A flower color protein, its encoding gene, and its application in petunia

Abstract

This invention discloses a petunia flower color protein, its encoding gene, and its applications, belonging to the field of plant genetic engineering technology. This invention clones a gene from petunias that may play an important regulatory role in flower color regulation. Its CDS coding sequence is shown in SEQ ID NO: 1, and the expressed protein is shown in SEQ ID NO: 2. This invention uses a recombinant VIGS (virus-induced gene silence) vector containing the CDS coding sequence of this petunia flower color gene to silence the gene in petunias via Agrobacterium-mediated transformation, obtaining gene-silenced plants. Compared to normal plants, the obtained gene-silenced plants have a deeper flower color, indicating that this gene plays an important role in regulating flower color. This invention provides a new gene resource for improving flower color and enhancing the ornamental qualities of plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a petunia flower color protein, its encoding gene, and its application. Background Technology

[0002] Petunia hybrida belongs to the Solanaceae family and the Petunia genus. It is an annual or biennial flowering plant widely used in home gardening and landscaping. Its flowers come in various colors, including white, pink, red, blue, and light purple. The main attraction of petunias lies in their vibrant flower colors, making the improvement of their coloring process significant.

[0003] The flower color of petunias is influenced by both internal factors such as genetics and external factors such as climate and human management. Among these factors, genetics is one of the key determinants of petunia flower color. The flower color of petunias is regulated by multiple genes. Producing more petunia plants with different flower colors can meet the market's demand for diverse flower colors. Furthermore, because petunias have a short growth cycle and a clear genetic background, they can be propagated through seeds, cuttings, and grafting. They can serve as model plants for studying the function of flower genes and the molecular mechanisms of flower development. Identifying the genes involved in petunia flower color formation can not only provide new ideas and methods for petunia breeding but also has significant implications for improving the plant's ornamental traits. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a petunia flower color protein, its encoding gene, and its application.

[0005] The first objective of this invention is to provide a color protein for petunias.

[0006] A second objective of this invention is to provide a gene encoding a flower color protein of petunia.

[0007] A third objective of this invention is to provide a recombinant vector containing a gene encoding a color protein of petunia flowers.

[0008] The fourth object of the present invention is to provide a recombinant bacterium with a recombinant vector containing a gene encoding a color protein of petunia flowers.

[0009] The fifth objective of this invention is to provide a method for changing the color of plant petals.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0011] A color protein of petunia, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0012] A gene encoding a flower color protein of petunia, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0013] A recombinant vector containing the gene encoding the flower color protein of the petunia flower.

[0014] A recombinant bacterium containing a recombinant vector encoding a color protein of the petunia flower.

[0015] The application of one or more of the following in altering the petal color of a plant should also be within the scope of protection of this invention: the use of the petunia chroma protein, the encoding gene of the petunia chroma protein, the recombinant vector of the encoding gene of the petunia chroma protein, and the recombinant bacteria containing the recombinant vector of the encoding gene of the petunia chroma protein.

[0016] Preferably, the plant is petunia.

[0017] Preferably, the recombinant vector is an overexpression vector or a silencing vector.

[0018] More preferably, the silencing carrier is a VIGS carrier.

[0019] Most preferably, the silencing vector is the pTRV2 vector.

[0020] Preferably, the alteration of the plant's petal color involves overexpressing the anthocyanin of the petunia and / or the gene encoding the anthocyanin of the petunia, thereby making the plant's petal color lighter.

[0021] More preferably, the alteration of the plant's petal color involves silencing and / or knocking out the encoding genes of the petunia's anthocyanin and / or anthocyanin, thereby darkening the plant's petal color.

[0022] A method for altering the color of plant petals, the method comprising: increasing the expression level of the flower color protein in petunias, thereby making the petal color lighter.

[0023] Preferably, the method for increasing the expression level of the anthocyanin in petunias is to overexpress the gene encoding the anthocyanin in the petunias.

[0024] A method for altering the color of plant petals, the method comprising: reducing the expression level of the flower color protein in petunias, thereby deepening the color of the plant petals.

[0025] Preferably, the method for reducing the expression level of the flower color protein in petunias is to silence and / or knock out the gene encoding the flower color protein in the petunias.

[0026] More preferably, the method for reducing the expression level of the flower color protein in petunias is to silence the gene encoding the flower color protein in petunias.

[0027] Preferably, the method for changing the color of plant petals includes the following steps:

[0028] S1. Construct a VIGS vector containing a gene encoding a flower color protein of petunia with a nucleotide sequence as shown in SEQ ID NO: 1;

[0029] S2. The VIGS vector constructed in S1 was transformed into Agrobacterium and then transformed into plants using the Agrobacterium-mediated transformation method to obtain transformed plants;

[0030] S3. Screen the transformed plants obtained in step S2 to obtain gene-silenced plants with deeper flower color.

[0031] Preferably, step S1 involves ligating the gene encoding the petunia flower color protein, whose nucleotide sequence is shown in SEQ ID NO: 1, into the VIGS vector to obtain a recombinant vector.

[0032] More preferably, step S1 specifically involves ligating the gene encoding the petunia flower color protein with the nucleotide sequence shown in SEQ ID NO: 1 into the pTRV2 vector to obtain a recombinant vector.

[0033] Preferably, step S2 involves: transferring the recombinant vector into Escherichia coli for amplification and propagation to obtain positive recombinant bacteria, extracting the recombinant plasmid, transferring it into Agrobacterium to obtain a recombinant strain, infecting the wounds of petunia plants with the recombinant strain, and culturing it in a constant temperature greenhouse.

[0034] More preferably, the Escherichia coli is Escherichia coli strain DH5α.

[0035] More preferably, the Agrobacterium is Agrobacterium strain GV3101.

[0036] Preferably, step S3 involves screening the transformed plants obtained in step S2 using qRT-PCR to obtain gene-silenced plants with deepened flower color.

[0037] More preferably, the transformed plants obtained in step S2 are screened by qRT-PCR detection using the detection primers with nucleotide sequences as shown in SEQ ID NO: 8-9, to obtain gene-silenced plants with deeper flower color.

[0038] More preferably, the transformed plants obtained in step S2 are screened by qRT-PCR detection using internal reference primers with nucleotide sequences as shown in SEQ ID NO: 10-11, to obtain gene-silenced plants with deeper flower color.

[0039] Preferably, in the method for changing the color of plant petals, the plant is a petunia.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The petunia flower color regulating gene PhDC provided by this invention is a novel flower color regulating gene that can regulate the depth of plant petal color, providing a new gene resource for petal color regulation.

[0042] The PhDC gene silencing in petunia plants provided by this invention results in a deeper flower color compared to normal plants. This gene can be applied to transgenic plants to improve flower color and enhance the ornamental qualities of the plants.

[0043] This invention also provides a method for deepening the color of plant petals. The method involves using a recombinant vector containing the CDS coding sequence of the PhDC gene (as shown in SEQ ID NO: 1) to silence the PhDC gene in petunias via Agrobacterium-mediated silencing, resulting in gene-silenced plants. Petunia plants treated using this method exhibit deeper petal color, which can improve the flower color of petunias and provide new gene resources and ideas for petunia flower color breeding. Attached Figure Description

[0044] Figure 1 Map of the recombinant vector pTRV2-PhDC that silences the PhDC gene;

[0045] Figure 2 Figure 1 shows the qRT-PCR results of PhDC in petunia corollas under different treatments.

[0046] Figure 3 PhDC silencing phenotypic analysis diagram: PhDC silencing and negative control flower phenotypic comparison.

[0047] Figure 4 Comparison of anthocyanin content between PhDC-silenced and negative control flowers. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Construction, transformation, and extraction of recombinant vectors and recombinant plasmids

[0050] I. Experimental Methods

[0051] Gene Source: Selected from the existing petunia transcriptome database of our research group, it was named PhDC, and its nucleotide sequence is shown in SEQ ID NO: 1. The DNA fragment encoding the CDS sequence of the PhDC gene was amplified using PCR technology, and the pTRV2-PhDC recombinant vector was constructed using enzyme digestion and ligation. The specific steps are as follows:

[0052] (1) Amplification of DNA fragments

[0053] mRNA was extracted from petunia hybrida and reverse transcribed into cDNA.

[0054] The DNA fragment encoding the CDS sequence of the PhDC gene was amplified using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase. Its nucleotide sequence is shown in SEQ ID NO: 3. The upstream primer for PCR was PhDC-pTRV2F (SEQ ID NO: 4), and the downstream primer was PhDC-pTRV2R (SEQ ID NO: 5). The specific nucleotide sequences are shown below:

[0055] Upstream primer PhDC-pTRV2F:

[0056] 5'-ACGCGTGAGCTCGGTACCGGATCCATGGCGATGAGGAACT-3';

[0057] Downstream primer PhDC-pTRV2R:

[0058] 5'-TCTGTGAGTAAGGTTACCGAATTCGCGACGAGGTAGGAGA-3'.

[0059] The PCR reaction system is shown in Table 1.

[0060] Table 1. Reaction system for amplifying the DNA fragment encoding the CDS sequence of the PhDC gene.

[0061]

[0062] PCR reaction procedure: pre-denaturation (95℃, 3 min); denaturation (95℃, 15 s), annealing (56-60℃, 15 s), extension (72℃, 90 s); 35 cycles, followed by complete extension (72℃, 5 min), and then hold at 16℃.

[0063] The PCR product is the amplification product containing the CDS coding sequence of the PhDC gene.

[0064] (2) Purification of amplification products

[0065] The amplification product containing the CDS coding sequence of the PhDC gene obtained by PCR amplification in step (1) was purified using a DNA purification and recovery kit. The specific steps are as follows:

[0066] Prepare an agarose gel with a concentration of 0.8% to 1%, and separate DNA fragments by electrophoresis. After the DNA fragments are separated, place the gel under a UV lamp and cut off the gel containing the target DNA fragment.

[0067] Weigh the gel and transfer it to a centrifuge tube. Calculate the gel ratio as 1 mg gel = 1 μL gel. Add Buffer GDP to the centrifuge tube at a ratio of gel:Buffer GDP = 1:2 (v / v). Incubate the centrifuge tube in a water bath at 50–55°C until the gel is completely dissolved. Centrifuge the tube and collect the droplets on the tube wall to obtain the sol solution.

[0068] Insert the DNA purification column into a 2 mL centrifuge tube, transfer the sol solution into the DNA purification column, centrifuge the tube at 8000×g for 30–60 s, discard the filtrate to obtain the remaining sol solution, insert the DNA purification column back into the 2 mL centrifuge tube, transfer the remaining sol solution into the DNA purification column, and centrifuge the tube at 8000×g for 30–60 s.

[0069] After centrifugation, discard the filtrate, reattach the DNA purification column to the 2 mL centrifuge tube, add 500 μL of Buffer DW2 to the DNA purification solution, centrifuge at 8000 × g for 30–60 s, and discard the filtrate. Repeat this step once.

[0070] Reinsert the DNA purification column into a 2 mL centrifuge tube, centrifuge at 10000×g for 2 min, discard the filtrate, insert the DNA purification column into a 1.5 mL centrifuge tube, and add Elution Buffer to the center of the DNA purification column membrane.

[0071] After the DNA purification column was allowed to stand for 2 minutes, it was centrifuged at 10000×g for 1 minute. The filtrate was discarded, and the purified amplified product containing the CDS coding sequence of the PhDC gene was obtained. The amplified product was sent to the Guangzhou branch of Beijing Qingke Xinyue Biotechnology Co., Ltd. for further sequencing verification. The correctly sequenced amplified product was stored at -20℃ for later use.

[0072] (3) Extraction of pTRV2 vector

[0073] The pTRV2 vector was extracted using a plasmid extraction kit. The multiple cloning site sequence of this vector is shown in SEQ ID NO: 12.

[0074] The specific steps are as follows:

[0075] Centrifuge the bacterial culture containing the pTRV2-PhDC vector at 10000×g for 1 min, discard the culture medium, and aspirate any remaining liquid by tapping the culture paper. Add 250 μL of a mixture of Buffer P1 and RNase A to the bacterial cells, vortex to resuspend the bacteria, add 250 μL of Buffer P2 to the resuspended culture, invert and mix, then add another 250 μL of Buffer P2 and invert and mix again.

[0076] When the solution becomes viscous and clear, add 350 μL of Buffer P3 to the solution, mix by inversion, and centrifuge at 13000–16000 × g for 2 min. Place the DNA purification column in a collection tube, transfer the supernatant after centrifugation to the DNA purification column, and centrifuge at 13000 × g for 30–60 s.

[0077] Discard the filtrate, reattach the DNA purification column to the collection tube, add 600 μL of Buffer PW2 to the DNA purification column, centrifuge at 13000×g for 30–60 s, and discard the filtrate. Repeat this step once.

[0078] Replace the DNA purification column back into the collection tube and centrifuge at 13000×g for 3 min.

[0079] After centrifugation, remove the DNA purification column and place it in a sterile 1.5 mL centrifuge tube. Add Elution Buffer to the center of the DNA purification column membrane.

[0080] After allowing the DNA purification column to stand for 2 minutes, centrifuge at 13000×g for 1 minute, discard the filtrate, and obtain the pTRV2 vector, which is stored at -20℃ for later use.

[0081] (4) Enzyme digestion and ligation of the pTRV2 vector with the amplified product containing the CDS coding sequence of the PhDC gene.

[0082] The pTRV2 vector obtained in step (3) and the amplification product obtained in step (2) were digested with restriction endonucleases (EcoRI and BamHI). The digestion reaction system of the pTRV2 vector is shown in Table 2.

[0083] Table 2 Enzyme digestion reaction system

[0084]

[0085]

[0086] The enzyme digestion conditions were: digestion at 37℃ for 4 hours. Electrophoresis was performed after the pTRV2 vector digestion reaction was completed.

[0087] The enzyme digestion products were recovered using a DNA purification and recovery kit to obtain the linearized pTRV2 vector and the purified amplified product containing the CDS coding sequence of the PhDC gene.

[0088] (5) Ligation of the linearized pTRV2 vector and the purified amplified product containing the CDS coding sequence of the PhDC gene.

[0089] The linearized pTRV2 vector obtained in step (4) and the purified amplified product containing the CDS coding sequence of the PhDC gene were ligated using homologous recombinase (Gib-seamless cloning). The ligation reaction system is shown in Table 3.

[0090] Table 3 Connection Reaction System

[0091]

[0092] After mixing the substances in the ligation reaction system in Table 3, the mixture was incubated at 50°C for 30 min. After incubation, the reaction solution was collected to obtain the ligation product of the purified amplification product and the linearized pTRV2 vector, namely the recombinant vector pTRV2-PhDC.

[0093] (6) Transformation and identification of Escherichia coli

[0094] Transformation of E. coli:

[0095] The recombinant vector pTRV2-PhDC obtained in step (5) was added to competent Escherichia coli cells, mixed well, and placed on ice for 30 min. Then, it was heat-shocked in a 42℃ water bath for 90 s. After the heat shock, it was transferred to an ice bath and left to stand for 2 min.

[0096] Next, 500 μL of antibiotic-free sterile culture medium LB was added to the centrifuge tube, the bacterial solution was mixed, and the bacteria were revived at 37°C and 110 rpm for 1 hour to obtain the transformed Escherichia coli.

[0097] Identification of positive clones:

[0098] The revived bacterial culture was evenly spread on solid LB medium containing kanamycin, and the plates were inverted and incubated overnight at 37°C.

[0099] After overnight culture, single clones were selected and cultured in 500 μL of LB medium containing kanamycin at 37°C and 200 rpm for 3–5 h. The culture medium was used as a PCR template for PCR reaction. The upstream primer for PCR was pTRV2F (SEQ ID NO: 6), and the downstream primer was pTRV2R (SEQ ID NO: 7). The specific nucleotide sequences are shown below.

[0100] Upstream primer pTRV2F (SEQ ID NO: 6):

[0101] 5'-TATTATTACGGACGAGTGGAC-3';

[0102] Downstream primer pTRV2R (SEQ ID NO: 7):

[0103] 5'-ACCTAAAACTTCAGACACGGA-3'.

[0104] The PCR reaction system (20 μL) for positive clone identification is shown in Table 4.

[0105] Table 4. PCR reaction system for identifying positive clones

[0106]

[0107] PCR reaction procedure: pre-denaturation (98℃, 3 min); denaturation (98℃, 10 s), annealing (56–72℃, 10 s), extension (72℃, 5–15 s / kb); 35 cycles, followed by complete extension (72℃, 2 min).

[0108] The PCR products were obtained and subjected to agarose gel electrophoresis. The PCR products with positive electrophoresis results were sent to the Guangzhou branch of Beijing Qingke Xinyue Biotechnology Co., Ltd. for further sequencing verification. The bacterial culture corresponding to the PCR product with correct sequencing (i.e., consistent with the CDS coding sequence SEQ ID NO: 3) was amplified and shaken, the bacterial culture was preserved and the plasmid was extracted to obtain the recombinant plasmid pTRV2-PhDC.

[0109] II. Experimental Results

[0110] The nucleotide sequence of the PhDC gene is shown in SEQ ID NO: 1; the amino acid sequence encoded by the CDS coding sequence of the PhDC gene is shown in SEQ ID NO: 2.

[0111] Sequencing results showed that the nucleotide sequence of the CDS coding sequence of the amplified PhDC gene is shown in SEQ ID NO: 3; the map of the synthesized recombinant plasmid pTRV2-PhDC is shown in... Figure 1 As shown.

[0112] Example 2: Cultivation and Identification of Gene-Silenced Plants

[0113] I. Experimental Methods

[0114] (1) Agrobacterium-mediated genetic transformation of petunia plants

[0115] Agrobacterium GV3101 containing pTRV2-PhDC, pTRV2-GFP (negative control recombinant vector), pTRV2-CHS (positive control recombinant vector), and pTRV1 (virus helper vector) were streaked in a clean bench to activate the Agrobacterium. The activated Agrobacterium was then inoculated into 10 mL of YEP liquid medium, 10 μL of Kana was added, and the medium was placed in a 28°C constant temperature shaker and cultured at 200 rpm for 18 h to prepare seed culture.

[0116] In a clean bench, inoculate 2-5 mL of seed culture at varying concentrations into liquid culture medium, add 10 μL of Kana, and incubate at 28°C with shaking at 100 rpm for approximately 12 hours to allow for large-scale culture. The volume of pTRV1 culture should be consistent with the sum of the volumes of pTRV2-PhDC, pTRV2-GFP, and pTRV2-CHS cultures. Measure the OD600 value of the culture; when it reaches 0.5-1.0, transfer the culture to a 50 mL centrifuge tube and centrifuge at room temperature at 4000 rpm for 10 minutes. Discard the supernatant, add an appropriate amount of MMA buffer (formulation shown in Table 5) to resuspend the precipitate, ensuring the OD600 value of the culture is within acceptable limits. 600 The value is between 2 and 4.

[0117] The bacterial culture was then placed in a 28°C incubator and allowed to stand for 2 hours. Agrobacterium GV3101 containing the helper vector pTRV1 was then mixed with OD at a 1:1 ratio. 600 Agrobacterium GV3101 strains with similar pTRV2-PhDC values ​​were mixed together. Healthy petunia plants with uniform growth were selected, excess branches and leaves were removed, and wounds were created on the stems and leaves. Using a pipette, the mixed bacterial solution was evenly applied to the wounds on the petunia plants.

[0118] Infected plants were cultured in a greenhouse at 20°C, 30% humidity, and a light / dark cycle of 16h / 8h. Negative control group petunia plants and PhDC gene-silenced plants were obtained.

[0119] Table 5 MMA buffer preparation formula

[0120]

[0121] (2) Identification of gene-silenced petunia plants

[0122] RNA was extracted from leaves of petunia plants in the negative control group and PhDC gene-silenced plants in step (1), and the expression level of PhDC in petunia plants was detected by qRT-PCR. The specific steps are as follows:

[0123] 1) Step (1) Extraction of leaf RNA from negative control petunia plants and PhDC gene-silenced plants

[0124] For example, RNA extraction from the negative control group petunia plants was performed by grinding the plant leaf tissue of the negative control group petunia plants into a fine powder using liquid nitrogen. 50-300 mg of the powder was weighed into a 1.5 mL pre-cooled centrifuge tube, 800 μL of Buffer RL was added to the centrifuge tube, and the mixture was vortexed at high speed for 15-30 s and then allowed to stand at room temperature for 3 min.

[0125] Then, centrifuge at 14000×g for 5 min at room temperature, collect the supernatant, place the gDNA filter column in a 2 mL collection tube, transfer the supernatant after centrifugation to the gDNA filter column, centrifuge at 14000×g for 2 min, collect the filtrate, and discard the gDNA filter column.

[0126] Add 350 μL of anhydrous ethanol to the filtrate and pipette 3–5 times to obtain a mixture. Place the RNA purification column in a 2 mL collection tube, transfer 700 μL of the mixture to the RNA purification column, centrifuge at 12000 × g for 30–60 s, discard the filtrate, and return the RNA purification column to the collection tube. Repeat this step once.

[0127] Then add 500 μL of Buffer RW1 to the RNA purification column, centrifuge at 10000×g for 30–60 s, discard the filtrate, and put the RNA purification column back into the collection tube.

[0128] Add 500 μL of Buffer RW2 to the RNA purification column, centrifuge at 12000×g for 30–60 s, and discard the filtrate. Repeat this step once.

[0129] Discard the filtrate, put the RNA purification column back into the collection tube, centrifuge at 12000×g for 2 min, and discard the filtrate.

[0130] Transfer the RNA purification column to a 1.5 mL centrifuge tube and add RNase-free water to the center of the membrane of the RNA purification column.

[0131] After allowing the RNA purification column to stand for 2 minutes, centrifuge at 12000×g for 1 minute, discard the filtrate, and obtain the RNA from the negative control group petunia plants. Store at -80℃ for later use.

[0132] RNA was extracted from 12 PhDC gene-silenced plants after undergoing the same treatment.

[0133] 2) Reverse transcription synthesis of cDNA

[0134] Taking the RNA from the negative control group petunia plants obtained in step 1) as an example: cDNA was synthesized using a reverse transcription kit, and the specific steps are as follows:

[0135] Prepare the mixtures shown in Table 6 in RNase-free centrifuge tubes.

[0136] Table 6 Composition of the Mixture

[0137]

[0138] Add the substances described in Table 6 to an RNase-free centrifuge tube, mix thoroughly with a pipette, and react at 42°C for 2 minutes to obtain a mixture.

[0139] Add 2 μL of 10×RT Mix and 2 μL of HiScript II Enzyme Mix to the mixture, mix well by pipetting, react at 50℃ for 15 min, and then react at 85℃ for 2 min. The reaction product is the cDNA of the negative control group petunia plants.

[0140] The RNA extracted from the 12 PhDC gene-silenced plants was processed in the same way to obtain the cDNA of the 12 PhDC gene-silenced plants.

[0141] 3) qPCR reaction of cDNA

[0142] Taking the cDNA from the negative control group petunia plants obtained in step 2) as an example: qRT-PCR was performed. The upstream primer for the qRT-PCR reaction of PhDC was PhDC-qPCRF (SEQ ID NO: 8), and the downstream primer was PhDC-qPCRR (SEQ ID NO: 9); the upstream primer for the qRT-PCR reaction of the internal reference gene CYCLOPHILIN was PhCYP-qPCRF (SEQ ID NO: 10), and the downstream primer was PhCYP-qPCRR (SEQ ID NO: 11).

[0143] The specific nucleotide sequences of the primers are shown below:

[0144] PhDC-qPCRF (SEQ ID NO: 8):

[0145] 5'-TTGCTTACGGTGCTCCTG-3';

[0146] PhDC-qPCRR (SEQ ID NO: 9):

[0147] 5'-CACATAGGCTGCCACTCA-3';

[0148] PhCYP-qPCRF (SEQ ID NO: 10):

[0149] 5'-AGGCTCATCATTCCACCGTGT-3';

[0150] PhCYP-qPCRR (SEQ ID NO: 11):

[0151] 5'-TCATCTGCGAACTTAGCACCG-3'.

[0152] The qRT-PCR reaction system of PhDC is shown in Table 7.

[0153] Table 7. qRT-PCR reaction system for PhDC

[0154]

[0155] The qRT-PCR reaction system for the internal reference gene CYCLOPHILIN is shown in Table 8.

[0156] Table 8. qRT-PCR reaction system for the internal reference gene CYCLOPHILIN

[0157]

[0158] qRT-PCR reaction program: pre-denaturation (95℃, 5 min, 1 cycle); cyclic reaction (95℃, 10 s, 60℃, 30 s, 40 cycles); melting curve (95℃, 15 s, 60℃, 60 s, 95℃, 15 s, 1 cycle).

[0159] The cDNA of the 12 PhDC gene-silenced petunia plant lines extracted in step 2) were subjected to the same treatment to obtain the corresponding qRT-PCR results.

[0160] II. Experimental Results

[0161] PhDC's qRT-PCR results are as follows Figure 2 As shown, the results indicated that, compared with the negative control group of petunia plants, the expression of the PhDC gene in the PhDC gene-silenced petunia plant lines was downregulated and the expression level was significantly reduced. This indicates that PhDC gene-silenced petunia plant lines with significantly reduced PhDC gene expression levels were successfully obtained.

[0162] Example 3: Phenotypic observation and statistical analysis of PhDC gene-silenced petunia plants

[0163] I. Experimental Methods

[0164] PhDC gene-silenced petunia plants and negative control group petunia plants were phenotyped, their phenotypes were recorded by photograph, and the petal color parameters and anthocyanin content of the petals were measured.

[0165] (1) Measurement of petal color parameters

[0166] The lightness (L*), red-green (a*), and yellow-blue (b*) values ​​of the petals were measured using a precision colorimeter (CS-210, Hangzhou Caipu Technology Co., Ltd.), with each petal observed five times. According to the formula... Calculate the chroma C* value, and calculate the hue angle H* value according to the formula H*=arctan(b* / a*).

[0167] (2) Determination of anthocyanin content in petals

[0168] Petals were collected from PhDC gene-silenced petunia plants and negative control petunia plants, with three biological replicates for each. Each sample was 0.3 g, flash-frozen in liquid nitrogen, and then placed in a -80°C freezer for 10–15 min in the dark. The mortar and pestle were pre-cooled with liquid nitrogen, and the sample was ground into powder. This process was performed in the dark as much as possible. 15 mL of 1% HCl methanol solution was added to the mortar and pestle to completely dissolve the sample powder. The solution was then transferred to a 50 mL centrifuge tube wrapped in aluminum foil and brought to a final volume of 30 mL. The mixture was incubated at 4°C in the dark for 2 h. Centrifuged at 10500 rpm for 10 min. The supernatant was quickly transferred to three 15 mL centrifuge tubes as three technical replicates.

[0169] The absorbance of the supernatant at 530 nm and 657 nm was measured using a spectrophotometer, according to the formula Q. Anthocyanins =(A 530 -0.25×A 657 )×M -1 Q Anthocyanins For anthocyanin content, A 530 The absorbance of anthocyanin is 0.25 × A. 657 denoted as chlorophyll absorbance, and M as the total mass of the sample.

[0170] II. Experimental Results

[0171] Phenotypic analysis statistical results are as follows: Figure 3 As shown, the results indicated that, compared with the negative control group of petunia plants, the petal color of the PhDC gene-silenced petunia plants was significantly darker. Figure 3The color parameters of the control and experimental groups showed that the flower color of the PhDC gene-silenced plants was darker, more reddish, and less blue compared to the control, while the color vibrancy of the two groups was similar (Table 9), consistent with the flower color deformation; the anthocyanin content results ( Figure 4 This indicates that the anthocyanin content was not significantly affected.

[0172] The results show that PhDC silencing can significantly deepen the flower color of petunia plants.

[0173] Table 9. Flower color parameters of PhDC gene-silenced petunias and negative control plants.

[0174]

[0175] Note: Data are expressed as mean ± SD (n = 5). Statistical analysis was performed using Student's t-test, with 5 biological replicates per trial. * indicates a statistically significant difference at ≤ 0.05.

[0176] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A phloretin of petunia hybrida, characterized in that, The amino acid sequence is shown as SEQ ID NO:

2.

2. A gene encoding a phytocyanin of Petunia hybrida, characterized in that, The anthocyanin protein is encoded by the gene.

3. A recombinant vector comprising the gene of claim 2.

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

5. Use of one or more of the anthocyanin of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3 and the recombinant bacterium of claim 4 for changing the petal color of a plant, characterized in that, The plant is petunia; The petal color of the plant is deepened.

6. A method of changing the petal color of a plant, comprising, The expression of the anthocyanin protein is reduced, and the petal color of the plant is deepened. The plant is petunia.

7. The method of claim 6, wherein, The method comprises the following steps: S1. Constructing a VIGS vector comprising the gene of claim 2; S2. Transforming the VIGS vector constructed in S1 into Agrobacterium, and introducing the VIGS vector into plants by Agrobacterium-mediated method to obtain transformed plants; S3. Screening the transformed plants obtained in S2 to obtain plants with deepened petal color.

8. The method of claim 7, wherein, The screening method is qRT-PCR detection using detection primers with nucleotide sequences shown as SEQ ID NO: 8-9.

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