Chrysanthemum constitutive high expression promoter and application thereof in gene editing

Abstract

The application discloses a chrysanthemum constitutive high-expression promoter and application thereof in gene editing. The nucleotide sequence of the promoter CmUbi is shown as SEQ ID NO. 4. Through double luciferase reporter experiments and stable genetic transformation experiments, it is proved that the expression activity of the CmUbi promoter in chrysanthemum protoplast and callus is significantly higher than that of commonly used CaMV 35S, AtRPS5a and corn ZmUbi promoters. A CRISPR / Cas9 gene editing vector is constructed by using the promoter, and a key gene of branch of hexaploid chrysanthemum is edited BRC1b , and a plurality of allele simultaneous mutation complete editing strains are successfully obtained, and the number of branches of the mutants is significantly increased. The endogenous high-activity promoter of the chrysanthemum provided by the application effectively solves the problems of low expression activity of an existing heterologous promoter in the chrysanthemum and poor gene editing efficiency of a polyploid, and provides an efficient and adaptive expression regulation element for chrysanthemum molecular breeding and functional genomics research.

Description

Technical Field

[0001] This invention relates to plant molecular biology techniques, specifically to a constitutive high-expression promoter for chrysanthemum and its application in gene editing. Background Technology

[0002] Plant growth, development, and physiological metabolic processes are finely regulated by gene expression regulatory networks. Promoters are crucial cis-regulatory elements controlling gene transcription. Constitutive promoters, as a type of promoter, can maintain the continuous and efficient expression of target genes in various tissues and organs of specific species and at different developmental stages, thus becoming important tools in plant genetic engineering research. Currently, widely used promoters in plant genetic engineering, especially in the field of genome editing, mainly include the Cauliflower mosaic virus 35S promoter (CaMV 35S), the Arabidopsis thaliana RPS5a promoter, the actin promoter, and the ubiquitin promoter. Chrysanthemum ( Chrysanthemum morifolium As an important ornamental crop worldwide, its molecular breeding research also mainly relies on the aforementioned heterologous promoters.

[0003] However, numerous studies have shown that the expression efficiency of the commonly used promoters mentioned above varies significantly across different plant species, exhibiting clear species specificity. For example, the expression efficiency of the CaMV 35S promoter is also limited in monocotyledons and callus tissue. Due to the high complexity of its genome and polyploid characteristics, chrysanthemum faces many challenges in the development of gene editing technology. In gene editing systems, the expression levels of Cas proteins and single-stranded guide RNAs (sgRNAs) are key factors determining editing efficiency, and the choice of promoter directly determines the transcriptional level of these core elements. The expression activity of exogenous promoters in chrysanthemum may be affected to varying degrees by genetic differences between species, thus limiting the expression level of exogenous genes and the overall efficiency of gene editing. Therefore, developing highly active constitutive promoters derived from chrysanthemum itself has significant theoretical and practical value for improving exogenous gene expression levels, optimizing transgenic screening efficiency, and enhancing the efficiency of CRISPR gene editing systems. This research direction will not only contribute to a deeper understanding of the gene expression regulation mechanism in chrysanthemum but will also provide more efficient and specific genetic manipulation tools for chrysanthemum molecular breeding. Summary of the Invention

[0004] Objective of this invention: The objective of this invention is to provide a constitutive high-expression promoter for chrysanthemum and its application in gene editing, solving the problems of low exogenous gene expression levels and poor polyploid gene editing efficiency caused by the lack of efficient endogenous chrysanthemum promoters in existing technologies. The promoter provided by this invention can replace commonly used exogenous constitutive promoters, improving the expression level of the target gene in chrysanthemum, thereby providing a new technical means to improve the expression level of exogenous genes and gene editing efficiency in chrysanthemum.

[0005] Technical solution: The constitutive high expression promoter CmUbi of chrysanthemum described in this invention has a nucleotide sequence of one of the following sequences: (1) The nucleotide sequence shown in SEQ ID NO.4; (2) The functional fragment of the nucleotide sequence shown in SEQ ID NO.4, and the fragment still has the promoter activity to drive the expression of the target gene.

[0006] The constitutive high expression promoter CmUbi of chrysanthemum is linked upstream of the target gene and is used to drive the expression of the target gene.

[0007] Preferably, the length of the functional segment is 2000~2500 bp.

[0008] On the other hand, the present invention provides an expression cassette comprising the aforementioned chrysanthemum constitutively high expression promoter and a target gene operatively linked thereto.

[0009] On the other hand, the present invention provides a plant expression vector comprising the aforementioned chrysanthemum constitutive high expression promoter for driving target gene expression.

[0010] Preferably, the target gene is a reporter gene, a screening marker gene, a functional gene, or a gene editing-related gene.

[0011] On the other hand, the present invention provides a plant gene editing vector comprising an sgRNA expression element and a constitutively high expression promoter for chrysanthemum, for driving the expression of Cas protein-encoding genes.

[0012] On the other hand, the present invention provides a host cell or plant cell containing the plant expression vector.

[0013] On the other hand, the present invention provides a transformant comprising the above-mentioned chrysanthemum constitutive high expression promoter CmUbi, or comprising the above-mentioned recombinant expression vector or expression cassette.

[0014] On the other hand, the present invention provides a method for improving the gene editing efficiency of chrysanthemum by utilizing a constitutively high expression promoter of chrysanthemum, including operatively linking the promoter to a Cas protein-encoding gene and transforming it into plant cells.

[0015] One possible approach includes the following steps: (1) Construct a plant gene editing vector, the vector containing an sgRNA expression element and a chrysanthemum constitutive high expression promoter, the promoter being used to drive the expression of the Cas protein encoding gene; (2) The plant gene editing vector was transformed into Agrobacterium to obtain recombinant Agrobacterium; (3) Gene-edited plants were obtained by transforming chrysanthemum explants with the recombinant Agrobacterium and then undergoing screening and regeneration culture.

[0016] On the other hand, the present invention provides the application of the constitutive high expression promoter of chrysanthemum, the expression cassette, the plant expression vector, the plant gene editing vector, the host cell or plant cell, or the transformant in plant gene expression regulation or CRISPR gene editing.

[0017] Preferably, it can improve the expression level of exogenous genes.

[0018] Preferably, it can enhance the expression efficiency of Cas protein and sgRNA in the CRISPR gene editing system.

[0019] Preferably, the above-mentioned constitutive high expression promoter CmUbi of chrysanthemum is linked upstream of the target gene in the vector to construct a recombinant expression vector, and the recombinant expression vector is transformed into plant cells, tissues or organs for culture.

[0020] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: By analyzing chrysanthemum transcriptome data, this invention screened a gene promoter, CmUbi, with high expression levels in multiple tissues; through dual-luciferase reporter assays in chrysanthemum leaf protoplasts, the driving ability of the CmUbi promoter was compared with that of CaMV 35S, AtRPS5a, and ZmUbi promoters, and the results showed that the CmUbi promoter has higher expression activity; the high expression activity of the CmUbi promoter in callus tissue was further verified through stable genetic transformation experiments of chrysanthemum callus tissue; the constitutive high-expression promoter CmUbi provided by this invention can be used to drive the expression of exogenous genes or gene editing-related elements, thereby improving the efficiency of plant genetic engineering and gene editing research, and providing an effective tool for the realization of chrysanthemum gene editing. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the CmUbi promoter-driven reporter gene expression vector of the present invention.

[0022] Figure 2The results show the relative expression activity of different promoters (CmUbi, CaMV 35S, AtRPS5a and ZmUbi) in chrysanthemum leaf protoplasts in a dual-luciferase assay.

[0023] Figure 3 The results of expression level analysis of reporter genes driven by different promoters (CmUbi, CaMV 35S, AtRPS5a and ZmUbi) in the stable transformation system of chrysanthemum callus.

[0024] Figure 4 This study analyzes the editing types of gene-edited lines obtained by gene editing using gene-editing vectors constructed with the CmUbi promoter.

[0025] Figure 5 The phenotype of gene-edited lines obtained by gene editing using gene-editing vectors constructed with the CmUbi promoter. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Example 1

[0028] Transcriptome data from different tissues and flower bud development stages of chrysanthemum were analyzed. Using a FPKM value greater than 100 for gene expression in each tissue and developmental stage as the screening criterion, genes stably and highly expressed in multiple chrysanthemum tissues were identified. Ubiquitin Gene( CmUbi The tissues include roots, stems, leaves, and floral organs, with the floral organs derived from tissues isolated from ray florets and disc florets. Expression analysis was performed on five stages of flower bud development, defined as flower bud diameters of less than 2 mm, 2 mm, 4 mm, 6 mm, and 8 mm. Transcriptome analysis results showed that the selected... CmUbi The gene showed high expression levels in the different tissues and flower bud development stages mentioned above. Therefore, the upstream sequence of the gene was selected as a candidate promoter for subsequent functional analysis.

[0029] CmUbi The gene sequence is shown in SEQ ID NO.1.

[0030] Based on target genes CmUbiAn upstream primer, CmUbi-F, was designed 3505 bp upstream of the translation start site, with the 5'-3' end sequence shown in SEQ ID NO.2; a downstream primer, CmUbi-R, was designed downstream of the translation start site, with the 5'-3' end sequence shown in SEQ ID NO.3, and PCR cloning was performed. The reaction program was as follows: 98℃ for 30 s; 98℃ for 10 s, 62℃ for 5 s, 72℃ for 25 s, for a total of 34 cycles. After agarose gel electrophoresis and gel extraction, the target band was successfully obtained from the PCR product and sent to a sequencing company for sequencing. The sequencing results, after sequence alignment, determined the final target promoter sequence, and the 5'-3' nucleotide sequence of CmUbipro is shown in SEQ ID NO.4.

[0031] Example 2

[0032] A schematic diagram of the CmUbipro-driven luciferase expression vector is shown below. Figure 1 As shown, the construction of the promoter luciferase expression vector includes the following steps: (1) Amplification and recovery of the target promoter fragment Based on the promoter length, upstream primers CmUbipro-0800-F with lengths of 2500bp, 2000bp, and 1500bp were designed for homologous recombination, with 5'-3' end sequences as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; downstream primer CmUbipro-0800-R, with 5'-3' end sequences as shown in SEQ ID NO.8.

[0033] Using a cloning vector containing the target promoter as a template, the target promoter fragment was amplified using the primers described above. The PCR product was subjected to agarose gel electrophoresis, and the target promoter fragment was recovered by gel excision.

[0034] (2) Use of restriction endonucleases Xho I and Bam The pGreenII-LUC vector was double-digested with HI (purchased from TaKara, catalog number 1635 / 1605). The target band was detected by agarose gel electrophoresis, and the linearized vector fragment was recovered from the agarose gel.

[0035] (3) The recovered target promoter fragment and linearized vector were ligated using the ClonExpress Ultra One Step Cloning Kit V3 (Vazyme, C117-01) homologous recombination kit. The total volume of the reaction system was 10 μL, including 100 ng of linearized vector fragment, 50 ng of promoter fragment, and 5 μL of 2×CE Mix V3, and the volume was made up with sterile distilled water. The reaction system was incubated at 50 °C for 5 min, and then placed on ice for 5 min to terminate the reaction.

[0036] (4) The recombinant product obtained in (3) was transformed into TOP10 competent cells, and positive single clones were screened using LB solid medium containing 50 mg / L kanamycin. Colony detection was performed using vector primers pGreenII-LUC-F and pGreenII-LUC-R. Single clones with correct colony detection were sequenced for verification, and the results were as expected. The recombinant vectors were named pGreenII-0800-CmUbipro2500, pGreenII-0800-CmUbipro2000, and pGreenII-0800-CmUbipro1500.

[0037] The 5'-3' end sequence of pGreenII-LUC-F is shown in SEQ ID NO.9, and the 5'-3' end sequence of pGreenII-LUC-R is shown in SEQ ID NO.10.

[0038] (5) The monoclonal bacteria that were verified by sequencing were inoculated into TB (Terrific Broth) liquid medium containing 50 mg / L kanamycin and cultured with shaking at 37℃ and 200 rpm for 12-14 h for expansion. After the culture was completed, endotoxin-free plasmids were extracted using the FastPure EndoFree Plasmid Midi Kit (Vazyme, DC205-01) according to the instructions. After the concentration of the extracted plasmids was detected, they were stored at -20℃ for later use.

[0039] Example 3

[0040] Chrysanthemum leaf mesophyll protoplast transformation includes the following steps: 1. Select robust materials from tissue culture flasks for protoplast preparation, and cut well-grown leaves from the middle and upper parts into strips less than 1 mm wide with a blade.

[0041] 2. Place the cut leaf strips into the pre-prepared enzymatic hydrolysate, use tweezers to completely immerse the leaves in the hydrolysate, and use a vacuum pump to evacuate the air in the dark (with the conical flask wrapped in aluminum foil) for 5 minutes.

[0042] 3. Enzymatic hydrolysis was carried out at 28°C in the dark with shaking at 50 rpm for 4 h.

[0043] 4. After enzyme digestion, add an equal volume of W5 solution to the conical flask, then gently shake by hand and wait 10 seconds to release the protoplasts.

[0044] 5. Wet a 40 µm cell sieve with W5 solution, filter the enzymatic hydrolysate containing protoplasts into a 50 mL round-bottom centrifuge tube, thoroughly wash the conical flask and undigested tissue with W5 solution, and gently shake the undigested tissue with tweezers (this will help increase protoplast yield), then filter it into a 50 mL round-bottom centrifuge tube.

[0045] 6. Centrifuge at 80×g, 25℃, acceleration 1 for 10 min to precipitate protoplasts.

[0046] 7. Discard the supernatant, add 1 mL of W5 solution using the cut pipette tip, gently shake to mix, and bring the volume up to 10 mL.

[0047] 8. 80×g, 25 ℃, acceleration of 1, centrifuge for 5 min.

[0048] 9. Discard the supernatant, add 1 mL of MMG solution using a cut pipette tip, gently mix, then add 3 mL of MMG solution. Use a hemocytometer to count the protoplasts and adjust the protoplast density to 2 × 10⁻⁶. 6 The protoplasts were counted at a rate of 1 / mL and incubated on ice for 30 min for transformation. The counting method and formula are as follows: Place a 24 mm × 24 mm coverslip on a 25 × 16 hemocytometer, add 5 µL of purified protoplasts, and count the number of protoplasts in the four corners and the center square of the counting area.

[0049] Protoplast density (units / mL) = Total number of protoplasts in the four corners and the five central square cells × 5 × 10 4 10. Aliquot protoplasts into 2 mL sterile centrifuge tubes, 100 µL per tube. Add 15 µg of the target promoter expression vectors pGreenII-0800-CmUbipro2500, pGreenII-0800-CmUbipro2000, and pGreenII-0800-CmUbipro1500 plasmids, and add (100 + plasmid volume) µL of PEG-Ca. 2+ Add the solution while gently tapping the tube wall with your hand to allow the PEG-Ca solution to dissolve. 2+ Mix with the protoplast solution and react for 15 min.

[0050] 11. Termination of reaction: Add 1 mL of W5, gently invert the centrifuge tube to completely terminate the reaction, centrifuge at 80×g, 25℃, with an acceleration of 1 for 3 min.

[0051] 12. Remove the supernatant, add 1 mL of WI solution, and incubate at 25°C in the dark (with aluminum foil) for 14 h.

[0052] Comparative Example 3.1 The positive control vector pGreenII-0800-CaMV 35S was used, and the chrysanthemum leaf protoplasts were transformed using the method described in Example 3 to obtain the transformed protoplasts.

[0053] Comparative Example 3.2 The control vector pGreenII-0800-AtRPS5a was used, and the chrysanthemum leaf protoplasts were transformed using the method described in Example 3 to obtain the transformed protoplasts.

[0054] Comparative Example 3.3 The control vector pGreenII-0800-ZmUbi was used, and the chrysanthemum leaf protoplasts were transformed using the method described in Example 3 to obtain the transformed protoplasts.

[0055] Experimental testing: (1) The fluorescence expression level in the transformed mesophyll protoplasts obtained in Example 3 and Comparative Examples 3.1-3.3 was detected by using the Dual-Luciferase Reporter Assay.

[0056] Protoplast cells were lysed using the Dual Luciferase Reporter Assay Kit (Vazyme, DL101-01) according to the manufacturer's instructions, and luciferase activity was measured. The expression intensity of different promoters was evaluated by detecting the luminescence signal of Firefly Luciferase (LUC), and Renilla Luciferase (REN) was used as an internal control to correct for transfection efficiency, thus eliminating the influence of differences in cell number and transformation efficiency between different samples. The relative expression activity of the promoters was finally expressed as the LUC / REN ratio.

[0057] The results of LUC gene expression level analysis in protoplasts transformed by each vector are as follows: Figure 2As shown in the figure. The results showed that the LUC expression level in protoplasts transformed with pGreenII-0800-CmUbipro2500 and pGreenII-0800-CmUbipro2000 vectors was significantly higher than that of the CaMV 35S, AtRPS5a and ZmUbi promoter control groups, indicating that the CmUbi promoter has higher driving expression activity in chrysanthemum mesophyll cells, and the functional fragment length is 2000~2500 bp.

[0058] Example 4

[0059] The recombinant vector containing the target promoter obtained in Example 3 was transformed into EHA105 competent Agrobacterium cells. The transformed bacterial culture was plated on YEB solid medium containing 50 mg / L kanamycin and 15 mg / L rifampin and cultured at 28°C for 2-3 days to screen for positive single clones.

[0060] Single colonies were selected and colony PCR was performed using vector-specific primers pGreenII-LUC-F and pGreenII-LUC-R. The amplification products were then detected by agarose gel electrophoresis to confirm the successful transformation of positive clones.

[0061] Correctly identified positive monoclonal antibodies were inoculated into YEB liquid medium containing 50 mg / L kanamycin and 15 mg / L rifampin for expansion culture to obtain Agrobacterium tumefaciens bacterial suspension containing the target promoter recombinant plasmid. The obtained bacterial suspension was mixed with sterile 50% (v / v) glycerol solution at a volume ratio of 1:1 and stored at -80°C for later use.

[0062] The obtained recombinant Agrobacterium bacteria carried expression vectors pGreenII-0800-CmUbipro2500, pGreenII-0800-CmUbipro2000, and pGreenII-0800-CmUbipro1500, respectively, for subsequent chrysanthemum genetic transformation experiments. Using aseptic tissue culture seedlings of the chrysanthemum variety "Shenma" as material, genetic transformation was carried out via Agrobacterium-mediated transformation. The specific steps are as follows: (1) Pre-culture (leaf disc pre-culture, 3 days) Select healthy, flat, and bright green leaves from wild-type tissue culture seedlings in a clean bench. Place the leaves on sterile filter paper and cut them into uniform leaf discs using a sterile scalpel, ensuring that cuts are made around the leaf discs.

[0063] The resulting leaf discs were inoculated onto MS medium plates containing 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA) at a concentration ratio of 1 mg / L:0.5 mg / L, and cultured upside down for 3 days as a pre-culture.

[0064] (2) Agrobacterium culture The expression vectors pGreenII-0800-CmUbipro2500, pGreenII-0800-CmUbipro2000, and pGreenII-0800-CmUbipro1500 containing the target promoter were inoculated and cultured in YEB liquid medium containing 50 mg / L kanamycin and 15 mg / L rifampin, and cultured overnight at 28°C with shaking to obtain bacterial culture in the logarithmic growth phase.

[0065] (3) Infection treatment Agrobacterium-mediated culturing to OD 600 The concentration was 0.6–0.8, and the cells were transferred to 50 mL centrifuge tubes and centrifuged at 28 °C and 4000 rpm for 15 min. After discarding the supernatant, the cells were resuspended in MS suspension at a 1:1 volume ratio.

[0066] Pour the resuspended bacterial solution into a sterile petri dish, add the pre-cultured leaf discs, ensuring the leaf discs are completely submerged in the bacterial solution, and gently rotate the petri dish to ensure the leaf discs are in full contact with the bacterial solution. Infect for 8 minutes. After infection, transfer the leaf discs to sterile filter paper to absorb excess bacterial solution from the surface, and then inoculate the leaf discs onto MS medium containing 1 mg / L 6-BA:0.5 mg / L NAA.

[0067] (4) Dark culture (co-culture, 3 days) The inoculated culture plates were inverted and cultured under dark conditions for 3 days.

[0068] (5) Sterilization culture (7 days) After co-culture, the leaf discs were transferred to a selection medium containing sterile antibiotics for further culture, with appropriate spacing between the leaf discs. They were inverted for 7 days to inhibit Agrobacterium growth and promote explant recovery.

[0069] Comparative Example 4.1 Using the positive control vector pGreenII-0800-CaMV 35S, chrysanthemum was genetically transformed according to the method described in Example 4 to obtain positively transformed callus tissue.

[0070] Comparative Example 4.2 Using the control vector pGreenII-0800-AtRPS5a, chrysanthemum was genetically transformed according to the method described in Example 4 to obtain positively transformed callus tissue.

[0071] Comparative Example 4.3 Using the control vector pGreenII-0800-ZmUbi, chrysanthemum was genetically transformed according to the method described in Example 4 to obtain positively transformed callus tissue.

[0072] Experimental testing: Expression levels driven by different promoters were analyzed using a luciferase activity assay. First, chrysanthemum callus tissue transformed with D-luciferin potassium salt solution was sprayed to allow for sufficient reaction. Subsequently, images were recorded using a Tanon 5200 chemiluminescence imaging system (Tanon Science & Technology), and quantitative statistical analysis of the image grayscale values ​​was performed using Fuji ImageJ software. Figure 3 As shown, the expression level of the LUC gene in chrysanthemum callus transformed with pGreenII-0800-CmUbipro2500 and pGreenII-0800-CmUbipro2000 vectors was significantly higher than that in the CaMV 35S, AtRPS5a, and ZmUbi promoter control groups. This result indicates that the CmUbi promoter has a strong ability to drive expression in chrysanthemum callus, and the functional fragment length is 2000-2500 bp, demonstrating that the CmUbi promoter provided by this invention can efficiently drive exogenous gene expression in chrysanthemum callus.

[0073] Example 5

[0074] The construction of the CmUbipro-driven gene editing vector includes the following steps: (1) Based on the promoter sequence obtained in Example 1, homologous recombination primers for gene editing vectors were designed. The upstream primer pChry-Ubi-CE-F has the 5'-3' end sequence as shown in SEQ ID NO.11; the downstream primer pChry-Ubi-CE-R has the 5'-3' end sequence as shown in SEQ ID NO.12.

[0075] Using a cloning vector containing the target promoter as a template, the target promoter fragment was amplified using the primers described above. The PCR product was subjected to agarose gel electrophoresis, and the target promoter fragment was recovered by gel excision.

[0076] (2) Use of restriction endonucleases Asc I and Sbf The pChry vector was double-digested using enzyme I (purchased from Yugong Biotechnology, catalog number EG15508S / EG15568S). The target band was detected by agarose gel electrophoresis, and the linearized vector fragment was recovered from the agarose gel.

[0077] (3) The recovered target promoter fragment and linearized vector were ligated using the ClonExpress Ultra One Step Cloning Kit V3 (Vazyme, C117-01) homologous recombination kit. The total volume of the reaction system was 10 μL, including 100 ng of linearized vector fragment, 50 ng of promoter fragment, and 5 μL of 2×CE Mix V3, and the volume was made up with sterile distilled water. The reaction system was incubated at 50 °C for 5 min, and then placed on ice for 5 min to terminate the reaction.

[0078] (4) The recombinant product obtained in (3) was transformed into db3.1 competent cells, and positive single clones were screened using LB solid medium containing 50 mg / L kanamycin. Colony detection was performed using vector primers pGEL-pro-seq-F and pGEL-pro-seq-R. Single clones with correct colony detection were sequenced for verification, and the results were as expected. The recombinant vector was named pChry-STU2.0. The 5'-3' end sequence of pGEL-pro-seq-F is shown in SEQ ID NO.13, and the 5'-3' end sequence of pGEL-pro-seq-R is shown in SEQ ID NO.14.

[0079] (5) The monoclonal bacteria that were verified by sequencing were inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 12-14 h for expansion. After the culture was completed, plasmids were extracted using the FastPure Plasmid Mini Kit (Vazyme, DC201-01) according to the instructions. The extracted plasmids were stored at -20°C after concentration detection for later use.

[0080] The gene editing vector containing the CmUbi promoter was constructed, ligated to the target site, and transformed into Agrobacterium, including the following steps: (1) Based on the Csy4-BRC1b four editing target sequence, Golden Gate primers were designed. The upstream primer BRC1b-golden gate-F has the 5'-3' end sequence as shown in SEQ ID NO.15; the downstream primer BRC1b-golden gate-R has the 5'-3' end sequence as shown in SEQ ID NO.16.

[0081] Using an sgRNA array cloning vector containing Csy4-BRC1b as a template, the fragment was amplified using the primers described above. The PCR product was subjected to agarose gel electrophoresis, and the fragment was excised and recovered from the gel.

[0082] (2) Use of restriction endonucleases BsaI and T4 DNA Ligase (Fast) (purchased from Yugong Biotechnology, catalog number EG15518S / EG15205S) were used in a Golden Gate reaction. The total reaction volume was 20 μL, including 200 ng of the vector fragment, 20 ng of the Csy4-BRC1b fragment, and 2 μL of 10×T4 DNA Ligase Buffer. Bsa Add 0.2 μL of I and 1 μL of T4 DNALigase, and make up the volume with sterile distilled water.

[0083] The reaction procedure was as follows: 37℃ for 1 min, 16℃ for 1 min, 30 cycles; 60℃ for 5 min.

[0084] (3) The recombinant product obtained in (2) was transformed into TOP10 competent cells, and positive single clones were screened using LB solid medium containing 50 mg / L kanamycin. The amplified product was sequenced and verified using Cas9-seq-F and HSP-seq-R primers, and the results were as expected. The recombinant vector was named pChry-STU2.0-BRC1b.

[0085] The Cas9-seq-F 5'-3' end sequence is shown in SEQ ID NO.17, and the HSP-seq-R 5'-3' end sequence is shown in SEQ ID NO.18.

[0086] (4) The monoclonal bacteria that were verified by sequencing were inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 12-14 h for expansion. Plasmids were extracted using the FastPure Plasmid Mini Kit (Vazyme, DC201-01) according to the instructions. After the concentration of the extracted plasmids was detected, they were stored at -20°C for later use.

[0087] Genetic transformation of chrysanthemums includes the following steps: (1) The obtained gene editing vector containing the target promoter was transformed into EHA105 competent Agrobacterium cells. Single colonies were picked and colony PCR was performed using vector-specific primers Cas9-seq-F and HSP-seq-R. The amplification products were detected by agarose gel electrophoresis to confirm the positive clones that had been successfully transformed.

[0088] (2) The correctly identified positive monoclonal clones were inoculated into YEB liquid medium containing 50 mg / L kanamycin and 15 mg / L rifampin for expansion culture to obtain recombinant Agrobacterium tumefaciens containing the target promoter. The cultured bacterial solution was mixed with sterile 50% (v / v) glycerol solution at a volume ratio of 1:1 and stored at -80℃ for later use.

[0089] (3) Genetic transformation of the chrysanthemum variety 'Shenma' was carried out using Agrobacterium-mediated transformation. Resistant plants were obtained after co-culture, screening, and regeneration culture, and roots were induced to form complete regenerated plants. T-DNA insertion identification was performed on the obtained rooted regenerated lines. Genomic DNA was extracted from the transgenic plants and PCR amplified using vector-specific primers Cas9-seq-F and HSP-seq-R. The PCR products were detected by agarose gel electrophoresis to confirm whether the T-DNA had been successfully integrated into the chrysanthemum genome.

[0090] (4) For strains that tested positive by T-DNA insertion, gene-specific primers BRC1b-seq-F and BRC1b-seq-R were used to... BRC1b PCR amplification was performed on the target site region. The obtained PCR product was purified, ligated into a TA cloning vector, and transformed into *E. coli* TOP10 competent cells. Single colonies were then picked for sequencing analysis to identify the mutation type at the target site and assess gene editing efficiency. Various editing methods and their proportions are shown in [link to documentation]. Figure 4 .

[0091] The 5'-3' end sequence of BRC1b-seq-F is shown in SEQ ID NO.19, and the 5'-3' end sequence of BRC1b-seq-R is shown in SEQ ID NO.20.

[0092] (5) Positive lines with gene-editing mutations confirmed by sequencing underwent hardening treatment. Regenerated plants from the rooting medium were carefully removed, and residual medium was washed off the roots before transplanting them into nutrient pots containing substrate for acclimatization under high humidity conditions. The environmental humidity was gradually reduced to allow the plants to adapt to the external growth environment. Once the plants recovered normal growth, they were transferred to a greenhouse for routine cultivation and management. After the plants stabilized, phenotypic identification was performed on each edited line, and changes in plant type were observed and recorded. Figure 5 As shown brc1 Gene-edited chrysanthemum mutants had a significantly higher number of branches than wild-type chrysanthemums.

Claims

1. A constitutive high-expression promoter for chrysanthemum, characterized in that, The constitutive high expression promoter of chrysanthemum is CmUbi, and its nucleotide sequence is one of the following sequences: (1) The nucleotide sequence shown in SEQ ID NO.4; (2) The functional fragment of the nucleotide sequence shown in SEQ ID NO.4, and the fragment still has the promoter activity to drive the expression of the target gene.

2. The chrysanthemum constitutive high-expression promoter according to claim 1, characterized in that, The length of the functional segment is 2000~2500 bp.

3. An expression box, characterized in that, It includes the constitutive high expression promoter of chrysanthemum as described in claim 1 or 2 and the target gene operatively linked thereto.

4. A plant expression vector, characterized in that, It includes the chrysanthemum constitutive high expression promoter as described in claim 1 or 2, for driving the expression of the target gene.

5. The plant expression vector according to claim 4, characterized in that, The target gene is a reporter gene, a screening marker gene, a functional gene, or a gene editing-related gene.

6. A plant gene editing vector, characterized in that, It includes an sgRNA expression element and the constitutive high-expression promoter of chrysanthemum as described in claim 1 or 2, said promoter being used to drive the expression of the Cas protein-encoding gene.

7. A transformant, characterized in that, It includes the chrysanthemum constitutive high expression promoter as described in claim 1 or 2, or the expression cassette as described in claim 3, or the plant expression vector as described in claim 4 or 5, or the plant gene editing vector as described in claim 6.

8. A host cell or plant cell, characterized in that, It includes the plant expression vector of claim 4 or 5, or the plant gene editing vector of claim 6.

9. A method for improving chrysanthemum gene editing efficiency using the constitutive high-expression promoter of chrysanthemum as described in claim 1 or 2, characterized in that, This includes operatively linking the promoter to the Cas protein-encoding gene and transforming it into plant cells.

10. The application of the chrysanthemum constitutive high expression promoter as described in claim 1 or 2, the expression cassette as described in claim 3, the plant expression vector as described in claim 4 or 5, the plant gene editing vector as described in claim 6, the transformant as described in claim 7, or the host cell or plant cell as described in claim 8 in plant gene expression regulation or CRISPR gene editing.

Images