CN116463342A - Application of carnation DcU6 promoter in CRISPR/Cas9 gene editing system and gene editing method - Google Patents
Application of carnation DcU6 promoter in CRISPR/Cas9 gene editing system and gene editing method Download PDFInfo
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Abstract
The invention provides an application of carnation DcU6 promoter in a CRISPR/Cas9 gene editing system and a gene editing method. The first aspect of the invention provides an application of carnation DcU promoter in a CRISPR/Cas9 gene editing system, wherein the nucleotide sequence of carnation DcU promoter is shown as SEQ ID NO. 1-2. The invention uses carnation DcU6 promoter to improve the editing efficiency of CRISPR/Cas9 gene editing system, can be used for carnation CRISPR/Cas9 gene editing system, and drives the high-efficiency expression of sgRNA, and has important significance for realizing carnation breeding.
Description
Technical Field
The invention relates to application of carnation DcU6 promoter in a CRISPR/Cas9 gene editing system and a gene editing method, and relates to the technical field of genetic engineering.
Background
Carnation (Dianthus Caryophyllus L.) has high ornamental value, is one of the four most commonly used cut flowers, is the ornamental flower with the largest yield and highest yield value in the world, and is commonly used in landscaping. In the breeding process of carnation, the traditional breeding technology still occupies an important position, but the traditional breeding means have the defects of long period and low efficiency; artificial mutagenesis improves mutation frequency, but directional breeding accuracy is low; although the cross breeding can make up for the defects of the two, the cross breeding also faces the problems of distant hybridization incompatibility and difficulty in breaking the reproductive isolation of species. The CRISPR-Cas9 gene editing system is beneficial to creating flower germplasm resources with specific characters but without exogenous genes, eliminates safety apprehension of transgenic plants, and provides a brand-new research means for directionally cultivating carnation new varieties with novel flower colors, flower fragrance and prolonged flowering phase by virtue of high-speed development of biotechnology represented by gene editing, and improves the efficiency and accuracy of carnation directional breeding.
The CRISPR-Cas9 gene editing technology is characterized in that a target gene is identified through an artificially designed sgRNA (guide RNA), and Cas9 protease is guided to cut the target gene to form double-strand breaks, and repair after damage realizes knockout or insertion of the gene. However, during gene editing of carnation, it was found that promoters that initiate transcription of sgrnas are different, and that the transcription levels of sgrnas are different, and the search for promoters with higher transcriptional activity is of continued interest to those skilled in the art.
Disclosure of Invention
The DcU promoter can be used for carnation CRISPR/Cas9 gene editing systems, can efficiently drive sgRNA to express, improves the editing efficiency of the CRISPR/Cas9 gene editing systems, and has important significance for realizing carnation breeding.
The first aspect of the invention provides an application of carnation DcU promoter in a CRISPR/Cas9 gene editing system, wherein the nucleotide sequence of carnation DcU promoter is shown as SEQ ID NO. 1-2.
Further, the nucleotide sequence of the carnation DcU promoter is shown as SEQ ID NO. 2.
In a second aspect, the invention provides a gene editing vector comprising a carnation DcU6 promoter as described in any of the preceding paragraphs.
The gene editing vector as described above, further comprising a sgRNA for targeting a gene of interest and a nucleotide sequence capable of editing cas protein for knocking out the gene of interest.
The above gene editing vector, wherein the nucleotide sequence capable of editing cas protein for knocking out the target gene is a nucleotide sequence encoding cas protein in the genome of Arabidopsis thaliana.
The third aspect of the present invention provides a method for constructing a gene editing vector, comprising the steps of:
step 1, constructing a DcU promoter sequence and a tracrRNA sequence on a pUC19 vector, and introducing a BsbI enzyme cutting site between the DcU promoter sequence and the tracrRNA sequence to construct a pUC19-proDcU6-BsbI-tracrRNA vector;
step 2, respectively recombining pro35S, a gene encoding arabidopsis AtCas9 and a Tnos terminator to an XbaI enzyme cleavage site of the pCAMBIA1300 vector to construct a pCAMBIA1300-35S-AtCas9 knockout vector;
step 3, designing a target gene to target an sgRNA sequence according to the target gene, synthesizing a primer with a BsbI restriction enzyme site sequence, annealing to form an sgRNA double-strand, connecting to a BsbI site of a pUC19-proDcU6-BsbI-tracrRNA vector through a Golden bratid system, designing a primer and amplifying to obtain a pUC19-proDcU6-sgRNA-tracrRNA fragment;
and 4, respectively connecting the pUC19-proDcU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector by utilizing recombinase, and constructing to obtain the gene editing vector.
The fourth aspect of the present invention provides a CRISPR/Cas9 gene editing method for carnation, comprising the steps of:
constructing a gene editing vector;
obtaining carnation protoplast cells;
and transiently transforming the carnation protoplast cell with the gene editing vector to enable the gene editing vector to be expressed in the carnation protoplast cell, so as to realize editing of the target gene.
The method comprises the following steps of:
step 1, constructing a DcU promoter sequence and a tracrRNA sequence on a pUC19 vector, and introducing a BsbI enzyme cutting site between the DcU promoter sequence and the tracrRNA sequence to construct a pUC19-proDcU6-BsbI-tracrRNA vector;
step 2, respectively recombining pro35S, a gene encoding arabidopsis AtCas9 and a Tnos terminator to an XbaI enzyme cleavage site of the pCAMBIA1300 vector to construct a pCAMBIA1300-35S-AtCas9 knockout vector;
step 3, designing a target gene to target an sgRNA sequence according to the target gene, synthesizing a primer with a BsbI restriction enzyme site sequence, annealing to form an sgRNA double-strand, connecting to a BsbI site of a pUC19-proDcU6-BsbI-tracrRNA vector through a Golden bratid system, designing a primer and amplifying to obtain a pUC19-proDcU6-sgRNA-tracrRNA fragment;
and 4, respectively connecting the pUC19-proDcU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector by utilizing recombinase, and constructing to obtain the gene editing vector.
The method for obtaining carnation protoplast cells comprises the following steps:
cutting carnation leaves with good growth state into strips with the thickness of 0.5-1mm, and placing the strips into enzymolysis liquid; vacuumizing the leaves and the enzymolysis liquid, standing and digesting under the dark condition, and obtaining the enzymolysis digestion liquid comprising the protoplast cells after the leaves and the strips are basically disappeared.
The method comprises the steps of transiently transforming the carnation protoplast cell with the gene editing vector, enabling the gene editing vector to be expressed in the carnation protoplast cell, and editing the target gene, wherein the method specifically comprises the following steps:
diluting the enzyme with an equal volume of W5 solutionDigesting the solution, centrifuging, removing supernatant, collecting protoplast, and re-suspending with W5 solution to control the concentration of protoplast cells to 2×10 4 /mL；
Mixing 10 μl of gene editing carrier with 100 μl of protoplast cell heavy suspension, adding 110 μl of PEG solution, mixing, standing at room temperature for 10min, adding 440 μl of W5 solution to dilute the reaction solution, mixing, centrifuging, and discarding supernatant;
the protoplast cells were resuspended using 1mL of WI solution and incubated at room temperature for 12-16 hours under dark conditions;
wherein the PEG solution comprises 40% PEG4000, 0.2M mannitol and 0.1M CaCl 2 ；
The WI solution included 4mM MES, 0.4M mannitol and 15mM MgCl 2 。
The DcU promoter for the carnation CRISPR/Cas9 gene editing system provided by the invention can drive the high-efficiency expression of sgRNA, improves the editing efficiency of the CRISPR/Cas9 gene editing system, and has important significance for realizing carnation breeding.
Drawings
FIG. 1 shows carnation DcU6-1 and DcU6-2 promoter sequence analysis;
FIG. 2 is a construction diagram of pUC19-proDcU6-1-BsbI-tracrRNA vector;
FIG. 3 is a construction diagram of pUC19-proDcU6-2-BsbI-tracrRNA vector;
FIG. 4 is a construction diagram of pCAMBIA1300-35S-AtCas9 vector;
FIG. 5 is a construction diagram of pDBG1- Ω 1-proNOS-gluc-pro 35S-nanoLUC vector;
fig. 6 is a graph of validation of editing efficiency of CRISPR/Cas9 gene editing systems in protoplasts.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental procedure, in which no specific conditions are noted in the examples below, is generally followed by conventional conditions, such as molecular cloning by Sambrook et al: the laboratory manual is found in New York: cold Spring Harbor Laboratory Press, or as recommended by the manufacturer.
The Agrobacterium tumefaciens GV3101, plasmid pCAMBIA1300, is commercially available from commercial sources, such as from CAMBIA, australia.
EXAMPLE 1 cloning of carnation DcU6 promoter
1. Extraction of carnation genomic DNA
Carnation genomic DNA was extracted using a plant genomic DNA extraction kit (CW 0553) provided by Beijing for century Biotech Co., ltd, comprising the steps of:
fresh carnation leaves were taken at about 100mg and added to liquid nitrogen for sufficient milling. The ground powder was collected in a centrifuge tube, 400. Mu.L Buffer LP1 and 6. Mu.L RNase A (10 mg/ml) were added, vortexed for 1 minute, and left at room temperature for 10 minutes to allow sufficient lysis. 130. Mu.L Buffer LP2 was added, mixed well and vortexed for 1 minute. Centrifuge at 12,000rpm (13,400 Xg) for 5 minutes and transfer the supernatant to a new centrifuge tube. 1.5 volumes of Buffer LP3 (absolute ethanol was added before use) was added and mixed well (e.g., 500. Mu.L filtrate added to 750. Mu.L Buffer LP 3). The resulting solution and precipitate were all added to an adsorption column (Spin Columns DM) loaded into a collection tube, centrifuged at 12,000rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube. 500. Mu.L Buffer GW2 (checked before use whether absolute ethanol has been added) was added to the adsorption column, centrifuged at 12,000rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube and repeated once. Centrifuge at 12,000rpm for 2 minutes and pour out the waste liquid in the collection tube. The column was left at room temperature for several minutes to dry thoroughly. Placing the adsorption column into a new centrifuge tube, suspending and dripping 100 μl of sterilized water into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm for 1 min, and collecting DNA solution.
2. Obtaining carnation U6 promoter sequence
The carnation genome database was aligned with the Arabidopsis AtU sequence to obtain a plurality of carnation U6 promoter sequences, wherein two sequences have conserved elements of the U6 promoters, named DcU-1 and DcU-2, respectively, the U6 promoter is a DNA sequence recognized, combined and transcribed by RNA polymerase, dc is carnation Latin short, and DcU6 is denoted as carnation's U6 promoter.
3. PCR amplification
The carnation U6 promoter sequence is amplified by PCR method using genomic DNA as template. Primers were designed based on the DcU-1 and DcU-6-2 promoter sequences (the nucleotide sequences of the primers are shown in SEQ ID NOS.3-6, respectively). The PCR reaction system is shown in Table 1, and the PCR conditions are: pre-denaturation at 94℃for 10min;94 ℃ 40s,55 ℃ 40s,68 ℃ 2min,34 cycles; extending at 68℃for 10min. The PCR products were electrophoretically detected in a 1% agarose gel, specific bands were recovered and ligated into pLB vector for sequencing to obtain DcU6-1 and DcU6-2 promoter sequences.
TABLE 1PCR reaction System
The cloned DcU-1 and DcU-2 promoters and the Arabidopsis AtU and cotton GhU6.9 promoter sequences are subjected to comparison analysis, the analysis results are shown in figure 1, and the results show that carnation DcU6 promoter, arabidopsis AtU promoter and cotton GhU6.9 promoter sequences contain TATA box and USE (upstream sequence element) elements, and the sequences and positions of the two upstream cis-acting elements are relatively conserved.
Example 2 construction of carnation CRISPR/Cas9 Gene editing vector
1. The DcU-1 and DcU-6-2 promoter sequences were ligated to the tracrRNA sequence and introduced into the BsbI cleavage site, and constructed on the pUC19 vector to give the pUC19-proDcU6-1-BsbI-tracrRNA vector and the pUC19-proDcU6-2-BsbI-tracrRNA vector.
The construction schemes of pUC19-proDcU6-1-BsbI-tracrRNA and pUC19-proDcU6-2-BsbI-tracrRNA vectors are shown in FIGS. 2-3, respectively.
the tracrRNA is a shorthand for tracr-activating crRNA, and tracrRNA and pre-crRNA are combined by base pairing, trimmed by RNaseIII, and combined with Cas9 to form a complex with DNA cleavage ability.
2. By utilizing a multi-fragment recombinase, pro35S (promoter), atCas9 (AtCAS 9 is an Arabidopsis Cas9 gene) and Tnos (terminator) are recombined to an XbaI enzyme cleavage site of the pCAMBIA1300 vector to construct the pCAMBIA1300-35S-AtCas9 vector, and a construction schematic diagram of the pCAMBIA1300-35S-AtCas9 vector is shown in figure 4.
3. The Golden brain system was used to express the NanoLUC and Fluc in protoplast cells simultaneously, the reporter gene NanoLUC was edited by the gene editing vector, which resulted in the inability of the NanoLUC to encode normal proteins, the inability to catalyze substrates, the knockout efficiency of the vector compared to the catalytic value of the internal standard gene, and Tnos (terminator), and pro35S (promoter), nanoLUC (reporter) and T35S (terminator) were constructed on the pDBG1- Ω 1 vector, and the construction of the vector was schematically shown in fig. 5.
4. The sgRNA targeting the reporter gene nanoLUC was designed, primers were synthesized, annealed to form a double strand of sgRNA, and ligated to the BsbI site of pUC19-proDcU6-1-BsbI-tracrRNA and pUC19-proDcU6-2-BsbI-tracrRNA vector.
5. Amplifying the proDcU6-1-sgRNA-tracrRNA and the proDcU6-2-sgRNA-tracrRNA fragments, connecting to the KpnI enzyme cutting site of the pCAMBIA1300-35S-AtCas9 vector, constructing a NanoLUC knockout vector, and sequencing.
TABLE 2 sequences involved in example 2
EXAMPLE 3 extraction of endotoxin-free plasmid
The plasmids pCAMBIA1300, pDBG 1-OMEGA 1-proNOS-Fluc-pro35S-NanoLUC, pCAMBIA1300-proDcU6-1-sgRNA-35S-AtCas9, pCAMBIA1300-proDcU6-1-sgRNA-35S-AtCas9 and a control vector pCAMBIA1300-proAtU6-sgRNA-35S-AtCas9 are extracted by using an OMEGA company endotoxin-free plasmid large-drawing kit.
1. 200mL of LB medium containing the screening antibiotics is added into a culture flask with the volume of 500mL, bacteria containing the target plasmid are added, and then the culture flask is placed at 37 ℃ for shaking culture for 12-16 hours.
2. And (3) centrifuging at room temperature for 10min at a concentration of 3,500-5,000Xg to collect bacterial precipitate.
3. 10ml Solution I/RNase A was added and the resuspended cells were vortexed or blown up and down with a pipette.
4. 10ml Solution II was added and the mixture was gently mixed back and forth 10-15 times to give a clear lysate.
5. 5mL Buffer N3 was added and mixed thoroughly by gently inverting it over several times until a white flocculent precipitate appeared. Standing at room temperature for 2-3min.
6. Preparing a binding column: adding 5mL of Buffer GPS into the binding column, standing at room temperature for 3-10min, centrifuging at room temperature for 5min at 3,000-5,000Xg, and discarding the filtrate.
7. The lysate and pellet were all transferred to a syringe filter and a clean 50ml centrifuge tube was prepared to collect the filtrate.
8. Equal volumes of ETR Binding Buffer (25 mL) were added to the binding column and inverted 7-10 times repeatedly.
9. The lysate was carefully transferred to a HiBind binding column, centrifuged at 3,000-5,000Xg for 3-5min, the liquid filtered through the column and the filtrate discarded. This step was repeated until all lysates were filtered, and the filtrate was discarded.
10. 10, 10mL ETR Wash Buffer was added to the binding column and centrifuged at 3,00-5,000Xg for 3-5min, the whole liquid was filtered through the column and the filtrate was discarded.
11. 10mL Buffer EHB was added to the binding column and the whole was filtered through the column by centrifugation at 3,000-5,000Xg for 3-5min, and the filtrate was discarded.
12. Adding 15. 15mL DNA Wash Buffer to the binding column, centrifuging at 3,000-5,000Xg for 3-5min to filter the whole liquid through the column, and discarding the filtrate. This step is repeated.
13. The empty column is sleeved back into the centrifuge tube, and the maximum rotation speed is centrifuge for 10-15min to fully dry the column.
14. The binding column is placed in a new 50mL centrifuge tube, 1-3mL Endotoxin-Free Elution Buffer (or deionized water) is added to the membrane of the binding column, the binding column is placed at room temperature for 2-5min, and the plasmid DNA is eluted by centrifugation at the highest rotational speed for 5 min.
EXAMPLE 4 carnation protoplast preparation and transformation
1. Cutting carnation leaf with good growth state into 0.5-1mM strips, adding 10ml enzymolysis solution (20 mM MES (pH 5.7), 1.5% (W/V) cellulase R-10,0.4% (W/V) educing enzyme R-10,0.4M mannitol, inactivating DNase with a temperature of 55deg.C for 10min, adding CaCl with final concentration of 10mM 2 0.1% BSA,5mM mercaptoethanol).
2. In a vacuum drier, vacuuming for 10 minutes under dark conditions, then standing and digesting for more than 3 hours under dark conditions, and after light shaking, the strip-shaped leaves are almost disappeared, at which time the release of the protoplasts can be observed under a microscope.
3. The enzymatic digest was diluted with an equal volume of W5 solution (2 mM MES (pH 5.7), 154mM NaCl,125mM CaCl2,5mM KCl), filtered through a 75 μm nylon membrane, and the filtrate was centrifuged at 100g for 2 minutes at room temperature, the supernatant was aspirated off, and the protoplasts were resuspended in a small amount of W5 solution.
4. The protoplast cells were counted under a microscope with a hemocytometer and finally the protoplast solution was suspended in a W5 solution to a concentration of 2X 10 5 /mL. Place on ice for 30 minutes and then remove the W5 solution as much as possible with a pipette.
5. MMG solution (4 mM MES (pH 5.7), 0.4M mannitol, 15mM MgCl) stored at room temperature 2 ) Suspending protoplasts to a concentration of 2X 10 5 /ml。
6. mu.L of endotoxin-free plasmid (control: pCAMBIA1300 and pDBG1- Ω 1-proNOS-Fluc-pro35S-nanoLUC, group 2: pCAMBIA1300-proDcU6-1-sgRNA-35S-AtCas9 and pDBG1- Ω 1-proNOS-Fluc-pro35S-nanoLUC, group 3: pCAMBIA1300-proDcU6-1-sgRNA-35S-AtCas9 and pDBG1- Ω 1-proNOS-Fluc-pro35S-nanoLUC, group 4: pCAMBIA1300-proAtU6-sgRNA-35S-AtCas9 and pDBG1- Ω 1-proNOS-Fluc-pro 35S-nanoLUC) was added to each of 2mL of EP tubes, 100. Mu.L of protoplast cells (2X 10 BG 1-2-G) 4 Per mL), 6 replicates per group, gently mixed, and added with 110. Mu.L of an as-prepared PEG solution (40% PEG4000, 0.2M mannitol, 0.1M CaCl) 2 ) And gently flick the EP tube to mix, leave it at room temperature for 10 minutes, slowly add 440. Mu.L of W5 solution to dilute the reaction, gently invert the mix, centrifuge 100g for 2 minutes, carefully pipette off the supernatant.
7. 1mL of WI solution (4 mM MES (pH 5.7), 0.4M mannitol, 15mM MgCl) was added 2 ) Protoplast cells were resuspended and transferred to 6-well plates (pre-treated with 5% calf serum for 5 seconds) and incubated at room temperature for 12-16 hours in the dark.
Example 5 Dual luciferase reporter assay
Protoplast cells cultured in a 6-well plate were transferred to a 2-mL round bottom centrifuge tube, and centrifuged at 200g at room temperature for 5min at a speed of 3 using a high-speed refrigerated centrifuge, and after removing the supernatant, 100. Mu.L of lysate was added, and after mixing, the mixture was lysed at room temperature for 10min. mu.L of the lysate was pipetted into a centrifuge tube and 20. Mu.L of Dual-port-Luciferase Reagent, after mixing, reacting for 10min, and measuring fLuc fluorescence value by using Promega GloMax; subsequently, 20. Mu.L of Dual-/was added to the measured liquid>Stop&/>Reagent, after mixing, reacted for 10min, and the NanoLuc fluorescence value was measured with Promega GloMax. ResultsAs shown, when the fluorescence value of pDIAL-Luc was taken as 100% and fLuc was taken as an internal reference, the editing efficiency of pCAMBIA1300-proDcU6-2-sgRNA vector was higher, 21.52%, while the editing efficiency of pCAMBIA1300-proDcU6-1-sgRNA vector and pCAMBIA1300-proAtU6-sgRNA vector was lower, 5.83% and 3.27%, respectively. Thus, the proDcU6-2 promoter can be used in carnation CRISPR/Cas9 gene editing systems to drive expression of sgRNA guide sequences.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The application of the carnation DcU promoter in a CRISPR/Cas9 gene editing system is characterized in that the nucleotide sequence of the carnation DcU6 promoter is shown as SEQ ID NO. 1-2.
2. The use according to claim 1, wherein the carnation DcU promoter has the nucleotide sequence shown in SEQ ID No. 2.
3. A gene editing vector comprising the carnation DcU6 promoter of any one of claims 1-2.
4. A gene editing vector according to claim 3, further comprising a sgRNA for targeting a gene of interest and a nucleotide sequence capable of editing cas protein for knocking out the gene of interest.
5. The gene editing vector according to claim 4, wherein the nucleotide sequence capable of editing cas protein for knocking out the target gene is a nucleotide sequence encoding cas protein in the genome of Arabidopsis thaliana.
6. The method for constructing a gene editing vector according to any one of claims 3 to 5, comprising the steps of:
step 1, constructing a DcU promoter sequence and a tracrRNA sequence on a pUC19 vector, and introducing a BsbI enzyme cutting site between the DcU promoter sequence and the tracrRNA sequence to construct a pUC19-proDcU6-BsbI-tracrRNA vector;
step 2, respectively recombining pro35S, a gene encoding arabidopsis AtCas9 and a Tnos terminator to an XbaI enzyme cleavage site of the pCAMBIA1300 vector to construct a pCAMBIA1300-35S-AtCas9 knockout vector;
step 3, designing a target gene to target an sgRNA sequence according to the target gene, synthesizing a primer with a BsbI restriction enzyme site sequence, annealing to form an sgRNA double-strand, connecting to a BsbI site of a pUC19-proDcU6-BsbI-tracrRNA vector through a Golden bratid system, designing a primer and amplifying to obtain a pUC19-proDcU6-sgRNA-tracrRNA fragment;
and 4, respectively connecting the pUC19-proDcU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector by utilizing recombinase, and constructing to obtain the gene editing vector.
7. A CRISPR/Cas9 gene editing method for carnation, comprising the steps of:
constructing a gene editing vector;
obtaining carnation protoplast cells;
and transiently transforming the carnation protoplast cell with the gene editing vector to enable the gene editing vector to be expressed in the carnation protoplast cell, so as to realize editing of the target gene.
8. The method according to claim 7, wherein the construction of the gene editing vector comprises the steps of:
step 1, constructing a DcU promoter sequence and a tracrRNA sequence on a pUC19 vector, and introducing a BsbI enzyme cutting site between the DcU promoter sequence and the tracrRNA sequence to construct a pUC19-proDcU6-BsbI-tracrRNA vector;
step 2, respectively recombining pro35S, a gene encoding arabidopsis AtCas9 and a Tnos terminator to an XbaI enzyme cleavage site of the pCAMBIA1300 vector to construct a pCAMBIA1300-35S-AtCas9 knockout vector;
step 3, designing a target gene to target an sgRNA sequence according to the target gene, synthesizing a primer with a BsbI restriction enzyme site sequence, annealing to form an sgRNA double-strand, connecting to a BsbI site of a pUC19-proDcU6-BsbI-tracrRNA vector through a Golden bratid system, designing a primer and amplifying to obtain a pUC19-proDcU6-sgRNA-tracrRNA fragment;
and 4, respectively connecting the pUC19-proDcU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector by utilizing recombinase, and constructing to obtain the gene editing vector.
9. The method of claim 7, wherein obtaining carnation protoplast cells comprises the steps of:
cutting carnation leaves with good growth state into strips with the thickness of 0.5-1mm, and placing the strips into enzymolysis liquid; vacuumizing the leaves and the enzymolysis liquid, standing and digesting under the dark condition, and obtaining the enzymolysis digestion liquid comprising the protoplast cells after the leaves and the strips are basically disappeared.
10. The method of claim 9, wherein transiently transforming the carnation protoplast cell with the gene editing vector to allow the gene editing vector to be expressed in the carnation protoplast cell, and wherein editing the target gene is achieved, specifically comprising:
diluting the enzymatic digestion solution with an equal volume of W5 solution, centrifuging, removing supernatant, collecting protoplasts, and re-suspending with W5 solution to control the concentration of protoplast cells to 2×10 4 /mL；
Mixing 10 μl of gene editing carrier with 100 μl of protoplast cell heavy suspension, adding 110 μl of PEG solution, mixing, standing at room temperature for 10min, adding 440 μl of W5 solution to dilute the reaction solution, mixing, centrifuging, and discarding supernatant;
the protoplast cells were resuspended using 1mL of WI solution and incubated at room temperature for 12-16 hours under dark conditions;
wherein the PEG solution comprises 40% PEG4000, 0.2M mannitol, and 0.1M CaCl 2 ；
The WI solution included 4mM MES, 0.4M mannitol and 15mM MgCl 2 。
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