Use of protein mtNPHE1 in cultivating alfalfa

Abstract

This invention discloses the application of the protein MtNPHE1 in alfalfa breeding to enhance phosphorus accumulation, regulate alfalfa growth and development, or increase alfalfa yield. By using the protein MtNPHE1 in alfalfa breeding, this invention improves the crop's ability to absorb and utilize phosphorus, thereby increasing nitrogen and phosphorus nutrient efficiency and yield. Through molecular breeding to improve plants, this not only enhances plant yield and quality but also has significant implications for environmental protection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of a protein, MtNPHE1, in the cultivation of alfalfa. Background Technology

[0002] Phosphorus and nitrogen are essential macronutrients for plant growth, playing a crucial role in plant development and crop yield. Phosphorus is absorbed by plants in its inorganic form, but because inorganic phosphorus readily forms precipitates with metal ions such as calcium and magnesium ions or is fixed by soil particles, it is difficult for phosphorus to diffuse in the soil, often leading to low phosphorus stress in plants. Low phosphorus stress not only directly affects plant growth but also impacts nitrogen absorption and utilization, such as affecting nitrogen fixation through nodule formation in legumes. Alfalfa, a legume, can form root nodules for biological nitrogen fixation; phosphorus deficiency severely inhibits its growth and development.

[0003] To increase crop yields, the application of phosphate and nitrogen fertilizers has been increasing year by year. However, due to the inherent characteristics of phosphorus, even with the application of large amounts of phosphate fertilizer to the soil, the concentration of inorganic phosphorus that plants can absorb and utilize remains very low. Therefore, identifying key genes involved in plant phosphorus absorption and utilization, and improving the ability of crops to absorb and utilize phosphorus, is the most direct and effective way to improve crop nitrogen and phosphorus nutrition efficiency and yield. Improving plants through molecular breeding can not only increase plant yield and quality, but also has important significance for environmental protection. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the protein MtNPHE1 in alfalfa cultivation, so as to overcome the defects of the existing technology.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Applications of protein MtNPHE1 in alfalfa breeding to improve phosphorus accumulation, regulate alfalfa growth and development, or increase alfalfa yield.

[0007] Furthermore, the MtNPHE1 protein is derived from alfalfa and is a protein as shown in (1) or (2) or (3) or (4) or (5) below:

[0008] (1) A protein consisting of the amino acid sequence shown in Sequence 2 or a protein containing the amino acid sequence;

[0009] (2) Proteins containing the amino acid sequence shown in Sequence 2 with a tag;

[0010] (3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of sequence 2.

[0011] (4) Proteins that have 75% or more homology with the amino acid sequence shown in Sequence 2;

[0012] (5) The amino acid sequence shown in Sequence 2 can be synthesized artificially or expressed by gene.

[0013] Furthermore, the protein is encoded by the CDS sequence shown in Sequence 1.

[0014] Furthermore, the MtNPHE1 nucleic acid sequence is as shown in (A1) or (A2) or (A3) or (A4):

[0015] (A1) A CDS consisting of the base sequence shown in Sequence 1 or a nucleic acid containing that sequence;

[0016] (A2) A nucleic acid sequence containing the CDS sequence shown in Sequence 1;

[0017] (A3) The CDS sequence or genomic DNA shown in sequence 1;

[0018] (A4) CDS or genomic DNA that has 75% or more homology with the nucleic acid sequence shown in Sequence 1.

[0019] Applications of biomaterials related to the protein MtNPHE1 in improving phosphorus accumulation, regulating growth and development, or increasing alfalfa yield in alfalfa breeding.

[0020] Furthermore, the biological material is any one of the following (B1) to (B7):

[0021] (B1) Nucleic acid molecules encoding the amino acid sequence shown in sequence 2 or nucleic acid molecules that are 75% or more homologous to the nucleic acid sequence shown in sequence 1;

[0022] (B2) A carrier containing the nucleic acid molecule described in (B1);

[0023] (B3) Microorganisms containing the nucleic acid molecules described in (B1);

[0024] (B4) Microorganisms containing the carrier described in (B2);

[0025] (B5) Transgenic plants containing the nucleic acid molecules described in (B1);

[0026] (B6) A transgenic plant containing the vector described in (B2);

[0027] (B7) Contains the nucleic acid sequence or part of the sequence described in (B1) for editing (or constructing Crispr / Cas9 materials) of recombinant vectors / recombinant microorganisms / recombinant plant cell lines used in alfalfa.

[0028] Compared with the prior art, the advantages of the present invention are as follows: The application of the protein MtNPHE1 provided by the present invention in the cultivation of alfalfa improves the crop's ability to absorb and utilize phosphorus, increases the crop's nitrogen and phosphorus nutrition efficiency and yield, and improves the plant through molecular breeding. This not only improves the yield and quality of the plant, but also has important significance for environmental protection. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a tissue expression analysis diagram of the MtNPHE1 gene in this invention.

[0031] Figure 2 This is the phenotypic detection diagram in this invention.

[0032] Figure 3 This is a graph showing the determination of inorganic phosphorus content in this invention. Detailed Implementation

[0033] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0034] The experimental methods involved in this embodiment are all conventional methods, and the reagents and consumables were ordered from a reagent and consumables company.

[0035] 1. Cloning of the alfalfa MtNPHE1 gene

[0036] The CDS sequence of the MtNPHE1 gene was retrieved using the Ensembl Plants website (https: / / plants.ensembl.org / index.html) via gene ID MTR_1g093080. This sequence corresponds to the A17 ecotype background. The CDS sequence of the MtNPHE1 gene in the R108 ecotype was then compared using this sequence on the website https: / / medicagohapmap2.org / . Primers Primer1 and Primer2 were designed at the 5' and 3' ends of this CDS sequence, respectively. Using cDNA from wild-type R108 as a template, the CDS sequence of the MtNPHE1 gene in the R108 ecotype was amplified.

[0037] Amplification system (50 μL): 5 μL 10×Taq Buffer, 0.5 μL ExTaq, 4 μL dNTPs, 2.5 μL Primer 1, 2.5 μL Primer 2, 35 μL ddH2O, 1 μL template.

[0038] Primer1:5'-ATGCAGCGTTTTAAAATGGATT-3'

[0039] Primer2: 5'-ATAGCCAGGG GAAGTATTTGTA-3'

[0040] Amplification program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min 20 s, 35 cycles of amplification, 72℃ extension for 10 min.

[0041] 2. Constructing overexpression vectors

[0042] The overexpression vector pCAMBIA3301 was used to construct the MtNPHE1 gene overexpression material. Wild-type R108 cDNA was used as a template, and the MtNPHE1 gene CDS sequence was amplified using primers Primer3 and Primer4 with adapters, ensuring that the recovered target CDS had adapters at both ends (the adapters are sequences on the vector located at the BglII and NcoI restriction sites). The vector was then linearized by digesting it with both BglII and NcoI restriction enzymes. Homologous recombination was used to ligate the adapter-containing CDS sequence into the linearized vector.

[0043] Primer3: 5'-gaacacgggggactcttgaccATGCAGCGTTTTAAAATGGATT-3'

[0044] Primer4: 5'-agaaatttaccctcagatctaccatATAGCCAGGGGAAGTATTTGTA-3'

[0045] Among them, lowercase letters are connector sequences.

[0046] Gene cloning: The MtNPHE1 gene was cloned with an adapter CDS sequence. The amplification system and procedure were the same as those in section 1, "Cloning of the Alfalfa MtNPHE1 Gene".

[0047] Vector linearization system: 5 μL 10× QuickCut Buffer, 1 μL QuickCut BglII, 1 μL QuickCut NcoI, plasmid ≤1 μg, ddH2O added to 50 μL.

[0048] Carrier linearization conditions: incubation at 37℃ for 5 min.

[0049] The steps for recovering the target gene and linearized vector are as follows:

[0050] (1) Cut agarose gel containing the CDS band of the MtNPHE1 gene and the linearized vector pCAMBIA3301 band respectively, and put them into a clean centrifuge tube;

[0051] (2) Add three times the volume of GSB, place it in a clean EP tube, and sol-gel at 55°C until the gel is completely melted.

[0052] (3) Add the melted gel to the centrifuge column, let stand for 1 min, centrifuge at 10,000g for 1 min, and discard the effluent.

[0053] (4) Add 650 μL of WB (Wash Buffer) to the centrifuge column, centrifuge at 10,000 g for 1 min, and discard the effluent.

[0054] (5) Centrifuge at 10,000g for 1-2 minutes to completely remove residual WB.

[0055] (6) Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 minute. Add 30-50 μL ddH2O to the center of the centrifuge column and let it stand for 1 minute.

[0056] (7) Centrifuge at 10,000g for 1 min and store the eluted DNA at -20℃.

[0057] Ligation system: 5 μL 2×Cloning Master Mix, 10-500 ng linearized vector, 30-1500 ng target gene recovery product, and ddH2O to bring the reaction system to 10 μL.

[0058] Connection conditions: After mixing the system, place it in a 50℃ air bath for 30 minutes for connection.

[0059] 3. Obtaining genetically modified alfalfa

[0060] The overexpression vector 35S:MtNPHE1 was transformed into Agrobacterium EHA105, following the Agrobacterium transformation method of Beijing Huayueyang Biotechnology Co., Ltd. Following the infection method described in a published article (Cosson, V., Durand, P., d'Erfurth, I., Kondorosi, A. and Ratet, P. (2006). Medicago truncatula transformation using leaf explants. Methods Mol. Biol. 343:115-127), wild-type Alfalfa R108 was infected with recombinant Agrobacterium. R108 leaves or cotyledons and hypocotyls were used as infection materials. Transgenic lines 35S:MtNPHE1-1 and 35S:MtNPHE1-2 were obtained through glufosinate resistance screening, utilizing plant cell dedifferentiation and redifferentiation.

[0061] 4. Analysis of MtNPHE1 expression in different tissues

[0062] RNA was extracted from alfalfa root nodules, roots, stems, unexpanded leaves, expanded leaves, flower buds, and flowers according to the Promega RNA Extraction Kit instructions. 1000 ng of RNA was reverse-transcribed into cDNA according to the Thermo Fisher Scientific Reverse Transcription Kit instructions. Using the 5-fold diluted cDNA as a template, Primer 5 and Primer 6 as primers, and MtActin11 as an internal control gene, quantitative real-time PCR was used to detect the expression of the MtNPHE1 gene in different tissues, indicating the sites where MtNPHE1 plays a major role. The detection results are shown below. Figure 1 As shown, MtNPHE1 is expressed at extremely high abundance in root nodules compared to other tissues, indicating that MtNPHE1 plays an important role in root nodules.

[0063] Primer5:5'-GTTGCACAAGCGATTCTTGA-3'

[0064] Primer6: 5'-AGGACAAAAGGTGCTGCTGT-3'

[0065] MtActin11-F: 5'-CAAAAGATGGCAGATGCTGAGGAT-3'

[0066] MtActin11-R: 5'-CATGACACCGGTATGACGAGGTCG-3'

[0067] The quantitative PCR system consists of: 1 μL Primer5 (1 μM), 1 μL Primer6 (1 μM), 1 μL cDNA, 10 μL quantitative enzyme, and 7 μL ddH2O.

[0068] Simultaneously, the expression of the internal reference gene MtActin11 in various tissue materials was detected using primers MtActin11-F and MtActin11-R in the same system.

[0069] Reaction program: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 sec, 60℃ annealing for 1 min, 60℃ extension for 1 min, 40 cycles.

[0070] 5. Phenotypic detection of transgenic alfalfa

[0071] Seeds of R108, 35S:MtNPHE1-1, and 35S:MtNPHE1-2 were sanded to remove the hardened kernels, then laid flat on moist filter paper and hydrated in a 4°C refrigerator for 2 days. Afterward, they were placed in a light incubator to continue growing for 3 days until the two cotyledons unfolded. The hypocotyls of the seedlings were wrapped with sponge and fixed onto a hydroponic board, then placed in the hydroponic solution for further cultivation. After one week of growth in a light incubator, photographs were taken, and the phenotype was as follows. Figure 2 As shown in A. Simultaneously, seedlings with unfolded cotyledons were planted in soil and placed in a greenhouse to continue growing for one month. Photos were taken, and the phenotype was as follows. Figure 2 As shown in Figure B. The hydroponic seedlings were removed with tweezers, dried, and placed on weighing paper. They were then dried in an 80℃ oven until constant weight, and the dry weight was measured using an analytical balance. The results showed that the biomass of the 35S:MtNPHE1-1 and 35S:MtNPHE1-2 overexpression materials was significantly higher than that of the wild-type control. Figure 2 C), which indicates that increasing the expression of MtNPHE1 can increase alfalfa biomass. Culture conditions in the light incubator and greenhouse: photoperiod of 16h (light) / 8h (dark), humidity of 70%, and temperature of 24℃.

[0072] Hydroponic solution formula: 2.5mM KNO3, 0.25mM CaCl2, 1mM Mg2SO4, 0.5mM KH2PHO4, 0.03mM M3BO3, 0.005mM MnSO4, 0.001mM ZnSO4, 0.001mM CuSO4, 0.0007mM Na2MoO4, 0.1mM FeSO4, 0.1mM EDTA-Na2, solvent is single-distilled water, pH adjusted to 5.95 with KOH.

[0073] Soil formula for soil cultivation: Mix imported soil, vermiculite and Northeast black soil in a 1:1:1 ratio, and then divide into flower pots.

[0074] 6. Determination of Inorganic Phosphorus Content in Transgenic Alfalfa

[0075] (1) R108, 35S:MtNPHE1-1 and 35S:MtNPHE1-2 were hydrated on filter paper for 2 days, placed in a light incubator and continued to grow for 3 days until the two cotyledons unfolded. Then the whole plant was harvested, and at least three biological replicates were taken for each material and frozen in liquid nitrogen for preservation.

[0076] (2) Prepare a 1.5mL EP tube in advance and add 100μL of inorganic phosphorus extraction solution to the tube.

[0077] (3) Grind the material from (1) into powder while frozen. Take about 100 mg of the powder into the EP tube from (2) using a pre-cooled steel spoon, weigh the fresh weight of the powder, mix it with a shaker, add 900 μL of glacial acetic acid to the EP tube and mix by inverting. React in a water bath at 42°C for 30 min. Centrifuge at 1,2000 rpm for 15 min at 4°C.

[0078] (4) Take 250 μL of the supernatant obtained in step (3) and add it to 350 μL of colorimetric solution. Mix by inverting and mixing. At the same time, prepare a standard curve and react in a water bath at 42°C for 30 min.

[0079] (5) Take the reaction solution from step (4) and use a spectrophotometer to determine the wavelength and concentration at 820 nm.

[0080] The results are as follows Figure 3 As shown, the inorganic phosphorus content of the 35S:MtNPHE1-1 and 35S:MtNPHE1-2 overexpression materials was significantly higher than that of the wild-type control, indicating that increasing the expression of MtNPHE1 can enhance phosphorus accumulation in alfalfa.

[0081] Inorganic phosphorus extract formulation (1L): 10mL 1M Tris-HCl (pH 8.0), 2mL 0.5M EDTA (pH 8.0), 5.844g NaCl, 700μL β-mercaptoethanol, 10mL 100mM PMSF (prepared fresh), and diluted to volume with ddH2O.

[0082] Colorimetric solution formulation (1L): Dissolve 25g ammonium molybdate in 400mL ddH2O, dissolve 1.25g ammonium metavanadate in 300mL boiling ddH2O, cool, add 250mL concentrated nitric acid, add the ammonium molybdate solution to the ammonium metavanadate solution, pour slowly while stirring constantly, make up to volume with ddH2O, store in the dark at room temperature.

[0083] Although embodiments of the present invention have been described in conjunction with the accompanying drawings, the patent owner may make various modifications or alterations within the scope of the appended claims, as long as they do not exceed the protection scope described in the claims of the present invention, they shall be within the protection scope of the present invention.

Claims

1. Application of protein MtNPHE1 in increasing phosphorus accumulation, regulating growth and development, or increasing alfalfa yield in alfalfa cultivation; The protein MtNPHE1 is derived from alfalfa, and its amino acid sequence is shown in Sequence 2.

2. The application according to claim 1, characterized in that, The protein MtNPHE1 is encoded by the CDS sequence shown in Sequence 1.

3. The application according to claim 1, characterized in that, MtNPHE1 The nucleic acid sequence is the CDS sequence shown in Sequence 1.

4. Application of biomaterials related to protein MtNPHE1 in alfalfa cultivation to improve phosphorus accumulation, regulate alfalfa growth and development, or increase alfalfa yield, wherein the protein MtNPHE1 is derived from alfalfa and its amino acid sequence is shown in Sequence 2. The biological material is any one of the following (B1) to (B7): (B1) A nucleic acid molecule encoding the amino acid sequence shown in sequence 2; (B2) A carrier containing the nucleic acid molecule described in (B1); (B3) Microorganisms containing the nucleic acid molecules described in (B1); (B4) Microorganisms containing the carrier described in (B2); (B5) Transgenic plants containing the nucleic acid molecules described in (B1); (B6) A transgenic plant containing the vector described in (B2); (B7) contains the nucleic acid described in (B1) and is used for editing recombinant vectors / recombinant microorganisms / recombinant plant cell lines for alfalfa.

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