Rose JAZ3 protein and its application in regulating petal color

Abstract

The present invention belongs to the field of plant genetic engineering technology, specifically relating to the rose JAZ3 protein and its use in regulating petal color. The technical problem to be solved by the present invention is to provide a new option for creating roses with different flower colors. The present invention also provides the use of the rose JAZ3 protein in regulating rose petal color. The present invention achieves precise editing of the rose RhJAZ3 gene for the first time, obtaining T0-generation mutants using an optimized CRISPR / Cas9 system. The anthocyanin content in the petals of the mutant plants decreased by 63%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to rose JAZ3 protein and its application in regulating petal color. Background Art

[0002] Rose (Rosa hybrida), a core economic species in the global ornamental flower industry, has long been hampered by its complex genetic background and inefficient genetic transformation systems. Agrobacterium-mediated genetic transformation efficiency in roses is only 2.7-3.1% (based on the 2023 International Horticultural Society database), far lower than that of the model plant Arabidopsis thaliana (85-90%) and the horticultural crop tomato (75-80%). To date, only our team has successfully reported gene editing in roses (granted patent CNCN116083339A, a CRISPR system targeting the PDS gene, with a T0 generation mutation efficiency of 78.4%), demonstrating that further significant technological breakthroughs are needed in rose molecular breeding.

[0003] JAZ proteins, key repressors of the jasmonic acid (JA) signaling pathway, have highly conserved functions in regulating plant secondary metabolism. Under high light stress, a knockout mutant of the AtJAZ3 gene (jaz3-1) in Arabidopsis thaliana exhibited a 2.5-fold increase in anthocyanin content compared to wild-type plants (P<0.001). This phenotype is associated with derepression of the transcription factor MYB75 / PAP1, which directly activates genes involved in anthocyanin biosynthesis. In tomato, JAZ4 influences anthocyanin accumulation through the co-regulation of the MYC2-PUB22–JAZ4 module. In apple, the JAZ1–TRB1–MYB9 module dynamically regulates JA-mediated anthocyanin and proanthocyanidin accumulation. In woody ornamental plants, TIFY 5A, a member of the grape jasmonic acid ZIM domain (JAZ) family, interacts with DWF4 and inhibits the expression of dihydroflavonol-4-reductase (DFR), which plays a key role in anthocyanin synthesis. These data establish a universal molecular paradigm: "JAZ deficiency leads to enhanced anthocyanin synthesis."

[0004] The global cut flower market is experiencing an annual growth rate of 14.6% for specialized flower colors (such as blue-purple and gradient bicolors). Traditional hybrid breeding or chemical mutagenesis to select plants for color variants relies on natural or random mutations, resulting in a long cycle (5-8 years) and low efficiency (<1%). The success rate of traditional hybrid breeding is only 0.68% (International Flower Trade Association 2023 White Paper). Molecular breeding techniques for roses rely excessively on overexpression of MYB transcription factors, leading to imbalances in flavonoid metabolism. Gene editing technology for roses has long stagnated, resulting in low genetic transformation efficiency (somatic embryo regeneration rate <10%) and a lack of precise editing of color-regulating genes (such as JAZ3). Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new option for creating roses with different colors.

[0006] The technical solution of the present invention is the rose JAZ3 protein, whose amino acid sequence is shown in SEQ ID No.1.

[0007] SEQ ID No. 1, rose JAZ3 protein:

[0008] MAERSNFAQTCNLLSQYLKEKRSQYLQGDSFGVKPAPATMNLLNTMEAGPPAAASTPVGQAPDQPRSAPMTIFFGGQVLVFNDVSAEKAKEIMGLATKGSAVVSSTESNVVVKQQQQPPPPQAVGSDLPIARRASLHKFLAKRKERVTAIAPYQLNHIQRAASPKAEEPVGAQSSKQLELSL.

[0009] The present invention also provides a gene encoding the rose JAZ3 protein, the nucleotide sequence of which is shown in SEQ ID No.2.

[0010] SEQ ID No. 2, gene encoding rose JAZ3 protein:

[0011] .

[0012] The present invention also provides the use of the rose JAZ3 protein in regulating the color of rose petals.

[0013] Furthermore, the regulation is negative regulation.

[0014] The present invention also provides the use of the rose JAZ3 protein in regulating the anthocyanin content in rose petals.

[0015] Specifically, the regulation is negative regulation.

[0016] The present invention also provides a method for creating light-colored roses, which is achieved by blocking or weakening the expression of the gene encoding the JAZ3 protein in the roses.

[0017] Specifically, the method for blocking or weakening the expression of the gene encoding the JAZ3 protein in rose is at least one of genome editing, homologous recombination or random insertion mutagenesis.

[0018] Furthermore, the genome editing method includes at least one of a meganuclease method, a ZFN method, a TALEN method or a CRISPR-Cas method.

[0019] In particular, the CRISPR-Cas method comprises the following steps:

[0020] a. Design sgRNA targeting the gene encoding JAZ3 protein;

[0021] b. Construction of Cas editing expression vector expressing sgRNA;

[0022] c. Transform the Cas editing expression vector into rose somatic embryos.

[0023] Wherein, the CRISPR-Cas method is CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas12a or CRISPR-Cas12b.

[0024] In particular, the sgRNA sequence targeting the gene encoding JAZ3 protein in step a is shown as SEQ ID No. 24.

[0025] Beneficial Effects of the Present Invention: This invention achieves precise editing of the rose RhJAZ3 gene for the first time, generating T0-generation mutants using an optimized CRISPR / Cas9 system. Quantitative analysis revealed a 63% decrease in anthocyanin content in the petals of the mutant plants, with discernible color differences in petal color. This phenotype significantly conflicts with the reported JAZ functional model, suggesting that rose JAZ3 may positively influence anthocyanin synthesis through a novel regulatory network. This discovery diverges from the classical theoretical framework of JAZ proteins and opens up new avenues for studying jasmonic acid signaling in Rosaceae plants. Therefore, by establishing a "JAZ3 dosage-flower color linear regulation" model, the present invention enables precise control of chromaticity values, shortening the breeding cycle compared to traditional techniques. This invention provides a new option for creating roses with light-colored petals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 , RhU6-29 promoter and AtU6-29 promoter sequence alignment.

[0027] Figure 2 , schematic diagram of the functional region structure of pYLsgRNA-RhU6-29.

[0028] Figure 3 , schematic diagram of the complete vector structure of pYLsgRNA-RhU6-29.

[0029] Figure 4 Detection of GFP insertion in DNA of JAZ3-OE transformed plants. M: Marker, Genstar @ D2000; NT: stands for No transformed plant, which refers to the plant that was not transformed with GFP; the 11 lanes to the right of NT represent JAZ3-overexpressing plants JAZ3-OE 1 to 11 transformed with the JAZ3-GFP vector; +: positive control, the template is the JAZ3-GFP plasmid.

[0030] Figure 5 , Cas9 insertion detection in CR-JAZ3 transformed plant DNA. M: stands for Marker, Genstar @ D2000; NT: stands for No transformed plant, which refers to the plant that has not been transformed with CRISPR-JAZ3; the 12 lanes to the right of NT represent the JAZ3 gene-edited plants CR-JAZ3 1 to 12 transformed with the CRISPR-JAZ3 vector; +: positive control, the template is the CRISPR-JAZ3 plasmid.

[0031] Figure 6 NGS sequencing comparison of NT and CR-JAZ3 plants.

[0032] Figure 7 , detection of sequence editing frequency in the target region of CR-JAZ3 plants.

[0033] Figure 8 , Examples of CR-JAZ3 plant editing types.

[0034] Figure 9 Relative expression levels of RhJAZ3 in NT, CR-JAZ3 and JAZ3-OE plants.

[0035] Figure 10 , NT, CR-JAZ3 and JAZ3-OE plants.

[0036] Figure 11 , NT, CR-JAZ3 and JAZ3-OE plant flower colors.

[0037] Figure 12 , NT, CR-JAZ3 and JAZ3-OE plants anthocyanin content.

[0038] Figure 13 , schematic diagram of the CRISPR Cas9 IAZ3-gt1 vector structure. DETAILED DESCRIPTION

[0039] JAZ3 is an interacting gene with EIN2, a gene previously known to control petal senescence. Its origin has no direct relationship with flower color. However, after cloning, gene editing, and overexpression of this gene, JAZ3 was found to be associated with the phenotype of lighter petal color.

[0040] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0041] Example 1 Construction of JAZ3 gene editing vector

[0042] 1. Extraction of rose genomic DNA

[0043] Rose genomic DNA was extracted using the CTAB method. Crush the rose leaves, add the extraction solution preheated at 65°C, and rapidly vortex to mix. Incubate the sample in a 65°C water bath for 30 minutes, mixing by inversion every 10 minutes. Extraction was performed with a chloroform / isoamyl alcohol (24:1) solution. Centrifuge at 12,000 rpm for 30 minutes at room temperature. Transfer the supernatant to a new 2 mL centrifuge tube, add twice the volume of anhydrous ethanol, and precipitate in a -20°C refrigerator for at least 2 hours. After precipitation, centrifuge at 12,000 rpm for 30 minutes at room temperature, discard the supernatant, and wash with pre-cooled 75% ethanol. Centrifuge at 12,000 rpm for 15 minutes at room temperature, and discard the supernatant. After the ethanol in the centrifuge tube evaporates, add 50 μL of ddH2O to dissolve the precipitate. This is the rose DNA solution.

[0044] 2. Construction of pYLsgRNA-RhU6-29 plasmid

[0045] PCR amplification of the RhU6-29 promoter was performed. The reaction system consisted of 1 µL template, 2 µL each of 10 µM F / R primers, 25 µL of 2 × PhantaUniFi Master Mix (Dye Plus, Vazyme™), and 20 µL of ddH2O. The template was genomic DNA from the rose 'Samantha'; the primers were proRhU6-29-F and proRhU6-29-R. PCR reaction conditions were: 98°C for 30 seconds, 35 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 1 minute, followed by 72°C for 5 minutes and 4°C for 10 minutes. Agarose gel electrophoresis confirmed that the band size was between 300 and 400 bp. The PCR product was sent for analysis (Qingke Biotechnology Co., Ltd.). The primers used were identical to those used for the amplification. Sequencing confirmed the presence of the U6-29 promoter's structural elements, the USE and TATA box. Figure 1 ).

[0046] For amplification of the RhU6-29 promoter, proRhU6-29-F: agaaaacacagtgcaccac (SEQ ID No. 4);

[0047] Used to amplify the RhU6-29 promoter, proRhU6-29-R: aactctactgcgaagcattcg (SEQ ID No. 5).

[0048] PCR amplify the gRNA scaffold region. The system and reaction conditions were the same as above. The template was the pYLsgRNA-AtU6-29 plasmid (SEQ ID No. 6), and the primers were 2 µL each of sgRNA-F and sgRNA-R. Agarose gel electrophoresis was used to confirm that the band size was between 100 and 200 bp. For sgRNA amplification, the primers were sgRNA-F: ggtctcggttttagagctag (SEQ ID No. 7); for sgRNA amplification, the primers were sgRNA-R: tcctttgctgccgattcca (SEQ ID No. 8).

[0049] SEQ ID No.6 pYLsgRNA-AtU6-29

[0050]

[0051] Ligate the gRNA scaffold region to RhU6-29 and add a BsaI restriction site. System: 1 µL RhU6-29 template and 1 µL sgRNA each; 10 mM primers, 2 µL each of sgRNA-F and BsaI-RhU6-29-R, 1 µL each of sgRNA-RhU6-29-F and RhU6-29-sgRNA-R; 25 µL 2 × Phanta UniFi Master Mix (Dye Plus, Vazyme™); 17 µL ddH2O. Reaction conditions are the same as above. The primers used to connect sgRNA and RhU6-29 are sgRNA-RhU6-29-F: tggaatcggcagcaaaggaagaaaacacagtgcaccaca (SEQ ID No. 9); the primers used to connect sgRNA and RhU6-29 are RhU6-29-sgRNA-R: tgtggtgcactgtgttttcttcctttgctgccgattcca (SEQ ID No. 10); and the primer used to add a BsaI restriction enzyme cutting site is BsaI-RhU6-29-R: ggtctcaactctactgcgaa (SEQ ID No. 11).

[0052] The sgRNA-RhU6-29 fragment and expression cassette were recombined. The pYLsgRNA-AtU6-29 vector backbone was removed by digestion with BsaI (ThermoScientific™). Digestion efficiency was confirmed by agarose gel electrophoresis, and the digested vector fragment was recovered using a gel extraction kit (Vazyme™, FastPure Gel DNA Extraction Mini Kit). The gRNA scaffold-RhU6-29 fragment and the digested pYLsgRNA-AtU6-29 vector backbone were then constructed into the pYLsgRNA-RhU6-29 plasmid (Vazyme™, ClonExpress II One Step Cloning Kit) using homologous recombination.

[0053] SEQ ID No.3, gRNA scaffold-RhU6-29 sequence:

[0054] .

[0055] 3. Plasmid transformation into E. coli and verification

[0056] Pipette 5 µL of the ligation product into competent DH5α E. coli and mix thoroughly by pipetting. Place the culture on ice for 5 minutes, then in a 42°C water bath for 90 seconds, and then on ice for 2 minutes. Add 500 µL of LB liquid medium to the clean bench and incubate at 37°C in a shaker at 200 rpm for 45-60 minutes. Pipette 200 µL of the bacterial solution onto a solid LB culture dish containing ampicillin and incubate in an incubator at 37°C overnight.

[0057] 4. Colony PCR detection

[0058] Mark the numbers of individual colonies with a marker. Use a sterile toothpick to pick a portion of the colonies and use them as templates for PCR amplification. Colony PCR reaction system: 10 μL of 2× Taq Master Mix (Vazyme™), 1 μL each of the F and R primers, a small number of colonies, and 8 μL of ddH2O. Primers are proRhU6-29-F and M13F. PCR program: 94°C for 2 minutes; 28 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; 72°C for 5 minutes; and 4°C for 10 minutes. After completion of PCR, check on a 1% agarose gel. For colony PCR detection of pYLsgRNA-RhU6-29-positive colonies, M13F: gtaaaacgacggccagt (SEQ ID No. 12).

[0059] 5. Extract plasmid and detect sequence

[0060] Colonies with correct band verification were inoculated into liquid LB medium containing 50 mg / L ampicillin and cultured at 37°C for 12 hours to extract the plasmid. Plasmid DNA was extracted according to the instructions of the Vazyme™ 8 min FastPure Plasmid Mini Kit. The extracted plasmid was sent to Qingke Biotechnology Co., Ltd. for sequencing verification. The schematic diagram of the modified functional region structure is shown below. Figure 2 The complete vector diagram is shown in Figure 3 shown.

[0061] 6. Construction of editing vector

[0062] Using genomic DNA as a template, the RhJAZ3 DNA sequence (SEQ ID No. 2) was cloned using gene-specific primers (RhJAZ3-clone-F and RhJAZ3-clone-R). Primers were designed based on the homologous gene RchiOBHm_Chr2g0146371 in the 'Yueyuehong' genome (https: / / lipm-browsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). The reaction system and conditions were the same as previously described. The PCR product was ligated into the pMD18-T vector (Takara) and proofread for sequencing.

[0063] The sequence used to clone the RhJAZ3 DNA was RhJAZ3-clone-F: ATGGCTGAGAGATCCAAC (SEQ ID No. 13); the sequence used to clone the RhJAZ3 DNA was RhJAZ3-clone-R: CTATAGACTGAGTTCAAGCTGC (SEQ ID No. 14).

[0064] Based on the corrected sequence, the online tool CRISPR-P (HYPERLINK "http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR" http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR ) was used to design a sgRNA with a target site (SEQ ID No. 24, JAZ-gt1: TGGAGAAGATGGGGCGGAG). Following the multiple sgRNA Golden Gate Cloning assembly protocol, the corresponding CRISPR / Cas9 vector, named CRISPR Cas9 IAZ3-gt1, was generated using the binary vector pYLCRISPR / Cas9Pubi-H and the helper plasmid pYLsgRNA-RhU6-29. Figure 13 ).

[0065] Multiplex sgRNA Golden Gate Cloning Assembly Protocol Reference: Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., et al. (2015) A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant, 8, 1274-1284.

[0066] Construction of the binary vector pYLCRISPR / Cas9Pubi-H. References: Ma, X., Zhu, Q., Chen, Y. and Liu, YG (2016) CRISPR / Cas9 Platforms for Genome Editing in Plants: Developments and Applications. Mol Plant, 9, 961-974.

[0067] The plasmid was subsequently transformed into Escherichia coli and verified, subjected to colony PCR detection, and the plasmid was extracted and sequenced before being used in the following experiments.

[0068] Example 2 Construction of JAZ3 overexpression vector

[0069] 1. Cloning of rose JAZ3 CDS sequence

[0070] Total RNA was extracted from roses using the FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics – Rich (Vazyme™)) and reverse transcribed using HiScript III All-in-one RTSuperMix Perfect for qPCR (Vazyme™) to obtain total cDNA. Primers were designed based on the homologous gene RchiOBHm_Chr2g0146371 in the 'Yueyuehong' genome (https: / / lipm-browsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). PCR products were cloned into the pMD18-T vector (Takara) and proofread for sequencing.

[0071] 2. Construction of super1300-JAZ3-GFP expression vector

[0072] The super1300-GFP vector (Zhou XF, Jin YH, Yoo CY, Lin XL, Kim WY, Yun DJ,Bressan RA, Hasegawa PM, Jin JB. CYCLIN H;1 regulates drought stress responses and blue light-induced stomatal opening by inhibiting reactive oxygen species accumulation in Arabidopsis. Plant Physiol. 2013 Jun;162(2):1030-41. doi: 10.1104 / pp.113.215798. Epub 2013 May 8. PMID: 23656895; PMCID:PMC3668038.) was double-digested with XbaI and KpnI (Thermo Fisher Scientific). Scientific™), and then used gene cloning PCR to add restriction sites to both ends of JAZ3. It was also double-digested with XbaI and KpnI, and then recombinase was used (same as above) to construct a super1300-JAZ3-GFP expression vector. Subsequently, the plasmid was transformed into Escherichia coli and verified, tested by colony PCR, and the plasmid was extracted and sequenced before being used in the following experiments.

[0073] Example 3 Genetic transformation and plant regeneration

[0074] 1. Agrobacterium infection of somatic embryos

[0075] Transformation of Agrobacterium

[0076] Transform the correctly identified plasmid into competent Agrobacterium EHA105 (DaLing DLC303 DL-EHA105). Mix 5 μL of plasmid with competent EHA105, incubate on ice for 5 minutes, place in liquid nitrogen for 5 minutes, then place in a 37°C water bath for 5 minutes, and then incubate on ice for another 5 minutes. Add 700 μL of antibiotic-free liquid LB medium and incubate on a shaker at 28°C for 3 hours. Then, plate 100 μL of the bacterial suspension onto solid LB medium containing kanamycin (50 mg / L) and rifampicin (25 mg / L) and incubate at 28°C for 2 days. After identifying a single colony, pick and incubate in 5 mL of liquid LB medium (with the same antibiotic type and concentration as above) for 12 hours. Then, add 100 μL of the bacterial suspension to 250 mL of liquid LB medium (with the same antibiotic type and concentration as above) and incubate for 12 hours. The primers used to identify positive Agrobacterium clones of CR-JAZ3 were proRhU6-29-F (SEQ ID No. 4) and SP-R, and the primers used to identify positive Agrobacterium clones of super1300-JAZ3-GFP were RhJAZ3-clone-F (SEQ ID No. 13) and GFP-R.

[0077] For the identification of Agrobacterium-positive clones of CR-JAZ3, SP-R: CCCGACATAGATGCAATAACTTCG (SEQ ID No. 15); for the detection of Agrobacterium-positive clones of super1300-JAZ3-GFP, GFP-R: CGCTTCTCGTTGGGGTCTTT (SEQ ID No. 16).

[0078] Somatic embryos were induced from leaves of sterile 'Samantha' seedlings. After 30 days of pre-culture, the leaves were co-infected with Agrobacterium EHA105 (OD600 = 0.6) carrying the CR-JAZ3 (vector CRISPRCas9 IAZ3-gt1) or JAZ3-GFP (vector super1300-JAZ3-GFP) plasmid for 45 minutes. After 3 days of co-cultivation (MS + 60 g / L glucose + 0.05 mg / L 6-BA + 1.0 mg / L 2,4-D + 2.5 g / L Gel + 100 mg / L acid hydrolyzed casein + 100 μM AS, pH 6.0), the cells were transferred to somatic embryo proliferation medium (4.43 g l-1 MS, 1.0 mg l-1 2,4-D, 1.0 mg l-1 6-BA, 6% glucose, 0.3% phytagel, pH 5.8) containing 20 mg / L hygromycin (Roche). After 4 weeks of selection, the cells were transferred to germination medium (1 / 2 MS + 30 g / L glucose + 1.0 mg / L 6-BA + 0.01 mg / L NAA + 0.5 mg / L GA3 + 2.5 g / L Gel + 300 mg / L cephalexin + 30 mg / L dapoxetine) containing 20 mg / L hygromycin. Hygromycin + 100 mg / L acid hydrolyzed casein + 20 g / L mannitol, pH 6.0), and the culture medium was replaced every four weeks with the same formula until regenerated plants were produced.

[0079] 2. Detection of DNA editing in transformed plants

[0080] DNA was extracted from the leaves of the regenerated plants for testing.

[0081] GFP detection primers (GFP-F / GFP-R) were used to detect plants transformed with super1300-JAZ3-GFP. The PCR system consisted of 10 μL of 2 × Taq Master Mix (Vazyme™), 1 μL of each F / R primer, 1 μL of template, and 8 μL of ddH2O. The program was as follows: 94°C for 2 min; 30 s at 4°C, 30 s at 55°C, 30 s at 72°C, 30 cycles; 5 min at 72°C, and 10 min at 4°C. After PCR, the results were detected on a 1% agarose gel. Untransformed plants germinated from somatic embryos (hereinafter referred to as NT, no transgenic) were used as negative controls, and the super1300 - JAZ3 - GFP plasmid was used as a positive control. The results are shown in Figure 2. Figure 4 GFP was not detected in the untransformed plants, while GFP was detected in the transformed positive plants 1-11, as with the plasmid. The positive plants were designated JAZ3-OE. The insertion of GFP into the DNA was detected using the GFP-F construct: GACCACATGAAGCAGCACGA (SEQ ID No. 17).

[0082] Plants transformed with the JAZ3 CRISPR / Cas9 vector were detected using Cas9 detection primers (Gas9-F / Cas9-R). The detection system and conditions were the same as those for GFP insertion detection. NT was used as a negative control, and the JAZ3 CRISPR / Cas9 vector was used as a positive control. For detecting Gas9 insertion in DNA, Gas9-F: GTTACACCGGTTGGGGTAGG (SEQ ID No. 18); for detecting Gas9 insertion in DNA, Gas9-R: TGTTCTTGTCGGACCTCGTG (SEQ ID No. 19). The results are shown in Figure 2. Figure 5 , Cas9 could not be detected in the non-transformed plants, while Cas9 could be detected in the transformed positive plants 1-12 just like the plasmid.

[0083] PCR amplification of the JAZ3 target region (primers seq-JAZ3-F / seq-JAZ3-R) was performed using 1 μL of template, 2 μL of each 10 μM F / R primer, 25 μL of 2× Phanta UniFi Master Mix (Dye Plus, Vazyme™), and 20 μL of ddH2O. PCR reaction conditions were: 98°C for 30 seconds, followed by 30 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 1 minute; 72°C for 5 minutes, and 4°C for 10 minutes. For amplification of approximately 300 bp upstream and downstream of the target site, the sequence for seq-JAZ3-F was: CTACCTTGTTTGACTCCCCCA (SEQ ID No. 20); for amplification of approximately 300 bp upstream and downstream of the target site, the sequence for seq-JAZ3-R was: TCACTCCGAAGCTATCTCCCT (SEQ ID No. 21).

[0084] Use Qingke's NGS sequencing to sequence the gene about 300bp upstream and downstream of the target. Figure 6 This is the peak diagram of NGS sequencing. The results show that compared with NT, there are overlapping peaks near the target sites of the two strains, indicating that gene editing has occurred in this area. Figure 7 As shown in the figure, after analyzing the mutation frequency (M), deletion frequency (D) and base substitution (I) of each base site in the amplified region, it was found that the probability of various base mutations occurring near the target site was higher. After analyzing 5,000 test results, it was found that Figure 8 The indicated editing types resulted in premature termination of JAZ3 protein translation. Positive plants were designated CR-JAZ3.

[0085] 3. Detection of JAZ3 RNA expression levels in positive plants

[0086] Total RNA from positive plants and NT was extracted and reverse transcribed into cDNA using the same method as above. The expression of JAZ3 was detected by qRT-PCR (see the HiScript II One Step qRT-PCR SYBR Green Kit instructions for system conditions). Detection primers qRT-JAZ3-F / qRT-JAZ3-R were designed for the sequence after the editing target site. The results are shown in Figure 2. Figure 9 For the detection of JAZ3 expression by qRT-PCR, qRT-JAZ3-F: CCAGATCTGCTCCCATGACC (SEQ ID No. 22); for the detection of JAZ3 expression by qRT-PCR, qRT-JAZ3-R: AGGTGGTGGTGGTTGTTGTT (SEQ ID No. 23).

[0087] Example 4 Phenotypic Analysis

[0088] The NT, CR-JAZ3 and JAZ3-OE plants that germinated at the same time were transferred to plant nutrient soil and cultured under greenhouse conditions (temperature: 22±1℃, photoperiod 16 h / 8 h (light / dark), relative humidity 50~60%). After a period of time, it was found that the flower colors of the three genotypes were different, such as Figure 10 and Figure 11 shown.

[0089] After anthocyanin extraction and testing using the Micro Plant Anthocyanin Assay Kit, it was found that the anthocyanin content of CR-JAZ3 decreased by 58% compared with NT, while the anthocyanin content of JAZ3-OE increased significantly. Figure 12 shown.

[0090] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Rose JAZ3 protein, characterized by: Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the rose JAZ3 protein according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

2.

3. The use of the rose JAZ3 protein according to claim 1 in regulating the color of rose petals, characterized in that: The regulation is to achieve lighter color of rose petals by blocking or weakening the expression of the gene encoding JAZ3 protein in rose.

4. A method for producing light-colored roses, characterized in that: This is achieved by blocking or weakening the expression of the gene encoding the JAZ3 protein in rose; the amino acid sequence of the JAZ3 protein is shown in SEQ ID No.

1.

5. The method according to claim 4, characterized in that: The method for blocking or weakening the expression of the gene encoding the JAZ3 protein in rose is at least one of genome editing, homologous recombination or random insertion mutagenesis.

6. The method according to claim 5, characterized in that: The genome editing method is at least one of a meganuclease method, a ZFN method, a TALEN method or a CRISPR-Cas method.

7. The method according to claim 6, characterized in that: The CRISPR-Cas method comprises the following steps: a. Design sgRNA targeting the gene encoding JAZ3 protein; b. Construction of Cas editing expression vector expressing sgRNA; c. Transform the Cas editing expression vector into rose somatic embryos.

8. The method according to claim 7, characterized in that: The CRISPR-Cas method is CRISPR-Cas9, CRISPR-Cas12a or CRISPR-Cas12b.

9. The method according to claim 7, characterized in that: The sequence of the sgRNA in step a is shown as SEQ ID No. 24.

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