Application of wheat gene TaPHR1 in improving plant salt tolerance

Abstract

The application discloses application of a wheat gene TaPHR1 in improving plant salt tolerance, and belongs to the technical field of functional genomics. The nucleotide sequence of the wheat gene TaPHR1 is shown in SEQ ID NO:2, and overexpression of the gene can improve the salt stress tolerance of plants, wherein the plants are wheat or Arabidopsis thaliana. The application has the advantages that the wheat gene TaPHR1 is transformed into the model plant Arabidopsis thaliana, and the salt tolerance of the Arabidopsis thaliana can be obviously improved; if the wheat gene TaPHR1 is transformed into plants such as wheat, rice, corn and cauliflower, new germplasm with salt tolerance characteristics can be obtained, and the application has important significance for cultivation of excellent crop new varieties and wide application of the excellent crop new varieties in production.

Description

TECHNICAL FIELD

[0001] The application relates to application of a gene, in particular to application of a wheat gene TaPHR1 in improving plant salt tolerance, and belongs to the technical field of functional genomics. BACKGROUND

[0002] Salt stress is a serious adverse environmental factor, which can cause osmotic stress, cell ion toxicity and oxidative stress on crops, and further causes seed germination inhibition, photosynthetic performance decline, growth and development stagnation and the like, and seriously affects crop yield. Soil salinization has become one of important adverse factors affecting wheat growth and yield increase. Therefore, cultivating salt-tolerant varieties is an effective measure to improve wheat salt tolerance, and further mining and identifying key salt-tolerant genes is a scientific and reasonable biological technology means for cultivating salt-tolerant varieties. SUMMARY

[0003] The application screens and clones a wheat gene TaPHR1 from Chinese spring (CS) wheat, and proves that the gene can effectively improve salt tolerance of Arabidopsis.

[0004] In order to achieve the above target, the application adopts the following technical scheme:

[0005] The application of the wheat gene TaPHR1 in improving plant salt tolerance, the nucleotide sequence of the wheat gene TaPHR1 is shown as SEQ ID NO: 2, and overexpression of the gene can improve the salt stress tolerance of plants, and the plants are wheat or Arabidopsis.

[0006] Preferably, the application comprises the following steps:

[0007] (1) PCR amplification and cloning of the wheat gene TaPHR1;

[0008] (2) connecting the cloned wheat gene TaPHR1 into an expression vector to obtain an overexpression recombinant vector;

[0009] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0010] (4) transforming the overexpression recombinant strain into plants, and screening and obtaining an overexpression strain of the wheat gene TaPHR1.

[0011] More preferably, the expression vector is super1300 (GFP-C), and the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0012] The wheat gene TaPHR1 is transformed into a model plant Arabidopsis thaliana, and the salt tolerance of the Arabidopsis thaliana is obviously improved; the wheat gene TaPHR1 is transformed into wheat, rice, corn, cauliflower and other plants, and new germplasm with salt tolerance characteristics can be obtained, which has important significance for the cultivation of excellent crop new varieties and the wide application of excellent crop new varieties in production. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a structural schematic diagram of the target vector super1300 (GFP-C);

[0014] Figure 2 is a PCR identification diagram of the TaPHR1 overexpression transgenic Arabidopsis thaliana strains (OE1, OE2, OE3);

[0015] Figure 3 is a relative expression amount statistical diagram of the gene TaPHR1 of the TaPHR1 overexpression transgenic Arabidopsis thaliana strains (OE1, OE2, OE3) and the wild type (WT) of the Columbia ecotype Arabidopsis thaliana;

[0016] Figure 4 is a germination rate statistical diagram of the TaPHR1 overexpression transgenic Arabidopsis thaliana seeds (OE1, OE2, OE3) and the wild type Arabidopsis thaliana seeds (WT) of the Columbia ecotype Arabidopsis thaliana under normal conditions and after salt stress treatment;

[0017] Figure 5 is a comparison diagram of the root system growth of each strain under normal conditions (control group);

[0018] Figure 6 is a root length statistical diagram of each strain under normal conditions (control group);

[0019] Figure 7 is a comparison diagram of the root system growth of each strain under salt stress treatment;

[0020] Figure 8 is a root length statistical diagram of each strain under salt stress treatment;

[0021] Figure 9 is a comparison diagram of the plant height of each strain under normal conditions and under salt stress treatment;

[0022] Figure 10 is a plant height statistical diagram of each strain under normal conditions and under salt stress treatment;

[0023] Figure 11 is a comparison diagram of the growth of each strain under normal conditions and under salt stress treatment;

[0024] Figure 12is the fresh weight statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0025] Figure 13 is the dry weight statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0026] Figure 14 is the relative conductivity statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0027] Figure 15 is the SOD activity statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0028] Figure 16 is the POD activity statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0029] Figure 17 is the proline content statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0030] Figure 18 is the H2O2 content statistics chart of each strain under normal conditions and under salt stress treatment conditions;

[0031] Figure 19 is the MDA content statistics chart of each strain under normal conditions and under salt stress treatment conditions. DETAILED DESCRIPTION

[0032] The present application will be described in detail below in conjunction with the accompanying drawings and examples. The reagents, materials and instruments used in the examples can be obtained from commercial channels unless otherwise specified. The terms used in the examples have the meanings generally understood by those of ordinary skill in the art unless otherwise specified. The experimental methods used in the examples are conventional methods unless otherwise specified.

[0033] Example 1: Cloning of wheat gene TaPHR1

[0034] Wheat is an important food crop, and has a wide planting range, and is also a crop with strong adaptability to harsh external environment. The application attempts to screen a salt-tolerant gene from wheat, and finally screens a salt-tolerant gene from Chinese Spring (CS) wheat, wherein the gene number of the gene in the Ensembl Plants database is TraesCS6D02G097000.1, the gene is located on the 6D chromosome of wheat, the ORF (open reading frame) is 1161 bp, the mRNA length is 1352 bp, the nucleotide sequence is shown as SEQ ID NO: 1, the size of the coding product is 386 amino acids, the molecular weight is 42631.63 g / mol, and the isoelectric point is 6.7343. The application names the gene as TaPHR1, and clones the gene by using a cloning technique, and the steps are specifically as follows:

[0035] Taking the water-cultivated Chinese Spring (CS) wheat for 7 days, the total RNA in the leaf is extracted by using the Trizol method, and the cDNA is obtained by reverse transcription with the HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcriptase after purification. Taking the cDNA as a template, the gene TaPHR1 is amplified by using the upstream primer L1 shown in SEQ ID NO: 3 and the downstream primer R1 shown in SEQ ID NO: 4, and the nucleotide sequence of the target gene is determined by sequencing the amplification product.

[0036] The nucleotide sequences of the upstream primer L1 and the downstream primer R1 are as follows:

[0037] The upstream primer L1 is 5'-ATGAGCACACAGAGTGTAATTC-3' (SEQ ID NO: 3);

[0038] The downstream primer R1 is 5'-TTCGGTGTCTGAAATTTTACGC-3' (SEQ ID NO: 4).

[0039] The PCR reaction system for amplifying the gene TaPHR1 is as follows: ddH2O 8 μL, 2×Phanta Max Master Mix (DyePlus) 12.5 μL, the upstream primer L1 1 μL, the downstream primer R1 1 μL, and the cDNA template 2.5 μL.

[0040] The PCR reaction conditions for amplifying the gene TaPHR1 are as follows: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 20 s, and repeating 34 cycles; 72 ℃ extension for 5 min, and finally 4 ℃ preservation.

[0041] The obtained PCR amplification product was electrophoresed in a 1% agarose gel, and the fragment with a sequence length of about 1158 bp was recovered, and then ligated to pCE2 TA / Blunt-Zero by 5 min TM TA / Blunt-Zero Cloning Kit was connected to pCE2 TA / Blunt-Zero, and E. coli DH5α competent cells were transformed, and positive clones were selected for sequencing.

[0042] The sequencing results showed that a DNA fragment with a sequence length of 1158 bp was inserted into the pCE2 TA / Blunt-Zero vector, and the nucleotide sequence of the DNA fragment was shown as SEQ ID NO: 2, which was consistent with the nucleotide sequence of the gene with the gene number TraesCS6D02G097000.1 in the Ensembl Plants database without a stop codon, and it was determined to be the ORF sequence of the wheat gene TaPHR1.

[0043] Example 2: Construction of a recombinant expression vector

[0044] 1. Amplification of the target gene

[0045] XbaI restriction sites were added to the 5' ends of the upstream primer L1 and the downstream primer R1 to obtain the upstream primer L2 and the downstream primer R2. The nucleotide sequences of the upstream primer L2 and the downstream primer R2 are shown as follows, respectively:

[0046] Upstream primer L2:

[0047] 5'-atacaccaaatcgactctagaATGAGCACACAGAGTGTAATTCCTG-3' (SEQ ID NO: 5);

[0048] Downstream primer R2:

[0049] 5'-cataggtacccgggctctagaTTCGGTGTCTGAAATTTTACGCT-3' (SEQ ID NO: 6).

[0050] The positive E. coli liquid containing the pCE2 TA / Blunt-Zero recombinant vector obtained in Example 1 was used as a template for PCR amplification.

[0051] The PCR reaction system was as follows: ddH2O 8 μL, 2×Phanta Max Master Mix (Dye Plus) 12.5 μL, upstream primer L2 1 μL, downstream primer R2 1 μL, and template 2.5 μL.

[0052] PCR reaction program: 95℃ pre-denaturation 3 min; 95℃ denaturation 30 s, 65℃ annealing 30 s, 72℃ extension 30 s, repeat 34 cycles; 72℃ extension 5 min, finally 4℃ preservation.

[0053] PCR product (target gene) with sequence length of about 1158 bp was recovered for subsequent reaction.

[0054] 2. Vector linearization

[0055] QuickCut TM The target vector super1300 (GFP-C) was linearized by XbaI restriction endonuclease, and the structure of the target vector super1300 (GFP-C) is shown in Figure 1 , to obtain a linearized cloning vector.

[0056] Enzyme digestion system: 1 μg of plasmid, 2 μL of 10×QuickCut Green Buffer, 1 μL of QuickCut TM XbaI, and ddH2O to 20 μL.

[0057] Reaction conditions: 37℃ incubation for 30 min.

[0058] 3. Connection of target gene and cloning vector

[0059] The target gene obtained in step 1 was connected to the linearized cloning vector obtained in step 2 by using CloneExpress II One step Cloning Kit (Vayme) to obtain a recombinant expression vector super1300 (GFP-C)-TaPHR1.

[0060] The connection system is as follows: 2 μL of linearized super1300 (GFP-C), 2 μL of target gene, 2 μL of 5×CE II Buffer, 1 μL of Exnase II, and 3 μL of ddH2O.

[0061] Reaction conditions: gently blow the connection system with a pipette to mix, centrifuge briefly to collect the liquid at the bottom of the tube, and react at 37℃ for 30 min.

[0062] 4. Screening and identification of target gene cloning

[0063] The screening and identification steps of target gene cloning are as follows:

[0064] (1) 10 μL of recombinant reaction product obtained in step 3 was used to transform the competent cells of E. coli DH5α, and the cells were incubated at 37℃ for 12 h (the culture medium was LB solid medium with the addition of 50 μg / mL kanamycin sulfate);

[0065] (2) Pick the monoclonal obtained in step (1) and culture at 37°C and 220 rpm for 3h (the culture medium is LB liquid medium, supplemented with 50 μg / mL kanamycin sulfate);

[0066] (3) Use the bacterial solution obtained in step (2) as a template, and perform PCR identification using the upstream primer L2 and the downstream primer R2;

[0067] (4) Send the bacterial solution with positive PCR identification in step (3) to Shengong Biotech (Shanghai) Co., Ltd. for sequencing.

[0068] The sequencing result of the TaPHR1 construct vector is shown in SEQ ID NO: 7. The reference sequence of the TaPHR1 construct vector is shown in SEQ ID NO: 8. The sequencing result shows that the present application obtains a recombinant expression vector containing the target gene (TaPHR1), i.e., the recombinant expression vector super1300 (GFP-C)-TaPHR1, and the recombinant expression vector super1300 (GFP-C)-TaPHR1 is a linearized vector super1300 (GFP-C) to which a TaPHR1 sequence is added at the XbaI site.

[0069] The plasmid extracted from the bacterial solution with correct sequencing is stored at -20°C and used for subsequent Agrobacterium transformation experiments.

[0070] Example 3: Transformation of Arabidopsis thaliana with the wheat gene TaPHR1

[0071] 1. Construction of recombinant Agrobacterium

[0072] The recombinant expression vector super1300 (GFP-C)-TaPHR1 prepared in Example 2 is transformed into competent cells of Agrobacterium tumefaciens GV3101, and the cells are screened and cultured in LB solid medium containing 50 μg / mL kanamycin sulfate and 20 μg / mL rifampicin at 28°C.

[0073] Pick the positive monoclonal and culture at 28°C and 220 rpm for 12h (the culture medium is LB liquid medium, supplemented with 50 μg / mL kanamycin sulfate and 20 μg / mL rifampicin).

[0074] Absorb 1 μL of the bacterial solution and perform PCR identification using the upstream primer L2 and the downstream primer R2.

[0075] The bacterial solution with positive PCR identification is the recombinant Agrobacterium tumefaciens containing the recombinant expression vector super1300 (GFP-C)-TaPHR1, and the recombinant Agrobacterium tumefaciens is named GV3101 / super1300 (GFP-C)-TaPHR1.

[0076] 2. Obtaining TaPHR1 overexpression transgenic Arabidopsis

[0077] The method for obtaining TaPHR1 overexpression transgenic Arabidopsis is as follows:

[0078] (1) Plant preparation before infection: Wild-type seeds of Columbia ecotype Arabidopsis were vernalized at 4°C for 72 h, sowed in MS solid medium, and cultured in a culture room at 22°C, 16 h light / 8 h darkness, and humidity of 60%-70%, and transplanted into planting pots containing mixed culture medium (nutrient soil and vermiculite mixed at a mass ratio of 3:1) when two true leaves were grown. After the plants flowered, the top of the main branch was cut off (to promote the development of lateral branches), and sufficient water was poured the day before infection;

[0079] (2) Activation of Agrobacterium: 1 mL of recombinant Agrobacterium tumefaciens GV3101 / super1300 (GFP-C)-TaPHR1 bacterial solution was poured into a sterilized 250 mL conical flask in a clean bench, 150 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and 20 μg / mL rifampicin was added, and the mixture was cultured in a 28°C full-temperature shaker for 16 h, until the OD 600 reached 0.8. The bacterial solution was divided into three 50 mL centrifuge tubes, centrifuged at 8000 rpm for 10 min, and the supernatant was discarded, and the bacterial cells were retained;

[0080] (3) Preparation of resuspension solution: 2.5 g of sucrose was dissolved in 50 mL of distilled water, and 10 μL of Silwet-L77 was added;

[0081] (4) The resuspension solution was added to the activated Agrobacterium bacterial cells in batches, 10 mL each time, and the OD 600 was measured until it reached 0.8, and the resuspended bacterial solution was obtained;

[0082] (5) Dipping flower method to infect Arabidopsis: at 9 o'clock in the morning (when flowering is most vigorous, which is conducive to infection), 1 mL of mixed resuspended bacterial solution was dropped on the inflorescences of Arabidopsis using a pipette gun head each time, and the inflorescences were immersed after the whole inflorescences were immersed, and then dark treatment was performed for 1 d, and water was poured during light, and secondary infection was performed after one week;

[0083] (6) Harvesting seeds of infected Arabidopsis plants: the Arabidopsis plants treated by secondary immersion were cultured to fruiting using a conventional method, and mature T0 generation seeds were harvested;

[0084] (7) Culturing T0 positive seedlings: the T0 seeds are sterilized and then evenly spread on MS solid medium containing 100 μL / 100 mL hygromycin, and the seedlings are cultured until the true leaves of the seedlings grow, and the true leaves of some seedlings grow healthily and the roots grow obviously longer into the medium (about 14 days), and the T0 positive seedlings are obtained. The T0 positive seedlings are moved to a planting pot containing mixed culture medium (nutrient soil and vermiculite mixed in a mass ratio of 3:1) for culture, and the mature T1 seeds are harvested from a single plant;

[0085] (8) The T1 seeds are cultured into T1 lines containing hygromycin resistance according to the method described in step (7), and if the ratio of positive seedlings to dead seedlings is about 3:1, it is a single copy line. Then the plants in the planting pot are cultured to bear fruit by using a conventional method, and the mature T2 seeds borne by each single plant in the T1 line are harvested;

[0086] (9) 10 T2 seeds are randomly taken and subjected to hygromycin resistance screening according to the same method, and no hygromycin resistance separation is obtained, which is a homozygous line. Finally, three T2 homozygous lines of TaPHR1 overexpression transgenic Arabidopsis are obtained.

[0087] (10) The seeds of the T2 homozygous lines of TaPHR1 overexpression transgenic Arabidopsis (T3 generation, denoted as OE1, OE2 and OE3, respectively) are harvested for the next step of phenotype identification and analysis.

[0088] T0 generation represents the seeds and plants grown from the transformed generation; T1 generation represents the seeds and plants grown from the selfing of T0 generation; T2 generation represents the seeds and plants grown from the selfing of T1 generation; T3 generation represents the seeds and plants grown from the selfing of T2 generation; line represents the seeds or plant population produced by the selfing of the same plant of the previous generation.

[0089] Example 4: PCR identification of TaPHR1 overexpression transgenic Arabidopsis

[0090] The leaves of the three T2 homozygous lines of TaPHR1 overexpression transgenic Arabidopsis (OE1, OE2 and OE3) obtained in Example 3 are taken, the DNA in the leaves is extracted by CTAB method, and PCR amplification is performed by using the upstream primer L1 and the downstream primer R1.

[0091] The electrophoresis result of the PCR product is shown in Figure 2 The PCR identification result shows that the line OE1, the line OE2 and the line OE3 are all TaPHR1 overexpression transgenic Arabidopsis lines.

[0092] Example 5: Real-time fluorescent quantitative PCR detection of TaPHR1 overexpression transgenic Arabidopsis

[0093] The roots of T3 generation homozygous transgenic lines (OE1, OE2, OE3) and the roots of wild-type lines of Columbia ecotype Arabidopsis thaliana (denoted as line WT) were photographed, and then total RNA was extracted by Trizol method, and cDNA was obtained by reverse transcription with HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcriptase after purification. The Arabidopsis AtActin gene was used as an internal reference gene, and the wheat gene TaPHR1 was detected by real-time fluorescent quantitative PCR using qRT-PCR primers.

[0094] The real-time fluorescent quantitative PCR instrument model was ABI QuantStudio3, ChamQTMSYBR Color qPCR Master Mix reagent was used, the expression level of Arabidopsis AtActin gene was used as an internal reference, the comparative threshold method was used for quantitative analysis of real-time fluorescent quantitative PCR results, the fluorescence domain value was set, and the cycle number Ct value under the fluorescence domain value was determined. According to the Ct value, the C value was calculated, C=2 -△Ct ,△Ct=Ct 目的基因 -Ct 内参基因 , and the average value of the C values of three repetitions was calculated as the relative expression amount of the target gene.

[0095] The reaction system was: 2×ChamQ SYBR Color qPCR Master Mix 5μL, 50×ROX Reference Dye II 0.2μL, upstream primer (10μM) 0.2μL, downstream primer (10μM) 0.2μL, first-strand cDNA solution diluted 3 times 0.5μL, ddH2O 3.9μL.

[0096] The qRT-PCR amplification program used a three-step method:

[0097] (1) Pre-denaturation: 95℃ for 3min;

[0098] (2) Cycle reaction: 95℃ for 15s, 59℃ for 20s, a total of 40 cycles;

[0099] (3) Melting curve: 95℃ for 15s, 60℃ for 60s, 95℃ for 15s.

[0100] The primer sequences used for real-time fluorescent quantitative PCR in this step are as follows:

[0101] Actin-L: 5'-ATGAGCCAGTACGATCACAA-3' (SEQ ID NO: 9);

[0102] Actin-R: 5'-TTAGAAGCAAATGTCCAGGG-3' (SEQ ID NO: 10);

[0103] qRT-L: 5'-ATGGCGGAAACTCTACGG-3' (SEQ ID NO: 11);

[0104] qRT-R: 5'-CGGTGTCTGAAATTTTACGC-3' (SEQ ID NO: 12).

[0105] The qRT-PCR amplification results are shown below. Figure 3 .Depend on Figure 3 It can be seen that the expression level of exogenous gene TaPHR1 in lines OE1, OE2 and OE3 is significantly higher than that in line WT, that is, the expression level of exogenous gene TaPHR1 in transgenic Arabidopsis plants is significantly higher than that in wild-type plants.

[0106] Example 6: Phenotypic identification of TaPHR1 overexpressing transgenic Arabidopsis plants

[0107] 1. Phenotypic identification of germination rate during germination of TaPHR1-overexpressing transgenic Arabidopsis lines

[0108] Salt stress treatment medium: Add 39.9g of 1 / 2MS solid dry powder to two Erlenmeyer flasks containing 1L of distilled water, then add 8.775g of NaCl and 11.7g of NaCl respectively, stir thoroughly, adjust the pH to 6.0, sterilize at 121℃ for 15min, and pour the medium into round petri dishes when it cools to 40-50℃ to prepare salt stress treatment medium with a NaCl concentration of 150mM (150mM NaCl group) and salt stress treatment medium with a NaCl concentration of 200mM (200mM NaCl group). The control group (CK group) did not add NaCl.

[0109] The TaPHR1 overexpression transgenic Arabidopsis seeds (OE1, OE2, OE3) obtained in Example 3 and the wild-type seeds (WT) of Colombian ecotype Arabidopsis were spread evenly on salt stress treatment medium containing 150 mM NaCl and 200 mM NaCl, respectively, and a control group (CK group) was set up. The germination of seeds in each group was observed, and the germination rate was statistically analyzed after 10 days.

[0110] The germination rates of TaPHR1-overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) and wild-type seeds (WT) of the Colombian ecotype Arabidopsis thaliana under salt stress treatment are statistically shown in the table below. Figure 4 .

[0111] From Figure 4 It can be seen that, under normal conditions (CK group), the germination rate of TaPHR1 overexpression transgenic Arabidopsis seeds (OE1, OE2, OE3) had no significant difference compared with that of wild-type Arabidopsis seeds (WT); under low concentration salt stress (150 mM NaCl group), the germination rate of TaPHR1 overexpression transgenic Arabidopsis seeds (OE1, OE2, OE3) was significantly increased by 11.36%, 12.12% and 13.26% respectively compared with that of wild-type Arabidopsis seeds (WT); under high concentration salt stress (200 mM NaCl group), the germination rate of TaPHR1 overexpression transgenic Arabidopsis seeds (OE1, OE2, OE3) was significantly increased by 5.88%, 10.59% and 5.10% respectively compared with that of wild-type Arabidopsis seeds (WT).

[0112] The above results show that the transgenic lines have higher tolerance to salt stress during seed germination.

[0113] 2. Phenotypic identification of TaPHR1 overexpression transgenic Arabidopsis lines at seedling stage

[0114] Square plate salt stress treatment medium: 39.9 g of 1 / 2MS solid dry powder and 8.775 g of NaCl were added to 1 L of distilled water, fully stirred and mixed, the pH value was adjusted to 6.0, 121℃ high temperature sterilization for 15 min, when the medium was cooled to 40-50℃, pour the plate (square plate), and the square plate salt stress treatment medium (square plate salt stress group) with a NaCl concentration of 150 mM was prepared. The control group (CK group) did not add NaCl.

[0115] The TaPHR1 overexpression transgenic Arabidopsis lines (OE1, OE2, OE3) and the wild-type strain of Columbia ecotype Arabidopsis (WT) were cultured in a incubator under normal conditions for 5 days, then transferred to square plate salt stress treatment medium added with NaCl for vertical culture (OE1-NaCl group, OE2-NaCl group, OE3-NaCl group, WT-NaCl group), and the control group (OE1-CK group, OE2-CK group, OE3-CK group, WT-CK group) was set, and after 10 days of culture, the plants were photographed and the root length was statistically analyzed.

[0116] The root growth of the control group of each strain is shown in Figure 5 , and the root length statistical results are shown in Figure 6 .

[0117] From Figure 5 and Figure 6It can be seen that, under normal conditions, the root length of TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) has no significant difference compared with that of wild type Arabidopsis thaliana line (WT).

[0118] The root growth of each line of square plate salt stress group is shown in Figure 7 , and the root length statistical results are shown in Figure 8 .

[0119] It can be seen from Figure 7 and Figure 8 that, under salt stress, the root length of TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is significantly increased by 8.36%, 15.07% and 22.24% respectively compared with that of wild type Arabidopsis thaliana line (WT).

[0120] 3. Phenotypic identification of TaPHR1 overexpression transgenic Arabidopsis thaliana lines at the adult stage

[0121] The TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and wild type Arabidopsis thaliana line (WT) of Columbia ecotype were cultured in a round culture dish under normal conditions for 7 days, then transferred to a plastic pot containing mixed culture medium (nutrient soil and vermiculite mixed at a mass ratio of 3:1) and covered with plastic wrap for 5 days, then the plastic wrap was removed and cultured under normal conditions for 10 days, followed by NaCl (200 mM) stress treatment (NaCl group) and normal treatment (CK group). On the 12th day of NaCl stress treatment, the plants were photographed and the plant height was statistically analyzed.

[0122] The growth of each group of plants is shown in Figure 9 , and the plant height statistical results are shown in Figure 10 .

[0123] It can be seen from Figure 9 and Figure 10 that, under normal conditions, the plant height of TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) has no significant difference compared with that of wild type Arabidopsis thaliana line (WT); under salt stress conditions, the plant height of TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is significantly increased by 24.16%, 15.73% and 16.85% respectively compared with that of wild type Arabidopsis thaliana line (WT).

[0124] The TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and the wild type line of Columbia ecotype Arabidopsis thaliana (WT) are cultured in a round culture dish under normal conditions for 7 days, then are transferred to a plastic pot containing mixed culture medium (nutrient soil and vermiculite mixed in a mass ratio of 3:1) and cultured for 5 days with a preservative film, then the film is removed, and the plants are cultured under normal conditions for 10 days, then are subjected to NaCl (300 mM) stress treatment (NaCl group) and normal treatment (CK group), and during this period, the bolting is cut off, and only the rosette leaves are reserved. On the 12th day of NaCl stress treatment, the plants are photographed, and the fresh weight, dry weight, relative conductivity, SOD and POD activities, proline content, hydrogen peroxide and MDA contents of the plants in each group are determined.

[0125] The growth conditions of the plants in each group are shown in Figure 11 . It can be seen from Figure 11 that under normal conditions, the growth conditions of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) are not significantly different from those of the wild type line of Arabidopsis thaliana (WT); under salt stress conditions, the growth conditions of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) are better than those of the wild type line of Arabidopsis thaliana (WT).

[0126] The fresh weight statistical results of the plants in each group are shown in Figure 12 . It can be seen from Figure 12 that under normal conditions, the fresh weight of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is not significantly different from that of the wild type line of Arabidopsis thaliana (WT); under salt stress conditions, the fresh weight of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is significantly increased by 109.66%, 127.52% and 124.52% respectively compared with that of the wild type line of Arabidopsis thaliana (WT).

[0127] The dry weight statistical results of the plants in each group are shown in Figure 13 . It can be seen from Figure 13 that under normal conditions, the dry weight of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is not significantly different from that of the wild type line of Arabidopsis thaliana (WT); under salt stress conditions, the dry weight of the TaPHR1 overexpression transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) shows an increasing trend compared with that of the wild type line of Arabidopsis thaliana (WT), and is significantly increased by 59.56%, 57.44% and 58.80% respectively.

[0128] The determination results of the plant height, fresh weight and dry weight show that the TaPHR1 overexpression transgenic Arabidopsis thaliana lines have higher tolerance to salt stress.

[0129] The statistical results of the relative electrical conductivity of each group of plants are shown in the figure. Figure 14 .Depend on Figure 14 It can be seen that under normal conditions, the relative electrical conductivity of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of the wild-type Arabidopsis thaliana line (WT); under salt stress, the relative electrical conductivity of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly lower than that of the wild-type Arabidopsis thaliana line (WT), decreasing by 48.40%, 48.29%, and 50.13%, respectively.

[0130] When subjected to salt stress, cells are damaged, leading to electrolyte leakage and impairing vital functions, manifested as an increase in relative conductivity. The results showed that under salt stress, the TaPHR1-overexpressing transgenic Arabidopsis thaliana lines suffered less cell damage and less electrolyte leakage than wild-type Arabidopsis thaliana lines, indicating that the former exhibited stronger salt tolerance.

[0131] The results of SOD activity assays for each group of plants are shown below. Figure 15 .Depend on Figure 15 It can be seen that under normal conditions, the SOD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the SOD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 67.82%, 94.47%, and 85.35% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0132] The results of POD activity assays for each group of plants are shown below. Figure 16 .Depend on Figure 16 It can be seen that under normal conditions, the POD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the POD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 104.63%, 117.97%, and 129.38% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0133] SOD and POD are the main components of the enzymatic antioxidant system that scavenge reactive oxygen species (ROS). Under salt stress, the SOD and POD activities of TaPHR1 overexpressing transgenic Arabidopsis lines (OE1, OE2, OE3) were significantly higher than those of wild-type Arabidopsis line (WT), indicating that the former has a stronger ability to scavenge ROS and can effectively reduce membrane lipid peroxidation levels, thereby mitigating the harm of salt stress to plants.

[0134] The results of proline content determination in each group of plants are shown below. Figure 17 .Depend on Figure 17 It can be seen that under normal conditions, the proline content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the proline content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 120.08%, 85.92%, and 90.08% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0135] Proline is an important osmotic regulator. Under salt stress, the proline activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly higher than that of wild-type Arabidopsis thaliana line (WT). This indicates that the former can more effectively reduce cell osmotic potential, enhance cell water absorption capacity, maintain cell volume stability under salt stress, protect protein and cell membrane structure, and thus help the plant adapt to the salt stress environment.

[0136] The results of H2O2 content determination for each group of plants are shown below. Figure 18 .Depend on Figure 18 It can be seen that under normal conditions, the H2O2 content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the H2O2 content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly reduced by 56.71%, 60.88%, and 60.29% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0137] The results of MDA content determination for each group of plants are shown below. Figure 19 .Depend on Figure 19 It can be seen that under normal conditions, the MDA content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the MDA content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly reduced by 61.09%, 60.67%, and 50.04% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0138] H2O2 is a reactive oxygen species (ROS) and its over-concentration can cause oxidative damage to plant cells, affecting their normal growth and development. MDA is the final product of membrane lipid peroxidation, and its increased content mainly harms plants in terms of cell membrane system damage, inhibited photosynthesis and decreased antioxidant capacity. The contents of H2O2 and MDA in the TaPHR1 overexpression transgenic Arabidopsis lines (OE1, OE2, OE3) under salt stress were significantly lower than those in the wild-type Arabidopsis line (WT), indicating that the former enhanced the antioxidant capacity of Arabidopsis, reduced oxidative stress damage, and thus reduced the harm of salt stress to the plants.

[0139] In summary, the wheat gene TaPHR1 transformed into the model plant Arabidopsis can significantly improve the salt tolerance of Arabidopsis.

[0140] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to enumerate all the embodiments here. Any obvious changes or modifications derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. The application of the wheat gene TaPHR1 in improving plant salt tolerance, characterized in that, The nucleotide sequence of the wheat gene TaPHR1 is shown in SEQ ID NO:

2. Overexpression of this gene can improve the plant's tolerance to salt stress. The plant is wheat or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The application includes the following steps: (1) PCR amplification and cloning of the wheat gene TaPHR1; (2) The cloned wheat gene TaPHR1 was ligated into an expression vector to obtain an overexpression recombinant vector; (3) The overexpression recombinant vector was transformed into Agrobacterium to obtain the overexpression recombinant strain; (4) Transform the overexpressing recombinant strain into plants, screen and obtain wheat gene TaPHR1 overexpressing lines.

3. The application according to claim 2, characterized in that, The expression vector is super1300 (GFP-C).

4. The application according to claim 2, characterized in that, The Agrobacterium is Agrobacterium tumefaciens GV3101.

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