PsWRKY33 gene for regulating aging of peony petals, vector, recombinant bacterium and application of PsWRKY33 gene

Abstract

The invention belongs to the technical field of biology, and particularly relates to a PsWRKY33 gene for regulating aging of peony petals, a carrier, recombinant bacteria and application of the PsWRKY33 gene. The nucleotide sequence of the PsWRKY33 gene is as shown in SEQ ID NO. 1. 1, and the coding amino acid sequence is shown as SEQ ID NO. 2. The expression of the PsWRKY33 gene is up-regulated along with the aging of petals; silencing PsWRKY33 in peony obviously delays aging of peony petals, overexpression of PsWRKY33 promotes aging of peony petals, expression of malondialdehyde (MDA) and aging-related genes is obviously increased, the content of hydrogen peroxide and superoxide anions is also obviously increased, and it is indicated that the PsWRKY33 gene participates in the aging process of peony petals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a PsWRKY33 gene, a vector, a recombinant bacterium and applications thereof for regulating peony petal senescence. Background Art

[0002] The peony (Paeonia suffruticosa Andr.) is a perennial woody plant of the genus Paeonia in the Ranunculaceae family. With a cultivated history of over 1,500 years in my country, it is beloved worldwide for its vibrant colors and diverse varieties, and is widely used for garden planting and ornamental purposes (He et al., 2025; Meng et al., 2012). In addition to being an important ornamental crop, peony roots, flowers, seeds, and leaves possess significant edible and medicinal value, offering promising development prospects. For example, peony root bark is a common Chinese herbal remedy used in clinical medicine, boasting benefits such as promoting blood circulation and removing blood stasis, clearing away heat and detoxifying, and providing sedative and analgesic properties. Peony flower and seed oil are rich in essential nutrients and trace elements, making them a valuable natural health supplement. Peony leaves also offer a potential medicinal resource, exhibiting significant inhibitory effects against some bacteria (Ma et al., 2023; Wang et al., 2017). In recent years, peonies have garnered widespread attention as a novel fresh cut flower. However, due to their highly seasonal cultivation, their flowering period is short and relatively concentrated. A single flower blooms and withers in less than a week, and cut flowers wither even faster after being removed from the body. This has severely impacted the development and economic benefits of the peony industry. Therefore, extending the flowering period of peonies is an urgent issue (Meng et al., 2012). However, due to their strong seasonality and low reproductive coefficient, conventional breeding methods are difficult to implement to extend their flowering period (Meng et al., 2012; Zhang et al., 2024). Therefore, elucidating the molecular mechanisms of petal senescence and identifying genes associated with petal senescence will provide a theoretical basis and foundation for breeding new peony varieties with longer flowering periods and developing novel flower preservation technologies, thus possessing significant application value (Zhang et al., 2024; Wang et al., 2022; Jiang et al., 2023).

[0003] Senescence is an adaptive mechanism formed through long-term plant evolution and natural selection. It generally refers to the process by which plants, at the cellular, tissue, organ, and individual levels, gradually decline in physiological function and eventually die with aging (Qiu et al., 2022). Flowers are the organs of sexual reproduction in angiosperms. Typical floral organs are composed primarily of sepals, petals, stamens, and carpels. Petals, in particular, produce unique colors and fragrances that attract pollinators for pollination and fertilization (Zhang et al., 2018). During flower senescence, petals (tepals) are the first floral organs to show signs of senescence, characterized by color change and wilting. Therefore, petal senescence largely determines a flower's ornamental and economic value (Miao et al., 2016). Petal senescence is the final stage of flowering and is a precisely controlled and irreversible process that is influenced by multiple autonomous and environmental factors. It is usually mediated by the combined effects of exogenous stresses (drought, high temperature, nutritional stress, and biotic stress) and endogenous factors (growth and development, plant hormones, sugar starvation, etc.) (Zhang et al., 2018; Lin et al., 2023).

[0004] WRKY transcription factors (WRKYs) are the second largest family of transcription factors involved in aging and participate in various signaling pathways in plants. Key features of WRKYs are the highly conserved "WRKYGQK" heptapeptide sequence unique to plants at the N-terminus of their DNA-binding domains and the presence of C2H2 or C2HC zinc finger structures at the C-terminus. The WRKY gene family can be divided into three major groups: I, II, and III, based on the number of WRKY domains or zinc finger structures. Group II is further divided into five subgroups (IIa-e) based on amino acid sequence differences. WRKYs can specifically bind to cis-acting elements on downstream gene promoters, including the W-box (TTGACC / T), PRE4 (Pathogen responsive element 4, TGCGCTT), WLE1 (W-box like element 1, TGACA), and the sugar-responsive element (TAAAGATTACTAATAGGAA), through their domains, thereby activating or repressing the expression of related genes (Yang et al., 2023; Deng et al., 2017; Luo et al., 2017). Studies have shown that the promoter regions of genes downstream of WRKY transcription factors mostly contain a W-box (TTGACC / T). TGAC is a highly conserved core sequence of the W-box, and changes in any nucleotide sequence can affect the ability of WRKY proteins to bind to downstream genes (Li et al., 2021).

[0005] The expression of WRKY transcription factors is rapidly and strongly induced by environmental and internal factors. They are widely involved in plant growth and development, aging, hormone signaling, and the synthesis of carbohydrates and defense chemicals. They play a vital role in plant growth metabolism, signaling, and disease defense (Luo et al., 2017). In recent years, research on WRKY transcription factors has focused on functional analysis and the study of their specific regulatory mechanisms, with significant progress achieved (Li et al., 2021). For example, TgWRKY75 in tulips enhances ABA and SA biosynthesis by binding to and activating the expression of key genes in the SA and ABA biosynthesis pathways, TgICS1, TgPAL1, and TgNCED3, thereby promoting petal senescence (Li et al., 2024). In carnation, DcEIL3-1 interacts with DcWRKY75 and, by inducing DcWRKY75 expression, co-activates the expression of ethylene biosynthesis genes and senescence-related genes, thereby accelerating petal senescence (Xu et al., 2024). In Arabidopsis, AtWRKY22 is phosphorylated by type I casein kinases, AtAELs, to enhance its transcriptional activity, regulating leaf senescence by promoting ethylene biosynthesis (Zhu et al., 2024). These results suggest that WRKY transcription factors participate in plant hormone signaling and responses to abiotic stresses, influencing floral lifespan. However, the regulatory mechanisms of WRKY transcription factors in peony, particularly in peony flower senescence, remain largely unreported. Summary of the Invention

[0006] In order to solve the technical problems of delaying petal senescence and extending the flowering period, the present invention provides a PsWRKY33 gene for regulating peony flower senescence, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007]

[0008] Furthermore, the amino acid sequence of the PsWRKY33 protein encoded by the PsWRKY33 gene is shown in SEQ ID NO. 2. (SEQ ID NO. 2).

[0009] The present invention determined the transcriptome associated with peony petal senescence and found that the expression of the PsWRKY33 gene was upregulated with petal senescence; silencing PsWRKY33 in tree peonies significantly delayed peony petal senescence, while overexpressing PsWRKY33 accelerated peony petal senescence. The expression of malondialdehyde (MDA) and senescence-related genes was significantly increased, as were the levels of hydrogen peroxide and superoxide anions, indicating that the PsWRKY33 gene is involved in the senescence process of tree peonies.

[0010] The first object of the present invention is to provide a PsWRKY33 gene for regulating peony petal senescence, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] The second object of the present invention is to provide a protein encoded by the PsWRKY33 gene, whose amino acid sequence is shown in SEQ ID NO.2.

[0012] The third object of the present invention is to provide a vector containing the PsWRKY33 gene and used for regulating peony petal senescence.

[0013] The fourth object of the present invention is to provide an Agrobacterium containing a PsWRKY33 gene vector and used for regulating peony petal senescence.

[0014] The present invention provides an application of the PsWRKY33 gene in delaying peony petal senescence.

[0015] For example, the application of PsWRKY33 gene in delaying the senescence of peony petals is achieved by silencing the PsWRKY33 gene.

[0016] The present invention provides application of the PsWRKY33 gene in promoting peony petal senescence.

[0017] For example, the PsWRKY33 gene is used to promote the senescence of peony petals by overexpressing the PsWRKY33 gene.

[0018] The beneficial effects of the present invention are as follows: the present invention found that the expression of the PsWRKY33 gene is upregulated with the aging of petals; silencing PsWRKY33 in tree peonies significantly delayed the aging of peony petals, overexpressing PsWRKY33 promoted the aging of peony petals, the expression of malondialdehyde (MDA) and aging-related genes was significantly increased, and the levels of hydrogen peroxide and superoxide anions were also significantly increased, indicating that the PsWRKY33 gene is involved in the aging process of peony petals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Cluster-3322.19119 protein sequence conserved domain analysis;

[0020] Figure 2 Phylogenetic analysis of PsWRKY33 and WRKYs protein sequences in Arabidopsis;

[0021] Figure 3 Analysis of conserved domains between PsWRKY33 and its homologous protein sequences in different species

[0022] In the figure: the black underlines mark the WRKYGQ domain and the C2H2 (C-X4-C-X23-H-X1-H) type zinc finger structure respectively;

[0023] Figure 4 Spatiotemporal expression patterns of the PsWRKY33 gene in 'Snow Reflecting Peach Blossoms'

[0024] Figure A. Flower status at different stages S1-S5, Bar = 3 cm, B. Expression levels of PsWRKY33 at different flowering stages, C. Expression patterns of PsWRKY33 in different tissues, one-way ANOVA and Waller-Duncan (W) multiple comparison analysis were performed.

[0025] Figure 5 Construction of pBI121-PsWRKY33 vector

[0026] M: DL 2000 Marker; P: positive; N: negative;

[0027] In the figure: A. pBI121-PsWRKY33 ORF amplification, B. pBI121-PsWRKY33 Escherichia coli culture PCR, C. pBI121-PsWRKY33 Agrobacterium culture PCR;

[0028] Figure 6 Functional characterization of overexpressed PsWRKY33 in peony petal senescence

[0029] Figure A. Phenotypic observation of PsWRKY33 overexpressing 'Xueyingtaohua', B. Time required for petals to fall off in peony with pBI121-PsWRKY33, C. qRT-PCR analysis of the expression level of PsWRKY33 in overexpressing petals, D. qRT-PCR detection of the expression levels of senescence-related genes PsSAG13 and PsSAG40.1 in overexpressing petals;

[0030] Figure 7 Phenotypic observation of floral discs overexpressing PsWRKY33 and determination of related physiological indices

[0031] Figure A. NBT staining, the staining degree represents the accumulation level of reactive oxygen species, Bar = 1 cm, B. DAB staining, the staining degree represents the accumulation level of hydrogen peroxide, Bar = 1 cm, C. Changes in MDA (malondialdehyde) content in petals overexpressing PsWRKY33 and the control group, D. Changes in SOD (superoxide dismutase) activity in petals overexpressing PsWRKY33 and the control group;

[0032] Figure 8 TRV2-PsWRKY33 vector construction

[0033] M: DL 2000 Marker; P: positive; N: negative;

[0034] In the figure: A. TRV2-PsWRKY33 ORF amplification, B. TRV2-PsWRKY33 Escherichia coli culture PCR, C. TRV2-PsWRKY33 Agrobacterium culture PCR;

[0035] Figure 9 Functional characterization of silencing PsWRKY33 in the senescence process of peony petals

[0036] Figure A. Phenotypic observation of 'Xueying Taohua' after silencing PsWRKY33, B. Time required for petals to fall off after silencing PsWRKY33, C. qRT-PCR analysis of the expression level of PsWRKY33 in silenced petals, D. qRT-PCR detection of the expression levels of senescence-related genes PsSAG13 and PsSAG40.1 in silenced petals;

[0037] Figure 10 Phenotypic observation of floral discs after silencing PsWRKY33 and determination of related physiological indices

[0038] Figure A. DAB staining, the degree of staining represents the accumulation level of hydrogen peroxide, Bar = 1 cm, B. NBT staining, the degree of staining represents the accumulation level of reactive oxygen species, Bar = 1 cm, C. Changes in MDA (malondialdehyde) content in petals of silencing PsWRKY33 and control group, D. Changes in SOD (superoxide dismutase) activity in petals of silencing PsWRKY33 and control group. DETAILED DESCRIPTION

[0039] 1. Materials

[0040] 1.1 Material handling

[0041] In mid-to-late October, four-year-old tree peonies, 'Snow Reflecting Peach Blossoms,' were individually potted and grown in a 20°C greenhouse for two weeks. Afterwards, they were transferred to a 2-4°C cold storage for 28 days and then transferred to a 20°C greenhouse (13 hours light / 11 hours dark). When the buds were soft and the sepals were loosely everted, they were cut along with the pedicels and immediately placed in water. Petals were collected at S1, S2, S3, S4, and S5, quickly frozen in liquid nitrogen, and stored at -80°C until ready for use.

[0042] 1.2 Commonly used vectors and strains

[0043] Escherichia coli (E. coli) strain DH5ɑ, Agrobacterium tumefaciens strain GV3101, Agrobacterium tumefaciens strain EHA105, pBI121 vector, pSuper1300-GFP vector (deposited by the Plant Genetics and Development Laboratory).

[0044] 1.3 Main enzymes and reagents

[0045] QuickCut restriction endonuclease, 5×ABscriptⅢRT Mix (purchased from ABdonal), 2×Flash MasterMix (purchased from Vazyme), 2×Rapid Taq Master Mix (purchased from Vazyme), SteadyPure Plant RNAExtraction Kit (Accurate Biology, AG21019, China) (purchased from Accurate), EVOM-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd., China) (purchased from Accurate), qPCRSYBR Green Premix Pro Taq HS qPCR KitⅡ (Accurate Biology, AG11701, China) (purchased from Accurate).

[0046] LB Broth, LB Broth with Agar, ampicillin, kanamycin, rifampicin, acetosyringone, dimethyl sulfoxide, MES.

[0047] Preparation of antibiotics:

[0048] Kan (kanamycin): Weigh 1 g of powder and dissolve in ddH2O, then dilute to 20 mL. Filter and aliquot to a concentration of 50 mg / mL.

[0049] Amp (Ampicillin): Weigh 1 g of powder and dissolve in ddH2O, then dilute to 20 mL. Filter and aliquot to a concentration of 50 mg / mL.

[0050] Rif (Rifampicin): Weigh 500 mg of powder and dissolve in DMSO to 25 mL. Filter with an organic filter membrane and dispense into aliquots at a concentration of 50 mg / mL.

[0051] Preparation of culture medium:

[0052] LB liquid medium: Weigh 25 g of LB broth powder and add 1 L of ddH2O. Sterilize at 121°C for 20 min. Add antibiotics as needed and store at 4°C.

[0053] LB solid medium: Weigh 40 g of LB broth with agar powder and add 1 L of ddH2O. Sterilize at 121°C for 20 min. Add appropriate antibiotics as needed. Pour into plates, seal, and store at 4°C.

[0054] Infection solution: Weigh 2.132 g MES, 0.9522 g MgCl2, and 0.5 M AS, add ddH2O and make up to 1 L. Prepare and use immediately.

[0055] 2 Main instruments

[0056] UV clean bench, UV spectrophotometer, high-speed refrigerated centrifuge (Heal Force), pipette (Eppendorf), fluorescence real-time quantitative PCR instrument, pH meter, Leica laser confocal microscope (Leica TCS-SP2), constant temperature and light incubator.

[0057] 3 Experimental methods

[0058] 3.1 Cloning and Analysis of PsWRKY33

[0059] 3.1.1 Mining and Analysis of PsWRKY33

[0060] A significantly differentially expressed gene (ID: Cluster-3322.19119) was identified in the peony senescence-related transcriptome. The full-length cDNA sequence of this gene was retrieved from the local 'Xueyingtaohua' floral development transcriptome database (Blast). Based on bioinformatics analysis, it was named PsWRKY33. After downloading its base sequence, the amino acid sequence was translated using DNAMAN.

[0061] 3.1.2 RNA extraction from peony flowers

[0062] Total RNA was extracted from peony flowers at different stages according to the instructions of the SteadyPure Plant RNA Extraction Kit.

[0063] 3.1.3 cDNA Synthesis

[0064] Refer to the EVO M-MLV Mix Kit with gDNA Clean for qPCR kit instructions and follow the two-step method to remove genomic DNA and perform reverse transcription reaction.

[0065] 3.2 Analysis of PsWRKY33 expression patterns

[0066] 3.2.1 qRT-PCR primer design

[0067] Using the actin gene as an internal reference, real-time quantitative primers (Table 1) were designed online using NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and synthesized by Bioengineering (Shanghai).

[0068] Table 1 Real-time quantitative primers for PsWRKY33 gene

[0069]

[0070] 3.2.2qRT-PCR reaction

[0071] According to the qPCR SYBR Green Premix Pro Taq HS qPCR Kit II, 20 μL of real-time quantitative fluorescence PCR reaction system was prepared. The reaction system is shown in Table 2. Three sample replicates and three technical replicates were set. The reaction was performed in Applied Biosystems Quant Studio TM 5. Fluorescence quantitative PCR system operation, using 2 -ΔΔ The data were analyzed by CT method for relative expression.

[0072] Table 2 PCR amplification system

[0073]

[0074] Functional Identification of PsWRKY33 in Peony Flower Senescence

[0075] 3.3.1 Overexpression of PsWRKY33 in Peony Petals

[0076] 3.3.1.1 Construction of pBI121-PsWRKY33 overexpression vector

[0077] 3.3.1.1.1 Primer design

[0078] Specific primers for the recombination of the PsWRKY33 gene with the pBI121 vector were designed using CE Design V1.04. The primer sequences are shown in Table 3.

[0079] Table 3 Primers required for PsWRKY33 ligation to pBI121 vector

[0080] Primer Sequence (5′-3′) pBI121-PsWRKY33-F gagaacacgggggactctagaATGTCTTCCTCCGCTAGCAGTC(XbaI) pBI121-PsWRKY33-R ataagggactgaccacccgggTCATAGAAAGGACTCAAAAAATGTGTC(SmaI)

[0081] 3.3.1.1.2 Amplification and recovery of target genes

[0082] cDNA from petals of 'Xueying Taohua' at different stages was used as a template for amplification of the open reading frame of the PsWRKY33 gene using 2× Flash Master Mix. Amplification was performed according to the reagent instructions, and the amplified products were detected by gel electrophoresis. The detected bands were consistent in size with the target gene product.

[0083] Cut the above strips, refer to Fast The product was purified according to the instructions of Gel DNA Extraction Mini Kit (Vazyme, Nanjing).

[0084] 3.3.1.1.3 Enzyme Digestion

[0085] pBI121 was double-digested using the XbaI and SmaI restriction sites, and the mixture was placed in a PCR instrument for constant enzyme digestion at 37°C for 30 minutes and inactivated at 80°C for 20 minutes.

[0086] 3.3.1.1.4 Connection

[0087] The target gene was recombined with the enzyme digestion solution using 5×ABscript III RT Mix. The reaction system is shown in Table 4. The reaction conditions were 50°C for 15 min.

[0088] Table 4 Recombination reaction system

[0089]

[0090] 3.3.1.1.5 Transformation into E. coli DH5ɑ competent cells

[0091] (1) Take out the competent DH5ɑ from the -80℃ freezer and thaw on ice;

[0092] (2) Transfer 10 μL of recombinant plasmid to DH5ɑ competent medium using a pipette;

[0093] (3) Ice bath for 30 min, heat shock in a 42°C water bath for 45 sec, and ice bath for 2 min;

[0094] (4) Add 800 μL of LB liquid medium without antibiotics;

[0095] (5) Place the transformed bacterial solution in a 37°C shaker at 200 rpm for 60 min.

[0096] (6) Centrifuge the bacterial solution at 5000 rpm for 30 seconds, retain 50 μL of the supernatant, resuspend it, and spread it on LB solid medium containing Kan for overnight culture.

[0097] 3.3.1.1.6 Bacterial liquid PCR and plasmid extraction

[0098] Single clones were picked for culture, turbid strains were identified by PCR, strains corresponding to positive bands were sent for sequencing and sequence comparison, strains corresponding to sequences consistent with the PsWRKY33 sequence were expanded overnight, and recombinant plasmids were extracted.

[0099] 3.3.1.2 Transformation into Agrobacterium EHA105 competent cells

[0100] (1) Take out the EHA105 competent cells from the -80°C freezer and thaw on ice;

[0101] (2) 1 μg of the above plasmid was transferred to the competent EHA105 cells;

[0102] (3) ice bath for 5 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, ice bath for 5 min;

[0103] (4) Add 800 μL of LB liquid medium without antibiotics, place the transformed bacterial solution in a 28°C shaker at 200 rpm, and let it recover for 2-3 hours;

[0104] (5) Centrifuge at 5000 rpm for 30 seconds, retain 50 μL of supernatant, resuspend, and apply to LB solid medium containing Kan + Rif, and invert and culture for 48-72 hours;

[0105] (6) Pick a single colony for bacterial liquid PCR, detect the amplified product by gel electrophoresis, and select the bacterial liquid with the same size as the target band for amplification and preservation.

[0106] 3.3.1.3 Agrobacterium preparation

[0107] Inoculate 200 μL of each pBI21 and pBI21-PsWRKY33 Agrobacterium strain into 50 mL of LB+Kan+Rif medium and culture overnight for activation. Inoculate the overnight culture into LB+Kan+Rif liquid medium at a ratio of 1:210 and culture at 28°C with a shaker at 210 rpm for 8-12 hours until the OD value reaches 1.0-1.2. Centrifuge at 4000 rpm for 8 minutes to collect the bacteria. Discard the supernatant and resuspend the cells in infection solution (see 2.3) to an OD value of 1.0-1.2. Use the pBI21 resuspension as the control group and the pBI21-PsWRKY33 resuspension as the experimental group. Place them in a large beaker for later use.

[0108] 3.3.1.4 Infection of peony buds

[0109] Prepare peony buds according to the experimental material processing method. Pre-prick the top of the bud with a needle several times. Place the treated peony bud upside down in a beaker filled with infection solution. Immerse the entire bud in the infection solution. Place the beaker in a vacuum pump and pump air at 0.1 atmosphere for 20 minutes, then slowly release the air. Repeat this three times. Rinse twice with tap water and once with bacterial solution. Incubate in the dark at 8°C for 2-3 days, then transfer to normal culture (16 hours daytime / 8 hours nighttime) for 2-3 days.

[0110] 3.3.1.5 Detection of the expression levels of PsWRKY33 and senescence marker genes PsSAG13 and PsSAG40.1

[0111] Peony buds that had been kept in the dark for 3 days at 8°C were removed and cultured under normal light conditions. The start of normal light exposure was designated as day 0. On day 2, RNA was extracted from peony petals and reverse-transcribed to obtain cDNA. The expression of PsWRKY33 in the peony buds was then analyzed using qRT-PCR. Buds with higher PsWRKY33 expression than the control were selected as successful overexpression buds for subsequent experiments. The expression levels of the senescence marker genes PsSAG13 and PsSAG40.1 were also measured using qRT-PCR in both control and successfully overexpressed peony buds.

[0112] 3.3.1.6 DAB and NBT staining

[0113] Petal discs were obtained by punching holes at the same location on the petals of the control and overexpression peony plants. These discs were then placed in 50 mL centrifuge tubes. DAB and NBT dye solutions (Coollebo, Beijing) were added, respectively. Vacuum pumps were used to evacuate the solution for 30 minutes, followed by slow degassing. The staining was allowed to proceed overnight at room temperature. The next day, the stained petals were decolorized with 95% ethanol in an 80°C water bath until clear.

[0114] 3.3.1.7 Determination of MDA content and SOD activity

[0115] According to the operating instructions of the MDA content detection kit, the MDA content of the peony petals in the experimental group and the control group were determined; according to the operating instructions of the SOD activity detection kit, the SOD activity of the peony petals in the experimental group and the control group were determined.

[0116] 3.3.2 VIGS silencing of PsWRKY33 in peony petals

[0117] 3.3.2.1 Construction of TRV2-PsWRKY33 vector

[0118] Gene-specific sequences were retrieved using the SGN VIGS Tool (https: / / vigs.solgenomics.net / ?tdsourcetag=s_pctim_aiomsg). Specific primers (Table 6) were designed to amplify the open reading frame of PsWRKY33. Following the steps for vector construction and transformation in 3.3.1 Overexpression Vector, the TRV2-PsWRKY33 vector was constructed and transformed into Agrobacterium tumefaciens EHA105.

[0119] Table 6 Primers required for PsWRKY33 ligation to TRV2 vector

[0120] Primer Sequence (5′-3′) TRV2-PsWRKY33-F aaggttaccgaattctctagaCAAGTGAAAGGAAGTGAGAATCCG(XbaI) TRV2-PsWRKY33-R gagacgcgtgagctcggtaccGCCTTGATCAAAGTCATCCTCTCC(KpnI)

[0121] 3.3.2.2 Agrobacterium preparation

[0122] Take 200 μL of each of TRV1, TRV2 and TRV2-PsWRKY33 Agrobacterium and inoculate them into 50 mL of LB+Kan+Rif medium, and culture them overnight for activation. Take the overnight cultured bacterial solution and inoculate it into LB+Kan+Rif liquid medium at a ratio of 1:210, and culture it in a shaking incubator at 210 rpm at 28°C for 8-12 hours until the OD value reaches 1.0-1.2. Centrifuge at 4000 rpm for 8 minutes to collect the bacteria. Pour out the supernatant and resuspend the bacteria with infection solution (see 2.3) until the OD value reaches 1.0-1.2. Mix equal volumes of TRV1 and TRV2 infection solutions as a control group, and mix equal volumes of TRV1 and TRV2-PsWRKY33 as an experimental group. Place it in a large beaker in the dark and let it stand for 3-5 hours for later use.

[0123] 3.3.2.3 Infection of peony buds

[0124] Prepare peony buds according to the experimental material processing method. Pre-prick the top of the bud with a needle several times. Place the treated peony bud upside down in a beaker filled with infection solution. Immerse the entire bud in the infection solution. Place the beaker in a vacuum pump and pump air at 0.1 atmosphere for 20 minutes, then slowly release the air. Repeat this three times. Rinse twice with tap water and once with bacterial solution. Incubate in the dark at 8°C for 2-3 days, then transfer to normal culture (16 hours daytime / 8 hours nighttime) for 2-3 days.

[0125] 3.3.2.4 Detection of the expression of PsWRKY33 senescence-related genes PsSAG13 and PsSAG40.1

[0126] Peony buds that had been kept in the dark for 3 days at 8°C were removed and cultured under normal light conditions. The start of normal light exposure was designated as day 0. On day 2, RNA was extracted from peony petals and reverse-transcribed to obtain cDNA. The expression of PsWRKY33 in the peony was then analyzed using qRT-PCR. Buds with lower PsWRKY33 expression than the control were selected as successfully silenced buds for subsequent experiments. qRT-PCR was used to determine the expression levels of the senescence-related genes PsSAG13 and PsSAG40.1 in both control and successfully silenced peony buds.

[0127] 3.3.2.5 DAB and NBT staining

[0128] According to the method in 3.3.1.6, the peony petals of the control group and the silenced group were stained and decolorized until clear.

[0129] 3.3.2.6 Determination of MDA content and SOD activity

[0130] The MDA content and SOD activity of peony petals in the control group and silenced group were determined according to the method in 3.3.1.7.

[0131] 4 Results and Analysis

[0132] 4.1 Screening, cloning, and analysis of PsWRKY33

[0133] A significantly differentially expressed gene (Cluster-3322.19119) was found in the peony senescence-related transcriptome. Based on its sequence, a local blast was performed in the 'Xueyingtaohua' transcriptome database to obtain the full cDNA sequence of the gene. The obtained gene was translated into a protein sequence using DANMAN software and then analyzed for conserved domains on the NCBI website ( Figure 1 ), Cluster-3322.19119 was found to belong to WRKYs transcription factors. At the same time, phylogenetic analysis was performed with all WRKY proteins in Arabidopsis ( Figure 2 ) and found that the Cluster-3322.19119 protein sequence was highly homologous to the AtWRKY33 protein sequence in Arabidopsis thaliana. Therefore, the Cluster-3322.19119 sequence was named PsWRKY33, which belongs to the Group I WRKY transcription factor. The open reading frame (ORF) of PsWRKY33 is 1662 bp, encoding a 553-amino acid polypeptide with a molecular weight of 62.02 kDa and a theoretical isoelectric point (PI) of 6.33. The PsWRKY33 protein sequence was blasted from the NCBI website to identify homologous proteins, and the conserved domains were analyzed using DANMAN software ( Figure 3 ), found that PsWRKY33 has two WRKYGQ domains and a C2H2 (C-X4-C-X23-H-X1-H) type zinc finger structure.

[0134] 4.2 Spatiotemporal expression analysis of PsWRKY33 in ‘Xueyingtaohua’

[0135] The expression levels of PsWRKY33 gene in roots, stems, leaves, sepals, bracts, stamens and petals were detected by real-time fluorescence quantitative PCR. The results showed that the expression level of PsWRKY33 gene was high in sepals of 'Xueyingtaohua' and moderate in petals. Figure 4 C). Then, the expression of PsWRKY33 in the petals of 'Xueying Taohua' at different developmental stages was detected. It was found that the expression level of PsWRKY33 gradually increased from S1 to S5, and reached the highest level in S5 ( Figure 4 B).

[0136] Functional Identification of PsWRKY33 in Peony Flower Senescence

[0137] 4.3.1 Overexpression of PsWRKY33 in Peony Petal Discs

[0138] 4.3.1.1 Construction of pBI121-PsWRKY33 Overexpression Vector

[0139] The PsWRKY33 sequence was amplified using specific primers and detected by gel electrophoresis. The electrophoresis results showed that the target band size was consistent with the expected ( Figure 5 A). Following the seamless cloning method, the target band was connected to the vector and transformed into the DH5ɑ E. coli strain. The transformed DH5ɑ was then spread on a plate containing antibiotics and a well-growing monoclonal strain was selected. To avoid strains with empty vectors self-ligated, PCR identification was performed and strains with the target band size consistent with PsWRKY33 were selected and sent to the company for sequencing ( Figure 5 B), the sequencing results were compared with the target sequence to confirm that the clone was correct. Then, the recombinant plasmid was extracted and transformed into Agrobacterium EHA105 strain, the strain was spread on a medium containing Kan and Rif, and a single clone was selected for PCR identification to screen out the positive strain ( Figure 5 C).

[0140] 4.3.1.2 PsWRKY33 overexpression peony bud phenotype

[0141] After overexpression of PsWRKY33 in peony buds, the overexpression efficiency of PsWRKY33 in peony buds was detected by qRT-PCR. The results showed that the expression level of PsWRKY33 was significantly increased in the overexpressed peony buds, indicating that the PsWRKY33 gene was successfully overexpressed in peony. Figure 6 C).

[0142] Phenotypic observation of peony buds overexpressing PsWRKY33 revealed that petals in peony flowers overexpressing PsWRKY33 began to fall on the 3rd day and were almost completely detached on the 6th day, while petals in the control group began to fall on the 4th day and were almost completely detached on the 7th day. Figure 6 A, B), indicating that overexpression of PsWRKY33 can promote the shedding of peony petals. qRT-PCR detection of the expression levels of PsSAG13 and PsSAG40.1 genes in the peony buds with successful overexpression found that the expression levels of PsSAG13 and PsSAG40.1 were significantly upregulated ( Figure 6 D), further indicating that PsWRKY33 can promote the shedding and senescence of peony petals.

[0143] To investigate how silencing of PsWRKY33 affects the accumulation of reactive oxygen species and superoxide anions, DAB and NBT staining were performed on the peony petals in the control and overexpression groups. The results showed that the petals in the control group were lighter in color, while the petals overexpressing PsWRKY33 were darker in color, indicating that the accumulation of reactive oxygen species and superoxide anions increased after overexpression of PsWRKY33 ( Figure 7 A, B). The SOD and MDA contents in the peony petals of the control group and the overexpression group were detected, and it was found that overexpression of PsWRKY33 could reduce the activity of superoxide dismutase (SOD) and promote the production of malondialdehyde (MDA) ( Figure 7 C, D).

[0144] 4.3.2 Silencing PsWRKY33 in Peony Petals

[0145] 4.3.2.1 Construction of TRV2-PsWRKY33 Vector

[0146] The PsWRKY33 sequence was amplified using specific primers and detected by gel electrophoresis. The electrophoresis results showed that the target band size was consistent with the expected ( Figure 8 A). Following the seamless cloning method, the target band was connected to the vector and transformed into the DH5ɑ E. coli strain. The transformed DH5ɑ was then spread on a plate containing antibiotics and a well-growing monoclonal strain was selected. To avoid strains with empty vectors self-ligated, PCR identification was performed and strains with the target band size consistent with PsWRKY33 were selected and sent to the company for sequencing ( Figure 8 B), the sequencing results were compared with the target sequence to confirm that the clone was correct. Then, the recombinant plasmid was extracted and transformed into Agrobacterium EHA105 strain, the strain was spread on a medium containing Kan and Rif, and a single clone was selected for PCR identification to screen out the positive strain ( Figure 8 C).

[0147] 4.3.2.2 PsWRKY33 silencing peony bud phenotype

[0148] After silencing PsWRKY33 in peony buds, the silencing efficiency of PsWRKY33 in peony buds was detected by qRT-PCR. The results showed that the expression level of PsWRKY33 was significantly reduced in silenced peony buds, indicating that the PsWRKY33 gene was successfully silenced in peony. Figure 9 C).

[0149] Phenotypic observation of peony buds with PsWRKY33 silenced revealed that petals in peony flowers with PsWRKY33 silenced began to fall on the 4th day and were almost completely detached on the 6th day, while petals in the control group began to fall on the 3rd day and were almost completely detached on the 5th day. Figure 9A, B), indicating that silencing PsWRKY33 can delay the shedding of peony petals. qRT-PCR detection of the expression levels of PsSAG13 and PsSAG40.1 genes in peony buds with successful silencing found that the expression levels of PsSAG13 and PsSAG40.1 were significantly downregulated ( Figure 9 D), further indicating that silencing PsWRKY33 can delay the shedding and senescence of peony petals.

[0150] To investigate how PsWRKY33 silencing affects the accumulation of reactive oxygen species and superoxide anions, DAB and NBT staining were performed on the peony petals in the control and silenced groups. The results showed that the petals in the control group were stained darker, while the petals in the PsWRKY33 silenced group were stained lighter, indicating that the accumulation of reactive oxygen species and superoxide anions increased and decreased after PsWRKY33 silencing treatment ( Figure 10 A, B). The SOD and MDA contents in the peony petals of the control group and the overexpression group were detected, and it was found that silencing PsWRKY33 could increase the activity of superoxide dismutase (SOD) and reduce the production of malondialdehyde (MDA) ( Figure 10 C, D), indicating that PsWRKY33 can promote the abscission and senescence of peony petals.

Claims

1. A PsWRKY33 gene that regulates peony petal senescence, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the PsWRKY33 gene according to claim 1, wherein: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A carrier, characterized in that: The vector contains the PsWRKY33 gene according to claim 1 and is used to regulate peony petal senescence.

4. An Agrobacterium, characterized in that: The Agrobacterium contains the PsWRKY33 gene vector according to claim 3 and is used to regulate the senescence of peony petals.

5. Use of the PsWRKY33 gene according to claim 1 in delaying senescence of peony petals.

6. The use according to claim 5, characterized in that: This was achieved by silencing the PsWRKY33 gene.

7. Use of the PsWRKY33 gene according to claim 1 in promoting senescence of peony petals.

8. The use according to claim 7, characterized in that: This was achieved by overexpressing the PsWRKY33 gene.

Images