Application of PhZPT2-5 gene in regulating anthocyanin synthesis in petunia

Abstract

This invention discloses the application of the PhZPT2-5 gene in regulating anthocyanin synthesis in petunias, belonging to the field of plant genetic engineering technology. This invention discovers that the PhZPT2-5 gene is significantly correlated with anthocyanin synthesis in petunias. Experiments show that silencing the PhZPT2-5 gene reduces anthocyanin synthesis, while overexpressing the PhZPT2-5 gene promotes anthocyanin synthesis. By regulating the expression of the PhZPT2-5 gene in petunias, different colored petunias can be obtained, laying the foundation for breeding new petunia varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the PhZPT2-5 gene in regulating anthocyanin synthesis in petunias. Background Technology

[0002] Zinc-finger proteins (ZFPs) are an important family of proteins characterized by the presence of zinc finger domains. ZFPs participate in various biological processes, including gene expression regulation and protein-protein interactions, through interactions with DNA, RNA, and proteins. The ZFP family contains numerous genes, many of which have been verified to be related to anthocyanin synthesis. For example, overexpression of AtZAT12 in Arabidopsis thaliana promotes anthocyanin accumulation, resulting in thicker, darker green leaves. AtZAT12 directly or indirectly regulates the expression of anthocyanin biosynthesis genes, leading to increased anthocyanin content in transgenic plants. AtZAT6 plays a crucial role in H2O2-activated anthocyanin synthesis by directly regulating the transcription of genes such as CHS / TT4, F3H / TT6, F3′H / TT7, and DFR / TTD. Overexpression of PbZAT12 in pear significantly upregulated the expression levels of CHS, DFR1, ANS1, UFGT2, and GST, promoting anthocyanin accumulation in pear peel. Silencing the PbZAT12 gene led to a significant decrease in the expression levels of anthocyanin synthesis-related genes PAL, UFGT2, MYB10, MYB10b, bHLH3, and bHLH33. PpZAT5 inhibited anthocyanin synthesis in pears by negatively regulating the expression of PpBBX18. Transcriptomic data from two different tropical water lilies (Nymphaea tetragona) under cold stress showed that the expression level of NlZAT12 was closely related to anthocyanin accumulation. ZPT2-5 is a gene in the zinc finger protein family, but there are currently no reports on its association with anthocyanin synthesis. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the PhZPT2-5 gene in regulating anthocyanin synthesis in petunias, in order to solve the problems existing in the prior art. By regulating the expression of the PhZPT2-5 gene in petunias, anthocyanin synthesis can be reduced or promoted, thereby obtaining petunias of different colors and laying the foundation for enriching the varieties of petunias.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the application of the PhZPT2-5 gene in any of the following:

[0006] (1) Application in regulating anthocyanin synthesis in petunia;

[0007] (2) Application in the breeding of purple petunia flowers;

[0008] The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1:

[0009] .

[0010] This invention also provides the use of the protein encoded by the PhZPT2-5 gene in any of the following:

[0011] (1) Application in regulating anthocyanin synthesis in petunia;

[0012] (2) Application in the breeding of purple petunia flowers;

[0013] The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0014] MVAPSTKREREEDNFYSITTMANYLMLLSRQANEHFDKNMNNNSSTSRVFECKTCNR RFSSFQALGGHRASHKKPRLMGDLHNLQLFHELPKRKTHECSICGLEFAIGQALGGHMRRH RAVMNDKNVQAPDDQHAPVVKKANGRRILSLDLNLTPLENDLEFDLRKSNTAPMVDCFL*.

[0015] This invention also provides the use of biological samples containing the PhZPT2-5 gene in any of the following:

[0016] (1) Application in regulating anthocyanin synthesis in petunia;

[0017] (2) Application in the breeding of purple petunia flowers;

[0018] The biological sample contains the nucleotide sequence of the PhZPT2-5 gene integrated into its gene sequence, and the nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1.

[0019] Optionally, the biological sample includes an expression vector, an expression cassette, and a microbial strain.

[0020] Optionally, silencing the PhZPT2-5 gene reduces anthocyanin synthesis in petunia flowers; overexpressing the PhZPT2-5 gene promotes anthocyanin synthesis in petunia flowers.

[0021] The present invention also provides a method for reducing anthocyanin synthesis in petunia flowers, comprising the step of silencing the PhZPT2-5 gene in petunias to reduce anthocyanin synthesis in petunias, wherein the nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.1.

[0022] The present invention also provides a method for promoting anthocyanin synthesis in petunia flowers, comprising the step of overexpressing the PhZPT2-5 gene in petunias to promote anthocyanin synthesis in petunias, wherein the nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.1.

[0023] The present invention also provides a breeding method for light purple petunia flowers, comprising the step of silencing the PhZPT2-5 gene in petunias to reduce anthocyanin synthesis in petunias and obtain light purple petunias flowers, wherein the nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.1.

[0024] This invention also provides a breeding method for deep purple petunia flowers, comprising the step of overexpressing the PhZPT2-5 gene in petunias to promote anthocyanin synthesis in petunias and obtain deep purple petunias flowers, wherein the nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.1.

[0025] The present invention discloses the following technical effects:

[0026] This invention discovered that the PhZPT2-5 gene is significantly associated with anthocyanin synthesis in petunia flowers. Experiments showed that silencing the PhZPT2-5 gene can reduce anthocyanin synthesis, while overexpressing the PhZPT2-5 gene can promote anthocyanin synthesis. By regulating the expression of the PhZPT2-5 gene in petunia flowers, petunia flowers of different colors can be obtained, laying the foundation for breeding new varieties of petunias. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Sequence analysis of PhZPT2-5; (A) Phylogenetic tree of ZPT2-5; (B) ZPT2-5 protein sequence alignment (domains are indicated by red boxes);

[0029] Figure 2 The results of the expression level analysis of PhZPT2-5 in different tissues of petunia; lowercase letters (ac) indicate significant differences (p<0.05);

[0030] Figure 3 The results of photoperiod regulation of anthocyanin synthesis and PhZPT2-5 expression were presented. (A) Phenotypic analysis under different photoperiod treatments; (B) Comparison of anthocyanin content under 16h / 8h and 10h / 14h treatments; (C) Comparison of PhZPT2-5 expression levels under 16h / 8h and 10h / 14h treatments; (D) Comparison of PhCHS expression levels under 16h / 8h and 10h / 14h treatments. Lowercase letters (ab) indicate significant differences (p<0.05).

[0031] Figure 4PhZPT2-5 virus-silenced plant phenotype analysis results; (A) PhZPT2-5 virus-silenced plant phenotype; (B) Anthocyanin extracts from control plants and PhZPT2-5 virus-silenced plants; (C) Anthocyanin content determination; (D) PhZPT2-5 expression level detection; Control is the control, TRV2-0 is the plant transformed with the empty vector, and Line1 and Line are PhZPT2-5 virus-silenced plants; lowercase letters (ab) indicate significant differences (p<0.05);

[0032] Figure 5 Phenotypic analysis of PhZPT2-5 overexpressing transgenic plants; (A) Phenotypic comparison between wild-type and PhZPT2-5 overexpressing transgenic plants, where Control represents wild-type plants and OE 1 and OE 2 represent different transgenic lines; (B) Comparison of leaves between wild-type and transgenic plants; (C) Detection of PhZPT2-5 expression level;

[0033] Figure 6 Phenotypic analysis results of petunia PhZPT2-5 transient injection plants; Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] 1. Experimental Methods

[0041] 1.1 Extraction and concentration determination of total RNA from various tissues of petunia

[0042] use RNA extraction using the Universal Plant Total RNA Isolatin Kit was performed according to the instructions. The specific steps are as follows: Take an appropriate amount of plant tissue that has been ground in liquid nitrogen and immediately add 600 μL of Buffer EL. Vortex vigorously for 30 seconds to ensure thorough mixing with the lysis buffer. Centrifuge at 12,000 rpm for 5 minutes. Transfer approximately 500 μL of the supernatant to FastPure gDNA-Filter Columns III. Centrifuge at 12,000 rpm for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate. Add 0.5 times the volume of the filtrate to the collection tube and vortex to mix. Transfer the mixture to FastPure RNA Columns V. Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate. Add 700 μL of Buffer RWA to FastPure RNA Columns V. Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate. Add 500 μL of Buffer RWB to FastPureRNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Return FastPureRNA Columns V to the collection tube and centrifuge at 12,000 rpm for 2 minutes. Transfer FastPure RNA Columns V to a new RNase-free 1.5 mL centrifuge tube, add 30–100 μL of RNase-free ddH₂O dropwise to the center of the adsorption column membrane, and centrifuge at 12,000 rpm for 1 minute to obtain the extracted RNA.

[0043] 1.2 cDNA reverse transcription

[0044] Using MonScript from Promed Precision Medical Technology (Beijing) Co., Ltd. TM The RTIII All-in-One MixwithdsDNase kit was used according to the instructions. The specific steps are as follows: Take 50 ng to 1 μg of RNA and add MonScript sequentially. TM dsDNase, EX 1μL, MonScript TM Add 1 μL of 10×dsDNase Buffer and 10 μL of Nuclease-Free Water, then incubate at 37°C for 2 min to remove genomic DNA contamination; incubate at 55°C for 5 min to inactivate dsDNase, then place on ice. Add 1 μL of EDTA, mix well, and incubate on ice for 5 min. Incubate in a 65°C water bath for 10 min, then immediately place on ice for 2 min. Add MonScript to the product obtained in the first step. TM Mix 4 μL of 5×RTⅢSuper Mix gently by pipetting; incubate at 50°C for 15 min; incubate at 85°C for 5 min to terminate the reaction; store the obtained cDNA at -20°C.

[0045] 1.3 PCR and qRT-PCR

[0046] The PCR reaction system consisted of: cDNA (1 μL); Forward Primer (1 μL); Reverse Primer (1 μL); 2×PCR Master Mix (10 μL); ddH2O (7 μL); and a total volume of 20 μL. The PCR program was as follows: 94℃, 3 min; 94℃, 30 s; 58℃, 30 s; 72℃, 2 min; 35 cycles; 72℃, 10 min extension; 4℃, Forever. The amplified products were detected on a 1% agarose gel, stained with Goldview stain, and observed under UV light.

[0047] qRT-PCR: using Novoprotein's NovoPCR. Add the following to a 96-well plate sequentially: SYBR qPCR Super Mix Plus kit; cDNA (2 μL); forward and reverse primers (0.8 μL each); RNase-free water (6.4 μL); and NovoRapids. SYBR qPCR Super Mix Plus (10 μL). The reaction program was as follows: 95℃, 1 min; 95℃, 20 s; 55℃, 20 s; 72℃, 30 s; 40 cycles. GAPDH gene was used as an internal control. Relative expression levels were measured using 2... -ΔΔCtThe data were calculated using SPSS software and statistically analyzed. All primers and their sequences used in this invention are shown in Table 1.

[0048] Table 1: Primer Table

[0049]

[0050]

[0051] 1.4 Detection of gene expression levels

[0052] Petunia plants of similar growth and age (100 days) were selected, and samples were taken from their roots, stems, leaves, leaf axils, and flowers. Three technical replicates were taken from each sample. RNA was extracted, reverse transcribed into cDNA, diluted 5 times, and then detected by qRT-PCR.

[0053] Petunia plants of similar growth age (60 days) were selected. After different photoperiod and high-temperature treatments, petals with significant color changes were collected, and RNA was extracted. The extracted RNA was then subjected to cDNA reverse transcription and finally detected by qRT-PCR.

[0054] Construction and transformation of 1.5PhZPT2-5 overexpression vector

[0055] (1) Primers PhZPT2-5-1300-F / PhZPT2-5-1300-R were designed using Primer Premier 5.0 to amplify the target gene and then perform gel recovery.

[0056] (2) pSuper1300-GFP was double-digested with HindIII and SalI. The digestion system was as follows: 3 μL plasmid DNA, 1 μL each restriction enzyme, 2 μL 10×Qcut Buffer, and 13 μL ddH2O. Digestion conditions: 37℃, 40 min, 50℃, 5 min.

[0057] (3) Combine the amplified target gene fragment with the vector's double enzyme digestion fragment reference kit. Follow the instructions for using the plusOne step PCR Cloning Kit to perform recombination reactions;

[0058] (4) Add 10 μL of recombinant product to 50 μL of LDH5α competent cells, mix well, and place on ice for 30 min; incubate in a 42℃ water bath for 30 s to 1 min, then immediately place on ice for 2 to 3 min; add 500 μL of LB solution, and place in a shaker at 37℃ for 1 h until the bacterial culture becomes turbid; centrifuge at 6000 rpm for 3 min, and after mixing well, spread the 250 μL of supernatant evenly on an LB plate and incubate in an inverted incubator at 37℃ overnight.

[0059] (5) Select positive plaques for culture and sequencing. Add the correctly sequenced recombinant plasmid to 100 μL of GV3101 Agrobacterium competent cells and incubate on ice for 5 min; incubate in liquid nitrogen for 5 min, incubate in water at 28℃ for 5 min, and incubate on ice for 5 min; add 500 μL of liquid medium to the sterilizer and incubate at 28℃ with shaking for 2-3 h; centrifuge at 6000 rpm for 1 min to collect the cells, spread them evenly on YEB solid medium containing Rif and Kan resistance, and incubate at 28℃ for 2 days to identify positive clones for later use.

[0060] Cloning of the 1.6VIGS gene fragment

[0061] 1.6.1 Construction of the pTRV2-PhZPT2-5 vector

[0062] Using petunia cDNA as a template, primers ZPT2-5-VIGS-F / ZPT2-5-VIGS-R containing two restriction enzyme sites, EcoRI and BamHI, were designed. Amplification was performed using KOD high-fidelity enzyme, followed by gel electrophoresis. The target gene was then recovered from the gel. The target fragment product and the pTRV2 silencing vector were double-digested with EcoRI and BamHI, respectively, at 37℃ for 30 min and 80℃ for 5 min. The gel was then recovered, and the brightness was detected by gel electrophoresis. The double-digested gene target fragment and vector product were ligated using T4 DNA ligase in the specified ratio. The ligation system consisted of: T4 DNA Ligase (1 μL), T4 DNA Ligase Buffer (10 μL), double-digested target fragment (3 μL), double-digested plasmid vector fragment (3 μL), and ddH2O (4 μL). Ligate at 4℃, then transform the ligation product into Escherichia coli (DH5α), and extract the plasmid after shaking the positive clone and transform it into Agrobacterium (GV3101).

[0063] 1.6.2pTRV2-PhZPT2-5 instantaneous conversion of petunias

[0064] (1) Buffer preparation: When preparing mixed solutions of MES (1 mol / L), MgCl2 (1 mol / L) and acetylsuccinone (AS, 50 mg / L), prepare the following solutions: MgCl2 (1 mL), MES (1 mL), AS (80 μL) and ddH2O (98 mL);

[0065] (2) Take 1 mL each of TRV1 bacterial suspension and the constructed silent vector bacterial suspension, add them to 100 mL of liquid LB medium containing Rif and Kan, and culture at 28°C with shaking until the bacterial suspension becomes turbid.

[0066] (3) Collect bacterial cells, using buffer solution as a blank control, and measure the OD of the resuspended bacterial solution using a UV spectrophotometer. 600The value is approximately between 0.8 and 1.0; mix at a volume ratio of 1:1;

[0067] (4) After resuspending the TRV1 bacterial solution and the silent carrier bacterial solution, mix them at a volume ratio of 1:1 and then protect them from light for 3 hours. Select petunia plants with uniform growth and an age of 20-30 days and inject them into the back of the leaves with a syringe.

[0068] 1.7 Determination of anthocyanins

[0069] (1) Weigh about 1.0g of sample, cut it into pieces and put it into a test tube, and add 10mL of extraction solution;

[0070] (2) Extraction in the dark at 4℃ for 24 hours;

[0071] (3) Centrifuge at 12000 rpm for 5 min and collect the supernatant;

[0072] (4) Colorimetry: Measure the absorbance at 530 nm, 620 nm and 650 nm using a spectrophotometer;

[0073] (5) The anthocyanin content is calculated as follows:

[0074] Anthocyanin density: ODλ=A 530 -A 620 -0.1×(A 650 -A 620 );

[0075] Anthocyanin content = ODλ / ξλ × V / m × 10 6 (ξλ=4.62×10 4 );

[0076] Anthocyanin molecules' extinction coefficient at 530nm wavelength;

[0077] ODλ - anthocyanin optical density, V - extract volume, M - petal mass, ξλ - molar extinction coefficient, 10 6 -Unit conversion factor.

[0078] 1.8 Arabidopsis inflorescence staining and identification of positive plants

[0079] 1.8.1 Arabidopsis inflorescence staining

[0080] (1) Agrobacterium containing the pSuper1300::PhZPT2-5:GFP plasmid was shaken to OD. 600 =0.8-1.0, collect bacterial cells by centrifugation, resuspend the cells in infection buffer (2.215 g / L MS, 50 g / L sucrose, 0.5 g / L MES, pH = 5.8), and adjust OD. 600 =0.8;

[0081] (2) Immerse the Arabidopsis thaliana inflorescences in full bloom in the infection buffer for 2 min;

[0082] (3) Place it flat in a culture dish and cover it to keep it moist. After culturing in the dark for 24 hours, place it under normal conditions for further culture.

[0083] (4) Infect again after one week, and collect T0 generation seeds when the seeds mature.

[0084] 1.8.2 Identification of transgenic Arabidopsis thaliana

[0085] T0 Arabidopsis thaliana seeds were soaked in 75% ethanol (containing 0.02% Tween) for 1.5 min, rinsed once with sterile water, sterilized with 2% NaClO for 4-5 min, rinsed three times with sterile water, then suspended in sterile water, and dried on filter paper. They were then sown in MS medium (containing 25 mg / L Kan), treated at 4℃ for 2 days, and then placed in an incubator.

[0086] Leaves were harvested from resistant Arabidopsis plants, and DNA was extracted for PCR testing to determine successful transfection into Arabidopsis. T3 transgenic plants were selected for subsequent phenotypic analysis.

[0087] 1.8.3 Genetic transformation of petunias

[0088] Agrobacterium overexpressing PhZPT2-5 was activated, and the OD of the bacterial culture was measured when the culture was turbid. 600 Value, pending OD 600 When the concentration reaches 0.3, the bacterial culture is dispensed into 50mL centrifuge tubes, centrifuged at 5000rpm and 4℃ for 10min, the supernatant is discarded, and the bacterial cells are resuspended in the resuspended liquid using a pipette.

[0089] Using a sterilized blade, cut the leaves of sterile petunia seedlings into 0.5cm × 0.5cm cubes and place them on a symbiotic solid medium for pre-culture. After two days of dark incubation, place the leaf discs in the prepared infection solution. After 8 minutes, remove the petunia leaves from the bacterial solution and aspirate the bacterial solution from the leaves. Place the cleaned leaves, adaxially downwards, on the co-culture medium (1 / 2 MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 1.0 mg / L zeatin + 2.0% sucrose + 1% glucose), and arrange them closely on the symbiotic solid medium for further culture. Leave a portion of uninfected leaves as a control, with the leaf surface facing upwards, and place them loosely on the callus induction medium (1 / 2 MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 1.0 mg / L zeatin + 2% sucrose + 1% glucose + 250 mg / L cephalosporin + 100 mg / L kanamycin), keeping the culture conditions unchanged. After two days of dark culture, rinse the petunia leaves 5-6 times with sterile water. After 2-3 days, transfer the petunia leaves that have undergone dark culture to the selection medium (1 / 2 MS + 2% sucrose + 1% glucose + 250 mg / L cephalosporin + 100 mg / L kanamycin), seal and label them, and place them on a light-controlled tissue culture rack at 25℃. Change the medium every 12 days. When the resistant buds grow to about 1.5 cm, cut them off and place them in the rooting medium. After the petunia tissue culture seedlings have grown stably and large, harden them off and transplant them.

[0090] 1.9 PhZPT2-5 gene sequence analysis

[0091] (1) Search for homologous sequences of PhZPT2-5 from different species on NCBI (https: / / www.ncbi.nlm.nih.gov / ) and perform amino acid sequence alignment using DNAMAN 6 software;

[0092] (2) The protein sequences of ZPT2-5 family members of petunia and Arabidopsis were compared using MEGA 11 software, and a phylogenetic tree was constructed using the Neighbor Joining method and a Bootstrap value of 1000.

[0093] 1.10 Metabolomics Measurement

[0094] 1.10.1 Sample Extraction

[0095] (1) Biological samples were placed in a freeze dryer (Scientz-100F) for vacuum freeze drying;

[0096] (2) Grind into powder using a grinder (MM 400, Retsch) (30Hz, 1.5min);

[0097] (3) Weigh 50 mg of powder and dissolve it in 1.2 mL of 70% methanol extract;

[0098] (4) Vortex once every 30 minutes, each lasting 30 seconds, for a total of 6 vortices;

[0099] (5) After centrifugation (12000 rpm, 3 min), aspirate the supernatant and filter the sample using a microporous membrane (0.22 μm pore size);

[0100] (6) Store in a vial for UPLC-MS / MS analysis.

[0101] 1.10.2 Screening for Differential Metabolites

[0102] Based on the OPLS-DA results, the variable importance in projection (VIP) of the obtained multivariate OPLS-DA model was used to initially screen for metabolites with differences among different varieties or tissues. Simultaneously, the p-value or fold change (FC) of the univariate analysis was used to further screen for differentially expressed metabolites. A method combining the VIP value and FC of the OPLS-DA model was adopted to screen for differentially expressed metabolites.

[0103] 1.10.3 Transcriptome Determination

[0104] Total RNA was processed using either mRNA enrichment or rRNA removal methods. For mRNA enrichment, mRNA with polyA tails was enriched using magnetic beads with OligodT. For rRNA removal, rRNA was hybridized using a DNA probe. The DNA / RNA hybrid strands were selectively digested with RNase H, and the DNA probe was then digested with DNase I. After purification, the desired RNA was obtained. The obtained RNA was fragmented using a fragmentation buffer, and reverse transcription was performed using random N6 primers to synthesize cDNA double-stranded DNA. The ends of the synthesized double-stranded DNA were padded and phosphorylated at the 5' end, and a sticky end with a protruding "A" was formed at the 3' end. A bubble-shaped adapter with a protruding "T" at the 3' end was then ligated. The ligation product was amplified by PCR using specific primers. The PCR product was denatured into single strands by heat, and then circularized using a bridging primer to obtain a single-stranded circular DNA library, which was then sequenced.

[0105] Differential gene analysis was performed using DESeq, GO enrichment analysis was performed using Goseq, and KEGG analysis was performed using the KOBAS software.

[0106] 1.11 Screening and Detection of Interacting Proteins

[0107] 1.11.1 Screening of interacting proteins

[0108] Primers were designed to amplify the target gene. pGBKT7 was digested with EcoRI and SalI, and pGADT7 was digested with EcoRI and XhoI. The target gene was then ligated into the digested vector, and positive clones were identified.

[0109] 1.11.2 Detection of bait toxicity and self-activation

[0110] (1) Take the DNA vector (Carrier DNA, 10 mg / mL) out of the -20℃ freezer, boil it in water for 5 minutes, and then quickly place it on ice;

[0111] (2) Take 3 sterile EP tubes in a sterile table, add 5 μL of Carrier DNA to each, and add 1000 ng pGBKT7-GAL4, 1000 ng pGBKT7 and 1000 ng pGBKT7-PZPT2-5 respectively.

[0112] (3) Add 100 μL LY2Hgold competent cells and 500 μL PEG / LiAc conversion solution to the EP tube respectively, mix gently, and incubate at 30℃ for 30 min (mix once every 10 min);

[0113] (4) Add 20 μL DMSO, mix well, incubate in a 42℃ water bath for 20 min, centrifuge at 5.700g for 5 min, and discard the supernatant;

[0114] (6) Resuspend in 1 ml LYPD Plus Medium and incubate at 30°C with shaking for 90 min;

[0115] (7) Centrifuge at 700g for 5 min, discard the supernatant, and resuspend in 200 μL of 0.9% NaCl;

[0116] (8) Take 100 μL of each and spread it onto SD / -Trp plates and SD / -Trp / -His / -Ade / Xa-gal plates;

[0117] (9) Invert the above plates and incubate them in a 30℃ incubator for 3-5 days. Determine whether there is self-activation based on the growth of bacterial plaques in the defective culture medium.

[0118] 2. Results and Analysis

[0119] 2.1 PhZPT2-5 sequence analysis

[0120] Using the published petunia genome as a reference, primers were designed to clone the petunia ZPT2-5 gene, which is 531 bp in length and encodes 176 amino acids. A phylogenetic tree was constructed by combining PhZPT2-5 with 10 Arabidopsis ZAT proteins, and the results are as follows: Figure 1 As shown in Figure A, PhZPT2-5 and AtZAT12 cluster together, indicating that they are homologous genes. Using DNAMAN software, PhZPT2-5 was compared with ZPT2-5 homologous sequences from Arabidopsis thaliana, apple, and pear species, with the following results: Figure 1 As shown in Figure B, the PhZPT2-5 protein contains conserved domains of L-box, First zinc finger, Second zinc finger, and DLN-box, indicating that this gene is indeed a member of the C2H2 family.

[0121] 2.2 PhZPT2-5 Tissue Expression Analysis

[0122] RNA was extracted from the roots, stems, leaves, axillary buds, and flowers of petunia, and the expression pattern of the PhZPT2-5 gene was analyzed using qRT-PCR. The results are as follows: Figure 2 As shown, PhZPT2-5 was most highly expressed in petunia stems, followed by relatively high levels in roots and flowers, while the expression levels were lowest in leaves and leaf axils. The expression levels in stems, leaves, leaf axils, and flowers were 1.4, 0.16, 0.15, and 0.89 times higher than those in roots, respectively.

[0123] 2.3 Photoperiodic Regulation of PhZPT2-5 Expression

[0124] To analyze whether the PhZPT2-5 gene is regulated by photoperiod, petunia plants were subjected to photoperiod treatments of 16h / 8h (light / dark) and 10h / 14h, respectively. After 7 days, petals were collected for anthocyanin content determination and gene expression analysis. The results are as follows: Figure 3 The results showed that as the light exposure time decreased, the petal color gradually changed from dark purple to light purple, with white spots appearing at the petal edges. Anthocyanin content decreased to 0.4 times that of the control, and the expression level of PhCHS (a synthetic gene in the anthocyanin pathway, used as a control) decreased to 0.2 times that of the control. The expression level of PhZPT2-5 decreased to 0.5 times that of the control. These results indicate that photoperiod affects the formation of purple flowers and the expression of PhZPT2-5.

[0125] 2.4 Viral Silencing Phenotypic Analysis

[0126] To investigate the function of PhZPT2-5, the VIGS-PhZPT2-5 vector was constructed and transiently transformed into petunias. The results are as follows: Figure 4 As shown, PhZPT2-5 expression was significantly reduced after silencing, reaching 0.2 times that of the control. This was accompanied by a marked decrease in flower color and a reduction in anthocyanin content to 0.52 times that of the control. This indicates that the decrease in PhZPT2-5 expression led to a reduction in anthocyanin synthesis.

[0127] 2.5 Phenotypic Analysis of Arabidopsis PhZPT2-5 Overexpression Transgenic Plants

[0128] To further investigate the function of PhZPT2-5, a pSuper1300::PhZPT2-5:GFP overexpression vector was constructed and transformed into Arabidopsis thaliana. A total of 14 independent transgenic lines were obtained, screened up to the T3 generation, and two lines were selected for phenotypic analysis. Results are as follows: Figure 5 As shown, compared with the control, the leaves of the transgenic plants turned significantly purple. This result indicates that overexpression of PhZPT2-5 in Arabidopsis thaliana promotes anthocyanin accumulation, causing the leaves of Arabidopsis thaliana to turn purple.

[0129] 2.6 Phenotypic Analysis of Petunia Plants Injected with PhZPT2-5

[0130] To investigate the function of PhZPT2-5, the pSuper1300::PhZPT2-5:GFP vector was constructed and transiently transformed into petunias. The results are as follows: Figure 6 As shown, when Agrobacterium-overexpressing solution was injected into petunia 'Mirage Blue Light', the flower color of petunias (Line 1, Line 2, and Line 3) overexpressing PhZPT2-5 became significantly darker. This indicates that increased PhZPT2-5 expression levels lead to increased anthocyanin synthesis.

[0131] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of the PhZPT2-5 gene in any of the following: (1) Application in regulating anthocyanin synthesis in petunia; (2) Application in the breeding of purple petunia flowers; The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1; Silencing the PhZPT2-5 gene reduces anthocyanin synthesis in petunia flowers, resulting in light purple petunias; overexpressing the PhZPT2-5 gene promotes anthocyanin synthesis in petunias, resulting in dark purple petunias.

2. The application of the protein encoded by the PhZPT2-5 gene in any of the following: (1) Application in regulating anthocyanin synthesis in petunia; (2) Application in the breeding of purple petunia flowers; The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2; Silencing the PhZPT2-5 gene reduces anthocyanin synthesis in petunia flowers, resulting in light purple petunias; overexpressing the PhZPT2-5 gene promotes anthocyanin synthesis in petunias, resulting in dark purple petunias.

3. Application of biological samples containing the PhZPT2-5 gene in any of the following: (1) Application in regulating anthocyanin synthesis in petunia; (2) Application in the breeding of purple petunia flowers; The biological sample contains the nucleotide sequence of the PhZPT2-5 gene integrated into its gene sequence, and the nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.

1. Overexpression of the PhZPT2-5 gene promotes anthocyanin synthesis in petunia flowers, resulting in deep purple petunias.

4. Application of biological samples with silenced PhZPT2-5 gene in any of the following: (1) Application in regulating anthocyanin synthesis in petunia; (2) Application in the breeding of purple petunia flowers; The biological sample contains the nucleotide sequence of the PhZPT2-5 gene integrated into its gene sequence, and the nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.

1. Silencing the PhZPT2-5 gene reduces anthocyanin synthesis in petunia flowers, resulting in light purple petunias.

5. The application as described in claim 3 or 4, characterized in that, The biological samples include expression vectors, expression cassettes, and microbial strains.

6. A method for reducing anthocyanin synthesis in petunia flowers, characterized in that, The method includes a step of silencing the PhZPT2-5 gene in petunias to reduce anthocyanin synthesis in petunia flowers, the nucleotide sequence of which is shown in SEQ ID NO.

1.

7. A method for promoting anthocyanin synthesis in petunia flowers, characterized in that, The method includes the step of overexpressing the PhZPT2-5 gene in petunias to promote anthocyanin synthesis in petunia flowers, the nucleotide sequence of which is shown in SEQ ID NO.

1.

8. A breeding method for light purple petunias, characterized in that, The method includes the steps of silencing the PhZPT2-5 gene in petunias to reduce anthocyanin synthesis in petunia flowers and obtain light purple petunia flowers, wherein the nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.

1.

9. A breeding method for deep purple petunias, characterized in that, The method includes the steps of overexpressing the PhZPT2-5 gene in petunias to promote anthocyanin synthesis in petunia flowers and obtain deep purple petunia flowers, wherein the nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1.

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