amount of 75% ethanol, and rinse for 1 min; discard the ethanol, add 10 mL of 50% H2SO4, place on a shaker and shake at room temperature for 5 min; discard the concentrated H2SO4, wash with sterile water 3 - 5 times, add 20 mL of 5% sodium hypochlorite, place on a shaker and shake at room temperature for 40 min; discard the sodium hypochlorite, wash with sterile water 5 - 6 times to obtain the washed seeds. Place the washed seeds on the MS solid medium to germinate, and sterile seedlings will grow after 7 days. The solvent of the MS solid medium is water, and the solutes and their concentrations are MS basal medium (M519, Phyto Technology) 4.43 g / L, sucrose 30 g / L, agar 8 g / L, and pH 5.8.

[0088] 2. Take 100 µL of the stored recombinant Agrobacterium tumefaciens 35S: MtPIF4 CDS -GFP / EHA105 bacterial liquid into 20 mL of YEP liquid medium, shake culture at 28 °C and 220 rpm for 14 h, then add As to 150 µM, and shake for another 2 h until the OD value of the bacterial liquid 600 reaches 0.8 - 1.0, centrifuge at 3000 rpm for 10 min to collect the bacteria, and then resuspend with 20 mL of resuspension solution until the OD of the bacterial liquid 600The value reaches 0.5 - 0.6, and it is shaken at 28°C and 80 rpm for 2 h to obtain the prepared Agrobacterium liquid. The solvent of the resuspension solution is water, and the solutes and their concentrations are 20×N6 macro mother liquor 50 mL / L, 200×Ms micro mother liquor 5 mL / L, 200×iron salt mother liquor 5 mL / L, 1000×organic components 1 mL / L, 2,4-D 3 mg / L, KT 0.05 mg / L, MES 0.6 g / L, sucrose 30 g / L, and pH 5.4. Among them, the solvent of the 20×N6 macro mother liquor is water, and the solutes and their concentrations are KNO3 56.6 g / L, (NH4)2SO4 9.2 g / L, CaCl2·2H2O 3.32 g / L, MgSO4·7H2O 3.7 g / L, and KH2PO4 8 g / L. The solvent of the 200×Ms micro mother liquor is water, and the solutes and their concentrations are MnSO4·4H2O 4.46 g / L, ZnSO4·7H2O 1.72 g / L, CuSO4·5H2O 0.005 g / L, Na2MoO4·2H2O 0.05 g / L, CoCl2·6H2O 0.005 g / L, KI 0.166 g / L, and H3BO4 1.24 g / L. The solvent of the 200×iron salt mother liquor is water, and the solutes and their concentrations are FeSO4·7H2O 5.56 g / L and Na2EDTA 7.46 g / L. The solvent of the 1000×organic components is water, and the solutes and their concentrations are VB1 9.9 g / L, VB6 9.5 g / L, and nicotinic acid 4.5 g / L.

[0089] 3. Cut the cotyledons of sterile seedlings grown for 7 - 8 d into small pieces of 3 - 4 mm 2 , cut the hypocotyls into small segments of 2 - 3 mm, pour the prepared explants into the prepared Agrobacterium liquid, ice-bath for 20 min, vacuumize at -0.8 MPa for 10 min, and infect with shaking at 28°C and 80 rpm for 30 min.

[0090] 4. Discard the Agrobacterium liquid, transfer the explants to sterile filter paper, and dry slightly (for more than 30 min). Lay the explants flat on the co-culture medium. A layer of filter paper is laid on the medium in advance, and it is placed in the dark and cultured for 2 - 3 d until the appearance of initial bacterial plaques. The solvent of the co-culture medium is water, and the solutes and their concentrations are 20×N6 macro mother liquor 50 mL / L, 200×Ms micro mother liquor 5 mL / L, 200×iron salt mother liquor 5 mL / L, 1000×organic components 1 mL / L, 2,4-D 3 mg / L, KT 0.05 mg / L, MES 0.6 g / L, sucrose 30 g / L, agar 8 g / L, and pH 5.4.

[0091] 5. After the co-culture, the explants are washed 3 - 5 times with sterile water, air-dried on sterile filter paper, and then transferred to the callus induction medium. Two weeks later, the concentration of hygromycin in the medium is increased to 10 mg / L. The solvent of the callus induction medium is water, and the solutes and their concentrations are as follows: 20×N6 macroelement mother liquor 50 mL / L, 200×Ms microelement mother liquor 5 mL / L, 200×iron salt mother liquor 5 mL / L, 1000×organic components 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, ticarcillin 200 mg / L, hygromycin 5 mg / L, pH 5.8.

[0092] 6. Two weeks later, the green and sticky callus is inoculated onto the differentiation medium and subcultured every two weeks. The torpedo-shaped embryos formed continuously are inoculated onto the rooting medium. The solvent of the differentiation medium is water, and the solutes and their concentrations are as follows: 20×N6 macroelement mother liquor 50 mL / L, 200×Ms microelement mother liquor 5 mL / L, 200×iron salt mother liquor 5 mL / L, 1000×organic components 1 mL / L, KT 0.6 mg / L, acid-hydrolyzed casein 0.5 g / L, sucrose 20 g / L, ticarcillin 200 mg / L, hygromycin 3 mg / L, agar 8 g / L, pH 5.8. The solvent of the rooting medium is water, and the solutes and their concentrations are as follows: MS basal medium 4.43 g / L, sucrose 20 g / L, agar 8 g / L, ticarcillin 200 mg / L, hygromycin 2 mg / L, pH 5.8.

[0093] 7. When the seedlings on the rooting medium grow to a height of about 10 cm and have healthy roots, the sterile seedlings are taken out with forceps, the medium on the roots is washed off, and they are transferred to nutrient soil for further cultivation to obtain transgenic plants.

[0094] IV. Identification of Transgenic Plants

[0095] 1. Genomic DNA of transgenic plants is extracted, and PCR identification is carried out using the primer pair composed of primer ID-35S-F and ID-MtPIF4 CDS -R to obtain transgenic positive plants. The primer sequences are as follows:

[0096] ID-35S-F: 5’-CGACTCTAGAGGATCCCCGGGTACC-3’;

[0097] ID-MtPIF4 CDS -R: 5’-CAAGGTGATGAAACCTCGGAGCCG-3’.

[0098] The agarose gel electrophoresis detection results of the PCR amplification products are as Figure 1 shown in C.

[0099] After two consecutive generations of identification, three transgenic positive lines with stably inheritable phenotypes were obtained.

[0100] 2. Approximately 100 mg of leaves from transgenic positive lines and Medicago truncatula ecotype R108 were selected, ground in liquid nitrogen, and total RNA was extracted according to the Trizol method, reverse transcribed into cDNA, and then qRT-PCR analysis was performed using cDNA as a template. At the same time, actin was used as an internal reference gene to detect MtPIF4 the expression levels of genes in different plants. The primer sequences are as follows:

[0101] MtPIF4-F: 5’-TGGGATCTGGCATGACACCA-3’;

[0102] MtPIF4-R: 5’-AACGGACAGATCCAGAGGCA-3’;

[0103] actin-F: 5’-CAAAAGATGGCAGATGCTGAGGAT-3’;

[0104] actin-R: 5’-CATGACA CCAGTATGACGAGGTCG-3’.

[0105] The analysis results of qRT-PCR are as Figure 1 shown in D. The results show that: in transgenic MtPIF4 Medicago truncatula lines OE-MtPIF4#1 、 OE-MtPIF4#2, and OE-MtPIF4#3, the MtPIF4 expression levels were significantly higher than those in Medicago truncatula ecotype R108. Transgenic MtPIF4 Medicago truncatula lines OE-MtPIF4#1, OE-MtPIF4#2, and OE-MtPIF4#3 were selected for the following phenotypic and biological function analysis.

[0106] Example 3. Phenotypic analysis of transgenic MtPIF4 Medicago truncatula

[0107] Test materials: Medicago truncatula ecotype R108 (abbreviated as wild-type R108), transgenic MtPIF4 Medicago truncatula lines OE-MtPIF4#1, OE-MtPIF4#2, and OE-MtPIF4#3.

[0108] 1. The test materials were planted according to the conventional method. At the branching stage, leaves at the same position (the third true leaf from the top down) of the test materials were gently cut with scissors, then photographed, and the leaf area was statistically analyzed.

[0109] 2. Plant the test materials according to the conventional method, cut the terminal leaflets from the same part 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.

[0110] 3. Plant the test materials according to conventional methods, count the plant height, main stem diameter and number of main stem branches of the test materials during the branching period, and take photos.

[0111] 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 leaf cell size phenotype of 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 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.

[0112] As can be seen from the figure, compared with the wild type R108, the 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, turn 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 branches are obviously more ( Figure 2 I, J, Figure 7 ).

[0113] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of a protein or a biological material related to the protein in the following 1)-5): 1) Promoting plant leaf development; 2) Increasing plant height; 3) Increasing the number of plant branches; 4) Increasing plant stem diameter; 5) Cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thickened stem diameter; The protein is any one of a1) or a2): a1) A protein with an amino acid sequence shown in SEQ ID No.2; a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No.2; The biological material is a nucleic acid molecule encoding the protein, or an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule; The promotion of plant leaf development is manifested as increasing the size of plant leaves and / or making the individual cells of the leaves larger and / or increasing the number of cells contained in a single leaf; The plant is alfalfa.

2. The application according to claim 1, wherein: The nucleic acid molecule is a gene shown in the following B1) or B2): B1) A DNA molecule shown in SEQ ID No.1 or SEQ ID No.3; B2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in B1) and encoding the protein described in claim 1.

3. A method for cultivating transgenic plants with enlarged leaves and / or increased plant height and / or increased number of branches and / or thickened stem diameter, comprising the following steps: increasing the content of the protein described in 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 thickened stem diameter; the plant is alfalfa.

4. The method according to claim 3, wherein: The method for increasing the content of the protein described in claim 1 in the recipient plant is to overexpress the protein described in claim 1 in the recipient plant.

5. The method according to claim 4, wherein: The method for overexpression is to introduce the coding gene of the protein described in claim 1 into the recipient plant.

6. The method according to any one of claims 3-5, characterized in that: The coding gene sequence of the protein is shown in SEQ ID No.1 in the sequence listing.

Images