Application of chrysanthemum methyltransferase genes CmDRM2a and CmDRM2b in controlling flower color of chrysanthemum

Abstract

The invention discloses an application of methyltransferase genes CmDRM2a and CmDRM2b of chrysanthemum in controlling the flower color of chrysanthemum, and belongs to the field of genetic engineering technology. In order to explore the methyltransferase that can regulate the methylation level of the promoter of the CmMYB6 gene of chrysanthemum and breed the flower color of chrysanthemum, the invention clones the methyltransferase genes CmDRM2a and CmDRM2b that can regulate the methylation level of the promoter of the CmMYB6 gene of chrysanthemum, and obtains the truncated protein CmDRM2a-cd that only retains the active domain of the methyltransferase CmDRM2a, and then uses dCas9 fusion DNA methyltransferase to target and modify a specific region of the CmMYB6 promoter region, and determines the functions of the methyltransferase genes CmDRM2a, CmDRM2b and CmDRM2a-cd by a genetic transformation method, successfully creates a mutant showing a new flower color of chrysanthemum, and realizes epigenetic breeding of flower color.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to the application of chrysanthemum methyltransferase genes CmDRM2a and CmDRM2b in controlling the flower color of chrysanthemum. Background Art

[0002] The winters in Northeast China are long and cold, and the summers are short and warm, so there is a lack of greening flower resources suitable for cold-region cultivation. In order to improve the richness of cold-region greening flower resources, a lot of introduction and breeding work is needed. Field chrysanthemum is the main cold-region greening flower cultivated in the north. Flower color, as an important ornamental characteristic, is the main target trait for breeding. Traditional breeding methods include artificial hybridization, natural hybridization, and radiation mutagenesis. However, since field chrysanthemums are mostly polyploid and highly heterozygous, the degree of genetic separation is complex, and directional target breeding such as flower color is very difficult. Therefore, it is urgent to open up new breeding approaches to accelerate the directional breeding of field chrysanthemum flower color traits. Plant epigenetic breeding is a new method that has been developed recently to discover, create, and use "epigenetic allele" variations for plant improvement. It is an innovative model of plant breeding today. For garden plants with complex genetic backgrounds, it is a breeding technology system with great potential.

[0003] The breeding of field chrysanthemum flower color is mainly carried out through natural hybridization to obtain seeds, which are sown in the spring of the second year and screened for new flower colors in the autumn. Once a new field chrysanthemum strain with excellent flower color and resistance is selected, it can be propagated and applied in large quantities through cuttings. In the process of field chrysanthemum flower color breeding, field chrysanthemum plants with different colors on the same plant are often found. The previous research of the research group has analyzed the cause of the formation of the field chrysanthemum 'Golden Powder Double Butterfly' with different colors on the same plant. In the anthocyanin synthesis pathway, the promoter region of the transcription factor CmMYB6 gene is highly methylated, the CmMYB6 gene is not expressed, and the flower color is yellow; on the contrary, the promoter region is low methylated, the CmMYB6 gene is expressed, and the flower color is pink; and this methylation modification is heritable in sexually reproduced offspring and is universal in different field chrysanthemum strains. Therefore, the CmMYB6 gene is a naturally formed epiallele. However, if the promoter region of the CmMYB6 gene is methylated, it is not clear which type of methyltransferase is responsible.

[0004] DNA methylation is an important epigenetic mark that plays an important role in regulating gene expression, maintaining genome stability, and affecting multiple biological processes such as gene imprinting. Unlike animals, which only have CpG methylation, plants have three types of DNA methylation: CG, CHG, and CHH (H represents A, T, or C), with different DNA methylation mechanisms. Plant DNA methylation is mainly completed through three steps: de novo synthesis, maintenance, and active demethylation. Among them, de novo synthesis catalyzes the de novo methylation of cytosine through the RNA-mediated DNA methylation pathway (RdDM); active demethylation is completed by ROS1 / DME; during cell mitosis or meiosis, the maintenance of DNA methylation mainly depends on three different methyltransferases: MET1 (METHYLTRANSFERASE1), CMT3 / 2 (CHROMOMETHYLASE3 / 2), and DRM2 / CMT2 (CHROMOMETHYLASE2). Symmetrical CG methylation is maintained by MET1, which can recognize hemimethyl CGs through VIM (VARIANTINMETHYLATION) proteins; CHG methylation is maintained by CMTs, a class of enzymes widely present in plants and unique to plants; the enzyme mainly methylates DNA in heterochromatin regions, thereby maintaining the heterochromatin state of the genome; the formation of CHH methylation is affected by which enzyme, DRM2 or CMT2, depends on the genomic region. The CHH methylation state of the RdDM-targeted region is mainly maintained by DRM2, and these regions are mainly distributed in evolutionarily young transposons, short transposons, and other repetitive sequences on some autosomal arms. In addition, DRM2 also plays an important role in maintaining the methylation of the edge regions of long transposons in heterochromatin regions. CMT2 is mainly responsible for maintaining the CHH methylation state between heterochromatin and long transposons. Among them, CMT2-mediated methylation has high randomness and polymorphism, and it interacts with DDM1, an ATP-dependent chromatin modifier, especially in chromatin regions enriched in histone H1; while DRM2-mediated methylation has low randomness and heritability. DNA methylation regulates the expression of endogenous genes in plants and plays an important role in plant growth and development, biotic stress and abiotic stress.

[0005] Changes in DNA methylation can be inherited across generations, producing stable epialleles in plants. The generation of epialleles broadens genetic diversity and phenotypic diversity, helps to produce important agronomic traits of crops, and provides a new approach for crop breeding. At present, plant epigenetic breeding mainly uses the regulation of epialleles to improve various agronomic traits. It has been found that multiple naturally occurring epialleles are associated with important plant traits such as flower shape, sex determination, fruit ripening, starch synthesis, flowering time, plant type, biomass, yield, and root length. These epialleles play important biological functions in horticultural plants and crops. By changing the modification of epialleles, gene expression can be regulated to promote plant growth and development. At present, the commonly used methods are to produce gene defective mutants involved in the DNA methylation mechanism, or to use chemicals that inhibit methylation maintenance, such as 5-azacytidine or 4-deoxyuridine. In most crop species, it is still technically challenging to produce mutants of DNA methyltransferase and demethylase genes, and plants have difficulty adapting to drastic changes in DNA methylation. Chemical inhibitors, as analogs of cytosine, interfere with the maintenance of DNA methylation during DNA replication. This leads to epigenetic changes during the cell division cycle and may cause certain phenotypic variations. Although the demethylation effect of these inhibitors is usually short-lived, epigenetic marks are easily restored to their original state when the chemical treatment is stopped. However, there are also reports that changes in epigenetic modifications caused during mitotic development are stably present in the proliferation induced on the epicotyl. It is worth mentioning that genetic manipulation (by changing DNA methylation levels in this way) is particularly challenging in polyploid crops. Both genetic and chemical methods have the disadvantage of affecting DNA methylation on a genome-wide scale, making it difficult to study the effects of DNA methylation at specific sites. Therefore, it is very important to create tools in plants to modify DNA methylation levels at specific sites.

[0006] Initially, exogenous RNAi was used to try to induce hypermethylation of the promoter region of the target gene and trigger transcriptional silencing (TGS). In recent years, the rapid development of gene editing and its derivative technologies has provided more powerful tools for targeted epigenetic engineering. Zinc finger technology, as the first generation of gene editing technology, has been widely used in targeted methylation and demethylation of plants, which provides favorable evidence for epigenetic editing as a useful method for crop improvement. Although epigenetic editing based on zinc finger technology has become increasingly mature, its complex design, high cost and high off-target rate still limit its application. The CRISPR-dCas9 system stands out in epigenetic editing technology due to its simple design, high targeted modification efficiency and multi-target editing. The dCas9 protein can be directly fused with epigenetic effector proteins to control gene expression and silencing. Although the CRISPR-dCas9 epigenetic modification system has been successfully attempted in the model plant Arabidopsis, the application of this system in agricultural crops is limited, and its application in horticultural crops has not yet been reported. At the same time, the use of crop endogenous methylases / demethylases for targeted epigenetic editing has not been reported (endogenous editing may be more efficient and specific). In addition, the system can also be used to directly verify the function of epigenetic modification enzymes in vivo, which has not been reported yet, but there is no doubt that the CRISPR-dCas9 system has great application potential. Summary of the invention

[0007] In order to explore the methyltransferase that can regulate the methylation level of the CmMYB6 gene promoter of chrysanthemum, and then use the plant endogenous methylase / demethylase for targeted epigenetic editing to breed the flower color of chrysanthemum, the present invention cloned the methyltransferase genes CmDRM2a and CmDRM2b that can regulate the methylation level of the CmMYB6 gene promoter of chrysanthemum, and obtained the sequence of the truncated protein CmDRM2a-cd that only retains the active domain of CmDRM2a; used the inactivated Cas9 (dCas9) fusion DNA methyltransferase to target and modify a specific region of the CmMYB6 promoter region, and successfully obtained the corresponding mutants through the Agrobacterium-mediated genetic transformation method, thereby achieving the purpose of changing the flower color of chrysanthemum.

[0008] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0009] The first object of the present invention is to provide an application of a chrysanthemum methyltransferase gene CmDRM2a in controlling the color of chrysanthemum flowers. The nucleotide sequence of the CmDRM2a gene is shown in SEQ ID NO.39.

[0010] In one embodiment of the present invention, the CmDRM2a gene encodes the amino acid sequence shown in SEQ ID NO.40.

[0011] The second object of the present invention is to provide an application of the chrysanthemum methyltransferase gene CmDRM2b in controlling the flower color of chrysanthemum, wherein the nucleotide sequence of the CmDRM2b gene is shown in SEQ ID NO.41.

[0012] In one embodiment of the present invention, the CmDRM2b gene encodes the amino acid sequence shown in SEQ ID NO.42.

[0013] The third object of the present invention is to provide an application of the chrysanthemum methyltransferase gene CmDRM2a-cd in controlling the flower color of chrysanthemum, wherein the nucleotide sequence of the CmDRM2a-cd gene is shown in SEQ ID NO.43.

[0014] In one embodiment of the present invention, the CmDRM2a-cd gene encodes the amino acid sequence shown in SEQ ID NO.44.

[0015] The fourth object of the present invention is to provide a method for changing the color of chrysanthemum to light pink or white, the method comprising using a CRISPR / dCas9 gene editing vector containing the chrysanthemum methyltransferase gene CmDRM2a to target a specific sgRNA site in the CmMYB6 promoter to obtain CRISPR-dCas9-sgRNA-CmDRM2a, and then using an Agrobacterium-mediated genetic transformation method to transform CRISPR-dCas9-sgRNA-CmDRM2a into the genome of chrysanthemum to obtain transgenic chrysanthemum; the sgRNA is sgRNA2, sgRNA3, sgRNA4, sgRNA5 or sgRNA6, and the nucleotide sequences corresponding to sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQID NO.9 and SEQ ID NO.10, respectively.

[0016] The fifth object of the present invention is to provide a method for changing the color of chrysanthemum to dark pink, the method comprising using a CRISPR / dCas9 gene editing vector containing the chrysanthemum methyltransferase gene CmDRM2b to target a specific sgRNA site in the CmMYB6 promoter to obtain CRISPR-dCas9-sgRNA-CmDRM2b, and then using an Agrobacterium-mediated genetic transformation method to transform CRISPR-dCas9-sgRNA-CmDRM2b into the genome of chrysanthemum to obtain transgenic chrysanthemum; the sgRNA is sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 or sgRNA6, and the nucleotide sequences corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ IDNO.7, SEQ IDNO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0017] The sixth object of the present invention is to provide a method for changing the color of chrysanthemum flowers to light pink or white, the method comprising using a CRISPR / dCas9 gene editing vector containing the chrysanthemum methyltransferase gene CmDRM2a-cd to target a specific sgRNA site in the CmMYB6 promoter to obtain CRISPR-dCas9-sgRNA-CmDRM2a-cd, and then using an Agrobacterium-mediated genetic transformation method to transform CRISPR-dCas9-sgRNA-CmDRM2a-cd into the genome of chrysanthemum to obtain transgenic chrysanthemum; the sgRNA is sgRNA2, sgRNA3 or sgRNA4, and the nucleotide sequences corresponding to sgRNA2, sgRNA3 and sgRNA4 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0018] In one embodiment of the present invention, the Agrobacterium-mediated genetic transformation method is the floral dipping method for transient transfection of open-field chrysanthemum buds or the vacuum infiltration method for transfection of open-field chrysanthemum buds.

[0019] The seventh object of the present invention is to provide a preparation for changing the color of chrysanthemums to light pink or white, the active ingredient of the preparation comprising a CRISPR / dCas9 gene editing vector containing the above-mentioned chrysanthemum methyltransferase gene CmDRM2a.

[0020] The eighth object of the present invention is to provide a preparation for changing the color of chrysanthemum flowers to dark pink, the active ingredient of the preparation comprising a CRISPR / dCas9 gene editing vector containing the above-mentioned chrysanthemum methyltransferase gene CmDRM2b.

[0021] The ninth object of the present invention is to provide a preparation for changing the color of chrysanthemums to light pink or white, the active ingredient of the preparation comprising a CRISPR / dCas9 gene editing vector containing the above-mentioned chrysanthemum methyltransferase gene CmDRM2a-cd.

[0022] Beneficial effects of the present invention:

[0023] The invention preliminarily identifies the chrysanthemum DNA methyltransferase DRM2 by constructing an evolutionary tree using FastTree through databases such as tblastn and PFAM, clones the methyltransferase genes CmDRM2a and CmDRM2b capable of regulating the methylation level of the chrysanthemum CmMYB6 gene promoter, and obtains the sequence of the truncated protein CmDRM2a-cd retaining only the active domain of the methyltransferase CmDRM2a; then, the inactivated Cas9 (dCas9) is fused with the DNA methyltransferase to target and modify a specific region of the CmMYB6 promoter region, and the functions of the methyltransferase genes CmDRM2a, CmDRM2b and CmDRM2a-cd are determined by a protoplast transient transformation method mediated by Agrobacterium, and by McrBC-qPCR and bisulfite detection. In addition, through the transient transformation method (transient transfection of field chrysanthemum flower buds by dipping the flower) and the stable genetic transformation method (vacuum infiltration transfection of field chrysanthemum foot buds), it was found that the use of CRISPR-dCas9 vector to connect the plant's own methylase DRM2 can change the methylation state of the CmMYB6 gene, successfully creating a mutant showing a new flower color of field chrysanthemum, and realizing the epigenetic breeding of field chrysanthemum flower color.

[0024] This paper is the first to use epigenetic modification editing technology combined with endogenous DNA methyltransferase to modify the epigenetic allele CmMYB6 of different flower colors in chrysanthemum, achieving an important breakthrough in epigenetic breeding of flower colors. At the same time, an epigenetic breeding framework was established, opening up new ways to apply epigenetic breeding strategies to other asexually propagated crops and crops with complex genomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is the result of agarose gel electrophoresis identification of PCR products of CmDRM2a and CmDRM2b genes; Figure 1 A in the figure is the result of agarose gel electrophoresis identification of the PCR product of CmDRM2a gene. Figure 1 B in the figure is the result of agarose gel electrophoresis identification of the PCR product of CmDRM2b gene; M is Maker DL5000;

[0026] Figure 2The functional domain analysis results of CmDRM2a, CmDRM2b and CmDRM2a-cd genes are shown in the figure; UBA is the ubiquitin binding domain; Dcm is the site-specific DNA methyltransferase;

[0027] Figure 3 Schematic diagram of the CmMYB6 gene structure;

[0028] Figure 4 This is a schematic diagram of the dCas9-DNA methyltransferase vector structure;

[0029] Figure 5 The result of McrBC-qPCR detection of the methylation level of the CmMYB6 gene promoter target site in transgenic chrysanthemum protoplasts; Figure 5 A in the figure is the McrBC-qPCR analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2a transgenic lines. Figure 5 B in the figure is the McrBC-qPCR analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2b transgenic lines; the data are the mean ± SEM of three biological replicates, *P<0.05, ***P<0.001, ns indicates no significant difference;

[0030] Figure 6 The results of McrBC-qPCR analysis of the methylation level of the CmMYB6 promoter region in the WT and dCas9-CmDRM2a-cd transgenic lines; the data are the mean ± SEM of three biological replicates, ***P < 0.001, ns indicates no significant difference;

[0031] Figure 7 The figure is a statistical analysis result of the methylation degree of CmDRM2a, CmDRM2b and CmDRM2a-cd by bisulfite sequencing; wherein, Figure 7 A in the figure is the analysis result of DNA methylation degree of CG, CHG and CHH types in WT and dCas9-CmDRM2a transgenic lines. Figure 7 Figure B shows the results of DNA methylation analysis of CG, CHG and CHH types in WT and dCas9-CmDRM2b transgenic lines. Figure 7 C in the figure is the result of DNA methylation degree analysis of CG, CHG and CHH types in WT and dCas9-CmDRM2a-cd transgenic lines; data are the mean ± SEM of three biological replicates, ***P < 0.001, ns indicates no significant difference;

[0032] Figure 8This is the result of transiently transfecting field chrysanthemum buds with the dipping method to analyze the effect of CmDRM2a on field chrysanthemum flower color, CmMYB6 gene expression level and promoter methylation level; Figure 8 A in the figure is the result of PCR amplification of Hyg, dCas9, and sgRNAs to identify dCas9-CmDRM2a transgenic flower buds, M is a marker, - is a negative control, + is a positive control, 1, 2, 3, 4, 5, 6, and 7 are flower buds, Figure 8 B in the figure is the flower color image produced after vacuum infiltration of the dCas9-CmDRM2a vector targeting regions 1-6 into the flower buds of 'Pink Double Butterfly'. The scale bar is 1 cm. Figure 8 C in the figure is the analysis result of CmMYB6 gene expression level in WT and dCas9-CmDRM2a transgenic lines. Figure 8 D in the figure is the analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2a transgenic lines; Figure 8 C and Figure 8 The data in D are the mean ± SEM of three biological replicates, **P < 0.01,

[0033] ***P<0.001, ns indicates no significant difference;

[0034] Fig. 9 This is the result of transiently transfecting field chrysanthemum buds with the dipping method to analyze the effect of CmDRM2b on field chrysanthemum flower color, CmMYB6 gene expression level and promoter methylation level; Fig. 9 A in the figure is the result of PCR amplification of Hyg, dCas9, and sgRNAs to identify dCas9-CmDRM2b transgenic flower buds, M is a marker, - is a negative control, + is a positive control, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 are flower buds, Fig. 9 B in the figure is the flower color image produced after vacuum infiltration of the dCas9-CmDRM2b vector targeting regions 1-6 into the flower buds of 'Pink Double Butterfly'. The scale bar is 1 cm. Fig. 9 C in the figure is the analysis result of CmMYB6 gene expression level in WT and dCas9-CmDRM2b transgenic lines. Fig. 9 D in the figure is the analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2b transgenic lines; Fig. 9 C and Fig. 9 Data in D are the mean ± SEM of three biological replicates, *P < 0.05, **P < 0.01, ***P < 0.001;

[0035] Fig.10This is the result of transiently transfecting field chrysanthemum buds with the dipping method to analyze the effects of CmDRM2a-cd and CmDRM2b on field chrysanthemum flower color, CmMYB6 gene expression level and promoter methylation level; Fig.10 A in the figure is the result of PCR amplification of Hyg, dCas9, and sgRNAs to identify dCas9-CmDRM2a-cd transgenic flower buds. 1, 2, 3, 4, 5, 6, and 7 are flower buds. Fig.10 B in the figure is the result of PCR amplification of Hyg, dCas9, and sgRNAs to identify dCas9-CmDRM2b transgenic flower buds. 1 and 2 are flower buds. Fig.10 A and Fig.10 In B, M is a marker, - is a negative control, and + is a positive control. Fig.10 C in the figure is the flower color image produced by vacuum infiltration of the dCas9-CmDRM2a-cd vector targeting regions 2-5 into the flower buds of 'Pink Double Butterfly'. The scale bar is 1 cm. Fig.10 D in the figure is the flower color image produced after vacuum infiltration of the dCas9-CmDRM2b vector targeting regions 2-5 into the flower buds of 'Pink Double Butterfly'. The scale bar is 1 cm. Fig.10 E in the figure is the analysis result of CmMYB6 gene expression level of WT and dCas9-CmDRM2a-cd transgenic lines. Fig.10 F in the figure is the analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2b transgenic lines; Fig.10 E and Fig.10 The data in F are the mean ± SEM of three biological replicates, ***P < 0.001, ns indicates no significant difference;

[0036] Fig.11 This is the result of analyzing the effect of CmDRM2a on the flower color, petal anthocyanin content, CmMYB6 gene expression level and promoter methylation level of field chrysanthemum by vacuum infiltration transfection of field chrysanthemum buds; Fig.11 A in the figure shows the phenotype of flowers at six different developmental stages of WT and dCas9-CmDRM2a transgenic lines. The scale bar is 2 cm. Fig.11 Figure B shows the analysis results of anthocyanin content in petals of WT and dCas9-CmDRM2a transgenic lines. Fig.11 C in the figure is the analysis result of CmMYB6 gene expression level in WT and dCas9-CmDRM2a transgenic lines. Fig.11 D in the figure is the analysis result of the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2a transgenic lines; Fig.11 B in Fig.11 C and Fig.11The data in D are the mean ± SEM of three biological replicates, *P < 0.05, ***P < 0.001, ns indicates no significant difference. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and drawings. The experimental methods used in the following examples are conventional experimental methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and those skilled in the art can purchase them through commercial channels.

[0038] The 'Pin Shuang Die' line of inchrysanthemum used in the present invention is disclosed in an article entitled "Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis inchrysanthemum" (doi.org / 10.1111 / nph.18389), and the 'Pin Shuang Die' line is the pink flower in the article.

[0039] Example 1: Cloning of the DNA methyltransferase CmDRM2 gene of the CHH modified type of Cyperus rotundus

[0040] According to the CDS sequence and mRNA sequence of the screened CmDRM2 gene, primers were designed in the 5'UTR and 3'UTR regions using geneious software. The primer sequences are shown in Table 1. We used the cDNA of the petals of 'Pink Double Butterfly' as a template to clone the CmDRM2 gene. The amplification was performed according to the reaction system shown in Table 2 and under the specific reaction conditions shown in Table 3. The PCR product was detected by electrophoresis using 1% agarose gel, and the gel block with the correct size of the target band was cut out and sent to a sequencing company for sequencing.

[0041] We screened the CHH modification of the promoter region of the CmMYB6 gene and obtained the DRM2 gene of the chrysanthemum, and PCR cloned and obtained two DRM2s of the chrysanthemum (CmDRM2a and CmDRM2b, the nucleotide sequences are shown in SEQ ID NO.39 and SEQ ID NO.41, respectively) Figure 1The proteins encoded by CmDRM2a and CmDRM2b genes contain 662 amino acids and 352 amino acids, respectively (the amino acid sequences are shown in SEQ ID NO.40 and SEQ ID NO.42, respectively). Based on the functional domain analysis of DNA methyltransferase, the full-length CmDRM2a gene was truncated to retain only the methyltransferase active domain, and the truncated protein CmDRM2a-cd ( Figure 2 ), the nucleotide sequence of the truncated protein is shown in SEQ ID NO.43, and the amino acid sequence is shown in SEQ ID NO.44.

[0042] Table 1 Primer sequence information for CmDRM2 gene cloning

[0043]

[0044] Table 2 PCR reaction system

[0045]

[0046] Table 3 PCR reaction conditions

[0047]

[0048] Example 2: Construction of dCas9-DNA methyltransferase vector

[0049] In order to verify the methyltransferase activity of CmDRM2a, CmDRM2b and CmDRM2a-cd, the present invention fused CmDRM2a and CmDRM2b with dCas9 respectively, and designed six sgRNAs sites for the CmMYB6 promoter region (-2655 to -1 bp) ( Figure 3 ), and successfully constructed dCas9-CmDRM2a and dCas9-CmDRM2b vectors according to the dCas9-DNA methyltransferase vector structure diagram ( Figure 4 Then, the present invention fused CmDRM2a-cd and CmDRM2b with dCas9 respectively, and designed three sgRNAs (sgRNA2-4) sites for the CmMYB6 promoter region (-1989 to -1465 bp) ( Figure 3 ) and successfully constructed dCas9-CmDRM2a-cd and dCas9-CmDRM2b ( Figure 4 ), the specific vector construction method is as follows:

[0050] (1) Selection of target site in the promoter region of CmMYB6 gene and design of sgRNA

[0051] First, transposon prediction was performed on CmMYB6 on the CENSOR (https: / / www.girinst.org / censor / index.php) transposon prediction website. The recognition of the target site by sgRNA depends on the recognition of the PAM sequence. Generally, we take the 20bp upstream of PAM as the target site sequence, but the selection of the target site should take into account the principles of sgRNA design and off-target factors. The sgRNA sites provided by the CRISPR-GE (Genome Editing)-Liu YG Lab website are combined with the principles of sgRNA design to select the target site. A specific enzyme-cutting linker is added to the 5' end of the sgRNA sense oligonucleotide to synthesize the corresponding guide oligonucleotide sequence (see Table 4).

[0052] Table 4 Design of sgRNA guide oligonucleotides

[0053]

[0054] (2) Construction of dCas9-DNA methyltransferase vector

[0055] ① Dimerization of sgRNA-guided oligonucleotides: 5 μL (10 μM) of the synthesized sgRNA-guided oligonucleotide sense chain and antisense chain were taken in PCR tubes respectively, and the PCR program was set as follows: 37°C, 5 min; cooling from 95°C to 25°C, decreasing by 0.2°C per second, to synthesize dimerized sgRNA1, sgRNA2, ... sgRNA6.

[0056] ② Take the bacterial solution of 18T-AtU6 (published in the following literature: Li, Xueqi, et al. "Optimization of CRISPR-Cas9 system in Eustoma grandiflorum." Iscience 27.3(2024):109053.) cloning vector stored in the laboratory, shake the bacteria (Amp + , 50 μg·mL -1 ) cloning vector, shake the bacteria (Amp + , 50 μg·mL -1 ) to extract the plasmid, and the concentration was measured after the plasmid was extracted. Then, the plasmid of the 18T-AtU6 cloning vector was digested with Bbs I, and the enzyme digestion system was as shown in Table 5. After 30 minutes of enzyme digestion at 37°C, the target band was detected by 1% agarose gel electrophoresis, and the target fragment DNA was recovered and purified from the gel. The recovered DNA needed to be tested for concentration and stored in a -40°C refrigerator for later use.

[0057] Table 5 Bbs I restriction enzyme digestion reaction system

[0058]

[0059] ③ Connect and transform the target fragment DNA of the cloning vector and the products of dimerization of the oligonucleotide adapters guided by the six sgRNAs respectively. The reaction system of the connection is shown in Table 6. The specific steps of the connection and transformation are as follows:

[0060] Add samples according to the system in Table 6, and place in a 25℃ oven for connection for 30 minutes. Then add the connection sample to the competent E. coli at a ratio of 1:10, gently pipette and mix, and let it stand in an ice box for 30 minutes; put it in a 42℃ alcohol bath and heat shock for 1 minute; finally, place it on ice for 5 minutes; add 500μL of LB liquid culture medium without resistance to the clean bench, place it on a shaker at 37℃ and 200rpm for 1 hour; centrifuge it at 4000rpm for 1 minute, and evenly spread the bacterial liquid on the clean bench containing the corresponding resistance (Amp + , 50 μg·mL -1 ) on LB solid medium and cultured upside down in a 37°C oven overnight.

[0061] After successful transformation, single clones were selected for identification. The primers for bacterial solution PCR identification are shown in Table 7, and the reaction system is shown in Table 8. The bacterial solution with the correct target band was sent for sequencing, and the sequencing results were compared with the known sequence, and the bacterial solution corresponding to the correct sample was saved.

[0062] Table 6 T4 ligase ligation reaction system

[0063]

[0064] Table 7 Primer sequence information for bacterial liquid PCR identification

[0065]

[0066] Table 8 Colony PCR reaction system

[0067]

[0068] ④ Use BamH I to digest the p1300-dCas9 vector (the website of the article that published this vector is as follows:

[0069] https: / / doi.org / 10.1038 / s41477-020-0671-x), the cloned DNA methyltransferase gene sequence was designed into a homologous recombination linker, the primer sequence was shown in Table 9, and then the DNA methyltransferase was connected to the BamHI site using the homologous recombination method using the ClonExpressIIOneStepCloningKit to construct a CRISPR-dCas9-methyltransferase fusion expression protein. The homologous recombination reaction system is shown in Table 10.

[0070] Table 9 Homologous recombination primer sequence information

[0071]

[0072]

[0073] Table 10 Homologous recombination reaction system

[0074]

[0075] After successful transformation, perform PCR identification on the bacterial solution. The primers used are shown in Table 9. After electrophoresis detection, send the bacterial solution with the correct target band to the company for sequencing. After successful sequencing, add 40% glycerol to the correct strain and store it at -80°C.

[0076] ⑤Use Xma I to single-digest p1300-dCas9-CmDRM2a, p1300-dCas9-CmDRM2b, and p1300-dCas9-CmDRM2a-cd, design homologous recombination primers for U6 and U3 promoters and corresponding sgRNAs as shown in Table 9, and then use the homologous recombination kit to clone the sgRNAs fragments into the dCas9-DNA methyltransferase vector linearized with Xma I to generate CRISPR-dCas9-sgRNA-DNA methyltransferase (dCas9-DNA methyltransferase) vector.

[0077] The dCas9-CmDRM2a and dCas9-CmDRM2b vectors were designed to target the sgRNA1, sgRNA2, …, sgRNA6 sites in the CmMYB6 promoter, while dCas9-CmDRM2a-cd and dCas9-CmDRM2b were designed to target the sgRNA2, sgRNA3, and sgRNA4 sites.

[0078] Example 3: Analysis of CmDRM2a, CmDRM2b and CmDRM2a-cd methyltransferase activities in transgenic chrysanthemum protoplasts

[0079] (1) Establishment of a protoplast isolation system for Chrysanthemum vulgare leaves

[0080] Based on the Arabidopsis protoplast isolation method, the protoplast isolation technology of chrysanthemum was optimized. The concentration of cleavage enzyme was set to 0.6%, the concentration of cellulose was set to 1.0%, and the concentration of mannitol was set to 1.0M. Then 10mL CPW solution was added to dissolve them and incubated in a 55℃ water bath for 10 minutes. After cooling to room temperature, 100μL CaCl2 (1M) and 0.1% BSA were added; then the lower epidermis of the leaves of chrysanthemum 'Pink Double Butterfly' was torn off and completely immersed in the enzyme solution, and then placed in a constant temperature shaker at 25℃ and 60rpm for enzymatic hydrolysis for 7h; after the enzymatic hydrolysis was completed, it was filtered with 74μm nylon cloth, and the filtrate was centrifuged at 100g for 5min; it was washed once with WS washing solution, and then the protoplasts were collected for use. Fluorescein diacetate (FDA) staining was used to verify the activity of protoplasts: 0.1 mL of the protoplast suspension was taken out and placed in a centrifuge tube with a capacity of 1.5 mL, and an appropriate amount of FDA was added to make the final protoplast concentration reach 10 μg / mL; after mixing, it was allowed to stand for 5 minutes in the dark; and compared under natural light and fluorescence. Protoplasts that fluoresced indicated activity, while those that did not fluoresce indicated inactivity.

[0081] (2) Transient transformation of CRISPR / dCas9 vectors in protoplasts of Echeveria paniculata

[0082] The protoplast suspension was diluted with WS solution to 2 × 10 5 The protoplasts were then placed on ice for 30 min. The WS solution was then gently removed and the protoplasts were resuspended in MMG solution and diluted to a final concentration of 2 × 10 per ml. 5 protoplasts. Take a 2mL centrifuge tube and add 110μL of protoplast suspension, 10μL of 1μg / μL plasmid DNA (dCas9-DNA methyltransferase vector obtained in Example 2) and 120μL of 40% PEG, mix gently, and induce in a 28°C constant temperature incubator for 15min; then add 440μL WS, gently invert upside down to mix, centrifuge at 100g for 2min, and collect the protoplasts; finally, add 1mL WI solution to each protoplast, invert upside down to mix gently, and then transfer the mixture to a culture dish, incubate at 20°C in the dark for more than 48h, and then observe using a fluorescence microscope and a laser scanning confocal microscope.

[0083] (3) Analysis of methyltransferase activity of CmDRM2a, CmDRM2b and CmDRM2a-cd

[0084] The plasmids of each dCas9-DNA methyltransferase vector were transferred into the protoplasts of the 'Pink Double Butterfly' plant using the PEG-mediated method. After incubation for 60 hours, the methylation level of the CmMYB6 promoter target site region in the transfected protoplasts was detected by the McrBC-qPCR method. The specific McrBC-qPCR detection method of the CmMYB6 promoter region is as follows:

[0085] First, the transiently transformed protoplast DNA obtained in step (2) was extracted and subjected to RNase digestion reaction. Then, the DNA sample was subjected to enzymatic digestion reaction using the methylation-sensitive enzyme McrBC. The enzyme digestion and sample addition system is shown in Table 11. After sample addition, the reaction was carried out at 37°C for 1h and inactivated at 65°C for 20min. The treated samples were placed at -40°C.

[0086] Table 11McrBC enzyme digestion reaction system

[0087]

[0088] McrBC-qPCR detection primers were designed in the area near the target site according to the position of sgRNA using the IDT primer design website (https: / / sg.idtdna.com / pages / tools / primerquest). The primer sequences are shown in Table 12. Then, the McrBC digestion product was diluted with ddH2O at a ratio of 1:10. The diluted sample McrBC-qPCR reaction system is shown in Table 13, and the McrBC-qPCR reaction conditions are shown in Table 14.

[0089] Table 12McrBC-qPCR detection primer sequence information

[0090]

[0091] Table 13 McrBC-qPCR reaction system

[0092]

[0093] Table 14 McrBC-qPCR reaction conditions

[0094]

[0095]

[0096] After the reaction program is completed, the qPCR raw data is exported, and the relative DNA methylation level of the sample is calculated based on the values ​​corresponding to +GTP and -GTP. The calculated values ​​are then plotted as a bar graph using GraphPad.Prism.v8.0 software.

[0097] The results showed that CmDRM2a methyltransferase was able to methylate DNA and increase the methylation levels of different targeted regions (regions 3-6) of the CmMYB6 promoter ( Figure 5 A in Figure 1); For CmDRM2b, which only has a methyltransferase activity domain, experiments have shown that it can also significantly increase the methylation level of the CmMYB6 promoter target site region (regions 1-6) ( Figure 5 B in the figure). These results indicate that CmDRM2a and CmDRM2b exhibit methyltransferase activity at specific target sites. In order to further verify the catalytic activity of CmDRM2a, the present invention constructed a dCas9 vector with only the catalytic domain of the enzyme, namely dCas9-CmDRM2a-cd (where cd represents the catalytic domain). The results of McrBC-qPCR detection using the protoplast transient transformation method showed that the activity of CmDRM2a-cd was similar to that of CmDRM2a, and it still had DNA methyltransferase activity, which could increase the methylation level of the CmMYB6 promoter region (regions 2-5) ( Figure 6 ). The above experimental results show that the catalytic domain of DNA methyltransferase is the key domain that dominates its function.

[0098] Example 4: Statistical analysis of methylation levels of CmDRM2a, CmDRM2b and CmDRM2a-cd by bisulfite sequencing

[0099] To further confirm the type of DNA methylation modification catalyzed by CmDRM2a and CmDRM2a-cd, we targeted the promoter region 3-5 (-1832 to -1327 bp) of CmMYB6 ( Figure 3 ) were subjected to bisulfite sequencing. The results showed that CmDRM2a and CmDRM2a-cd only catalyzed CHH-type cytosine methylation ( Figure 7 A and Figure 7 In addition, to investigate the DNA methylation type catalyzed by CmDRM2b in the CmMYB6 promoter region (-1832 to -1327 bp, region 3-5) Figure 3 ), and bisulfite sequencing was also performed. The results showed that there was no difference in the degree of DNA methylation of CG and CHG types between the control group and the experimental group, while the degree of DNA methylation of CHH type in the experimental group was significantly higher than that in the control group ( Figure 7 The results further indicate that CmDRM2a and CmDRM2b can catalyze CHH-type DNA methylation.

[0100] Example 5: Analysis of the effects of CmDRM2a, CmDRM2b and CmDRM2a-cd on the color of field chrysanthemums, the expression level of the CmMYB6 gene and its promoter methylation level by transiently transfecting field chrysanthemum buds with the flower dipping method

[0101] The dCas9-DNA methyltransferase vector obtained in Example 2 was used to transform Agrobacterium GV3101, and 50 μL of the transformed Agrobacterium culture solution was inoculated into 5 mL LB liquid culture medium (25 μg / L rif + , 50μg / L kana + ) and cultured at 28°C and 180 rpm for 24 h; then, the Agrobacterium cultures of the six sgRNAs corresponding to each enzyme of dCas9-CmDRM2a and dCas9-CmDRM2b were mixed, and the Agrobacterium cultures of sgRNA2, sgRNA3, and sgRNA4 corresponding to dCas9-CmDRM2a-cd and dCas9-CmDRM2b were mixed, and 1 mL of the sgRNA strain corresponding to each enzyme was added to 200 mL of LB liquid culture medium (25 μg / L rif + , 50μg / L kana + ), and then cultured at 28°C and 180 rpm for 24 h, and then the concentration was measured and the flask was shaken until the OD 600nm The value is about 1.5-2.0, and then the bacterial liquid is collected by centrifugation at 4000rpm for 5 minutes. Resuspend the Agrobacterium bacterial liquid with 5% (W / V) sucrose solution in a ratio of 1:1, and add 0.02% SilwetL-77; select the unopened flower buds of 'Pink Double Butterfly', gently open the top of the flower bud with tweezers to facilitate Agrobacterium infection, soak the flower buds of 'Pink Double Butterfly' in the bacterial liquid, and then use the vacuum infiltration method to vacuum twice with a vacuum pump, each time for 3 minutes; after the infection is completed, each flower bud is cultured on 1 / 4 Hogland medium; wrap the flower buds of the chrysanthemum 'Pink Double Butterfly' that have completed the dipping with plastic wrap, cover them with a black bag, and culture them in the culture chamber for 16-24 hours; after the dark culture is completed, remove the plastic wrap and restore normal light. After the flower buds open, continuously observe the changes in the color of the petals, and record the number of open flower buds and the number of flower buds that have changed color.

[0102] After CmDRM2a methyltransferase methylated the 1-6 regions of the CmMYB6 promoter, the flower buds of the chrysanthemum 'Pink Double Butterfly' were infected with Agrobacterium containing the dCas9-CmDRM2a vector, and the transformed flower buds were identified as transgenic flower buds by PCR amplification of the Hyg, dCas9 genes and sgRNAs ( Figure 8 A in the figure), and observed that CmDRM2a caused the petal color to change from pink to light pink or white ( Figure 8B); RT-qPCR and McrBC-qPCR were used to detect the expression level of CmMYB6 gene and the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2a transgenic lines, respectively. The results showed that CmDRM2a reduced the expression level of CmMYB6 gene ( Figure 8 C), methylated CmMYB6 promoter regions 2, 4, 5, and 6 ( Figure 8 D) Comprehensive Figure 5 The results shown in A further prove that CmDRM2a has methyltransferase activity and can methylate other regions except regions 1 and 3, reduce the expression level of CmMYB6 gene, and make the petals of 'Pink Double Butterfly' lighter in color.

[0103] After CmDRM2b methyltransferase methylated the 1-6 regions of the CmMYB6 promoter, the flower buds of the chrysanthemum 'Pink Double Butterfly' were infected with Agrobacterium containing the dCas9-CmDRM2b vector, and the transformed flower buds were identified as transgenic flower buds by PCR amplification of the Hyg, dCas9 genes and sgRNAs ( Fig. 9 A in the figure), and observed that CmDRM2b changed the flower color to pink or dark pink ( Fig. 9 B); RT-qPCR and McrBC-qPCR were used to detect the expression level of CmMYB6 gene and the methylation level of CmMYB6 promoter region in WT and dCas9-CmDRM2b transgenic lines, respectively. The results showed that CmDRM2b could increase the expression of CmMYB6 gene ( Fig. 9 C in the figure) and can increase the methylation level of CmMYB6 promoter region 1-6 ( Fig. 9 D).

[0104] Combining the results of transient transformation of protoplasts and transient infection of flower buds, we found that CmDRM2a could not methylate regions 1 and 3 of the CmMYB6 promoter, but could increase the methylation levels of other regions, inhibiting the expression of the CmMYB6 gene; CmDRM2b could methylate regions 1-6, thereby promoting the expression of the CmMYB6 gene. This further proves that the difference between CmDRM2a and CmDRM2b in the expression of the CmMYB6 gene mainly lies in whether they can target and modify region 1.

[0105] After a comprehensive comparison of the functional domains of CmDRM2a and CmDRM2b, only the active domain of the methyltransferase was retained and fused with dCas9, respectively, targeting the modification region 2-5. The flower buds of the chrysanthemum 'Pink Double Butterfly' were infected with Agrobacterium containing dCas9-CmDRM2a-cd and dCas9-CmDRM2b vectors, and the Hyg, dCas9 genes and sgRNAs were amplified by PCR. The transformed flower buds were identified as transgenic flower buds ( Fig.10 The results showed that CmDRM2a-cd and CmDRM2b changed the flower color from pink to light pink or white ( Fig.10 C and Fig.10 D), which reduced the expression level of CmMYB6 ( Fig.10 E), which significantly increased the DNA methylation level in the CmMYB6 promoter region (regions 2, 3, and 5) ( Fig.10 F).

[0106] Example 6: Vacuum infiltration transfection of field chrysanthemum buds to analyze the effects of CmDRM2a on field chrysanthemum flower color, petal anthocyanin content, CmMYB6 gene expression level and promoter methylation level

[0107] Take 10 μL of Agrobacterium culture medium containing the modified vector and add it to 5 mL of LB liquid medium (rif + , kana + ), shake and culture in a shaker at 28°C for 24 h, then take 2 mL and add it to 200 mL LB liquid culture medium (rif + , kana + ) and cultured in a shaking incubator at 28°C until OD 600nm In the range of 1.5-2.0. Enrich the bacterial liquid and resuspend the Agrobacterium with 5% (W / V) sucrose solution, add SilwetL-77 to the resuspended bacterial liquid to a final concentration of 0.02% (V / V), and add acetosyringone (200μM) at a ratio of 1:1000 (V / V). Take the outdoor chrysanthemum foot buds, leaving only a pair of true leaves on the top of each foot bud, so that the foot buds have wounds, and completely immerse them in the prepared Agrobacterium resuspension, and vacuum three times, each time for 3 minutes. Finally, wrap it with plastic wrap and culture it in a dark environment in a culture chamber for 16-24 hours. After the dark culture is completed, carefully remove the plastic wrap wrapped around the plant and resume normal light culture. After the foot buds are infected, they need to be inserted into the sand, transplanted into the soil after rooting, and then placed in Percival for culture.

[0108] The results of protoplast transformation showed that CmDRM2a methylated regions 3-6 ( Figure 5A in the figure), resulting in the inhibition of CmMYB6 gene expression. Based on this, we developed a system, the inhibition system (RL system), in which dCas9-CmDRM2a was designed to target regions 1-6. Transgenic plants were successfully obtained by infecting the foot buds of the 'Pink Double Butterfly' line of the chrysanthemum. We found that compared with the WT, the color of the flowers of the transgenic plants in the RL system changed to yellow or white ( Fig.11 A in the figure); by analyzing anthocyanin content, CmMYB6 gene expression level and CmMYB6 promoter methylation level, it was found that in the RL system, anthocyanin content decreased ( Fig.11 B), and the expression of CmMYB6 decreased ( Fig.11 C), the methylation level of region 1 did not change, while the methylation levels of regions 2, 3, 4, and 6 increased ( Fig.11 D).

[0109] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Chrysanthemum methyltransferase gene CmDRM2a The application of the method in controlling the color of field chrysanthemums is characterized in that: CmDRM2a The nucleotide sequence of the gene is shown in SEQ ID NO.

39.

2. Chrysanthemum methyltransferase gene CmDRM2b The application of the method in controlling the color of field chrysanthemums is characterized in that: CmDRM2b The nucleotide sequence of the gene is shown in SEQ ID NO.

41.

3. Chrysanthemum methyltransferase gene CmDRM2a-cd The application of the method in controlling the color of field chrysanthemums is characterized in that: CmDRM2a-cd The nucleotide sequence of the gene is shown in SEQ ID NO.

43.

4. A method for changing the color of field chrysanthemums to light pink or white, characterized in that: Utilizing the method containing the chrysanthemum methyltransferase gene of claim 1 CmDRM2a CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmDRM2a , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmDRM2a Transfected into the buds of field chrysanthemum to obtain transgenic field chrysanthemum; The sgRNA is a combination of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the nucleotide sequences corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

5. A method for changing the color of field chrysanthemums to dark pink, characterized in that: Utilizing the method containing the chrysanthemum methyltransferase gene as claimed in claim 2 CmDRM2b CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmDRM2b , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmDRM2b Transfected into the flower buds of field chrysanthemum to obtain transgenic field chrysanthemum; the sgRNA is a combination of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the nucleotide sequences corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

6. A method for changing the color of field chrysanthemums to light pink or white, characterized in that: Utilizing the method containing the chrysanthemum methyltransferase gene as claimed in claim 3 CmDRM2a-cd CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmDRM2a-cd , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmDRM2a-cd Transfected into the flower buds of field chrysanthemum to obtain transgenic field chrysanthemum; the sgRNA is a combination of sgRNA2, sgRNA3 and sgRNA4, and the nucleotide sequences corresponding to sgRNA2, sgRNA3 and sgRNA4 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

7. A preparation for changing the color of field chrysanthemums to light pink or white, characterized in that: The active ingredients of the formulation include CRISPR- dCas9 -sgRNA- CmDRM2a , CRISPR- dCas9 -sgRNA- CmDRM2a The method comprises the methyltransferase gene of claim 1. CmDRM2a CRISPR / dCas9 gene editing vector targeting CmMYB6 A specific sgRNA site in the promoter is obtained, and the sgRNA is a combination of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the nucleotide sequences corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

8. A preparation for changing the color of field chrysanthemums to dark pink, characterized in that: The active ingredients of the formulation include CRISPR- dCas9 -sgRNA- CmDRM2b , CRISPR- dCas9 -sgRNA- CmDRM2b The method comprises the methyltransferase gene of claim 2 CmDRM2b CRISPR / dCas9 gene editing vector targeting CmMYB6 A specific sgRNA site in the promoter is obtained, and the sgRNA is a combination of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the nucleotide sequences corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

9. A preparation for changing the color of field chrysanthemums to light pink or white, characterized in that: The active ingredients of the formulation include CRISPR- dCas9 -sgRNA- CmDRM2a-cd , CRISPR- dCas9 -sgRNA- CmDRM2a-cd The method comprises the methyltransferase gene of claim 3. CmDRM2a-cd CRISPR / dCas9 gene editing vector targeting CmMYB6 A specific sgRNA site in the promoter is obtained, and the sgRNA is a combination of sgRNA2, sgRNA3 and sgRNA4. The nucleotide sequences corresponding to sgRNA2, sgRNA3 and sgRNA4 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

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