Chinese rose JAZ3 protein and application thereof in regulation and control of petal color

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Chinese rose JAZ3 protein and application thereof in regulation and control of petal color. The technical problem to be solved by the invention is to provide a new choice for creating Chinese roses with different flower colors. The invention also provides an application of the Chinese rose JAZ3 protein in regulating and controlling the color of Chinese rose petals. According to the invention, precise editing of the rosa chinensis RhJAZ3 gene is realized for the first time, a T0-generation mutant is obtained through an optimized CRISPR / Cas9 system, and the anthocyanin content of petal of a mutant plant is reduced by 63%.

Description

Technical Field

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

[0002] Rose (Rosa hybrida), as the core economic species in the global ornamental flower industry, has long been limited in its molecular breeding technology by the complex genetic background of its genome and the inefficient genetic transformation system. The genetic transformation efficiency mediated by Agrobacterium tumefaciens in roses is only 2.7 - 3.1% (based on the statistics of the International Society for Horticultural Science database in 2023), far lower than that of the model plant Arabidopsis thaliana (85 - 90%) and the horticultural crop tomato (75 - 80%). So far, only the present team has successfully reported on the gene editing research of roses (authorized patent CNCN116083339A, a CRISPR system targeting the PDS gene, with a T0 generation mutation efficiency of 78.4%), indicating that more major technological breakthroughs are needed in rose molecular breeding.

[0003] The key inhibitor JAZ protein of the jasmonic acid signaling pathway has a highly conserved function in the regulation of plant secondary metabolism. Under high - light stress conditions, compared with wild - type plants, the knockout mutant (jaz3 - 1) of the Arabidopsis AtJAZ3 gene had a 2.5 - fold increase in anthocyanin content (P < 0.001). This phenotype is related to the de - inhibition of the transcription factor MYB75 / PAP1, which can directly activate genes related to anthocyanin biosynthesis; in tomatoes, JAZ4 affects anthocyanin accumulation under the co - regulation of the MYC2 - PUB22–JAZ4 module; in apples, the JAZ1–TRB1–MYB9 module can dynamically regulate the accumulation of jasmonic acid - mediated anthocyanins and proanthocyanidins. In woody ornamental plants, TIFY 5 A of the grape jasmonic acid ZIM domain (JAZ) family can interact with DWF4, and TIFY 5 A inhibits the expression of dihydroflavonol - 4 - reductase (DFR), which plays a key role in the synthesis of anthocyanins. The above data together construct a general molecular paradigm of "JAZ deletion → enhanced anthocyanin synthesis".

[0004] The annual growth rate of the demand for special flower colors (such as blue-purple series and gradient bicolor) in the global cut flower market reaches 14.6%. Traditional cross-breeding or chemical mutagenesis for screening flower color mutant plants relies on natural mutations or random mutagenesis, with a long cycle (5 - 8 years) and low efficiency (<1%). Among them, the success rate of traditional cross-breeding is only 0.68% (2023 White Paper of the International Flower Trade Association). However, the molecular breeding technology of roses relies too much on the overexpression of MYB transcription factors, resulting in an imbalance in flavonoid metabolism. The gene editing technology of roses has been stagnant for a long time, with low genetic transformation efficiency (the somatic embryo regeneration rate <10%), and there is a lack of precise editing cases for flower color regulatory 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 flower colors.

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

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

[0008] The present invention also provides a gene encoding the rose JAZ3 protein, whose nucleotide sequence is shown in SEQ ID No.2.

[0009] SEQ ID No.2, gene encoding rose JAZ3 protein: ATGGCTGAGAGATCCAACTTTGCGCAGACCTGCAACCTCCTCAGCCAGTACTTGAAGGAGAAGAGAAGCCAGTACCTGCAGGGAGATAGCTTCGGAGTGAAGCCAGCCCCGGCGACCATGAACTTGCTCAACACCATGGAGGCCGGCCCTCCAGCGGCGGCGAGTACTCCAGTGGGCCAGGCTCCGGATCAGCCCAGATCTGCTCCCATGACCATCTTCTTCGGCGGGCAGGTCCTGGTTTTCAACGACGTTTCGGCTGAGAAGGCAAAGGAAATTATGGGATTGGCTACCAAAGGAAGCGCTGTGGTTTCCTCTACTGAATCGAATGTTGTAGTCAAACAACAACAACAACCACCACCACCTCAGGCTGTTGGGTCGGATTTGCCTATTGCAAGAAGGGCTTCGCTTCACAAGTTTCTGGCCAAGAGGAAAGAGAGGTAAATTCATTTCCCAAATTGGTTTTTGTTTGTTTGACTTAATTTATTCCCTGATTGGGTGAATATTATAGATGGGAGTTCTTATTGGGTTGTTTGAATTTCAGAGTGACTGCAATAGCTCCATACCAACTGAACCACATCCAGAGAGCAGCTTCTCCCAAGGCAGAGGAACCAGTAGGAGCGCAAAGTTCAAAGCAGCTTGAACTCAGTCTATAG。

[0010] The present invention also provides the use of the Rosa chinensis JAZ3 protein in regulating the color of Rosa chinensis petals.

[0011] Furthermore, the regulation is negative regulation.

[0012] The present invention also provides the use of the Rosa chinensis JAZ3 protein in regulating the anthocyanin content of Rosa chinensis petals.

[0013] Specifically, the regulation is negative regulation.

[0014] The present invention also provides a method for creating light-colored Rosa chinensis, which is achieved by blocking or weakening the expression of the gene encoding the JAZ3 protein in Rosa chinensis.

[0015] Specifically, the method for blocking or weakening the expression of the gene encoding JAZ3 protein in roses is at least one of genome editing method, homologous recombination method or random insertion mutation method.

[0016] Furthermore, the genome editing method includes at least one of meganuclease method, ZFN method, TALEN method or CRISPR-Cas method.

[0017] Particularly, the CRISPR-Cas method includes the following steps: a. Design an sgRNA targeting the gene encoding JAZ3 protein; b. Construct a Cas editing expression vector expressing the sgRNA; c. Transform the Cas editing expression vector into rose somatic embryos.

[0018] Among them, the CRISPR-Cas method is CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas12a or CRISPR-Cas12b.

[0019] Particularly, the sgRNA sequence targeting the gene encoding JAZ3 protein in step a is as shown in SEQ ID No. 24.

[0020] Beneficial effects of the present invention: The present invention realizes the precise editing of the rose RhJAZ3 gene for the first time, and obtains T0 generation mutants through the optimized CRISPR / Cas9 system. Quantitative analysis shows that the anthocyanin content in the petals of mutant plants decreases by 63%, and the color difference of the petals is distinguishable by the naked eye. This phenotype is significantly inconsistent with the reported JAZ functional model, suggesting that rose JAZ3 may positively affect the anthocyanin synthesis pathway through a new regulatory network. This discovery is different from the classical theoretical framework of JAZ proteins, opening up a new direction for the study of jasmonic acid signals in Rosaceae plants. Therefore, by establishing a "JAZ3 dose-color linear regulation" model, the present invention can achieve precise regulation of chromaticity values and shorten the breeding cycle compared with traditional technologies. The present invention provides a new option for creating roses with light-colored petals. Description of the Drawings

[0021] Figure 1 Sequence alignment of RhU6-29 promoter and AtU6-29 promoter.

[0022] Figure 2 Schematic diagram of the functional region structure of pYLsgRNA-RhU6-29.

[0023] Figure 3 Schematic diagram of the complete vector structure of pYLsgRNA-RhU6-29.

[0024] Figure 4, Detection of GFP insertion in the DNA of JAZ3-OE transgenic plants. M: represents Marker, Genstar @ D2000; NT: represents No transform plant, referring to plants not transformed with GFP; the 11 lanes to the right of NT respectively represent JAZ3 overexpression plants JAZ3-OE 1 to 11 transformed with the JAZ3-GFP vector; +: is the positive control, with the plasmid of JAZ3-GFP as the template.

[0025] Figure 5 , Detection of Cas9 insertion in the DNA of CR-JAZ3 transgenic plants. M: represents Marker, Genstar @ D2000; NT: represents No transform plant, referring to plants not transformed with CRISPR-JAZ3; the 12 lanes to the right of NT respectively represent JAZ3 gene-edited plants CR-JAZ3 1 to 12 transformed with the CRISPR-JAZ3 vector; +: is the positive control, with the plasmid of CRISPR-JAZ3 as the template.

[0026] Figure 6 , NGS sequencing comparison of NT and CR-JAZ3 plants.

[0027] Figure 7 , Detection of the sequence editing frequency in the target region of CR-JAZ3 plants.

[0028] Figure 8 , Examples of editing types in CR-JAZ3 plants.

[0029] Figure 9 , Relative expression levels of RhJAZ3 in NT, CR-JAZ3 and JAZ3-OE plants.

[0030] Figure 10 , Whole plants of NT, CR-JAZ3 and JAZ3-OE plants.

[0031] Figure 11 , Flower colors of NT, CR-JAZ3 and JAZ3-OE plants.

[0032] Figure 12 , Anthocyanin contents of NT, CR-JAZ3 and JAZ3-OE plants.

[0033] Figure 13 , Schematic diagram of the structure of the CRISPR Cas9 IAZ3-gt1 vector. Specific implementation methods

[0034] JAZ3 is an interacting gene of EIN2, a gene that controls petal senescence in the early stage, and its origin has no direct relationship with flower color. After cloning this gene and performing gene editing and overexpression, it was found that JAZ3 is related to the phenotype of lighter petal color.

[0035] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in combination with the accompanying drawings and preferred embodiments, describe in detail the specific implementation manners, structures, features and their effects according to the present invention as follows.

[0036] Example 1 Construction of JAZ3 gene editing vector 1. Extraction of Rosa chinensis genomic DNA The CTAB method was used to extract Rosa chinensis genomic DNA. After crushing Rosa chinensis leaves, the extraction solution preheated at 65°C was added, and the mixture was quickly shaken and mixed evenly. The sample was incubated in a water bath at 65°C for 30 min, and inverted and mixed every 10 min. Chloroform / isoamyl alcohol (24:1) solution was added for extraction. Centrifuge at 12000 rpm at room temperature for 30 min. The obtained supernatant was transferred to a new 2 mL centrifuge tube, and 2 volumes of absolute ethanol were added, and the mixture was placed in a -20°C refrigerator for precipitation for more than 2 h. After precipitation, centrifuge at 12000 rpm at room temperature for 30 min, discard the supernatant, wash with pre-cooled 75% ethanol, centrifuge at 12000 rpm at room temperature for 15 min, and discard the supernatant. After the ethanol in the centrifuge tube evaporated completely, 50 μL of ddH2O was added to dissolve the precipitate, and the solution was the Rosa chinensis DNA solution.

[0037] 2. Construction of pYLsgRNA-RhU6-29 plasmid The RhU6-29 promoter was amplified by PCR. Reaction system: 1 μL of template, 2 μL of 10 μM F / R primers each, 25 μL of 2 × PhantaUniFi Master Mix (Dye Plus, Vazyme™), 20 μL of ddH2O; the template was Rosa chinensis 'Samantha' genomic DNA; the primers were proRhU6-29-F and proRhU6-29-R. PCR reaction conditions: 98°C for 30 sec; 98°C for 10 sec, 60°C for 10 sec, 72°C for 1 min, 35 cycles; 72°C for 5 min; 4°C for 10 min. The size of the band was confirmed to be between 300 and 400 bp by agarose gel electrophoresis, and the PCR product was sent for sequencing (Qingke Biotechnology Co., Ltd.). The primers were the same as the amplification primers, and the USE and TATA box of the U6-29 promoter structure element were present in the sequenced and identified sequence ( Figure 1 )

[0038] For amplifying the RhU6-29 promoter, proRhU6-29-F: agaaaacacagtgcaccac (SEQ ID No.4); For amplifying the RhU6-29 promoter, proRhU6-29-R: aactctactgcgaagcattcg (SEQ ID No.5).

[0039] PCR amplifies the gRNA scaffold region. The system and reaction conditions are as before, with the template being the pYLsgRNA-AtU6-29 plasmid, the sequence being SEQ ID No.6, and the primers being 2 μL each of sgRNA-F and sgRNA-R. Use agarose gel electrophoresis to confirm that the band size is between 100 and 200 bp. For amplifying sgRNA, sgRNA-F: ggtctcggttttagagctag (SEQ ID No.7); For amplifying sgRNA, sgRNA-R: tcctttgctgccgattcca (SEQ ID No.8).

[0040] SEQ ID No.6 pYLsgRNA-AtU6-29

[0041] Link the gRNA scaffold region to RhU6-29 and add the BsaI restriction site. Reaction system: The template is 1 μL of RhU6-29 and 1 μL of each sgRNA; the primer concentration is 10 mM, 2 μL each of sgRNA-F and BsaI-RhU6-29-R, and 1 μL each of sgRNA-RhU6-29-F and RhU6-29-sgRNA-R; 25 μL of 2 × Phanta UniFi Master Mix (Dye Plus, Vazyme™); 17 μL of ddH2O. The reaction conditions are as before. The primers for linking sgRNA and RhU6-29 are: sgRNA-RhU6-29-F: tggaatcggcagcaaaggaagaaaacacagtgcaccaca (SEQ ID No.9); for linking sgRNA and RhU6-29, RhU6-29-sgRNA-R: tgtggtgcactgtgttttcttcctttgctgccgattcca (SEQ ID No.10); for adding the BsaI restriction site, BsaI-RhU6-29-R: ggtctcaactctactgcgaa (SEQ ID No.11).

[0042] Recombinant sgRNA-RhU6-29 fragment and expression cassette vector. Digest the vector backbone of the promoter AtU6-29 from pYLsgRNA-AtU6-29 with BsaI (ThermoScientific™), confirm the digestion efficiency by agarose gel electrophoresis after digestion, and recover the digested vector fragment using a gel extraction kit (Vazyme™, FastPure Gel DNA Extraction Mini Kit). Use homologous recombination to construct the gRNA scaffold-RhU6-29 fragment and the digested pYLsgRNA-AtU6-29 vector backbone into the pYLsgRNA-RhU6-29 plasmid (Vazyme™, ClonExpress II One Step CloningKit).

[0043] SEQ ID No.3, gRNA scaffold-RhU6-29 sequence: Ggtctcggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttcaagagcttggagtggatggaattttcctccgttttacctgtggaatcggcagcaaaggaagaaaacacagtgcaccacaccgactgggaatgcttcgtaaggtattttggagattgcaagaggtgaaatgcttgtgcttagaattgaatatatgaggaagaaagcaagttacgaatgtaggatctacatcaccaacttgatttggattctattaattcaaaacaaatacaatagtagtgtcacatgtataatcaattctactttcacggataagcagccaaagtttattagttttttcacctttactcgcatagagtcgcacttgagacgggacacgtgaggcacgtgcgcttttaaaacatttgtgataagaatcccacatcgagaaacagtggcaacctaatgctcttcatatacgaatgcttcgcagtagagttgagacc。

[0044] 3. Transformation of plasmid into Escherichia coli and verification Pipette 5 μL of the ligation product into the competent cells of Escherichia coli DH5α, and pipette and mix well. First, place it on ice for 5 min, then in a water bath at 42 °C for 90 s, and then on ice for 2 min. Add 500 μL of LB liquid medium in a laminar flow hood, and incubate it in a constant temperature shaker at 37 °C with an oscillation speed of 200 rpm for 45 - 60 min. Pipette 200 μL of the bacterial solution and spread it on an LB solid culture dish containing ampicillin antibiotic, and incubate it upside down in an incubator at 37 °C overnight.

[0045] 4. Colony PCR detection Mark the serial numbers of monoclonal colonies with a marker pen. Pick a part of the monoclonal colonies with a sterilized toothpick as a template for PCR amplification. Colony PCR reaction system: 2 × Taq Master Mix (Vazyme™) 10 μL, 1 μL each of F / R primers, a little colony, 8 μL of ddH2O. The primers are proRhU6-29-F and M13F. PCR program: 94°C for 2 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 28 cycles; 72°C for 5 min; 4°C for 10 min. After PCR, detect on 1% agarose gel. Used for the detection of pYLsgRNA-RhU6-29 positive clones in colony PCR, M13F: gtaaaacgacggccagt (SEQ ID No.12).

[0046] 5. Extract the plasmid and detect the sequence Inoculate the colonies with correct band verification into liquid LB medium containing 50 mg / L ampicillin and culture at 37°C for 12 h to extract the plasmid. The extraction of plasmid DNA is carried out according to the Vazyme™ 8 min FastPure Plasmid Mini Kit instruction manual. The extracted plasmid is sent to Tsingke Biotechnology Co., Ltd. for sequencing verification. The schematic diagram of the modified functional region structure is as Figure 2 shown, and the schematic diagram of the complete vector is as Figure 3 shown.

[0047] 6. Construction of editing vector Using genomic DNA as a template, gene-specific primers (RhJAZ3-clone-F, RhJAZ3-clone-R) are used to clone the RhJAZ3 DNA sequence SEQ ID No. 2. The primers are designed according to the homologous gene RchiOBHm_Chr2g0146371 in the 'Yueyuehong' genome (https: / / lipm-browsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). The reaction system and conditions are as before. Connect the PCR product to the pMD18-T vector (Takara) and perform sequencing correction.

[0048] For cloning the RhJAZ3 DNA sequence, RhJAZ3-clone-F: ATGGCTGAGAGATCCAAC (SEQ IDNo.13); for cloning the RhJAZ3 DNA sequence, RhJAZ3-clone-R: CTATAGACTGAGTTCAAGCTGC (SEQ IDNo.14).

[0049] According to the corrected sequence, the sgRNA (SEQ ID No.24, JAZ-gt1: TGGAGAAGATGGGGCGGAG) with the target site was designed using the online tool CRISPR-P (HYPERLINK"http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR" http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR ). According to the multiplex sgRNA Golden Gate Cloning assembly protocol, the corresponding CRISPR / Cas9 vector was generated using the binary vector pYLCRISPR / Cas9Pubi-H and the helper plasmid pYLsgRNA-RhU6-29, named CRISPR Cas9 IAZ3-gt1 ( Figure 13 ).

[0050] The reference for the multiplex sgRNA Golden Gate Cloning assembly protocol is Ma, X., Zhang, Q., Zhu,Q., Liu, W., Chen, Y., Qiu, R., et al. (2015) A Robust CRISPR / Cas9 System forConvenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Mol Plant, 8, 1274-1284. The reference for the construction of the binary vector pYLCRISPR / Cas9Pubi-H is Ma, X., Zhu, Q., Chen, Y. andLiu, Y. G. (2016) CRISPR / Cas9 Platforms for Genome Editing in Plants:Developments and Applications. Mol Plant, 9, 961-974. Subsequently, after the plasmid was transformed into Escherichia coli for verification, colony PCR detection, plasmid extraction and sequence detection, it was used for the following experiments.

[0051] Example 2 Construction of JAZ3 overexpression vector 1. Cloning of the Rosa chinensis JAZ3 CDS sequence Total RNA of Rosa chinensis was extracted using the FastPure Plant Total RNA Isolation Kit (Polysaccharides&Polyphenolics –rich (Vazyme™)). After reverse transcription with HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme™), total cDNA of Rosa chinensis was obtained. The primers were designed based on the homologous gene RchiOBHm_Chr2g0146371 in the ‘Yueyuehong’ genome (https: / / lipm-browsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). The PCR products were cloned into the pMD18-T vector (Takara) and sequenced for correction.

[0052] 2. Construction of super1300-JAZ3-GFP expression vector 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 Scientific™). Then, enzyme digestion sites were added to both ends of JAZ3 by gene cloning PCR and double digested with XbaI and KpnI as well. Subsequently, a super1300-JAZ3-GFP expression vector was constructed using recombinase (the same as above). After transforming the plasmid into Escherichia coli for verification, colony PCR detection, plasmid extraction, and sequence detection, it was used for the following experiments.

[0053] Example 3 Genetic Transformation and Plant Regeneration 1. Agrobacterium-mediated infection of somatic embryos Agrobacterium transformation Transform the correctly identified plasmid into Agrobacterium tumefaciens competent cells EHA105 (Daling DLC303 DL-EHA105). Mix 5 μL of the plasmid with the competent EHA105, let it stand on ice for 5 minutes, place it in liquid nitrogen for 5 minutes, then place it in a 37°C water bath for 5 minutes, and then let it stand on ice for 5 minutes. Add 700 μL of liquid LB medium without antibiotics and culture it on a shaker at 28°C for 3 hours. Then, spread 100 μL of the bacterial solution on an LB solid medium containing kanamycin (50 mg / L) and rifampicin (25 mg / L), and culture it upside down at 28°C for 2 days. After identifying the monoclonal colonies, pick a monoclonal colony and culture it in 5 mL of liquid LB medium (with the same types and concentrations of antibiotics as above) for 12 hours. Then, add 100 μL of the bacterial solution to 250 mL of liquid LB medium (with the same types and concentrations of antibiotics as above) and culture it for 12 hours. The primers for identifying the Agrobacterium positive clones of CR-JAZ3 are proRhU6-29-F (SEQ ID No. 4) and SP-R, and the primers for identifying the Agrobacterium positive clones of super1300-JAZ3-GFP are RhJAZ3-clone-F (SEQ ID No. 13) and GFP-R.

[0054] For the identification of Agrobacterium positive clones of CR-JAZ3, SP-R: CCCGACATAGATGCAATAACTTCG (SEQ ID No. 15); for detecting Agrobacterium positive clones of super1300-JAZ3-GFP, GFP-R: CGCTTCTCGTTGGGGTCTTT (SEQ ID No. 16).

[0055] Take the leaves of 'Samantha' sterile seedlings to induce somatic embryos. After 30 days of pre-culture, co-infect with Agrobacterium tumefaciens EHA105 (OD600 = 0.6) carrying the plasmid of CR-JAZ3 (vector CRISPRCas9 IAZ3-gt1) or JAZ3-GFP (vector super1300-JAZ3-GFP) for 45 minutes. After 3 days of co-culture (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 hydrolysate casein + 100 μM AS, pH 6.0), transfer to the 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% plant gel, pH 5.8) containing 20 mg / L hygromycin (Roche) for screening for 4 weeks, and then transfer to the 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 cefotaxime + 30 mg / L hygromycin + 100 mg / L acid hydrolysate casein + 20 g / L mannitol, pH 6.0) containing 20 mg / L hygromycin. Replace the fresh medium every four weeks with the same formula until regenerated plants are produced.

[0056] 2. Detect the editing of the DNA of the transformed plants Extract the DNA from the leaves of the regenerated plants for detection.

[0057] Detect the plants transformed with super1300-JAZ3-GFP using GFP detection primers (GFP-F / GFP-R). The PCR system is 2 × Taq Master Mix (Vazyme™) 10 μL, 1 μL each of F / R primers, 1 μL of template, and 8 μL of ddH2O. The program is: 94°C for 2 min; 4°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 5 min; 4°C for 10 min. After PCR, detect on 1% agarose gel. Use the plants germinated from somatic embryos without transformation (hereinafter referred to as NT, no transgenic) as the negative control and the super1300 - JAZ3 - GFP plasmid as the positive control. The results are as Figure 4 , GFP cannot be detected in the untransformed plants, while the transformed positive plants 1 - 11 can detect GFP as well as the plasmid. The positive plants are named JAZ3-OE. Used to detect the insertion of GFP in the DNA, GFP-F: GACCACATGAAGCAGCACGA (SEQ ID No.17).

[0058] Plants transformed with the CRISPR / Cas9 vector containing JAZ3 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 the negative control, and the CRISPR / Cas9 vector containing JAZ3 was used as the positive control. For detecting the insertion of Gas9 in DNA, Gas9-F: GTTACACCGGTTGGGGTAGG (SEQ ID No.18); for detecting the insertion of Gas9 in DNA, Gas9-R: TGTTCTTGTCGGACCTCGTG (SEQ ID No.19). The results were as follows Figure 5 , Cas9 was not detected in untransformed plants, while Cas9 could be detected in the transformed positive plants 1-12, the same as in the plasmid.

[0059] PCR amplification of the JAZ3 target region (primers seq-JAZ3-F / seq-JAZ3-R). The PCR system was 1 μL of template, 2 μL each of 10 μM F / R primers, 25 μL of 2 × Phanta UniFi Master Mix (Dye Plus, Vazyme™), and 20 μL of ddH2O. PCR reaction conditions: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 1 min, for 30 cycles; 72℃ for 5 min; 4℃ for 10 min. For amplifying sequences approximately 300 bp upstream and downstream of the target site, seq-JAZ3-F: CTACCTTGTTTGACTCCCCCA (SEQ ID No.20); for amplifying sequences approximately 300 bp upstream and downstream of the target site, seq-JAZ3-R: TCACTCCGAAGCTATCTCCCT (SEQ ID No.21).

[0060] NGS sequencing of Qingke was used to sequence genes approximately 300 bp upstream and downstream of the target site. Figure 6 This is the peak map of NGS sequencing. The results showed that compared with NT, there were overlapping peaks near the target sites in the two illustrated strains, indicating that gene editing occurred in this region. As Figure 7 shown, after analyzing the mutation frequency (M), deletion frequency (D), and base substitution (I) at each base site in the amplified region, it was found that the probability of various base variations was higher near the target site. After analyzing 5000 detection results, it was found that Figure 8 the editing types shown below caused premature termination of JAZ3 protein translation. The positive plants were named CR-JAZ3.

[0061] 3. Detect the JAZ3 RNA expression level in positive plants Total RNA of positive plants and NT was extracted and reverse transcribed into cDNA using the same method. The expression of JAZ3 was detected by qRT-PCR (the conditions of the system are shown in the instruction manual of HiScript II One Step qRT-PCR SYBR Green Kit). Detection primers qRT-JAZ3-F / qRT-JAZ3-R were designed for the sequence after the editing target. The results are as Figure 9 shown. For the detection of the expression level of JAZ3 by qRT-PCR, qRT-JAZ3-F: CCAGATCTGCTCCCATGACC (SEQ ID No.22); for the detection of the expression level of JAZ3 by qRT-PCR, qRT-JAZ3-R: AGGTGGTGGTGGTTGTTGTT (SEQ ID No.23).

[0062] Example 4 Phenotypic analysis NT, CR-JAZ3 and JAZ3-OE germinated at the same time were transferred to plant nutrient soil and cultured under greenhouse conditions (temperature: 22±1°C, photoperiod 16 h / 8 h (light / dark), relative humidity 50-60%) for a period of time. It was found that the flower colors of the plants of the three genotypes were different, as Figure 10 and Figure 11 shown.

[0063] After extracting and detecting anthocyanins using the Micro Plant Anthocyanin Assay Kit, it was found that compared with NT, the anthocyanin content of CR-JAZ3 decreased by 58%, while the anthocyanin content of JAZ3-OE increased significantly. As Figure 12 shown.

[0064] The above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the technical solution content of the present invention, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

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

1.

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

2.

3. Use of the Rosa chinensis JAZ3 protein according to claim 1 or the gene according to claim 2 in regulating the color of Rosa chinensis petals.

4. A method for creating light-colored roses, characterized in that: It is achieved by blocking or weakening the expression of the gene encoding the JAZ3 protein in Rosa chinensis.

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 Rosa chinensis is at least one of genome editing method, homologous recombination method or random insertion mutation method.

6. The method according to claim 5, wherein: The genome editing method includes at least one of meganuclease method, ZFN method, TALEN method or CRISPR-Cas method.

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

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

9. The method according to claim 7, wherein: The sgRNA sequence targeting the gene encoding the JAZ3 protein in step a is shown in SEQ ID No. 24.

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