Application of Chrysanthemum methyltransferase genes CmCMT2 and CmCMT2cd in controlling flower color of Chrysanthemum

Abstract

The invention discloses an application of methyltransferase genes CmCMT2 and CmCMT2cd of chrysanthemum in controlling the flower color of chrysanthemum, and belongs to the field of genetic engineering technology. In order to explore the DNA methyltransferase that can regulate the methylation level of the promoter of the CmMYB6 gene of chrysanthemum, and then breed the flower color of chrysanthemum, the invention clones the methyltransferase gene CmCMT2 that can regulate the methylation level of the promoter of the CmMYB6 gene of chrysanthemum, and obtains the sequence of the truncated protein CmCMT2cd that only retains the active domain of CmCMT2, and then uses the inactivated Cas9 to fuse CmCMT2 or CmCMT2cd, and targets and modifies the specific region of the promoter region of CmMYB6, and determines the functions of CmCMT2 and CmCMT2cd through the Agrobacterium-mediated genetic transformation method, and successfully creates a mutant showing a new flower color of chrysanthemum, and realizes the epigenetic breeding of the flower color of chrysanthemum.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a gene encoding a methyltransferase of chrysanthemum morifolium. CmCMT2 and CmCMT2cd Application in controlling the color of open-field chrysanthemums. 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] Field chrysanthemum flower color breeding 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 research team's previous research has analyzed the reasons for the formation of field chrysanthemum 'Golden Powder Double Butterfly' with different colors on the same plant. In the anthocyanin synthesis pathway, the transcription factor CmMYB6 Gene promoter region hypermethylation CmMYB6 If the gene is not expressed, the flower color is yellow; on the contrary, if the promoter region is hypomethylated, CmMYB6 Gene expression, flower color is pink; and this methylation modification is heritable in sexually reproduced offspring and is universal in different varieties of chrysanthemum. CmMYB6 Genes are naturally occurring epialleles. However, CmMYB6 If the gene promoter region is modified by methylation, 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, there are three types of plant 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, namely: MET1 (METHYLTRANSFERASE1), CMT3 / 2 (CHROMOMETHYLASE3 / 2), and DRM2 / CMT2 (CHROMOMETHYLASE2). Symmetrical CG methylation is maintained by MET1, which can recognize hemimethyl CGs through VIM (VARIANT INMETHYLATION) 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 stable 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 crop improvement method. 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 tried 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 yet 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 yet been reported, but there is no doubt that the CRISPR-dCas9 system has great application potential. Summary of the invention

[0007] In order to explore the regulation of CmMYB6 The invention discloses a DNA methyltransferase that regulates the methylation level of gene promoters, and then uses plant endogenous methylase / demethylase for targeted epigenetic editing to breed the flower color of chrysanthemum. The invention clones a gene that can regulate the methylation level of chrysanthemum CmMYB6 DNA methyltransferase genes with promoter methylation levels CmCMT2 , and obtained the sequence of the truncated protein CmCMT2cd that only retained the active domain of CmCMT2, and used inactivated Cas9 (dCas9) to fuse CmCMT2 or CmCMT2cd for targeted modification CmMYB6 The specific area of ​​the promoter region was successfully transformed by Agrobacterium-mediated genetic transformation to obtain the corresponding mutant, achieving the purpose of changing the color of field 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 a methyltransferase gene of chrysanthemum morifolium CmCMT2 Application in controlling the color of field chrysanthemums, CmCMT2 The nucleotide sequence of the gene is shown in SEQ ID NO.35.

[0010] In one embodiment of the present invention, CmCMT2 The gene encodes the amino acid sequence shown in SEQ ID NO.36.

[0011] The second object of the present invention is to provide a methyltransferase gene of chrysanthemum morifolium CmCMT2cd Application in controlling the color of field chrysanthemums, CmCMT2cd The nucleotide sequence of the gene is shown in SEQ ID NO.37.

[0012] In one embodiment of the present invention, CmCMT2cd The gene encodes the amino acid sequence shown in SEQ ID NO.38.

[0013] The third object of the present invention is to provide a method for changing the color of chrysanthemum to dark pink, said method utilizing a gene containing the chrysanthemum methyltransferase gene CmCMT2 CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmCMT2 , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmCMT2 transformed 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.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0014] In one embodiment of the present invention, CRISPR- dCas9 -sgRNA- CmCMT2 The transformation into the genome of Chrysanthemum chrysanthemum is specifically carried out by transiently transfecting the Chrysanthemum buds through the floral dip method.

[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 using a plant containing the chrysanthemum methyltransferase gene CmCMT2cd CRISPR / Cas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmCMT2cd, and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmCMT2cd The sgRNA is transformed into the genome of the 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.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0016] In one embodiment of the present invention, CRISPR- dCas9 -sgRNA- CmCMT2cd The transformation into the genome of Chrysanthemum chrysanthemum is specifically carried out by transiently transfecting the Chrysanthemum buds through the floral dip method.

[0017] The fifth object of the present invention is to provide a preparation for changing the color of chrysanthemum to dark pink, wherein the active ingredient of the preparation comprises the chrysanthemum methyltransferase gene CmCMT2 CRISPR / Cas9 gene editing vector.

[0018] The sixth object of the present invention is to provide a preparation for changing the color of chrysanthemum to light pink or white, wherein the active ingredient of the preparation comprises the chrysanthemum methyltransferase gene CmCMT2cd CRISPR / Cas9 gene editing vector.

[0019] Beneficial effects of the present invention:

[0020] The present invention uses tblastn, PFAM and other databases to construct an evolutionary tree using FastTree to preliminarily identify the DNA methyltransferase CMT2 of chrysanthemum, and clones a gene that can regulate the growth of chrysanthemum. CmMYB6 Gene promoter methylation levels of methyltransferase genes CmCMT2 , and obtained the sequence of the truncated protein CmCMT2cd that only retained the active domain of the methyltransferase CmCMT2; then the inactivated Cas9 (dCas9) was used to fuse CmCMT2 for targeted modification CmMYB6 Six specific regions in the promoter region were targeted for modification using inactive Cas9 (dCas9) fused to CmCMT2cd CmMYB6 Four specific regions in the promoter region were identified by Agrobacterium-mediated protoplast transient transformation, and the methyltransferase gene was identified by McrBC-qPCR and bisulfite detection. CmCMT2 and CmCMT2cdIn addition, through the transient transformation method (transient transfection of open-field chrysanthemum buds by the flower dipping method), it was found that the CRISPR-dCas9 vector was used to connect the plant's own methylase CmCMT2 or CmCMT2cd and target CmMYB6 Specific regions of the promoter region can be altered CmMYB6 By studying the methylation status of genes, we successfully created mutants that exhibited new flower colors of field chrysanthemums, realizing epigenetic breeding of field chrysanthemum flower colors.

[0021] This invention is the first to use epigenetic modification editing technology combined with endogenous DNA methyltransferase to modify the epigenetic alleles of different flower colors of chrysanthemum CmMYB6 The attempt achieved an important breakthrough in epigenetic breeding of flower color. 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

[0022] Figure 1 for CmCMT2 Agarose gel electrophoresis identification result of PCR product of gene; where M is MakerDL5000;

[0023] Figure 2 for CmCMT2 Genes and CmCMT2cd Functional domain analysis results of genes; Dcm is site-specific DNA methyltransferase, BAH is BAH domain, and Chromo is chromatin structure modification domain;

[0024] Figure 3 for CmMYB6 Schematic diagram of gene structure;

[0025] Figure 4 for dCas9- Schematic diagram of the DNA methyltransferase vector structure;

[0026] Figure 5 For WT and dCas9-CmCMT2 Transgenic lines CmMYB6 McrBC-qPCR analysis results of promoter region methylation level; data are the mean ± SEM of three biological replicates, ** P <0.01,*** P <0.001, ns means no significant difference;

[0027] Figure 6 For WT and dCas9-CmCMT2cd Transgenic lines CmMYB6 McrBC-qPCR analysis results of promoter region methylation level; data are the mean ± SEM of three biological replicates, * P <0.05,***P <0.001, ns means no significant difference;

[0028] Figure 7 This is a statistical analysis result of the methylation degree of CmCMT2 and CmCMT2cd by bisulfite sequencing; Figure 7 A in the equation is WT and dCas9-CmCMT2 Results of DNA methylation analysis of CG, CHG and CHH types in transgenic lines. Figure 7 B in the equation is WT and dCas9-CmCMT2cd Results of DNA methylation analysis of CG, CHG and CHH types in transgenic lines; data are the mean ± SEM of three biological replicates, *** P <0.001, ns means no significant difference;

[0029] Figure 8 To analyze the effect of CmCMT2 on the color and function of chrysanthemum flowers by transiently transfecting chrysanthemum buds with the dipping method. CmMYB6 The results of the influence of gene expression level and promoter methylation level; Figure 8 A in the figure represents PCR amplification Hyg , dCas9 , sgRNAs identification dCas9-CmCMT2 The result diagram of transgenic flower buds, M is Marker, - is negative control, + is positive control, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 are flower buds, Figure 8 B in the figure is for areas 1-6 dCas9-CmCMT2 Flower color diagram of 'Pink Double Butterfly' flower buds after the carrier was vacuum infiltrated. The scale bar is 1 cm. Figure 8 C in the equation is WT and dCas9-CmCMT2 Transgenic lines CmMYB6 Analysis results of gene expression levels. Figure 8 D in the equation is WT and dCas9-CmCMT2 Transgenic lines CmMYB6 Analysis results of promoter region methylation level; Figure 8 C and Figure 8 The data in D are the mean ± SEM of three biological replicates, * P <0.05,** P <0.01,*** P <0.001;

[0030] Fig. 9 To analyze the effect of CmCMT2cd on the color and growth of chrysanthemum flowers by transiently transfecting chrysanthemum buds with the dip method. CmMYB6 The results of the influence of gene expression level and promoter methylation level; Fig. 9 A in the figure represents PCR amplification Hyg , dCas9 , sgRNAs identification dCas9-CmCMT2cd The result diagram of transgenic flower buds, M is Marker, - is negative control, + is positive control, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 are flower buds, Fig. 9 B in the figure is for areas 2-5 dCas9-CmCMT2 Flower color diagram of 'Pink Double Butterfly' flower buds after the carrier was vacuum infiltrated. The scale bar is 1 cm. Fig. 9 C in the equation is WT and dCas9-CmCMT2cd Transgenic lines CmMYB6 Analysis results of gene expression levels. Fig. 9 D in the equation is WT and dCas9-CmCMT2cd Transgenic lines CmMYB6 Analysis results of promoter region methylation level; Fig. 9 C and Fig. 9 Data in D are the mean ± SEM of three biological replicates, *** P <0.001, ns indicates no significant difference. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] Example 1: DNA methyltransferase modified by CHH of chrysanthemum CmCMT2 Gene cloning

[0034] According to the filtered CmCMT2 The CDS sequence and mRNA sequence of the gene were determined. 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. CmCMT2The gene cloning was performed according to the reaction system shown in Table 2 and 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.

[0035] We target CmMYB6 Screening for CHH modification in the gene promoter region to obtain the CMT2 Gene, PCR clone and obtain a CMT2 endogenous DNA methyltransferase ( Figure 1 ). CmCMT2 The nucleotide sequence of the gene is shown in SEQ ID NO.35, and the protein it encodes contains 912 amino acids, and the amino acid sequence is shown in SEQ ID NO.36. Based on the functional domain analysis of DNA methyltransferase, the full-length CmCMT2 gene was truncated to retain only the methyltransferase active domain, and the truncated protein CmCMT2cd ( Figure 2 ), the nucleotide sequence of the truncated protein is shown in SEQ ID NO.37, and the amino acid sequence is shown in SEQ ID NO.38.

[0036] Table 1 CmCMT2 Primer sequence information for gene cloning

[0037]

[0038] Table 2 PCR reaction system

[0039]

[0040] Table 3 PCR reaction conditions

[0041]

[0042] Example 2: Construction of dCas9-DNA methyltransferase vector

[0043] In order to verify the methyltransferase activity of CmCMT2 and CmCMT2cd, the present invention fused CmCMT2 with dCas9 and targeted CmMYB6 Six sgRNAs sites were designed in the promoter region (-2655 to -1 bp) Figure 3 ), according to the dCas9-DNA methyltransferase vector structure diagram, was successfully constructed dCas9-CmCMT2 Vector ( Figure 4 Then the present invention fuses CmCMT2cd with dCas9 and targets CmMYB6 Three sgRNAs (sgRNA2, sgRNA3, and sgRNA4) were designed in the promoter region (-1989 to -1465 bp) ( Figure 3), successfully built dCas9-CmCMT2cd ( Figure 4 ), the specific vector construction method is as follows:

[0044] (1) CmMYB6 Selection of target sites in gene promoter regions and design of sgRNA

[0045] First, the transposon prediction website CENSOR (https: / / www.girinst.org / censor / index.php) CmMYB6 For transposon prediction, the recognition of the target site by sgRNA depends on the recognition of the PAM sequence. Generally, we take the 20 bp 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).

[0046] Table 4 Design of sgRNA guide oligonucleotides

[0047]

[0048] (2) Construction of dCas9-DNA methyltransferase vector

[0049] ① Dimerization of sgRNA-guided oligonucleotides: Take 5 μL (10 μM) of the synthesized sgRNA-guided oligonucleotide sense chain and antisense chain in a PCR tube respectively, and set the PCR program as follows: 37°C, 5 min; cool from 95°C to 25°C, reducing the temperature by 0.2°C per second, to synthesize dimerized sgRNA1, sgRNA2, ... sgRNA6.

[0050] ② Take the laboratory stored 18T-AtU6 (Published in the following document: Li, Xueqi, et al. "Optimization of CRISPR-Cas9 system in Eustoma grandiflorum." Iscience 27.3(2024): 109053.) The bacterial solution of the cloning vector was shaken (Amp + , 50 μg·mL -1 ) to extract the plasmid, and then measure the concentration after the plasmid is extracted. Bbs I digestion 18T-AtU6The plasmid of the cloning vector and the enzyme digestion system are shown in Table 5. After enzyme digestion at 37℃ for 30min, the target DNA fragment was recovered and purified by 1% agarose gel electrophoresis. The recovered DNA needed to be tested for concentration and stored in a -40℃ refrigerator for later use.

[0051] Table 5 Bbs I restriction enzyme reaction system

[0052]

[0053] 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:

[0054] 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 500 μL of LB liquid culture medium without resistance to the clean bench, and culture it on a shaker at 37℃ and 200rpm for 1 hour; centrifuge at 4000 rpm 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.

[0055] 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.

[0056] Table 6 T4 ligase ligation reaction system

[0057]

[0058] Table 7 Primer sequence information for bacterial liquid PCR identification

[0059]

[0060] Table 8 Colony PCR reaction system

[0061]

[0062] ④ Single restriction enzyme digestion using BamH I p1300-dCas9The vector was used to design homologous recombination linkers with the cloned DNA methyltransferase gene sequence. The primer sequences are shown in Table 9. Then, the DNA methyltransferase was linked to the ClonExpress II One Step Cloning Kit by homologous recombination. Bm I site, construct CRISPR- dCas9 -methyltransferase fusion expression protein, the homologous recombination reaction system is shown in Table 10.

[0063] Table 9 Homologous recombination primer sequence information

[0064]

[0065] Table 10 Homologous recombination reaction system

[0066]

[0067] 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℃.

[0068] ⑤Use X I single restriction enzyme digestion p1300-dCas9-CmCMT2 and p1300-dCas9-CmCMT2cd , 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 X I linearization dCas9 -DNA methyltransferase vector to generate CRISPR- dCas9 -sgRNA-DNA methyltransferase ( dCas9 -DNA methyltransferase) vector.

[0069] dCas9-CmCMT2 The vector is designed to target CmMYB6 sgRNA1, sgRNA2, ..., sgRNA6 sites in the promoter, and dCas9-CmCMT2cd It is designed to target sgRNA2, sgRNA3, and sgRNA4 sites.

[0070] Example 3: Analysis of CmCMT2 and CmCMT2cd methyltransferase activities in transgenic chrysanthemum protoplasts

[0071] (1) Establishment of a protoplast isolation system from leaves of Chrysanthemum vulgare

[0072] Based on the Arabidopsis protoplast isolation method, the protoplast isolation technology of Echeveria 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 1.0 M. Then 10 mL of CPW solution was added to dissolve them and incubated in a 55℃ water bath for 10 minutes. After cooling to room temperature, 100 μL CaCl2 (1 M) and 0.1% BSA were added. Then the lower epidermis of the leaves of Echeveria 'Pink Double Butterfly' was torn off and completely immersed in the enzyme solution, and then placed in a constant temperature shaker at 25℃ and 60 rpm for enzymatic hydrolysis for 7 hours. After the enzymatic hydrolysis was completed, it was filtered with 74 μm nylon cloth and the filtrate was centrifuged at 100 g for 5 minutes. 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 1.5 mL centrifuge tube, 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 min in the dark; and compared under natural light and fluorescence. Protoplasts that fluoresced indicated activity, while those that did not fluoresce indicated inactivity.

[0073] (2) Transient transformation of CRISPR / dCas9 vector in protoplasts of Cyperus rotundus

[0074] 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 Take a 2 mL centrifuge tube and add 110 μL of protoplast suspension, 10 μL of 1 μg / μL plasmid DNA (obtained in Example 2) dCas9 -DNA methyltransferase vector) and 120 μL 40% PEG, gently mixed, and induced in a 28°C constant temperature incubator for 15 min; then added 440 μL WS, gently inverted upside down to mix, centrifuged at 100 g for 2 min, and collected protoplasts; finally, added 1 mL WI solution to each protoplast, inverted upside down to mix gently, and transferred the mixture to a culture dish, incubated at 20°C in the dark for more than 48 h, and then observed using a fluorescence microscope and a laser scanning confocal microscope.

[0075] (3) Analysis of CmCMT2 and CmCMT2cd methyltransferase activity

[0076] Using the PEG-mediated method, each dCas9-DNA methyltransferase vector plasmids were respectively transferred into the protoplasts of 'Pink Double Butterfly'. After incubation for 60 h, McrBC-qPCR method was used to detect the expression of CmMYB6 Methylation levels in the promoter target site region. CmMYB6 The specific detection method of McrBC-qPCR in the promoter region is as follows:

[0077] 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 1 h and inactivated at 65°C for 20 min. After treatment, the sample was placed at -40°C.

[0078] Table 11 McrBC enzyme digestion reaction system

[0079]

[0080] 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.

[0081] Table 12 McrBC-qPCR detection primer sequence information

[0082]

[0083] Table 13 McrBC-qPCR reaction system

[0084]

[0085] Table 14 McrBC-qPCR reaction conditions

[0086]

[0087] 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.

[0088] The results of McrBC-qPCR confirmed that CmCMT2 is functional and can improve CmMYB6 The methylation level of promoter 1-6 region ( Figure 5 ), indicating that CmCMT2 exhibits methyltransferase activity at specific targets. In order to further verify the catalytic activity of CmCMT2, the present invention constructed a dCas9 Carrier, i.e. dCas9- CmCMT2cd (where cd stands for catalytic domain). Using the protoplast transient transformation method and McrBC-qPCR detection, the experimental results showed that CmCMT2cd can increase the expression of CmCMT2cd near the specific target site. CmMYB6 Promoter region methylation level ( Figure 6 ). The above experimental results show that the catalytic domain of DNA methyltransferase is the key domain that dominates its function.

[0089] Example 4: Statistical analysis of methylation levels of CmCMT2 and CmCMT2cd by bisulfite sequencing

[0090] In order to investigate the role of CmCMT2 and CmCMT2cd in CmMYB6 The types of DNA methylation catalyzed by the promoter region (-1832 to -1327 bp, region 3-5) Figure 3 ), the present invention uses bisulfite sequencing method for analysis. The results show that there is 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 is significantly higher than that in the control group. This result further shows that CmCMT2 can catalyze CHH type DNA methylation ( Figure 7 ).

[0091] Example 5: Analysis of the effects of CmCMT2 and CmCMT2cd on the color and color of chrysanthemums by transient transfection of chrysanthemum buds by the flower dipping method CmMYB6 Effects of gene expression levels and promoter methylation levels

[0092] Using Example 2 dCas9 -DNA methyltransferase vector was used to transform Agrobacterium GV3101, and 50 μL of the transformed Agrobacterium culture solution was inoculated into 5 mL LB liquid medium (25 μg / L rif + , 50 μg / L kana + ), incubated at 28°C, 180 rpm for 24 h; then dCas9-CmCMT2 The corresponding 6 sgRNA Agrobacterium bacterial solutions were mixed. dCas9- CmCMT2cdThe corresponding Agrobacterium bacterial cultures of sgRNA2, sgRNA3, and sgRNA4 were mixed, and 1 mL of each sgRNA strain corresponding to each enzyme was added to 200 mL LB liquid culture medium (25 μg / L rif + , 50 μg / L kana + ), cultured at 28°C and 180 rpm for 24 h, and then tested for concentration. 600nm The value is about 1.5-2.0, and then the bacterial liquid is collected by centrifugation at 4000 rpm for 5 min. The Agrobacterium bacterial liquid is resuspended with 5% (W / V) sucrose solution in a ratio of 1:1, and 0.02% SilwetL-77 is added; the unopened flower buds of 'Pink Double Butterfly' are selected, and the top of the flower buds are gently opened with tweezers to facilitate Agrobacterium infection, and the flower buds of 'Pink Double Butterfly' are immersed in the bacterial liquid, and then the vacuum infiltration method is used 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; the flower buds of the chrysanthemum 'Pink Double Butterfly' that have completed the dipping are wrapped with plastic wrap, covered with a black bag, and cultured in the culture chamber for 16-24 hours; after the dark culture is completed, the plastic wrap is removed and normal light is restored. After the flower buds open, the color changes of the petals are continuously observed, and the number of open flower buds and the number of flower buds that have changed color are recorded.

[0093] CmCMT2 methyltransferase methylation CmMYB6 After promoter region 1-6, use dCas9-CmCMT2 The Agrobacterium containing the vector was used to infect the flower buds of the chrysanthemum 'Pink Double Butterfly', 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 CmCMT2 changed the flower color to pink or dark pink ( Figure 8 RT-qPCR and McrBC-qPCR were used to detect WT and dCas9-CmCMT2 Transgenic lines CmMYB6 Gene expression levels and CmMYB6 The methylation level of the promoter region was found to be CmMYB6 Increased gene expression ( Figure 8 C in the CmMYB6 The methylation level of promoter region 1-6 increased ( Figure 8 D in.

[0094] Then, the present invention retains only the active domain of the methyltransferase and fuses it with dCas9 to target the modification region 2-5. dCas9-CmCMT2cdThe flower buds of chrysanthemum 'Pink Double Butterfly' were infected with Agrobacterium carrying the vector, and the Hyg, dCas9 genes and sgRNAs were amplified by PCR, and the transformed flower buds were identified as transgenic flower buds ( Fig. 9 The results showed that CmCMT2cd changes the flower color from pink to light pink or white ( Fig. 9 B in the CmMYB6 The expression level of Fig. 9 C in the CmMYB6 The DNA methylation level in the promoter region 3-5 was significantly increased ( Fig. 9 D in.

[0095] 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 CmCMT2 The application of the method in controlling the color of field chrysanthemums is characterized in that: CmCMT2 The nucleotide sequence of the gene is shown in SEQ ID NO.

35.

2. The use according to claim 1, characterized in that: CmCMT2 The gene encodes the amino acid sequence shown in SEQ ID NO.

36.

3. Chrysanthemum methyltransferase gene CmCMT2cd The application of the method in controlling the color of field chrysanthemums is characterized in that: CmCMT2cd The nucleotide sequence of the gene is shown in SEQ ID NO.

37.

4. The use according to claim 3, characterized in that: CmCMT2cd The gene encodes the amino acid sequence shown in SEQ ID NO.

38.

5. A method for changing the color of field chrysanthemums to dark pink, characterized in that: Utilizing the method containing the chrysanthemum methyltransferase gene of claim 1 CmCMT2 CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmCMT2 , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmCMT2 transformed into the genome of the chrysanthemum to obtain transgenic 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.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

6. The method according to claim 5, characterized in that Agrobacterium-mediated genetic transformation was used to introduce CRISPR- dCas9 -sgRNA- CmCMT2 The transformation into the genome of Chrysanthemum chrysanthemum is specifically carried out by transiently transfecting the Chrysanthemum buds through the floral dip method.

7. 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 CmCMT2cd CRISPR / dCas9 gene editing vector targeting CmMYB6 Specific sgRNA sites in the promoter to obtain CRISPR- dCas9 -sgRNA- CmCMT2cd , and then used Agrobacterium-mediated genetic transformation to transform CRISPR- dCas9 -sgRNA- CmCMT2cd The sgRNA is transformed into the genome of the chrysanthemum to obtain transgenic 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.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

8. The method according to claim 7, characterized in that Agrobacterium-mediated genetic transformation was used to introduce CRISPR- dCas9 -sgRNA- CmCMT2cd The transformation into the genome of Chrysanthemum chrysanthemum is specifically carried out by transiently transfecting the Chrysanthemum buds through the floral dip method.

9. A preparation for changing the color of field chrysanthemums to dark pink, characterized in that: The active ingredients of the preparation include CRISPR- dCas9 -sgRNA- CmCMT2 , CRISPR- dCas9 -sgRNA- CmCMT2 The method comprises the methyltransferase gene of claim 1. CmCMT2 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.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

10. A preparation for changing the color of field chrysanthemums to light pink or white, characterized in that: The active ingredients of the preparation include CRISPR- dCas9 -sgRNA- CmCMT2cd , CRISPR- dCas9 -sgRNA- CmCMT2cd The method comprises the methyltransferase gene of claim 3. CmCMT2cd 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.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

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