Wheat phosphorus absorption and utilization transcription factor as well as coding gene and application thereof

Abstract

Phosphorus is one of major elements necessary for growth and development of plants, and plays a crucial role in physiological processes and metabolic activities of the plants, such as energy metabolism, nucleic acid synthesis, stability of cell membrane structures, signal transduction and the like. The invention provides a wheat phosphorus absorption and utilization transcription factor TraesCS3D01G144700 and an application of a coding gene of the wheat phosphorus absorption and utilization transcription factor TraesCS3D01G144700 in response to low phosphorus stress. The invention provides new gene resources and technical means for improving the growth and yield of wheat under the condition of low-phosphorus soil and improving the phosphorus utilization efficiency and yield of wheat, and plays an important role in realizing the sustainable development of agriculture.

Description

Technical Field

[0001] The present invention relates to the fields of plant molecular biology and agricultural biotechnology, and particularly relates to a transcription factor bHLH for phosphorus uptake and utilization in wheat, its encoding gene, and the application of this gene in the processes of phosphorus uptake, transport, and metabolism in wheat. Background Art

[0002] Phosphorus is one of the macronutrients essential for plant growth and development and plays a crucial role in the life activities of plants. It is involved in numerous important physiological processes, including but not limited to energy metabolism, nucleic acid synthesis, maintenance of the structure and function of cell membranes, signal transduction, photosynthesis, and regulation of the activity of many enzymes. For the important food crop wheat ( Triticum aestivum L.), sufficient supply of phosphorus is even more indispensable for the formation of its yield and quality. However, the content of available phosphorus in the soil is often low, and the large application of phosphate fertilizers can lead to a series of environmental problems, such as eutrophication of soil and water bodies. At the same time, as a non-renewable resource, the reserves of phosphate rock resources are gradually decreasing. Therefore, improving the efficiency of wheat in absorbing and utilizing phosphorus in the soil is of great significance for ensuring global food security, achieving sustainable agricultural development, and environmental protection.

[0003] In plants, the absorption, transport, and metabolism of phosphorus are a complex process strictly regulated, involving the coordinated action of multiple genes and proteins. Among them, transcription factors, as important regulatory factors, play a key regulatory function in the process of plant absorption and utilization of phosphorus. Transcription factors can bind to the promoter region of target genes and regulate the expression of a series of downstream genes related to phosphorus absorption, transport, and metabolism, thereby regulating the plant's response and utilization of phosphorus at the transcriptional level. Among many transcription factor families, the bHLH (basic Helix-Loop-Helix) transcription factor family has been found to play an important role in various physiological processes and stress responses in plants. The bHLH transcription factor usually contains a conserved bHLH domain, which consists of an alkaline helix-loop-helix structure, enabling it to bind to specific DNA sequences and thus regulate gene expression.

[0004] In many plants, studies have shown that bHLH transcription factors are involved in the regulation of phosphorus uptake and utilization. However, relatively few studies have been conducted on wheat. For some model plants, such as Arabidopsis thaliana and rice, multiple bHLH transcription factors involved in phosphorus uptake and utilization have been identified, and it has been revealed that they affect the physiological state, growth, and development of plants under different phosphorus levels by interacting with other regulatory factors to activate or inhibit the expression of genes related to phosphorus transporters, phosphatases, etc. However, there are certain differences in genomic structure and physiological characteristics between wheat and these model plants, and its unique mechanism of phosphorus uptake and utilization may involve undiscovered bHLH transcription factors and their complex regulatory networks.

[0005] Existing studies on wheat phosphorus uptake and utilization have mainly focused on protein-coding genes directly involved in phosphorus uptake, transport, and metabolism, such as phosphorus transporters (e.g., the PHT family) and acid phosphatases. However, the research on transcription factors that regulate these processes at the transcriptional level, especially bHLH transcription factors, is not yet in-depth. For wheat, its large genome and complex genetic background pose certain challenges to the discovery and functional identification of transcription factors.

[0006] Therefore, the present invention aims to deeply study a wheat phosphorus uptake and utilization transcription factor TraesCS3D01G144700 and its coding gene. By identifying its characteristics, verifying its functions, analyzing its expression patterns under different phosphorus levels, and exploring its applications in wheat, it is expected to reveal its regulatory mechanism in the process of wheat phosphorus uptake and utilization, provide a theoretical basis and technical support for the development of new wheat varieties and agricultural cultivation techniques, improve the efficiency of wheat phosphorus uptake and utilization in low-phosphorus soils, and ultimately achieve high-quality and high-yield wheat and sustainable agricultural development.

[0007] There is no report on the function and application of the wheat TraesCS3D01G144700 transcription factor in the prior art. The present invention will deeply explore the TraesCS3D01G144700 transcription factor and its coding gene through a series of modern molecular biology techniques, including qRT-PCR technology, gene cloning, gene expression vector construction, transgenic technology, etc., to provide innovative solutions for wheat genetic improvement and agricultural production practices. Summary of the Invention

[0008] The key technical problem to be solved by the present invention is to provide the application of the wheat phosphorus uptake and utilization transcription factor TraesCS3D01G144700 and its coding gene in response to low phosphorus stress. To solve the above technical problems, the present invention adopts the following technical solutions: 1. Application of overexpressing the transcription factor TraesCS3D01G144700 in improving the low-phosphorus tolerance of Arabidopsis thaliana. The CDS sequence of the TraesCS3D01G144700 transcription factor is as shown in the attached Figure 12 and Sequence Listing SEQ No. 1.

[0009] 2. The application of overexpressing the transcription factor TraesCS3D01G144700 in improving the low-phosphorus tolerance of Arabidopsis thaliana according to claim 1. The improvement of the low-phosphorus tolerance of Arabidopsis thaliana means that under low-phosphorus conditions, Arabidopsis thaliana overexpressing TraesCS3D01G144700 has better growth.

[0010] 3. The application of overexpressing the transcription factor TraesCS3D01G144700 in improving the low-phosphorus tolerance of Arabidopsis thaliana according to claim 1. The TraesCS3D01G144700 transcription factor exerts its function in the nucleus.

[0011] 4. Transcription factor TraesCS3D01G144700. The CDS sequence of the TraesCS3D01G144700 transcription factor is as shown in Sequence Listing SEQ No. 1.

[0012] 5. Method for functional verification of the transcription factor TraesCS3D01G144700. The method includes: (1) Culturing of experimental materials and preparation of reagents; (2) RNA extraction and cDNA synthesis; (3) Cloning of the gene encoding the TraesCS3D01G144700 transcription factor; (4) Construction of subcellular localization vector and expression vector; (5) Agrobacterium transformation; (6) Genetic transformation of Arabidopsis thaliana.

[0013] Beneficial effects: In the present invention, the gene encoding the transcription factor TraesCS3D01G144700 is cloned, and further a subcellular localization vector and an overexpression vector are constructed. Subcellular localization in Arabidopsis thaliana and tobacco reveals that the transcription factor TraesCS3D01G144700 is mainly expressed in the nucleus. After successfully transforming Arabidopsis thaliana, the phenotype of the transgenic Arabidopsis thaliana transcription factor TraesCS3D01G144700 under low-phosphorus stress is observed. Description of the Drawings

[0014] Figure 1 A shows the expression pattern of the TraesCS3D01G144700 transcription factor in different tissues; Figure 1B represents the expression pattern under low phosphorus stress. Among them, Grain_z71, Grain_z75, and Grain_z85 represent the grains at 2, 14, and 20 days after flowering, respectively; Spike_z32, Spike_z39, and Spike_z65 represent the spikes at the internode interval, visible flag leaf stage, and flowering stage, respectively; Leaf_z10, Leaf_z23, and Leaf_z71 represent the leaves at the seedling stage, three-tiller stage, and 2 days after flowering, respectively; Stem_z30, Stem_z32, and Stem_z65 represent the stems at the heading stage, internode interval, and flowering stage, respectively; Root_z10, Root_z13, and Root_z39 represent the roots at the seedling stage, three-leaf stage, and visible flag leaf stage, respectively.

[0015] Figure 2 It is the qRT-PCR analysis of the TraesCS3D01G144700 transcription factor in the roots under low phosphorus stress for 3 days, 7 days, and 14 days. Among them, SW2 is a wheat variety with high phosphorus efficiency, and SW14 is a wheat variety with low phosphorus efficiency.

[0016] Figure 3 It is the phosphorus concentration in the roots and shoots of wheat varieties SW2 and SW14 under normal phosphorus and low phosphorus conditions.

[0017] Figure 4 It is the verification of the total RNA extraction from wheat roots.

[0018] Figure 5 It is the alignment of the sequencing results of the amino acid sequence cloned from the TraesCS3D01G144700 transcription factor.

[0019] Figure 6 It is the map of the pC1300S-GFP-TraesCS3D01G144700 vector.

[0020] Figure 7 It is the double digestion verification of the plasmid of the gene cloning vector. Note: In the figure, 1 is the plasmid synthesized by gene synthesis; 2 is the plasmid synthesized by gene synthesis verified by enzyme digestion; M is the KB Ladder, which are 10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1000bp, and 500bp from top to bottom in turn.

[0021] Figure 8 It is the bioinformatics analysis of the TraesCS3D01G144700 protein. Among them, A: Prediction of transmembrane domain; B: Prediction of hydrophilicity and hydrophobicity; C: Phosphorylation site; D: Prediction of signal peptide region; E: Prediction of secondary structure; F: Prediction of tertiary structure.

[0022] Figure 9Prediction analysis of the TraesCS3D01G144700 protein domain.

[0023] Figure 10 Subcellular localization analysis map of the TraesCS3D01G144700 transcription factor in Arabidopsis thaliana.

[0024] Figure 11 Growth status of overexpressed Arabidopsis thaliana and wild-type seedlings under different phosphorus conditions.

[0025] Figure 12 Gene sequence of TraesCS3D01G144700, with the start and stop codons underlined. Specific implementation method In the embodiments of this invention patent, unless otherwise specified, the methods and devices used are conventional technical means, and the equipment and reagents involved are standard products purchased from reagent companies. To make the purpose, technical solutions, and advantages of the present invention clearer, the following will elaborate on the implementation manners of the present invention in combination with specific embodiments. These preferred implementation manners will be demonstrated in specific examples. It should be particularly noted that, to avoid unnecessary details from affecting the clarity of the technical solutions of the present invention, only the technical content and processing steps closely related to the present invention are shown in the embodiments, and other details that are not relevant are omitted.

[0027] Example 1 This example provides a method for cloning, vector construction, and transformation of the wheat low-phosphorus tolerance transcription factor TraesCS3D01G144700, including: 1. Preparation of test materials and reagents The phosphorus-efficient wheat variety (SW2) and phosphorus-inefficient wheat variety (SW14) involved in the present invention were obtained through screening in previous laboratory research work. To ensure the controllability and consistency of experimental conditions, these wheat varieties were cultivated in an artificial climate incubator. During the experiment, a variety of advanced kits and reagents were used to ensure the smooth progress of the experiment and the accuracy of the data. The present invention uses a polysaccharide polyphenol plant total RNA extraction kit (TIANGEN, Beijing, DP441), qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). Restriction enzymes and ligases were purchased from Wuhan Tianwen Biotechnology Co., Ltd. Escherichia coli ( Eschrichia coli ) DH5α competent cells and Agrobacterium tumefaciens GV3101 competent cells (Qingke, Beijing).

[0028] 2. Determination of total phosphorus content Under different conditions of low phosphorus and normal phosphorus, there is a positive correlation between the expression of transcription factor TraesCS3D01G144700 and phosphorus uptake. To deeply explore this relationship, 3 biological replicates were set up, and the total phosphorus concentration of plant samples in each treatment group was measured using the molybdate blue colorimetric method. The specific operation steps were as follows: First, the plant samples were dried, then crushed into powder, and then digested with concentrated H2SO4 and H2O2. Finally, the determination of the total phosphorus concentration was completed for subsequent analysis.

[0029] 3. RNA Extraction and cDNA Synthesis Root tissues of phosphorus-efficient wheat variety (SW2) and phosphorus-inefficient wheat variety (SW14) under different phosphorus level treatments were collected, and RNA was extracted following the specific steps specified in the instruction manual of the polysaccharide polyphenol plant total RNA extraction kit (TIANGEN, Beijing, DP441). To ensure that the quality and concentration of the extracted RNA meet the requirements of subsequent experiments, a variety of detection methods were used. First, a Pultton P100 / P100 + ultra-micro spectrophotometer was used to accurately detect the concentration of RNA. Through this instrument, the concentration of RNA in the sample can be accurately measured, providing accurate data support for subsequent experiments. At the same time, using the UK UVITEC multi-color fluorescence / chemiluminescence gel imaging analysis system, the extracted RNA samples were subjected to 1% agarose gel electrophoresis to detect the quality of RNA. This operation helps to judge whether the extracted RNA is complete and whether there is degradation, etc., to ensure that it can meet the requirements of subsequent experiments. The extracted RNA was reverse transcribed into cDNA using the first-strand cDNA synthesis kit. The reverse transcription process was carried out strictly according to the operation instructions of the kit to ensure the efficiency and accuracy of the reverse transcription reaction. Finally, the obtained reverse transcription products were stored in an ultra-low temperature environment of -80 °C to ensure their stability and provide a reliable cDNA template for subsequent experiments.

[0030] 4. Cloning of Transcription Factor TraesCS3D01G144700, Construction of Subcellular Localization Vector and Expression Vector Through steps such as RNA extraction, cDNA reverse transcription, PCR amplification and product gel recovery, T cloning (pEASY®-Blunt Cloning Vector), transformation of recombinant plasmid into Escherichia coli DH5α, positive clone screening, PCR verification of bacterial liquid and sequencing, the bacterial liquid required for subsequent experiments was successfully obtained. After extracting the vector plasmid, it was transformed into Agrobacterium tumefaciens GV3101, and colony PCR verification and sequencing result comparison were carried out. The bacterial liquid containing the target gene was successfully obtained and verified by sequencing, as Figure 12 shown.

[0031] 5. Genetic Transformation of Arabidopsis thaliana The recombinant plasmid was introduced into the competent cells of Agrobacterium tumefaciens GV3101 by the freeze-thaw method. The transformed cells were cultured in an environment at 28 °C for 2 d, and monoclonal colonies were picked from the medium. The picked colonies were verified by colony PCR and sequenced. After confirmation, the positive bacterial liquid was stored in an ultra-low temperature refrigerator. The stored positive bacterial liquid was taken out for propagation culture. After the culture was completed, the Arabidopsis thaliana infection solution was prepared according to the established method. Before infecting Arabidopsis thaliana, sufficient water was added to the tray to allow the soil to fully absorb it, ensuring that the Arabidopsis thaliana plants were in the best growth state during infection. Arabidopsis thaliana was infected with the prepared infection solution. After the infection was completed, the residual infection solution on the surface of the plants was carefully wiped dry. The plants were cultured in the dark for 24 h and then transferred to grow under normal light conditions. To improve the transformation effect, Arabidopsis thaliana was reinfected according to the same infection steps after 7 d. After Arabidopsis thaliana matured, the seeds were harvested, and the transgenic Arabidopsis thaliana seeds were stored.

[0032] 6. Screening and Identification of Transgenic Arabidopsis thaliana Wild-type Arabidopsis thaliana seeds were planted and infected by the floral-dip method, and the T1 generation seeds were harvested. The T1 generation seeds were screened using a 1 / 2 MS medium containing 30 mg / L hygromycin. After transplantation and growth for about 14 d, leaves were collected for PCR positive identification. After the positive single plants set seeds, the T2 generation seeds were harvested. Screening of T2 generation lines: The T2 generation Arabidopsis thaliana was subjected to resistance screening, and the ratio of the number of normally growing seedlings to the number of seedlings that could not grow normally was observed. The lines that conformed to a ratio of 3:1 after chi-square test could be regarded as single-copy insertion lines. Harvest of T3 generation seeds: The seedlings of the screened T2 generation single-copy insertion lines were transplanted into the nutrient substrate, and high-expression lines were screened by qRT-PCR, and the T3 generation seeds were harvested from each single plant.

[0033] The results showed that the transcription factor TraesCS3D01G144700 had the highest expression level in roots, and the expression level was the highest at 10 d under low phosphorus stress ( Figure 1 ). In the low phosphorus tolerant material SW2 of wheat, the transcription factor TraesCS3D01G144700 was down-regulated from 3 to 7 d, and the expression level was higher under low phosphorus conditions than under severe low phosphorus conditions. It was up-regulated from 7 to 14 d, and the expression level was higher under severe low phosphorus conditions than under low phosphorus conditions. Overall, it showed an upward trend; in the phosphorus sensitive material SW2 of wheat, it was up-regulated from 3 to 7 d and down-regulated from 7 to 14 d, and the expression level was higher under low phosphorus conditions than under severe low phosphorus conditions. Overall, it showed a downward trend ( Figure 2 ) To study the correlation between the expression of the transcription factor TraesCS3D01G144700 and phosphorus uptake under low phosphorus and normal phosphorus conditions, it was observed that after 14 d of low phosphorus stress, the low phosphorus stress in the roots of the low phosphorus tolerant material SW2 and the phosphorus efficient material SW14 of wheat was significantly reduced. In the buds of wheat, there were significant differences from 3 to 14 d, and the root phosphorus concentration of SW2 reached the peak at the 14th d of low phosphorus stress (P <0.05). The results showed that within 14 days of phosphorus stress, SW2 showed stronger adaptability to low phosphorus stress compared with SW14 ( Figure 3 ).

[0034] Total RNA was extracted from the root tissues of the phosphorus-efficient wheat variety (SW2), reverse-transcribed into cDNA, and TraesCS3D01G144700 was amplified using the cDNA as a template. The PCR reaction product was detected by 1% agarose gel electrophoresis, and a fragment of about 768 bp consistent with the expectation was visible ( Figures 4 - 5 ). The target fragment recovered from the gel was constructed into the vector pC1300S-GFP. The recombinant plasmid was introduced into Escherichia coli by heat shock method, evenly spread on the solid medium containing Kan, and the transformed strains were screened for verification by colony PCR. Positive strains were selected for sequencing, and the CDS sequence of the wheat TraesCS3D01G144700 (768 bp) gene, encoding 255 amino acids, was obtained, and finally the construction of the vector was completed ( Figures 6 - 7 ).

[0035] Example 2 This example provides the wheat low-phosphorus tolerance transcription factor TraesCS3D01G144700, characterized in that the CDS sequence of the transcription factor TraesCS3D01G144700 is as shown in the appendix Figure 12 shown.

[0036] Example 3 This example provides a method for verifying the function of the wheat low-phosphorus tolerance gene TraesCS3D01G144700, including: 1. Characterization analysis of TraesCS3D01G144700 protein Based on the wheat whole-genome database, bioinformatics analysis was performed on the wheat transcription factor TraesCS3D01G144700. The CDS region was obtained using the online software ORFfinder (https: / / www.ncbi.nlm.nih.gov / orffinder / ), and amino acid translation was carried out using the ExPASy online tool (https: / / web.expasy.org / translate / ). The prediction of protein physical and chemical properties was completed using the online tool ProtParam (https: / / web.expasy.org / protparam / ); ProtScale (https: / / web.expasy.org / protscale / ) was used to predict its hydrophilicity and hydrophobicity; the phosphorylation site information of the protein was predicted using the Server software (http: / / www.cbs.dtu.dk / services / NetPhos-3.1 / ); the online software SignalP-5.0 (https: / / www.novopro.cn / tools / signalp) and TMHMM (https: / / services.healthtech.dtu.dk / ) were used to perform prediction analysis of the signal peptide region and transmembrane region respectively; SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html), Swiss-Model (https: / / swissmodel.expasy.org / interactive) and SMART (http: / / smart.embl-heidelberg.de / ) were used to complete the prediction of protein secondary structure, tertiary structure and domain in sequence; subcellular localization prediction was carried out using WoLF PSORT (https: / / wolfpsort.hgc.jp / ), and the cis-acting elements in the promoter region were predicted and analyzed using Plant CARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0037] 2. Subcellular localization analysis of TraesCS3D01G144700 According to the TraesCS3D01G144700 gene sequence in the wheat reference genome, specific primers were designed, and the full-length coding sequence (CDS) was amplified using the cDNA of wheat phosphorus-efficient SW2 roots as a template. The vector pC1300-GFP was used to construct a 35S::TraesCS3D01G144700-GFP fusion protein expression plasmid, and the PEG-mediated method was used to transform Arabidopsis protoplasts. A laser confocal microscope (Leica STELLARIS 8) was used to observe the subcellular localization signal and take pictures.

[0038] 3. Analysis of the low-phosphorus tolerance of TraesCS3D01G144700 in transgenic Arabidopsis under low-phosphorus stress T3 generation transgenic Arabidopsis and wild-type Arabidopsis were cultured under different phosphorus concentrations (phosphorus deficiency, low phosphorus, high phosphorus) in MS medium. Arabidopsis seeds were germinated on MS medium, where the MS powder was 4.74 g / L, 0.8% agar powder was 8 g / L, 1% sucrose was 10 g / L, and the pH was adjusted to 5.6 - 5.8. After vernalization at 4°C for 2 d, they were placed in a light incubator at 22°C with 16 h of light and 8 h of darkness for 7 d and then transplanted. They were treated with phosphorus deficiency (0 μM KH2PO4), low phosphorus (5 μM KH2PO4), and high phosphorus (500 μM KH2PO4) respectively. After growing on MS medium with three different phosphorus concentrations for 7 d, pictures were taken and recorded.

[0039] In this experiment, MS medium was used to perform different phosphorus concentration treatments on T3 generation transgenic Arabidopsis and wild-type Arabidopsis. First, Arabidopsis seeds were placed on MS medium for germination. The composition and content of the MS medium were as follows: the addition amount of MS powder was 4.74 g / L, the addition amount of 0.8% agar powder was 8 g / L, the addition amount of 1% sucrose was 10 g / L, and the pH value of the medium was adjusted to between 5.6 and 5.8 using an appropriate method to meet the optimal environmental conditions for Arabidopsis seed germination.

[0040] Vernalize Arabidopsis thaliana seeds to promote seed germination. Subsequently, transfer the vernalized seeds to a light incubator for cultivation. The environment of the light incubator is set at 22 °C, with a light duration of 16 hours per day and a dark duration of 8 hours per day. After 7 days of cultivation, transfer the seedlings to media with different phosphorus concentrations. For phosphorus deficiency treatment: Plant the seedlings on MS medium without KH2PO4 (i.e., 0 μM KH2PO4); for low phosphorus treatment: Plant the seedlings on MS medium containing 5 μM KH2PO4; for high phosphorus treatment: Plant the seedlings on MS medium containing 500 μM KH2PO4. The seedlings continue to grow on the above three different phosphorus concentration MS media for 7 days. After the growth cycle ends, take pictures of Arabidopsis thaliana in different treatment groups for subsequent observation and analysis of the effects of different phosphorus concentrations on the growth of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana.

[0041] The results showed that the full-length open reading frame encoded by the TraesCS3D01G144700 gene is 768 bp, encoding 255 amino acids, with a molecular weight of 26.88 kDa, and the molecular formula is C 1152 H 1878 N 362 O 362 S9, with a total of 3763 atoms; the isoelectric point (pI) of the encoded protein is approximately 7.70, which is a basic protein; the total number of positively charged residues (Arg + Lys) is 34, and the total number of negatively charged residues (Asp + Glu) is 28. The average hydrophilicity value (GRAVY) is -0.256, indicating that this protein is a hydrophilic protein and has serine and threonine phosphorylation sites. The signal peptide prediction results showed that the probability of this protein having a signal peptide is 0.190%, indicating that this protein has no signal peptide. Subcellular localization prediction showed that the protein encoded by the TraesCS3D01G144700 gene is localized in the nucleus. Secondary structure analysis showed that the proportion of α-helix is the largest, accounting for 47.06%, random coil accounts for 37.65%, and β-turn accounts for the least, 2.75%. The tertiary structure of the protein encoded by this gene is as Figure 8 shown. SMART analysis showed that amino acids 67 to 116 of the Tab HLH123-6A protein are the HLH conserved domain ( Figure 9). Prediction of cis-acting elements in the promoter region of TraesCS3D01G144700 revealed that there were 3 A-box elements related to meristem activity, 6 abscisic acid response elements ABRE, 5 cis-regulatory elements ARE involved in seed-specific regulation, 27 CAAT-box elements common to promoters and enhancers, 7 G-Box light response elements, 2 I-Box light response elements, 2 MYB protein-binding elements MYB, 2 cis-elements LTR involved in low-temperature response, 20 transcription start core promoter elements TATA-box, 2 auxin response elements TCA-element, 7 methyl jasmonate response elements TGACG-motif, and 1 WRKY protein-binding element W-box.

[0042] To verify the online prediction results of the subcellular localization of the gene-expressed protein, the subcellular localization of TraesCS3D01G144700 protein was studied. A TraesCS3D01G144700-GFP fusion protein expression vector was constructed and the empty vector pC1300S-GFP was used as a control. The GFP green fluorescence in protoplasts and leaf cells connected to the target protein TraesCS3D01G144700 was mainly distributed in the nucleus, and the GFP signal in the cell membrane was weak. The results showed that TraesCS3D01G144700 protein was localized in the nucleus and also distributed in the cell membrane ( Figure 10 ). To preliminarily identify the phosphorus utilization-related functions of wheat TraesCS3D01G144700, transgenic Arabidopsis thaliana TraesCS3D01G144700 was used as a material for functional verification under low-phosphorus stress. The growth conditions of overexpressed and wild-type Arabidopsis thaliana at the seedling stage under different phosphorus conditions showed that: under phosphorus-deficient conditions, the root lengths of the three transgenic Arabidopsis thaliana lines (T3-1, T3-2, T3-5) exceeded those of the wild type, and the number of their lateral roots was also more than that of the wild type, with the T3-5 line being the most obvious. Under low-phosphorus conditions, transgenic Arabidopsis thaliana showed better growth adaptability, and its root length was also better than that of wild-type Arabidopsis thaliana. Under high-phosphorus conditions, although both showed good growth momentum, transgenic Arabidopsis thaliana showed stronger growth ability ( Figure 11 ). In summary, overexpression of the TraesCS3D01G144700 transcription factor promoted phosphorus absorption and improved the low-phosphorus tolerance of wheat.

Claims

1. Application of overexpressing the transcription factor TraesCS3D01G144700 to improve the low-phosphorus tolerance of Arabidopsis thaliana, characterized in that The CDS sequence of the transcription factor TraesCS3D01G144700 is shown in Sequence Listing SEQ No.

1.

2. Use of overexpressing the transcription factor TraesCS3D01G144700 to improve the low phosphorus tolerance of Arabidopsis thaliana according to claim 1, characterized in that The improvement of the low-phosphorus tolerance of Arabidopsis thaliana means that under low-phosphorus conditions, Arabidopsis thaliana overexpressing TraesCS3D01G144700 has better growth.

3. Use of overexpressing the transcription factor TraesCS3D01G144700 to improve the low-phosphorus tolerance of Arabidopsis thaliana according to claim 1, characterized in that The transcription factor TraesCS3D01G144700 functions in the nucleus.

4. Transcription factor TraesCS3D01G144700, characterized in that The CDS sequence of the transcription factor TraesCS3D01G144700 is shown in Sequence Listing SEQ No.

1.

5. Method for functional verification of transcription factor TraesCS3D01G144700, characterized in that The method includes: (1) cultivation of experimental materials and preparation of reagents; (2) RNA extraction and cDNA synthesis; (3) cloning of the encoding gene of the transcription factor TraesCS3D01G144700; (4) construction of subcellular localization vectors and expression vectors; (5) Agrobacterium transformation; (6) genetic transformation of Arabidopsis thaliana.

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