Alfalfa mspt1 gene and application thereof
By cloning and regulating the MsPT1 gene in alfalfa, the plant's ability to absorb and utilize phosphorus was improved, solving the problem of phosphorus deficiency limiting alfalfa yield. This achieved efficient phosphorus utilization and yield increase, and has environmental protection value.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF ANIMAL SCIENCES OF CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2025-04-21
- Publication Date
- 2026-05-29
AI Technical Summary
Alfalfa has a high demand for phosphorus, and phosphorus deficiency severely limits its yield. Existing technologies that increase yield by increasing fertilizer application have limited effectiveness and do not comply with resource conservation policies.
The MsPT1 gene of alfalfa was cloned, and its expression was regulated to improve the plant's ability to absorb and utilize phosphorus. Recombinant vectors and recombinant microorganisms were constructed to overexpress or silence the MsPT1 gene, thereby improving the plant's efficiency in arsenate absorption and phosphorus utilization.
Molecular breeding has improved plants by increasing their phosphorus uptake rate, biomass, phosphorus content, and protein content, thereby enhancing the efficiency of nitrogen and phosphorus nutrition and yield in crops, which is of environmental significance.
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Figure CN120350023B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically, to an alfalfa MsPT1 gene and its applications. Background Technology
[0002] Phosphorus is one of the essential macronutrients for plant growth and development, but it is also one of the most difficult nutrients for plants to move and utilize in the soil. Phosphorus is absorbed by plants in the form of inorganic phosphorus. Although large amounts of phosphate fertilizer are applied to the soil annually, inorganic phosphorus is easily converted into organic phosphorus by microorganisms, which is difficult for plants to absorb. Furthermore, inorganic phosphorus has an extremely low diffusion coefficient in the soil, resulting in a severe deficiency of available phosphorus in the soil. Low phosphorus stress not only directly affects plant growth but also impairs nitrogen absorption and utilization, inhibiting nodule formation and nitrogen fixation in legumes. Alfalfa (Medicago sativa L.), as a legume, can fix nitrogen symbiotically through nodules and requires a high phosphorus content in the soil; severe phosphorus deficiency severely limits alfalfa yield. Increasing crop yield by continuously increasing fertilizer application is inconsistent with the basic policy of "resource conservation," and the yield-increasing effect of fertilizer is limited. Therefore, identifying key genes involved in phosphorus absorption and utilization in plants and improving the ability of crops to absorb and utilize phosphorus is the most direct and effective way to improve the efficiency of nitrogen and phosphorus nutrition and yield of crops. 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
[0003] To achieve the above objectives, the present invention first provides an isolated MsPT1 gene from alfalfa, the nucleotide sequence of which is shown in SEQ ID No. 1.
[0004] The present invention also provides an isolated alfalfa MsPT1 gene, the amino acid sequence of which is shown in SEQ ID No. 2.
[0005] The present invention also provides a polypeptide having the amino acid sequence shown in SEQ ID No. 2.
[0006] In some embodiments, the polypeptide is encoded by the nucleotide sequence shown in SEQ ID No. 1.
[0007] This invention also provides biological materials related to the MsPT1 gene described above, which are any one of B1) to B6) below:
[0008] B1) Expression cassettes containing the MsPT1 gene described above;
[0009] B2) Recombinant vectors containing the MsPT1 gene mentioned above.
[0010] B3) Recombinant microorganisms containing the aforementioned MsPT1 gene;
[0011] B4) A recombinant vector containing the expression cassette described in B1);
[0012] B5) Recombinant microorganisms containing the recombinant vector described in B2);
[0013] B6) Compositions containing the above-mentioned polypeptides.
[0014] The present invention also provides the application of the MsPT1 gene, the polypeptide, or the biological material described above in regulating the physiological process of plant flowering or in plant breeding.
[0015] In some embodiments, the plant includes alfalfa and / or Arabidopsis thaliana.
[0016] In some embodiments, the application is to construct high-quality plants.
[0017] In some embodiments, the high quality includes at least one of promoting arsenate absorption, increasing phosphorus absorption rate, increasing plant height, increasing plant vegetative biomass, phosphorus content, and / or protein content.
[0018] In some embodiments, the application is achieved by regulating the expression of the MsPT1 gene.
[0019] In some embodiments, the regulation of MsPT1 gene expression is described as either upregulating or downregulating MsPT1 gene expression.
[0020] In some embodiments, the regulation of MsPT1 gene expression is to silence MsPT1 gene expression or to overexpress MsPT1 gene.
[0021] The present invention also provides a method for detecting the arsenate uptake capacity of plants, the method comprising the step of quantitatively detecting the expression level of the MsPT1 gene.
[0022] In some embodiments, the plant includes alfalfa and / or Arabidopsis thaliana.
[0023] Finally, this invention provides a method for preparing high-quality alfalfa, the method comprising the step of regulating the MSPT1 gene.
[0024] In some embodiments, the regulation of MsPT1 gene expression is described as either upregulating or downregulating MsPT1 gene expression.
[0025] In some embodiments, the regulation of MsPT1 gene expression is to silence MsPT1 gene expression or to overexpress MsPT1 gene.
[0026] In some embodiments, the plant includes alfalfa and / or Arabidopsis thaliana.
[0027] Compared with the prior art, the present invention has at least the following beneficial effects:
[0028] This invention cloned the MsPT1 gene from alfalfa and demonstrated through biochemical experiments that MsPT1 promotes arsenate uptake in Arabidopsis thaliana (L.) Heynh., increases biomass and inorganic phosphorus content; overexpression of MsPT1 significantly increases alfalfa biomass, phosphorus content, and protein content. Alfalfa, as a legume, requires high phosphorus levels in the soil, and phosphorus deficiency severely limits its yield. This invention identifies the key plant gene MsPT1 and explores the most direct and effective way to improve crop nitrogen and phosphorus nutrient efficiency and yield by modifying the crop's ability to absorb and utilize arsenate. Improving plants through molecular breeding not only enhances yield and quality but also has significant implications for environmental protection. Attached Figure Description
[0029] Figure 1 This is a screening diagram of the MsPT1 gene in alfalfa; Figure 1 A is the gene clustering analysis diagram; Figure 1 B is a diagram of gene expression patterns; Figure 1 C represents the heatmap analysis result;
[0030] Figure 2 This is a graph showing the amplification results; Figure 2 A represents the DNA identification result; Figure 2 B represents the results of the expression level analysis;
[0031] Figure 3 This is a graph showing the effect of the MsPT1 gene on arsenate uptake in Arabidopsis thaliana; Figure 3 A represents the result for arsenate. Figure 3 B represents the green cotyledon percentage;
[0032] Figure 4 This is a graph showing the effect of the MsPT1 gene on biomass and inorganic phosphorus content in Arabidopsis thaliana. Figure 4 A is a biomass diagram of Arabidopsis thaliana; Figure 4 B is the graph showing the inorganic phosphorus content.
[0033] Figure 5 This is a graph showing the effect of the MsPT1 gene on plant height and biomass of alfalfa. Figure 5 A represents the relative expression level; Figure 5 B is the plant height diagram; Figure 5 C represents the biomass diagram; Figure 5 D represents the phosphorus content graph; Figure 5 E is a graph showing protein content. Detailed Implementation
[0034] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.
[0035] The nucleotide sequence of the MsPT1 gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the MsPT1 gene is shown in SEQ ID No. 2.
[0036] SEQ ID No. 1:
[0037]
[0038] SEQ ID No.2:
[0039] MSGELGVLNALDLAKTQLYHFTTIVIAGMGFFTDAYDLFCISLVTKLLGRIYYTEPNPTR
[0040] PGTLPPSAQSAVTGVALVGTLAGQLFFGWLGDKLGRKKVYGLTLILMVGCSVASGLSFGS
[0041] SPKGVMATLCFFRFWLGFGIGGDYPLSATIMSEYANKKTRGAFIAAVFAMQGFGILGGGI
[0042] VALIVASIFDHKYKVPTFEENPAASLLVPQFDYVWRLILMFGALPAALTYYWRMKMPETA
[0043] RYTALVAKNAKQAAADMSKVLQVELEVEEEKVQKMTSDKRNSYGLFTKQFAKRHGWA
[0044] LFGTCSTWFLLDIAFYSQNLFQKDIFSAIGWIPPAKEMNAIHEVYKIARAQTLIALCSTVPG
[0045] YWFTVAFIDYMGRFAIQMMGFFFMTVFMFALAIPYDHWSKEENRIGFVVMYSLTFFFAN
[0046] FGPNATTFVVPAEIFPARLRSTCHGISAAAGKAGAIVGAFGFLYAAQSKDPTKTDKGYPTGIGIKNSLIMLGVINFVGMLCTLLVPESKGKSLEELSGENEGEGAEATEQEGSRV;
[0047] Example 1. Screening of Medicago sativa MsPT1 gene
[0048] Alfalfa was treated with both high-phosphorus (500 μmol / L KH₂PO₄) and low-phosphorus (5 μmol / L KH₂PO₄) treatments. Root samples were selected for RNA sequencing, and differentially expressed genes were screened based on a p-value < 0.05 and a |log₂ FC| > 1 threshold. Under low-phosphorus treatment, a total of 5829 differentially expressed genes were identified in the roots, including 3504 upregulated genes and 2325 downregulated genes. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the sequencing results to screen for important genes in alfalfa's response to low-phosphorus stress.
[0049] The analysis results showed that the genes were divided into 28 modules. Figure 1 A), where the light blue module contains a large number of genes, and the gene expression pattern analysis results show that low phosphorus stress upregulates the gene expression in this module. Figure 1 (B) This result suggests that genes in the sky-blue module may play an important role in alfalfa's response to low phosphorus stress. Further analysis of the genes in this module was conducted to identify core genes. Pearson correlation analysis was performed on differentially expressed genes and characteristic genes in the module, identifying genes with |kME|>0.99 as core genes, resulting in a total of 753 genes. Among the core genes, a phosphorus transporter family coding gene, MsG0180001800_01, was discovered. Analysis of genes of this family detected in the roots revealed a total of 5 coding genes: in addition to MsG0180001800_01, they also included MsG0780040736_01, MsG0780040738_01, MsG0180001796_01, and MsG0180001797_01. Heatmap analysis showed that all five genes were induced to increase by low phosphorus stress, with MsG0180001800_01 showing the most significant increase. Figure 1 C) The gene was named MsPT1 (PhosphateTransporter 1).
[0050] Example 2: Cloning of the MsPT1 gene in alfalfa
[0051] Primers Primer1 and Primer2 were designed at the 5' and 3' ends of the MsPT1 gene CDS sequence to amplify the full-length MsPT1 gene CDS. RNA was extracted from the alfalfa variety “Zhongmu 1”, reverse-transcribed into cDNA, and then used as a template to amplify the full-length MsPT1 gene CDS sequence. RNA was extracted from “Zhongmu 1” leaves 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.
[0052] Amplification system (50L): 5L 10×Taq Buffer, 0.5L ExTaq, 4L dNTPs, 2.5L Primer 1, 2.5L Primer 2, 35L ddH2O, 1L template.
[0053] Primer1: 5'-ATGTCAGGAGAGCTAGGAGTGC-3' (SEQ ID No. 3);
[0054] Primer2: 5'-AACCCTT GATCCTTCTT GCTCA-3' (SEQ ID No. 4);
[0055] 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, followed by 72℃ extension for 10 min. After recovery, it was ligated to the T-vector.
[0056] Ligation of the T-vector: The amplification product is ligated to the intermediate vector pMD18-T for subsequent sequencing identification.
[0057] Connection system: 1L 10×Enhancer, 50-100ng of recovered product, 1L carrier, ddH2O to make up to 10L.
[0058] Connection conditions: Place at room temperature for 5 minutes.
[0059] Transformation of *E. coli* competent cells DH5α: The ligation product was added to *E. coli* competent cells using a pipette, incubated on ice for 30 min, heat-shocked at 42°C for 90 sec, and incubated on ice for 2-3 min. Recovery LB medium was added in a sterile workbench, mixed thoroughly, and then spread onto solid LB medium supplemented with ampicillin. The cells were inverted and incubated at 37°C. After approximately 12 h of culture, single clones appeared. Single clones were picked for sequencing to identify the MsPT1 gene CDS sequence. Sequencing results showed that the sequence of the cloned product was consistent with the nucleic acid sequence shown in SEQ ID No. 1, encoding the amino acid sequence shown in SEQ ID No. 2. DH5α competent cells were ordered from Novizan.
[0060] Example 3: Construction of recombinant overexpression vector
[0061] (1) Construction of recombinant overexpression vector
[0062] To demonstrate the function of the MsPT1 gene in plants, a recombinant vector capable of overexpressing the MsPT1 gene was constructed. This recombinant vector was introduced into recipient plants to obtain transgenic plants with overexpression of the MsPT1 gene. The vector used was Super1300, and the resulting recombinant expression vector was Super:MsPT1. Using cDNA from *Amyda sinensis* var. *zhongmu* as a template, the CDS sequence of the MsPT1 gene was amplified using primers Primer3 and Primer4 with adapters, ensuring that the amplified CDS sequence had adapters at both ends (the adapters are the sequences on the vector, located at both ends of the SmaI restriction site). Simultaneously, the vector was linearized by digesting it with SmaI restriction enzyme. Homologous recombination was then used to ligate the adapter-attached CDS sequence into the linearized vector.
[0063] Primer3: 5'-tctagaaagcttctgcaggggATGTCAGGAGAGCTAGGAGTGC-3' (SEQ IDNo. 5);
[0064] Primer4: 5'-gctcctcgcccttgctcaccatAACCCTTGATCCTTCTTGCTCA-3' (SEQ IDNo. 6);
[0065] Note: Lowercase letters are connector sequences.
[0066] Cloning the gene: The MsPT1 gene was cloned with an adapter CDS sequence. The amplification system and procedure were the same as those in Example 2 above, which described the cloning of the MsPT1 gene in alfalfa.
[0067] Vector linearization system: 5 μL 10×QuickCut Buffer, 1 μL QuickCut BglII, 1 μL QuickCut NcoI, plasmid ≤1 μg, ddH2O added to 50 μL. Vector linearization conditions: 37℃ for 5 min.
[0068] The cloned gene product and linearized vector backbone were recovered, and the recovered cloned gene product was ligated to the vector backbone using homologous recombination. Ligation system: 5 μL 2×Cloning Master Mix, 10-500 ng linearized vector, 30-1500 ng recovered target gene product, and ddH2O was used to bring the reaction volume to 10 μL. Ligation conditions: After mixing the system, it was incubated in a 50℃ air bath for 30 min.
[0069] After ligation, the product was transformed into competent E. coli DH5α cells, and single clones were selected for sequencing. The plasmid with the correct sequence was the CDS sequence shown in SEQ ID No. 1 inserted between SmaI cells. This recombinant vector was used for subsequent introduction into Agrobacterium and plants.
[0070] (2) Obtaining Arabidopsis thaliana transgenic with the MsPT1 gene
[0071] Cut off the top of the bolting Arabidopsis thaliana a week in advance to encourage the growth of lateral branches.
[0072] (2.1) Obtaining the conversion solution
[0073] Take approximately 100 μL of bacterial culture containing the positive clone of the recombinant expression vector Super1300:MsPT1 and inoculate it into 1 mL of LB liquid medium (50 μg / mL rifampicin and 50 μg / mL kanamycin). Incubate overnight at 28°C with a shaker at 200 rpm. (LB medium: 10 g / L peptone; 5 g / L yeast extract; 10 g / L NaCl) to complete the first activation.
[0074] Transfer 1 mL of activated bacterial culture to 200 mL of LB liquid medium (50 μg / mL rifampicin and 50 μg / mL kanamycin), and continue to incubate in a shaker at 28 °C until the OD600 value reaches 1.2-1.8, thus completing the second activation of Agrobacterium.
[0075] Centrifuge at 4000g for 15 min and discard the supernatant. Resuspend the precipitate in 500mL of conversion buffer and adjust the OD 600 to the range of 0.6-0.8. Add Silwet-77 at a ratio of 0.03%, invert and mix well to complete the bacterial culture collection and obtain the conversion buffer.
[0076] (2.2) Infection
[0077] Dispense the conversion solution into small bowls of approximately 200 mL each. Invert the Arabidopsis thaliana plants that have already bolted and produced flower buds into the conversion solution, ensuring the flower buds are fully in contact with the solution, for 4 minutes. (Arabidopsis thaliana conversion solution: 40× (macronutrient stock solution): 12.5 mL / L; 40× (MgSO4): 12.5 mL / L; 200× (micronutrient stock solution): 25 mL / L; sucrose: 50 g / L; Silwet-77: 300 μL / L).
[0078] The 1L mother liquor contains the following macro-elements: NH4NO3 33g; KNO3 38g; CaCl2 6.643g; MgSO4·7H2O 7.4g; KH2PO4 3.4g.
[0079] 1L of trace element stock solution contains: KI 0.083g; H3BO3 0.62g; MnSO4·H2O 1.74g; ZnSO4·7H2O 0.86g; Na2Mo4·2H2O 0.025g; CuSO4·5H2O 0.0025g; CoCl2·6H2O 0.0025g.
[0080] (2.3) Seed harvesting
[0081] After infection, Arabidopsis plants were placed flat on trays and cultured in the dark for 18-24 hours. The pots were then placed upright in an artificial climate chamber to allow them to grow normally. One month later, when the Arabidopsis plants matured, the seeds were harvested.
[0082] Eight positive strains were identified: OE-9, OE-11, OE-14, OE-18, OE-24, OE-25, OE-26, and OE-31. DNA identification results are shown below (...). Figure 2 As shown in A), the positive lines amplified a single band of uniform size, while the wild-type negative control (Col-0) did not amplify a band. Homozygous lines OE-9 and OE-26 were identified using hygromycin resistance for subsequent experiments. Expression levels of the selected homozygous OE-9 and OE-26 lines were analyzed using qRT-PCR. Figure 2 B) The results showed that the expression level of MsPT1 in both strains was significantly increased, which can be used for subsequent phenotypic analysis.
[0083] (3) Obtaining genetically modified alfalfa
[0084] The positive Agrobacterium tumefaciens EHA105 containing Super1300:MsPT1 was removed from the -80℃ freezer for activation. Specifically, approximately 60 μL was inoculated into 1.4 mL of LB liquid medium (50 μg / mL rifampicin and 50 μg / mL kanamycin), and incubated overnight at 28℃ with a shaker at 200 rpm to complete the first activation. 1 mL of the activated bacterial culture was then transferred to 30 mL of LB liquid medium (50 μg / mL rifampicin and 50 μg / mL kanamycin), and incubated at 28℃ with a shaker until the OD600 reached 0.6-0.8, completing the second activation. The bacterial culture was then collected.
[0085] Select healthy leaves and place them in a 50mL centrifuge tube containing 36mL of ddH2O. Add 4mL of sodium hypochlorite to the centrifuge tube, tighten the cap, and invert for 10 minutes. In a clean bench, pour out the sodium hypochlorite solution. Use tweezers to transfer the leaves to an Erlenmeyer flask and wash the leaves 6 times with sterile ddH2O, 1-2 minutes each time. Pour out the ddH2O and add SH3a liquid culture medium to the Erlenmeyer flask containing the leaves, ensuring the leaves are submerged. Cover the flask with sealing film and sonicate the leaves. Stop sonicating when 80% of the leaves show scattered dark green spots; this completes the leaf pretreatment.
[0086] Transfer the bacterial suspension with an OD600 value of 0.6-0.8 to a clean bench and pour it into a sterile 50mL centrifuge tube. Centrifuge at 4000 rpm for 10 min. Discard the supernatant in the clean bench, resuspend the bacterial suspension in 1mL of SH3a liquid medium, and add the resuspended bacterial suspension to an Erlenmeyer flask containing the leaflets, adjusting the OD600 value to 0.2-0.4. Place the Erlenmeyer flask in a vacuum pump and evacuate for 10 min. Place it in a 28℃ shaker at 70 rpm for 15 min to complete the infection.
[0087] In a clean bench, the leaves were removed, and the moisture on both sides of the leaves was blotted dry with sterilized filter paper. Using tweezers, the leaves were laid flat on SH3a solid medium with the upper side facing up and incubated in the dark for 48 hours. The leaves were then transferred to SH3a solid medium containing 2 mg / L cephalosporin and 5 mg / L hygromycin, with the medium changed every two weeks. After two months of dark incubation, once callus tissue appeared on the leaves, the callus tissue was transferred to MSBK solid medium containing 2 mg / L cephalosporin and 5 mg / L hygromycin for normal light culture.
[0088] Once green buds appear on the callus tissue, transfer the callus tissue with buds to SH9a solid medium containing 2 mg / L cephalosporin and 5 mg / L hygromycin, and culture under normal light until rooting and leaf growth. Transfer the seedlings from the medium to potting soil for positive identification.
[0089] The specific components of each culture medium in the above steps are as follows:
[0090] SH3a medium: S811: 13.2 g / L; SH vitamin solution: 1 mL / L; inositol: 2 mL / L; sucrose: 20 g / L; 2-4D (10 mg / mL): 0.5 mL / L; 6-BA (1 mg / mL): 0.25 mL / L; iron salt (FeSO4): 2 mL / L; Phytagel plant gel: 3 g / L;
[0091] MSBK medium: M519: 4.43 g / L; sucrose: 30 g / L; K483: 1 ml / L; 6-BA: 0.25 ml / L; Phytagel plant gel: 3 g / L;
[0092] SH9a medium: S811: 13.2 g / L; SH vitamin solution: 1 mL / L; inositol: 2 mL / L; sucrose: 20 g / L; iron salt (FeSO4): 2 mL / L; agar: 6 g / L;
[0093] Example 4: MsPT1 promotes arsenate absorption in Arabidopsis thaliana.
[0094] Arsenate germination experiments were conducted using the Colombian wild-type (Col-0) and MsPT1 overexpression lines OE-9 and OE-26. Arsenic and phosphorus belong to the same main group, and arsenates are structurally similar to phosphates. When plants absorb large amounts of arsenates, they exhibit toxic phenotypes. Seeds of Col-0, OE-9, and OE-26 were placed in 1.5 ml tubes and soaked in 6% sodium hypochlorite for 10 minutes to thoroughly sterilize the seeds. The seeds were then washed six times with sterile double-distilled water to remove the sodium hypochlorite. Finally, an appropriate amount of double-distilled water was added to the 1.5 ml tubes, and the tubes were placed in a 4°C refrigerator. Two days later, the seeds were removed from the refrigerator and spotted onto 1 / 2 MS medium and 1 / 2 MS medium + 200 mM sodium arsenate medium, respectively. The culture dishes were then placed flat in a light incubator to allow the seeds to germinate and grow. On arsenate-free 1 / 2 MS medium, there was no significant difference in leaf color and growth vigor between the OE-9 and OE-26 lines and wild-type Arabidopsis thaliana; on medium containing 200 μM arsenate, the MsPT1 overexpression lines exhibited a significant toxic phenotype, with severely inhibited germination. Figure 3 A). Statistical analysis of the green cotyledon percentage showed that, compared to the wild type, the green cotyledon percentage of the MsPT1 overexpression lines was reduced by 42% and 48%, respectively. Figure 3 B). The above results indicate that MsPT1 overexpression lines are more sensitive to arsenate, and overexpression of MsPT1 can increase the phosphorus uptake rate in Arabidopsis thaliana.
[0095] The composition of the plant 1 / 2MS solid medium was as follows: M 519: 2.2 g / L; sucrose: 15 g / L; agar: 8 g / L;
[0096] The composition of the externally supplemented arsenate medium was as follows: M519: 2.2 g / L; sucrose: 15 g / L; agar: 8 g / L; 100 mM sodium arsenate: 2 mL / L.
[0097] Example 5: MsPT1 overexpression can increase Arabidopsis biomass and inorganic phosphorus content.
[0098] Col-0, OE-9, and OE-26 seeds were spotted on MS medium, and the culture dishes were then placed vertically in a light incubator to allow for seed germination and growth. Biomass was collected after 7 days. The results showed that MsPT1 overexpression increased Arabidopsis biomass. Figure 4 A). The inorganic phosphorus content of MsPT1 overexpression materials OE-9 and OE-26, as well as the wild-type control, was detected using the molybdenum blue assay. The results showed that the inorganic phosphorus content of the MsPT1 overexpression materials was significantly higher than that of the wild-type control. Figure 4 B).
[0099] The molybdenum blue method for determining inorganic phosphorus content is as follows:
[0100] 1. Take whole seedlings that have germinated and grown for 7 days on MS medium, record the fresh weight of each group of materials, freeze them in liquid nitrogen, grind the samples with a grinder, and add 1% glacial acetic acid to 10mL EP tubes in advance for later use.
[0101] 2. Add inorganic phosphorus extraction solution to the sample tube at a ratio of 100L of extraction solution per 10mg sample (1L inorganic phosphorus extraction solution formula: 10mL 1M Tris-HCl (pH 8.0), 2mL 0.5M EDTA (pH 8.0), 5.844g NaCl, 700L β-mercaptoethanol, 10mL 100mM PMSF (freshly prepared)). For a 70mg sample, add 700L of extraction solution and mix by inverting. Pipe the sample into an EP tube containing 4.3mL glacial acetic acid. Since the original sample tube still contains sample, pipette another 1mL of glacial acetic acid into the sample tube, invert the tube to clean it, and then pipette the sample into a 10mL EP tube. Repeat this cleaning process twice; the volume of glacial acetic acid in the 10mL EP tube will now be 6.3mL. Carefully invert the tube to mix, and incubate at 42℃ for 30min. Centrifuge at 4000 rpm for 15 min at 4℃.
[0102] 3. Take the extract obtained in step 2 and determine the phosphorus content using the vanadium-molybdenum blue method. Colorimetric analysis: Pipette 150 L of supernatant into 350 L of colorimetric solution and mix thoroughly by inverting. Simultaneously, prepare a standard curve and react in a 42℃ water bath for 30 min.
[0103] Absorbance measurement: Pipette 200L of the reaction solution into an ELISA plate and measure the absorbance at a wavelength of 820nm using a continuous ELISA reader.
[0104] Example 6: MsPT1 overexpression can increase alfalfa plant height, biomass, and protein content.
[0105] Three alfalfa MsPT1 overexpression lines were selected for further research, named OE-1, OE-2, and OE-3. Expression level detection results showed that the expression levels of the three transgenic lines were significantly higher than the control (…). Figure 5 A). Phenotypic results showed that the three overexpression lines, OE-1, OE-2, and OE-3, exhibited better growth than the control. Figure 5 B), the biomass statistics were consistent with the phenotype, and the biomass of the overexpression lines was significantly higher than that of the control. Figure 5 C). Near-infrared spectroscopy was used to determine the phosphorus and protein content of each material. The results showed that the phosphorus content of MsPT1 transgenic alfalfa was significantly increased. Figure 5 D). Protein content is one of the important indicators for evaluating alfalfa quality. Protein content determination results showed that the protein content of MsPT1 transgenic alfalfa was significantly higher than that of the control. Figure 5 E). The above results indicate that overexpression of MsPT1 can significantly increase alfalfa biomass, phosphorus content, and protein content.
[0106] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A type of isolated alfalfa MsPT1 The application of genes in any of the following A1) to A5): A1) Increases the rate of phosphorus absorption by plants; A2) Increase plant height; A3) Increase the biomass of the plant's vegetative body; A4) Increase the phosphorus content in the plant's vegetative cells, and / or A5) Increase the protein content of the plant's vegetative tissues; in, The MsPT1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the polypeptide it encodes is shown in SEQ ID No. 2; The application is achieved through overexpression MsPT1 Genetic realization; The plants in question are alfalfa and / or Arabidopsis thaliana.
2. With isolated alfalfa MsPT1 Application of gene-related biological materials in any one of A1) to A5) below: A1) Increases the rate of phosphorus absorption by plants; A2) Increase plant height; A3) Increase the biomass of the plant's vegetative body; A4) Increase the phosphorus content in the plant's vegetative cells, and / or A5) Increase the protein content of the plant's vegetative tissues; in, The MsPT1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the polypeptide it encodes is shown in SEQ ID No. 2; The application is achieved through overexpression MsPT1 Genetic realization; The plants in question are alfalfa and / or Arabidopsis thaliana; The biomaterial is any one of B1) to B5) below: B1) Contains the contents of claim 1 MsPT1 Gene expression cassettes; B2) contains the contents of claim 1 MsPT1 Gene recombination vectors; B3) contains the contents of claim 1 MsPT1 Recombinant microorganisms; B4) A recombinant vector containing the expression cassette described in B1); B5) Recombinant microorganisms containing the recombinant vector described in B2).
3. A method for preparing high-quality alfalfa, characterized in that, The method involves overexpressing alfalfa. MsPT1 The steps of gene generation; The high quality refers to improving phosphorus absorption rate, increasing plant height, increasing plant vegetative biomass, increasing phosphorus content in plant vegetative tissue, and / or increasing protein content in plant vegetative tissue. Among them, the MsPT1 The nucleotide sequence of the gene is shown in SEQ ID No. 1.