Application of plant transcription factor PHR1 active site

Abstract

The invention relates to application of a plant transcription factor PHR1 active site. The active sites of the plant transcription factor PHR1 comprise serine at the 291th site in arabidopsis thaliana AtPHR1 and threonine at the 296th site in corn ZmPHR1. Through phosphorylation modification on the active site of the Arabidopsis thaliana transcription factor AtPHR1, the transcriptional activation activity of the transcription factor PHR1 can be enhanced, the protein stability of the AtPHR1 is improved, and the inorganic phosphorus content of plants is increased. According to amino acid sequence comparison, threonine at the 296th site of the corn ZmPHR1 and serine at the 291th site of the arabidopsis thaliana AtPHR1 are conserved in evolution, and phosphorylation modification of the threonine at the 296th site of the corn ZmPHR1 also promotes activation activity of transcription factors of the corn ZmPHR1. The active site of the plant transcription factor PHR1 provided by the invention can provide a direction for cultivating stress-resistant variety plants capable of efficiently absorbing and utilizing phosphorus and promote the development of modern basic biology and agricultural breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to an application of an active site of a plant transcription factor PHR1. Background Art

[0002] Phosphorus is an important component of various functional substances in living cells, such as nucleic acids, nucleoproteins, and phospholipids. It is closely related to almost all life processes, such as energy metabolism of living cells, synthesis and decomposition of various organic substances, cell signal transduction, and gene expression regulation. Phosphorus is a coenzyme for many enzymes, such as NAD + , NADP + Phosphorus is an important medium element for energy conversion in plants. The high-energy phosphate bonds in ADP and ATP are the source of energy for most biochemical metabolic reactions. Phosphorus is essential for the growth and development of plants. Plants absorb and utilize inorganic phosphorus from the soil. However, due to the assimilation of phosphorus in the soil by microorganisms and chelation by metal ions, a large amount of phosphorus fertilizer cannot be absorbed and utilized by plants. As a result, the effective phosphorus concentration in the soil is generally lower than 10μM, while the phosphorus concentration in plant cells is generally at the mM level. Plants often suffer from low phosphorus stress, which inhibits plant growth and development and seriously affects crop yield and quality. Therefore, soil phosphorus deficiency has become an important limiting factor in agricultural production.

[0003] Corn is an important food crop. It is the largest crop in the world and China. It has a wide range of applications and plays an important role in ensuring national food security. Corn is widely grown around the world and can be used for food, feed, biomass energy, etc.

[0004] Research on plant response to low phosphorus stress has mostly focused on the transcriptional regulation of downstream phosphorus starvation response genes and phosphorus transporter genes. The transcription factor PHR is a core component of the plant low phosphorus signaling pathway. PHR contains a MYB-type DNA binding domain and a coiled-coil binding domain, in which the DNA binding domain is responsible for binding to the target gene promoter, and the coiled-coil binding domain regulates protein interaction and dimer formation. PHR usually binds to the P1BS cis-acting element on the downstream gene promoter in the form of a dimer to positively regulate gene expression, promoting plants to enhance phosphorus absorption under low phosphorus conditions. PHR has a conserved function in plant phosphorus signaling and phosphorus homeostasis. Protein phosphorylation and dephosphorylation processes are widely present in plants, and they play an important role in regulating protein function. The transcription level of PHR1 does not respond to low phosphorus stress, and the mechanism involving the response of PHR1 protein level to low phosphorus stress is still unclear. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide an application of the active site of plant transcription factor PHR1.

[0006] Arabidopsis thaliana is used as a model plant for research due to its short growth cycle, many fruits, simple genome, obvious shape characteristics and other characteristics. The present invention first uses Arabidopsis thaliana as a research material, and identifies that the phosphorylation modification of the 291st serine of the Arabidopsis thaliana transcription factor AtPHR1 can improve its transcriptional activation activity, improve the protein stability of AtPHR1, and the phosphorylation modification of this site can increase the inorganic phosphorus content of the plant. Then, through sequence alignment, it is found that the 296th threonine of the corn transcription factor ZmPHR1 and the 291st serine of AtPHR1 are evolutionarily conservative, and the phosphorylation modification of the 296th threonine of the corn transcription factor ZmPHR1 can also improve the transcriptional activation activity. Therefore, the active site of the plant transcription factor PHR1 provided by the present invention can provide a direction for cultivating stress-resistant varieties of plants that efficiently absorb and utilize phosphorus, and promote the development of modern basic biology and agricultural breeding.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] An application of the active site of a plant transcription factor PHR1, characterized in that the application improves the transcriptional activation activity of the transcription factor PHR1, improves the stability of the PHR1 protein and the inorganic phosphorus content of the plant by phosphorylating the active site of the transcription factor PHR1.

[0009] The active site of the plant transcription factor PHR1 is located at:

[0010] (1) Serine at position 291 of Arabidopsis thaliana AtPHR1; the amino acid sequence of Arabidopsis thaliana AtPHR1 is shown in SEQ ID NO.3, and the gene sequence encoding the amino acid sequence is shown in SEQ ID NO.1;

[0011] or:

[0012] (2) Threonine at position 296 of maize ZmPHR1; the amino acid sequence of maize ZmPHR1 is shown in SEQ ID NO.4, and the gene sequence encoding the amino acid sequence is shown in SEQ ID NO.2.

[0013] The primers used to identify the active site in the Arabidopsis thaliana AtPHR1 of claim 1 are characterized in that the sequences of the primers are shown in SEQ ID NOs. 5-16.

[0014] The primers used to identify the active sites in the maize ZmPHR1 of claim 1 are characterized in that the sequences of the primers are shown in SEQ ID NOs. 17-28.

[0015] The application of the plant transcription factor PHR1 active site of the present invention has the following beneficial effects:

[0016] The present invention found that the phosphorylation level of the S291 site of Arabidopsis AtPHR1 was significantly increased under low-phosphorus conditions, and the phosphorylation modification of this site enhanced the transcriptional activation activity of AtPHR1, improved the protein stability of AtPHR1, and increased the inorganic phosphorus content of the plant. Through sequence alignment, the phosphorylation site T296 in corn ZmPHR1 that is conserved with S291 of AtPHR1 was found, and the phosphorylation modification of this site also significantly enhanced the transcriptional activation activity of ZmPHR1. The phosphorylation modification mechanism of ZmPHR1 broadens the regulatory network of plant response to low-phosphorus stress, and provides ideas for cultivating new stress-resistant varieties that efficiently absorb and utilize phosphorus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention has the following accompanying drawings:

[0018] Figure 1 The amplified AtPHR1 and downstream reporter gene fragments, where lane a is the wild-type AtPHR1 fragment and lane b is the AtPHR1 S291A Fragment, lane c is AtPHR1 S291E The fragments are all 1230 bp in size; lane d is the proAtIPS1 fragment, lane e is the proAtAT4 fragment, and lane f is the proAtPAP14 fragment, all of which are 2000 bp in size;

[0019] Figure 2 The results of detecting the effect of phosphorylation modification of AtPHR1 S291 site on its transcriptional activation activity in Arabidopsis protoplasts in Example 2 of the present invention; wherein, Empty vector means that Super1300 is empty, AtPHR1 means that no mutation occurs, and AtPHR1 S291A Represents a punctate form that mimics a persistent non-phosphorylated state, AtPHR1 S291E Indicates the punctate form simulating the continuous phosphorylation state. Statistical analysis was performed using ANOVA analysis method, and lowercase letters indicate P < 0.05.

[0020] Figure 3 In Example 3 of the present invention, phr1, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 S291A -GFP and PhR1 / ProPHR1:PHR1 S291E -GFP genetic material, and the total plant protein was extracted for Western detection of the protein level of AtPHR1.

[0021] Figure 4phr1, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 under sufficient and low phosphorus conditions S291A -GFP and PhR1 / ProPHR1:PHR1 S291E -GFP genetic material. Statistical analysis was performed using ANOVA, and lowercase letters indicate P < 0.05.

[0022] Figure 5 The results of amino acid sequence comparison between Arabidopsis thaliana AtPHR1 and maize ZmPHR1 are shown in Figure 1. The amino acid at position 291 of AtPHR1 is S, and the amino acid at position 296 of ZmPHR1 is T.

[0023] Figure 6 The amplified ZmPHR1 and downstream reporter gene fragments, where lane g is the wild-type ZmPHR1 fragment and lane h is the ZmPHR1 T296A Fragment, lane i is ZmPHR1 T296E The fragments are all 1353 bp in size; lane j is the proZmIPS1 fragment, lane k is the proZmSPX5 fragment, and lane l is the proZmPT4 fragment, all of which are 2000 bp in size.

[0024] Figure 7 The results of detecting the effect of phosphorylation modification of ZmPHR1 T296 site on its transcriptional activation activity in tobacco system in Example 6 of the present invention; wherein, Empty vector means Super1300 is empty, ZmPHR1 means no mutation occurs, and ZmPHR1 T291A Represents a punctate form that mimics a persistent non-phosphorylated state, ZmPHR1 T291E Indicates the punctate form simulating the continuous phosphorylation state. Statistical analysis was performed using ANOVA analysis method, and lowercase letters indicate P < 0.05. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The wild-type corn used in the following examples is corn ecotype B73; the Agrobacterium strain is EHA105; the main reagents used include: restriction endonucleases, DNA polymerases, T4 ligases, etc. from biological companies such as NEB and Toyobo; reverse transcription kits from Thermo; RNA extraction kits from Magen; quantitative PCR reagents from Taraka; plasmid extraction kits and DNA recovery kits purchased from Tiangen; agar powder, ampicillin, kanamycin, gentamicin sulfate, rifampicin and other antibiotics purchased from Sigma; various other chemical reagents used are imported or domestic analytical reagents.

[0028] Agrobacterium tumefaciens EHA105 is described in the following literature: Nyaboga et al. Agrobacterium-mediated genetic transformation of yam (Dioscorea arotundata): an important tool for functional study of genes and crop improvement. Frontiers in Plant Science 2014, 5: 463.

[0029] Example 1 Construction of vectors for AtPHR1, ZmPHR1 and related reporter genes

[0030] 1. Primer design and PCR amplification

[0031] According to the gene CDS sequence provided in the gene library, primers for amplifying the full-length fragments of AtPHR1 and ZmPHR1 and the 2000bp fragment of the downstream reporter gene promoter were designed upstream and downstream of the transcription start site (+1) of the gene using Primer Premier 5.0 software; primers for AtPHR1 and ZmPHR1 point mutations were designed to simulate the continuous phosphorylation form of AtPHR1 S291E 、ZmPHR1 T296E and mimicking a constitutively non-phosphorylated form of AtPHR1 S291A 、ZmPHR1 T296A The amplified fragment was homologously recombined with the Super1300 vector to obtain Super1300:AtPHR1 and Super1300:AtPHR1 S291E 、Super1300:AtPHR1 S291A and Super1300:ZmPHR1, Super1300:ZmPHR1 S296E、Super1300:ZmPHR1 S296A .

[0032] AtIPS1, AtAT4 and AtPAP14 as well as ZmIPS1, ZmSPX5 and ZmPT4 were selected as reporter genes, and primers were designed to amplify the promoter fragments about 2000 bp upstream of the 5' end of these genes. The fragments were homologously recombined with the pGreen0800 vector to obtain proAtIPS1:LUC, proAtAT4:LUC, proAtPAP14:LUC and proZmIPS1:LUC, proZmSPX5:LUC and proZmPT4:LUC.

[0033] The above primer sequences are as follows:

[0034]

[0035]

[0036]

[0037] The cDNA of Arabidopsis Col-0 and maize B73 was used as template to amplify AtPHR1, ZmPHR1 and the punctate fragments, and the genomic DNA of Arabidopsis Col-0 and maize B73 was used as template to amplify the promoter fragment of the reporter gene.

[0038] The experiment was carried out using the relevant reagents of X-ray Biotechnology Co., Ltd. The reaction system was 50 μL, consisting of 25 μL 2xApexHF CL Buffer (Mg 2+ and dNTP plus),

[0039] It consists of 2μL Primer F aqueous solution (concentration of 10μM), 2μL Primer R aqueous solution (concentration of 10μM), 1μL template, 1μL ApexHFHSDNA Polymerase CL and 19μL ddH2O.

[0040] The reaction program was: 94°C for 1 min; 98°C for 10 s; 60°C for 15 s; 68°C for 1 min / kb, 35 cycles; and extension at 68°C for 5 min.

[0041] 2. Connection of cloning vector

[0042] The amplified fragments were recovered from gel, AtPHR1, ZmPHR1 and the punctate fragments were connected to the Super1300 vector after restriction digestion, and the promoters of the reporter genes proAtIPS1, proAtAT4, proAtPAP14 and proZmIPS1, proZmSPX5, proZmPT4 were connected to the pGreen0800 vector after restriction digestion.

[0043] The experiment was carried out using relevant reagents from Acre Biotechnology Co., Ltd. The reaction system was 10 μL, consisting of 2 μL 5x OKClon Master Mix, 3 μL recovered fragments, 1 μL enzyme-cut vector, and 4 μL ddH2O.

[0044] The reaction procedure is: 50℃15min

[0045] 3. Transformation of E. coli competent cells and identification

[0046] Then, the connected vector was transformed into DH5α Escherichia coli competent cells, the bacterial solution was spread on plates, and cultured in a 37°C incubator for 12-16 hours. Single clones were picked, positive clones were identified, and plasmids were extracted by shaking to test the correctness of the sequence to obtain the recombinant vector.

[0047] Example 2 Detection of the transcriptional activation activity of AtPHR1 in Arabidopsis protoplasts

[0048] 1. Large-scale plasmid extraction

[0049] The kit used for large-scale plasmid extraction was purchased from Viagra, and the experiment was carried out according to the instructions. The steps are as follows:

[0050] (1) Replace the required Super1300:AtPHR1, Super1300:AtPHR1 S291E 、Super1300:AtPHR1 S291A , proAtIPS1:LUC, proAtAT4:LUC, proAtPAP14:LUC vector plasmids were used to transform Escherichia coli, and the culture was shaken until saturated. The bacterial solution was transferred to 150-200 mL of liquid LB medium with corresponding antibiotics for overnight culture.

[0051] (2) Collect the cells by centrifugation at 3320 g for 12 min at 4°C. Resuspend the cells in 5 mL of Buffer I and transfer the culture to a 50 mL centrifuge tube.

[0052] (3) Add 5 mL of Buffer II, mix by inversion, and place horizontally for 5 min to fully lyse the bacteria.

[0053] (4) Add 5 mL of Buffer III to the cells and mix thoroughly by inversion until a white flocculent precipitate is formed. Incubate on ice for 16 min.

[0054] (5) Centrifuge at 13500 g for 16 min at 4°C. Then, take another 50 mL centrifuge tube and add 12 mL of isopropanol. Transfer the supernatant after centrifugation to the isopropanol, mix well, and place on ice for 16 min.

[0055] (6) Centrifuge at 13500 g for 10 min at 4°C, discard the supernatant and retain the precipitate, drain the liquid in the tube. Add 500 μL of Buffer I to dissolve the precipitate, transfer the liquid to a 1.5 mL centrifuge tube, and continue to dissolve at room temperature for 20 min.

[0056] (7) Centrifuge at 13500g for 3 min at room temperature and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0057] (8) Add 100 μL of Buffer IV to the supernatant and mix gently. Centrifuge at 13500 g for 5 min. Transfer the supernatant to a 1.5 mL centrifuge tube and repeat this step.

[0058] (9) Add 70 μL of Buffer V to the supernatant from the previous step and mix well. Centrifuge at 13500 g for 5 min. Transfer the supernatant to a 1.5 mL centrifuge tube containing 600 μL of isopropanol.

[0059] (10) After inversion to mix, let stand at room temperature for 15 min. Centrifuge at 13500 g for 12 min and discard the supernatant.

[0060] (11) Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 13500 g for 12 min, discard the supernatant, centrifuge at 13500 g for 2 min, remove the residual liquid in the tube, and blow dry in a fume hood for 5 min.

[0061] (12) Preheat sterile ddH2O at 65°C, add 500 μL ddH2O to a centrifuge tube to dissolve the precipitate, add 300 μL Buffer VI, mix well, and place on ice for 30 min. Centrifuge at 13500 g for 20 min at room temperature and discard the supernatant.

[0062] (13) Wash the precipitate with 1 mL of 75% ethanol, centrifuge at 13500 g for 5 min, and repeat the wash once. Remove the residual liquid in the tube and blow dry in a fume hood. Add 100 μL of ddH2O to dissolve the plasmid and measure the concentration.

[0063] 2. Extraction and transformation of Arabidopsis protoplasts

[0064] (1) After Arabidopsis seedlings germinate and grow on MS medium for 15-20 days, cut the crown material and put it into 15 mL TVL solution. Chop the seedlings in a square dish and keep it in the dark for 30 minutes.

[0065] (2) Add 15 mL of the pre-prepared enzymatic hydrolysis solution to the culture dish and perform enzymatic hydrolysis at room temperature at 40 rpm in the dark for 4-5 hours.

[0066] (3) Filter the solution through a 200-mesh nylon membrane into a 50-mL centrifuge tube. Gently and slowly add 15 mL of W5 solution. Adjust the centrifuge speed to the lowest setting, centrifuge at 100 g for 5 min, and discard the supernatant.

[0067] (4) Carefully add 30 mL of W5 solution to suspend the protoplasts in the tube, centrifuge at 100 g for 3 min, and discard the supernatant.

[0068] (5) Add 30 mL of W5 solution to suspend the protoplasts and place on ice for 30 min;

[0069] (6) Prepare a 2 mL round-bottom centrifuge tube and add plasmid to the bottom of the tube. Combine transcription factor, empty vector, and promoter according to experimental needs, 8 μg each.

[0070] (7) Take out the protoplasts that have been left to stand on ice, centrifuge at 100g for 3 min, and discard the supernatant. Calculate the volume of MMG solution required based on 200 μL of protoplasts per reaction and the number of reactions, and add an appropriate amount of MMG solution to suspend the protoplasts.

[0071] (8) Add 200 μL of protoplasts to the centrifuge tube, mix well, add 220 μL of PEG solution, mix well, and place horizontally on the table for transformation for 15 minutes.

[0072] (9) Add 1 mL of W5 solution to terminate the transformation reaction. Mix carefully and centrifuge at 100 g for 5 min. Discard the supernatant.

[0073] (10) Add 1 mL of W5 solution to suspend the protoplasts, centrifuge at 100 g for 5 min, and discard the supernatant.

[0074] (11) Add 1 mL of W5 solution and incubate at 22°C in the dark for 14-16 h.

[0075] 3. Dual luciferase activity assay

[0076] The dual luciferase activity was determined using the Dual Luciferase Reporter Assay Kit from Novozymes. The experimental method was described in the instruction manual. The steps are as follows:

[0077] (1) Take out the protoplasts that have completed expression and carefully resuspend them. Adjust the centrifuge speed to the lowest setting, centrifuge at 4°C, 100 g for 5 min, and remove the supernatant.

[0078] (2) Mix 5x Cell Lysis Buffer and ddH2O in a ratio of 1:4, add 20 μL to each tube of sample, mix well, and place on ice for 10 min.

[0079] (3) Centrifuge at 12000g for 5 minutes in a 4℃ low-temperature centrifuge. Transfer the supernatant to a new 1.5mL centrifuge tube. Centrifuge again for 5 minutes and transfer the supernatant to a new 1.5mL centrifuge tube.

[0080] (4) Dispense Luciferase Substrate according to the number of samples, 50 μL per tube. (When using for the first time, pour all of Reaction Buffer II into the brown bottle of Luciferase Substrate containing the lyophilized substrate, mix thoroughly, and dispense according to usage requirements and store at -80°C away from light).

[0081] (5) When using, store Renilla Substrate on ice. Calculate the amount of Renilla Substrate required based on the number of samples. Dilute Renilla Substrate 50 times with Stop & Reation Buffer. Each reaction requires 50 μL of the substrate. Store at room temperature away from light.

[0082] (6) Using Promega 20 / 20 instrument for measurement. Turn on the machine for preheating, reset the system according to the following steps: Tools-Setting-Reset, set the dual luciferase program Protocol→Run promega protocol→DLR-O-INJ→OK.

[0083] (7) Take 10 μL of the supernatant from step (3) and carefully add it into a centrifuge tube containing 50 μL of Luciferase Substrate. Mix by gently pipetting for 5 times and place the tube into the instrument to measure the LUC value.

[0084] (8) Take out the sample tube, add 50 μL Stop&Glo Reagent, mix by gently pipetting five times, and return it to the instrument to measure the REN value. The instrument automatically calculates the relative fluorescence intensity LUC / REN Ratio.

[0085] The results are as follows Figure 2As shown: Wild-type AtPHR1 protein can activate the expression of proAtIPS1:LUC, proAtAT4:LUC and proAtPAP14:LUC, while AtPHR1 that mimics a persistent non-phosphorylated state S291A The transcriptional activation activity of AtPHR1 is significantly lower than that of AtPHR1, which mimics the constitutively phosphorylated form of AtPHR1. S291E The transcriptional activation activity of AtPHR1 was significantly higher than that of AtPHR1.

[0086] The above results indicate that phosphorylation modification of AtPHR1 S291 site can enhance the transcriptional activation activity of AtPHR1 in Arabidopsis protoplasts.

[0087] Example 3 Detection of AtPHR1 protein level in Arabidopsis AtPHR1 related genetic materials I. Cultivation of Arabidopsis

[0088] (1) Combine phr1, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 S291A -GFP and PhR1 / ProPHR1:PHR1 S291E -GFP Arabidopsis seeds were immersed in sodium hypochlorite seed disinfectant (6% NaClO aqueous solution plus 0.01% Triton X-100) for 10-15 min.

[0089] (2) Wash the seeds six times with sterile ddH2O in a clean bench, and finally add 1 mL of sterile ddH2O. Protect the seeds from light and place them in a 4°C incubator for vernalization for 3 days.

[0090] (3) The seeds were evenly sown on the MS medium, sealed with a sealing film, and kept at a temperature of 22°C, a photoperiod of 16 h (light) / 8 h (dark), and a light intensity of 70 μM·m -2 ·S -1 Culture in an incubator for 7 days.

[0091] (4) Before transferring to LP medium, take a portion of the plants and place them in a 1.5 mL centrifuge tube as the material for LP treatment on day 0.

[0092] (5) Arabidopsis seedlings were transferred to LP medium and cultured for another 3 days. A portion of the plants were placed in a 1.5 mL centrifuge tube and quickly frozen in liquid nitrogen to serve as the material for LP treatment for 3 days.

[0093] (6) The remaining seedlings were transferred to MS medium with or without CHX (CHX is a protein synthesis inhibitor) and cultured for another 3 days. Samples were collected, placed in 1.5 mL centrifuge tubes, and quickly frozen in liquid nitrogen.

[0094] 2. Plant total protein extraction

[0095] (1) The above-mentioned phr1, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 S291A -GFP and PhR1 / ProPHR1:PHR1 S291E -GFP was treated with LP for 0 days, LP for 3 days, and then transferred to MS medium with CHX and without CHX for 3 days after LP treatment. The materials were ground, added with appropriate amount of protein extract, mixed by vortexing, and placed on ice for 15 min.

[0096] (2) Centrifuge the sample at 12,000 rpm in a 4°C centrifuge for 10 min.

[0097] (3) Transfer the supernatant to a new 1.5 mL centrifuge tube and centrifuge again at 12,000 rpm in a 4°C centrifuge for 10 min.

[0098] (4) Transfer the supernatant to a new centrifuge tube, use Biorad 1×Dye Reagent to determine the protein concentration, calculate the volume corresponding to the required total protein amount, and carry out subsequent experiments.

[0099] 3. Western Detection of AtPHR1 Protein Levels in Different Materials

[0100] (1) Prepare SDS-PAGE protein gel.

[0101] (2) Add protein loading buffer to the above protein sample, denature the protein in boiling water for 10 min, immediately put it on ice, and centrifuge it at 12000 rpm in a 4°C centrifuge for 3 min.

[0102] (3) Use 1× electrophoresis buffer for electrophoresis. First, run the electrophoresis at a constant voltage of 90 V for 10 min. After the sample enters the separation gel, run the electrophoresis at a constant voltage of 130 V. Terminate the electrophoresis according to the size of the target protein.

[0103] (4) Remove the protein gel and cut off the concentrated gel.

[0104] (5) Precool the wet transfer solution and soak the PVDF membrane in anhydrous methanol for 3 minutes. The order from negative electrode to positive electrode is: sponge-filter paper-protein glue-PVDF membrane-filter paper-sponge. Constant voltage 100V transfer for 60 minutes. The whole process is carried out in an ice bath.

[0105] (6) Prepare a 5% skim milk solution with TBST buffer. After transfer, place the PVDF membrane in the skim milk and block it at room temperature for 2 h.

[0106] (7) Pour out the skim milk solution, wash the PVDF membrane once with TBST buffer, place the membrane in TBST buffer with a primary antibody (in this example, a commercial GFP antibody is used), and incubate at room temperature for 1 h or at 4°C overnight.

[0107] (8) Pour off the TBST buffer used for primary antibody incubation and wash the PVDF membrane three times with TBST buffer, each time for 10 min.

[0108] (9) Place the PVDF membrane in TBST buffer containing secondary antibody and incubate at room temperature for 45 min-1 h.

[0109] (10) Pour off the TBST buffer used for incubation with the secondary antibody and wash the PVDF membrane three times with TBST buffer, each time for 10 min.

[0110] (11) Chemiluminescence was performed using Super-Signal West Femto Trial Kit (Thermo) and X-ray film was used for pressing, developing and fixing.

[0111] The results are as follows Figure 3 As shown: the phr1 mutant material does not contain PHR1 protein. For the phr1 / proPHR1:PHR1-GFP material, after being transferred from MS to LP medium for 3 days, PHR1 protein accumulated. After the seedlings were transferred to MS medium without CHX for 3 days, PHR1 protein decreased. After being transferred to MS medium with CHX for 3 days, PHR1 protein further decreased. Compared with the phr1 / proPHR1:PHR1-GFP material, the phr1 / proPHR1:PHR1 S291A -GFP materials, the protein level of PHR1 was less than that of the phr1 / proPHR1:PHR1-GFP materials after culturing in MS medium supplemented with CHX for 3 days, while the protein level of PHR1 in the phr1 / proPHR1:PHR1-GFP materials was S291E The PHR1 protein level in the -GFP material was higher than that in the phr1 / proPHR1:PHR1-GFP material.

[0112] The above results indicate that phosphorylation modification of serine 291 of Arabidopsis transcription factor AtPHR1 enhances its protein stability under low-phosphate conditions.

[0113] Example 4 Detection of Inorganic Phosphorus Content in Arabidopsis AtPHR1 Related Genetic Materials

[0114] 1. Cultivation of Arabidopsis thaliana

[0115] (1) Combine phr1, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 S291A -GFP and PhR1 / ProPHR1:PHR1 S291E -GFP Arabidopsis seeds were immersed in sodium hypochlorite seed disinfectant (6% NaClO aqueous solution plus 0.01% Triton X-100) for 10-15 min.

[0116] (2) Wash the seeds six times with sterile ddH2O in a clean bench, and finally add 1 mL of sterile ddH2O. Protect the seeds from light and place them in a 4°C incubator for vernalization for 3 days.

[0117] (3) The seeds were evenly sown on the MS medium, sealed with a sealing film, and kept at a temperature of 22°C, a photoperiod of 16 h (light) / 8 h (dark), and a light intensity of 70 μM·m -2 ·S -1 Culture in an incubator for 7 days.

[0118] (4) The Arabidopsis seedlings were transferred to MS and LP medium respectively and cultured for 5 days.

[0119] (5) Wash the material with ddH2O, dry it with absorbent paper, place it in a 2 mL centrifuge tube with steel balls, record the fresh weight, and quickly freeze it in liquid nitrogen.

[0120] 2. Determination of inorganic phosphorus content

[0121] (1) Use a ball mill to grind the material into powder. Add inorganic phosphorus extract to the centrifuge tube at a ratio of 100 μL of extract for every 10 mg of material. For example, if the sample has a fresh weight of about 70 mg, add 700 μL of extract to the sample and mix thoroughly by inverting. Pipette the sample and extract mixture into a 10 mL centrifuge tube pre-added with 4.3 mL of 1% glacial acetic acid. Wash the 2 mL centrifuge tube containing the sample twice with 1 mL of 1% glacial acetic acid. At this time, the total volume of liquid in the 10 mL centrifuge tube is 7 mL.

[0122] (2) Place the centrifuge tube in a 42°C water bath for 30 min of extraction reaction and centrifuge at 4°C, 3320 g for 15 min.

[0123] (3) Prepare the colorimetric solution and dispense it into 1.5 mL centrifuge tubes, 350 μL / tube. Pipette 150 μL of the supernatant after centrifugation and add it to the colorimetric solution. At the same time, prepare a standard curve. After thorough inversion and mixing, perform the colorimetric reaction in a 42°C water bath for 30 min.

[0124] (4) Pipette 200 μL of sample into the ELISA plate and measure the absorbance at 820 nm using an ELISA reader.

[0125]

[0126] (5) Calculate the inorganic phosphorus content based on the standard curve.

[0127] The results are as follows Figure 4 As shown, under MS conditions, the inorganic phosphorus content of the phr1 mutant material was significantly lower than that of the wild type, phr1 / proPHR1:PHR1-GFP, phr1 / proPHR1:PHR1 S291A The inorganic phosphorus content of -GFP material was not significantly different from that of wild type, while phr1 / proPHR1:PHR1 S291E -GFP-12 strain had a significantly higher inorganic phosphorus content than the wild type. Under low phosphorus conditions, the inorganic phosphorus content of each material was significantly lower than that of the material cultured in MS. Among them, the inorganic phosphorus content of the phr1 mutant material was significantly lower than that of the wild type, and the inorganic phosphorus content of the phr1 / proPHR1:PHR1-GFP material had no significant difference from that of the wild type. In particular, the inorganic phosphorus content of the phr1 / proPHR1:PHR1 S291E -GFP material had a significantly higher inorganic phosphorus content than the wild type, while phr1 / proPHR1:PHR1 S291A The inorganic phosphorus content of -GFP material was significantly lower than that of wild type.

[0128] The above results indicate that phosphorylation modification of serine 291 of Arabidopsis transcription factor AtPHR1 increases the inorganic phosphorus content of the plant.

[0129] Example 5 Comparison of amino acid sequences of Arabidopsis thaliana AtPHR1 and maize ZmPHR1

[0130] The gene number of Arabidopsis AtPHR1 gene in Tair website (https: / / www.arabidopsis.org / ) is AT4G28610, which consists of 409 amino acids. The gene number of maize ZmPHR1 gene in MaizeGDB website (https: / / www.maizegdb.org / ) V3 version is GRMZM2G006477, and the gene number in V4 version is Zm00001d029020, which consists of 450 amino acids. The amino acid sequence was compared using software, and the results are as follows Figure 5 As shown, ZmPHR1 T291 is a conserved site of AtPHR1 S291.

[0131] Example 6 Detection of the transcriptional activation activity of ZmPHR1 in tobacco system

[0132] 1. Transformation of Agrobacterium

[0133] Super1300:ZmPHR1 and Super1300:ZmPHR1 were electroporated by S296E 、Super1300:ZmPHR1 S296A and proZmIPS1:LUC, proZmSPX5:LUC, and proZmPT4:LUC were transformed into Agrobacterium competent cells, the bacterial solution was spread on plates, and cultured upside down in a 28°C incubator for 2 days.

[0134] 2. Tobacco dual luciferase activity assay

[0135] (1) Place Agrobacterium into YEB liquid culture medium and incubate at 28°C overnight.

[0136] (2) Use tobacco transformation liquid to adjust the OD of bacterial liquid 600 The value is about 0.8. Mix different systems according to experimental requirements, place them at room temperature away from light for 2 hours, and then inject them into tobacco leaves.

[0137] (3) After 3 days of culture, use a hole puncher to punch holes near the injection hole. Take 4 holes from each leaf and place them in a 2 mL centrifuge tube with steel beads in advance, and quickly freeze them in liquid nitrogen.

[0138] (4) Grind the sample evenly using a ball mill. Add 100 μL of diluted Cell Lysis Buffer, mix well, and place on ice for 10 min.

[0139] (5) Centrifuge at 12000g for 5 minutes in a 4℃ low-temperature centrifuge. Transfer the supernatant to a new 1.5mL centrifuge tube. Centrifuge again for 5 minutes and transfer the supernatant to a new 1.5mL centrifuge tube.

[0140] (6) Dispense Luciferase Substrate according to the number of samples, 50 μL per tube. (When using for the first time, pour all of Reaction Buffer II into the brown bottle of Luciferase Substrate containing the lyophilized substrate, mix thoroughly, and dispense according to usage requirements and store at -80°C away from light).

[0141] (7) When using, store Renilla Substrate on ice. Calculate the amount of Renilla Substrate required based on the number of samples. Dilute Renilla Substrate 50 times with Stop & Reation Buffer. Each reaction requires 50 μL of the substrate. Store at room temperature away from light.

[0142] (8) Using Promega 20 / 20 instrument for measurement. Turn on the machine for preheating, reset the system according to the following steps: Tools-Setting-Reset, set the dual luciferase program Protocol→Run promega protocol→DLR-O-INJ→OK.

[0143] (9) Take 10 μL of the supernatant from step (5) and carefully add it into a centrifuge tube containing 50 μL of Luciferase Substrate. Mix by gently pipetting five times and place the tube into the instrument to measure the LUC value.

[0144] (10) Take out the sample tube, add 50 μL Stop&Glo Reagent, mix by gently pipetting five times, and return it to the instrument to measure the REN value. The instrument automatically calculates the relative fluorescence intensity LUC / REN Ratio.

[0145] The results are as follows Figure 7 As shown: Wild-type ZmPHR1 protein can activate the expression of proZmIPS1:LUC, proZmSPX5:LUC and proZmPT4:LUC, while ZmPHR1 that mimics a persistent non-phosphorylated state T296A The transcriptional activation activity of ZmPHR1 is significantly lower than that of ZmPHR1, which mimics the constitutively phosphorylated form of ZmPHR1. T296E The transcriptional activation activity of PHR1 was significantly higher than that of ZmPHR1.

[0146] The above results indicate that in tobacco cells, phosphorylation modification of the ZmPHR1 T296 site can enhance the transcriptional activation activity of ZmPHR1.

[0147] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. An application of a plant transcription factor PHR1 active site, characterized in that: The application improves the transcriptional activation activity of the transcription factor PHR1, the stability of the PHR1 protein and the inorganic phosphorus content of the plant by phosphorylating the active site of the transcription factor PHR1; The active site of the plant transcription factor PHR1 is located at: (1) Serine at position 291 of Arabidopsis thaliana AtPHR1; the amino acid sequence of Arabidopsis thaliana AtPHR1 is shown in SEQ ID NO.3, and the gene sequence encoding the amino acid sequence is shown in SEQ ID NO.1; or: (2) Threonine at position 296 of maize ZmPHR1; the amino acid sequence of maize ZmPHR1 is shown in SEQ ID NO.4, and the gene sequence encoding the amino acid sequence is shown in SEQ ID NO.

2.

2. A primer for identifying the active site in AtPHR1 of Arabidopsis thaliana according to claim 1, characterized in that: The sequences of the primers are shown in SEQ ID NO.5-16.

3. A primer for identifying the active site in the maize ZmPHR1 of claim 1, characterized in that: The sequences of the primers are shown in SEQ ID NO.17-28.

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