Grape vvlbdid5 gene and application thereof

Abstract

The application discloses a grape VvLBDId5 gene and application thereof, relates to the technical field of plant genetic engineering, and has the technical scheme as follows: the grape VvLBDId5 gene can regulate drought resistance of plants, the nucleotide sequence of the VvLBDId5 gene is shown in SEQ ID NO. 1, and the amino acid sequence coded by the nucleotide sequence of the VvLBDId5 gene is shown in SEQ ID NO. 2.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to an LBD-type transcription factor gene VvLBDd5 obtained from grapes, the protein encoded by the gene, a recombinant expression vector containing the gene, genetic transformation of transgenic plants, and the application of the gene to improve the drought resistance of tobacco. Background Technology

[0002] Grapes are a fruit tree of significant economic value worldwide, but their cultivars are generally sensitive to drought stress, severely restricting their industrial development in arid and semi-arid regions. Currently, due to the diversity of grape's wild ancestors, hybridization and selection during long-term human domestication, and countless historical mutations solidified and accumulated through asexual reproduction, the genetic background of grapes is complex, leading to relatively slow progress in screening for drought-resistant genes. Furthermore, drought resistance is a trait controlled by multiple genes, further increasing the difficulty of research. The LBD transcription factor family plays a crucial regulatory role in plant responses to biotic and abiotic stresses, but reports on the specific functions and applications of grape LBD genes in regulating drought resistance are scarce. Therefore, discovering new gene resources that can significantly improve grape drought resistance is of great importance. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the first objective of this invention is to provide a grape VvLBDId5 gene that has the advantage of responding to drought stress.

[0004] To achieve the first objective mentioned above, the present invention provides the following technical solution: a grape VvLBDId5 gene that can regulate the drought resistance of plants, wherein the nucleotide sequence of the VvLBDId5 gene is shown in SEQ ID NO.1.

[0005] The present invention also provides a protein obtained by translating the VvLBDId5 gene, wherein the amino acid sequence encoded by the nucleotide sequence of the VvLBDId5 gene is shown in SEQ ID NO. 2.

[0006] An expression vector containing the VvLBDId5 gene as described above, said expression vector can regulate drought resistance in plants.

[0007] A genetically engineered bacterium was obtained by transferring the above expression vector into Agrobacterium.

[0008] An expression vector is used to regulate plant drought resistance by overexpressing the VvLBDId5 gene in plants.

[0009] The application of a genetically engineered bacterium in regulating plant drought resistance was achieved by overexpressing the VvLBDId5 gene in plants.

[0010] According to the application described above, the primers have sequences as shown in SEQ ID NO.3-4.

[0011] A method for screening drought-resistant grapes involves detecting the expression level of the VvLBDId5 gene.

[0012] Furthermore, the plant is grape or tobacco.

[0013] In summary, the present invention has the following beneficial effects:

[0014] This invention improves plant drought resistance through plant genetic engineering. A complete DNA fragment encoding a drought-related gene was isolated and cloned from Cabernet Sauvignon grape tissue culture seedlings. Phenotypic observations and related physiological index measurements of wild-type WT and transgenic tobacco under drought stress revealed that the relative expression level of the VvLBDId5 gene significantly increased under drought stress, indicating that the VvLBDId5 gene can respond to drought stress. After drought stress treatment, the MDA content and relative conductivity of transgenic tobacco leaves were significantly lower than those of wild-type (WT), indicating that the membrane system of transgenic tobacco is more stable under drought stress. Utilizing its function, it was ultimately found that overexpression significantly improved the drought resistance of transgenic tobacco. After drought stress treatment, the SOD and POD enzyme activities in transgenic tobacco were significantly higher than those in wild-type (WT). VvLBDId5 effectively scavenges excess ROS by enhancing the activity of antioxidant enzymes in tobacco, thereby improving the plant's resistance to drought stress. Attached Figure Description

[0015] Figure 1 The sequence structure and phylogenetic relationships of the grape VvLBDId5 protein were compared. Among them, (A) the sequence comparison of VvLBDId5 with the LBD protein sequences of 12 other species including apple, tomato, and Arabidopsis thaliana (maize ZmLBD18, wheat Ta1DLBD8, rice OsLBD5-2, maize ZmLBD20, wheat Ta3DLBD22, Arabidopsis thaliana AtLBD13, wheat AtLBD15, tomato SlLBD13-3, tomato SlLBD14, apple MdLBD8, soybean GmLBD26, and soybean GmLBD19); (B) the similarity comparison between VvLBD15 and AtLBD15 proteins.

[0016] Figure 2 The relative expression levels of the VvLBDId5 gene in Cabernet Sauvignon grape tissue culture seedlings after 0, 4, 8, 12, and 16 days of drought stress were calculated.

[0017] Figure 3 To identify VvLBDId5-overexpressing transgenic tobacco plants using qRT-PCR;

[0018] Figure 4 The study aimed to analyze the overexpression of the VvLBDId5 gene in tobacco and its resistance to drought stress. In this study, (A) and (B) represent the control and drought treatment of WT and transgenic tobacco, respectively, and (CD) represent the analysis of MDA content (C) and relative conductivity (D) of WT and transgenic tobacco after the two treatments.

[0019] Figure 5 The relationship between VvLBDId5 gene overexpression in tobacco and antioxidant enzyme activity and ROS accumulation in plants; where (AB) represents... Figure 4 Histochemical staining of DAB and NBT in leaves of WT and VvLBDId5 transgenic tobacco; H2O2 content (C) and O2 content in WT and transgenic lines. - Production rate (D), SOD enzyme activity (E), and POD enzyme activity (F) were analyzed. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] The reagents or raw materials used in this invention are all commercially available. Unless otherwise specified, the plasmids, vectors, etc., described in this invention are all commercially available. Methods or processes not described in detail can be performed using conventional practices in the field.

[0022] The Cabernet Sauvignon grape tissue culture seedlings used in this invention were obtained from Shandong Qingda Seedling Co., Ltd., and Escherichia coli competent cells DH5α and Agrobacterium tumefaciens EHA105 were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0023] The culture medium involved in this invention is as follows:

[0024] Tobacco co-culture medium: MS + 6-BA (2 mg / L) + NAA (1 mg / L);

[0025] Tobacco differentiation medium: MS + 6-BA (2 mg / L) + NAA (1 mg / L) + Kan (50 mg / L);

[0026] Tobacco rooting medium: 1 / 2 MS + IAA (0.1 mg / L).

[0027] Example 1: Cloning of the grape VvLBDId5 gene

[0028] I. Total RNA extraction from Cabernet Sauvignon grape tissue culture seedlings

[0029] 1. Total RNA was extracted from Cabernet Sauvignon grape tissue culture seedlings using a kit method, as follows:

[0030] (1) Homogenization treatment: Weigh about 0.1 g of Cabernet Sauvignon grape tissue culture seedlings, grind them thoroughly in liquid nitrogen, transfer them into a pre-cooled 1.5 mL centrifuge tube, add 0.5 mL of lysis buffer SL (add β-mercaptoethanol to SL to a final concentration of 5% before use), and immediately vortex vigorously to mix.

[0031] (2) Centrifuge at 12,000 rpm for 2 min.

[0032] (3) Transfer the supernatant to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm for 2 min, and carefully aspirate the supernatant from the collection tube into a new RNase-free centrifuge tube. Try to avoid contact between the pipette tip and the cell debris in the collection tube.

[0033] (4) Slowly add 0.4 times the volume of supernatant of anhydrous ethanol, mix well. Precipitation may occur at this time. Transfer the obtained solution and precipitate together into the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0034] (5) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0035] (6) Preparation of DNaseI working solution: Take 10 μL of DNaseI stock solution and put it into a new RNase-Free centrifuge tube. Add 70 μL of RDD buffer and mix gently.

[0036] (7) Add 80 μL of DNaseI working solution to the center of the adsorption column CR3 and let it stand at room temperature for 15 min.

[0037] (8) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0038] (9) Add 500 μL of washing solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0039] (10) Repeat step 9.

[0040] (11) Centrifuge at 12,000 rpm for 2 min, place the adsorption column CR3 into a new RNase-Free centrifuge tube, add 30-50 μL of RNase-Free ddH2O to the middle of the adsorption membrane, place at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min to obtain the RNA solution, and store at -80℃.

[0041] II. Reverse transcription to synthesize the first strand of cDNA

[0042] Follow the instructions provided with the DNA reverse transcription kit manufactured by Aibimeng Biotechnology Co., Ltd., and keep the entire process on ice. The specific steps are as follows:

[0043] (1) Prepare the reaction system in a 0.2 mL PCR tube according to Table 1, and mix well:

[0044] Table 1 Reverse transcription system

[0045]

[0046] X must ensure that the RNA concentration is ≤1 μg.

[0047] (2) Mix the above system thoroughly, centrifuge, and set the PCR instrument program to 37℃ for 10 min, 55℃ for 15 min, and 95℃ for 3 min. Then incubate on ice. The newly synthesized cDNA can be used directly for RT-PCR or stored at -20℃ for a long time.

[0048] III. Obtaining the full-length cDNA sequence

[0049] (1) Take a 0.2 mL PCR tube and add the following components in sequence, as shown in Table 2:

[0050] Table 2 Amplification reaction system

[0051]

[0052] The primer sequences are as follows:

[0053] VvLBDId5-F: 5'-ATGTCAAGAGAAAGGGAGAG-3', SEQ ID NO.3;

[0054] VvLBDId5-R: 5'-ACCGAAGTAGGAGACATTTTC-3', SEQ ID NO. 4.

[0055] (2) The PCR reaction program is 98℃ pre-denaturation for 10 s, 98℃ denaturation for 10 s, 58℃ annealing for 5 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 1 min.

[0056] (3) After the PCR reaction was completed, 1.0% agarose gel electrophoresis was performed for detection. If the bands matched, the PCR reaction solution was recovered using a PCR reaction solution recovery kit (Aibimeng Biotechnology Co., Ltd.), the vector was ligated, transformed (transformed into E. coli competent cells DH5α, cultured overnight at 37°C on LB agar plates), and sequenced. Single colonies were picked and sequenced at Sangon Biotech Co., Ltd. to obtain the VvLBDId5 gene, whose nucleotide sequence is shown in SEQ ID NO.1; its amino acid sequence is shown in SEQ ID NO.2. Wherein, SEQ ID NO.1;

[0057] MSRERERFDEIGKKIKRETDAFTQMGRRHMLGPPGTLNSITPCAACKLLRRRCAQECPFSPYFSPHEPQKFASVHKVFGASNVSKMLMEVPESQRADAANSLVYEANVRLRDPV YGCMGAISALQQQVQSLQAELNAVRAEILKYKYREANIIPPSHIALLSSGAVSVAAPPPAQPPAQAPPPPPPPPPIPLPLPPNSSSSMYTQPSSSADYSTISSENVSYFG,SEQ ID NO.2.

[0058] Example 2

[0059] Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of the VvLBDId5 gene in Cabernet Sauvignon grapes under drought stress.

[0060] (1) Select Cabernet Sauvignon grape tissue culture seedlings of uniform size, and after hardening, subject them to 0 d, 4 d, 8 d, 12 d and 16 d drought treatment (without watering), collect the plants and quick-freeze them with liquid nitrogen.

[0061] (2) Grape cDNA after drought treatment was obtained by total RNA extraction and cDNA reverse transcription according to the method in Example 1.

[0062] (3) Preparation of the real-time PCR reaction system: Prepare the master mix in sterile low-adsorption centrifuge tubes under ice and light-protected conditions according to the table below. Set up 3 technical replicates for each sample.

[0063] Table 3 Amplification reaction system

[0064]

[0065] Reaction conditions: (Pre-denaturation) 95℃ pre-denaturation for 10 min; 95℃ denaturation for 5-10 s, 60℃ annealing / extension for 30 s, 40 cycles; 95℃, 15 s, 60℃, 60 s (melting curve analysis).

[0066] (4) Using the VvActin gene as a control, according to 2^ -ΔΔCt The relative expression level of the VvLBDId5 gene was calculated using this method.

[0067] The primer sequences are as follows:

[0068] VvLBDId5 (qRT-PCR)-F: 5'-AAGACACATGTTGGGGCCTC-3';

[0069] VvLBDId5 (qRT-PCR)-R: 5'-GGCTTTTCCGGGACTTCCAT-3';

[0070] VvActin (qRT-PCR)-F: 5'-TCACCACTACTGCTGAACGG-3';

[0071] VvActin (qRT-PCR)-R: 5'-ATTCCTGCAGCTTCCATCCC-3'.

[0072] Depend on Figure 2 It can be seen that the relative expression level of the VvLBDId5 gene is relatively stable in the first 8 days under drought stress, increases significantly at around 12 days, and then decreases slightly. This indicates that the VvLBDId5 gene can respond to drought stress, and its expression level increases significantly under drought stress.

[0073] Example 3

[0074] Obtaining genetically modified tobacco and identifying VvLBDId5 genetically modified tobacco

[0075] (1) Use a sterilized toothpick to pick up a single colony of Agrobacterium tumefaciens containing the VvLBDId5 recombinant plant expression vector stored at 4℃, place it in 8 mL of resistant LB liquid medium (containing 100 mg / mL Kan and 100 mg / mL Rif), and put the centrifuge tube into a constant temperature shaking incubator at 28℃ with a rotation speed of 200 rpm for overnight culture.

[0076] (2) The next day, take out 1 mL of the shaken bacterial solution and place it in 50 mL of fresh liquid LB medium (containing 100 mg / mL Kan, 100 mg / mL Rif and 100 μM acetylsuccinone), put it back into the shaking incubator and incubate for 4-5 h, shaking until the bacterial solution concentration OD600≈0.7.

[0077] (3) Place the bacterial solution in a 50 mL sterile centrifuge tube, adjust the centrifuge temperature to 26℃, centrifuge at 6000 rpm for 5 min.

[0078] (4) Discard the supernatant in the clean bench, resuspend the cells in 10 mL of sterile water, centrifuge, and wash the cells.

[0079] (5) Open the centrifuge tube in the clean bench, add 1 mL of MS liquid medium, and mix well by pipetting. Add the resuspended bacterial solution to the tissue culture flask containing MS liquid medium and adjust the concentration OD600 to between 0.6 and 0.8.

[0080] (6) In a clean bench, use sterile scissors to cut sterile tobacco leaves and place them on a petri dish. Use a sterile scalpel to remove the veins and cut them into pieces about 1 cm long. 2 The square pieces are placed in a petri dish containing sterilized water and left to use.

[0081] (7) After cutting an appropriate amount of leaves, put them into a tissue culture bottle containing an appropriate concentration of Agrobacterium tumefaciens solution, wrap them with tin foil and newspaper to protect them from light, and shake them at 120 rpm at room temperature.

[0082] (8) After 15 minutes, use filter paper (sterilized and cooled) to absorb the bacterial solution on the leaves in a clean bench, spread them evenly on the tobacco subculture medium (lower epidermis facing up), and culture them in the dark at room temperature.

[0083] (9) Two days later, the leaves were picked up with sterile tweezers in the clean bench and transferred to the tobacco differentiation medium and cultured until new shoots emerged.

[0084] (10) Cut off the callus tissue with a sterilized scalpel in a clean bench and place it in a new tobacco differentiation medium for further culture. After the seedlings grow, cut them off and place them in a tobacco rooting medium for culture.

[0085] (11) After the tobacco plants grow roots, RNA is extracted for identification. Positive plants are transferred to nutrient pots for growth after hardening, and seeds are harvested and planted for second-generation screening.

[0086] Following the method in Example 2, total RNA was extracted from WT and transgenic tobacco and reverse transcribed to obtain cDNA. Using the tobacco NtActin gene as a control, the reaction was carried out according to 2^ -ΔΔCt The relative expression levels of the VvLBDId5 gene in WT and transgenic tobacco were calculated.

[0087] The primer sequences are as follows:

[0088] VvLBDId5 (qRT-PCR)-F: 5'-AAGACACATGTTGGGGCCTC-3';

[0089] VvLBDId5 (qRT-PCR)-R: 5'-GGCTTTTCCGGGACTTCCAT-3';

[0090] NtActin (qRT-PCR)-F: 5'-AGTGGAGGTTCTACCATGTTTCCT-3';

[0091] NtActin (qRT-PCR)-R: 5'-CACTGTATTTCCTTTTCAGGTGGTG-3'.

[0092] Depend on Figure 3 It can be seen that three VvLBDId5 tobacco lines were obtained through genetic transformation. The expression level of the VvLBDId5 gene in the three tobacco lines was significantly higher than that in the wild type (WT), indicating that three VvLBDId5 transgenic tobacco lines were successfully obtained.

[0093] Example 4

[0094] Verification of drought resistance function of VvLBDId5 gene in tobacco:

[0095] Phenotypic observation and related physiological index determination of WT and transgenic tobacco under drought stress:

[0096] Four-week-old WT and transgenic tobacco plants with uniform growth were selected as control (watered) and drought stress treatment (no watering). After 7 days, the growth of tobacco was observed, and the MDA content and relative conductivity were measured.

[0097] Depend on Figure 4 As shown in A, WT and transgenic tobacco showed no significant phenotypic difference under control conditions, both exhibiting bright green, well-developed, and lush leaves; however, under drought stress conditions, such as Figure 4 As shown in Figure B, the leaves of wild-type tobacco (WT) withered and had a dull color, while the leaves of transgenic tobacco remained largely unfolded and grew vigorously. The degree of wilting of transgenic tobacco leaves was significantly lower than that of WT.

[0098] Drought stress leads to excessive accumulation of reactive oxygen species (ROS) in plants, causing severe oxidative damage. This, in turn, induces membrane lipid peroxidation, generating harmful degradation products such as molecularly active organic oxidase (MDA), and exacerbates electrolyte leakage. The more severe the stress, the higher the MDA content and relative conductivity. Figure 4 As shown in C and 4D, after drought stress treatment, the MDA content and relative conductivity of transgenic tobacco leaves were significantly lower than those of WT, indicating that the membrane system of transgenic materials is more stable under drought stress conditions.

[0099] The above results indicate that overexpression of VvLBDId5 in tobacco significantly enhances the tobacco's tolerance to drought stress.

[0100] Performance testing

[0101] 1. Method for determining MDA content

[0102] (1) Prepare a 10% trichloroacetic acid solution (10 g TCA, dissolved in distilled water and brought to a final volume of 100 mL) and a 0.6% thiobarbituric acid solution (0.6 g TBA, dissolved in 10% TCA solution and brought to a final volume of 100 mL).

[0103] (2) Weigh about 0.2-0.5 g of fresh plant tissue, freeze it quickly with liquid nitrogen, and place it in a pre-cooled mortar. Add a small amount of quartz sand and 2-3 mL of pre-cooled 10% TCA, and grind it into a homogenate under ice bath conditions. Transfer the entire homogenate to a centrifuge tube, and then rinse the mortar with a small amount of extraction solution. Combine the rinsing solutions (usually control the tissue mass: extraction solution volume = 1:10).

[0104] (3) Centrifuge the centrifuge tube at 4℃ and 10000-12000 rpm for 15-20 min, and carefully aspirate the supernatant. This is the extract of the sample to be tested.

[0105] (4) Take an appropriate amount of the test solution, add an equal volume of 0.6% (w / v) thiobarbituric acid solution, mix well, and react accurately in a 95℃ water bath for 30 min. Immediately stop the reaction in an ice bath (use an equal volume of extract as a blank control).

[0106] (5) After cooling the reaction solution to room temperature, measure its absorbance at wavelengths of 532 nm, 600 nm and 450 nm.

[0107] (6) MDA concentration C (µmol / L) = [6.45 × (A 532 -A 600 -0.56×A 450 × Dilution factor;

[0108] MDA content (µmol / g FW) = [C(µmol / L)×Vtotal(L)] / [W(g)×1000].

[0109] 2. Methods for measuring relative conductivity

[0110] (1) Select tobacco leaves from the control and drought treatment, wash their surface with deionized water, and take samples using a punch (avoiding the midrib). Accurately weigh 0.2 g of the sample and place it in a clean test tube or beaker.

[0111] (2) Add 20 mL of deionized water to the test tube to ensure that the sample is completely submerged. Let it stand at room temperature for 1 h, gently shaking it during the process. Then use a conductivity meter to measure the conductivity value of the soaking solution and record it as EC1.

[0112] (3) Seal the test tube containing the sample and soaking solution, place it in a boiling water bath and heat for 30 min, take it out and cool it to room temperature, shake it again, measure the conductivity value and record it as EC2.

[0113] (4) Using an equal amount of deionized water as a blank, the boiling and cooling steps were performed in the same way, and the conductivity value EC0 was measured.

[0114] Relative conductivity = [(EC1-EC0) / (EC2-EC0)]×100%

[0115] 3. DAB and NBT staining methods

[0116] Mature leaves of WT and transgenic tobacco with consistent physiological states were selected. Uniformly sized circular leaves were removed using a perforator. The leaves were placed in DAB and NBT solutions and treated in a vacuum desiccator for 20 min. After treatment, they were allowed to stand in the dark for 8 h or 4 h, and then decolorized in an 85℃ water bath until chlorophyll was completely removed and the background was transparent. The DAB product H2O2 was brownish-red, and the NBT product O2... - It is blue.

[0117] 4. H2O2 Determination Method

[0118] (1) Extraction and processing: Accurately weigh 0.5 g of fresh tobacco leaves and immediately place them in a pre-cooled mortar. Add 5 mL of pre-cooled acetone and grind rapidly into a homogenate in an ice bath. Transfer the homogenate to a centrifuge tube, rinse the mortar with 2-3 mL of extraction solution and combine the solutions. Centrifuge at 10,000 g for 15 min at 4℃, and collect the supernatant as the extract to be tested.

[0119] (2) Preparation of the reaction system: Take a stoppered centrifuge tube and add 1.0 mL of the extract to be tested, 0.1 mL of 20% (v / v) titanium sulfate (a sulfuric acid solution of titanium tetrachloride), and 0.2 mL of concentrated ammonia in sequence. Vortex the mixture after each addition. Adding ammonia will form H2O2. - Yellow precipitate of titanium complex.

[0120] (3) Precipitation and washing: Centrifuge the above reaction solution at 10,000 g for 10 min at 4℃ and carefully discard the supernatant. Gently wash the yellow precipitate 2-3 times with 3-5 mL of pre-cooled acetone to remove interfering pigments and impurities. Centrifuge and discard the supernatant after each wash.

[0121] (4) Dissolution and determination: Add 3.0 mL of 1 M sulfuric acid to the washed precipitate, and shake or vortex until the precipitate is completely dissolved. Transfer the solution to a cuvette, and using 1 M sulfuric acid as a reference, measure its absorbance value A at a wavelength of 415 nm using a spectrophotometer. 415 .

[0122] (5) Prepare a standard curve: Prepare a series of H2O2 standard solutions of known concentrations (e.g., 0, 5, 10, 20, 40, 60 μM). Take 1.0 mL of the standard solution to replace the sample extract and follow steps (2)-(4) exactly. Plot the H2O2 concentration as the abscissa (X, μM) and the corresponding absorbance A. 415Using the ordinate (Y), a standard curve is plotted to obtain the linear regression equation Y=aX+b.

[0123] (6) Calculate the sample concentration: Calculate the measured sample absorbance A 415 Substitute the values ​​into the standard curve equation to calculate the concentration C (in μM) of H2O2 in the sample extract. Note: If the sample absorbance exceeds the linear range of the standard curve, it needs to be appropriately diluted with the extract and retested, and multiplied by the dilution factor D during the calculation.

[0124] (7) Calculate the content (μmol / g FW): Calculate the hydrogen peroxide content per gram of fresh weight sample using the following formula:

[0125]

[0126] In the formula:

[0127] C: H2O2 concentration in the extract (μmol / mL) calculated from the standard curve.

[0128] Vt: Total volume of sample extract (mL).

[0129] m: Fresh weight of the sample (g).

[0130] 5. O2 - Generation rate measurement method

[0131] (1) Extraction and incubation: Prepare 50 mM, pH 7.8 phosphate buffer (pre-cooled). Accurately weigh 0.5 g of fresh tobacco leaves and add pre-cooled phosphate buffer at a mass (g): volume (mL) ratio of 1:10. Grind the mixture in an ice bath to homogenize. Centrifuge the homogenate at 12,000 g for 20 min at 4°C and collect the supernatant. Immediately take 1.0 mL of the supernatant, add 1.0 mL of 50 mM phosphate buffer (pH 7.8) and 1.0 mL of 1 mM hydroxylamine hydrochloride (prepared with the same buffer), mix well, and incubate precisely at 25°C for 1 h. In this step, the endogenously generated O2 in the sample... - It reacts quantitatively with hydroxylamine to produce nitrite.

[0132] (2) Colorimetric reaction: After incubation, add 1.0 mL of 17 mM p-aminobenzenesulfonic acid (dissolved in glacial acetic acid:water = 3:1) and 1.0 mL of 7 mM α-naphthylamine (dissolved in glacial acetic acid:water = 3:1) to the above reaction solution in sequence. Mix thoroughly and let stand at 25℃ in the dark for 20 min to develop color.

[0133] (3) Measurement of absorbance: Transfer the colorimetric reaction solution to a cuvette, and use a sample tube without hydroxylamine hydrochloride (replaced with an equal volume of buffer solution) as a reference to measure the absorbance value A at a wavelength of 530 nm. 530 .

[0134] (4) Set up controls: Set up a sample background control (without adding hydroxylamine during incubation) and a reagent blank control (without adding sample supernatant) to correct non-specific color development.

[0135] (5) Prepare a standard curve: Prepare a series of standard solutions (e.g., 0, 5, 10, 20, 40, 60 μmol / L) using sodium nitrite. Take 1.0 mL of the standard solution and follow steps (1)-(3) exactly. Plot the NaNO2 concentration (X, μmol / L) on the x-axis, corresponding to A 530 Using (Y) as the ordinate, a standard curve is plotted, yielding the regression equation Y = aX + b.

[0136] (6) Calculate NO2 - Production volume: Net absorbance value ΔA after subtracting background and blank from the sample tube. 530 Substituting into the standard curve equation, the amount of NO2 generated in the incubation reaction solution was calculated. - Concentration C (unit: μmol / L).

[0137] (7) Calculate the superoxide anion generation rate [μmol / (min·g) FW]: Calculate the O2 generated per gram of fresh weight sample per minute using the following formula. - rate:

[0138]

[0139] In the formula:

[0140] C: NO2 obtained in step (6) - Concentration (μmol / L);

[0141] Vi: Total volume of the incubation reaction system (L);

[0142] Vt: Total volume of sample extract (L);

[0143] Vs: Volume of sample supernatant used for incubation reaction (L);

[0144] T: Incubation time (min);

[0145] m: Fresh weight of the sample (g).

[0146] 6. SOD enzyme activity assay:

[0147] (1) Extraction of crude SOD enzyme solution: Prepare extraction solution (0.1 M, pH 7.8 phosphate buffer, containing 2% PVP and 1 mM EDTA). Accurately weigh 0.5 g of fresh tobacco leaves and add them to the pre-cooled extraction solution at a ratio of mass (g):volume (mL) = 1:10. Grind and homogenize in an ice bath. Centrifuge at 12,000 g for 20 min at 4℃, collect the supernatant and place it on ice for testing.

[0148] (2) Preparation of reaction system: Add the following to a cuvette (optical path 1 cm, total volume 3.0 mL): 1.8 mL 50 mM phosphate buffer (pH 7.8), 0.3 mL 10 mM xanthine, 0.3 mL 7.5 mM NBT solution, and 0.1 mL crude enzyme supernatant.

[0149] (3) Start the reaction: After incubating the above mixture at 25°C for 5 min, add 0.5 mL of 0.4 U / mL xanthine oxidase working solution and mix thoroughly immediately to start the reaction.

[0150] (4) Measurement: Quickly place the reaction system in a 25℃ constant temperature spectrophotometer, monitor and record the change in absorbance within 20 min after the start of the reaction at a wavelength of 560 nm.

[0151] (5) Set up controls: Set up two control tubes: a) Maximum reduction tube (V0): Use an equal volume of extract instead of crude enzyme solution; b) Blank tube (V b ): Replace xanthine oxidase with an equal volume of buffer solution.

[0152] (6) Calculate the reaction rate and inhibition rate: Calculate the slope of the absorbance change of each tube over time, i.e., the reaction rate (ΔA). 560 / min). Calculate the inhibition rate of the sample on the NBT reduction reaction according to the formula:

[0153]

[0154] in, , , These represent the reaction rates of the maximum reduction tube, the sample tube, and the blank tube, respectively.

[0155] (7) Calculation of enzyme activity (U / mg FW): The amount of enzyme required to inhibit 50% of the photochemical reduction of NBT is defined as one unit of enzyme activity (U). Calculate the SOD activity per milligram of fresh weight sample using the following formula:

[0156]

[0157] In the formula:

[0158] Inhibition rate: The result of step (6) (%);

[0159] V t : Total volume of crude enzyme extract (mL);

[0160] D: The possible dilution factor of the crude enzyme solution during the assay (if undiluted, then D=1);

[0161] V s : Volume of crude enzyme solution added to the reaction system (mL);

[0162] m: Fresh weight of the sample (g).

[0163] 7. POD enzyme activity assay:

[0164] (1) Extraction of crude POD enzyme solution: Prepare extraction solution (0.1 M, pH 6.0 phosphate buffer, containing 2% PVP and 1 mM EDTA). Accurately weigh 0.5 g of fresh tobacco leaves and add them to the pre-cooled extraction solution at a mass (g): volume (mL) ratio of 1:10. Homogenize in an ice bath. Centrifuge at 12000 g for 20 min at 4℃, collect the supernatant and place it on ice for testing.

[0165] (2) Preparation of reaction system: Add the following to the cuvette (optical path 1 cm, total volume 3.0 mL): 2.7 mL 50 mM phosphate buffer (pH 6.0), 0.1 mL 150 mM guaiacol, and 0.1 mL crude enzyme supernatant.

[0166] (3) Start-up and measurement: Finally, add 0.1 mL of 50 mM H2O2 to start the reaction and mix immediately. Quickly place the system in a spectrophotometer at 30℃, and monitor and record the absorbance (A) within the first 1-3 minutes at a wavelength of 470 nm. 470 The linear change of ).

[0167] (4) Set up a background control: Set up a reaction tube and use an equal volume of buffer solution instead of H2O2 or use inactivated enzyme solution to correct for background changes.

[0168] (5) Calculate the reaction rate: Calculate the slope of the absorbance of the sample tube (after subtracting the background) over time, i.e., V = ΔA 470 / min.

[0169] (6) Calculate enzyme activity (U / mg FW): Calculate the POD activity per milligram of fresh weight of plant tissue using the following formula.

[0170]

[0171] In the formula:

[0172] V: Reaction rate, ΔA 470 / min;

[0173] Vt: Total volume of the reaction system (L);

[0174] ε: Molar extinction coefficient of the product, 26.6 L·mmol -1 ·cm -1 ;

[0175] d: Cuvette path length (cm);

[0176] Vs: Volume of crude enzyme solution added (L);

[0177] m: Fresh weight of the sample (g).

[0178] DAB and NBT staining are based on specific ROS-mediated redox reactions, producing insoluble colored precipitates. The accumulation of these precipitates is positively correlated with staining depth / area. DAB specifically detects H₂O₂ accumulation, while NBT specifically detects O₂. - Accumulation. To verify whether VvLBDId5 overexpression affects ROS accumulation in tobacco, DAB and NBT specific staining techniques were used to analyze H2O2 and O2 in WT and transgenic tobacco lines. - The results showed no significant difference between WT and transgenic tobacco in the control group, but under drought stress treatment, WT leaves stained significantly darker (5A, B), indicating that its reactive oxygen species accumulation was higher than that of transgenic tobacco. Quantitative detection further confirmed that after drought stress treatment, the H2O2 content and O2 content in WT increased significantly. - The generation rate was significantly higher than that of the transgenic lines ( Figure 5 C, D). Antioxidant enzyme systems are crucial for ROS scavenging. Experimental results showed that after drought stress treatment, the activities of SOD and POD enzymes in transgenic tobacco were significantly higher than those in WT (C, D). Figure 5 E, F). In summary, these results indicate that VvLBDId5 effectively scavenges excess ROS by enhancing the activity of antioxidant enzymes in tobacco, thereby improving the plant's resistance to drought stress.

[0179] In conclusion, the results of the verification of the stress resistance function of the VvLBDId5 gene in tobacco fully demonstrate that the VvLBDId5 gene is a drought-related gene, and that overexpression of this gene can improve the drought resistance of the plant.

[0180] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A grape VvLBDId5 gene, characterized in that, The VvLBDId5 gene can regulate drought resistance in plants, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein, characterized in that, The protein is obtained by translating the VvLBDId5 gene, the amino acid sequence encoded by the nucleotide sequence of the VvLBDId5 gene being shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, Containing the VvLBDId5 gene as described in claim 1, the expression vector can regulate the drought resistance of plants.

4. A genetically engineered bacterium, characterized in that, The expression vector as described in claim 3 was transferred into Agrobacterium to obtain the product.

5. The application of an expression vector in regulating plant drought resistance, characterized in that, This is achieved by overexpressing the VvLBDId5 gene as described in claim 1 in plants.

6. The application of a genetically engineered bacterium in regulating plant drought resistance, characterized in that, This is achieved by overexpressing the VvLBDId5 gene as described in claim 1 in plants.

7. The application according to claim 5 or 6, characterized in that, The primers have sequences as shown in SEQ ID NO.3-4.

8. The application according to claim 5 or 6, characterized in that, The plant in question is either grape or tobacco.

9. A method for screening drought-resistant grapes, characterized in that, Screening was performed by detecting the expression level of the VvLBDId5 gene.

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