Method for establishing trifolium repens protoplast separation and instantaneous conversion system

Abstract

The invention discloses a method for establishing a trifolium repens protoplast separation and instantaneous transformation system. The method comprises the following steps: selecting tender and disease-free trifolium repens leaves for enzymolysis; washing the protoplast liquid with a pre-cooled modified W5 solution, and centrifuging the protoplast liquid to obtain a protoplast precipitate; mixing the plasmid with the protoplast, and adding a PEG-Ca < 2 + > solution; standing for 5 minutes at room temperature, so that the plasmids are in full contact with the protoplast; adding a W5 solution and a protoplast suspension, and incubating at room temperature in darkness; and observing the conversion effect through a confocal microscope. According to the method, the separation efficiency and purity of the trifolium repens protoplast are remarkably improved, the yield of the protoplast is improved, the biological activity of the protoplast and the reliability of subsequent experiments are guaranteed, and more efficient and stable technical support is provided for research and application of the trifolium repens; according to the method, the efficiency and success rate of instantaneous transformation of the protoplast are improved, and a more efficient and stable method is provided for molecular biology research and genetic improvement of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cell engineering, and relates to a method for separating white clover protoplasts and establishing an instantaneous transformation system. Background Art

[0002] Plant protoplasts refer to plant cells after the cell wall is removed, which contain structures such as cell membrane, cytoplasm, nucleus and organelles. Protoplast technology has important applications in plant cell engineering and is widely used in gene transformation, cell fusion, genetic improvement and functional genomics research. By isolating and culturing protoplasts, researchers can gain a deeper understanding of the physiological characteristics of plant cells, gene expression and their response to the environment.

[0003] White clover (Trifolium repens) is an important leguminous forage grass, widely used in agriculture and ecosystems in temperate regions. It has good nutritional value, can provide abundant protein for herbivores, and improve soil fertility through symbiotic nitrogen fixation with rhizobia. White clover also plays an important role in controlling soil erosion, inhibiting weed growth, and urban greening. With the adjustment of agricultural structure and the emphasis on ecological environment, the research and application of white clover has received more and more attention.

[0004] Existing protoplast separation technologies mainly include mechanical methods and enzymatic methods. The mechanical method destroys the cell wall by physical means, but often leads to cell damage and low yield. The enzymatic method is to use specific enzymes (such as cellulase and pectinase) to decompose the cell wall and release the protoplasts. Although the enzymatic method is relatively mild and can obtain protoplasts with higher purity, the prior art still has some shortcomings in enzymatic conditions, enzymatic solution components and operating steps, resulting in problems such as low protoplast yield, low purity and complicated operation.

[0005] Protoplast transformation technology is a key step in introducing foreign genes into plant cells. The transformation efficiency is affected by many factors, including the quality of protoplasts, the selection of plasmids and the transformation conditions. At present, the transformation efficiency is generally low, and the survival rate and gene expression level of transformed cells are unstable, which limits its application in plant genetic improvement and functional research. Summary of the invention

[0006] In view of the limitations of the prior art, the present invention aims to provide an efficient and simple method for isolating white clover protoplasts and establishing an instantaneous transformation system. By optimizing the enzymatic hydrolysis conditions, the formula of the modified W5 solution and the transformation steps, the present invention hopes to improve the yield and purity of protoplasts, enhance the transformation efficiency, and provide reliable technical support for the gene function research and genetic improvement of white clover. This will help promote the application of white clover in the fields of agriculture and ecology and promote sustainable development.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is to provide a method for isolating protoplasts of white clover, comprising the following steps:

[0008] Select young and disease-free white clover leaves and cut them into thin strips; select an enzymatic solution in which non-specific enzymes have been inactivated;

[0009] Enzymatic hydrolysis was performed at 25 °C with shaking at 50 rpm for 6 h;

[0010] After enzymolysis, W5 solution was added to terminate the enzymolysis reaction, and shaking was continued for 50 to 60 minutes to obtain protoplast fluid;

[0011] Filter the protoplast fluid through a 75 μm filter and wash 2 to 3 times with precooled modified W5 solution;

[0012] Centrifuge at 100 × g for 2 min, remove the supernatant, and obtain the protoplast pellet;

[0013] The precipitated protoplasts are resuspended in 3-10 ml of MMg solution, mixed and allowed to stand, and then separated to obtain the protoplasts.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention significantly improves the separation efficiency and purity of white clover protoplasts. First, young and disease-free leaves are selected as starting materials to ensure the quality and activity of protoplasts. Secondly, an enzymatic solution in which non-specific enzymes have been inactivated is used to avoid unnecessary cell damage and ensure the efficiency of enzymatic hydrolysis. Under mild conditions of 25°C, the solution is shaken at a speed of 50 rpm for 6 hours. This mild enzymatic hydrolysis treatment not only fully releases the protoplasts, but also maintains the integrity of its structure. After enzymatic hydrolysis, W5 solution is added to terminate the enzymatic hydrolysis reaction, and the shaking is continued. This step further ensures the release and stability of the protoplasts. Filtering through a 75μm filter effectively removes incompletely dissociated cell fragments, and washing with a pre-cooled modified W5 solution further improves the purity of the protoplasts. Finally, centrifugation is performed at 100×g for 2 minutes, the supernatant is quickly removed, and a pure protoplast precipitate is obtained. This series of optimized steps not only improves the yield of protoplasts, but also ensures its biological activity and the reliability of subsequent experiments, providing more efficient and stable technical support for the research and application of white clover.

[0016] On the basis of the above technical solution, the present invention can also be improved as follows:

[0017] Furthermore: the enzymatic solution comprises the following components: MES buffer, mannitol, cellulase R10, enzyme R10, pectinase, mercaptoethanol, KCl, CaCl2, BSA. Before adding BSA, CaCl2 and KCl solution, the enzyme solution is heat pretreated at 55°C for 10 minutes to inactivate non-specific enzymes.

[0018] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0019] By experimentally selecting and combining the components of the enzymatic solution, efficient and specific decomposition of the cell wall of white clover leaves was achieved. The addition of MES buffer and mannitol provided a suitable pH and osmotic pressure environment for the enzymatic hydrolysis process, which helped maintain the stability and activity of the protoplasts. Cellulase R10 and pectinase specifically targeted the main components of the cell wall and effectively promoted the decomposition of the cell wall, while the addition of enzyme R10 further improved the efficiency of the enzymatic hydrolysis. As a reducing agent, mercaptoethanol can protect the proteins in the protoplasts from oxidative damage and maintain their functions. The addition of KCl and CaCl2 not only provided the necessary ionic environment, but also helped to stabilize the cell membrane structure. The addition of BSA further protected the protoplasts from mechanical damage during the enzymatic hydrolysis process. Heat pretreatment at 55°C for 10 minutes effectively inactivated nonspecific enzymes, reduced nonspecific degradation, and improved the purity and activity of the protoplasts. These optimization measures worked together to not only improve the yield of protoplasts, but also ensure its biological activity and the reliability of subsequent experiments, providing more efficient and stable technical support for the research and application of white clover.

[0020] Further: the modified W5 solution comprises the following components:

[0021] 5 mM MES, pH 5.7,

[0022] 75mM CaCl2,

[0023] 154mM NaCl,

[0024] 5 mM KCl,

[0025] 0.4M maltose,

[0026] 0.5 mM ascorbic acid.

[0027] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0028] The optimized components of the modified W5 solution significantly improved the separation and purification of white clover protoplasts. By precisely adjusting the concentration and pH value of the MES buffer, the solution can better maintain the stability of the protoplasts, while providing a suitable chemical environment to reduce cell damage that may occur during enzymatic hydrolysis. The optimization of the concentrations of CaCl2 and NaCl helps maintain the ion balance inside and outside the cells, which is essential for the integrity and activity of the protoplasts. The addition of KCl further stabilizes the intracellular and extracellular environment, while the addition of maltose, as an osmotic agent, helps regulate the osmotic pressure of the solution and prevents the protoplasts from swelling or shrinking during separation and washing, thereby improving the survival rate of the protoplasts. The addition of ascorbic acid, as an antioxidant, effectively reduces the damage of oxidative stress to the protoplasts and further improves the quality and stability of the protoplasts. These improvements make the modified W5 solution more efficient in washing and purifying protoplasts, reduce cell loss during the operation, improve the yield and purity of protoplasts, and provide higher quality protoplast materials for subsequent experiments such as gene transformation and functional analysis.

[0029] The present invention also provides a method for establishing a white clover protoplast transient transformation system, comprising the following steps:

[0030] The plasmid was mixed with the protoplasts obtained by the white clover protoplast isolation method, and PEG-Ca was added. 2+ Solution;

[0031] Let stand at room temperature for 5 minutes to allow the plasmid to fully contact the protoplasts;

[0032] W5 solution and protoplast suspension were added, and after centrifugation for 2 min, the microspheres were gently suspended in W5 solution;

[0033] The protoplast suspension was transferred to a tube precoated with 1% BSA and incubated at room temperature in the dark for 10–24 h;

[0034] The transformation effect was observed by confocal microscopy.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] First, by mixing the plasmid with protoplasts and PEG-Ca 2+ The addition of PEG-Ca solution optimizes the binding efficiency between plasmid and protoplast and improves the transformation efficiency. 2+The use of the solution promotes the fusion of plasmid DNA with the protoplast membrane, while the operation of standing for 5 minutes at room temperature simplifies the transformation steps and ensures sufficient contact between the plasmid and the protoplast. Second, the addition of W5 solution and the optimization of the centrifugation step enable the protoplasts to be better suspended and survive after transformation, reducing cell damage caused by operation. Third, the protoplast suspension is transferred to a test tube pre-coated with 1% BSA and incubated under dark conditions, which provides an environment for protoplasts to reduce stress and optimize expression, and helps to improve the expression efficiency and stability of genes after transformation. Fourth, the transformation effect can be observed by confocal microscopy, which can intuitively and accurately evaluate the transformation efficiency and gene expression, providing reliable data support for subsequent gene function research and genetic improvement. Overall, this technical solution improves the efficiency and success rate of protoplast transient transformation through a series of optimization steps, providing a more efficient and stable method for plant molecular biology research and genetic improvement.

[0037] On the basis of the above technical solution, the present invention can also be improved as follows:

[0038] Further: the PEG-Ca 2+ The solution contains the following components:

[0039] PEG4000 at 40% wt / vol concentration;

[0040] 100 mM CaCl2;

[0041] 0.3 M mannitol.

[0042] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0043] The PEG-Ca 2+ The solution can significantly improve the instantaneous transformation efficiency of white clover protoplasts. PEG4000 is a commonly used chemical fusion agent, and its 40% concentration is optimized to promote the fusion of plasmid DNA and protoplast membrane, which helps to improve the transformation efficiency. The addition of CaCl2 helps to stabilize the binding of DNA to the cell membrane, while mannitol, as an osmotic pressure regulator, helps to protect the integrity of protoplasts during the transformation process and prevent excessive cell expansion or contraction. This specific component of PEG-Ca 2+ The solution can more effectively promote the uptake of exogenous DNA while reducing the damage and death of protoplasts, thereby improving the success rate and stability of transformation and providing a more reliable basis for subsequent gene expression analysis and functional studies.

[0044] Furthermore: the plasmid is pCombia1300-GFP.

[0045] The present invention also discloses a method for studying the gene function of white clover, which comprises the following steps:

[0046] Isolating protoplasts using the white clover protoplast isolation method;

[0047] Transforming protoplasts using the white clover protoplast transient transformation system establishment method;

[0048] Transformed protoplasts were used for gene expression and functional analysis.

[0049] Furthermore: the gene is the TrATAF1 gene, and the nucleotide sequence of the TrATAF1 gene is shown in SEQ ID NO.1.

[0050] Compared with the prior art, the beneficial effects of this technical solution are:

[0051] By combining the optimization method of white clover protoplast isolation and transient transformation system, the present invention provides a method for efficiently studying the gene function of white clover. In particular, by using the nucleotide sequence specific to the TrATAF1 gene, researchers can more accurately study the function and mechanism of action of the gene. The beneficial effect of this method is that it not only improves the efficiency and accuracy of gene function analysis, but also allows in-depth research on specific genes, thereby accelerating the development of gene function analysis and application. In addition, the application of this method can be extended to the study of other genes, providing a powerful tool for genetic improvement and molecular breeding of white clover and other plants, helping to improve crop yield, quality and stress resistance, and thus promoting the development of agricultural biotechnology.

[0052] The present invention also provides a method for genetic improvement of white clover, comprising the following steps:

[0053] Introducing foreign genes into the white clover protoplasts separated by the white clover protoplast separation method;

[0054] Screening and characterization of genetically improved white clover plants.

[0055] Furthermore: the gene is the TrATAF1 gene, and the nucleotide sequence of the TrATAF1 gene is shown in SEQ ID NO.1.

[0056] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0057] By introducing a specific exogenous gene, the TrATAF1 gene (its nucleotide sequence is shown in SEQ ID NO.1), into white clover protoplasts, this method can improve the genetic characteristics of white clover in a targeted manner. Compared with the prior art, the beneficial effect of this improvement method is that it can more accurately control the gene editing process, reduce the interference of non-target genes, and improve the accuracy and efficiency of genetic improvement. In addition, by screening and identifying genetically improved white clover plants, plants with desired traits can be quickly identified and selected, accelerating the breeding process. The application of this method can not only improve the agronomic traits of white clover, such as yield, quality and stress resistance, but also provide a new strategy for plant genetic improvement, which will help promote sustainable agricultural development and the application of biotechnology in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The effect of the enzymatic solution containing pectinase Y-23 on the degree of leaf dissociation at different stages. Figure 1 In the figure, a is the leaves of mature seedlings, and b is the leaves of seedlings.

[0060] Figure 2 This is the state after enzymatic hydrolysis and filtration of the protoplasts of white clover in the embodiment, and the enzymatic hydrolyzate contains pectinase Y-23.

[0061] Figure 3 This is the condition after enzymatic hydrolysis and filtration of the protoplasts of white clover in the embodiment, and the enzymatic hydrolyzate does not contain pectinase Y-23.

[0062] Figure 4 The figure shows the effects of five PEG4000 concentration gradients (wt / vol), namely 10% (Figure a), 20% (Figure b), 30% (Figure c), 40% (Figure d), and 50% (Figure e) on the transformation efficiency of protoplast plasmids.

[0063] Figure 5 The graph shows the luminescence of GFP, RFP, Bright Field, and Merged when the gene vector pCombia1300-GFP was empty and transformed into protoplasts of white clover in the example.

[0064] Figure 6The luminescence of the homologous recombination product of the target gene pCombia1300-TrATAF1-GFP transformed into white clover protoplasts in GFP, RFP, Bright Field, and Merged.

[0065] Figure 7 This is the subcellular localization of TrATAF1.

[0066] Figure 8 This is the electrophoresis diagram of the gene TrATAF1 amplification in protoplasts of white clover transformed in the example.

[0067] Fig. 9 Bioinformatics analysis of TrATAF1 in white clover. Fig. 9 In the figure, a is the protein domain; b is the TrATAF1 sequence map; c is the protein phosphorylation site prediction; d is the protein transmembrane region prediction; e is the protein signal peptide prediction; f is the protein disordered region analysis; g is the protein hydrophobicity analysis; h is the protein secondary structure prediction. DETAILED DESCRIPTION

[0068] The following describes the invention in conjunction with specific embodiments.

[0069] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0070] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0071] 1. Preparation

[0072] Material selection: Select healthy, disease-free young leaves and mature leaves of Haifa white clover as controls, preferably the top leaves about 10-12 days after leaf budding, and remove the tip and base of the leaves.

[0073] Leaf pretreatment: The selected leaves were rinsed with sterile water, then disinfected with 70% ethanol for 30 seconds, and finally rinsed three times with sterile water to remove residual ethanol on the surface.

[0074] Leaf cutting: Cut the disinfected leaves into 0.2-0.3 mm wide filaments using sterile scissors.

[0075] 2. Enzymatic hydrolysis stage

[0076] Enzyme solution preparation: Prepare enzymatic solution containing MES buffer, mannitol, cellulase R10, enzyme R10, pectinase, mercaptoethanol, KCl, CaCl2, and BSA according to Table 1. Before adding BSA, CaCl2, and KCl, the enzyme solution was heat-pretreated at 55°C for 10 minutes to inactivate nonspecific enzymes.

[0077] Table 1 Composition of reagents used in this example

[0078]

[0079]

[0080] Note: Except for special instructions in this example, the solution was sterilized by high pressure steam.

[0081] Enzymatic hydrolysis: The cut leaf filaments were placed in a sterile container containing 10 ml of enzyme solution, vacuum infiltrated (0.1 MPa) for 1 hour, and then shaken at 25°C and 50 rpm for 6 hours for enzymatic hydrolysis.

[0082] 3. Termination of enzymatic digestion and washing phase

[0083] Termination of enzymatic hydrolysis: After 6 hours of enzymatic hydrolysis, add W5 solution to terminate the enzymatic hydrolysis reaction and continue shaking for 50-60 minutes.

[0084] Filtration and washing: The protoplast fluid after enzymatic hydrolysis was filtered through a 75 μm nylon mesh and washed 2-3 times with pre-cooled modified W5 solution. After each wash, centrifuge at 100 × g for 2 minutes and remove the supernatant.

[0085] Preparation of modified W5 solution

[0086] A modified W5 solution was prepared containing 5 mM MES (pH 5.7), 75 mM CaCl2, 154 mM NaCl, 5 mM KCl, 0.4 M maltose, 0.5 mM ascorbic acid.

[0087] Solution adjustment: According to experimental needs, adjust the concentration of each component in the modified W5 solution to optimize the stability of protoplasts and experimental effects.

[0088] 4. Protoplast Collection

[0089] Protoplast precipitation: The washed protoplast fluid was centrifuged at 100 × g for 2 minutes, and the supernatant was removed to obtain the protoplast precipitate.

[0090] Protoplast resuspension: resuspend the precipitated protoplasts in pre-cooled modified W5 solution, let stand on ice for about 1 hour or at 4°C overnight to allow the protoplasts to precipitate, and then remove the supernatant.

[0091] The precipitated protoplasts are resuspended in 3-10 ml of MMg solution, then mixed, allowed to stand, and separated to obtain protoplasts.

[0092] The effects of materials at different times on the degree of protoplast dissociation are shown in the following table. Figure 1 As shown, the view is with a 10x lens.

[0093] The presence or absence of pectinase had a significant effect on the dissociation of clover bioplastids. Figure 2 and Figure 3 As shown, the view is with a 10x lens.

[0094] 5. Plasmid Preparation

[0095] Empty plasmid pCombia1300-GFP and plasmid pCombia1300-TrATAF1-GFP containing target gene TrATAF1 were selected as transformation plasmids. Vector primers pCombia1300-T rATAF1-GFP F: 5'-GGGGCCCGGGGTCGACATGGCATCCGAGCTTCA ATTG-3'; pCombia1300-TrATAF1-GFP R: 5'-TACCGGATCCACTAGTTCAGAAGGATTTGGGTAGATACATGA-3'.

[0096] Ensure that the plasmid DNA is of high purity and free of impurities.

[0097] The sequence of the gene TrATAF1 is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.2.

[0098] 6. Mixing of plasmid and protoplasts

[0099] Mixing ratio: 1-10 μg of plasmid DNA and 200 μl of protoplast suspension at room temperature.

[0100] PEG-Ca 2+ Solution preparation: Prepare PEG-Ca according to the composition in Table 1. 2+The solution contained 40% wt / vol PEG4000, 100mM CaCl2 and 0.3M mannitol. For comparison, five PEG4000 concentration gradients (wt / vol) were set up simultaneously in this example, namely 0.1M, 0.2M, 0.3M, 0.4M, and 0.5M, to illustrate their effects on the transformation efficiency of protoplast plasmids. The results are shown in FIG. Figure 4 As shown, the field of view scale is 100 μm.

[0101] Plasmid transformation: Add an equal amount of PEG-Ca to the mixture of plasmid and protoplasts. 2+ The solution was gently mixed and allowed to stand for 5 minutes to promote the binding of plasmid DNA to protoplasts.

[0102] Figure 4 As shown in Figure a, when the concentration of PEG-4000 was 0.1 M, there were 257 intact single molecules in 4 fields of view, 39 of which were successfully transformed, with a transformation efficiency of 15.1%.

[0103] Figure 4 As shown in Figure b, when the concentration of PEG-4000 was 0.2 M, there were 531 intact single molecules in 4 fields of view, 116 intact single molecules were successfully transformed, and the transformation efficiency was 21.8%.

[0104] Figure 4 As shown in Figure c, when the concentration of PEG-4000 was 0.3 M, there were 347 intact single molecules in 4 fields of view, 91 of which were successfully transformed, with a transformation efficiency of 26.2%.

[0105] Figure 4 As shown in Figure d, when the concentration of PEG-4000 was 0.4 M, there were 514 intact single molecules in 4 fields of view, 193 of which were successfully transformed, with a transformation efficiency of 37.5%.

[0106] Figure 4 As shown in Figure e, when the concentration of PEG-4000 was 0.5 M, there were 486 intact single molecules in 4 fields of view, 87 of which were successfully transformed, with a transformation efficiency of 17.9%.

[0107] 7. Post-conversion processing

[0108] Washing and resuspension: Add 3 ml of W5 solution to the transformed protoplast suspension, mix gently and centrifuge at 100 × g for 2 minutes, remove the supernatant and gently suspend the microspheres in 0.5 ml of W5 solution.

[0109] Incubation conditions: The resuspended protoplast suspension was transferred to a 1.5 ml tube pre-coated with 1% BSA to reduce protoplast adhesion to the tube wall, and then incubated at room temperature in the dark for 10-24 hours to allow expression of the plasmid DNA.

[0110] 8. Observation of conversion effect

[0111] Microscope observation: Use confocal microscopy to observe the transformed protoplasts, especially the luminescence under GFP, RFP, Bright Field, and Merged channels to evaluate the transformation efficiency and gene expression. Figures 5 to 7 shown. Figure 5 It is the luminescence of GFP, RFP, Bright Field and Merged after transforming the gene vector pCombia1300-GFP into protoplasts of white clover. Figure 6 It is the luminescence of the homologous recombination product of the target gene pCombia1300-TrATAF1-GFP transformed into white clover protoplasts in GFP, RFP, Bright Field, and Merged. Figure 7 It is the subcellular localization of TrATAF1 (the gene is located in the cell membrane and cytoplasm).

[0112] Data Analysis: Record and analyze the observed transformation effects, including the number, brightness, and distribution of GFP-expressing cells, to assess the efficiency of the transient transformation system.

[0113] The above steps provide a detailed specific implementation method of the method for establishing the transient transformation system of white clover protoplasts, which can be appropriately adjusted according to the experimental conditions and purposes. Through this method, the transient expression of foreign genes in white clover protoplasts can be achieved, providing an effective tool for studying gene function and carrying out genetic improvement.

[0114] Cloning and bioinformatics analysis of Clover TrATAF1

[0115] The coding sequence of TrATAF1 was extracted in the preliminary analysis. The TrATAF1 gene sequence is shown in SEQ ID NO.1, and its encoded protein sequence is shown in SEQ ID NO.2.

[0116] Combined GWAS, Sweep and BSA were used to explore important candidate genes for yield-related traits of white clover. 300 core germplasms of white clover were resequenced with an average coverage depth of 10X, and 2,250,001 high-quality SNPs were obtained. Based on these SNPs, the population structure analysis was performed, and the 300 white clover germplasm resources could be divided into three subpopulations. The Fst value was calculated to analyze the population differentiation of the wild subpopulations and introduced subpopulations of white clover, and a large number of regions related to genetics and differentiation were explored. Based on the SNPs obtained from resequencing, GWAS analysis of plant height and petiole length was performed, and two significant loci, chr5P_20531104 and chr5P_9062595, were found in plant height; a significant locus, chr3P_40484643, was identified in petiole length, which overlapped with the Sweep result and BSA positioning region; analysis found that there was a gene chr11.jg6047 encoding NAC protein in the candidate interval of the significant locus chr3P_40484643, which may regulate the growth and development of white clover. chr11.jg6047 was renamed TrATAF1.

[0117] Amplification primers were designed using Primer Premier 5: upstream primer: 5'-ATGGCATCCGAGCTTCAATTG-3'; downstream primer R: 5'-TCAGAAGGATTTGG GTAGATACATGA-3'.

[0118] Refer to the instructions of the high-fidelity Taq enzyme 2×Phanta Max Master Mix (Dye Plus) kit, use the cDNA of young leaves of white clover as a template for PCR amplification, and use 1% agarose gel electrophoresis for detection. Figure 8 The DNA was purified and recovered using the HiPureGel Pure DNA Mini Kit (Guangzhou Meiji Biotechnology Co., Ltd., D2111), and the purified product was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing.

[0119] Using the CDD conserved functional region analysis in NCBI, we found that the chr11.jg6047 protein has a typical NAC (NAM) domain ( Fig. 9 The NAC domain of the protein is distributed in the region of 9aa to 131aa ( Fig. 9(b) This is a family of transcription factors that are widely present in plants and are involved in a variety of biological processes, including developmental regulation and stress response. NAC transcription factors are generally involved in regulating plant growth and development processes, including cell differentiation, organ development, and overall plant morphological construction. Many NAC family transcription factors are involved in plant responses to various stresses, such as drought, salt stress, low temperature, and high heat. They may enhance plant stress resistance by regulating the expression of downstream genes.

[0120] The coding sequence of chr11.jg6047 gene is 876bp in length, with molecular formula C1499H2275N399O436S12, encoding 291 amino acids. The total number of atoms is 4621, the molecular weight is 33246.69Da, the theoretical isoelectric point (pI) is 7.66, the fat solubility index is 62.03, the total average hydrophilicity coefficient is -0.716, the instability index (II) is 56.93, the total number of positively charged residues (Arg+Lys) is 38, the total number of negatively charged residues (Asp+Glu) is 37, the aliphatic index is 62.03, and the average hydrophilicity coefficient of the protein (GRAVY) is -0.716.

[0121] Protein phosphorylation site prediction results show ( Fig. 9 c), there are 33 possible phosphorylation sites in its amino acid sequence, including 20 serine (S) phosphorylation sites, 6 threonine (T) phosphorylation sites, and 7 tyrosine (Y) phosphorylation sites, with serine phosphorylation being the main site. The protein transmembrane region prediction results show that there is no transmembrane domain ( Fig. 9 d), signal peptide prediction protein does not have a signal peptide ( Fig. 9 e), the results show that the protein is not a secretory protein. Protein disordered region analysis results ( Fig. 9 f) shows that the amino acid sequence of the protein contains 5 disordered regions. Hydrophobicity analysis results ( Fig. 9 g) shows that the hydrophobicity of its amino acids is mostly negative, belonging to hydrophilic proteins. Subcellular localization prediction results show that the protein is located in chloroplasts. The secondary structure consists of 17.87% α-helix, 12.37% extended chain, 3.44% β-turn structure and 66.32% random coil ( Fig. 9 h).

[0122] Studying the function of the TrATAF1 gene is of great significance for revealing the molecular mechanism of plant growth and development, improving crop stress tolerance, and improving crop varieties. Through gene editing, overexpression or RNA interference and other technologies, researchers can further explore the specific role and regulatory network of TrATAF1 in plants.

[0123] Transforming the TrATAF1 gene plasmid into white clover protoplasts can explore the function and mechanism of action of this gene in plant cells. Through this transient transformation system, researchers can directly observe and analyze the expression pattern, protein function, stress response and interaction with other biological molecules of the TrATAF1 gene in protoplasts. This method not only helps to reveal the specific role of TrATAF1 in plant growth and development and stress adaptation, but also provides experimental data for the genetic improvement of white clover, which helps to breed new varieties with excellent traits. In addition, this transformation technology also provides an effective experimental platform for studying gene regulatory networks, signal transduction pathways, protein stability and subcellular localization, thereby promoting research progress in the fields of plant molecular biology and genetic engineering.

[0124] In the description of the present invention, it should be understood that "-" and "~" represent the range between two values, and the range includes the endpoints. For example: "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0125] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0126] In the description of the invention, the numerical values ​​of time, temperature, ratio, mass, etc. involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0127] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0128] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for isolating protoplasts of white clover, characterized in that: The following steps are involved: Select young and disease-free white clover leaves and cut them into thin strips; select an enzymatic solution in which non-specific enzymes have been inactivated; Enzymatic hydrolysis was performed at 25 °C with shaking at 50 rpm for 6 h; After enzymolysis, W5 solution was added to terminate the enzymolysis reaction, and shaking was continued for 50 to 60 minutes to obtain protoplast fluid; Filter the protoplast fluid through a 75 μm filter and wash 2 to 3 times with precooled modified W5 solution; Centrifuge at 100 × g for 2 min, remove the supernatant, and obtain the protoplast pellet; The precipitated protoplasts are resuspended in 3-10 ml of MMg solution, mixed and allowed to stand, and then the protoplasts are obtained after separation.

2. The method according to claim 1, characterized in that The enzymatic solution comprises the following components: MES buffer, mannitol, cellulase R10, enzyme R10, pectinase, mercaptoethanol, KCl, CaCl2, and BSA. Before adding BSA, CaCl2, and KCl solutions, the enzyme solution is heat-pretreated at 55° C. for 10 minutes to inactivate nonspecific enzymes.

3. The method according to claim 1, characterized in that The modified W5 solution comprises the following components: 5 mM MES, pH 5.7, 75mM CaCl2, 154mM NaCl, 5 mM KCl, 0.4M maltose, 0.5 mM ascorbic acid.

4. A method for establishing a transient transformation system of white clover protoplasts, characterized in that: The steps include: The plasmid was mixed with the protoplasts obtained by the method of claim 1, and PEG-Ca was added. 2+ Solution; let stand at room temperature for 5 minutes to allow the plasmid and protoplasts to fully contact; W5 solution and protoplast suspension were added, and after centrifugation for 2 min, the microspheres were gently suspended in W5 solution; The protoplast suspension was transferred to a tube precoated with 1% BSA and incubated at room temperature in the dark for 10–24 h; The transformation effect was observed by confocal microscopy.

5. The method according to claim 4, characterized in that The PEG-Ca 2+ The solution contains the following components: PEG4000 at 40% wt / vol concentration; 100 mM CaCl2; 0.3 M mannitol.

6. The method according to claim 4, characterized in that The plasmid is pCombia1300-GFP.

7. A method for studying the gene function of white clover, characterized in that: The steps include: Isolating protoplasts using the method according to any one of claims 1 to 3; Transforming protoplasts using the method according to any one of claims 4 to 6; Transformed protoplasts were used for gene expression and functional analysis.

8. The method according to claim 7, characterized in that The gene is the TrATAF1 gene, and the nucleotide sequence of the TrATAF1 gene is shown in SEQ ID NO.

1.

9. A method for genetic improvement of white clover, characterized in that: The following steps are involved: Introducing a foreign gene into the protoplasts of white clover isolated by the method according to any one of claims 1 to 3; Screening and characterization of genetically improved white clover plants.

10. The improved method according to claim 9, characterized in that: The gene is the TrATAF1 gene, and the nucleotide sequence of the TrATAF1 gene is shown in SEQ ID NO.1.

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