Method for rapidly detecting editing efficiency of plant DNA methylation appearance editor

Abstract

The invention discloses a method for rapidly detecting the editing efficiency of a plant DNA methylation appearance editor, and belongs to the technical field of molecular biology and genetic engineering. According to the method, a transgenic plant cell of a tdTomato fluorescence reporter gene driven by an FIE1 promoter and a transgenic plant cell of a tdTomato fluorescence reporter gene driven by a 35S promoter are constructed, the FIE1 promoter region has a relatively high DNA methylation level, and the 35S promoter region has a relatively low DNA methylation level. The method comprises the following steps: introducing a to-be-detected DNA methylation appearance removing editor into a plant cell of a fluorescence reporter gene driven by an FIE1 promoter, introducing a to-be-detected DNA methylation appearance adding editor into a plant cell of a fluorescence reporter gene driven by a 35S promoter, and evaluating the editing efficiency of the DNA methylation appearance editor by detecting the intensity change of a fluorescence signal. The method is easy and convenient to operate, the result is visual, the editing efficiency of the DNA methylation editor can be efficiently and rapidly identified, and powerful support is provided for development and application of the appearance editor.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and genetic engineering technology, and specifically relates to a method for rapidly detecting the editing efficiency of plant DNA methylation epigenetic editors. Background Technology

[0002] Epigenetics, as a regulatory mechanism that can be stably inherited without alteration of DNA sequence, plays a crucial role in plant growth and development, environmental adaptation, and the formation of agronomic traits. Among these, DNA methylation is one of the most widely studied and functionally well-defined epigenetic modifications. Its dynamic changes in the promoter region can directly regulate gene transcriptional activity, thereby affecting plant phenotype. Numerous studies have shown that many key agronomic traits in crops, such as plant architecture, disease resistance, and reproductive development, are finely regulated by DNA methylation status. For example, the expression levels of genes such as RAV6, PigmS, and IPA1 in rice are closely related to the methylation status of their upstream regulatory regions, while hypomethylation of the FIE1 gene promoter region can lead to its ectopic expression, causing dwarfism and sterility. Importantly, certain DNA methylation variations in plants not only have a high spontaneous mutation rate but can also be stably inherited in offspring without transgenic residues. This provides a novel strategic approach for targeted crop improvement through artificial intervention in epigenetic states.

[0003] In recent years, with the maturation of the CRISPR-Cas9 technology system, epigenetic editing tools built on this platform have gradually become a research hotspot. By fusing catalytically inactivated Cas9 proteins with DNA methyltransferase or demethylase domains, researchers have successfully achieved targeted methylation or demethylation of specific genomic sites in Arabidopsis and rice. For example, Gallego-Bartolomé et al. first achieved targeted demethylation of the FWA site in Arabidopsis using the dCas9-TET1 system, inducing a heritable late-flowering phenotype; Papikian et al. constructed a targeted methylation tool using dCas9-DRM. Our laboratory has also pioneered the establishment of an epigenetic editing system for targeted removal of FIE1 promoter methylation in rice, and confirmed that the obtained hypomethylated state and its related phenotypes can be stably maintained in transgenic segregating progeny, fully validating the application potential of epigenetic editing in crop breeding. However, despite the increasingly diverse construction strategies of epigenetic editors, their practical application still faces a fundamental bottleneck: the lack of a technical means to quickly, efficiently, and intuitively evaluate their editing efficiency.

[0004] The root cause lies in the fact that existing methods for detecting DNA methylation status generally suffer from problems such as long processing times, cumbersome operations, low throughput, or insufficient sensitivity. While traditional bisulfite sequencing can provide methylation information at single-base resolution, its process involves DNA degradation, multiple rounds of PCR amplification, and cloning sequencing, taking several days to weeks, making it difficult to meet the needs of high-throughput screening or real-time evaluation. Techniques based on methylation-sensitive restriction endonucleases or methylated DNA immunoprecipitation are limited by site specificity or insufficient resolution, failing to accurately reflect the overall methylation dynamics of the target region. More importantly, none of these methods can establish a direct, visual correlation between changes in methylation status and gene expression levels, making it difficult to quickly assess the editor's functionality in the early stages. This deficiency is particularly prominent in the optimization of epigenetic editors—since different sgRNA designs, fusion protein conformations, or delivery methods can significantly affect editing efficiency, the inability to obtain reliable efficiency feedback in a short time will greatly delay tool iteration and application.

[0005] Against this backdrop, there is an urgent need for a detection system that can directly couple DNA methylation status with reporter gene expression intensity. Ideally, this system should possess the following characteristics: first, reporter gene expression should be strictly regulated by the methylation status of the target promoter; second, changes in methylation levels should be rapidly converted into quantifiable and visualized signal outputs; and third, the entire detection process should be completed at the cellular or tissue level, avoiding cumbersome DNA extraction and processing steps. It is worth noting that although the 35S promoter is a strongly constitutive promoter, it is susceptible to transcriptional silencing mediated by DNA methylation, while the OsFIE1 promoter is naturally in a hypermethylation-repressed state. These two represent typical regulatory modes of "being silenced by methylation" and "being activated by demethylation," respectively. If these two promoters can drive fluorescent reporter genes separately and be combined with a protoplast transient transformation system, it is hoped that a dual-modal detection platform can be constructed that can detect both methylation addition efficiency and demethylation effects. However, ensuring that the strength changes of the reported signals in the platform accurately and specifically reflect the methylation editing efficiency of the target promoter, rather than being affected by other epigenetic or transcriptional interference factors, remains a core challenge in the technical implementation.

[0006] Therefore, how to construct a rapid detection method based on endogenous promoter methylation sensitivity that can intuitively reflect the editing efficiency of DNA methylation epigenetic editors and achieve efficient parallel evaluation of targeted methylation and demethylation tools has become a key challenge and a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a rapid and efficient method for detecting the editing efficiency of DNA methylation epigenetic editors. The method is based on a rice protoplast transient transformation system, combining two promoters with different methylation sensitivity characteristics to drive the same fluorescent reporter gene, constructing a dual-modality detection platform to achieve synchronous, intuitive, and efficient evaluation of the efficiency of targeted methylation addition and demethylation editing.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a method for rapidly detecting the editing efficiency of plant DNA methylation epigenetic editors, comprising the following steps: S1. Provide transgenic plant cells containing a fluorescent reporter gene driven by a first promoter and transgenic plant cells containing a fluorescent reporter gene driven by a second promoter, wherein the DNA methylation level of the first promoter in the transgenic plant is relatively higher than that of the second promoter. S2. The DNA methylation removal epigenetic editor to be tested is imported into the transgenic plant cells of the fluorescent reporter gene driven by the first promoter, and the DNA methylation addition epigenetic editor to be tested is imported into the transgenic plant cells of the fluorescent reporter gene driven by the second promoter. S3. Culture the transformed plant cells and evaluate the editing efficiency of the DNA methylation epigenetic editor by detecting changes in fluorescence signal intensity.

[0009] Furthermore, the first promoter is rice. OsFIE1 The first promoter is a gene promoter, the nucleotide sequence of which is shown in SEQ ID NO.1; the second promoter is the tobacco mosaic virus (CaMV) 35S promoter, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] Furthermore, the fluorescent reporter gene is the tdTomato fluorescent protein gene, the nucleotide sequence of which is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0011] Furthermore, in step S2, the DNA methylation removal epigenetic editor is for targeted removal. FIE1 An epigenetic modification editor for gene promoter DNA methylation.

[0012] Furthermore, the targeted removal FIE1 The expression vector for the epigenetic modification editor of gene promoter DNA methylation was constructed using the following method: Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (nucleotide sequence shown in SEQ ID NO.15) and the reverse primer FIE1-sgRNA-R (nucleotide sequence shown in SEQ ID NO.16); the second fragment was amplified using the forward primer FIE1-sgRNA-F (nucleotide sequence shown in SEQ ID NO.17) and the reverse primer CP9789 (nucleotide sequence shown in SEQ ID NO.18); the amplified first and second fragments were ligated into the Zmubi-Zmubi-Suntag-14aa vector to obtain targeted removal FIE1 Expression vectors for epigenetic modification editors of promoter DNA methylation.

[0013] Furthermore, in step S2, the DNA methylation addition epigenetic editor is an epigenetic modification editor that targets and adds 35S promoter DNA methylation.

[0014] Furthermore, the expression vector for the epigenetic modification editor that targets and adds 35S promoter DNA methylation is constructed using the following method: Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (nucleotide sequence shown in SEQ ID NO.15) and the reverse primer 35S-sgRNA-R (nucleotide sequence shown in SEQ ID NO.19); the second fragment was amplified using the forward primer 35S-sgRNA-F (nucleotide sequence shown in SEQ ID NO.20) and the reverse primer XP073 (nucleotide sequence shown in SEQ ID NO.21); the amplified first and second fragments were ligated into the Zmubi-SunTag14aa-DRM2 vector to obtain an expression vector for an epigenetic modification editor that targets the addition of 35S promoter DNA methylation.

[0015] Furthermore, in step S3, when the fluorescence signal intensity of the fluorescent reporter gene driven by the first promoter increases, it indicates that the DNA methylation removal epigenetic editor has high editing efficiency; when the fluorescence signal intensity of the fluorescent reporter gene driven by the second promoter decreases, it indicates that the DNA methylation addition epigenetic editor has high editing efficiency.

[0016] Furthermore, the plant cells are rice protoplasts. Beneficial effects

[0017] This invention provides a method for efficiently and rapidly identifying the editing efficiency of DNA methylation editors, allowing for intuitive judgment of editing effects without complex sequencing analysis; it establishes a DNA methylation editing efficiency detection platform suitable for rice systems, providing technical support for the application of epigenetic editors in rice; and it also provides new tools and methods for DNA methylation epigenetics research. Attached Figure Description

[0018] Picture 1 for OsFIE1:tdTomato (a) and 35S:tdTomato (b) Schematic diagram of the carrier Picture 2 For transformation OsFIE1:tdTomato and 35S:tdTomato A schematic diagram of DNA methylation detection results for the strain.

[0019] Picture 3 The results of red fluorescence observation for the protoplast-transformed DNA methylation epieditor. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0021] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., 3rd edition, 2001, NY, Cold Spring Harbor).

[0022] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials in this invention. In fact, the sources of the biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted according to the instructions in the embodiments. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0023] The nucleotides involved in the following examples can all be synthesized using existing technologies.

[0024] Example 1: OsFIE1:tdTomato and 35S:tdTomato Carrier construction In this embodiment OsFIE1:tdTomato and 35S:tdTomatoThe vector was modified from the pBI101-tdTomato vector.

[0025] Specifically, the forward primer CP9993 (SEQ ID NO.7) and the reverse primer CP9995 (SEQ ID NO.8) were used to fertilize rice ( Oryza Sativa L.) Genomic DNA was used as a template for amplification. FIE1 The promoter sequence was ligated to the BamHI (NEB, R3136V) and SalI (NEB, R3138V) restriction sites of the pBI101-tdTomato vector to obtain a new vector, which was named [name missing]. OsFIE1:tdTomato The schematic diagram of the vector's plasmid structure is shown below. Picture 1 As shown in Figure a. Using forward primer CP9996 (SEQ ID NO. 9) and reverse primer CP9997 (SEQ ID NO. 10) as a template, the 35S promoter sequence of tobacco mosaic virus was amplified. This sequence was then ligated to the BamHI (NEB, R3136V) and SalI (NEB, R3138V) restriction sites of the pBI101-tdTomato vector to obtain a new vector, named 35S:tdTomato. A schematic diagram of the vector's plasmid structure is shown in Figure a. Picture 1 As shown in b.

[0026] The PCR system for vector construction consisted of: 10 ng plasmid template or 100 ng rice genomic DNA, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix (10 mMeach), 1 μL PhantaMax Super-Fidelity DNA Polymerase (1 unit / μL) (Nanjing Novizan Biotechnology Co., Ltd., P505-d1), and ddH2O to a final volume of 50 μL. The PCR reaction program was as follows: 94℃ for 2 min; (94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 32 cycles; 72℃ for 10 min, and then held at 4℃.

[0027] The enzyme digestion system was as follows: 5 μL of 10×CutSmart Buffer, 1 μL of each restriction endonuclease (BamHI, 20 units / μL, SalI, 20 units / μL), 1 μg of the vector to be digested, and ddH2O to bring the total volume to 50 μL. Digestion was carried out at 37°C for at least 8 hours.

[0028] Example 2: Detection of DNA methylation levels in transgenic lines Using the system constructed in Example 1 OsFIE1:tdTomato and 35S:tdTomato The transformation of the rice variety Kitaake was performed using Agrobacterium-mediated transformation and screening, following previously reported methods (Hiei, Y. et al. Efficient transformation of rice). Oryza sativa L.) Genomic DNA was extracted from transgenic T2 generation rice using the CTAB (Hexadecyl trimethylammonium bromide) method and dissolved in 100 μl ddH2O. (1994) [The results were mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6,271-282, doi:10.1046 / j.1365-313x.1994.6020271.x (1994)).

[0029] Bisulfite treatment deamination of unmethylated cytosine in DNA converts it to uracil, while methylated cytosine remains unchanged. Following PCR amplification, all uracil is converted to thymine. Finally, the PCR products are Sanger sequenced and compared with the untreated sequence to determine whether cytosine methylation has occurred.

[0030] We took 500 ng DNA from each of two independent transgenic lines (F-1, F-2 and 35S-1, 35S-2) of each vector and treated them with bisulfite, following the instructions of the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH, D5005). The final product was dissolved in 15 μL of ddH2O. OsFIE1:tdTomato Transgenic lines were amplified by PCR using forward primer XP2769 (SEQ ID NO.11) and reverse primer XP2770 (SEQ ID NO.12). For the 35S:tdTomato transgenic line, PCR amplification was performed using forward primer XP2771 (SEQ ID NO.13) and reverse primer XP2770 (SEQ ID NO.12).

[0031] PCR reaction system: 30 ng of bisulfite-treated genomic DNA, 25 μL of 2×PCR buffer for KOD-Multi&Epi-, 1.5 μL of forward primer (10 μM), 1.5 μL of reverse primer (10 μM), 1 μL of KOD-Multi&Epi-(KME-101, TOYOBO), and ddH2O to a final volume of 50 μL. PCR reaction program: 94℃ for 2 min; (98℃ for 10 s, 55℃ for 30 s, 68℃ for 30 s) for 40 cycles; 68℃ for 10 min, then hold at 4℃.

[0032] After separation of the PCR products using a 1% agarose gel, the PCR amplified bands were recovered using the Zymoclean Gel DNA Recovery Kit (ZYMORESEARCH, D4008) and dissolved in 15 μl ddH2O. 50 ng of the recovered product was ligated to 1 μL of the pEASY-Blunt Cloning vector using the pEASY-Blunt Cloning Kit (Beijing TransGen Biotech Co., Ltd., CB101-01).

[0033] The ligation method was performed according to the instruction manual. The ligation product was transformed into *E. coli* DH5α and cultured overnight at 37°C on kanamycin-resistant plates. Ten single colonies were picked and cultured in liquid medium containing kanamycin until OD500. 600 =2.0, sequencing was performed using the M13R primer (SEQ ID NO.14) on the vector. The sequencing results were analyzed using the web-based Kismeth software (http: / / katahdin.mssm.edu / kismeth / revpage.pl), and the changes in methylation levels after removing identical clones were statistically analyzed.

[0034] The results are as follows Picture 2 As shown, each row represents a sequenced single clone, with solid circles representing methylated cytosine (C) and hollow circles representing unmethylated C. The results indicate that transformation... OsFIE1:tdTomato The FIE1 transgenic line has a high level of DNA methylation in its promoter region, while the transformed... 35S:tdTomato The transgenic lines exhibited low DNA methylation levels in the 35S promoter region. This result indicates that... OsFIE1:tdTomato and 35S:tdTomato Two independent transgenic lines selected from two transgenic vectors can be used to detect the editing efficiency of epigenetic editors targeting DNA methylation removal and DNA methylation addition, respectively.

[0035] Example 3: Construction of a DNA methylation epigenetic editor In this embodiment, the vector for the epigenetic modification editor that targets the addition of DNA methylation was modified from the vector Zmubi-SunTag14aa-DRM2. In this embodiment, the vector for the epigenetic modification editor that targets the removal of DNA methylation was modified from the vector Zmubi-Suntag-14aa.

[0036] Specifically, OsFIE1 The promoter sequence (SEQ ID NO.1) was submitted to the online website https: / / crispr.dbcls.jp / for target design. A suitable target sequence (SEQ ID NO.5) with a low off-target probability and moderate GC content was selected. Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (SEQ ID NO.15) and the reverse primer FIE1-sgRNA-R (SEQ ID NO:19), and the second fragment was amplified using the forward primer FIE1-sgRNA-F (SEQ ID NO:20) and the reverse primer CP9789 (SEQ ID NO:21). The two fragments were then ligated to the PmeI (NEB, R0560V) restriction site of Zmubi-Zmubi-Suntag-14aa via homologous recombination to construct an expression vector for an epigenetic modification editor that targets and removes DNA methylation from the FIE1 promoter.

[0037] Will 35S The promoter sequence (SEQ ID NO.2) was submitted to the online website https: / / crispr.dbcls.jp / for target design. A suitable target sequence (SEQ ID NO.6) with a low off-target probability and appropriate GC content was selected. Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (SEQ ID NO.15) and the reverse primer 35S-sgRNA-R (SEQ ID NO.19), and the second fragment was amplified using the forward primer 35S-sgRNA-F (SEQ ID NO.20) and the reverse primer XP0730 (SEQ ID NO.21). The two fragments were then ligated to the PmeI restriction site of Zmubi-SunTag14aa-DRM2 via homologous recombination to construct an expression vector targeting the methylation of DNA by the 35S promoter.

[0038] The PCR system for vector construction consisted of: 10 ng plasmid template, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix (10 mM each), 1 μL PhantaMax Super-Fidelity DNA Polymerase (1 unit / μL) (Nanjing Novizan Biotechnology Co., Ltd., P505-d1), and ddH2O to a final volume of 50 μL. The PCR reaction program was as follows: 94℃ for 2 min; (94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 32 cycles; 72℃ for 10 min, and then held at 4℃.

[0039] The enzyme digestion system was as follows: 5 μL of 10×CutSmart Buffer, 1 μL of each restriction endonuclease (PmeI, 10 units / μL), 1 μg of the vector to be digested, and ddH2O to bring the total volume to 50 μL. Digestion was carried out at 37°C for at least 8 hours.

[0040] Example 4: Transformation of rice protoplasts using a DNA methylation epigenetic editor Transformation at two weeks old OsFIE1:tdTomato and 35S:tdTomato The rice seedlings were cut into 1 mm pieces with a blade and placed in a petri dish containing 0.6 M mannitol. The enzymatic hydrolysate was prepared by adding 0.3 g of Cellulase R-10 (Yakult Pharmaceutical Industry Co., Ltd. Code.No: L0012), 0.15 g of Macerozyme R-10 (Yakult Pharmaceutical Industry Co., Ltd. Code.No: L0021), 2.1844 g of Mannitol, and 0.04264 g of MES (sodium methyl ester sulfonate of fatty acids, Sigma-Aldrich Code.No: M2933) to a final volume of 20 mL. The pH of the solution was adjusted to 5.8 with KOH and the solution was incubated in a 55 °C water bath for 10 min. 0.02 g of CaCl2 and 0.02 g of BSA (bovine serum albumin) were then added.

[0041] Transfer rice seedling segments from the petri dish to the enzymatic hydrolysate and enzymatically hydrolyze at 28℃, 50 rpm, and in the dark for about 3 hours. Add 1 / 2 volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 4 mM MES, pH 5.7) to a small beaker to dilute the enzymatic hydrolysate containing protoplasts. Rinse a 100-mesh filter with W5 solution and filter the enzymatic hydrolysate to remove undissolved leaves (handle gently). Rinse the leaves in the filter with 1 / 2 volume of W5 solution and collect the filtrate.

[0042] Aliquot the filtrate into 50 mL centrifuge tubes, centrifuge at 100-200 g (acceleration set to 0 or 1) for 5 min to precipitate protoplasts, remove as much supernatant as possible, gently resuspend the protoplasts in pre-cooled W5 solution on ice, and let stand on ice for 30 min.

[0043] Centrifuge at 100-200 g for 5 min at room temperature (acceleration set to 0 or 1) to allow protoplasts to precipitate at the bottom of the tube. Remove as much W5 solution as possible without losing the protoplasts. Then resuspend the protoplasts in an appropriate amount of MMG solution (4 mL of 1 M Mannitol, 0.15 mL of 1 M MgCl2, and 0.2 mL of 200 mM MES).

[0044] Take a new 2.0 mL centrifuge tube and add 20 μg of the successfully constructed FIE1-targeted DNA demethylation vector and 35S-targeted DNA methylation-adding vector. Add 100 μL of OsFIE1:tdTomato and 35S:tdTomato protoplasts (obtained in step (4.3)) respectively, and mix gently.

[0045] Add 100 μL of PEG4000 solution, gently invert the centrifuge tube to mix completely; induce transformation at room temperature in the dark for 30 min.

[0046] The conversion reaction was terminated at room temperature with 1 mL of W5 solution. Then, the centrifuge tube was gently inverted and shaken to mix the contents and terminate the conversion reaction. The tube was centrifuged at 100 g for 5 min at room temperature, the supernatant was removed, and the reaction was repeated once.

[0047] The protoplasts were gently resuspended in 200 μL of W5 solution and cultured at 28 °C for 48 h in the dark. The intensity of the red fluorescence signal of the protoplasts was observed using a fluorescence microscope (531 nm). The results are as follows: Picture 3 As shown, the results indicate that... OsFIE1:tdTomatoThe protoplasts without plasmid exhibited a weak red fluorescence signal, while the fluorescence signal significantly increased after the addition of the epigenetic editing tool targeting and removing FIE1 promoter DNA methylation, indicating that the system has high editing efficiency for targeting and removing DNA methylation. 35S:tdTomato The system exhibits a strong red fluorescence signal in protoplasts without plasmid addition, but the fluorescence signal significantly weakens after the addition of the targeted 35S promoter DNA methylation epiediting tool, indicating that the system has a high editing efficiency for targeted DNA methylation addition.

[0048] In summary, this invention utilizes the rice protoplast system to efficiently and rapidly identify the editing efficiency of DNA methylation editors, providing strong support for the future development of epigenetic editors.

Claims

1. A method for rapidly detecting the editing efficiency of a plant DNA methylation epigenetic editor, characterized in that, Includes the following steps: S1. Provide transgenic plant cells containing a fluorescent reporter gene driven by a first promoter and transgenic plant cells containing a fluorescent reporter gene driven by a second promoter, wherein the DNA methylation level of the first promoter in the transgenic plant is relatively higher than that of the second promoter. S2. The DNA methylation removal epigenetic editor to be tested is imported into the transgenic plant cells of the fluorescent reporter gene driven by the first promoter, and the DNA methylation addition epigenetic editor to be tested is imported into the transgenic plant cells of the fluorescent reporter gene driven by the second promoter. S3. Culture the transformed plant cells and evaluate the editing efficiency of the DNA methylation epigenetic editor by detecting changes in fluorescence signal intensity.

2. The method according to claim 1, characterized in that, The first promoter is rice OsFIE1 The first promoter is a gene promoter, the nucleotide sequence of which is shown in SEQ ID NO.1; the second promoter is the tobacco mosaic virus (CaMV) 35S promoter, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The fluorescent reporter gene is the tdTomato fluorescent protein gene, whose nucleotide sequence is shown in SEQ ID NO.3, and whose encoded protein amino acid sequence is shown in SEQ ID NO.

4.

4. The method according to claim 1, characterized in that, In step S2, the DNA methylation removal epigenetic editor is used for targeted removal. FIE1 An epigenetic modification editor for gene promoter DNA methylation.

5. The method according to claim 4, characterized in that, The targeted removal FIE1 The expression vector for the epigenetic modification editor of gene promoter DNA methylation was constructed using the following method: Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (nucleotide sequence shown in SEQ ID NO.15) and the reverse primer FIE1-sgRNA-R (nucleotide sequence shown in SEQ ID NO.16); the second fragment was amplified using the forward primer FIE1-sgRNA-F (nucleotide sequence shown in SEQ ID NO.17) and the reverse primer CP9789 (nucleotide sequence shown in SEQ ID NO.18); the amplified first and second fragments were ligated into the Zmubi-Zmubi-Suntag-14aa vector to obtain targeted removal FIE1 Expression vectors for epigenetic modification editors of promoter DNA methylation.

6. The method according to claim 1, characterized in that, In step S2, the DNA methylation addition epigenetic editor is an epigenetic modification editor that targets and adds 35S promoter DNA methylation.

7. The method according to claim 6, characterized in that, The expression vector for the epigenetic modification editor that targets 35S promoter DNA methylation was constructed using the following method: Using the OsU6-sgRNA plasmid as a template, the first fragment was amplified using the forward primer CP9788 (nucleotide sequence shown in SEQ ID NO.15) and the reverse primer 35S-sgRNA-R (nucleotide sequence shown in SEQ ID NO.19); the second fragment was amplified using the forward primer 35S-sgRNA-F (nucleotide sequence shown in SEQ ID NO.20) and the reverse primer XP073 (nucleotide sequence shown in SEQ ID NO.21); the amplified first and second fragments were ligated into the Zmubi-SunTag14aa-DRM2 vector to obtain an expression vector for an epigenetic modification editor that targets the addition of 35S promoter DNA methylation.

8. The method according to claim 1, characterized in that, In step S3, when the fluorescence signal intensity of the fluorescent reporter gene driven by the first promoter increases, it indicates that the DNA methylation removal epigenetic editor has high editing efficiency; when the fluorescence signal intensity of the fluorescent reporter gene driven by the second promoter decreases, it indicates that the DNA methylation addition epigenetic editor has high editing efficiency.

9. The method according to claim 1, characterized in that, The plant cells are rice protoplasts.

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