Application of gene ghwrky46 in regulating plant growth and development

Abstract

The application belongs to the technical field of bioengineering, and relates to application of a gene GhWRKY46 in regulating plant growth and development. The application provides application of the gene GhWRKY46 in regulating plant growth and development. The application clones the GhWRKY46 gene from Gossypium hirsutum, constructs an overexpression vector, and heterogeneously expresses the overexpression transgenic strain in Arabidopsis thaliana. Compared with wild type, the overexpression transgenic strain blooms earlier, which indicates that the GhWRKY46 gene plays an important regulating role in controlling the flowering period of cotton. The application provides important application value for cotton variety improvement and breeding.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology, specifically involving the application of the gene GhWRKY46 in regulating plant growth and development. Background Technology

[0002] The WRKY transcription factor family is a class of plant-specific transcription factors, with 75 members found in the model plant Arabidopsis thaliana. These transcription factors are named WRKY transcription factors because they contain a highly conserved WRKYGQK amino acid sequence at their N-terminus. WRKY transcription factors regulate various plant developmental processes, including dormancy, germination, morphogenesis, flowering, gametophyte development, and seed development. In recent years, there have been increasing reports on WRKY transcription factors in regulating plant flowering time. For example, AtWRKY34 binds to the promoter of CUL3A, activating its expression. The accumulated CUL3A interacts with FRI, degrading FRI and thus inhibiting FLC expression, participating in the vernalization pathway to mediate flowering (Hu et al., 2014). AtWRKY71 and AtWRKY75 directly bind to the W-box motif on the FT promoter, activating FT expression and accelerating flowering (Yu et al., 2016; Zhang et al., 2018). Under short-day conditions, AtWRKY12 and AtWRKY13 participate in the gibberellin pathway to regulate flowering through interaction with DELLAs proteins. Furthermore, their expression patterns are closely related to plant age; as plant age increases, the expression level of WRKY13 gradually decreases, while the expression level of WRKY12 steadily increases (Li et al., 2016). WRKY63 plays a dual role in flowering regulation. It inhibits flowering by activating AtFLC under non-vernalization conditions, but promotes flowering by activating the inhibitors COOLAIR and COLDAIR of AtFLC during vernalization. These two inhibitors are derived from the 3' end and the first intron of AtFLC, respectively.

[0003] Cotton is an important economic crop globally and boasts a variety of varieties with distinct characteristics. Early-maturing cotton, in particular, is characterized by its short growing season and rapid growth, mitigating the negative impacts on cotton quality caused by unfavorable light and heat conditions and significant temperature variations in high-latitude regions (such as cotton-producing areas like Liaoning, Gansu, and Xinjiang). Therefore, identifying cotton flowering-related genes, elucidating their regulatory mechanisms, and creating superior early-maturing cotton germplasm resources are of great significance for the healthy and stable development of the cotton industry. Summary of the Invention

[0004] This application cloned the GhWRKY46 gene from upland cotton. By constructing an overexpression vector, the transgenic lines heterologously expressed in Arabidopsis thaliana flowered earlier than the wild type, indicating that the GhWRKY46 gene plays an important regulatory role in controlling cotton flowering time. This provides significant application value for cotton variety improvement and breeding.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the application of the gene GhWRKY46 in regulating plant growth and development.

[0007] This invention also provides the application of overexpressing the gene GhWRKY46 in promoting plant growth and development.

[0008] This invention also provides the application of overexpressing the gene GhWRKY46 in promoting early maturity of plants.

[0009] This invention also provides the application of overexpressing the gene GhWRKY46 in promoting the expression of GhFT in plants.

[0010] This invention also provides the application of overexpressing the gene GhWRKY46 in advancing the flowering period of plants.

[0011] In this invention, the aforementioned plants and / or plants include cotton. Attached Figure Description

[0012] Figure 1 Quantitative results for GhWRKY46;

[0013] Figure 2 For dual-luciferase reporter experiments;

[0014] Figure 3 To promote early flowering in transgenic Arabidopsis thaliana that has been overexpressed with GhWRKY46. Detailed Implementation

[0015] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0016] Example

[0017] 1. Cotton material

[0018] The cotton materials selected for this application were the early-maturing upland cotton variety Zhongmian Institute 36 and the late-maturing variety Guoxin Cotton 11. Zhongmian Institute 36 and Guoxin Cotton 11 showed highly significant differences in flowering time and growth period. They were planted in the experimental field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, located in Baibi Town, Anyang City, Henan Province, and managed under normal field management practices. Sampling was performed on young shoots of both cotton varieties at the one-leaf to five-leaf stage, which were placed in liquid nitrogen and stored at -80℃ before RNA extraction.

[0019] 2. Reagents and consumables

[0020] (1) Restriction endonucleases, modifying enzymes, PCR reaction system-related enzymes, homologous recombinases, gel extraction kits, cloning kits, and plasmid miniprep kits were purchased from Novizan Biotechnology Co., Ltd., the real-time fluorescence kit was purchased from Yisheng Biotechnology Co., Ltd., and the RNA extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd.

[0021] (2) Other medicines

[0022] Agarose was a Spanish original product; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestically produced analytical grade; kanamycin, etc. were from Solarbio Biotechnology Co., Ltd.; and Escherichia coli competent cells DH5α and Agrobacterium competent cells were purchased from Weidi Company.

[0023] (3) Culture medium

[0024] LB liquid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L;

[0025] LB solid medium: Tryptone 10g / L, Yeast extract 5g / L, Sodium chloride (NaCl) 10g / L, Agar powder 15g / L, bring to a final volume of 1L;

[0026] LB selective medium: Before plating LB, add the appropriate concentration of antibiotics when the medium has been autoclaved and cooled to 55 degrees Celsius, shake well and then plating.

[0027] 1 / 2MS solid medium: 1 / 2MS 22g / L, agar powder 8g / L, sucrose 30g / L;

[0028] (4) Main instruments: PCR amplification instrument (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), real-time PCR instrument (ABI7500), electric thermostatic incubator (Shanghai Senxin), thermostatic incubator shaker (Shanghai Zhicheng), artificial climate test chamber (Saifu), artificial climate chamber.

[0029] 3. Experimental methods and results

[0030] 3.1 Gene cloning and sequence analysis

[0031] The CDS sequence and encoded amino acid sequence of the GhWRKY46 (Gh_D07G1384) gene were obtained from CottonFGD (http: / / www.cottonfgd.org / ). Its open reading frame is 1008 bp and encodes 335 amino acids. The gene was named GhWRKY46 and its function was studied.

[0032] The open reading frame sequence of GhWRKY46 is:

[0033]

[0034] The amino acid sequence encoded by GhWRKY46 is as follows:

[0035] MEKTMGWEQNTLLNELAQGRDFTNMLRKHLHPSSSPETRQVLLDKILCSYDKALSLLNCSRFMVETKPRVRTLGSPENDASDNKDMFKKRKTSSGWSEQIRVCSAMSLEGPLDDGYCWRKYGQKDILGSNFPRAYYRCTHRYSQGCLAGKQVQRSDEDPTIFEVKYRGR HACNQVPHLVATPKEKGNHYREKQQVEEKQKQSKEMLLSFETGLKVKTEDLDNREDIFPSFSFPIESEEVQNGLLLNSLMKNMSPAFVSPATSESNYFSVSAFHMGNFDFGQNVQTSESELTEIISAPASVTNSPIVDLDISSLEKLELDQSFPYDNPEFFTNFLQ, SEQ ID NO.2.

[0036] 3.2 Pattern Analysis

[0037] Studies have shown that cotton flower bud differentiation is closely related to early maturity and is a marker of the transition from vegetative to reproductive growth in cotton, directly affecting flowering time. Early-maturing variety Zhongmian Suo 36 and late-maturing variety Guoxin 11 were selected, and flower bud RNA was extracted from the one-leaf to five-leaf stages. The expression level of GhWRKY46 was detected using qRT-PCR technology. The results showed that the expression level of this gene in the early-maturing variety 36 from the one-leaf to five-leaf stages was significantly higher than that in the late-maturing variety Guoxin 11.

[0038] 3.2.1 Sampling and Grinding

[0039] Terminal buds of Zhongmian Institute 36 and Guoxin 11 from the one-leaf to five-leaf stage were placed in liquid nitrogen and ground into powder using a mortar and pestle. Approximately 1g of the sample was placed in a 1.5ml centrifuge tube.

[0040] 3.2.2 Extraction

[0041] RNA extraction was performed using the FastPure Universal Plant Total RNA Isolation Kit (Novozymes, Nanjing, China). The specific steps are as follows:

[0042] (1) The experiment was carried out at room temperature. 600 μl of Buffer PSL (Polyphenol Polysaccharide Plant) was immediately added to the centrifuge tube containing plant tissue. The sample was vortexed vigorously for 30 seconds to ensure that the sample and lysis buffer were thoroughly mixed. The sample was centrifuged at 12,000 rpm (134,00×g) for 5 min and then the subsequent operations were carried out immediately.

[0043] (2) Take about 500 μl of the supernatant into FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, and collect the filtrate.

[0044] (3) Add 0.5 times the volume of the filtrate to the collection tube, and vortex to mix for 15 seconds. Transfer the mixture to FastPure RNAColumns V (FastPure RNAColumns V is already in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, and discard the filtrate.

[0045] (4) Add 700 μl of Buffer RWA to FastPure RNAColumns V, centrifuge at 12,000 rpm (13,400×g) for 30 seconds, and discard the filtrate.

[0046] (5) Add 500 μl of Buffer RWB to FastPure RNAColumns V (please check before use whether 48 ml of anhydrous ethanol has been added, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate).

[0047] (6) Repeat step (5).

[0048] (7) Put FastPure RNAColumns V back into the collection tube and centrifuge at 12,000 rpm (13,400×g) for 2 min.

[0049] (8) Transfer FastPure RNAColumns V to a new RNase-free Collection Tubes 1.5ml centrifuge tube, add 30-100μl of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000rpm (13,400×g) for 1min.

[0050] *The recommended elution volume is no less than 30 μl; a volume that is too small will affect the efficiency of nucleic acid recovery.

[0051] *The following steps can help increase RNA product concentration: Add RNase-free ddH2O and let stand at room temperature for 5 minutes; add the first elution buffer back into the adsorption column for elution.

[0052] (9) The extracted RNA can be used directly for downstream experiments or stored at -85 to -65℃.

[0053] 3.2.3 Synthesis of reverse-transcribed cDNA

[0054] Reverse transcription cDNA synthesis kit The IIQ RT SuperMix for qPCR (+gDNAwiper) (Novozymes, Nanjing, China) was used. The reaction can be divided into two parts: removal of genomic gDNA and reverse transcription of RNA. The reaction was carried out on ice, and the steps are as follows:

[0055] (1) Removal of genomic gDNA

[0056] Table 1 Reaction system configuration

[0057] reagents Dosage <![CDATA[RNase-freeddH2O]]> to 16μl 4×gDNAwiperMix 4μl template RNA 1 pg~1 μg

[0058] Mix gently by pipetting. Incubate at 42°C for 2 minutes.

[0059] (2) Preparation of reverse transcription reaction system

[0060] Table 2 Reverse Transcription Reaction System

[0061] reagents Dosage The reaction solution in the first step 16μl 5×HiScriptIIqRTSuperMixII 4.0μl

[0062] Gently pipette to mix thoroughly. Incubate 20 μl of the mixture in a PCR instrument at 50°C for 15 min, then at 85°C for 5 sec. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months.

[0063] 3.2.4 Quantitative Real-Time PCR

[0064] (1) Specific primers for the GhWRKY46 gene were designed using Oligo 7 software, and the cotton His3 gene was used as an internal reference gene.

[0065] Table 3 Specific primers

[0066]

[0067] (2) Real-time PCR

[0068] The procedure was performed using the Cwbio (China) UltraSYBR Mixture (Low ROX) kit and an AppliedBiosystems 7500 instrument. The specific steps are as follows:

[0069] 1) Dilute the above-mentioned cDNA stock solution 5 times;

[0070] 2) Preparation of the reaction system (operation on ice).

[0071] Table 4. Configuration of the reaction system

[0072]

[0073]

[0074] The prepared system was thoroughly mixed and centrifuged until no air bubbles remained. Then, quantitative real-time PCR was performed using Applied Biosystems 7500. The PCR program was set up using a two-step method: pre-denaturation: 95℃ for 2 min; 95℃ for 5 s; 60℃ for 34 s (fluorescence signal was collected in this step), for 40 cycles. Finally, melting curve analysis was performed: 95℃ for 15 s; 60℃ for 20 s; 95℃ for 15 s. Data were processed using Microsoft Excel 2019 to calculate gene expression levels, and plotted using Origin 2022.

[0075] 3.2.5 Quantitative Analysis of GhWRKY46

[0076] The results of fluorescence quantification are categorized according to 2 -ΔC t-calculation yielded the relative expression level of GhWRKY46 (e.g., Figure 1 (As shown). By Figure 1 It can be seen that during the one-leaf to five-leaf stage of plant development, the expression level of GhWRKY46 in the early-maturing variety Zhongmian50 is significantly higher than that in the late-maturing variety Guoxinmian11.

[0077] 3.3 Dual-luciferase assay and Arabidopsis heterologous expression

[0078] pGreen II 62-SK-GhWRKY46 and pGreen II 0800-LUC-qGhFT were constructed to transiently transform tobacco. The injected leaves were cut and coated with dual-luciferase substrate, and then left to stand in the dark for 10 min. After standing, the fluorescence of tobacco was observed using a fully automated chemiluminescence image analysis system, and the results were recorded by taking pictures.

[0079] The full-length GhWRKY46 CDS sequence was ligated into the pCambia2300-HA vector to construct a 35S promoter vector. The 35S::GhWRKY46 recombinant vector was then used to infect Arabidopsis thaliana using the Arabidopsis inflorescence infection method. Positive selection and purification of progeny seeds were performed, followed by multiple generations to obtain T3 pure lines. Phenotypic and expression level analysis of the progeny plants revealed that overexpression of GhWRKY46 promoted early flowering in Arabidopsis thaliana.

[0080] 3.3.1 Gene Primer Design

[0081] Based on the principles of homologous recombination primer design, specific primers were designed using Oligo 7 software to amplify the full-length coding region of the GhWRKY46 gene. According to the gene's CDS sequence, corresponding restriction enzyme sites were added at the start codon (ATG) and stop codon to ensure the target gene fragment had the same terminal sequence as the linearized vector after restriction enzyme digestion. EcoRI and KpnRI were selected as restriction enzyme sites for the pCambia2300-HA vector; BamHI and Xhol were selected as restriction enzyme sites for the pGreen II 62-SK and pGreen II 0800 vectors. Both the cDNA and DNA templates used were from *Uplandia TM-1*.

[0082] The 35S::GhWRKY46 specific primer sequence is shown below.

[0083] Table 5. Specific primer sequences for GhWRKY46

[0084]

[0085] The specific primer sequences for pGreen II 0800-LUC-GhFT and pGreen II 62-SK-GhWRKY46 are shown below.

[0086] Table 6 Specific primer sequences

[0087]

[0088]

[0089] 3.3.2 Gene Cloning PCR System, Procedure and Product Detection

[0090] (1) The reaction was carried out on ice. The reaction system was designed according to the PhantaMax Super-Fidelity DNAPolymerase kit (Novozymes, Nanjing) as follows.

[0091] Table 7 Reaction System

[0092] Reagent Name Reagent dosage <![CDATA[ddH2O]]> up to 50 μl 2×PhantaMaxBuffera 25μl dNTPMix(10mMeach) 1μl Upstream primer (10 μM) 2μl Downstream primer (10 μM) 2μl PhantaMaxSuper-FidelityDNAPolymerase 1μl Template DNA 1μl

[0093] (2) PCR reaction procedure

[0094] Table 8 PCR reaction procedure

[0095]

[0096] (3) Detection of PCR products

[0097] Take 2 μl of PCR product, add 2 μl of 5×Loading Buffer, mix well, and spot onto a 1% agarose gel. Electrophoresis is used to check whether the band size meets the requirements.

[0098] (4) Gel recovery of PCR products

[0099] The Vazyme product purification kit was used, and the steps are as follows:

[0100] 1) After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is recommended to blot the surface liquid of the gel with a paper towel and cut it into small pieces, removing as much excess gel as possible. Weigh the gel (excluding the weight of the empty tube). 100 mg of gel is equivalent to 100 μl of volume, which is taken as one gel volume.

[0101] 2) Add an equal volume of Buffer GDP, incubate in a water bath at 50–55°C for 7–10 minutes. Adjust the time according to the size of the gel to ensure complete dissolution. Invert the container twice during the water bath to accelerate the dissolution process.

[0102] 3) Briefly centrifuge to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column in a 2ml Collection Tube, transfer ≤700μl of the sol to the adsorption column, and centrifuge at 12,000×g for 30–60 seconds. If the sol volume is greater than 700μl, place the adsorption column in the collection tube, transfer the remaining sol to the adsorption column, and centrifuge at 12,000×g for 30–60 seconds.

[0103] 4) Discard the filtrate and place the adsorption column in the collection tube. Add 300 μl of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12,000 × g for 30–60 sec.

[0104] 5) Discard the filtrate and place the adsorption column in the collection tube. Add 700 μl of Buffer GW (with anhydrous ethanol added) to the adsorption column. Centrifuge at 12,000 × g for 30–60 seconds.

[0105] 6) Repeat step 5.

[0106] 7) Discard the filtrate and place the adsorption column in the collection tube. Centrifuge at 12,000 × g for 2 min.

[0107] 8) Place the adsorption column in a 1.5 ml sterile centrifuge tube, add 20–30 μl of sterile water to the center of the column, and let stand for 2 min. Centrifuge at 12,000 X g for 1 min. Discard the adsorption column and store the DNA at -20℃.

[0108] 3.3.3 Construction of pGreen II 62-SK, pGreen II 0800-LUC, and pCambia2300-HA plant expression vectors

[0109] (1) Double enzyme digestion and gel recovery of plasmids

[0110] The plasmid was double-digested with pGreen II 62-SK, pGreen II 0800-LUC, and pCambia2300-HA, and the vector product was recovered by electrophoresis. The enzyme digestion reaction system is as follows.

[0111] Table 9 Enzyme digestion reaction system

[0112] Reagent Name Reagent dosage Enzyme 1 1μl enzyme 2 1μl CutSmart 5μl plasmid 1μg <![CDATA[ddH2O]]> Up to 50μl

[0113] (2) Ligation of PCR gel recovery products and linearized plasmids

[0114] The PCR product with adapter and the linearized plasmid were mixed with Novitane recombinase reagent. When connecting the One Step Cloning Kit, the connection response is as follows:

[0115] The system was configured on ice.

[0116] Table 10

[0117] Reagent Name Reagent dosage 5×CEⅡBuffer 2μl Exnase II 1μl Linearized carrier 25~100ng PCR fragments 10~100ng <![CDATA[ddH2O]]> Up to 10 μl

[0118] After the system is completed, mix the components by blowing, react at 37°C for 30 min, immediately in an ice-water bath for 5 min, and then convert or store at -20°C.

[0119] (3) Transformation of the ligation product into Escherichia coli

[0120] 1) Add 100 μl of E. coli DH5α competent cells to the ligation reaction system and incubate on ice for 30 min;

[0121] 2) 42℃ water bath heat shock for 45-90 seconds;

[0122] 3) Ice bath for 2 min; add 900 μl of antibiotic-free LB liquid medium, incubate at 37℃ and 190 rpm for 1 h;

[0123] 4) Centrifuge at 4000 rpm for 3 min, discard the supernatant, keep about 100 μl, mix well and spread on LB plates containing kanamycin resistance.

[0124] 5) Incubate overnight at 37℃;

[0125] (4) Detection and sequencing of positive clones

[0126] 1) Pick white colonies from the transformation plate and place them in liquid LB medium containing Kans, and incubate at 37°C in a shaker for 8 hours;

[0127] 2) Verify positive clones by colony PCR. Send the verified single clones to Shangya Biotechnology Co., Ltd. for sequencing, and sequence each sequence in 3 replicates.

[0128] (5) Preservation of positive bacterial culture

[0129] Add a certain amount of glycerol to the bacterial culture that has been verified by PCR and has been correctly sequenced, bringing the final glycerol concentration to approximately 20%, and store at -80℃. Return the correctly sequenced plasmid for transfection with Agrobacterium.

[0130] (6) Transformation of Agrobacterium tumefaciens

[0131] The Agrobacterium tumefaciens GV3101 competent cells were transformed using the freeze-thaw method. The specific transformation process is as follows:

[0132] 1) Melt Agrobacterium at -80℃, then insert it into ice in a mixture of ice and water.

[0133] 2) Add 0.01-1 μg of plasmid DNA to 100 μl competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 min, liquid nitrogen for 5 min, 37℃ for 5 min, and ice bath for 5 min in sequence.

[0134] 3) Add 700 μl of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2–3 h.

[0135] 4) Take 100-150 μl of bacterial culture onto an LB agar plate containing kanamycin and rifampin, and invert it in an incubator at 28°C for 2-3 days.

[0136] 5) Select positive clones and incubate them at 28°C for 48 hours on LB liquid medium with added antibiotics. Verify the correct bacterial PCR bands. The final concentration of glycerol in the bacterial culture should be around 20%. Store at -80°C for later use.

[0137] 3.3.4 Dual-luciferase reporter assay

[0138] (1) Tobacco cultivation: Sow a number of tobacco seeds and cultivate the tobacco under long-day conditions for one month for use in experiments.

[0139] (2) Agrobacterium containing pGreen II 62-SK-GhWRKY46, pGreen II 0800-LUC-qGhFT, pGreen II 62-SK, and pGreen II 0800-LUC plasmids was activated and cultured for 12–24 h. 200 μL of the activated broth was added to 30 mL of double-antibiotic liquid LB medium and incubated at 28 °C for approximately 16 h. The culture was continued until the bacterial culture reached OD500. 600 The pH value is approximately between 1.0 and 1.5 (approximately 18 to 20 hours). Centrifuge at 5000g for 8 minutes, discard the supernatant, and collect the bacterial cells.

[0140] (3) Resuspension: The bacterial cells were resuspended in a suspension of 10 mM MgCl2 and 10 mM MgCl2 (containing 120 μMAS, pH = 5.8), and the OD was adjusted. 600 Up to around 0.8;

[0141] (4) Injection: Select tobacco plants with good growth, use a 1ml syringe with the nozzle removed to inject into the lower epidermis of the tobacco leaves, and make a label.

[0142] (5) Cut off the injected leaves, apply dual-luciferase substrate, and let stand in the dark for 10 minutes. After standing, observe the fluorescence of tobacco using a fully automated chemiluminescence image analysis system and take pictures to record the results.

[0143] 3.3.5 Results of Dual-Luciferase Reporter Experiment

[0144] To verify the function of GhWRKY46 in promoting early flowering in cotton, it was found that GhWRKY46 can bind to the promoter of GhFT to promote GhFT expression in cotton. The results of the dual-luciferase reporter assay are as follows: Figure 2 As shown.

[0145] 3.3.6 Agrobacterium-mediated Arabidopsis inflorescence infection

[0146] (1) Arabidopsis thaliana culture

[0147] Colombian wild-type Arabidopsis thaliana were cultured under long-day conditions (16 hours of light and 8 hours of darkness). Healthy strains around 5 weeks old were selected, their siliques were removed, and the strains were watered the day before infection to ensure their condition and humidity.

[0148] (2) Infection of Arabidopsis thaliana inflorescences

[0149] 1) Activation of bacterial culture: Take 20 μl of Agrobacterium culture corresponding to the recombinant vector stored at -80℃, inoculate it into 1 ml of LB liquid medium (with the corresponding antibiotics: kanamycin, rifampin and streptomycin added), and incubate at 28℃ and 180 rpm for 14 to 18 h.

[0150] 2) Propagation: Add 200 μl of the activated bacterial culture to 50 ml of LB liquid medium (with the corresponding antibiotic added); incubate at 28°C and 180 rpm until the bacterial culture reaches OD500. 600 The pH value is approximately between 1.2 and 1.6 (approximately 18 to 20 hours). Centrifuge at 5000g for 8 minutes, discard the supernatant, and collect the bacterial cells.

[0151] 3) Preparation of the infection and transformation medium: 1 / 2 MS (half the amount), 6% sucrose, 0.02% Silwet L-77, and adjust the pH to 5.6-5.7 with NaOH;

[0152] 4) Suspend the above bacterial cells in transformation medium and convert OD0.05 600 Adjust to 0.6-0.8;

[0153] 5) Inoculation: Place Arabidopsis inflorescences (mainly unopened buds) in the transformation medium for 30-50 seconds. After inoculation, place the Arabidopsis flat under weak light or dark conditions for 24 hours.

[0154] 6) Place the treated Arabidopsis thaliana under normal conditions for cultivation, and spray water on the leaves of Arabidopsis thaliana every day for one week after infection; to improve the conversion efficiency, repeated infection can be carried out after about one week.

[0155] 7) After maturity, harvest the Arabidopsis seeds, which are the transgenic T0 generation seeds.

[0156] 3.3.7 Phenotypic identification of transgenic Arabidopsis plants

[0157] (1) After disinfecting the harvested seeds, plant them on 1 / 2 MS containing kanamycin, then vernalize them at 4℃ for 2 days, and then transfer them to an artificial climate test chamber. After about 10 days, the positive plants will grow normally, while the negative plants will have yellow leaves and stop growing.

[0158] (2) The positive Arabidopsis thaliana plants were transplanted into small flower pots and planted. After one month of growth, DNA was extracted and tested by PCR.

[0159] The primers used in the detection are shown in the table below.

[0160] Table 11

[0161] 35S gacgcacaatcccactatcc SEQ ID NO.13 GhWRKY46-R ttactgtaaaaaattagtgaagaac SEQ ID NO.14

[0162] (3) Transgenic T3 generation plants and non-transgenic plants were sterilized and cultured on 1 / 2 MS medium. After vernalization at 4℃ for two days, Arabidopsis seedlings were transplanted into small flower pots after about 10 days when they grew true leaves. Under the same planting and cultivation conditions, phenotypic observation showed that non-transgenic Arabidopsis flowered significantly later than overexpressing transgenic Arabidopsis. Figure 3This indicates that overexpression of GhWRKY46 significantly promotes flowering and reproductive growth and development in Arabidopsis thaliana.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpressed genes GhWRKY46 Its application in advancing the flowering period of plants is characterized by... The gene GhWRKY46 The open reading frame sequence is shown in SEQ ID NO.1, gene. GhWRKY46 The encoded amino acid sequence is shown in SEQ ID NO.2; The plant in question is Arabidopsis thaliana.

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