CN118726410A - WRKY40 transcription factor of peanut for promoting drought tolerance and early flowering in plants and its application - Google Patents

Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a cranberry WRKY40 transcription factor for promoting drought tolerance and early flowering of plants and application thereof. The nucleotide sequence of the cranberry WRKY40 transcription factor is shown in SEQ ID NO. 1; through over-expressing the cranberry WRKY40 transcription factor in the plant, the drought tolerance of the transgenic plant is improved, and the flowering time is advanced; the plant comprises arabidopsis thaliana. The invention can provide theoretical support for the subsequent elucidation of the regulation and control mechanism of the plant responding to drought stress and early flowering, and also lays a foundation for drought tolerance, flowering habit genetic improvement and new germplasm creation.

Description

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

Technical Field

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

Background Art

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 Peanut has guiding significance for improving the drought resistance of cultivated peanuts. In addition, if Peanut as a lawn grass can be made to bloom earlier, its ornamental value can be improved, thereby increasing its commercial value.

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.

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

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:

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

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

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

There are differences in flowering time between wild-type and transgenic plants overexpressing the WRKY40 transcription factor in peanut. Under 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.

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.

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

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 can lead to stomatal closure, increase the germination rate of the plant, reduce the water loss rate, but do 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 drought tolerance and promote flowering in Arabidopsis, 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 genetic improvement of drought tolerance and flowering habits and creation of new germplasm.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2 is a statistical graph of the 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 the wild type and transgenic strains under normal growth conditions, B is a germination phenotype graph of the wild type and transgenic strains under drought conditions, C is a statistical graph of the germination rate of the wild type and transgenic strains under normal growth conditions, and D is a statistical graph of the germination rate of the 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.

FIG3 is a statistical graph showing the 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.

4 is a graph showing the results of drought tolerance related index determination of AdWRKY40 overexpressing transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana under drought treatment conditions, wherein A is a statistical graph of O ₂ ^- concentration, B is a statistical graph of H ₂ O ₂ concentration, C is a statistical graph of SOD content, D is a statistical graph of CAT content, and E is a statistical graph of proline content.

FIG5 is a graph 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.

FIG. 6 is a statistical graph showing the survival rates of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana treated with long-term drought in the present invention.

FIG. 7 is a graph showing the flowering phenotype and flowering time of transgenic Arabidopsis thaliana overexpressing AdWRKY40 and wild-type Arabidopsis thaliana in the present invention, wherein A is a graph showing the flowering phenotype and B is a graph showing the flowering time. FIG.

DETAILED DESCRIPTION

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.

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:

ATGTCTGATGAAGCTAAACAACTTCTCTACCAAGACCTTATTCTAGATCATCATCAGAATCAGAATATTATTGCAGGAGGAGGAAGATTATCCAACAACATGTTCTGTGAGAAGCAGTTTCCATCATCATCATCATCACAAGTAGCGTTTGATCCATCTTCATACATGAGCTTCACTGAATGCCTTCAAGGAGGGATGGACTATAACTCGCTTGCAACTTCTTTTGGTTTGTCTCCTTCTTCATCGGAGGTCTTTTCATCCAT CGAAG GCAACAATAACAATCAAAAGCCCGAAGGTGATGTATTAGGAACTGGTGGTGGTGGTGGTGGAGGTAGTGAAACCCTTGCAACCCTGAACTCATCGATCTCTTCGTCATCATCTGAAGCCGGGGGCGAAGAAGATTCTGGAAAGAGCAACAAAGATAGCCAGGTCAAAGAAGAAGCAGCAGGAGAAACCTCTAAGAAGGGGAACAAGGAGAATAATAATAACAAGAAGAAAGGGGAGAAGAAGCAGAAGGAGCCAAGGTTTGCG TTCATGACAAAG ACGAGGTTGATCATCTTGAAGATGGATACCGATGGAGAAAATACGGACAGAAAGCCGTTAAGAATAGCCCTTTATCCAAGGAGCTACTACAGATGCACGACACAGAAGTGCAGTGTGAAGAAACGTGTGGAGAGGTGTTATCGGGATCCAACGACTGTGATAACAACCTATGAAGGTCAACACAACCATCCAGTCCCCACTTCTTTGAGAGGGAATGCGGCTTCAATGTTCACACCTTCCATGCTCTCCATCTCCGCCCC ACTCCCCTCCTCTC ATCCGCCGGACATCACGACCTCGCCCTTTTTGCTCCTCTCTCTCACCACCACAACCAATATTCCGCTGCTTCTGGATCACAATCGTCCTTATTGTTTCATCACCATCAGAGTATTAACAACAACGTTAATAACAACAATATTAATAATAATAATAACTCTCTTCTTCTTCAATCATCATCATCATCCGTATAATAATCAGCAGCTTCCTCCTGAATATGGCCTCCTTCAAGACATGCTCCCTTCCATGTTCCTCAAGCAAGAGCCATGA .

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

MSDEAKQLLYQDLILDHHQNQNIIAGGGRLSNNMFCEKQFPSSSSSSSQVAFDPSSYMSFTECLQGGMDYNSLATSFGLSPSSSEVFSSIEGNNNNQKPEGDVLGTGGGGGGGSETLATLNSSSSSEAGGEEDSGKSNKDSQVKEEAAGETSKKGNKENNNNKKKGEKKQKEPRFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPYPRSYYRCTTQ KCSVKKRVERCYRDPTTVITTYEGQHNHPVPTSLRGNAASMFTPSMLSISAPTPLLSSAGHHDLALPHAPLSHHHNQYSAASGSQSSLLFHHHQSINNNVNNNNINNNNNNSLLLLNHHHHPYNNQQLPPEYGLLQDMLPSMFLKQEP.

Example 1: Cloning of the Peanut AdWRKY40 gene

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

1. Primer design

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:

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

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

2. RNA Extraction

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.

The reverse transcription reaction procedure is as follows:

The first step is to denature the RNA template. After adding 1 μL RNA and 7 μL ddH ₂ O to an RNase-free centrifuge tube, the tube is heated at 65°C for 5 min, quickly cooled on ice, and left standing on ice for 2 min.

In the second step, genomic DNA was removed by adding 2 μL of 5×gDNA wiper Mix to the mixture in the previous step, mixing by pipetting, and reacting at 42°C for 2 min.

The third step is to prepare the first-strand cDNA synthesis reaction solution, add 2μL 10×RT Mix, 2μL HiScript III Enzyme Mix, 1μL Oligo(dT) ₂₀ VN and 5μL ddH ₂ O to the mixture in the previous step, and mix it by gently pipetting. Finally, react in RT-PCR instrument at 50℃ for 45min.

3. Gene cloning

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.

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 ddH ₂ O to make up to 10 μL;

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 products were subjected to agarose gel electrophoresis.

4. Gel recovery and sequencing

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

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.

Example 2: Construction of recombinant plant overexpression vector

1. Double enzyme digestion of overexpression vector

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.

Use gel electrophoresis to detect the restriction 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.

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

2. Homologous recombination connects the target gene and the overexpression vector

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.

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

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

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

3. Transformation of recombinant plasmid into Escherichia coli

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.

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

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.

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

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

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

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

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

LB medium: LB broth, model HB0128, Soao, Qingdao, China.

4. Transformation of recombinant plasmid into Agrobacterium

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.

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

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

1. Arabidopsis plant preparation

Place 200 Arabidopsis seeds in a 1.5mL centrifuge tube in a clean bench, add 1mL ddH ₂ O, 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 ddH ₂ O. Add 10% NaClO to disinfect for 6min, and shake the centrifuge tube by hand during this period. After washing 3 times with ddH ₂ O, add 1mL ddH ₂ O to soak and swell for 45min. Use a 1mL sterile pipette tip to absorb Arabidopsis seeds and evenly spread them on 1/2MS solid culture medium. 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.

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

2. Preparation of infection solution

Pick the Agrobacterium cells containing the pBI121-AdWRKY40 recombinant vector, and culture them overnight on a 28°C constant temperature shaker at 200 rpm using LB liquid culture medium containing 50 mg/L kanamycin and 50 mg/L rifampicin until OD ₆₀₀ is about 0.8. Take 200 mL of the shaken bacterial solution, put it into a centrifuge tube, and centrifuge it at 24°C and 8000 rpm for 10 minutes to collect the bacteria. Pour out the supernatant after centrifugation, add the 5% sucrose solution prepared in advance, mix it and resuspend it to OD ₆₀₀ of about 1.0 to prepare a resuspended bacterial solution. Then add 0.03% of the surfactant sliwet-77 to obtain the infection solution for use.

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.

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

3. Flower dip method to infect Arabidopsis

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.

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

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

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:

Table 1 Nutrient solution formula

4. Identification of transgenic plants

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.

The steps of extracting DNA by CTAB method are as follows:

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

(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.

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

(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.

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

(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.

(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.

(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 ddH ₂ O after air-drying.

(9) Incubate at 4°C overnight to obtain DNA, then store at -20°C until needed.

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.

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

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.

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

The identification results are shown in Figure 1. 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.

The specific propagation steps are as follows: Place 200 Arabidopsis seeds in a 1.5mL centrifuge tube in a clean bench, add 1mL ddH ₂ O, 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 ddH ₂ O. Add 10% NaClO to disinfect for 6min, and shake the centrifuge tube by hand during this period. After washing ddH ₂ O three times, add 1mL ddH ₂ O 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.

5. Evaluation of drought tolerance and flowering habits of transgenic Arabidopsis

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 cotyledons of the seeds turned green and were identified as germination. The germination rate was counted for 10 consecutive days, and 3 biological replicates were set up, 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 200min cumulatively, and the relative water loss rate was calculated. Four-week-old wild-type and transgenic Arabidopsis were treated with PEG6000 and 100µM ABA at a mass volume concentration of 10%, respectively. According to the instructions for use of the kit of Beijing Solebow Technology Co., Ltd., the contents of hydrogen peroxide H ₂ O ₂ , superoxide anion O ^2- , superoxide dismutase SOD, catalase CAT and proline were measured before and after stress. 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 leaves of the wild-type and transgenic Arabidopsis were measured before and after PEG6000 treatment 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.

As shown in Figure 2, under normal growth conditions, there was no difference in germination rate between wild-type and transgenic Arabidopsis, but under 300mM mannitol stress, the germination rate of transgenic Arabidopsis was higher than that of wild-type Arabidopsis. As shown in Figure 3, the relative water loss rate of transgenic Arabidopsis was lower than that of wild-type Arabidopsis. As shown in Figure 4, under drought stress, transgenic Arabidopsis accumulated high concentrations of O ^2- , low concentrations of H ₂ O ₂ , and high levels of SOD and CAT enzymes. Compared with wild-type Arabidopsis, transgenic Arabidopsis contained high concentrations of proline under drought stress. As shown in Figure 5, under normal growth conditions and 10% PEG6000 treatment for 3d, the stomatal conductance of transgenic Arabidopsis was significantly lower than that of wild-type Arabidopsis. As shown in Figure 6, after long-term drought treatment of wild-type and transgenic Arabidopsis, the survival rate of transgenic Arabidopsis was significantly higher than that of wild-type Arabidopsis after rehydration.

The 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 the seed dormancy, and placed in an incubator with 16h/8h light and dark alternation for germination determination. When the cotyledons of the seeds turn green, it is considered to be germination, 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.

The results are shown in FIG7 . Under normal growth and drought treatment conditions, AdWRKY40 transgenic plants flowered earlier than wild-type plants.

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.

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

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.

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.

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 (8)

1. A cranberry WRKY40 transcription factor for promoting drought tolerance and early flowering of plants is characterized in that the nucleotide sequence of the cranberry WRKY40 transcription factor is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising the cranberry WRKY40 transcription factor of claim 1.

3. The recombinant expression vector according to claim 2, wherein said cranberry WRKY40 transcription factor is inserted between the Nco I and Pml I sites of the pBI121 overexpression vector plasmid.

4. A recombinant engineering bacterium comprising the recombinant expression vector of claim 3.

5. The recombinant engineering bacterium according to claim 4, wherein the recombinant expression vector is obtained by transferring the recombinant expression vector into an agrobacterium competent cell EHA 105.

6. Use of the cranberry WRKY40 transcription factor of claim 1, for promoting drought tolerance and early flowering in a plant, wherein said plant comprises arabidopsis thaliana.

7. Use of the recombinant expression vector according to any one of claims 2 to 3 for promoting drought tolerance and early flowering in plants, wherein the plants comprise arabidopsis thaliana.

8. The use of the recombinant engineering bacterium according to any one of claims 4-5 for promoting drought tolerance and early flowering in plants, wherein the plants comprise arabidopsis thaliana.