Application of TaTCP7-D gene in plant drought tolerance

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses application of a TaTCP7-D gene in drought tolerance of plants, 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 plant drought tolerance gene. The TaTCP7-D gene is over-expressed through a genetic engineering means, so that the drought 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 drought tolerance. Background Art

[0002] Drought stress severely affects plant growth and development through multiple mechanisms, including damage to plant cell structure, inhibition of photosynthesis, disruption of metabolic balance, and induction of oxidative damage. These are manifested in physiological and biochemical abnormalities such as increased cell membrane permeability, growth stagnation, decreased photosynthetic efficiency, accumulation of reactive oxygen species, and hormonal imbalances.

[0003] Wheat is one of the world's three major grain crops, and drought stress severely impacts its yield and quality. Drought leads to insufficient soil moisture, making it impossible for roots to absorb enough water to compensate for transpiration, disrupting the plant's water balance and inhibiting photosynthesis and nutrient synthesis. Drought reduces wheat's resistance to drought, while high temperatures encourage pests such as aphids and spider mites and increase the risk of fusarium head blight outbreaks. Sustained drought weakens cellular stability (e.g., insufficient accumulation of osmotic regulators) and exacerbates the effects of high temperatures or dry, hot winds. With the rapid advancement of genetic engineering in improving plant abiotic stress resistance, transcription factor genes may become a practical and effective target for wheat stress resistance. Therefore, it is necessary to explore new approaches to improving plant drought tolerance. Summary of the Invention

[0004] To develop a new approach to improve plant drought tolerance, the present invention provides TaTCP7-D Application of genes in plant drought tolerance. The present invention overexpresses TaTCP7-D genes, thereby improving drought tolerance in plants (Arabidopsis or wheat).

[0005] The present invention provides TaTCP7-D Application of genes in plant drought 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.

[0006] The present invention overexpresses by genetic engineering TaTCP7-D By testing drought-resistant wheat varieties, we can determine the TaTCP7-D Genes are highly expressed, indicating overexpression TaTCP7-D Genes help improve the plant's drought tolerance.

[0007] Furthermore, overexpression in plants through genetic engineering TaTCP7-D Genes improve plant drought tolerance.

[0008] Furthermore, the genetic engineering method is to construct an overexpression vector to transform the plant to construct an overexpression vector. TaTCP7-D transgenic plants of the gene, thereby obtaining a drought-tolerant plant gene variety.

[0009] Further, the construction method of the overexpression vector is as follows: connecting the gene to the TaTCP7-D gene to the Pst I and Kpn I sites of the pSuper1300 vector.

[0010] Further, the plant is Arabidopsis thaliana or wheat.

[0011] The application further provides a recombinant expression vector, which is obtained by connecting the TaTCP7-D gene to the Pst I and Kpn I sites of the pSuper1300 vector.

[0012] The application further provides a recombinant bacterium, which is obtained by transforming the agrobacterium with the recombinant expression vector.

[0013] The application further provides a method for improving the drought tolerance of plants, which comprises the following steps: transforming the recombinant expression vector into agrobacterium to obtain a recombinant bacterium, and culturing the bacterium to obtain a bacterium liquid to infect plants, and screening transgenic plants with drought tolerance.

[0014] The application further provides the use of the recombinant expression vector or the recombinant bacterium in the drought tolerance of plants.

[0015] Further, the plant is Arabidopsis thaliana or wheat.

[0016] Compared with the prior art, the application has the beneficial effects that: The application provides the use of the TaTCP7-D gene in the drought tolerance of plants, and overexpression of the TaTCP7-D gene in wheat or Arabidopsis thaliana can significantly improve the drought tolerance of wheat or Arabidopsis thaliana.

[0017] The application is based on the in-depth and systematic researches of stress resistance function identification, transgenic Arabidopsis thaliana, etc. TaTCP7-D The application discloses the molecular basis for regulating the drought tolerance of wheat, and provides a theoretical basis for improving the drought tolerance of wheat and a gene resource for wheat stress resistance breeding. TaTCP7-D The application discloses the molecular basis for regulating the drought tolerance of wheat, and provides a theoretical basis for improving the drought tolerance of wheat and a gene resource for wheat stress resistance breeding. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only show some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0019] 图1 Effects of different concentrations of mannitol on transgenic Arabidopsis under drought stress; In the figure, A is the effect of 0 mM mannitol on transgenic Arabidopsis under drought stress; B is the effect of 300 mM mannitol on transgenic Arabidopsis under drought stress; C is the effect of 350 mM mannitol on transgenic Arabidopsis under drought stress; 图2 Effects of different concentrations of mannitol on the root length of transgenic Arabidopsis under drought stress.

[0020] 图3 Effects of natural drought treatment on transgenic Arabidopsis; In the figure, A is the phenotype of wild type (WT) and transgenic Arabidopsis under normal treatment; B is the phenotype of wild type (WT) and transgenic Arabidopsis after 18 days of natural drought treatment.

[0021] 图4 Effects of natural drought treatment on the enzyme activity of wild type (WT) and transgenic Arabidopsis; In the figure, A is the effect of natural drought treatment on the peroxidase (POD) activity in the leaves of wild type (WT) and transgenic Arabidopsis; B is the effect of drought treatment on the proline (PRO) activity in the leaves of wild type (WT) and transgenic Arabidopsis; C is the effect of drought treatment on the soluble sugar (SS) activity in the leaves of wild type (WT) and transgenic Arabidopsis; D is the effect of drought treatment on the hydrogen peroxide (H2O2) activity in the leaves of wild type (WT) and transgenic Arabidopsis; E is the effect of drought treatment on the malondialdehyde (MDA) activity in the leaves of wild type (WT) and transgenic Arabidopsis; F is the effect of drought treatment on the superoxide anion (O2 - ) activity in the leaves of wild type (WT) and transgenic Arabidopsis.

[0022] 图5 Effects of drought stress treatment on the relative expression amount of genes in different parts of Qingmai No. 6 seedlings; TaTCP7-D ​A: different time periods of drought stress treatment of roots and leaves of Qingmai No. 6 at seedling stage TaTCP7-D Relative expression amount of genes B: aboveground and underground parts of Qingmai No. 6 at seedling stage under drought stress treatment TaTCP7-D Relative expression amount of genes C: roots and stems of Qingmai No. 6 at jointing stage under drought stress treatment TaTCP7-D Relative expression amount of genes D: roots, stems and husks of Qingmai No. 6 at mature stage under drought stress treatment TaTCP7-D Relative expression amount of genes DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0024] Example 1 TaTCP7-D Application of genes in drought tolerance of wheat

[0025] TaTCP7-D The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded TaTCP7-D protein is shown as SEQ ID NO. 2.

[0026] SEQ ID NO. 1 ATGACCTCCCACGACATCGCCGCCGCCTCCAGCCCTTTCCACGTCTACCAGCAGCACCTACCGATGACGGTGGCGCCCGCCGCCGCCTCCGCCGACGCTGGCTTGGCTCCGGCGGCGAGCAAGAAGGCTGGTGGCAGCGTGAAGGACCGGCACAGCAAGGTGAACGGGCGCGGTCGGCGCGTGCGCATGCCGATCGTGTGCGCCGCGCGCGTGTTCCAGCTCACGCGCGAGCTGGGGCTCAAGTCCGACGGCCAGACCATCGAGTGGCTGCTCCGGCAGGCGGAGCCCTCGATCCTCGCCGCCACGGGGACCGGCACCACCCCCGCCGCCTTCGTCTCCTCCTCCGCGCCATCCACCTCCTCCTCCTCCTCCTTCTCGTACCCACACACCCTCCTCGGCAAGCGCCCGCGCGAGGAGGAGAACGAAGCCTTCGGCGCGGGGGCTACAACGTCGGCCTTCTGGGCGGCGCTGCAGGCGCCGCCGCGGCAGGACACGTGGGGGTTCTCGCCGCTGGAGGCGCAGGCCGCGTACATGCCGATGGCGCAGGTCCACCACCACCACCTCAACCTTGTCGCCGCGCTCTCCGGCGCCGCTCGGCGCACCGAGGAGGAGACCCGGTGA.

[0027] SEQ ID NO. 2: MTSHDIAAASSPFHVYQQHLPMTVAPAAASADAGLAPAASKKAGGSVKDRHSKVNGRGRRVRMPIVCAARVFQLTRELGLKSDGQTIEWLLRQAEPSILAATGTGTTPAAFVSSSAPSTSSSSSFSYPHTLLGKRPREEENEAFGAGATTSAFWAALQAPPRQDTWGFSPLEAQAAYMPMAQVHHHHLNLVAALSGAARRTEEETR.

[0028] I. Extraction of RNA and obtaining of cDNA 1. Extraction of RNA Take the wheat 6 as material, when the wheat grows to two leaves one heart stage, take down 1 g of wheat leaves with tweezers, extract RNA with plant RNA extraction kit purchased from Aikuerui biological company, the specific extraction method is as follows.

[0029] (1) Lysis of plant tissue Transfer the plant tissue sample (wheat leaves) to a pre-cooled mortar in liquid nitrogen, grind the plant tissue with a pestle, and continuously add liquid nitrogen to the mortar during grinding until it is ground into powder.

[0030] Transfer 80 mg of the sample ground into powder to a 1.5 mL centrifuge tube (RNase free) containing 500 μl of lysis buffer Buffer RLS (50×DTT Solution has been added to Buffer RLS before use), immediately vortexed at high speed or repeatedly blown with a pipette until the sample is fully lysed (no obvious precipitate), and the sample lysis solution is obtained.

[0031] After the above sample lysis solution is placed at room temperature for 2 minutes, centrifuge at 4°C, 12,000 rpm for 5 minutes.

[0032] After centrifugation, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube (RNase free).

[0033] (2) Purification Add 1 / 2 volume of anhydrous ethanol to the supernatant and mix well with a pipette. If there is obvious thickening or precipitation, use the pipette to blow and disperse the precipitate several times to obtain a mixture.

[0034] Transfer the above mixture to the Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 2 minutes, and discard the filtrate (if the volume of the mixture is greater than 700 μL, add it in batches).

[0035] 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.

[0036] Add 750 μl of Buffer RWB (Buffer RWB has been added with a 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.

[0037] DNase I digestion: ① Prepare DNase I reaction solution according to the following table and mix well. Add 50 μL DNase I reaction solution to the center of the membrane of Plant RNA Mini Column, and stand at room temperature for 15 minutes.

[0038] Table 1 DNase I reaction solution formula ② Add 350 μL Buffer RWB to the center of the membrane of Plant RNA Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0039] 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.

[0040] Place the adsorption column of Plant RNA Mini Column on a new 2.0 mL Collection Tube, and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0041] Place the adsorption column of Plant RNA Mini Column on a new RNase Free Tube, add 150 μL of RNase Free Water to the center of the adsorption column membrane, stand at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes at room temperature to elute the RNA, which can be used for subsequent experiments. If the subsequent experiments are not performed immediately, the dissolved RNA can be stored in -80°C.

[0042] 2. Obtain cDNA by reverse transcription (1) Prepare the RNA template solution on ice according to Table 2 below, and perform denaturation and annealing reactions in a PCR instrument to obtain the denatured and annealed reaction solution.

[0043] Table 2 Reaction system (10 μL) Denaturation and annealing reaction conditions: 65°C for 5 min, 4°C storage.

[0044] (2) Prepare the reverse transcription reaction system according to Table 3 to synthesize cDNA Table 3 Reverse transcription reaction system cDNA synthesis reaction conditions: 30°C for 10 min; 42°C for 50 min; 95°C for 5 min; 4°C storage.

[0045] (3) PCR amplification The cDNA solution obtained above can be directly used for subsequent PCR amplification. The amount of reaction solution should not exceed 1 / 10 of the PCR reaction system.

[0046] two, TaTCP7-D Gene cloning The cDNA of the leaves of Qingmai No. 6 obtained above was used as a template and the cDNA of the Chinese Spring variety in the WheatOmics1.0 database was used as a template. TaTCP7-D Targeted primers were designed based on the gene sequence: TaTCP7-DF primer shown in SEQ ID NO.3 and TaTCP7-DR primer shown in SEQ ID NO.4 were used for PCR amplification and cloned using Novozymes P525 polymerase. TaTCP7-D Gene sequence. The PCR amplification reaction system is shown in Table 4, and the PCR amplification reaction procedure is shown in Table 5. After amplification, agarose gel electrophoresis was performed.

[0047] SEQ ID NO. 3: 5'-ACTCCCTCCAACGTACAGCGA-3'.

[0048] SEQ ID NO. 4: 5'-AGTACACCCAGGATCCTAATA-3'.

[0049] Table 4 PCR amplification reaction system Table 5 PCR amplification reaction program Gel excision and recovery: Use 1% agarose gel electrophoresis to verify gene integrity. Use a 2000 bp marker at 120 volts for 15 minutes. Select the correct fragment from the electrophoretic band and excise the gel. The resulting amplified product is the target gene. Specific procedures are similar to those in the Novozymes DC301 kit.

[0050] Connect the intermediate vector: connect the target gene with pEASY-Blunt Zero (Beijing Quanshijin) to obtain the recombinant vector pEASY-TaTCP7-D.

[0051] 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 incubate 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.

[0052] Sequencing: PCR verification screening out the correct band liquid sent to Sheng Gong Company for gene sequence identification. After identification, the sequence of the correct bacteria was retained for subsequent experiments.

[0053] Plasmid extraction: the correct bacteria liquid and LB liquid medium were expanded with a volume ratio of 1:100 mL, cultured at 37°C, 200 rpm for 12 hours, and then plasmid extraction was performed. Refer to the method of Nuowei DC201 kit. The extracted plasmid was stored at -20°C for subsequent experiments.

[0054] Three, TaTCP7-D Construction of gene recombinant expression vector The Super1300-TaTCP7-D expression vector was constructed, and the homologous arm primers were designed according to the pSuper1300 vector multiple cloning site selection enzyme cutting site: the Super1300-TaTCP7-D-F primer shown in SEQ ID NO. 5 and the Super1300-TaTCP7-D-R primer shown in SEQ ID NO. 6. 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 pSuper1300 vector, construction of Super1300-TaTCP7-D expression vector, the specific reaction system is as follows.

[0055] SEQ ID NO. 5: gactctagaaagcttctgcagATGACCTCCCACGACATCGC.

[0056] SEQ ID NO. 6: gcccttgctcaccatggtaccTCACCGGGTCTCCTCCTCGGT.

[0057] Table 6 pSuper1300 vector enzyme cutting system Table 7 TaTCP7-D amplification system Four, transformation of Agrobacterium (1) The successfully constructed pSuper1300-TaTCP7-D recombinant expression vector was introduced into Agrobacterium, and the specific method was as follows: 1. Take the Agrobacterium competent cells stored at -80°C on ice, and under sterile conditions, add 10 μL pSuper1300-TaTCP7-D recombinant expression vector plasmid DNA to the competent cells, mix gently, and ice bath for 5 min.

[0058] 2, Place the competent cells in liquid nitrogen for 5 minutes, quickly place them in a 37°C water bath for 5 minutes, do not shake the water surface, and then place the centrifuge tube in an ice water bath for 5 minutes.

[0059] 3, Under sterile conditions, add 800 mL of LB liquid medium, and incubate at 28°C for 3 hours to recover the bacterial cells.

[0060] 4, Centrifuge at 5000 rpm for 1 minute to collect the bacteria, leave about 100 μL of supernatant, gently blow and mix, and spread on LB solid plates containing Kan and Rif antibiotics, and incubate for 48 hours.

[0061] 5, After identifying positive colonies by PCR, the correct Agrobacterium liquid is expanded and reserved.

[0062] (II) Agrobacterium inflorescence infection (1) Prepare the bacterial solution: inoculate the above Agrobacterium solution into 10 mL of LB liquid medium containing Kan and Rif antibiotics, and incubate at 28°C in a 220 rpm shaker for 12 hours to obtain the bacterial solution.

[0063] (2) Select 1 mL of bacterial solution and inoculate into 100 mL of LB liquid medium containing Rif and Kan antibiotics, and incubate for 18 hours. Under the same culture conditions, the OD 600 is increased to 1.2. Then, centrifuge at 5000 rpm for 10 minutes, remove the supernatant, collect the sinking Agrobacterium, resuspend the bacterial cells with Arabidopsis transformation infiltration solution, and then adjust the OD 600 to 1.0. The formula of Arabidopsis infiltration solution is shown in Table 8.

[0064] Table 8 Formula of Arabidopsis infiltration solution Soak the Arabidopsis flower buds in the infiltration solution for 12 seconds, avoiding contact between the Agrobacterium infiltration solution and the soil, which may adversely affect the tender roots of Arabidopsis. After infection, place the Arabidopsis in a dark environment for 48 hours, and then continue to culture in a light incubator, with a temperature of 23°C, 16h / 8h, light / dark. After the Arabidopsis matures, collect the T0 generation seeds from each plant.

[0065] Five, homologous selection of transgenic Arabidopsis After disinfection, sow the T0 generation seeds evenly on 1 / 2MS medium containing 15 mg / L hygromycin for T1 generation Arabidopsis culture, select transgenic plants that grow well under hygromycin selection pressure, transplant them to soil for single plant culture, and collect the T1 generation seeds from each plant after the Arabidopsis matures.

[0066] The T1 generation seeds were cultured in the T2 generation of Arabidopsis by the same method, and the transgenic plants that grew well under the selection pressure of hygromycin were selected, transplanted into soil for single plant culture, and the DNA and RNA of each Arabidopsis plant were extracted when the Arabidopsis plants reached the bolting stage. The obtained DNA was used as a template, and PCR amplification was performed using the Super1300-F primer described by SEQ ID NO. 7 and the Super1300-TaTCP7-DR primer described by SEQ ID NO. 6. The TaTCP7-D gene insertion into Arabidopsis was confirmed by 1% agarose gel electrophoresis. The correct strain was selected by using RNA for fluorescence quantitative detection, and the three strains with the highest TaTCP7-D expression were further cultured. After the Arabidopsis matured, the T2 generation seeds were collected.

[0067] SEQ ID NO. 7: aatctcgatacaccaaatcg.

[0068] The T2 generation seeds of the three selected strains were used, 100 seeds were taken for disinfection, and were evenly sowed on 1 / 2MS medium containing 25 mg / L hygromycin using toothpicks. The culture was carried out according to the above method, and the positive seedlings with consistent growth were selected from the 4-leaf plants. After the seedlings could normally root on the medium, they were transplanted into soil and cultured in a light incubator. The conditions of the incubator were 23°C, 16h / 8h, light / dark. After the Arabidopsis matured, the T3 generation seeds were collected from single plants, and three homozygous transgenic Arabidopsis seeds were obtained, which were named OE-5, OE-11 and OE-12, respectively.

[0069] Six, drought tolerance detection of homozygous transgenic Arabidopsis 1. Plate simulation drought stress experiment The cotyledon-unfolding and growth-consistent seedlings of the TaTCP7-D overexpression Arabidopsis strains (OE-5, OE-11 and OE-12) and the wild type strain (WT) were selected after being cultured on 1 / 2MS medium, and were vertically cultured on normal 1 / 2MS medium, 1 / 2MS medium containing 300 mM mannitol, and 1 / 2MS medium containing 350 mM mannitol for 10 days, respectively.

[0070] As shown in 图1 , there was no obvious difference in the growth of Arabidopsis plants on normal 1 / 2MS medium, the overexpression Arabidopsis strains (OE-5, OE-11 and OE-12) grew better than the wild type strain (WT) on 1 / 2MS medium containing 300 mM mannitol and 1 / 2MS medium containing 350 mM mannitol, TaTCP7-D , and the overexpression of the gene improved the drought resistance of Arabidopsis. TaTCP7-D

[0071] ​The results are as follows 图2 As shown in the figure, there is no significant difference in the growth of Arabidopsis plants grown on normal 1 / 2MS medium, and there is no significant difference in the growth of Arabidopsis plants grown on 1 / 2MS medium containing 300mM mannitol and 350mM mannitol. TaTCP7- The root length of the D-overexpressing Arabidopsis strain was better than that of the wild-type strain. TaTCP7-D Overexpression improved drought resistance in Arabidopsis.

[0072] From the above, we can see that under simulated drought stress conditions, TaTCP7-D Transgenic Arabidopsis thaliana with overexpressed genes has drought stress tolerance.

[0073] 2. Natural drought treatment Natural drought treatment: Sterilized seeds of the wild-type (WT) and three transgenic lines (designated OE-5, OE-11, and OE-12) were sown on 1 / 2 MS medium. After 3 days of vernalization, the seeds were placed in a 22°C constant-temperature greenhouse with a 16-h light / 8-h dark photoperiod. Ten days later, the plants were transplanted into pots filled with nutrient soil. Six Arabidopsis plants were transplanted into each pot and subjected to natural drought treatment (continuously without watering). Phenotypic differences between the transgenic lines and the wild type were observed during this period. After 10 days, the phenotypes were recorded and photographed. Subsequently, Arabidopsis leaves were collected for various physiological indicators (POD, SOD, soluble sugar content, proline content, MDA, and hydrogen peroxide superoxide anion content). (Physiological indicator kits were purchased from Grace Biotechnology.) A normal control group was watered 75 mL every three days.

[0074] The results are as follows 图3 As shown, from 图3 It can be seen that after 10 days of natural drought, Arabidopsis plants overexpressing OE-5, OE-11, and OE-12 showed some wilting. Comparison of the overexpressing Arabidopsis with wild-type Arabidopsis showed that the phenotype of the overexpressing Arabidopsis was better than that of the wild-type Arabidopsis. This shows that the transgenic Arabidopsis has significantly improved drought resistance compared to the wild-type line.

[0075] The results are as follows 图4 As shown, when the distilled water treatment is normally applied, TaTCP7-D There were no significant differences in POD activity, proline, and soluble sugar content between the overexpression lines and the wild-type plants. When treated with drought, POD activity, proline, and soluble sugar content increased, and compared with the wild-type WT plants, TaTCP7-D The POD activity, proline and soluble sugar content of the overexpression strain increased more significantly, indicating that TaTCP7-D Overexpression improved drought resistance in Arabidopsis.

[0076] During normal processing, TaTCP7-D MDA, H2O2, O2 in overexpressed Arabidopsis and wild-type plants -No significant difference between the contents; under drought treatment, compared with wild type WT plants, TaTCP7-D MDA, H2O2, O2 - contents were smaller, indicating TaTCP7-D Overexpression improved drought resistance of Arabidopsis.

[0077] Seven, the expression pattern analysis of Qingmai No. 6 1. Experimental method Qingmai No. 6 was cultured with Hoagland nutrient solution to the two-leaf-one-heart stage, the experimental group was treated with 15% PEG600, the control group was normally cultured, and the aboveground and root parts were taken at 0h, 4h, 6h, 8h, 12h, 24h, 48h, 72h Different time period preservation. Qingmai No. 6 was continued to soil culture to the jointing stage, the control group maintained soil moisture content of 70%, the experimental group was naturally droughted for seven days, and the stems and leaves were taken for preservation. The culture method of Qingmai No. 6 at the mature stage was the same as above, and the stems, leaves and husks were taken after drought treatment and stored at -80℃ for qPCR fluorescence quantification experiment. The primers used for fluorescence quantification are shown in Table 9.

[0078] Table 9 Primers used for fluorescence quantification 2. Experimental results The results are shown in 图5 8h TaTCP7-D Gene expression was highest in leaves, which was 3.75 times higher than that at 0h without treatment. After stress treatment at the jointing stage and mature stage, TaTCP7-D Significantly up-regulated expression in all tissues 。 It can be seen that TaTCP7-D The gene is a drought resistance functional gene of Qingmai No. 6 plants.

[0079] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept.

[0080] Obviously, those skilled in the art can make various modifications and changes to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and changes of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and changes.

Claims

1. TaTCP7-D The application of a gene in plant drought 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 a gene in plant drought tolerance is characterized in that Overexpression in plants through genetic engineering TaTCP7-D Genes improve plant drought tolerance.

3. according to claim 2 TaTCP7-D The application of a gene in plant drought tolerance is characterized in that The genetic engineering method is to transform plants by constructing an overexpression vector TaTCP7-D Gene-transgenic plants are produced to obtain drought-tolerant plant gene varieties.

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

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

6. A recombinant expression vector, characterized in that: The recombinant expression vector is transformed into TaTCP7-D Gene ligation into pSuper1300 vector Pst I and Kpn Obtained in site I.

7. A recombinant bacterium, characterized in that The recombinant bacteria are obtained by transforming Agrobacterium with the recombinant expression vector according to claim 6.

8. A method for improving drought tolerance of plants, characterized in that: The steps include: The recombinant expression vector according to claim 6 is transformed into Agrobacterium to obtain recombinant bacteria, and the bacterial solution cultured with the recombinant bacteria is used to infect plants to screen transgenic plants with drought resistance.

9. Use of the recombinant expression vector according to claim 6 or the recombinant bacterium according to claim 7 in plant drought tolerance.

10. The use according to claim 9, characterized in that The plant is Arabidopsis thaliana or wheat.

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