Application of PhZPT2-5 gene in regulation and control of anthocyanin synthesis of petunia hybrida

Abstract

The invention discloses application of a PhZPT2-5 gene in regulation and control of anthocyanin synthesis of petunia hybrida, and belongs to the technical field of plant gene genetic engineering. The invention finds that the PhZPT2-5 gene is remarkably related to the anthocyanin synthesis of petunia, and experiments find that the anthocyanin synthesis can be reduced by silencing the PhZPT2-5 gene, the anthocyanin synthesis can be promoted by overexpressing the PhZPT2-5 gene, petunia with different colors can be obtained by regulating the expression of the PhZPT2-5 gene in the petunia, and a foundation is laid for breeding new varieties of petunia.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly to the application of the PhZPT2-5 gene in regulating anthocyanin synthesis in petunias. Background Art

[0002] The zinc-finger protein (ZFP) family is an important family of proteins, and its main characteristic is that they contain zinc-finger domains. Zinc-finger proteins participate in various biological processes such as gene expression regulation and protein interaction by interacting with DNA, RNA, and proteins. The zinc-finger protein family contains numerous genes, and there are already genes verified to be related to anthocyanin synthesis. For example, overexpression of Arabidopsis AtZAT12 promotes anthocyanin accumulation, making the leaves thicker and dark green. AtZAT12 directly or indirectly regulates the expression of anthocyanin biosynthesis genes, resulting in an increase in anthocyanin content in transgenic plants. AtZAT6 plays an important 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 pears significantly upregulates the expression levels of CHS, DFR1, ANS1, UFGT2, and GST, promoting anthocyanin accumulation in pear peels. Silencing of the PbZAT12 gene leads to a significant decrease in the expression levels of anthocyanin synthesis-related genes PAL, UFGT2, MYB10, MYB10b, bHLH3, and bHLH33. PpZAT5 inhibits anthocyanin synthesis in pears by negatively regulating the expression of PpBBX18. Transcriptomic data of two different tropical water lilies (Nymphaea tetragona) under cold stress show that the expression level of NlZAT12 is closely related to anthocyanin accumulation. ZPT2-5 is a gene of the zinc-finger protein family genes, but there is currently no report on its relationship with anthocyanin synthesis. Summary of the Invention

[0003] The object of the present invention is to provide the application of the PhZPT2-5 gene in regulating anthocyanin synthesis in petunias, so as to solve the problems existing in the above-mentioned prior art. By regulating the expression of the PhZPT2-5 gene in petunia flowers, the reduction or promotion of anthocyanin synthesis is achieved, and then petunia flowers of different colors can be obtained, laying a foundation for enriching petunia varieties.

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

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

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

[0007] (2) Application in breeding of Petunia hybrida purple flowers;

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

[0009] ATGGTGGCTCCATCAACGAAGAGAGAAAGAGAAGAAGATAACTTTTACAGCATAACAACCATGGCAAATTACTTGATGTTACTCTCACGCCAAGCAAATGAACATTTTGACAAGAATATGAACAACAACTCAAGTACTAGTCGAGTTTTCGAGTGCAAGACTTGTAATCGCCGGTTTTCATCTTTTCAAGCACTAGGTGGCCATAGAGCAAGTCACAAGAAGCCAAGATTAATGGGAGACTTGCATAACTTGCAATTATTTCATGAATTGCCTAAACGTAAAACTCACGAGTGCTCCATTTGTGGGCTTGAGTTCGCCATTGGGCAAGCTTTAGGAGGACATATGAGAAGGCATAGAGCTGTGATGAATGATAAAAATGTTCAAGCTCCTGATGATCAACATGCTCCTGTCGTCAAAAAAGCAAATGGTCGGAGAATTTTGTCCTTGGATTTGAACTTGACGCCATTGGAAAATGACTTAGAATTTGATTTGCGAAAGAGTAATACTGCTCCTATGGTCGATTGCTTTTTATGA.

[0010] The present invention also provides the application of the protein encoded by the PhZPT2-5 gene in any of the following:

[0011] (1) Application in regulating the synthesis of anthocyanin in Petunia hybrida;

[0012] (2) Application in breeding of Petunia hybrida purple flowers;

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

[0014] MVAPSTKREREEDNFYSITTMANYLMLLSRQANEHFDKNMNNNSSTSRVFECKTCNRRFSSFQALGGHRASHKKPRLMGDLHNLQLFHELPKRKTHECSICGLEFAIGQALGGHMRRHR AVMNDKNVQAPDDQHAPVVKKANGRRILSLDLNLTPLENDLEFDLRKSNTAPMVDCFL*。

[0015] The present invention also provides the use of a biological sample containing the PhZPT2-5 gene in any one of the following:

[0016] (1) Use in regulating the synthesis of petunia anthocyanins;

[0017] (2) Use in breeding purple petunias;

[0018] The nucleotide sequence of the PhZPT2-5 gene is integrated into the gene sequence of the biological sample, and the nucleotide sequence of the PhZPT2-5 is shown as 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 the synthesis of anthocyanins in petunia flowers; overexpressing the PhZPT2-5 gene promotes the synthesis of anthocyanins in petunia flowers.

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

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

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

[0024] The present invention also provides a breeding method for deep purple petunias, which includes the steps of overexpressing the PhZPT2-5 gene in petunias to promote anthocyanin synthesis in petunia flowers and obtaining deep purple petunias. The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.1.

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

[0026] The present invention discovers that the PhZPT2-5 gene is significantly correlated with anthocyanin synthesis in petunia flowers. Experimental findings show 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, petunias of different colors can be obtained, laying a foundation for breeding new varieties of petunias. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

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

[0030] Figure 3 For the results of photoperiod regulation of anthocyanin synthesis and the expression of PhZPT2-5; (A) Phenotype analysis under different photoperiod treatments; (B) Comparison of anthocyanin contents under 16h / 8h and 10h / 14h treatments; (C) Comparison of the expression levels of PhZPT2-5 under 16h / 8h and 10h / 14h treatments; (D) Comparison of the expression levels of PhCHS under 16h / 8h and 10h / 14h treatments; lowercase letters (a-b) indicate significant differences (p < 0.05);

[0031] Figure 4Results of phenotypic analysis of PhZPT2-5 virus-silenced plants; (A) Phenotype of PhZPT2-5 virus-silenced plants; (B) Extraction solutions of anthocyanins from control plants and PhZPT2-5 virus-silenced plants; (C) Determination of anthocyanin content; (D) Detection of PhZPT2-5 expression level; Control is the control, TRV2-0 is the plant transformed with the empty vector, Line1 and Line are PhZPT2-5 virus-silenced plants; lowercase letters (a-b) indicate significant differences (p<0.05);

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

[0033] Figure 6 Results of phenotypic analysis of Petunia hybrida PhZPT2-5 transient injection plants; Detailed implementation manners

[0034] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0038] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are intended to include but not be limited to.

[0039] Example 1

[0040] 1. Experimental method

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

[0042] Using Universal Plant Total RNAIsolatin Kit to extract RNA, operating according to the instructions. The specific steps are as follows: Take an appropriate amount of plant tissue ground in liquid nitrogen and immediately add 600 μL of Buffer EL, vortex vigorously for 30 sec to fully mix the sample with the lysate, and centrifuge at 12,000 rpm for 5 min. Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter ColumnsⅢ, centrifuge at 12,000 rpm for 30 sec, discard FastPure gDNA-FilterColumnsⅢ, and collect the filtrate. Add anhydrous ethanol with a volume 0.5 times that of the filtrate to the collection tube and mix well by shaking. Transfer the above mixture to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 sec, and discard the filtrate. Add 700 uL of Buffer RWA to FastPure RNAColumns V, centrifuge at 12,000 rpm for 30 sec, and discard the filtrate. Add 500 μL of Buffer RWB to FastPureRNAColumns V, centrifuge at 12,000 rpm for 30 sec, and discard the filtrate. Place FastPureRNAColumns V back into the collection tube and centrifuge at 12,000 rpm for 2 min. Transfer FastPure RNA Columns V to a new RNase-free 1.5 mL centrifuge tube, suspend and drop 30 - 100 μL of RNase-free ddH2O in the center of the adsorption column membrane, and centrifuge at 12,000 rpm for 1 min to obtain the extracted RNA.

[0043] 1.2 cDNA reverse transcription

[0044] Using MonScript of Promai Jingyi Technology (Beijing) Co., Ltd. TM For the RTIII All-in-One Mix with dsDNase kit of TM , operate according to the instruction manual. The specific steps are as follows: Take 50 ng - 1 μg of RNA, and sequentially add MonScript TM dsDNase, EX 1 μL, MonScript TM 10×dsDNase Buffer 1 μL, supplement with Nuclease-Free Water 10 μL, then place at 37°C for 2 min to remove genomic DNA contamination; incubate at 55°C for 5 min to inactivate dsDnase, and place on ice. Add 1 μL of EDTA, mix well and place on ice for 5 min. Place in a 65°C water bath for 10 min, then quickly place on ice for 2 min. Add MonScript TM 5×RTⅢ Super Mix 4 μL to the product obtained in the first step, gently pipette and mix well; 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 is as follows: cDNA (1 μL); Forward Primer (1 μL); Reverse Primer (1 μL); 2×PCR Master Mix (10 μL); ddH2O (7 μL); total volume 20 μL. PCR program: 94°C, 3 min; 94°C, 30 s; 58°C, 30 s; 72°C, 2 min; cycle 35 times; 72°C, 10 min for extension; 4°C, Forever. The amplified products are detected on a 1% agarose gel, stained with Goldview, and observed under ultraviolet light.

[0047] qRT-PCR: Use the Novo SYBR qPCR Super Mix Plus kit of novoprotein company. Sequentially add cDNA (2 μL); upstream and downstream primers each (0.8 μL), RNase Free Water (6.4 μL); Novo SYBR qPCR Super Mix Plus (10 μL) in a 96-well plate. The reaction program is as follows: 95°C, 1 min; 95°C, 20 s; 55°C, 20 s; 72°C, 30 s; 40 cycles. The GAPDH gene is used as an internal reference. The relative expression level is calculated using 2 -ΔΔCtCalculated by the method, SPSS software was used to statistically analyze the data. All primers and their sequences used in the present invention are shown in Table 1.

[0048] Table 1: Primer Table

[0049]

[0050]

[0051] 1.4 Detection of gene expression level

[0052] Petunias of the same growth vigor at 100 days old were selected, and their roots, stems, leaves, leaf axils and flowers were sampled respectively. Three technical replicates were taken for each sample, RNA was extracted, reverse transcribed into cDNA, and diluted 5-fold for qRT-PCR detection.

[0053] Petunias of the same growth vigor at 60 days old were selected. After different photoperiod treatments and high-temperature treatments, petals with obvious color changes were taken, RNA was extracted, the extracted RNA was reverse transcribed into cDNA, and finally qRT-PCR detection was carried out.

[0054] 1.5 Construction and transformation of PhZPT2-5 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 gel recovery was performed;

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

[0057] (3) The amplified target gene fragment and the double-digested fragment of the vector were used for recombination reaction with reference to the instruction manual of the plusOne step PCR Cloning Kit; plusOne step PCR Cloning Kit;

[0058] (4) 10 μL of the recombinant product was taken and added to 50 μL of DH5α competent cells, pipetted and mixed well, and placed on ice for 30 min; incubated in a 42°C water bath for 30 s - 1 min, and immediately placed on ice for 2 - 3 min; 500 μL of LB solution was added, and the mixture was placed in a 37°C shaker for 1 h until the bacterial solution became turbid; centrifuged at 6000 rpm for 3 min, 250 μL of the supernatant was left, pipetted and mixed well, and evenly spread on the LB plate, and incubated overnight in an inverted position in a 37°C constant temperature incubator;

[0059] (5) Pick positive plaques for culture and sequencing. Add the correctly sequenced recombinant plasmid to 100 μL of Agrobacterium tumefaciens GV3101 competent cells, and let it stand on ice for 5 min; place it in liquid nitrogen for 5 min, in a 28 °C water bath for 5 min, and then in an ice bath for 5 min; add 500 μL of LB liquid medium on a sterile bench, and culture it with shaking at 28 °C for 2 - 3 h; centrifuge at 6000 rpm for 1 min to collect the bacteria, spread them evenly on YEB solid medium containing Rif and Kan resistance, and culture at 28 °C for 2 d to identify positive clones for standby.

[0060] 1.6 Cloning of VIGS gene fragments

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

[0062] Using petunia cDNA as a template, design primers ZPT2 - 5 - VIGS - F / ZPT2 - 5 - VIGS - R containing two restriction enzyme sites, EcoR I and BamHI, amplify with KOD high - fidelity enzyme, and detect by gel electrophoresis. Recover the target gene from the gel. Perform double digestion of the target fragment product and the pTRV2 silencing vector with EcoR I and BamHI respectively, react at 37 °C for 30 min, and at 80 °C for 5 min, then recover the gel and detect its brightness by gel electrophoresis. Ligate the double - digested gene target fragment and vector product in proportion with T4 DNA ligase. Ligation system: T4 DNA Ligase (1 μL), T4DNA Ligase Buffer (10 μL), double - digested target fragment (3 μL), double - digested plasmid vector fragment (3 μL), ddH2O (4 μL). Ligate at 4 °C, then transfer the ligation product into Escherichia coli (DH5α), culture the positive clones in liquid, extract the plasmid, and transfer it into Agrobacterium tumefaciens (GV3101).

[0063] 1.6.2 Transient transformation of petunia with pTRV2 - PhZPT2 - 5

[0064] (1) Buffer preparation: Prepare a mixed solution of MES (1 mol / L), MgCl2 (1 mol / L), and acetosyringone (AS, 50 mg / L) according to MgCl2 (1 mL), MES (1 mL), AS (80 μL), and ddH2O (98 mL);

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

[0066] (3) Collect the bacteria, use a UV - visible spectrophotometer with the buffer as a blank control to measure, so that the OD of the resuspended bacterial solution 600The value is approximately between 0.8 and 1.0; mix in a volume ratio of 1:1;

[0067] (4) Mix the resuspended TRV1 bacterial solution and the silencing vector bacterial solution in a volume ratio of 1:1, keep in the dark for 3 h, select petunia plants at the age of 20 - 30 d with consistent growth, and inject the back of the leaves with a syringe.

[0068] 1.7 Determination of anthocyanin

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

[0070] (2) Extract in the dark at 4 °C for 24 h;

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

[0072] (4) Colorimetric analysis, measure the absorbance values at 530 nm, 620 nm, and 650 nm with a spectrophotometer;

[0073] (5) The calculation of anthocyanin content is 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] Extinction coefficient of anthocyanin molecule at 530 nm wavelength;

[0077] ODλ - Anthocyanin optical density, V - Volume of extraction solution, M - Mass of petals, ξλ - Molar extinction coefficient, 10 6 - Unit conversion coefficient.

[0078] 1.8 Inflorescence infiltration of Arabidopsis thaliana and identification of positive plants

[0079] 1.8.1 Inflorescence infiltration of Arabidopsis thaliana

[0080] (1) Shake the Agrobacterium containing the pSuper1300::PhZPT2 - 5:GFP plasmid until OD 600 = 0.8 - 1.0, centrifuge to collect the bacterial cells, resuspend the bacterial cells with the 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 inflorescences of Arabidopsis thaliana at the full-bloom stage into the infiltration buffer for 2 min;

[0082] (3) Place them flat in a culture dish, cover for moisture retention, and after dark incubation for 24 h, transfer them to normal conditions for cultivation;

[0083] (4) Infiltrate again after one week. When the seeds are mature, harvest the T0 generation seeds.

[0084] 1.8.2 Identification of transgenic Arabidopsis thaliana

[0085] Soak the T0 Arabidopsis thaliana seeds in 75% alcohol (containing 0.02% Tween) for 1.5 min, and rinse once with sterile water; add 2% NaClO for sterilization for 4 - 5 min, rinse 3 times with sterile water, then suspend with sterile water, pipette and blow dry on filter paper. Then sow them in MS medium (containing 25 mg / L Kan), after 2 days of low-temperature treatment at 4°C, transfer them to the culture room.

[0086] Cut the leaves of the resistant Arabidopsis thaliana plants, extract DNA for PCR detection to determine whether the transformation into Arabidopsis thaliana is successful. Screen until the T3 transgenic plants are obtained for subsequent phenotypic analysis.

[0087] 1.8.3 Petunia hybrida genetic transformation

[0088] Activate the overexpression Agrobacterium tumefaciens solution of PhZPT2-5. When the solution becomes turbid, measure the OD 600 value of the solution. When OD 600 reaches 0.3, aliquot the solution into 50 mL centrifuge tubes, centrifuge at 5000 rpm and 4°C for 10 min, pour out the supernatant, and use a pipette to aspirate the resuspension to resuspend the bacteria.

[0089] Use a sterilized blade to cut the leaves of Petunia aseptic seedlings into small squares of 0.5 cm×0.5 cm and place them on the symbiotic solid medium for pre-culture. After two days of dark culture, place the leaf discs in the prepared infection solution. After 8 minutes, clamp out the Petunia leaves from the bacterial solution and blot dry the bacterial solution on the leaves. Place the blotted leaves with the adaxial side down in the co-culture medium (1 / 2MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 1.0 mg / L zeatin + 2.0% sucrose + 1% glucose), arrange them closely on the symbiotic solid medium for culture, and leave a part of the non-infected leaves as a control, place them loosely with the leaf surface up on the callus induction medium (1 / 2MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 1.0 mg / L zeatin + 2% sucrose + 1% glucose + 250 mg / L cefotaxime + 100 mg / L kanamycin), and keep the culture conditions unchanged. After two days of dark culture, rinse with sterile water 5 - 6 times. After 2 - 3 days, transfer the Petunia leaves that have undergone dark culture to the selection medium (1 / 2MS + 2% sucrose + 1% glucose + 250 mg / L cefotaxime + 100 mg / L kanamycin), seal and label, and place them on the light tissue culture rack at 25°C for culture; 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 for culture. After the Petunia tissue culture seedlings grow stably and grow up, carry out acclimatization and transplantation.

[0090] 1.9 PhZPT2-5 Gene Sequence Analysis

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

[0092] (2) Use MEGA 11 software to compare the protein sequences of ZPT2-5 family members in Petunia and Arabidopsis, and construct a phylogenetic tree by the Neighbor Joining method and Bootstrap value of 1000.

[0093] 1.10 Metabolome Determination

[0094] 1.10.1 Sample Extraction

[0095] (1) Place biological samples in a freeze dryer (Scientz-100F) for vacuum freeze-drying;

[0096] (2) Grind (30 Hz, 1.5 min) to a powdery state using a grinder (MM 400, Retsch);

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

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

[0099] (5) After centrifugation (at a speed of 12,000 rpm for 3 minutes), aspirate the supernatant and filter the sample with a microporous membrane (pore size 0.22 μm);

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

[0101] 1.10.2 Screening of differential metabolites

[0102] Based on the OPLS-DA results, from the variable importance in projection (VIP) of the obtained multivariate analysis OPLS-DA model, metabolites with differences between different varieties or tissues were preliminarily screened. At the same time, the p-value or differential fold change value (Fold Change, FC) of univariate analysis was combined to further screen out differential metabolites. The method of combining the VIP value and FC of the OPLS-DA model was adopted to screen differential metabolites.

[0103] 1.10.3 Transcriptome determination

[0104] Total RNA was processed by mRNA enrichment method or rRNA removal method. mRNA enrichment: Use magnetic beads with OligodT to enrich mRNA with polyA tails; rRNA removal. Hybridize rRNA with a DNA probe, selectively digest the DNA / RNA hybrid strand with RNaseH, and then digest the DNA probe with DNaseI. After purification, the required RNA is obtained. Fragment the obtained RNA with fragmentation buffer, perform reverse transcription with random N6 primers, and then synthesize the second strand of cDNA to form double-stranded DNA. Fill in the ends of the synthesized double-stranded DNA and phosphorylate the 5' end to form a sticky end with a protruding "A" at the 3' end, and then ligate a bulged adapter with a protruding "T" at the 3' end. The ligation product is amplified by PCR with specific primers. The PCR product is heat-denatured into single-stranded DNA, and then a single-stranded circular DNA library is formed by circularizing the single-stranded DNA with a bridging primer, and then sequenced on the machine.

[0105] The method used for differential gene analysis is DESeq, the software method used for GO enrichment analysis is Goseq, and KEGG uses the software KOBAS for data analysis.

[0106] 1.11 Screening and detection of interacting proteins

[0107] 1.11.1 Interaction protein screening

[0108] Design primers to amplify the target gene. Treat pGBKT7 with double digestion using EcoRI and SalI, and treat pGADT7 with double digestion using EcoRI and XhoI. Subsequently, ligate the target gene with the digested vector and perform positive clone identification.

[0109] 1.11.2 Bait toxicity and self-activation detection

[0110] (1) Take out the DNA vector (Carrier DNA, 10 mg / mL) from the -20 °C refrigerator, boil it in water for 5 min, and quickly place it on ice;

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

[0112] (3) Add 100 μL of Y2Hgold competent cells and 500 μL of PEG / LiAc transformation solution to the EP tubes respectively, and gently mix. Incubate at 30 °C for 30 min (mix once every 10 min);

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

[0114] (6) Resuspend with 1 mL of YPD Plus Medium and incubate with shaking at 30 °C for 90 min;

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

[0116] (8) Take 100 μL of each and spread them on SD / -Trp plates and SD / -Trp / -His / -Ade / X-a-gal plates;

[0117] (9) Invert the above plates and incubate them in a 30 °C incubator for 3 - 5 d. Judge whether there is self-activation phenomenon according to the growth of colonies on the defective medium plates.

[0118] 2. Results and analysis

[0119] 2.1 PhZPT2-5 sequence analysis

[0120] Using the published petunia genome as a reference, design primers to clone the petunia ZPT2-5 gene. The full length of this gene is 531 bp and it encodes 176 amino acids. Construct a phylogenetic tree of PhZPT2-5 and 10 Arabidopsis ZAT proteins. The results are asFigure 1 As shown in A, PhZPT2-5 and AtZAT12 clustered into one branch, indicating that they are homologous genes. The homologous sequences of ZPT2-5 from species such as Arabidopsis thaliana, apple, and pear were compared with PhZPT2-5 using the DNAMAN software. The results are as Figure 1 shown in B. It was found that the PhZPT2-5 protein contains conserved domains such as 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 Tissue expression analysis of PhZPT2-5

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

[0123] 2.3 Photoperiod regulation of the expression of PhZPT2-5

[0124] To analyze whether the PhZPT2-5 gene is regulated by the light cycle, petunia plants were treated with photoperiods of 16 h / 8 h (light / dark) and 10 h / 14 h respectively. After 7 days, the petals of petunia were taken for determination of anthocyanin content and gene expression analysis. The results are as Figure 3 shown: As the light exposure time decreased, the color of petunia petals gradually changed from dark purple to light purple, and white spots appeared at the petal edges. The anthocyanin content decreased to 0.4 times that of the control, and the expression level of PhCHS (this gene is a synthetic gene in the anthocyanin pathway and was 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. This result indicates that the photoperiod affects the formation of purple flowers and the expression of PhZPT2-5.

[0125] 2.4 Analysis of virus silencing phenotypes

[0126] To study the function of PhZPT2-5, the VIGS-PhZPT2-5 vector was constructed and transiently transformed into petunia. The results are as Figure 4 shown. After PhZPT2-5 was silenced, the expression level decreased significantly, to 0.2 times that of the control. And along with the obvious lightening of the flower color, the anthocyanin content decreased to 0.52 times that of the control. This indicates that the decrease in the expression level of PhZPT2-5 leads to a reduction in anthocyanin synthesis.

[0127] 2.5 Phenotypic analysis of Arabidopsis thaliana PhZPT2-5 overexpression transgenic plants

[0128] To further study 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 and screened to the T3 generation. Two lines were selected for phenotypic analysis. The results are as Figure 5 shown. Compared with the control, the leaves of transgenic plants turned significantly purple. This result indicates that overexpression of PhZPT2-5 in Arabidopsis thaliana promotes the accumulation of anthocyanins, causing the leaves of Arabidopsis thaliana to turn purple.

[0129] 2.6 Phenotypic analysis of Petunia hybrida PhZPT2-5 transient injection plants

[0130] To study the function of PhZPT2-5, a pSuper1300::PhZPT2-5:GFP vector was constructed and transiently transformed into Petunia hybrida. The results are as Figure 6 shown. When the overexpression Agrobacterium liquid was injected into Petunia hybrida 'Mirage Blue Light', the flower color of Petunia hybrida overexpressing PhZPT2-5 (Line1, Line2, Line3) became significantly darker. This shows that an increase in the expression level of PhZPT2-5 leads to an increase in anthocyanin synthesis.

[0131] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of PhZPT2-5 gene in any of the following: (1) Application in regulating the synthesis of anthocyanins in petunia; (2) Application in petunia purple flower breeding; The nucleotide sequence of PhZPT2-5 is shown in SEQ ID NO.

1.

2. Use of the protein encoded by the PhZPT2-5 gene in any of the following: (1) Application in regulating the synthesis of anthocyanins in petunia; (2) Application in petunia purple flower breeding; 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.

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

1.

4. The use according to claim 3, characterized in that The biological sample includes an expression vector, an expression cassette and microbial strains.

5. The use according to any one of claims 1 to 4, characterized in that: Silencing the PhZPT2-5 gene reduces the synthesis of anthocyanins in petunia flowers; overexpressing the PhZPT2-5 gene promotes the synthesis of anthocyanins in petunia flowers.

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

1.

7. A method for promoting anthocyanin synthesis in petunia, characterized in that: The method comprises the steps of overexpressing the PhZPT2-5 gene in petunia to promote the synthesis of anthocyanins in petunia flowers. The nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.

1.

8. A method for breeding light purple petunia, characterized in that: The method comprises the steps of silencing the PhZPT2-5 gene in petunia, reducing anthocyanin synthesis in petunia flowers, and obtaining light purple petunia flowers. The nucleotide sequence of the PhZPT2-5 is shown in SEQ ID NO.

1.

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

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