Application of TaTCP7-D gene in plant salt tolerance

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses application of a TaTCP7-D gene in plant salt tolerance, the nucleotide sequence of the TaTCP7-D gene is shown as SEQ ID NO.1, and the amino acid sequence of TaTCP7-D protein coded by the TaTCP7-D gene is shown as SEQ ID NO.2. The invention further discloses a method for preparing the TaTCP7-D gene. The TaTCP7-D gene is over-expressed through a genetic engineering means, so that the salt tolerance of arabidopsis thaliana and wheat is improved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, specifically to TaTCP7-D Application of genes in plant salt tolerance. Background Art

[0002] Salt stress can inhibit plant growth, affect plant root development, and significantly affect plant physiological metabolism. Triticumaestivum Wheat (Triticum aestivum L.) is an annual or biennial herbaceous plant in the genus Triticum, Poaceae, and one of the world's three major grain crops. Salt stress severely affects wheat yield and quality. With the rapid development of genetic engineering to enhance plant abiotic stress tolerance, transcription factor genes may become a practical and effective target for wheat stress tolerance. It is necessary to explore new approaches to improve plant salt tolerance. Summary of the Invention

[0003] To develop a new approach to improve plant salt tolerance, the present invention provides TaTCP7-D The present invention uses gene to overexpress in plant salt tolerance by genetic engineering. TaTCP7-D genes, thereby improving salt tolerance in Arabidopsis and wheat.

[0004] The present invention provides TaTCP7-D Application of genes in plant salt tolerance, the TaTCP7-D The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the TaTCP7-D protein encoded by the gene is shown in SEQ ID NO.2.

[0005] The present invention overexpresses by genetic engineering TaTCP7-D By testing salt-tolerant wheat varieties, we can determine the TaTCP7-D Genes are highly expressed, indicating overexpression TaTCP7-D Genes are beneficial for improving salt tolerance in Arabidopsis and wheat.

[0006] Furthermore, overexpression in plants through genetic engineering TaTCP7-D Genes improve plant salt tolerance.

[0007] Furthermore, the genetic engineering method is to construct an overexpression vector to transform the plant to obtain the overexpression TaTCP7-D Genetically modified plants.

[0008] Furthermore, the overexpression vector is constructed by: TaTCP7-D Gene ligated into pSuper1300 vector Pst I. Kpn I site is obtained.

[0009] Furthermore, the plant is wheat or Arabidopsis thaliana.

[0010] Furthermore, the TaTCP7-D Gene overexpression was used to increase POD activity, proline, and soluble sugar content in Arabidopsis lines under salt stress.

[0011] Furthermore, the TaTCP7-D Gene overexpression was used to reduce MDA, H2O2, and O in Arabidopsis lines under salt stress. 2- content.

[0012] The present invention also provides a method for improving plant salt tolerance, comprising the following steps: Constructed according to the method of claim 4 TaTCP7-D Gene overexpression vector; after the overexpression vector is transformed into Agrobacterium, it infects the plant, making the plant TaTCP7-D Genes are overexpressed, thereby improving plant salt tolerance; The plant is wheat or Arabidopsis thaliana.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention overexpresses by genetic engineering TaTCP7-D By testing salt-tolerant wheat varieties, we can determine the TaTCP7-D Genes are highly expressed, indicating overexpression TaTCP7-D Genes are beneficial for improving salt tolerance in Arabidopsis and wheat.

[0014] The present invention has carried out in-depth and systematic research on stress resistance function identification and transgenic Arabidopsis thaliana. TaTCP7-D The molecular basis for regulating salt tolerance in wheat TaTCP7-D It provides a theoretical basis for improving wheat salt tolerance and offers genetic resources for wheat stress resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] 图1 The effect of different concentrations of NaCl simulating salt stress on transgenic Arabidopsis thaliana; In the figure, A shows the effect of 0 mM NaCl-simulated salt stress on transgenic Arabidopsis; B shows the effect of 100 mM NaCl-simulated salt stress on transgenic Arabidopsis; C is the effect of 125 mM NaCl simulated salt stress on transgenic Arabidopsis thaliana.

[0017] 图2 This is a quantitative diagram of the root length of transgenic Arabidopsis thaliana, showing the effect of different concentrations of NaCl simulating salt stress on the root length.

[0018] 图3 Effects of salt treatment on enzyme activities of wild-type (WT) and transgenic Arabidopsis thaliana; In the figure, A shows the effect of salt treatment on peroxidase (POD) activity in leaves of wild-type (WT) and transgenic Arabidopsis thaliana; B Effects of salt treatment on proline (PRO) activity in leaves of wild-type (WT) and transgenic Arabidopsis thaliana; Effects of C salt treatment on soluble sugar (SS) activity in leaves of wild-type (WT) and transgenic Arabidopsis thaliana; D Effects of salt treatment on hydrogen peroxide (H2O2) activity in wild-type (WT) and transgenic Arabidopsis leaves; Effects of E salt treatment on malondialdehyde (MDA) activity in leaves of wild-type (WT) and transgenic Arabidopsis thaliana; Superoxide anion (O) in wild type (WT) and transgenic Arabidopsis leaves after F salt treatment 2- ) activity.

[0019] 图4 The salt stress treatment of the aboveground part, underground part and various parts of Qingmai No. 6 seedlings TaTCP7-D relative expression level; In the figure, A shows the leaves and roots of Qingmai No. 6 at different time periods under salt stress during the seedling stage. TaTCP7-D relative expression level; B shows the aboveground and underground parts of Qingmai 6 treated with salt stress at the seedling stage TaTCP7-D relative expression level; C is the salt stress treatment of Qingmai No. 6 at the seedling stage, and the salt stress treatment of roots and stems at the jointing stage. TaTCP7-D relative expression level; D is the root, stem and husk of Qingmai 6 treated with salt stress at seedling stage TaTCP7-D Relative expression level. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0021] 实施例1: TaTCP7-D 基因在小麦耐盐性中的应用。

[0022] TaTCP7-D 基因的核苷酸序列如SEQ ID NO.1所示,其编码的TaTCP7-D蛋白的氨基酸序列如SEQ ID NO.2所示。

[0023] SEQ ID NO.1: ATGACCTCCCACGACATCGCCGCCGCCTCCAGCCCTTTCCACGTCTACCAGCAGCACCTACCGATGACGGTGGCGCCCGCCGCCGCCTCCGCCGACGCTGGCTTGGCTCCGGCGGCGAGCAAGAAGGCTGGTGGCAGCGTGAAGGACCGGCACAGCAAGGTGAACGGGCGCGGTCGGCGCGTGCGCATGCCGATCGTGTGCGCCGCGCGCGTGTTCCAGCTCACGCGCGAGCTGGGGCTCAAGTCCGACGGCCAGACCATCGAGTGGCTGCTCCGGCAGGCGGAGCCCTCGATCCTCGCCGCCACGGGGACCGGCACCACCCCCGCCGCCTTCGTCTCCTCCTCCGCGCCATCCACCTCCTCCTCCTCCTCCTTCTCGTACCCACACACCCTCCTCGGCAAGCGCCCGCGCGAGGAGGAGAACGAAGCCTTCGGCGCGGGGGCTACAACGTCGGCCTTCTGGGCGGCGCTGCAGGCGCCGCCGCGGCAGGACACGTGGGGGTTCTCGCCGCTGGAGGCGCAGGCCGCGTACATGCCGATGGCGCAGGTCCACCACCACCACCTCAACCTTGTCGCCGCGCTCTCCGGCGCCGCTCGGCGCACCGAGGAGGAGACCCGGTGA。

[0024] SEQ ID NO.2: MTSHDIAAASSPFHVYQQHLPMTVAPAAASADAGLAPAASKKAGGSVKDRHSKVNGRGRRVRMPIVCAARVFQLTRELGLKSDGQTIEWLLRQAEPSILAATGTGTTPAAFVSSSAPSTSSSSSFSYPHTLLGKRPREEENEAFGAGATTSAFWAALQAPPRQDTWGFSPLEAQAAYMPMAQVHHHHLNLVAALSGAARRTEETR.

[0025] 1. RNA extraction and cDNA acquisition 1. RNA Extraction Using Qingmai No. 6 as the material, when the wheat grows to the two-leaf and one-heart stage, 1 g of wheat leaves are removed with tweezers, and RNA is extracted using a plant RNA extraction kit purchased from Aikerui Biological Company. The specific extraction method is as follows.

[0026] (1) Lysis of plant tissue Transfer the plant tissue sample (wheat leaf) to a liquid nitrogen pre-cooled mortar and grind the plant tissue with a pestle. During the grinding process, liquid nitrogen needs to be continuously added to the mortar until it is ground into powder.

[0027] Transfer 80 mg of the powdered sample into a 1.5 ml centrifuge tube (RNase-free) containing 500 μl of Buffer RLS (make sure 50× DTT Solution has been added to Buffer RLS before use). Immediately mix by high-speed vortexing or repeatedly pipetting until the sample is fully lysed (no obvious precipitation) to obtain the sample lysate.

[0028] The sample lysate was allowed to stand at room temperature for 2 minutes and then centrifuged at 4°C, 12,000 rpm for 5 minutes.

[0029] After centrifugation, carefully pipette the supernatant into a new 1.5 ml centrifuge tube (RNase-free).

[0030] (2) Purification Add 1 / 2 volume of anhydrous ethanol to the supernatant and mix well with a pipette. If obvious sticky matter or precipitation appears, pipette several times to break up the precipitation to obtain a mixed solution.

[0031] Transfer the entire mixture to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 2 minutes at room temperature, and discard the filtrate (if the volume of the mixture is greater than 700 μl, add in batches).

[0032] Add 600 μl of Buffer RWA to the Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the filtrate.

[0033] Add 750 μl of Buffer RWB (Buffer RWB has been added with the specified volume of 100% ethanol) to the Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the filtrate.

[0034] DNase I digestion: ① Prepare DNase I reaction solution according to the table below and mix thoroughly. Add 50 μl of DNase I reaction solution to the center of the Plant RNA Mini Column membrane and let it stand at room temperature for 15 minutes.

[0035] Table 1 DNase I reaction solution formula ② Add 350 μl of Buffer RWB to the center of the Plant RNA Mini Column membrane. Centrifuge at 12,000 rpm for 1 minute at room temperature and discard the filtrate.

[0036] Add 750 μl of Buffer RWB to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0037] Place the Plant RNA Mini Column adsorption column on a new 2.0 ml Collection Tube and centrifuge at 12,000 rpm at room temperature for 2 minutes.

[0038] Place the Plant RNA Mini Column onto a new RNase-Free Tube. Add 150 μl of RNase-Free Water to the center of the column membrane. Let stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 2 minutes to elute the RNA, which can be used for subsequent experiments. If not immediately proceeding with further experiments, store the dissolved RNA at -80°C.

[0039] 2. Reverse transcription to obtain cDNA (1) Prepare the RNA template solution on ice according to Table 2 below, place it in a PCR instrument for denaturation and annealing reaction, and obtain the denatured and annealed reaction solution.

[0040] Table 2 Reaction system (10 μL) Denaturation, annealing reaction conditions: 65°C for 5 min, 4°C for preservation.

[0041] (2) According to Table 3, the reverse transcription reaction system was prepared, and cDNA was synthesized Table 3 Reverse transcription reaction system Synthesis of cDNA reaction conditions: 30°C, 10 min; 42°C, 50 min; 95°C, 5 min; 4°C preservation.

[0042] (3) The cDNA solution obtained above can be directly used for subsequent PCR amplification, and the amount of reaction solution should not exceed 1 / 10 of the PCR reaction system.

[0043] Two, TaTCP7-D Gene cloning The cDNA of Qingmai No. 6 leaf obtained above was used as a template, and the TaTCP7-D-F primer shown in SEQ ID NO. 3 and the TaTCP7-D-R primer shown in SEQ ID NO. 4 were used as primers for PCR amplification. The gene sequence was cloned by using the Nuzyne P525 polymerase. TaTCP7-D The PCR amplification reaction system is shown in Table 4, and the PCR amplification reaction program is shown in Table 5. After amplification, agarose gel electrophoresis detection was performed. TaTCP7-D

[0044] SEQ ID NO. 3: 5'-ACTCCCTCCAACGTACAGCGA-3'.

[0045] SEQ ID NO. 4: 5'-AGTACACCCAGGATCCTAATA-3'.

[0046] Table 4 PCR amplification reaction system Table 5 PCR amplification reaction program Gel recovery: The gene structure integrity was detected by 1% agarose gel electrophoresis, 2000bp maker was used, and electrophoresis was performed at 120 volt for 15 minutes. The correct fragment in the electrophoresis band was selected for gel recovery. The obtained amplification product was the target gene. The specific method referred to the Nuzyne DC301 kit method.

[0047] Intermediate vector ligation: the target gene was ligated with pEASY-Blunt Zero (Beijing Quanshi Gold), to obtain the recombinant vector pEASY-TaTCP7-D.​

[0048] Transform competent E. coli cells with the recombinant vector pEASY-TaTCP7-D and select a single colony: In a sterile laminar flow hood, select a single colony of excellent appearance and culture it in 1 mL of LB liquid medium (50 mg / L AMP) at 37°C for 3 hours to obtain a colony mixture. Use 1 μl of the colony mixture as a template for PCR analysis.

[0049] Sequencing: PCR-verified bacterial cultures with the correct bands are sent to a biotechnology company for gene sequence identification. Once identified, the cultures with the correct sequence are retained for subsequent experiments.

[0050] Plasmid extraction: A culture of the correctly sequenced bacterial strain was cultured with LB liquid medium at a volume ratio of 1:100 ml. Incubate at 37°C, 200 rpm for 12 hours before plasmid extraction. Follow the instructions in the Novozymes DC201 kit. Store the extracted plasmid at -20°C for subsequent experiments.

[0051] three, TaTCP7-D Construction of gene recombinant expression vector The Super1300-TaTCP7-D expression vector was constructed, and the homology arm primers were designed based on the restriction enzyme cutting site selected according to the multiple cloning site of the pSuper1300 vector: the Super1300-TaTCP7-DF primer shown in SEQ ID NO.5 and the Super1300-TaTCP7-DR primer shown in SEQ ID NO:6. The high-fidelity enzyme (KOD-Plus-Neo) was used for amplification. TaTCP7-D gene, select enzyme cutting site ( Pst I and Kpn I) Double-enzyme digestion of the pSuper1300 vector was performed to construct the Super1300-TaTCP7-D expression vector. The specific reaction system is shown in the table below.

[0052] SEQ ID NO. 5: gactctagaaagcttctgcagATGACCTCCCACGACATCGC.

[0053] SEQ ID NO: 6: gcccttgctcaccatggtaccTCACCGGGTCTCCTCCTCGGT.

[0054] Table 6 pSuper1300 vector enzyme digestion system Table 7 TaTCP7-D amplification system 4. Agrobacterium Transformation (1) The successfully constructed pSuper1300-TaTCP7-D recombinant expression vector was introduced into Agrobacterium as follows: 1. Thaw the competent Agrobacterium cells stored at -80°C on ice. Under sterile conditions, add 10 μL of pSuper1300-TaTCP7-D recombinant expression vector plasmid DNA to the competent cells, mix gently, and let stand on ice for 5 minutes.

[0055] 2. Quickly freeze the competent cells in liquid nitrogen for 5 minutes, then quickly place them in a 37°C water bath for 5 minutes without shaking the water surface, and then place the centrifuge tube in an ice water bath for 5 minutes.

[0056] 3. Under sterile conditions, add 800 mL of LB liquid medium and culture at 28°C with shaking for 3 hours to allow the bacteria to recover.

[0057] 4. Centrifuge at 5000 rpm for 1 min to collect the bacteria, retain about 100 μL of the supernatant and gently pipette to mix, then spread on LB solid plates containing Kan and Rif resistance antibiotics and culture for 48 h.

[0058] 5. After PCR identification of positive colonies, the correct Agrobacterium culture solution was amplified and shaken for later use.

[0059] (2) Agrobacterium inflorescence infection (1) Preparation of bacterial suspension: Inoculate the above-mentioned Agrobacterium bacterial suspension into 10 mL of LB liquid culture medium containing Kan and Rif resistance antibiotics, and culture in a shaker at 220 rpm at 28°C for 12 h to obtain bacterial suspension.

[0060] (2) Select 1 mL of bacterial solution and inoculate it into 100 mL of LB liquid medium containing Rif and Kan resistance antibiotics, culture for 18 hours, and proliferate to OD 600 The mixture was centrifuged at 5000 rpm for 10 min, the supernatant was removed, the Agrobacterium that had settled was collected, and the Agrobacterium that had settled was resuspended in the Arabidopsis transformation infiltration solution, and then adjusted to OD 600 The formula of the Arabidopsis thaliana infiltration solution is shown in Table 8.

[0061] Table 8 Arabidopsis infiltration solution formula The flower buds of Arabidopsis thaliana were briefly immersed in the infiltration solution for 12 seconds. During the process, the Agrobacterium infiltration solution was prevented from contacting the soil to avoid adverse effects on the tender roots of Arabidopsis thaliana. After infection, the Arabidopsis thaliana was placed in a dark environment for 48 hours and then placed in a light incubator for further cultivation. The incubator was maintained at 23°C, 16 h / 8 h, light / dark. After the Arabidopsis thaliana matured, T0 generation seeds were collected from individual plants.

[0062] 5. Homologous breeding of transgenic Arabidopsis After the T0 generation seeds are disinfected, they are evenly sown with a pipette tip on 1 / 2MS medium containing 15 mg / L hygromycin to culture T1 generation Arabidopsis. Transgenic plants that grow well under hygromycin selection pressure are selected and transplanted into soil for single plant culture. After the Arabidopsis matures, the T1 generation seeds are collected from the single plants.

[0063] The T1 generation seeds were cultured into T2 generation Arabidopsis using the same method. Transgenic plants that grew well under hygromycin selection pressure were selected and transplanted into soil for individual plant culture. DNA and RNA were extracted from each Arabidopsis plant at the bolting stage. The obtained DNA was used as a template for PCR amplification using the TaTCP7-D cross-vector-specific primers: Super1300-F described in SEQ ID NO. 7 and Super1300-TaTCP7-DR described in SEQ ID NO. 6. Insertion of the TaTCP7-D gene into Arabidopsis was confirmed by 1% agarose gel electrophoresis. Lines with correct bands were subjected to RNA fluorescence quantitative detection, and the three lines with the highest TaTCP7-D expression levels were selected for further cultivation. After the Arabidopsis matured, T2 generation seeds were collected.

[0064] SEQ ID NO. 7: aatctcgatacaccaaatcg.

[0065] 100 T2 generation seeds of three selected strains were taken out for disinfection and evenly sown with toothpicks on 1 / 2 MS medium containing 25 mg / L hygromycin. The seeds were cultured according to the above method. When they grew to 4 leaves, positive seedlings with consistent growth were selected. After they could take root normally on the culture medium, they were transplanted into soil and continued to be cultured in a light incubator. The incubator conditions were 23°C, 16h / 8h, light / dark. After the Arabidopsis matured, T3 generation seeds were collected from each plant to obtain three homozygous transgenic Arabidopsis seeds, which were designated as OE5, OE-11 and OE-12.

[0066] VI. Salt tolerance testing of homozygous transgenic Arabidopsis 1. Flat plate simulated salt stress experiment TaTCP7-D-overexpressing Arabidopsis lines (OE5, OE-11, and OE-12) and the wild-type line (WT) were cultured on 1 / 2MS medium. The seedlings with expanded cotyledons and consistent growth were transferred to normal 1 / 2MS medium, 1 / 2MS medium containing 100 mM NaCl, and 1 / 2MS medium containing 125 mM NaCl for upright culture for 10 days.

[0067] The results are as follows 图1As shown in the phenotype results, there was no significant difference in the growth of Arabidopsis plants grown on normal 1 / 2MS medium. The TaTCP7-D overexpressing Arabidopsis lines (OE5, OE-11, and OE-12) grown on 1 / 2MS medium containing 100 mM NaCl and 125 mM NaCl had better growth than the wild-type line. This shows that overexpression of TaTCP7-D improves the salt tolerance of Arabidopsis.

[0068] The results are as follows 图2 As shown in the results, there was no significant difference in the growth of Arabidopsis plants grown on normal 1 / 2MS medium. The TaTCP7-D overexpressing Arabidopsis lines (OE5, OE-11 and OE-12) grown on 1 / 2MS medium containing 100mM NaCl and 125mM NaCl had better growth and longer root length than the wild-type line (WT), indicating that overexpression of TaTCP7-D improved the salt tolerance of Arabidopsis.

[0069] From the above, we can see that under the simulated salt stress conditions, TaTCP7-D Transgenic Arabidopsis thaliana with overexpressed gene has salt stress tolerance.

[0070] 2. Soil salt treatment Sterilized WT and transgenic seeds were sown on 1 / 2 MS medium. After 3 days of vernalization, they were placed in a 22°C constant-temperature greenhouse (with a 16-h light / 8-h dark photoperiod). Ten days later, they were transplanted into pots filled with nutrient soil (Qingdao Yongtu). Approximately 4 weeks later, experiments were conducted using well-grown Arabidopsis plants. Five Arabidopsis plants were transplanted into each pot, and the bottoms were sealed with plastic wrap. Salt treatments were applied to transgenic and untransgenic Arabidopsis (WT) plants, with equal amounts of 200 mmol / L and 250 mmol / L NaCl solutions. Each salt treatment was repeated 2 days apart, with three replicates of five Arabidopsis seedlings per replicate. Morphological observations were performed on the transgenic and WT plants to compare the growth differences between the two transgenic and WT plants. Phenotypes were recorded and photographed after three salt treatments at each stage. After the salt stress tolerance experiment, several Arabidopsis leaves were collected and tested for physiological indicators (POD, SOD, soluble sugar content, proline content, MDA, and hydrogen peroxide and superoxide anion content). Physiological indicator tests were performed using physiological indicator kits purchased from Grace Biotechnology.

[0071] The results are as follows 图3As shown in the data, when treated with 0 mM NaCl (normal), there was no significant difference in POD activity, proline, and soluble sugar content between the TaTCP7-D overexpression line and the wild-type plants; when treated with 250 mM NaCl, POD activity, proline, and soluble sugar content increased, and the POD activity, proline, and soluble sugar content of the TaTCP7-D overexpression line increased more significantly than those of the wild-type WT plants, indicating that TaTCP7-D improves the salt tolerance of Arabidopsis.

[0072] When treated with 0 mM NaCl (normal), the MDA, H2O2, and O 2- There was no significant difference in the content between the two groups; when treated with 250 mM NaCl, the MDA, H2O2, and O 2- The content was smaller, indicating that TaTCP7-D improved the salt tolerance of Arabidopsis.

[0073] 0. Analysis of tissue expression patterns of Qingmai No. 6 1. Experimental methods Green Wheat No. 6 was cultured in Hoagland nutrient solution until it reached the two-leaf, one-heart stage. The experimental group was treated with 200 mM NaCl, while the control group was cultured normally. Aerial and root samples were collected and preserved at 0, 4, 6, 8, 12, 24, 48, and 72 hours. Green Wheat No. 6 was soil-cultured until the jointing stage. The control group was watered with distilled water as usual, while the experimental group was watered with 250 mM saline every three days. After three waterings, stems and leaves were collected and preserved. Green Wheat No. 6 was cultured in the same manner at maturity. After saline treatment, stems, leaves, and glumes were collected and stored at -80°C for quantitative real-time PCR (qPCR) analysis. qPCR primers are listed in Table 9.

[0074] Table 9 Primers used for fluorescence quantification 2. Experimental results The results are as follows 图4 As shown, hydroponic 200mM NaCl treatment for 6h TaTCP7-D The gene expression level was highest in leaves, which was 3.75 times higher than that at 0h without treatment. TaTCP7-D Significantly upregulated expression in all tissues 。

[0075] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0076] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. TaTCP7-D The application of the gene in plant salt tolerance is characterized in that, described TaTCP7-D The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the TaTCP7-D protein encoded by the gene is shown in SEQ ID NO.

2.

2. according to claim 1 TaTCP7-D The application of the gene in plant salt tolerance is characterized in that, Overexpression in plants through genetic engineering TaTCP7-D Genes improve plant salt tolerance.

3. according to claim 2 TaTCP7-D The application of the gene in plant salt tolerance is characterized in that, The genetic engineering method is to construct an overexpression vector to transform the plant to obtain an overexpression TaTCP7-D Genetically modified plants.

4. according to claim 3 TaTCP7-D The application of the gene in plant salt tolerance is characterized in that, The method for constructing the overexpression vector is as follows: TaTCP7-D Gene ligation into pSuper1300 vector Pst I. Kpn Obtained in site I.

5. The method according to any one of claims 1 to 4 TaTCP7-D The application of the gene in plant salt tolerance is characterized in that, The plant is wheat or Arabidopsis thaliana.

6. according to claim 5 TaDREB44B The application of the gene in plant salt tolerance is characterized in that, described TaDREB44B Gene overexpression was used to increase POD activity, proline, and soluble sugar content in Arabidopsis lines under salt stress.

7. according to claim 5 TaDREB44B The application of the gene in plant salt tolerance is characterized in that, described TaDREB44B Gene overexpression was used to reduce MDA, H2O2, and O in Arabidopsis lines under salt stress. 2- content.

8. A method for improving plant salt tolerance, characterized in that: The steps include: Constructed according to the method of claim 4 TaTCP7-D Gene overexpression vector; after the overexpression vector is transformed into Agrobacterium, it infects the plant, making the plant TaTCP7-D Genes are overexpressed, thereby improving plant salt tolerance; The plant is wheat or Arabidopsis thaliana.

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