WRKY40 transcription factor of peanut for promoting drought tolerance and early flowering in plants and its application

Abstract

The present invention belongs to the field of molecular biology and genetic engineering technology, and specifically relates to a WRKY40 transcription factor of peanut that promotes drought tolerance and early flowering in plants and its application. The nucleotide sequence of the WRKY40 transcription factor of peanut is shown in SEQ ID NO.1; by overexpressing the WRKY40 transcription factor of peanut in plants, the drought tolerance of transgenic plants is improved and the flowering time is advanced; the plants include Arabidopsis. The present invention can provide theoretical support for the subsequent clarification of the regulatory mechanism of plant response to drought stress and early flowering, and also lay a foundation for the genetic improvement of drought tolerance and flowering habits and the creation of new germplasm.

Description

Technical Field

[0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to a peanut WRKY40 transcription factor for promoting drought resistance and early flowering of plants and an application thereof. Background Art

[0002] Wild plants of the genus Arachis have good environmental adaptability and ornamental value, and are often used as lawn grass; because their aboveground parts have high crude protein and low crude fiber content, they can also be used as forage. Peanut is a perennial wild diploid plant of the genus Arachis, one of the ancestral species of cultivated peanuts, and has good drought resistance. Exploring and utilizing the drought-resistant genes of peanuts has guiding significance for improving the drought resistance of cultivated peanuts. In addition, if the peanuts used as lawn grass can be made to bloom earlier, their ornamental value can be improved, thereby increasing their commercial value.

[0003] Drought stress affects the yield of peanuts. Drought stress forces peanuts to shorten their roots, reduce leaf area, close stomata, decrease glandular density, and reduce transpiration. Field studies have found that drought stress affects the reproductive growth of peanuts, mainly affecting the development of fruit needles, pods, and biomass accumulation during the filling period, ultimately reducing peanut production by about 30%. By evaluating the responses of different peanut varieties in my country to drought, it was found that drought stress reduced the plant height, yield, and stem and leaf growth of peanuts. Drought stress significantly affects the leaf tissue structure, reduces the leaf area per plant, functional leaf area, stomatal conductance, photosynthetic rate, and transpiration rate, and increases specific leaf weight. Therefore, it is urgent to improve the drought tolerance of peanuts.

[0004] WRKY is an important transcription factor in plants and is widely involved in plant physiological development as well as biotic and abiotic stress processes. However, it is still unclear whether WRKY in peanut has the function of regulating plant drought tolerance and flowering habits. Summary of the invention

[0005] In order to solve the problem of lagging application of WRKY transcription factors in regulating plant drought tolerance and flowering habits, the present invention aims to clarify the function of WRKY40 transcription factors in promoting plant drought tolerance and early flowering. To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an Arachis WRKY40 transcription factor that promotes drought tolerance and early flowering of plants. The nucleotide sequence of the Arachis WRKY40 transcription factor is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a recombinant expression vector comprising the peanut WRKY40 transcription factor.

[0008] Furthermore, the recombinant expression vector is obtained by inserting the AdWRKY40 gene between the Nco I and Pml I sites of the pBI121 overexpression vector plasmid.

[0009] In a third aspect, the present invention provides a recombinant bacterium comprising the recombinant expression vector.

[0010] Furthermore, the recombinant bacteria are obtained by transferring the recombinant expression vector into Agrobacterium competent cells EHA105.

[0011] In a fourth aspect, the present invention provides the use of the peanut WRKY40 transcription factor in promoting drought tolerance and early flowering in plants, wherein the plants include Arabidopsis thaliana.

[0012] In a fifth aspect, the present invention provides the use of the recombinant expression vector in promoting drought tolerance and early flowering in plants, wherein the plants include Arabidopsis thaliana.

[0013] In a sixth aspect, the present invention provides the use of the recombinant engineered bacteria in promoting drought tolerance and early flowering of plants, wherein the plants include Arabidopsis thaliana.

[0014] The research group of the applicant of the present invention found through preliminary research that peanut was subjected to different degrees of drought stress treatment, and the expression pattern of some members of the WRKY gene family was analyzed by qRT-PCR. The results showed that the peanut WRKY40 transcription factor was upregulated and differentially expressed after drought stress. Therefore, the applicant speculates that the peanut WRKY40 transcription factor can participate in the drought tolerance process, and Arabidopsis thaliana overexpressing the peanut WRKY40 transcription factor shows an early flowering phenotype under long-day conditions. This study lays a theoretical foundation for genetic improvement and new germplasm creation to improve plant drought tolerance and promote flowering.

[0015] The coding region of the WRKY40 transcription factor of peanut is 1098 bp in length and encodes 365 amino acids. The present invention uses a bioinformatics prediction method to identify the peanut WRKY40 transcription factor in response to drought stress in the peanut response RNA-seq data.

[0016] The engineered bacteria can be understood as the engineered bacteria used by those skilled in the art in the transgenic process, such as Agrobacterium competent cells EHA105. However, with the development of science and technology, the selection of the engineered bacteria may change, or the application fields for non-transgenic purposes also involve the use of vectors and engineered bacteria, but only those containing the genes or vectors of the present invention are within the scope of protection of the present invention.

[0017] The peanut WRKY40 transcription factor improves the drought resistance of plants by regulating the stomatal conductance, germination rate, proline content and active oxygen content of plants.

[0018] Under drought stress conditions, compared with wild-type Arabidopsis, the germination rate of transgenic Arabidopsis overexpressing the WRKY40 transcription factor was increased, thereby reducing the water loss rate; the stomatal conductance of the transgenic Arabidopsis was significantly lower than that of the wild-type Arabidopsis; the content of reactive oxygen in the transgenic Arabidopsis was reduced, and the content of proline was increased.

[0019] There are differences in flowering time between wild-type and transgenic plants overexpressing the WRKY40 transcription factor in peanut. Under both normal growth and drought treatment conditions, transgenic plants overexpressing the WRKY40 transcription factor in peanut flowered earlier than wild-type plants. However, drought treatment delayed the flowering time of transgenic plants compared with transgenic plants under normal growth conditions. In contrast, there was no significant difference in flowering time between wild-type plants under the two treatment conditions.

[0020] The method for constructing a transgenic plant overexpressing the peanut WRKY40 transcription factor in the present invention specifically includes cloning a target gene, constructing a target gene overexpression vector, transforming the target gene into Agrobacterium, transforming the target gene into Arabidopsis, identifying positive strains, observing phenotypes, and determining early flowering and drought-resistant materials.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention is the first to discover the role of overexpressed WRKY40 transcription factor in regulating drought tolerance and early flowering of Arabidopsis. By overexpressing the WRKY40 transcription factor in Arabidopsis, the tolerance of transgenic Arabidopsis to drought stress is enhanced and early flowering occurs. By comparing Arabidopsis plants overexpressing the gene with wild-type Arabidopsis plants, it was found that overexpression of the WRKY40 transcription factor in the peanut will lead to stomatal closure, which can increase the germination rate of the plant and reduce the water loss rate, but does not affect the development of root length; transgenic Arabidopsis can remove ROS, promote stomatal closure, accumulate proline, and bloom early. It shows that overexpression of the WRKY40 transcription factor in the peanut can effectively enhance the drought tolerance of Arabidopsis and promote flowering, which can provide theoretical support for the subsequent clarification of the regulatory mechanism of plant response to drought stress and early flowering, and also lay the foundation for the genetic improvement of drought tolerance and flowering habits and the creation of new germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis diagram is the PCR identification electrophoresis diagram of the transgenic plant overexpressing AdWRKY40 in the present invention. Band 1 is the DNA molecule marker; bands 2-7 are AdWRKY40 transgenic positive plants.

[0024] Figure 2It is a statistical graph of germination rate of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana in the present invention, wherein A is a germination phenotype graph of wild-type and transgenic strains under normal growth conditions, B is a germination phenotype graph of wild-type and transgenic strains under drought conditions, C is a statistical graph of germination rate of wild-type and transgenic strains under normal growth conditions, and D is a statistical graph of germination rate of wild-type and transgenic strains under drought conditions. In A and B, 1-4 are wild-type Arabidopsis thaliana CK, transgenic strain Ad40-2, transgenic strain Ad40-4, and transgenic strain Ad40-5, respectively.

[0025] Figure 3 The figure is a statistical graph of water loss rate of detached leaves of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana under drought treatment conditions in the present invention.

[0026] Figure 4 This is a graph showing the results of measuring drought tolerance related indicators of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana under drought treatment conditions, wherein A is O2 - A is a statistical chart of H2O2 concentration, C is a statistical chart of SOD content, D is a statistical chart of CAT content, and E is a statistical chart of proline content.

[0027] Figure 5 Graphs showing the stomatal conductance measurement results of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana in the present invention, wherein A is a stomatal conductance phenotype graph, and B is a stomatal aspect ratio statistical graph.

[0028] Figure 6 This is a statistical diagram of the survival rates of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana treated with long-term drought in the present invention.

[0029] Figure 7 Statistical graphs of flowering phenotype and flowering time of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana in the present invention, wherein A is a flowering phenotype graph and B is a flowering time statistical graph. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] The present invention provides a new use of the peanut WRKY40 transcription factor. The peanut WRKY40 transcription factor is also called the AdWRKY40 gene. The nucleotide sequence of the AdWRKY40 gene is shown in SEQ ID NO.1:

[0032]

[0033] The amino acid sequence of the protein expressed by the AdWRKY40 gene is shown in SEQ ID NO.2:

[0034] MSDEAKQLLYQDLILDHHQNQNIIAGGGRLSNNMFCEKQFPSSSSSSSQVAFDPSSYMSFTECLQGGMDYNSLATSFGLSPSSSEVFSSIEGNNNNQKPEGDVLGTGGGGGGGSETLATLNSSSSSEAGGEEDSGKSNKDSQVKEEAAGETSKKGNKENNNNKKKGEKKQKEPRFAFMTK SEVDHLEDGYRWRKYGQKAVKNSPYPRSYYRCTTQKCSVKKRVERCYRDPTTVITTYEGQHNHPVPTSLRGNAASMFTPSMLSISAPTPLLSSAGHHDLALFAPLSHHHNQYSAASGSQSSLLFHHHQSINNNVNNNNINNNNNNSLLLLNHHHHPYNNQQLPPEYGLLQDMLPSMMFLKQEP.

[0035] Example 1: Cloning of the Peanut AdWRKY40 gene

[0036] The present invention uses peanut as the test material, which is provided by the laboratory of Grassland College of Qingdao Agricultural University.

[0037] 1. Primer design

[0038] Primer 5.0 software was used to analyze and design primers for amplifying the AdWRKY40 gene. The designed primers and their nucleotide sequences are shown below:

[0039] AdWRKY40-F: 5′-ATGTCTGATGAAGCTAAACAACTTCTC-3′, as shown in SEQ ID NO. 3;

[0040] AdWRKY40-R: 5′-TCATGGCTCTTGCTTGAGGAA-3′, as shown in SEQ ID NO.4.

[0041] 2. RNA Extraction

[0042] The total RNA of the test materials was extracted using the FastPure Plant Total RNA Isolation Kit. The entire operation process was carried out according to the instructions of the RNA extraction kit, and then the extracted total RNA was used as a template for reverse transcription to obtain cDNA.

[0043] The reverse transcription reaction procedure is as follows:

[0044] The first step was to denature the RNA template by adding 1 μL RNA and 7 μL ddH2O to an RNase-free centrifuge tube, heating at 65°C for 5 min, rapidly cooling on ice, and leaving it on ice for 2 min.

[0045] The second step is to remove genomic DNA. Add 2 μL of 5×gDNA wiper Mix to the mixture in the previous step, mix well with a pipette, and react at 42°C for 2 minutes.

[0046] Step 3: Prepare the first-strand cDNA synthesis reaction solution. Add 2 μL 10×RT Mix, 2 μL HiScript III Enzyme Mix, and 1 μL Oligo(dT) to the mixture in the previous step. 20 VN and 5 μL ddH2O were added, and the mixture was mixed by gently pipetting. Finally, the reaction was carried out in an RT-PCR instrument at 50°C for 45 min.

[0047] 3. Gene cloning

[0048] The obtained cDNA was used as a template and PCR amplification was performed using primers AdWRKY40-F and AdWRKY40-R to obtain a target fragment of 1098 bp.

[0049] The PCR reaction system: 1 μL of cDNA template, 5 μL of 2×Phanta Flash Master Mix, 0.5 μL of forward and reverse primers, and finally add ddH2O to 10 μL;

[0050] PCR reaction program: 98℃ pre-denaturation for 30 sec; 98℃ denaturation for 10 sec, 60℃ annealing for 5 sec, 72℃ extension for 4 sec / kb, 34 cycles; 72℃ full extension for 5 min. The product was subjected to agarose gel electrophoresis.

[0051] 4. Gel recovery and sequencing

[0052] The PCR amplification product was recovered by gel gel and sequenced. The sequencing sequence is shown in SEQ ID NO.1.

[0053] The coding region sequences of the AdWRKY40 genes were aligned and the amino acid sequence of the encoded protein was shown in SEQ ID NO.2.

[0054] Example 2: Construction of recombinant plant overexpression vector

[0055] 1. Double enzyme digestion of overexpression vector

[0056] After gel recovery, the recovered PCR amplification product and pBI121 were double-digested with Nco I and Pml I restriction endonucleases, respectively. For specific steps, please refer to the NEB manual.

[0057] Use gel electrophoresis to detect the enzyme cleavage sequence, and use a product purification kit to purify and recover the target band. For specific operations, refer to the instructions of the product purification kit. Store the purified PCR amplification product in a -20°C refrigerator.

[0058] Among them, product purification kit: FastPure® Gel DNA Extraction Mini Kit Product purification kit: Novazonics, Nanjing, China.

[0059] 2. Homologous recombination connects the target gene and the overexpression vector

[0060] Use a homologous recombination kit to connect the target gene fragment to the overexpression vector after double restriction digestion. For specific steps, refer to the instructions of the homologous recombination kit.

[0061] Among them, homologous recombination kit: ClonExpress® Ultra One Step Cloning Kit Homologous recombination kit: Novozymes, Nanjing, China.

[0062] PCR reaction system: 2 μL target fragment, 3 μL linear vector, 5 μL 2*ClonExpress Mix;

[0063] PCR reaction program: 50℃ for 30min, then immediately incubate at 4℃.

[0064] 3. Transformation of recombinant plasmid into Escherichia coli

[0065] The target band was connected to the double-enzyme-digested pBI121 vector, and the ligation product was transferred into the E. coli competent cell DH5α. For specific steps, please refer to the instruction manual of the E. coli competent cell DH5α.

[0066] Among them, Escherichia coli competent cells DH5α were provided by Shanghai Weidi Biotechnology Co., Ltd.

[0067] After transformation, the E. coli liquid was spread on LB solid medium containing 50 mg / L kanamycin and inverted in a 37°C incubator for 36 hours. Single clones were picked with a sterile pipette tip and identified and sequenced by PCR using pBI121-F / R universal primers.

[0068] Among them, the pBI121-F / R universal primer and its nucleotide sequence are as follows:

[0069] pBI121-F: 5′-GATATCTCCACTGACGTAAGG-3′, as shown in SEQ ID NO. 5;

[0070] pBI121-R: 5′-GCGATCCAGACTGAATGC-3′, as shown in SEQ ID NO.6.

[0071] The monoclonal bacterial solution with correct sequencing was activated, preserved and plasmid extracted. For specific steps, please refer to the instructions for plasmid extraction.

[0072] Among them, FastPure Plasmid Mini Kit plasmid extraction kit: Novozymes, Nanjing, China.

[0073] LB medium: LB broth, model HB0128, Soao, Qingdao, China.

[0074] 4. Transformation of recombinant plasmid into Agrobacterium

[0075] The AdWRKY40 overexpression vector plasmid was transferred into the Agrobacterium competent cell EHA105. For specific steps, refer to the instructions of the Agrobacterium competent cell EHA105. The transformed Agrobacterium bacterial solution was spread on the LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and inverted in a 28°C incubator for 36 hours. Single clones were picked with a sterile pipette tip and identified by bacterial solution PCR using pBI121-F / R universal primers. The correctly identified single clone colonies were activated and maintained to obtain Agrobacterium containing the pBI121-AdWRKY40 recombinant vector.

[0076] Among them, Agrobacterium competent cells EHA105 were provided by Shanghai Weidi Biotechnology Co., Ltd.

[0077] Example 3: Screening and phenotypic analysis of AdWRKY40 transgenic Arabidopsis plants

[0078] 1. Arabidopsis plant preparation

[0079] Place 200 Arabidopsis seeds in a 1.5mL centrifuge tube in a clean bench, add 1mL ddH2O, shake and oscillate, and then place in a small centrifuge for centrifugation. Subsequently, add 1mL of 75% alcohol to disinfect for 30s, and wash again with ddH2O. Add 10% NaClO to disinfect for 6min, and shake the centrifuge tube by hand during this period. After washing 3 times with ddH2O, add 1mL ddH2O to soak and swell for 45min. Use a 1mL sterile pipette tip to absorb Arabidopsis seeds, evenly spread them on 1 / 2MS solid culture medium, and after germination at 4℃ for 3d, transfer the culture dish to a light incubator at 24℃, 16h light / 8h dark, and culture it. After growing 3 true leaves, it can be transplanted into the soil. Arabidopsis continues to grow in the soil, with normal watering and management during the period, until bolting, in preparation for genetic transformation.

[0080] Among them, 1 / 2MS solid culture medium; Agar powder: Bio-Toda, Beijing, China; MS powder: Model M519, PhytoTech, USA.

[0081] 2. Preparation of infection solution

[0082] The Agrobacterium cells containing the pBI121-AdWRKY40 recombinant vector were picked up and cultured overnight in LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin on a constant temperature shaker at 28°C and 200 rpm until the OD 600 OD = 0.8. Take 200 mL of the shaken bacterial solution and put it into a centrifuge tube. Centrifuge at 24°C and 8000 rpm for 10 min to collect the bacteria. Pour away the supernatant after centrifugation and add the 5% sucrose solution prepared in advance. Mix and resuspend until the OD is 0.8. 600 The pH value was about 1.0, and a resuspended bacterial solution was prepared. Then, 0.03% of the surfactant Sliwet-77 was added to obtain an infection solution for use.

[0083] Among them, the 5% sucrose solution is prepared from the 1 / 2MS solution. The formula registration form of the 1 / 2MS solution is to add 2.22g of MS powder and 8g of sucrose to a beaker, and then add ultrapure water to make up to 1L. Stir with a magnetic stirrer to fully dissolve it, and adjust the pH to 5.85.

[0084] Surfactant sliwet-77: Model S9430, Solebao, Beijing, China.

[0085] 3. Flower dip method to infect Arabidopsis

[0086] The Arabidopsis inflorescence was immersed in the infection solution for 60 seconds, taken out immediately, cultured in the dark for 24 hours, and then transferred to normal conditions for continued growth. After 7 days, a cotton swab was used to dip the infection solution and smeared on the Arabidopsis inflorescence for secondary infection.

[0087] After infection, Arabidopsis thaliana was managed normally until the seeds matured, and the mature seeds of transgenic Arabidopsis thaliana were harvested.

[0088] Among them, the Arabidopsis inflorescence refers to removing the fruit pods and blooming flowers, leaving only the unopened buds.

[0089] The normal management method / conditions of Arabidopsis thaliana are to cultivate Arabidopsis thaliana plants at 24°C, 16h light / 8h dark conditions, and use Hoagland nutrient solution to replenish water during this period. The formula of Hoagland nutrient solution is shown in Table 1:

[0090] Table 1 Nutrient solution formula

[0091]

[0092] 4. Identification of transgenic plants

[0093] After sterilizing the mature transgenic Arabidopsis seeds, evenly spread them on 1 / 2MS solid medium containing 50 μg / mL kanamycin. The specific steps are the same as those of the above-mentioned Arabidopsis planting method. After the Arabidopsis seedlings grow to 6 leaves, take the leaves with good growth, extract DNA using the CTAB method, use the wild-type Arabidopsis plants as negative controls, use AdWRKY40-F / pBI121-R primers, and identify positive strains by PCR amplification and agarose gel electrophoresis.

[0094] Among them, the steps of CTAB method for extracting DNA are as follows:

[0095] (1) Mix 40 mL of CTAB preheated at 65°C with 20 μL of RNase in a volume ratio of 1:2000.

[0096] (2) Take 0.2 g of leaves and put them into a 2 mL centrifuge tube, beat them into a homogenate, centrifuge briefly, and throw all the homogenate at the mouth of the centrifuge tube to the bottom of the centrifuge tube. Then add 700 μL of CTAB.

[0097] (3) Keep the tube in a 65°C water bath for 30 min, inverting the tube three times.

[0098] (4) Add 700 μL of DNA extraction solution and shake thoroughly to mix thoroughly. This step needs to be kept away from light to prevent DNA decomposition.

[0099] (5) Centrifuge the mixture at 13,000 rpm for 15 min at room temperature using a high-speed refrigerated centrifuge.

[0100] (6) Take 600 μL of supernatant, transfer it to a new centrifuge tube, add 400 μL of isopropanol, mix well and place at -20°C for 15 min.

[0101] (7) Centrifuge the above reaction mixture at 4°C and 13,000 rpm for 15 min. Remove the supernatant, add 400 μL of 75% ethanol, and wash three times.

[0102] (8) Centrifuge at 13,000 rpm for 5 min at room temperature to remove the upper layer of isopropanol and aspirate the excess liquid. Air dry in a clean bench and add 100 μL of ddH2O after air drying.

[0103] (9) Incubate at 4°C overnight to obtain DNA, then store at -20°C for later use.

[0104] The conditions for PCR amplification were as follows: Arabidopsis DNA was used as a template and 2× Phanta Flash Master Mix kit was used for RT-PCR identification.

[0105] PCR reaction system: Take 1 μL of DNA template, 5 μL of 2×Phanta Flash Master Mix, 0.5 μL each of AdWRKY40-F and pBI121-R, and finally add ddH2O to make up to 10 μL.

[0106] PCR reaction procedure: the first step, pre-denaturation at 98°C for 30 seconds; the second step, denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 5 seconds / kb, for 34 cycles; finally, complete extension at 72°C for 5 minutes.

[0107] The wild-type Arabidopsis plants were provided by the Laboratory of Grassland College of Qingdao Agricultural University.

[0108] The identification results are as follows Figure 1 As shown in the figure, the transgenic line showed a 1098 bp fragment, while the wild-type line did not, indicating that the AdWRKY40 overexpression vector had been successfully introduced into the Arabidopsis genome. Three homozygous transgenic lines were randomly selected for subsequent experiments.

[0109] The specific propagation steps are as follows: Place 200 Arabidopsis seeds in a 1.5mL centrifuge tube in a clean bench, add 1mL ddH2O, shake and oscillate, and then place in a small centrifuge for centrifugation. Subsequently, add 1mL of 75% alcohol to disinfect for 30s, and wash again with ddH2O. Add 10% NaClO to disinfect for 6min, and shake the centrifuge tube by hand during this period. After washing ddH2O 3 times, add 1mL ddH2O to soak and swell for 45min. Use a 1mL sterile pipette tip to absorb Arabidopsis seeds, evenly spread them on 1 / 2MS solid culture medium, and after germination at 4℃ for 3d, transfer the culture dish to a light incubator at 24℃, 16h light / 8h dark, and culture it. After growing 3 true leaves, it can be transplanted into the soil. Arabidopsis continues to grow in the soil, with normal watering and management during the period until the seeds mature, and the transgenic Arabidopsis seeds are harvested.

[0110] 5. Evaluation of drought tolerance and flowering habits of transgenic Arabidopsis

[0111] Wild-type and positive homozygous transgenic Arabidopsis seeds were surface disinfected with 10% sodium hypochlorite for 10 minutes, placed in 1 / 2MS medium containing 0mM, 100mM, 200mM and 300mM mannitol, cultured at 4℃ for 3 days, breaking seed dormancy, and placed in an incubator with 16h / 8h light and dark alternation for germination determination. The seeds were identified as germination when the cotyledons turned green. The germination rate was counted for 10 consecutive days, and 3 biological replicates were set, with 20 samples in each replicate. 0.5g of rosette leaves of the same size of wild-type and transgenic Arabidopsis were weighed and placed in a tissue culture incubator with a humidity of 28% and a light of 66%, for a total of 3 replicates, weighed every 20min, and the relative water loss rate was calculated for a total of 200min. Four-week-old wild-type and transgenic Arabidopsis were treated with PEG6000 at a mass volume concentration of 10% and 100µM ABA, respectively. According to the instructions of the kit of Beijing Solebaugh Technology Co., Ltd., hydrogen peroxide H2O2 and superoxide anion O were measured before and after stress. 2- , superoxide dismutase SOD, catalase CAT and proline content. The wild-type Arabidopsis was used as the control group, and three biological replicates were set. In addition, the length and width of the stomata of the lower epidermal cells of the wild-type and transgenic Arabidopsis leaves before and after PEG6000 treatment were measured using a fluorescent inverted microscope, and the fluorescence inverted microscope was used to take pictures, and 50 biological replicates were set. The four-week-old wild-type and transgenic Arabidopsis were subjected to 30 days of natural drought treatment, followed by 7 days of rehydration culture, and the survival rate of the wild-type and transgenic Arabidopsis was counted.

[0112] The results are as follows Figure 2As shown in the figure, under normal growth conditions, there was no difference in germination rate between wild-type and transgenic Arabidopsis, but under 300 mM mannitol stress, the germination rate of transgenic Arabidopsis was higher than that of wild-type Arabidopsis. Figure 3 As shown in Figure 2, the relative water loss rate of transgenic Arabidopsis is lower than that of wild-type Arabidopsis. Figure 4 As shown in Figure 2, under drought stress, transgenic Arabidopsis accumulated high concentrations of O 2- , low concentration of H2O2, high content of SOD enzyme and CAT enzyme. Compared with wild-type Arabidopsis, transgenic Arabidopsis contains high concentration of proline under drought stress. Figure 5 As shown in Figure 2, under normal growth conditions and 10% PEG6000 treatment for 3 days, the stomatal conductance of transgenic Arabidopsis was significantly lower than that of wild-type Arabidopsis. Figure 6 As shown, the wild-type and transgenic Arabidopsis were subjected to long-term drought treatment. After rehydration, the survival rate of the transgenic Arabidopsis was significantly higher than that of the wild-type Arabidopsis.

[0113] Wild-type and positive homozygous transgenic Arabidopsis seeds were surface-sterilized with 10% sodium hypochlorite for 10 minutes, placed in 1 / 2MS medium, cultured at 4°C for 3 days, breaking seed dormancy, and placed in an incubator with 16h / 8h light-dark alternation for germination determination. When the cotyledons of the seeds turn green, it is considered to have germinated, which is the first day of the growth cycle. After 1 week of normal growth, the seedlings were transplanted to the greenhouse and soil cultured. The flowering time of wild-type and transgenic plants was counted, and photos were taken at 3 weeks of age.

[0114] The results are as follows Figure 7 As shown, under normal growth and drought treatment conditions, AdWRKY40 transgenic plants flowered earlier than wild-type plants.

[0115] The above results indicate that AdWRKY40 is an important gene involved in regulating drought tolerance and early flowering in Arabidopsis, and can be used to regulate drought tolerance and early flowering in other plants.

[0116] Example 4: Application of the AdWRKY40 gene in genetic improvement breeding of flowering drought tolerance in peanut

[0117] In production practice, the above genes can be transformed into peanut or peanut explant cells, and then the transformed callus tissue can be cultivated into plants. Through transgenic methods, plant expression vectors can be used to transform explant cells to cultivate drought-resistant and early-flowering peanuts or peanuts.

[0118] In production practice, the above genes can also be used to enhance the efficiency and accuracy of breeding targets through molecular marker-assisted selection breeding methods. For example, molecular markers are used to associate the target gene with the drought-resistant and early flowering traits of peanut, and the presence of the target gene is detected to achieve the purpose of selecting the target trait. The present invention discovered the function and specific application of the AdWRKY40 gene in regulating drought-resistant and early flowering of plants, which provides a new gene resource for drought-resistant and early flowering breeding of peanut, and also lays a foundation for further analyzing the molecular mechanism of AdWRKY40 regulating drought-resistant and early flowering of plants.

[0119] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the technical equivalents of the present invention.

Claims

1. An application of peanut WRKY40 transcription factor in promoting early flowering of Arabidopsis thaliana, characterized in that: The nucleotide sequence of the peanut WRKY40 transcription factor is shown in SEQ ID NO.

1. By overexpressing the peanut WRKY40 transcription factor in Arabidopsis thaliana, the flowering of Arabidopsis thaliana is advanced.

2. The use according to claim 1, characterized in that: Overexpression of the peanut WRKY40 transcription factor in Arabidopsis thaliana is achieved by transforming Arabidopsis thaliana with a recombinant engineering bacterium containing the peanut WRKY40 transcription factor.

3. The use according to claim 2, characterized in that: The recombinant engineering bacteria is obtained by transferring the recombinant expression vector containing the peanut WRKY40 transcription factor into Agrobacterium competent cells EHA105.

4. The use according to claim 3, characterized in that: The recombinant expression vector is obtained by inserting the peanut WRKY40 transcription factor into the Nco I and Pml I sites of the pBI121 overexpression vector plasmid.

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