The wheat is TaPHT1; 36 gene and application thereof

Abstract

The invention provides a wheat TaPHT1; according to the application of the gene 36 under low-phosphorus stress, through a sequence published in a wheat genome database, a low-phosphorus-resistant spring wheat material SW2 root system total RNA is taken as a template, cDNA is prepared through reverse recording, and TaPHT1 is cloned by taking the cDNA as the template; the method comprises the following steps of: constructing an overexpression vector, carrying out gene function verification on arabidopsis thaliana through transgenosis genetic transformation, and finding transgenosis TaPHT1 compared with wild type arabidopsis thaliana WT; the gene 36 can improve the low-phosphorus resistance, and provides a theoretical basis and gene resources for cultivating low-phosphorus-resistant wheat.

Description

Technical Field

[0001] The present invention belongs to the fields of plant bioengineering and transgenic technology, and particularly relates to an application of a wheat phosphate transporter and its encoding gene TaPHT1;36 in response to low phosphorus stress. Background Art

[0002] Food security is a severe challenge faced globally and is the foundation of human survival and development (Cordell, Drangert, and White 2009; Rengasamy 2016). Wheat (Triticum aestivum L.) is one of the important food crops widely cultivated globally and plays a significant role in food production and food security (Godfray et al. 2010). In the actual process of wheat cultivation, the availability of phosphorus in the soil is low. Most of the phosphorus in the soil is easily adsorbed and fixed by soil particles, forming insoluble phosphorus, resulting in a relatively low content of available phosphorus that can be absorbed and utilized by wheat roots. To increase its yield, a large amount of phosphate fertilizer is often applied to promote wheat maturation and enhance the agronomic quality of wheat (Shukla et al. 2016). Phosphorus is easily fixed in the soil and has low mobility, making it difficult for wheat to effectively absorb phosphorus, leading to phosphorus deficiency in wheat, resulting in problems such as slow growth, short plants, reduced tillering, and decreased yield. Phosphate transporters (PHTs) are responsible for the absorption and transport of phosphorus in plants. By studying wheat phosphate transporters, the molecular mechanism of wheat's absorption and utilization of phosphorus can be deeply understood, new wheat varieties with high phosphorus use efficiency can be cultivated, the dependence on phosphate fertilizer can be reduced, and the agricultural production cost can be lowered. Since the earliest identification of the high-affinity PHT gene PHO84 in yeast, more and more PHT families have been reported in various plants such as Arabidopsis thaliana (Muchhal, Pardo, and Raghothama 1996), rice (Oryza sativa) (Paszkowski et al. 2002), apple (Malus domestica) (Sun et al. 2017), Populus simonii (Zhang et al. 2016), Medicago truncatula (Harrison, Dewbre, and Liu 2002), Solanum tuberosum (Liu et al. 2017), Lupinus albus (Aslam et al. 2022), Brassica napus (Yang et al. 2020), Capsicum annuum (Ahmad et al. 2021), Saccharum (Murugan et al. 2022), etc., which play a key role in the process of phosphorus absorption. Understanding the mechanism of wheat phosphate transporters and their encoding genes can improve wheat varieties and enhance the efficiency of phosphorus absorption and utilization.This background technology not only helps to improve the productivity of wheat, but also provides a new idea for addressing the challenges of global food security. Therefore, the identification and functional analysis of genes related to low-phosphorus stress tolerance in wheat is of great practical significance for improving the effective utilization of phosphorus by wheat and achieving its high and stable yield. This experiment uses spring wheat as the research object to explore key genes related to low-phosphorus tolerance and provide the necessary genetic resources and technical support for breeding new low-phosphorus tolerant wheat varieties. Problems with the existing technology: There are no reports in the existing technology on the application of wheat TaPHT1;36 gene in responding to low-phosphorus stress. Summary of the invention

[0003] The key technical problem to be solved by the present invention is to provide the application of wheat phosphate transporter and its encoding gene TaPHT1;36 in responding to low phosphorus stress. To solve the above technical problem, the present invention adopts the following technical solution:

[0004] 1. Application of overexpression of wheat TaPHT1;36 gene to promote low phosphorus tolerance in Arabidopsis thaliana, wherein the CDS sequence of TaPHT1;36 gene is as shown in the attached Figure 22 And as shown in the sequence listing SEQ No.1.

[0005] 2. The use of overexpressing wheat TaPHT1;36 gene to promote low-phosphorus tolerance in Arabidopsis according to claim 1, wherein the root growth of Arabidopsis overexpressing TaPHT1;36 gene in a low-phosphorus environment is greater than that of wild-type Arabidopsis and shows better growth potential.

[0006] 3. Use of overexpression of wheat TaPHT1;36 gene to promote low-phosphorus tolerance in Arabidopsis according to claim 1, wherein the TaPHT1;36 protein functions in the plasma membrane.

[0007] 4. Wheat low-phosphorus tolerance gene TaPHT1;36, the CDS sequence of TaPHT1;36 is shown in the sequence listing SEQ ID NO: 1.

[0008] . A method for verifying the function of the wheat low-phosphorus tolerance gene TaPHT1;36, the method comprising: (1) subcellular localization of TaPHT1;36; (2) verification of the TaPHT1;36 gene in transgenic Arabidopsis under low-phosphorus stress.

[0009] Beneficial effects: The present invention obtains the low-phosphorus-tolerant wheat gene TaPHT1;36 through wheat PHT gene family analysis, clones the gene, further constructs a subcellular localization vector and an overexpression vector, and finds that the TaPHT1;36 protein is mainly expressed in the plasma membrane in subcellular localization of rice protoplasts; after successful transformation of Arabidopsis thaliana, the phenotype of transgenic Arabidopsis thaliana TaPHT1;36 under low-phosphorus stress is studied. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Phosphorus concentrations in roots and shoots of wheat varieties SW2 and SW14 under normal phosphorus and low phosphorus conditions.

[0011] Figure 2 Expression patterns of TaPHT genes in different tissues.

[0012] Note: Grain_z71, Grain_z75, and Grain_z85 represent grains at 2, 14, and 20 days after flowering, respectively; Spike_z32, Spike_z39, and Spike_z65 represent spikes at the internode interval, visible flag leaf stage, and flowering stage, respectively; Leaf_z10, Leaf_z23, and Leaf_z71 represent leaves at the seedling stage, three-tiller stage, and 2 days after flowering, respectively; Stem_z30, Stem_z32, and Stem_z65 represent stems at the heading stage, internode interval, and flowering stage, respectively; Root_z10, Root_z13, and Root_z39 represent roots at the seedling stage, three-leaf stage, and visible flag leaf stage, respectively.

[0013] Figure 3 Expression patterns of TaPHT genes under low phosphorus stress.

[0014] Note: -P-S-10d: Buds under low phosphorus stress for 10 days; S-0d: Buds under low phosphorus stress for 0 days; P-R-10d: Roots under low phosphorus stress for 10 days; R-0d: Roots under low phosphorus stress for 0 days.

[0015] Figure 4 qRT-PCR analysis of TaPHT genes in roots at 0d, 0.25d, 0.5d, 1d, 3d, 7d, 14d, and 21d under low phosphorus stress. Among them, SW2 is a low phosphorus tolerant wheat variety and SW14 is a phosphorus sensitive wheat variety. LSD represents the significance test of different varieties at different time points at the 0.05 level.

[0016] Figure 5 qRT-PCR analysis of TaPHT genes in roots at 0d, 0.25d, 0.5d, 1d, 3d, 7d, 14d, and 21d under low phosphorus stress. Among them, SW2 is a low phosphorus tolerant wheat variety and SW14 is a phosphorus sensitive wheat variety. LSD represents the significance test of different varieties at different time points at the 0.05 level.

[0017] Figure 6 Total RNA extraction from wheat shoots.

[0018] Note: M: DL 2000 DNA Marker; Lanes 1-3: TaPHT1; 36 Shoot RNA.

[0019] Figure 7 For the extraction of total RNA from wheat roots.

[0020] Note: M: DL 2000 DNA Marker; Lanes 1-3: TaPHT1;36 root RNA.

[0021] Figure 8 For the PCR amplification of TaPHT1;36.

[0022] Note: M: DL15000 + 2000 DNA Marker.

[0023] Figure 9 For the identification of positive clones of TaPHT1;36.

[0024] Note: M: 2000 DNA Marker.

[0025] Figure 10 For the amino acid sequence alignment of TaPHT1;36 protein.

[0026] Figure 11 For the vector map of pC1300S-GFP-TaPHT1;36.

[0027] Figure 12 Identification of recombinant positive clones of TaPHT1;36.

[0028] Figure 13 For the double digestion verification of the gene cloning vector plasmid.

[0029] Note: M is KB Ladder 1: gene plasmid; 2. gene double digestion verification.

[0030] Figure 14 For the comparison of the amino acid sequence sequencing results of TaPHT1:36 gene cloning.

[0031] Figure 15 For the resistance screening of TaPHT1;36 positive transgenic Arabidopsis thaliana T 1 generation seeds.

[0032] Figure 16 For the resistance screening of TaPHT1;36 positive transgenic Arabidopsis thaliana T 2 generation seeds.

[0033] Figure 17 For the PCR positive detection of transformed plants.

[0034] Figure 18 For bioinformatics analysis software and website.

[0035] Figure 19 For the bioinformatics analysis of TaPHT1;36 protein.

[0036] Among them, A: Prediction of the transmembrane domain of TaPHT1;36 protein; B: Prediction of the hydrophilicity and hydrophobicity of TaPHT1;36 protein; C: Phosphorylation sites of TaPHT1;36 protein; D: Prediction of the signal peptide region of TaPHT1;36 protein; E: Prediction of the secondary structure of TaPHT1;36 protein; F: Prediction of the tertiary structure of TaPHT1;36 protein; G: Transmembrane topology of TaPHT1;36 protein.

[0037] Figure 20 It is the subcellular localization analysis after transient expression of TaPHT1;36 gene in rice protoplasts.

[0038] Figure 21 It is the growth status of overexpressing Arabidopsis thaliana and wild-type seedlings under different phosphorus conditions.

[0039] Figure 22 It is the CDS sequence of TaPHT1;36 gene, with the start and stop codons underlined. Specific implementation method

[0041] In the following embodiments of this invention patent, if not otherwise specified, the methods and devices used are conventional methods and devices, and the equipment and reagents used are also conventional equipment and reagents purchased from reagent companies. In order to more clearly present the purpose, technical solutions and advantages of this invention patent, the following will combine specific embodiments to elaborate in detail on the specific implementation manners of this invention patent. These preferred implementation manners are all exemplified in the specific embodiments. Here, it should be particularly noted that to prevent the technical solutions of this invention patent from being unclear due to unnecessary details, only the technical solutions and / or processing steps closely related to the solutions of this invention patent are shown in the embodiments, while other details with little relevance are discarded.

[0042] Example 1

[0043] This example provides a screening method for the wheat low-phosphorus tolerance gene TaPHT1;36, including:

[0044] 1. Based on the low-phosphorus tolerant wheat variety (SW2) and phosphorus-sensitive wheat variety (SW14) previously screened in the laboratory, select plump and uniform grains. After 4 days of cultivation, transplant them into a hydroponic box containing 900 mL and cultivate for 2 days, and place them in an artificial climate chamber with a relative humidity of 60%. The light condition is white fluorescent lamp irradiation intensity of 300 μmol / m 2 / s, and adopt a day-night cycle mode: 16 h of light (25 °C) and 8 h of darkness (18 °C) for phosphorus treatment. In the wheat hydroponics experiment, an improved Hoagland nutrient solution is used, and the nutrient solution is changed every 3 days, including macronutrients: 4 mM Ca(NO3 ) 2 · 4H 2 O, 5 mM KNO 3 , 1 mM NH 4 NO 3 , 2 mM MgSO 4 · 7H 2 O; Trace elements: 200 μM Na 2 -EDTA, 200 μM FeSO 4 · 7H 2 O, 10 μM H 3 BO 3 , 13 μM MnSO 4 · H 2 O, 0.01 μM CuSO 4 · 5H 2 O, 3 μM ZnSO 4 · 7H 2 O, 0.02 μM (NH 4 ) 6 Mo 7 O 24 · 4H 2 O. Normal phosphorus (+P) treatment: 500 mM KH 2 PO 4 ; Low phosphorus (-P) treatment: 6.25 mM KH 2 PO 4 , 247 mM K 2 SO 4 . Samples were collected at different time points (0 d, 0.25 d, 0.5 d, 1 d, 3 d, 7 d, 14 d, 21 d) after transplantation. After removing excess moisture by gently pressing with filter paper, the roots and shoots were placed separately into 5-ml cryotubes and quickly frozen in liquid nitrogen. Some samples were used for RNA extraction, and the remaining samples were stored at -80 °C in the refrigerator for later use.

[0045] 2. Total RNA of wheat roots and leaves was extracted using a polysaccharide polyphenol plant total RNA extraction kit (TIANGEN, Beijing, DP441). RNA samples were reverse-transcribed into cDNA according to the manufacturer's instructions of qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan).

[0046] 3. To explore the positive correlation between TaPHT expression and phosphorus uptake under low phosphorus and normal phosphorus conditions, total phosphorus concentration in plant samples of each treatment was determined with three biological replicates. The total phosphorus concentration was measured using the molybdate blue colorimetric method. The dried samples were ground, and treated with concentrated H 2 SO 4 and H 2O 2 Digestion. To study the expression of TaPHT genes in response to phosphate starvation, the relative expression levels of two wheat varieties, SW2 and SW14, were compared by qRT-PCR under two growth conditions: normal phosphorus (+P) and low phosphorus (-P). To understand the response of TaPHT to low phosphorus stress at the transcriptional level, 180 TaPHT genes were retrieved from the wheat expression database, combined with the tissue expression patterns of wheat roots, leaves, stems, spikes and grains and the expression levels under low phosphorus stress treatment. According to the predicted expression levels, 3-5 genes were selected from each TaPHT subfamily, and the expression levels of 30 TaPHT genes in wheat roots and shoots were detected by qRT-PCR under low phosphorus stress. Among them, 6 genes were not expressed or abnormally expressed, and the expression levels of 24 genes in roots and shoots were analyzed. Fluorescent quantitative primers were designed according to the non-conserved sequence at the N-terminus of TaPHT genes (Table 1). Using the cDNA of the low-phosphorus tolerant spring wheat SW2 root system as a template, the SYBR-Green Prime Script RT-PCR kit (Takara Biotechnology, Japan) was used for qRT-PCR analysis of the QuantStudio TM 5 System real-time system (Table 1). A 0.2 ml 96-well plate was used for Comparative Ct (ΔΔCt). The thermal cycling parameters were as follows: The Hold Stage had an initial denaturation at 95 °C for 30 s, and then the amplification stage (PCR Stage) had denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, for 40 cycles. The Melt Curve Stage had denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and dissociation at 95 °C for 15 s. Three biological replicates were applied for each treatment, and the expression level was calculated as the average signal intensity of the three replicates, using the 2(-ΔΔCt) method. The data were the mean ± standard deviation of three independent experiments, and the error bars represented the standard error from three independent experiments. GenStat 12th Edition was used for least significant difference (LSD) analysis test (P<0.05) to evaluate the expression differences of TaPHT after low phosphorus treatment.

[0047] Table 1 TaPHT gene-specific primer sequences for qRT-PCR expression analysis

[0048]

[0049] Table 2 qRT-PCR reaction system

[0050]

[0051]

[0052] To study the correlation between TaPHT expression and phosphorus uptake under low-phosphorus and normal-phosphorus conditions, the phosphorus contents in the roots and shoots of low-phosphorus tolerant spring wheat material (SW2) and phosphorus-sensitive spring wheat material (SW14) were measured. After 14 days of low-phosphorus stress, it was observed that the low-phosphorus stress in the roots of SW2 and SW14 decreased significantly. There were significant differences in the shoots from 3 to 14 days, and the phosphorus concentration in the roots of SW2 reached a peak on the 14th day of low-phosphorus stress (P<0.05). The results showed that within 14 days of phosphorus stress, the adaptability of SW2 to low-phosphorus stress was stronger than that of SW14( Figure 1 ).

[0053] To understand the response of TaPHT to low-phosphorus stress at the transcriptional level, 180 TaPHT genes were retrieved from the wheat expression database, combined with the tissue expression patterns and expression levels under low-phosphorus stress treatment in wheat roots, leaves, stems, spikes and grains( Figure 2 ). The results showed that under low-phosphorus stress, the TaPHT1 and TaPHT3 subfamilies were up-regulated in roots and down-regulated in shoots, while the TaPHT2 and TaPHT4 subfamilies were up-regulated in shoots and down-regulated in roots. Most of the TaPHT5 subfamily was up-regulated in shoots, and TaPHO1 was mostly up-regulated in roots( Figure 3 ). Among the six subfamilies, the TaPHT1 subfamily had the highest expression level in roots, and the TaPHT2 subfamily had the highest expression level in shoots.

[0054] In the TaPHT1 subfamily, the initial response was from 0 to 3 days. The members of the PHT1 gene family in wheat roots quickly responded to low-phosphorus stress, and the relative expression level began to rise. With the continuous low-phosphorus stress, the relative expression level of the PHT1 gene in wheat roots continued to rise and slowly decreased from 14 to 21 days. Figure 4LSD indicates significant differences based on LSD (P < 0.05), and the specific LSD values are shown in Table 3. Among them, the relative expression level of the TaPHT1;36 gene in the roots was the highest at 7 days, reaching 114.727, and then reached a relatively stable state. This is a process in which plants gradually adapt to and optimize the phosphorus absorption mechanism under long-term low-phosphorus stress. The results show that when phosphate in the soil is limited, these up-regulated TaPHT1 genes may be involved in phosphate absorption, thereby alleviating the physiological stress caused by low phosphorus. The roots of the TaPHT2 subfamily were slightly up-regulated within 0 - 21 days of low-phosphorus stress and gradually adapted to the low-phosphorus environment after reaching the peak at 7 days. The up-regulation of the TaPHT2 gene expression indicates that wheat can sense the lack of phosphorus in the environment. In the TaPHT3 subfamily, except that the relative expression level of TaPHT3;36 reached the peak value of 2.207 at 7 days, the relative expression levels of other genes increased slowly, indicating that the physiological stress caused by low phosphorus was slightly alleviated by increasing the expression of TaPHT3 genes. Under low-phosphorus conditions, wheat optimizes the absorption, transport, and allocation of phosphorus by adjusting the expression of TaPHT4 gene family members to adapt to the low-phosphorus environment. Among the 5 tested genes of TaPHT4, TaPHT4;10 and TaPHT4;13 were highly expressed in wheat roots, while the relative expression levels of TaPHT4;7 and TaPHT4;20 were relatively low in wheat roots, indicating that the up-regulation of TaPHT4 helps plants enhance phosphorus absorption in the low-phosphorus environment. The TaPHT5 gene was slightly up-regulated in wheat roots. Among them, the expression level of TaPHT5;9 had a peak value of 3.841 at 7 days, and the other two genes remained relatively stable after 7 days of low-phosphorus treatment. The results show that the up-regulation of the TaPHT5 gene helps plants enhance phosphorus absorption in the low-phosphorus environment. In the TaPHO1 subfamily, different from other genes, the relative expression level of TaPHO1;H11 in SW14 was higher than that in SW2, and was significantly higher than that of the other 2 tested genes. This may be because the gene expression varies among different varieties. With the prolongation of low-phosphorus stress time, the relative expression level of the TaPHO1 gene in wheat roots gradually stabilized, reducing the dependence on phosphorus nutrition. Except for TaPHO1;H11, the relative expression level of the PHTs gene family in wheat roots was higher in SW2 than in SW14.

[0055] Figure 5For the above-ground parts of wheat, except for TaPHT1;36, the relative expression levels of TaPHT1 subfamily genes ranged from 0.2 to 1.2 within 0 - 21 days. Combining with the relative expression levels in wheat roots indicated that TaPHT1 subfamily genes were expressed in wheat roots. The relative expression levels of TaPHT2 subfamily in the above-ground parts of wheat at the initial stage of low phosphorus treatment did not change significantly, only showing a slight up-regulation. This is because when plants face low phosphorus stress, they need a certain amount of time to perceive and respond to this stress and adjust the corresponding gene expression. With the prolongation of low phosphorus treatment time, the expression levels of TaPHT2 subfamily in the above-ground parts of wheat increased, attempting to increase the ability to absorb and transport phosphorus. Under longer-term low phosphorus treatment, the relative expression levels gradually decreased. The expression level of TaPHT2;1 was the highest at 7 days, reaching 64.985. With the prolongation of stress time, the expression level of TaPHT2;1 in leaves may gradually stabilize, adapting to low phosphorus stress and attempting to enhance the transport of phosphorus from roots to leaves. This is because the PHT2 subfamily is expressed in leaves, and as the main part of plants for photosynthesis and material accumulation, the gene expression pattern is usually more conservative and stable to maintain the normal physiological functions of plants. The PHT3 gene family mainly participates in the transport and distribution of phosphorus in plants, but its specific expression regulation mechanism varies due to different plant species and growth conditions, playing different roles in wheat roots and above-ground parts. Except for TaPHT3;36 among the tested genes, the relative expression levels of the other 4 TaPHT3 genes in the above-ground parts of wheat were relatively low. At the initial stage of low phosphorus treatment, the relative expression levels of TaPHT4 subfamily in the above-ground parts of wheat did not change significantly. Among them, the relative expression level of TaPHT4;20 reached the peak at 7 days and then gradually decreased because the changes in other physiological and molecular mechanisms in plants after long-term low phosphorus treatment further affected the relative expression level of TaPHT4;20. The relative expression levels of TaPHT5 subfamily genes in the above-ground parts of wheat were the highest at the 7th day. After a long-term response to low phosphorus stress, the expression of TaPHT5 subfamily genes decreased to a certain extent. Among them, the relative expression level of TaPHT5;7 in SW14 was higher than that in SW2 after 3 days, which may be affected by various factors, including phosphorus transport, recycling, and the physiological state of leaves themselves, reflecting the mechanism of plants adapting to low phosphorus. Among the TaPHO1 subfamily, the relative expression level of TaPHO1;H11 was significantly higher than that of the other two tested genes. The above-ground parts of wheat immediately sensed the phosphorus deficiency in the environment under low phosphorus conditions, thus initiating the corresponding stress response. With the prolongation of treatment time, the expression of TaPHO1 subfamily genes continued to increase and reached the peak at 7 days. This reflects that during the process of plants adapting to low phosphorus environment, the expression of corresponding genes was enhanced to promote the absorption and utilization of phosphorus by roots. In the later stage of low phosphorus stress, the expression of TaPHO1 subfamily genes in the above-ground parts of wheat decreased to a certain extent, reflecting the adaptability of wheat.At this time, wheat may activate other metabolic pathways to cope with the challenge of long-term low phosphorus. The above-ground part of wheat is not the main part directly sensing low-phosphorus stress, so the response of its gene expression is relatively lagged, but the overall expression is up-regulated, and the relative expression level of the TaPHT gene family in the above-ground part of wheat is higher in wheat variety SW2.

[0056] Table 3 LSD values of TaPHT family genes in roots and above-ground parts

[0057]

[0058]

[0059] Example 2

[0060] This example provides the wheat low-phosphorus tolerance TaPHT1;36 gene, and the CDS sequence of the TaPHT1;36 is as shown in the appendix. Figure 22 as shown.

[0061] Example 3

[0062] This example provides a method for cloning, vector construction and transformation of the wheat low-phosphorus tolerance gene TaPHT1;36, including:

[0063] 1. Preparation of test materials and reagents

[0064] The low-phosphorus tolerance wheat variety (SW2) and phosphorus-sensitive wheat variety (SW14) used in the present invention were screened in the early stage of the laboratory and planted in an artificial climate incubator. The present invention uses a polysaccharide polyphenol plant total RNA extraction kit (TIANGEN, Beijing, DP441), qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan), SYBR-Green Prime Script RT-PCR kit (Takara Biotechnology, Japan). Restriction enzymes and ligases were purchased from Wuhan Tianwen Biotechnology Co., Ltd. Escherichia coli DH5α competent cells and Agrobacterium tumefaciens GV3101 competent cells (Qingke, Beijing).

[0065] 2. RNA extraction and cDNA synthesis

[0066] Collect the root and leaf tissues of low phosphorus tolerant wheat variety (SW2) and phosphorus sensitive wheat variety (SW14) under different phosphorus level treatments. The specific extraction steps are carried out according to the instructions of the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, DP441). Use a Pultton P100 / P100+ ultra-micro spectrophotometer to detect the RNA concentration, and use a 1% agarose gel electrophoresis with a UK UVITEC multi-color fluorescence / chemiluminescence gel imaging analysis system to detect the RNA quality. Reverse transcribe the RNA into cDNA using a First Strand cDNA Synthesis Kit, and store the reverse transcription product at -80 °C.

[0067] 3. Cloning of TaPHT1;36 gene

[0068] Extract the total RNA from the shoots and roots of wheat. After detection by 1% agarose gel electrophoresis, 3 electrophoretic bands are obtained. Use an ultra-micro ultraviolet spectrophotometer to measure the OD260 / 280 value to be 1.94, indicating that the total RNA from the shoots and roots of wheat, which is intact and of high purity, can be used for subsequent experiments ( Figures 6 - 7 ). Using the cDNA of wheat roots under low phosphorus stress for 7 days as a template, perform PCR amplification with specific primers to obtain an expected band of 1323 bp ( Figure 8 ). Purify the successfully amplified TaPHT1;36 gene, ligate it to the cloning vector pMDTM19-T, transform the recombinant plasmid into Escherichia coli DH5α competent cells, screen for positive clones and verify by colony PCR ( Figure 9 ). Use DNAMAN 9.0 software for alignment and find that the similarity between the sequencing result and the gene sequence in the wheat genome database is 100% ( Figure 10 ), indicating that the gene sequence is correct, and the bacterial solution required for subsequent experiments is successfully obtained and stored in a -80 °C refrigerator for later use. At this time, the wheat TaPHT1;36 gene is successfully cloned. 4. Construction of subcellular localization vector and expression vector

[0069] According to the sequence information of the wheat TaPHT1;36 gene and the plant expression vector pC1300S-GFP, use SnapGene software to select appropriate restriction enzyme sites KpnI (5' end) and BamHI (3' end).

[0070] (1) Activation of pC1300S-GFP vector: Using pC1300S-GFP as the overexpression vector, prepare an LBA plate containing 50 μg / mL Kan in a laminar flow hood. After the medium solidifies, gently streak the vector bacterial solution on the plate using the "Z" method and seal it. Incubate the strain at 37 °C in the dark. After white single colonies form on the surface of the medium, randomly select 10 colonies and inoculate them into 10 mL of LB liquid medium containing 50 μg / mL Kan for amplification culture. Seal the medium well and incubate it at 37 °C, 200 rpm in the dark for 12 - 16 h.

[0071] (2) Activation of the ligated cloning vector bacterial solution: Pipette 10 μL of the bacterial solution with an ideal sequencing result and transfer it to 10 mL of LB liquid medium containing 100 μg / mL Amp. Seal the medium and incubate it in a shaker at 37 °C, 200 rpm in the dark for 12 - 16 h.

[0072] (3) Plasmid extraction: Use a plasmid miniprep kit (TIANGEN, Beijing) to extract the plasmid from the bacterial solution containing the target gene and the overexpression vector. After the plasmid concentration is detected to be qualified, store the plasmid at -20 °C.

[0073] (4) Construction of the overexpression vector: Select restriction endonucleases (KpnI and BamHI) to perform double digestion on the TaPHT1;36 gene plasmid and the overexpression vector plasmid. After overnight digestion, perform 1% agarose gel electrophoresis analysis. When the bands of the target gene and the overexpression vector are consistent with the theoretical values, perform gel cutting and DNA purification operations, and conduct secondary electrophoresis detection on the recovered product to ensure the accuracy of the experimental results.

[0074] Use T-4 DNA ligase to ligate the TaPHT1;36 gene fragment with the gel recovery product of the overexpression vector. Take out the ligation product the next day and transform it into Escherichia coli, and pick monoclonal colonies. Perform PCR verification on the bacterial solution containing the recombinant transformant. Prepare glycerol tubes for the bacterial solution with bands meeting the expectations. Store a part at -80 °C, and use the remaining bacterial solution for plasmid extraction. Use Hind III and EcoR I to perform double digestion verification on the plasmid. Store the plasmid successfully verified by 1% agarose electrophoresis at -20 °C in the refrigerator.

[0075] The results showed that: Activate and ligate the TaPHT1;36 positive bacterial solution and the pC1300S-GFP vector bacterial solution. The size of the whole vector is 11970 bp, and the size of the foreign fragment is 3064 bp. The sequencing results indicate that the gene is part of the foreign fragment. Judging from the length, the "Insert Sequence" is about 1300 bp ( Figure 11)。Extract the plasmid, double-digest the TaPHT1;36 gene and the vector with the restriction enzymes KpnI and BamHI, ligate the purified products, transform DH5α, and perform colony PCR detection after screening for blue and white colonies to obtain a band of 1323 bp size( Figure 12 )。Perform double-digest identification with Hind III and EcoR I to successfully obtain a 3064 bp target band containing TaPHT1;36( Figure 13 )。Extract the vector plasmid and transform Agrobacterium tumefaciens GV3101, perform colony PCR verification and sequence alignment of the sequencing results to successfully obtain the bacterial liquid containing the target gene( Figure 14 )。

[0076] Extract total RNA from the root tissues of the low-phosphorus tolerant wheat variety (SW2), reverse-transcribe it into cDNA, and use the cDNA as a template to amplify the TaPHT1;36 gene. The PCR reaction product was detected by 1% agarose gel electrophoresis, and a fragment of about 1323 bp consistent with the expectation was visible. Recover the target fragment from the gel and construct it onto the vector pC1300S-GFP. Use the heat shock method to introduce the recombinant plasmid into Escherichia coli, evenly coat it on the solid medium containing Kana, screen the transformed strains for colony PCR verification, select the positive strains for sequencing, obtain the CDS sequence of the wheat TaPHT1;36 (1323 bp) gene, which encodes 440 amino acids, and finally complete the construction of the vector.

[0077] 5. Genetic transformation of Arabidopsis thaliana

[0078] Using the freeze-thaw method, introduce the recombinant plasmid into the competent cells of Agrobacterium tumefaciens GV3101. Incubate the transformed cells at 28 °C for 2 days, and then pick monoclonal colonies from the medium. Perform colony PCR verification and sequencing of the picked colonies. After confirmation, store the positive bacterial liquid in an ultra-low temperature freezer. Take out the stored positive bacterial liquid for expansion culture. After the culture is completed, prepare the Arabidopsis thaliana infection solution according to the established method. Before infecting Arabidopsis thaliana, sufficient water needs to be added to the tray to allow the soil to fully absorb it to ensure that the Arabidopsis thaliana plants are in the best growth state during infection. Use the prepared infection solution to infect Arabidopsis thaliana. After the infection operation is completed, carefully dry the residual infection solution on the surface of the plants. Then, keep the plants in the dark for 24 h and then transfer them to grow under normal light conditions. To improve the transformation effect, infect Arabidopsis thaliana again according to the same infection steps after 7 days. Harvest the seeds when Arabidopsis thaliana is mature and store the transgenic Arabidopsis thaliana seeds.

[0079] 6. Screening and identification of transgenic Arabidopsis thaliana

[0080] Plant the wild-type Arabidopsis thaliana seeds, infect them by the floral dip method, and harvest the T 1 -generation seeds, and use 1 / 2 MS medium containing hygromycin (30 mg / L) to screen the T1 The T0 generation seeds were transplanted, and after about 30 days of growth, the leaves were collected for PCR positive identification. After the positive single plants produced seeds, the T1 generation seeds were harvested. 2 For the T1 generation of Arabidopsis thaliana, resistance screening was carried out. The ratio of the number of seedlings that could grow normally 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. The seedlings of the T1 generation single-copy insertion lines screened out were transplanted into the nutrient substrate, and high-expression lines were screened by qRT-PCR. The T2 generation seeds were harvested from each single plant ( 2 ) 2 In the process of antibiotic screening, false-positive transgenic Arabidopsis thaliana plants may be produced, which need to be further verified by PCR method. After obtaining the transformed plants by hygromycin resistance screening, they were transplanted into the culture substrate and subcultured in the Arabidopsis thaliana culture room. After growing for 14 days, the leaf DNA of the T1 generation transformed plants and the wild-type control was extracted, and PCR amplification was carried out using specific primers. The specific primers of the resistance gene Hpt (F: 5’-GAAGTGCTTGACATTGGGGAGT-3’; R: 5’-AGATGTTGGCGACCTCGTATT-3’) were used to detect the PCR positivity of 8 transgenic transformed plants. Among them, 7 were positive transformed plants ( 3 ), and the numbers were 1, 3, 4, 5, 6, 7, and 8 respectively. At the same time, no amplification products were shown in the wild-type Arabidopsis thaliana DNA and ddH Figures 15 - 16 2O samples used as negative controls, confirming the successful acquisition of positive plants of Arabidopsis thaliana transformed with the TaPHT1;36 gene.

[0081] Example 4 1 This example provides a method for verifying the function of the wheat low phosphorus tolerance gene TaPHT1;36, including: Figure 17 1. Characterization analysis of TaPHT1;36 protein 2 The prediction of protein physical and chemical properties was completed using the online tool ProtParam; the transmembrane region was predicted and analyzed using the online software TMHMM; its hydrophilicity and hydrophobicity were predicted using ProtScale; the information of protein phosphorylation sites was predicted using the Server software; the signal peptide region was predicted using SignalP-5.0; SOPMA, Swiss-Model, and SMART were used to complete the prediction of protein secondary structure, tertiary structure, and domain in turn; Phyre2 was used to predict its transmembrane topology; subcellular localization prediction was carried out using WoLFPSORT, and the website link is shown in

[0082]

[0083]

[0084]

[0085] Figure 18

[0085] Figure 18 Figure 18 .

[0086] 2. Subcellular localization analysis of TaPHT1;36 protein

[0087] According to the TaPHT1;36 gene sequence in the wheat reference genome, specific primers were designed, and the cDNA of the wheat low-phosphorus tolerant SW2 root system was used as a template to amplify the full-length coding region (CDS) sequence. The vector pC1300S-GFP was used to construct the 35S::TaPHT1;36-GFP fusion protein expression plasmid, and the rice protoplasts were transformed by PEG-mediated method. The subcellular localization signal was observed and photographed using a laser confocal microscope (Leica STELLARIS 8).

[0088] 3. Analysis of low-phosphorus tolerance of TaPHT1;36 in transgenic Arabidopsis thaliana under low-phosphorus stress

[0089] Different phosphorus concentrations (phosphorus deficiency, low phosphorus, and high phosphorus) were used to culture T. 3 The transgenic Arabidopsis and wild-type Arabidopsis of the same generation were germinated on MS medium, which contained 4.74 g / L MS powder, 8 g / L 0.8% agar powder, 10 g / L 1% sucrose, and the pH was adjusted to 5.6-5.8. After vernalization at 4°C for 2 days, the seeds were placed in a light incubator at 22°C with 16 h light and 8 h dark for 7 days, and then transplanted. The seeds were subjected to phosphorus deficiency (0 μM KH 2 PO 4 ), low phosphorus (5 μM KH 2 PO 4 ), high phosphorus (500μM KH 2 PO 4 ) treatment. After growing on MS medium at three different phosphorus concentrations for 7 days, photos were taken. The results showed that the open reading frame encoded by the TaPHT1;36 gene was 1323 bp in length, encoding 440 amino acids. The isoelectric point (pI) of the encoded protein was about 7.70, which was an alkaline protein. The molecular weight was 48.14 kDa, and the molecular formula was C 2211 H 3367 N 561 O 596 S 25 The total number of atoms is 6760; the fat index is 86.8, the instability index is 32.61, and it is speculated to be a stable protein (II<40); the average hydrophilicity (GRAVY) is 0.36, indicating that this protein is a hydrophobic protein ( Figure 19B). The total number of positively charged residues (Arg + Lys) is 34, and the total number of negatively charged residues (Asp + Glu) is 33. Among the 440 amino acids, 204 are located in the transmembrane region, embedded internally, and there are 9 transmembrane domains, with specific positions at amino acid sites 23 - 35, 55 - 71, 92 - 121, 142 - 203, 233 - 251, 273 - 280, 299 - 310, 338 - 348, 371 - 389( Figure 19 A, G); The protein encoded by the TaPHT1;36 gene has 64 serine, tyrosine, and threonine phosphorylation sites( Figure 19 C); The signal peptide prediction results show that the probability of this protein having a signal peptide is 74.523%, and the signal peptide type: SP (Sec / SPI)( Figure 19 D). Secondary structure analysis shows that the proportion of α - helix is the largest, accounting for 51.36%, random coil accounts for 30.23%, and β - turn accounts for the least, 4.55%( Figure 19 E), and the predicted model of the tertiary structure of the protein encoded by this gene is as Figure 19 shown in F. Subcellular localization prediction indicates that the protein encoded by the gene TaPHT1;36 is mainly localized on the plasma membrane.

[0090] To verify the online prediction results of the subcellular localization of the gene - expressed protein and study the subcellular localization of TaPHT1;36 protein, a TaPHT1;36 - GFP fusion protein expression vector was constructed, with the empty vector pC1300S - GFP as a control. According to the results of rice protoplasts, GFP green fluorescence signals were detected throughout the cells of the GFP empty - vector control protoplasts, without obvious organelle localization specificity. The GFP green fluorescence linked to the target protein TaPHT1;36 was mainly distributed in the plasma membrane( Figure 20 ). The results show that the TaPHT1;36 protein is localized in the plasma membrane, which is consistent with previous studies and online prediction results.

[0091] To preliminarily identify the phosphorus - transport - related function of wheat TaPHT1;36, transgenic Arabidopsis thaliana TaPHT1;36 was used as a material for functional verification under low - phosphorus stress. The growth conditions of over - expressed and wild - type Arabidopsis thaliana at the seedling stage under different phosphorus conditions showed that: in the T 3 - 3 lines, the shoots of wild - type and over - expressed Arabidopsis thaliana showed dark green color under phosphorus - deficient (0 μM KH 2 PO 4 ) conditions; The T 3 - 4 and T 3 - 8 lines showed dark green color in shoots under phosphorus - deficient (0 μM KH 2 PO 4 ), low - phosphorus (5 μM KH 2 PO 4) Under such conditions, the aerial parts of wild-type and overexpressing Arabidopsis thaliana were dark green, and the dark green color of the leaves of wild-type Arabidopsis thaliana was more obvious under phosphorus treatment. At the same time, the number of lateral roots of overexpressing plants was reduced compared with that of wild-type plants, and the length difference was not significant( Figure 21 ). Under phosphorus-deficient conditions, the root lengths of the three transgenic Arabidopsis thaliana lines were longer than those of wild-type Arabidopsis thaliana, and the number of lateral roots of transgenic Arabidopsis thaliana was more than that of wild-type Arabidopsis thaliana. Under low-phosphorus conditions, transgenic Arabidopsis thaliana showed better growth adaptability, with longer root lengths than wild-type Arabidopsis thaliana and better growth vigor; under high-phosphorus conditions, both showed good growth, but transgenic Arabidopsis thaliana showed a higher growth level, and the leaves of Arabidopsis thaliana were significantly larger than those under phosphorus-deficient and low-phosphorus conditions. In summary, the TaPHT1;36 gene improved the low-phosphorus tolerance of wheat.

[0092] References:

[0093] [1] Cordell, Dana, Jan-Olof Drangert, and Stuart White. 2009. 'The story of phosphorus: Global food security and food for thought', Global Environmental Change, 19: 292 - 305.

[0094] [2] Rengasamy, Pichu. 2016. "Soil Salinization." In.: Oxford University Press.

[0095] [3] Godfray, H. Charles J., John R. Beddington, Ian R. Crute, Lawrence Haddad, David Lawrence, James F. Muir, Jules Pretty, Sherman Robinson, Sandy M. Thomas, and Camilla Toulmin. 2010. 'Food Security: The Challenge of Feeding 9 Billion People', Science, 327: 812 - 18.

[0096] [4]Shukla, Vishnu, Mandeep Kaur, Sipla Aggarwal, Kaushal Kumar Bhati, Jaspreet Kaur, Shrikant Mantri, and Ajay K. Pandey. 2016. 'Tissue specific transcript profiling of wheat phosphate transporter genes and its association with phosphate allocation in grains', Scientific Reports, 6:39293.

[0097] [5]Muchhal, U.S., J.M. Pardo, and K.G. Raghothama. 1996. 'Phosphate transporters from the higher plant Arabidopsis thaliana', Proceedings of the National Academy of Sciences, 93:10519 - 23.

[0098] [6]Paszkowski, Uta, Scott Kroken, Christophe Roux, and Steven P. Briggs. 2002. 'Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis', Proceedings of the National Academy of Sciences, 99:13324 - 29.

[0099] [7]Sun, Tingting, Mingjun Li, Yun Shao, Lingyan Yu, and Fengwang Ma. 2017. 'Comprehensive Genomic Identification and Expression Analysis of the Phosphate Transporter (PHT) Gene Family in Apple', Frontiers in Plant Science, 8.

[0100] [8]Zhang, Chunxia, Sen Meng, Mingjun Li, and Zhong Zhao. 2016. 'Genomic Identification and Expression Analysis of the Phosphate Transporter Gene Family in Poplar', Frontiers in Plant Science, 7.

[0101] [9]Harrison, Maria J., Gary R. Dewbre, and Jinyuan Liu. 2002. 'A Phosphate Transporter from Medicago truncatula Involved in the Acquisition of Phosphate Released by Arbuscular Mycorrhizal Fungi', The Plant Cell, 14:2413 - 29.

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[10] Liu, Bailin, Shuo Zhao, Xiaofei Wu, Xiaoyu Wang, Yunyou Nan, Dongdong Wang, and Qin Chen. 2017. 'Identification and characterization of phosphate transporter genes in potato', Journal of Biotechnology, 264:17 - 28.

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[11] Aslam, Mehtab Muhammad, Muhammad Waseem, Xu Weifeng, and Muhammad Tahir ul Qamar. 2022. 'Identification and expression analysis of phosphate transporter (PHT) gene family in Lupinus albus cluster root under phosphorus stress', International Journal of Biological Macromolecules, 205: 772 - 81.

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[12] Yang, Jin, Jie Zhou, Hong-Jun Zhou, Mang-Mang Wang, Ming-Ming Liu, Yun-Zhuo Ke, Peng-Feng Li, Jia-Na Li, and Hai Du. 2020. 'Global Survey and Expressions of the Phosphate Transporter Gene Families in Brassica napus and Their Roles in Phosphorus Response', International Journal of Molecular Sciences, 21: 1752.

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[13] Ahmad, Ilyas, Abdul Rawoof, Khushbu Islam, John Momo, and Nirala Ramchiary. 2021. 'Identification and expression analysis of phosphate transporter genes and metabolites in response to phosphate stress in Capsicum annuum', Environmental and Experimental Botany, 190: 104597.

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[14] Murugan, Naveenarani, Vignesh Palanisamy, Mahadevaiah Channappa, Valarmathi Ramanathan, Manimekalai Ramaswamy, Hemaprabha Govindakurup, and Appunu Chinnaswamy. 2022. 'Genome-Wide In Silico Identification, Structural Analysis, Promoter Analysis, and Expression Profiling of PHT Gene Family in Sugarcane Root under Salinity Stress', Sustainability, 14: 15893.

Claims

1. Application of overexpression of wheat TaPHT1;36 gene to promote low phosphorus tolerance in Arabidopsis, characterized in that The CDS sequence of the TaPHT1;36 gene is shown in the sequence table SEQ No.

1.

2. The use of overexpressing wheat TaPHT1;36 gene to promote low-phosphorus tolerance in Arabidopsis according to claim 1, characterized in that The root growth of the Arabidopsis thaliana overexpressing the TaPHT1;36 gene is greater than that of the wild-type Arabidopsis thaliana under a low-phosphorus environment, and the Arabidopsis thaliana exhibits better growth potential.

3. The use of overexpressing wheat TaPHT1;36 gene to promote low-phosphorus tolerance in Arabidopsis according to claim 1, characterized in that The TaPHT1;36 protein functions in the plasma membrane.

4. Wheat low phosphorus tolerance gene TaPHT1;36, characterized by The CDS sequence of TaPHT1;36 is shown in the sequence listing as SEQ ID NO:

1.

5. A method for verifying the function of wheat low-phosphorus tolerance gene TaPHT1;36, characterized in that The method includes: (1) subcellular localization of TaPHT1;36; and (2) verification of the TaPHT1;36 gene in transgenic Arabidopsis under low-phosphorus stress.

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