Medicago sativa MsPT1 gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a medicago sativa MsPT1 gene and application thereof, the nucleotide sequence of the MsPT1 gene is shown as SEQ ID No.1, and the coding amino acid sequence of the MsPT1 gene is shown as SEQ ID No.2. The MsPT1 gene can promote arsenate absorption of plants and improve biomass and inorganic phosphorus content, is the most direct and effective way for improving crop absorption and utilizing arsenate to improve crop nitrogen and phosphorus nutrition efficiency and yield, improves plants through molecular breeding, not only can improve the yield and quality of the plants, but also can improve the yield and quality of the plants through molecular breeding. And the method also has important significance on environmental protection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a Medicago sativa MsPT1 gene and its applications. Background Art

[0002] Phosphorus is one of the macronutrients essential for plant growth and development, and it is also the most difficult nutrient element to move and be utilized by plants in the soil. Phosphorus is absorbed by plants in the form of inorganic phosphorus. Although a large amount of phosphate fertilizer is applied to the soil every year, inorganic phosphorus is easily transformed into organic phosphorus that is difficult for plants to absorb by microorganisms, and the diffusion coefficient of inorganic phosphorus in the soil is extremely low. Therefore, soil available phosphorus is severely deficient. Low phosphorus stress not only directly affects plant growth, but also affects the absorption and utilization of nitrogen by plants, and inhibits nodule formation and nodule nitrogen fixation in leguminous plants. Alfalfa (Medicago sativa L.), as a leguminous plant, can carry out symbiotic nitrogen fixation through nodules and has a relatively high requirement for the phosphorus content in the soil. Phosphorus deficiency severely limits alfalfa yield. Increasing the amount of fertilizer applied continuously to increase crop yield does not conform to the basic policy of "resource conservation", and the yield increase effect of fertilizers is limited. Therefore, exploring 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 the nitrogen and phosphorus nutrient efficiency and yield of crops. Improving plants through molecular breeding can not only increase the yield and quality of plants, but also has important significance for environmental protection. Summary of the Invention

[0003] To achieve the above object, the present invention first provides an isolated Medicago sativa MsPT1 gene, and the nucleotide sequence of the MsPT1 gene is as shown in SEQ ID No.1.

[0004] The present invention also provides an isolated Medicago sativa MsPT1 gene, and the encoded amino acid sequence of the MSPT1 gene is as 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 certain embodiments, the polypeptide is encoded by the nucleotide sequence shown in SEQ ID No.1.

[0007] The present invention also provides biological materials related to the above MsPT1 gene, which are any one of the following B1) to B6):

[0008] B1) An expression cassette containing the above MsPT1 gene;

[0009] B2) A recombinant vector containing the above MsPT1 gene,

[0010] B3) A recombinant microorganism containing the above MsPT1 gene;

[0011] B4) A recombinant vector containing the expression cassette described in B1);

[0012] B5) A recombinant microorganism containing the recombinant vector described in B2);

[0013] B6) A composition containing the above polypeptide.

[0014] The present invention also provides the use of the above MsPT1 gene, the above polypeptide or the above biological material in regulating the physiological process of plant flowering or in plant breeding.

[0015] In certain embodiments, the plants include alfalfa and / or Arabidopsis thaliana.

[0016] In certain embodiments, the use is to construct high-quality plants.

[0017] In certain embodiments, the high quality includes at least one of promoting arsenate absorption, increasing the phosphorus absorption rate, increasing plant height, increasing the biomass of the vegetative part of the plant, phosphorus content and / or protein content.

[0018] In certain embodiments, the use is achieved by regulating the expression of the MsPT1 gene.

[0019] In certain embodiments, the regulation of the expression of the MsPT1 gene is upregulating the expression of the MsPT1 gene or downregulating the expression of the MsPT1 gene.

[0020] In certain embodiments, the regulation of the expression of the MsPT1 gene is silencing the expression of the MsPT1 gene or overexpressing the MsPT1 gene.

[0021] The present invention also provides a method for detecting the arsenate absorption ability of plants, the method comprising the step of quantitatively detecting the expression level of the MsPT1 gene.

[0022] In certain embodiments, the plants include alfalfa and / or Arabidopsis thaliana.

[0023] The present invention finally provides a method for preparing high-quality alfalfa, the method comprising the step of regulating the MSPT1 gene.

[0024] In certain embodiments, the regulation of the expression of the MsPT1 gene is upregulating the expression of the MsPT1 gene or downregulating the expression of the MsPT1 gene.

[0025] In certain embodiments, the regulation of the expression of the MsPT1 gene is silencing the expression of the MsPT1 gene or overexpressing the 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] The present invention cloned the alfalfa MsPT1 gene, and through biochemical experiments, it was proved that MsPT1 promotes the absorption of arsenate by Arabidopsis thaliana ((Arabidopsis thaliana (L.) Heynh.)), can increase the biomass and inorganic phosphorus content of Arabidopsis thaliana; overexpression of MsPT1 can significantly increase the biomass, phosphorus content and protein content of alfalfa. As a leguminous plant, alfalfa has relatively high requirements for the phosphorus content in the soil, and phosphorus deficiency severely limits the yield of alfalfa. The present invention excavated the key gene MsPT1 involved in plants, and the most direct and effective way to improve the nitrogen and phosphorus nutrient efficiency and yield of crops is to improve the ability of crops to absorb and utilize arsenate. Improving plants through molecular breeding can not only increase the yield and quality of plants, but also has important significance for environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the screening diagram of the alfalfa MsPT1 gene; Figure 1 A is the gene clustering analysis diagram; Figure 1 B is the gene expression pattern diagram; Figure 1 C is the heat map analysis result;

[0030] Figure 2 is the amplification result diagram; Figure 2 A is the DNA identification result; Figure 2 B is the expression level analysis result;

[0031] Figure 3 is the diagram of the effect of the MsPT1 gene on the absorption of arsenate by Arabidopsis thaliana; Figure 3 A is the arsenate result. Figure 3 B is the result of the green cotyledon rate;

[0032] Figure 4 is the diagram of the effect of the MsPT1 gene on the biomass and inorganic phosphorus content of Arabidopsis thaliana; Figure 4 A is the Arabidopsis thaliana biomass diagram; Figure 4 B is the inorganic phosphorus content diagram.

[0033] Figure 5 is the diagram of the effect of the MsPT1 gene on the plant height and biomass of alfalfa; Figure 5 A is the relative expression level diagram; Figure 5 B is the plant height diagram; Figure 5 C is the biomass diagram; Figure 5 D is the phosphorus content diagram; Figure 5 E is the protein content diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0035] The nucleotide sequence of the MsPT1 gene is shown as SEQ ID No.1, and the amino acid sequence encoded by the MsPT1 gene is shown as 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] Taking alfalfa as the material, sufficient phosphorus (500 μmol / L KH2PO4) and low phosphorus (5 μmol / L KH2PO4) treatments were carried out. The roots were selected for RNA sequencing, and the differentially expressed genes were screened according to the thresholds of P < 0.05 and |log2 FC| > 1. Under low phosphorus treatment, there were 5829 differentially expressed genes in the roots, including 3504 up-regulated genes and 2325 down-regulated genes. Weighted gene co-expression network analysis (WGCNA) was performed on the sequencing results to screen important genes during the process of alfalfa responding to low phosphorus stress.

[0049] The analysis results showed that the genes were divided into 28 modules ( Figure 1 A), among which the sky blue module aggregated more genes. At the same time, the analysis results of the gene expression pattern map showed that the gene expression in this module was up-regulated under low phosphorus stress ( Figure 1 B), which implied that the genes in the sky blue module might play an important role when alfalfa responded to low phosphorus stress. Further analysis was carried out on the genes in this module to find the core genes. Pearson correlation analysis was performed on the differential genes and the characteristic genes in the module, and the genes with |kME| > 0.99 were defined as core genes, and a total of 753 genes were screened. A gene encoding a phosphate transporter family, MsG0180001800_01, was found among the core genes. Analysis of the genes of this family detected in the roots showed that there were 5 genes encoding this family. In addition to MsG0180001800_01, it also included MsG0780040736_01, MsG0780040738_01, MsG0180001796_01, and MsG0180001797_01. Heat map analysis showed that the expression of these 5 genes was induced to increase under low phosphorus stress, and MsG0180001800_01 was the most significantly induced by low phosphorus ( Figure 1 C), and this gene was named MsPT1 (Phosphate Transporter 1).

[0050] Example 2: Cloning of alfalfa MsPT1 gene

[0051] Primers Primer1 and Primer2 were designed at the 5' end and 3' end of the CDS sequence of the MsPT1 gene for amplifying the full-length CDS of the MsPT1 gene. RNA of alfalfa variety "Zhongmu No. 1" was extracted, and after reverse transcription into cDNA, this cDNA was used as a template to amplify the full-length CDS sequence of the MsPT1 gene. The leaves of "Zhongmu No. 1" were cut to extract RNA, and RNA was extracted according to the instructions of Promega RNA extraction kit. 1000 ng of RNA was reverse transcribed into cDNA according to the instructions of Thermo Fisher reverse transcription kit.

[0052] Amplification system (50 μL): 5 μL of 10× Taq Buffer, 0.5 μL of ExTaq, 4 μL of dNTPs, 2.5 μL of primer Primer1, 2.5 μL of primer Primer2, 35 μL of ddH₂O, 1 μL of template.

[0053] Primer1: 5’-ATGTCAGGAGAGCTAGGAGTGC-3’ (SEQ ID No.3);

[0054] Primer2: 5’-AACCCTT GATCCTTCTT GCTCA-3’ (SEQ ID No.4);

[0055] Amplification program: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 20 s, 35 cycles of amplification, and extension at 72°C for 10 min. After recovery, it is ligated to the T-vector.

[0056] Ligation to the T-vector: The amplified product is ligated to the intermediate vector pMD18-T vector for subsequent sequencing and identification.

[0057] Ligation system: 1 μL of 10× Enhancer, 50 - 100 ng of recovered product, 1 μL of vector, supplemented with ddH₂O to 10 μL.

[0058] Ligation condition: Incubate at room temperature for 5 min.

[0059] Transformation of Escherichia coli competent cell DH5α: Use a pipette to add the ligation product into the Escherichia coli competent cells, incubate on ice for 30 min, heat shock at 42°C for 90 sec, incubate on ice for 2 - 3 min, add recovery LB in a sterile workbench, mix well and spread on a solid LB medium supplemented with ampicillin, invert and place it in a 37°C incubator. After culturing for about 12 h, monoclonal colonies appear. Pick monoclonal colonies for sequencing and identification of the CDS sequence of the MsPT1 gene. The sequencing results show that the sequence of the cloned product is consistent with the nucleic acid sequence shown in SEQ ID No.1 and encodes the amino acid sequence shown in SEQ ID No.2. The DH5α competent cells are ordered from Novoprotein.

[0060] Example 3. Construction of recombinant overexpression vector

[0061] (1) Construction of recombinant overexpression vector

[0062] To prove the role of the MsPT1 gene in plants, a recombinant vector capable of overexpressing the MsPT1 gene was constructed and introduced into recipient plants to obtain transgenic plants with overexpression of the MsPT1 gene. The vector used was Super1300, and the obtained recombinant expression vector was Super:MsPT1. Using the cDNA of "Zhongmu No. 1" as a template, the CDS sequence of the MsPT1 gene was amplified with primers Primer3 and Primer4 with adapters, so that both ends of the amplified CDS sequence carried adapters (the adapter is the sequence on the vector, located at both ends of the SmaI restriction site). At the same time, the vector was digested with SmaI endonuclease to linearize the vector. The CDS sequence with adapters was ligated to the linearized vector by homologous recombination method.

[0063] Primer3: 5’-tctagaaagcttctgcaggggATGTCAGGAGAGCTAGGAGTGC-3’ (SEQ ID No.5);

[0064] Primer4: 5’-gctcctcgcccttgctcaccatAACCCTTGATCCTTCTTGCTCA-3’ (SEQ ID No.6);

[0065] Note: The lowercase letters are adapter sequences.

[0066] Cloning the gene: Clone the CDS sequence of the MsPT1 gene with adapters. The amplification system and procedure are the same as those in the "Cloning of the Medicago sativa MsPT1 gene" in Example 2 above.

[0067] Vector linearization system: 5 μL of 10×QuickCut Buffer, 1 μL of QuickCut BglII, 1 μL of QuickCut NcoI, plasmid ≤1 μg, supplemented with ddH2O to 50 μL. Vector linearization condition: Incubate at 37°C for 5 min.

[0068] Recover the cloned gene product and the linearized vector backbone, and ligate the recovered product of the cloned gene with the vector backbone by homologous recombination method. Ligation system: 5 μL of 2×Cloning Master Mix, 10 - 500 ng of linearized vector, 30 - 1500 ng of the recovered product of the target gene, and supplement the reaction system to 10 μL with ddH2O. Ligation condition: Mix the system and place it in a 50°C air bath for ligation for 30 min.

[0069] After the connection is completed, the product is transformed into Escherichia coli competent DH5α, and monoclonal colonies are picked for sequencing. The correct plasmid for sequencing is the one with the CDS sequence shown in SEQ ID No.1 inserted between SmaI. This recombinant vector is used for subsequent transformation into Agrobacterium and plants.

[0070] (2) Obtaining of MsPT1 gene - transformed Arabidopsis thaliana

[0071] Cut off the top of the bolting Arabidopsis thaliana one week in advance to increase the growth of lateral branches.

[0072] (2.1) Obtaining of transformation solution

[0073] Take out the bacterial liquid containing the positive clone of the recombinant expression vector Super1300:MsPT1, and pipette about 100 μL and inoculate it into a liquid medium containing 1 mL of LB (rifampicin 50 μg / mL and kanamycin 50 μg / mL). Incubate overnight at 28°C on a shaker at 200 r (LB medium: peptone 10 g / L; yeast extract: 5 g / L; NaCl: 10 g / L), to complete the first activation.

[0074] Transfer all 1 mL of the activated bacterial liquid to 200 mL of LB liquid medium (rifampicin 50 μg / mL and kanamycin 50 μg / mL), and continue to culture in a 28°C shaker until the OD600 value reaches 1.2 - 1.8, to complete the second activation of Agrobacterium.

[0075] Centrifuge at 4000 g for 15 min, pour off the supernatant. Resuspend the precipitate with 500 mL of transformation solution, and adjust the OD600 to the range of 0.6 - 0.8. Add Silwet - 77 at a ratio of 0.03%, invert and mix well to complete the collection of the bacterial liquid and obtain the transformation solution.

[0076] (2.2) Infection

[0077] Dispense the transformation solution into small pots of about 200 mL, invert the Arabidopsis thaliana plants with bolts and flower buds into the transformation solution, and let the flower buds fully contact the transformation solution for 4 min. (Arabidopsis thaliana transformation solution: 40×(macroelement mother liquor): 12.5 mL / L; 40×(MgSO4): 12.5 mL / L; 200×(trace element mother liquor): 25 mL / L; sucrose: 50 g / L; Silwet - 77: 300 μL / L).

[0078] Among them, 1 L of the macroelement mother liquor contains: NH4NO3 33 g; KNO3 38 g; CaCl2 6.643 g; MgSO4·7H2O 7.4 g; KH2PO4 3.4 g.

[0079] The 1L trace element mother liquor contains: 0.083 g of KI; 0.62 g of H3BO3; 1.74 g of MnSO4·H2O; 0.86 g of ZnSO4·7H2O; 0.025 g of Na2Mo4·2H2O; 0.0025 g of CuSO4·5H2O; 0.0025 g of CoCl2·6H2O.

[0080] (2.3) Seed harvest

[0081] Lay the infected Arabidopsis plants flat on a tray and culture them in the dark for 18 - 24 h. Then stand the flower pots upright and place them in a phytotron for normal growth. After one month, when the Arabidopsis is mature, collect the seeds.

[0082] After identification, a total of 8 positive strains were screened: OE - 9, OE - 11, OE - 14, OE - 18, OE - 24, OE - 25, OE - 26, and OE - 31. The DNA identification results are as shown in ( Figure 2 A). A single and consistent - sized band can be amplified in the positive strains, and no band is amplified in the wild - type negative control (Col - 0). The homozygous strains OE - 9 and OE - 26 were identified by hygromycin resistance and used for subsequent experiments. The expression levels of the screened homozygous strains OE - 9 and OE - 26 were analyzed by qRT - PCR Figure 2 B). The results show that the expression levels of MsPT1 in these two strains increased significantly and can be used for subsequent phenotypic analysis.

[0083] (3) Obtaining transgenic alfalfa

[0084] Take out the positive Agrobacterium tumefaciens EHA105 containing Super1300:MsPT1 from the - 80°C refrigerator for activation. Specifically: Pipette about 60 μL and inoculate it into a liquid medium containing 1.4 mL of LB (rifampicin 50 μg / mL and kanamycin 50 μg / mL), and culture it overnight at 28°C on a shaker at 200 r to complete the first activation; Transfer all 1 mL of the activated bacterial solution to 30 mL of LB liquid medium (rifampicin 50 μg / mL and kanamycin 50 μg / mL), and continue to culture it in a 28°C shaker until the OD600 value reaches 0.6 - 0.8 to complete the second activation, and then collect the bacterial solution.

[0085] Select leaves with better growth and place them in a 50 mL centrifuge tube containing 36 mL of ddH2O. Add 4 mL of sodium hypochlorite to the centrifuge tube, tighten the lid, and invert it for 10 min. Pour out the sodium hypochlorite solution in the laminar flow hood, use forceps to clamp the leaves into a conical flask, and wash the leaves 6 times with sterilized ddH2O for 1 - 2 min each time. Pour out the ddH2O, pour the SH3a liquid medium into the conical flask containing the leaves, and ensure that the SH3a liquid medium submerges the leaves. Cover the bottle mouth with sealing film and perform ultrasonic treatment on the leaves. Stop the ultrasonic treatment when 80% of the leaves show dotted dark green, and complete the pretreatment of the leaves.

[0086] Take the bacterial solution with an OD600 value of 0.6 - 0.8 to the laminar flow hood and pour it into a sterilized 50 mL centrifuge tube. Centrifuge at 4000 r / min for 10 min. Pour out the supernatant in the laminar flow hood, aspirate 1 mL of SH3a liquid medium to resuspend the bacterial solution precipitate, and add the resuspended bacterial solution to the conical flask containing the leaves to make the OD600 value reach 0.2 - 0.4. Place the conical flask in a vacuum pump and evacuate for 10 min. Place it in a shaker at 28 °C and 70 rpm for 15 min to complete the infection.

[0087] In the laminar flow hood, take out the leaves, dry the moisture on both sides of the leaves with sterilized filter paper, use forceps to lay the leaves face up flat on the SH3a solid medium, and perform dark culture for 48 h. Transfer the leaves to the SH3a solid medium containing 2 mg / L cefotaxime and 5 mg / L hygromycin, and change the medium every two weeks. Perform dark culture for 2 months. After callus appears on the leaves, transfer the callus to the MSBK solid medium containing 2 mg / L cefotaxime and 5 mg / L hygromycin, and perform normal light culture.

[0088] When green bud points appear on the callus, transfer the callus with bud points to the SH9a solid medium containing 2 mg / L cefotaxime and 5 mg / L hygromycin, and perform normal light culture to wait for rooting and leaf growth. Transfer the seedlings in the medium to nutrient soil for positive identification.

[0089] Among them, the specific components of each 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 the absorption of arsenate by Arabidopsis thaliana

[0094] Using the Columbia wild type (Col-0) and the MsPT1 overexpression lines OE-9 and OE-26 as materials, an arsenate germination experiment was carried out. Arsenic and phosphorus are in the same main group, and arsenate is structurally similar to phosphate. When plants absorb more arsenate, they will show toxic phenotypes. Put the seeds of Col-0, OE-9 and OE-26 in 1.5 ml tubes, soak them in 6% sodium hypochlorite for 10 minutes, disinfect the seeds thoroughly, then wash the seeds 6 times with sterilized double-distilled water to wash off the sodium hypochlorite, and finally add an appropriate amount of double-distilled water to the 1.5 ml tubes and place the tubes in a 4°C refrigerator. After 2 days, take the seeds out of the refrigerator and spread them on 1 / 2 MS medium and 1 / 2 MS + 200 mM sodium arsenate medium respectively, and then place the culture dishes flat in a light incubator to germinate and grow the seeds. On the 1 / 2 MS medium without arsenate, there was no obvious difference in the leaf color and growth of the OE-9 and OE-26 lines and the wild-type Arabidopsis thaliana; on the medium containing 200 μM arsenate, the MsPT1 overexpression lines showed obvious toxic phenotypes and germination was severely inhibited ( Figure 3 A). The green cotyledon rate was statistically analyzed, and the results showed that the green cotyledon rates of the MsPT1 overexpression lines were reduced by 42% and 48% respectively compared with the wild type ( Figure 3 B). The above results indicate that the MsPT1 overexpression lines are more sensitive to arsenate, and overexpressing MsPT1 can increase the phosphorus absorption rate of Arabidopsis thaliana.

[0095] Among them, the components of the 1 / 2 MS solid medium for plants are: M 519: 2.2 g / L; sucrose: 15 g / L; agar: 8 g / L;

[0096] The components of the externally supplemented arsenate medium are: M519: 2.2 g / L; sucrose: 15 g / L; agar: 8 g / L; 100 mM sodium arsenate: 2 mL / L.

[0097] Example 5. Overexpression of MsPT1 can increase the biomass and inorganic phosphorus content of Arabidopsis thaliana

[0098] Col-0, OE-9, and OE-26 seeds were sown on MS medium, and then the culture dishes were placed vertically in a light incubator for seed germination and growth. After 7 days, the materials were collected and the biomass was counted. The results showed that overexpression of MsPT1 could increase the biomass of Arabidopsis thaliana ( Figure 4 A). The inorganic phosphorus contents of the MsPT1 overexpression materials OE-9 and OE-26 and the wild type were detected by the molybdenum blue method. The detection results showed that the inorganic phosphorus contents of the MsPT1 overexpression materials were significantly higher than those of the wild type control ( Figure 4 B).

[0099] The specific method for detecting the inorganic phosphorus content by the molybdenum blue method is as follows:

[0100] 1. The whole seedlings germinated and grown on MS medium for 7 days were collected, the fresh weight values of each group of materials were recorded, and they were stored in liquid nitrogen. The samples were crushed with a grinder. 1% glacial acetic acid was added to a 10 mL EP tube in advance for standby.

[0101] 2. The inorganic phosphorus extraction solution was added to the sample tube according to the ratio of 100 μL of extraction solution per 10 mg of sample (the formula of 1 μL of inorganic phosphorus extraction solution: 10 mL of 1 M Tris-HCl (pH 8.0), 2 mL of 0.5 M EDTA (pH 8.0), 5.844 g of NaCl, 700 μL of β-mercaptoethanol, 10 mL of 100 mM PMSF (prepared freshly before use)). Taking 70 mg of sample as an example, 700 μL of extraction solution was added, and it was mixed well by inverting up and down. The sample was aspirated into an EP tube containing 4.3 mL of glacial acetic acid with a pipette. Since there was still sample adhering to the original sample tube at this time, 1 mL of glacial acetic acid was aspirated into the sample tube again, and the EP tube was washed by inverting up and down. The sample was aspirated into a 10 mL EP tube, and the washing was repeated twice. At this time, the volume of glacial acetic acid in the 10 mL EP tube was 6.3 mL. It was carefully mixed by inverting, and reacted in a water bath at 42 °C for 30 min. Centrifuge at 4 °C and 4000 rpm for 15 min.

[0102] 3. Take the leaching solution obtained in step 2 and determine the phosphorus content by the vanadium molybdenum blue method. Color development: Aspirate 150 μL of the supernatant and add it to 350 μL of the color developing solution, and mix well by inverting. At the same time, prepare a standard curve and react in a water bath at 42 °C for 30 min.

[0103] Absorbance measurement: Aspirate 200 μL of the reacted solution into an enzyme-linked immunosorbent assay (ELISA) plate and measure it at a wavelength of 820 nm with a continuous ELISA reader.

[0104] Example 6: Overexpression of MsPT1 can increase the plant height, biomass, and protein content of alfalfa

[0105] Three alfalfa MsPT1 overexpression lines were selected for subsequent research. The three lines were named OE-1, OE-2, and OE-3. The detection results of the expression levels showed that the expression levels of the three transgenic lines were significantly higher than those of the controlFigure 5 A). The results of phenotypic detection showed that the growth of the three overexpression lines OE-1, OE-2 and OE-3 was better than that of the control ( Figure 5 B). The results of biomass statistics were consistent with the phenotypes, and the biomass of the overexpression lines was significantly higher than that of the control ( Figure 5 C). The phosphorus content and protein content of each material were measured by near-infrared. The results of phosphorus content measurement 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 the quality of alfalfa. The results of protein content measurement 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 the biomass, phosphorus content and protein content of alfalfa.

[0106] The above are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An isolated alfalfa MsPT1 gene, characterized in that, The nucleotide sequence of the MsPT1 gene is shown in SEQ ID No.

1.

2. An isolated Medicago sativa MsPT1 gene, characterized in that, The MsPT1 gene encodes a polypeptide having the amino acid sequence shown in SEQ ID No.

2.

3. A polypeptide, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID No. 2; Preferably, the polypeptide is encoded by the nucleotide sequence shown in SEQ ID No.

1.

4. A biological material related to the MsPT1 gene according to claim 1 or 2, which is any one of the following B1) to B6): B1) An expression cassette containing the MsPT1 gene according to claim 1 or 2; B2) A recombinant vector containing the MsPT1 gene according to claim 1 or 2, B3) A recombinant microorganism containing the MsPT1 gene according to claim 1 or 2; B4) A recombinant vector containing the expression cassette described in B1); B5) A recombinant microorganism containing the recombinant vector described in B2); B6) A composition containing the polypeptide according to claim 3.

5. Use of the MsPT1 gene according to claim 1 or 2, the polypeptide according to claim 3, or the biological material according to claim 4 in regulating the physiological process of arsenate absorption in plants or in plant breeding; Preferably, the plants include alfalfa and / or Arabidopsis thaliana.

6. The application according to claim 5, characterized in that, The use is to construct high-quality plants; Preferably, the high quality includes at least one of promoting arsenate absorption, increasing the phosphorus absorption rate, increasing plant height, increasing the biomass of the vegetative part of the plant, increasing the phosphorus content of the vegetative part of the plant, and / or increasing the protein content of the vegetative part of the plant.

7. The application according to claim 5, characterized in that, The use is achieved by regulating the expression of the MsPT1 gene.

8. The application according to claim 7, characterized in that The regulation of the expression of the MsPT1 gene is to up-regulate the expression of the MsPT1 gene or down-regulate the expression of the MsPT1 gene; or The regulation of the expression of the MsPT1 gene is to silence the expression of the MsPT1 gene or over-express the MsPT1 gene.

9. A method for detecting the arsenate absorption ability of plants, characterized in that, The method includes the step of quantitatively detecting the expression level of the MsPT1 gene; Preferably, the plants include alfalfa and / or Arabidopsis thaliana.

10. A method for preparing high-quality alfalfa, characterized in that, The method includes the step of regulating the MsPT1 gene; Preferably, the regulation of the expression of the MsPT1 gene is to up-regulate the expression of the MsPT1 gene or down-regulate the expression of the MsPT1 gene; Preferably, the regulation of the expression of the MsPT1 gene is to silence the expression of the MsPT1 gene or over-express the MsPT1 gene; Preferably, the plants include alfalfa and / or Arabidopsis thaliana.

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