A gene for controlling chrysanthemum flowering and its use

Abstract

The application discloses a gene for controlling the flowering of Dendranthema grandiflorum and application thereof, and belongs to the technical field of controlling the flowering period of chrysanthemum.The application aims to provide a method for controlling the flowering period of Dendranthema grandiflorum.A gene for controlling the flowering of chrysanthemum, wherein the nucleic acid molecule of the gene is shown as SEQ ID NO.3.The application provides a more solid theoretical basis for controlling the flowering period of Dendranthema grandiflorum.

Description

Technical Field

[0001] This invention belongs to the field of chrysanthemum flowering period control technology, specifically relating to a gene for controlling the flowering of open-field chrysanthemums and its application. Background Technology

[0002] Chrysanthemums are short-day plants with a relatively concentrated flowering period. Currently, how to modify or regulate the flowering period has become a hot topic in chrysanthemum research. Flower induction is a crucial stage in the plant life cycle. In recent years, with in-depth research into plant flowering mechanisms, it has been found that epigenetic modifications play an important role in the plant flowering pathway. Targeted breeding using epigenetic alleles has wide applications in crop quality improvement. However, field chrysanthemums have high heterozygosity and a complex genetic background, making targeted breeding difficult using traditional methods.

[0003] Ground cover chrysanthemum is a perennial herbaceous plant belonging to the cultivated chrysanthemum category. The plant is small and sturdy, not only cold-resistant and drought-resistant, and resistant to pests and diseases, but also boasts vibrant colors and diverse flower shapes, blooming profusely even in open-air environments. As early as 1990, Wang Pengwei et al. bred the first batch of ground cover chrysanthemums. From 1992 to 1995, Liu Xiaodong et al. conducted cultivation experiments in four regions of Heilongjiang Province, creating new varieties suitable for local cultivation and capable of overwintering in open environments, simply referred to as 'ground cover chrysanthemum'. As a common ornamental plant, its unique beauty and cold resistance make it popular among horticulture enthusiasts and professional growers. Most ground cover chrysanthemums possess a complex hexaploid genome structure, resulting in a rich variety of strains with high value in both biological research and commercial applications. This genetic diversity provides endless possibilities for the genetic improvement and creation of new varieties of chrysanthemums, contributing to their increasingly important position in the global flower market.

[0004] Chrysanthemums can be categorized by their natural flowering period into spring chrysanthemums (late April to late May), summer chrysanthemums (late May to August), summer-autumn chrysanthemums (August to September), autumn chrysanthemums (late October to late November), and winter chrysanthemums (early December to February of the following year). Most chrysanthemum varieties belong to the autumn chrysanthemum category, with a relatively concentrated flowering period, a late natural flowering time, and a short duration, making it difficult to meet market demand. Outdoor chrysanthemums are short-day plants with photoperiod dependence, with their flowering period concentrated between September and October, lasting approximately 40 to 50 days. However, due to Heilongjiang Province's high latitude, the peak flowering period of outdoor chrysanthemums often clashes with the time of frost. When temperatures drop, the plants are prone to premature withering, severely impacting their ornamental value. Therefore, how to extend or regulate their ornamental period through molecular breeding has become a hot topic in outdoor chrysanthemum research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating the flowering period of *Chrysanthemum indicum*.

[0006] This invention provides a gene for controlling chrysanthemum flowering, the nucleic acid molecule of which is shown in SEQ ID NO.3.

[0007] The present invention provides a protein for controlling chrysanthemum flowering, the sequence of which is shown in SEQ ID NO.4.

[0008] This invention provides the application of a protein with an amino acid sequence as shown in SEQ ID NO.4 in the delayed flowering of Chrysanthemum in open field.

[0009] This invention provides the application of nucleotides with gene sequences as shown in SEQ ID NO.3 in delayed flowering of *Chrysanthemum indicum*.

[0010] This invention provides the application of a recombinant vector containing the nucleotides shown in SEQ ID NO.3 in delayed flowering of *Chrysanthemum indicum*.

[0011] Further specifying, the starting vector of the recombinant vector is either the pCAMBIA1305 vector or the pFGC5941 vector.

[0012] This invention provides the application of recombinant microbial cells containing the nucleotides shown in SEQ ID NO.3 in the delayed flowering of Chrysanthemum in open field.

[0013] Further specifying, the recombinant microbial cells are eukaryotic microbial cells or prokaryotic microbial cells.

[0014] This invention provides a method for delaying the flowering of chrysanthemums in open fields, the steps of which are as follows:

[0015] Step 1: The nucleotide shown in SEQ ID NO.3 is ligated into the pCAMBIA1305 vector to obtain the recombinant vector;

[0016] Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0017] Step 3: Transfer the recombinant Agrobacterium obtained in Step 3 into Chrysanthemum indicum to obtain transgenic Chrysanthemum indicum.

[0018] This invention provides a method for promoting the flowering of chrysanthemums in open fields, the steps of which are as follows:

[0019] Step 1: The nucleotides shown in SEQ ID NO.3 are ligated into the pFGC5941 vector to obtain the recombinant vector;

[0020] Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0021] Step 3: Transfer the recombinant Agrobacterium obtained in Step 3 into Chrysanthemum indicum to obtain transgenic Chrysanthemum indicum.

[0022] Beneficial effects: CRISPR-dCas9-TET1cd-sgRNA can reduce the DNA methylation level in the CmFDa gene promoter region. *Chrysanthemum indicum* transfected with the CRISPR-dCas9-TET1cd-sgRNA vector failed to bud, exhibiting delayed flowering. Using the dCas9-SunTag-NtDRM2cd system to direct NtDRMcd to the CmFDa promoter region induced early flowering in *Chrysanthemum indicum*. CmFDa overexpression leads to delayed flowering in *Chrysanthemum indicum*. CmFDa gene silencing also leads to delayed flowering in *Chrysanthemum indicum*. Attached Figure Description

[0023] Figure 1 This is a diagram showing the cloning results of the coding region of the CmFDa gene;

[0024] Figure 2 Phylogenetic tree diagram of CmFDa and the FD gene in other species;

[0025] Figure 3 This is a schematic diagram of the CmFDa-OE overexpression vector;

[0026] Figure 4 Figure 1 shows the identification results of the CmFDa-OE transgenic line. Figure 2 shows the identification results of the CmFDa–OE transgenic plant by PCR, with primer JC-F1 / R1 located on the selection gene HYG. Figure 3 shows the identification results of the CmFDa–OE transgenic plant by PCR, with primer JC-F2 / R2 located at both ends of the CmFDa insertion site. Figure 4 shows the identification results of the CmFDa–OE transgenic plant by Western blot.

[0027] Figure 5 Figure 1 shows how overexpression of CmFDa inhibits the expression of CmSOC1a and CmAP1a genes and delays flowering and fruiting; a) CmFDa gene overexpression delays flowering, Bar = 2cm; b) CmFDa gene upregulated expression in CmFDa-OE overexpressing plants.

[0028] Figure 6 Figure showing the primer positions in the promoter region of the CmFDa gene;

[0029] Figure 7 A schematic diagram of the CRISPR-dCas9-TET1cd-sgRNA vector;

[0030] Figure 8Figure 1 shows the identification results of the CRISPR-dCas9-TET1cd-sgRNA transgenic line. a) Primer hyg-F / hyg-R is located on the selection gene HYG. b) Primer dCas9-F / dCas9-R is located on the dCas9 gene. c) Primers U6-F / UBQ-R are located on the U6 and UBQ promoters, respectively. d) Primers U3-F / UBQ-R are located on the U3 and UBQ promoters, respectively.

[0031] Figure 9 Figure 1 shows the effect of CmFDa DNA demethylation on upregulating CmFDa expression and causing delayed flowering and fruiting. a) Delayed flowering is caused by CmFDa DNA demethylation using the CRISPR-dCas9-TET1cd-sgRNA system. Bar = 2 cm. b) Upregulated CmFDa gene expression in CRISPR-dCas9-TET1cd-sgRNA transgenic plants. Data are mean ± SD of three biological replicates. Asterisks indicate significant differences (***P < 0.001; t-test). c) McrBC-PCR shows decreased CmFDa promoter region DNA methylation levels in CRISPR-dCas9-TET1cd-sgRNA transgenic plants.

[0032] Figure 10 Figure 1 shows the comparison results between epilate and dCas9-NtDRMcd-sgRNA transgenic lines; Figure 2 shows the phenotypic comparison results between epilate and dCas9-NtDRMcd-sgRNA transgenic lines (#1, #2, #3); Figure 3 shows the comparison results of CmFDa expression in epilate and dCas9-NtDRMcd-sgRNA transgenic plants; Figure 4 shows the comparison results of CmFDa promoter regions in epilate and dCas9-NtDRMcd-sgRNA transgenic plants; Figure 5 shows the quantitative comparison of the total number of leaves in the second-generation plants produced by asexual reproduction of epilate and dCas9-NtDRMcd-sgRNA transgenic lines. N = 9 biological replicates. Asterisks indicate significant differences (***P < 0.001; t.test);

[0033] Figure 11Figure 1 shows the comparison results of epilate and CmFDa-RNAi transgenic plants; (a) shows the phenotypic comparison results of epilate and CmFDa-RNAi transgenic plants, with a scale bar of 2 cm; (b) shows the relative expression levels of CmFDa in epilate and CmFDa-RNAi transgenic plants, with gene expression levels normalized relative to CmEF expression, and data are the mean ± standard deviation of three independent biological replicates; (c) shows the quantitative comparison of the total number of leaves in the second-generation plants produced by asexual reproduction of epilate and CmFDa-RNAi transgenic plants, with N = 9 biological replicates. Asterisks indicate significant differences (***P < 0.001; t.test). Detailed Implementation

[0034] The CRISPR-dCas9-TET1cd vector is described in Li, J., Yang, DL., Huang, H. et al. Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation. Nat. Plants 6, 661–674 (2020).

[0035] The dCas9-SunTag-NtDRMcd vector is documented in Papikian, A., Liu, W., Gallego-Bartolomé, J., and Jacobsen, SE (2019) Site-specific manipulation of Arabidopsis lociusing CRISPR-Cas9 SunTag systems.

[0036] Nat Commun, 10, 729.

[0037] The description of *Chrysanthemum 'YP-Y'* is found in the article "Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum".

[0038] 18T-AtU6-chim and 18T-AtU3b-chim are described in Zhang, Z. et al. A multiplex CRISPR / Cas9 platform for fast and efcient editing of multiple genes in Arabidopsis. Plant Cell Rep. 35, 1519–1533 (2016).

[0039] Example 1. Cloning of the CDS coding region of the CmFDa gene

[0040] (1) RNA extraction and reverse transcription

[0041] Total RNA was extracted from the leaves of *Chrysanthemum indicum* 'YP-Y' and reverse transcribed into cDNA as a PCR template.

[0042] (2) RCR amplification

[0043] Primers were designed using Geneious software based on the CmFDa gene CDS sequence obtained from local blast radiometry. Primer sequences are detailed in Table 1, and the PCR loading system and reaction conditions are as follows:

[0044] Table 1. PCR reaction system and procedure

[0045] reagents Sample loading amount Genomic DNA 1μL Primer-F / R 2μL 2×Phanta Max Master Mix 25μL <![CDATA[ddH2O]]> Fill to 50μL

[0046] The primer sequences are as follows:

[0047] CmFDA-F:ATGAAACCAACAGATGACGTATGGAA (SEQ ID NO. 1);

[0048] CmFDa-R: TTAAAATGGGGCACTTTTGGTTCTGT (SEQ ID NO. 2);

[0049] CmFDa's CDS: (SEQ ID NO.3)

[0050] ATGAAACCAACAGATGACGTATGGAAAGACATAAGTAGCCTCTCATCTCCGATCAC

[0051] CAACCCCTTCCCAAACTTCCTTCCACCGTCACTCCGGCCACCGGAAACCACCACTATGT

[0052] TCTTTCCACCAACACCACCATTAACAACCAATAACATGCTAACCCTACTTACACCTACTA

[0053] GTGACCCCCTTAATGAAAACCCTAATAAGAAACAAAAACCAAACCCACCACAACCATT

[0054] ACTTTACACCTCAAGGGAGAGTTCTCCACCGGAGTACTCTGAGAAGTTTCGGCGGCTG

[0055] ATGAAAAATAGGGAGTCTGCTGCTCGATCTAGAGCTAGAAAACAGGCTCGTGCAGATG

[0056] AGCTGGAGCAAGAAGTAATGCGTCTAGCAAAGGAAAATGCTAAGCTTAAAAGGCTGCA

[0057] AAAAGAAGCATGTGTCTCCCAGCTTGAAAAAAAATCTAGACTTCACAGAACCAAAAGTGCCCCATTTAA;

[0058] CmFDa protein: (SEQ ID NO.4)

[0059] MKPTDDVWKDISSLSSPITNPFPNFLPPSLRPPETTTMFFPPTPPLTTNNMLTLLTPTSDP

[0060] LNENPNKKQKPNPPQPLLYTSRESSPPEYSEKFRRLMKNRESAARSRARKQARADELEQEVMRLAKENAKLKRLQKEACVSQLEKKSRLHRTKSAPF.

[0061] (3) Recovery of the target fragment using CmFDa CDS: A 1% agarose gel was prepared for electrophoresis detection of the above PCR products (5 μL of amplified product was used for detection). Electrophoresis was performed at 120V for 20 min to determine the homogeneity of the PCR products. After identifying the target fragment, the remaining PCR products were spotted onto a 1% agarose gel, run at 100V for 30 min, and then the target band was cut for recovery. The recovery of the target fragment was performed using the Gel Extraction Kit.

[0062] (4) Connection and Transformation

[0063] The purified target gene fragment was ligated with the pCE2 TA / Blunt-Zero Vector (5min TA / Blunt-Zero Cloning Kit, Vazyme), as shown in Table 2 below:

[0064] Table 2 Connection Reaction System

[0065] reagents Sample loading amount 5×TA / Blunt-Zero Cloning Mix 1μL PCR purification and product recovery 25ng <![CDATA[ddH2O]]> Make up to 5μL

[0066] A. Incubate at 37°C for 5 minutes. After the reaction, immediately place the centrifuge tubes on ice. The competent cells used in this experiment were Escherichia coli strain DH5α.

[0067] (5) Identification of positive clones

[0068] Propagate positive monoclonal colonies: Pick at least 5 monoclonal colonies (white, round, with clear edges, moderate size, and healthy condition) from each plate, and add 1 mL of Kanamycin-containing liquid culture medium to each colony. + ) Shaking of bacteria. Colony PCR was performed using universal primers M13-F and M13-R on the pCE2TA / Blunt-Zero Vector. Clones with electrophoresis results of about 500 bp were selected, and 500 μL of the corresponding bacterial culture was sent to the company for sequencing.

[0069] (6) Extraction of plasmid DNA

[0070] E. coli transformed with the successfully identified vector were cultured in LB broth (Kana) + Incubate overnight at 37°C. The kit used in this experiment was the Plasmid Mini Kit (OMEGA). The plasmid DNA extraction steps are as follows (note before use: add anhydrous ethanol to wash buffer PW and add RNase A to buffer P1).

[0071] Results: CmFDa cloning and sequence homology analysis: Based on the genome sequence, specific primers were designed, and PCR amplification was performed using cDNA from *Chrysanthemum indicum* 'YP-Y' leaves as a template. Specific bands of similar size to the target fragment were amplified. Figure 1 A CDS region of 477 bp was obtained, encoding 158 amino acids (aa).

[0072] To further understand the homology of CmFDa in eukaryotes, amino acid sequences of FD from different species were downloaded from the NCBI database, and a phylogenetic tree was constructed using the maximum likelihood method. Figure 2The results showed that the proteins most homologous to *C. sarsaparilla* CmFDa were *C. chamaejasminoides* CsFD and *Artemisia annua* AaFD.

[0073] Example 2. Construction of CmFDa overexpression vector and obtaining transgenic *Chrysanthemum indicum*

[0074] (1) Vector double digestion and homologous recombination cloning of CmFDa gene

[0075] Based on the vector sequence of the plant overexpression vector pCAMBIA1305-3×FLAG, homologous recombination primers for the CmFDa gene were designed using CE Design V1.04 software with Pst I and Hind III double restriction sites.

[0076] The pCAMBIA1305-3×FLAG vector plasmid was double-digested with restriction endonucleases Pst I and Hind III. The target fragment and the linear vector were then used to construct the CmFDa gene expression vector using the ClonExpress II One Step Cloning Kit (Vazyme). The reaction conditions were 37°C for 30 min, followed immediately by placing the sample on ice. The homologous recombination reaction system is shown in Table 3 below. After successful vector construction and sequencing analysis, the overexpression vector pCAMBIA1305-CmFDa-3×FLAG was transformed into Agrobacterium GV3101 and flash-frozen in 15% glycerol liquid nitrogen at -80°C.

[0077] Table 3 Homologous recombination reaction system

[0078] Reagent Name Dosage Recombinase Exnase II 1μL Target fragment product (20 ng / μL) 5μL 5×CE II Buffer 2μL Vector digested with enzymes (10 ng / μL) 2μL

[0079] (2) Transformation of Agrobacterium tumefaciens using the repeated freeze-thaw method;

[0080] (3) Using vacuum transgenic technology to infect 'YP-Y';

[0081] Results: CmFDa overexpression delayed flowering of *Chrysanthemum indicum*: To fully investigate the biological function of the CmFDa gene in flowering of *Chrysanthemum indicum*, the CmFDa gene CDS sequence was fused with a 3×FLAG tag and constructed into the pCAMBIA1305-3×FLAG expression vector. Figure 3 They then used Agrobacterium-mediated genetic transformation to transfer the gene into the 'YP-Y' strain background, and obtained a stable overexpression (CmFDa-OE) transgenic line in the 'YP-Y' strain background.

[0082] DNA was extracted from leaves of 'YP-Y' wild-type (WT), pCAMBIA1305-3×FLAG empty vector transgene (EV), and CmFDa-OE overexpressing transgenes, respectively. The transgenes were validated by PCR. Primers (JC-F1 / R1 and JC-F2 / R2) were designed at the HYG selection gene and at both ends of the CmFDa insertion site, respectively. Figure 3 The results showed that OE#1, OE#5, OE#19, and OE#22 were double-positive plants. Figure 4 (a and b in the original text). Further identification of the CmFDa-OE transgenic line was performed using Western blotting. Figure 4 (c)

[0083] JC-F1: AATCTCGTGCTTTCAGCTTCGATGTAGGA (SEQ ID NO.5);

[0084] JC-R1: CTATCGGCGAGTACTTCTACACAGCCATC (SEQ ID NO. 6);

[0085] JC-F2: AGCGACAATTTCACACAGGA (SEQ ID NO.7);

[0086] JC-R2: AGGGGAACCCTGTGGTTGGCA (SEQ ID NO.8);

[0087] Phenotypic observation of the CmFDa-OE transgenic lines revealed that CmFDa overexpressing plants flowered later than the control group (EV). Figure 5 RNA was extracted from photoperiod samples of CmFDa overexpressing plants (CmFDa-OE) and control plants (EV), respectively. The expression level of CmFDa was identified by RT-qPCR. The results showed that the expression level of CmFDa in the overexpressing plants was higher than that in the control group (a). Figure 5 (b) The primers are as follows (EF is the internal reference gene), which are used to calculate the quantitative expression level of the internal reference gene:

[0088] EF-F:GCAGCCAGTTTCACTTC(SEQ ID NO.9);

[0089] EF-R: TACCAGCATCACCATTC (SEQ ID NO. 10);

[0090] qFDa-F: GCTCGTGCAGATGAGCTGGAGCA (SEQ ID NO. 11);

[0091] qFDa-R: TGGGGCACTTTTGGTTCTGTGAAGTCT (SEQ ID NO. 12);

[0092] Example 3. Obtaining the CRISPR-dCas9-TET1cd-sgRNA vector and transgenic *Chrysanthemum indicum*

[0093] Transposon insertion sites are sites enriched by DNA methylation modifications. Transposons include DNA transposons and retrotransposons. Analysis of the repetitive sequences in the CmFDa gene promoter region was based on the known repetitive sequence database RepBase (http: / / www.girinst.org / repbase) using CENSOR software (http: / / www.girinst.org / censor / index.php).

[245] Predict sequences that are similar to known repeating sequences.

[0094] Dot-plot analysis was performed using the online software dotmatcher (http: / / emboss.bioinformatics.nl / cgi-bin / emboss / dotmatcher / ). The analysis window size was 200 bp, and the recognition threshold was 23.

[0095] 1. Construction of the CRISPR-dCAS9-TET1cd vector with demethylated promoter region of the CmFDa gene of *Chrysanthemum indicum*

[0096] To construct a DNA demethylation vector targeting CmFDa, sgRNA dimerization primers were designed at six sites targeting the CmFDa gene promoter region sequence. Figure 6 The specific primer sequences are as follows.

[0097] CmFDa gene promoter sequence (SEQ ID NO.13):

[0098] Primers for obtaining sgRNA1-6:

[0099] FD1-dSg1-U6-F: GATTGCGTCGTTGTATCGTGATGCA (SEQ ID NO. 14);

[0100] FD1-dSg1-U6-R: AAACTGCATCACGATACAACGACGC (SEQ ID NO. 15);

[0101] FD1-dSg2-U3-F:GATTGCGCATTACAACCGTTCGAAA(SEQ ID NO.16);

[0102] FD1-dSg2-U3-R:AAACTTTCGAACGGTTGTAATGCGC(SEQ ID NO.17);

[0103] FD1-dSg3-U3-F:GGTCATCTTGTGCACGCCTGACAAA(SEQ ID NO.18);

[0104] FD1-dSg3-U3-R:AAACTTTGTCAGGCGTGCACAAGAT(SEQ ID NO.19);

[0105] FD1-dSg4-U6-F:GGTCACTTGGCTAGACAATCCAAGT(SEQ ID NO.20);

[0106] FD1-dSg4-U6-R:AAACACTTGGATTGTCTAGCCAAGT(SEQ ID NO.21);

[0107] FD1-dSg5-U6-F:GATTGGTAAATGGTGGTCTATTGTG(SEQ ID NO.22);

[0108] FD1-dSg5-U6-R:AAACCACAATAGACCACCATTTACC(SEQ ID NO.23);

[0109] FD1-dSg6-U3-F:GGTCATTGATTATTCTTGTCGGCGG(SEQ ID NO.24);

[0110] FD1-dSg6-U3-R:AAACCCGCCGACAAGAATAATCAAT(SEQ ID NO.25);

[0111] sgRNA1-6:

[0112] AtU6-sgRNA1:CGCATTACAACCGTTCGAAA(SEQ ID NO.26);

[0113] AtU3-sgRNA2:TCTTGTGCACGCCTGACAAA(SEQ ID NO.27);

[0114] AtU6-sgRNA3: GTAAATGGTGGTCTATTGTG (SEQ ID NO. 28);

[0115] AtU3-sgRNA4: GCAACATAGACACCACCAAA (SEQ ID NO. 29);

[0116] AtU6-sgRNA5: CGTCGTTGTATCGTGATGCA (SEQ ID NO. 30);

[0117] AtU3-sgRNA6: TTGATTATTCTTGTCGGCGG (SEQ ID NO. 31);

[0118] (1) Primer dimerization

[0119] Mix equal volumes of sgRNA-F and sgRNA-R, and perform a dimerization reaction using a PCR instrument, as shown in Table 5 below:

[0120] Table 4

[0121] 95℃ 3min 95～25℃ -1℃ / 20s 4℃ ∞

[0122] (2) Construction of cloning vector

[0123] The 18T-AtU6-chim and 18T-AtU3b-chim cloning vectors were digested with BbsI and reacted at 37°C for 4 h. The six sgRNA dimerization products were then ligated into the 8T-AtU6-chim and 18T-AtU3b-chim cloning vectors, respectively. Fusion fragments of AtU3-sgRNA1, AtU3-sgRNA2, AtU6-sgRNA3, AtU3-sgRNA4, AtU6-sgRNA5, and AtU6-sgRNA6 were constructed. The reaction system is shown in Table 5 below.

[0124] Table 5. Reaction system with connecting support

[0125]

[0126] (3) Construction of expression vector

[0127] Homologous recombination primers were designed to amplify the target fragment in the cloning vector using a high-fidelity enzyme. The expression vector CRISPR-dCas9-TET1cd was digested with the restriction endonuclease XmaⅠ and recovered. The target fragment was ligated into the expression vector. The reaction program was 37℃ for 30 min, and the reaction system is shown in Table 6 below.

[0128] Table 6 Homologous Recombination System

[0129]

[0130] The ligation product was transformed into competent E. coli DH5α cells, and single clones were selected for sequencing to identify whether the expression vector was correctly constructed for subsequent genetic transformation experiments.

[0131] result:

[0132] (4) Transformation of Agrobacterium GV3101

[0133] The six successfully constructed 1300-CRISPR-dCas9-TET1cd-sg1 / 2 / 3 / 4 / 5 / 6 demethylated vectors were transformed into Agrobacterium GV3101 competent cells to obtain Agrobacterium strains.

[0134] (5) Using vacuum transgenic technology to infect 'YP-Y'

[0135] Agrobacterium GV3101 containing 1300-CRISPR-dCas9-TET1cd-sg1 / 2 / 3 / 4 / 5 / 6 demethylated vectors with sgRNA was added to LB liquid medium (containing the antibiotics rifampin and kanamycin) and incubated in the dark at 28°C and 180 rpm for 24 h. OD 600 Approximately 1.5-2.0. After bacterial enrichment, the mixture was mixed 1:1 to infect the peduncles.

[0136] (6) ChIP-qPCR was used to identify the level of histone modification in the promoter region of the CmFDa gene. The primers used for qPCR are as follows, showing different positions on the promoter.

[0137] ChIP-pFDa#1-F: CACCCACATCTCCTCCAAAG (SEQ ID NO.32);

[0138] ChIP-pFDa#1-R: CCAACGTGTAGGGACAAAGT (SEQ ID NO. 33);

[0139] ChIP-pFDa#2-F: CGTCAACGTAGCAATGTGTTAAA (SEQ ID NO. 34);

[0140] ChIP-pFDa#2-R: CGAGAACTGAGCGTAACCTATT (SEQ ID NO.35);

[0141] ChIP-pFDa#3-F:ATGCATATCGTAGTCAAACTTTCTG (SEQ ID NO.36);

[0142] ChIP-pFDa#3-R:GGTGGTGTCTATGTTGCTAAGT (SEQ ID NO. 37);

[0143] Results: The level of DNA methylation modification in the promoter region of the CmFDa gene is heritable during asexual reproduction.

[0144] CRISPR-dCas9 demethylation led to increased CmFDa gene expression: sgRNAs designed with the CmFDa promoter region as a reference were ligated into AtU6-18T and AtU3b-18T vectors, respectively. Fusion fragments linked to different sgRNAs (AtU3-sgRNA1, AtU3-sgRNA2, AtU6-sgRNA3, AtU3-sgRNA4, AtU6-sgRNA5, and AtU6-sgRNA6) were ligated into the 1300-CRISPR-dCas9-TET1cd vector to construct a CRISPR-dCas9-TET1cd-sgRNA CmFDa-directed demethylation vector. Figure 7 The demethylation vector was then transformed into Agrobacterium GV3101 strain.

[0145] Agrobacterium GV3101 containing the demethylation vector plasmid was transfected into the basal shoots of Chrysanthemum 'YP-Y'. The transfected transgenic plants were then processed... Figure 7 The primers shown were used for molecular identification of positive plants. The primer sequences are as follows, and positive plants were identified using CRISPR-dCas9-TET1cd-#1 and CRISPR-dCas9-TET1cd-#3. Figure 8 The control group 'YP-Y' plants were also transfected with Agrobacterium GV3101 strain, which did not contain the demethylation vector plasmid.

[0146] Hyg-F: AATCTCGTGCTTTCAGCTTCGATGTAGGA (SEQ ID NO. 38);

[0147] Hyg-R: CTATCGGCGAGTACTTCTACACAGCCATC (SEQ ID NO. 39);

[0148] U3-F: CTCGAGCCTGTGATGGATAAC (SEQ ID NO. 40);

[0149] U6-F: TGTCCCAGGATTAGAATGATTAGGCATCG (SEQ ID NO. 41);

[0150] UBQ-R: CTTGCTGATGATGAGGCGATTGACAAGAG (SEQ ID NO. 42);

[0151] Phenotypic observation showed that when the control group 'YP-Y' budded, the *Chrysanthemum indicum* transformed with the CRISPR-dCas9-TET1cd-sgRNA vector did not bud, exhibiting delayed flowering. Figure 9 (a) RT-qPCR analysis showed that the expression level of the CmFDa gene in leaves transfected with the targeted DNA demethylation vector was significantly higher than that in the control group. Figure 9 (b) McrBC-PCR results showed that the degree of DNA methylation modification in the CmFDa gene promoter region was significantly lower in *Chrysanthemum indicum* transformed with the CRISPR-dCas9-TET1cd-sgRNA vector than in the control group, indicating that CRISPR-dCas9-TET1cd-sgRNA can reduce the degree of DNA methylation modification in the CmFDa gene promoter region. Figure 9 (c)

[0152] Example 4. A method for advancing the flowering of outdoor chrysanthemums using the dCas9-SunTag-NtDRMcd system.

[0153] A similar strategy was used to construct the dCas9-SunTag-NtDRMcd-sgRNA plasmid. AtU6-sgRNA1, AtU3b-sgRNA2, AtU6-sgRNA3, AtU3b-sgRNA4, AtU6-sgRNA5, and AtU3b-sgRNA6 were amplified using sequence-specific primers, and then cloned into the enzyme-digested dCas9-SunTag-NtDRM2cd vector (pEG302 22aa SunTag NtDRMcd(noNLS)nog vector, derived from Steven Jacobsen (Addgene plasmid #119554; http: / / n2t.net / addgene:119554; RRID:Addgene_119554 32), ultimately generating six dCas9-suntag-ntdrm2cd-sgrna vectors.

[0154] We induced DNA methylation in the CmFDa promoter region by transforming epilate plants using the dCas9-SunTag-NtDRM2cd methylation system. We obtained three genome-edited plants that, compared to untransformed epilate lines, all exhibited increased DNA methylation levels in the CmFDa promoter, decreased CmFDa expression, and earlier flowering. Figure 10(a, b, c in the original text). A quantitative comparison of the total number of leaves in the second-generation plants produced asexually from epilate and dCas9-SunTag-NtDRM2cd transgenic lines revealed that the dCas9-SunTag-NtDRM2cd transgenic plants had more leaves. Figure 10 (d) Using the dCas9-SunTag-NtDRM2cd system, NtDRMcd was directed to the CmFDa promoter region to induce early flowering of epilate.

[0155] The conversion of the dCas9-SunTag-NtDRM2cd methylation system to "YP-Y" will also cause the variety to flower earlier.

[0156] Methods for constructing epilate plants:

[0157] The epigenetic inhibitor 5-AzaC was dissolved in dimethyl sulfoxide (DMSO) and then added to 1× phosphate-buffered saline (PBS) at a final concentration of 50 μM. To improve solute penetration, 0.05% (v / v) of L-77 surfactant was added to the prepared solution. Chrysanthemum 'YP-Y' pedicels were collected from the plant, washed under running water for 1 h, and placed in the prepared solution. Vacuum impregnation was performed as before. Simply put, a vacuum pump was used to evacuate the buds for 15 min, followed by rapid release of air. This process was repeated three times to ensure sufficient penetration of the epigenetic inhibitor into the bud tissue. After treatment, the pedicels were incubated in a sand basket in the dark for 24 h, and then transferred to light conditions. Once roots developed, they were transplanted into soil baskets. A mutant variety, epilate, was found in the offspring, exhibiting delayed flowering compared to the wild type.

[0158] Example 5. A method for advancing the flowering of *Chrysanthemum indicum* using an RNAi system.

[0159] The purified CmFDa CDS product was inserted near the NcoI and AscI recognition sites of the pFGC5941 plasmid to construct an RNAi knockdown CmFDa expression vector. Then, the purified CmFDa reverse fragment was inserted near the XbaI and BamHI recognition sites of the pFGC5941 plasmid. Finally, the CmFDa RNAi vector was constructed.

[0160] It was transferred into an epilate plant background. The CmFDa-rnai transgenic plant had fewer leaves than the epilate plant. Figure 11 The earlier flowering (ac) indicates that CmFDa plays an important role in flower transition. Transitioning to "YP-Y" also causes the variety to flower earlier.

Claims

1. Application of a protein with the amino acid sequence shown in SEQ ID NO.4 in delaying flowering of Chrysanthemum in open field.

2. Application of the gene with the nucleotide sequence shown in SEQ ID NO.3 in delaying flowering of Chrysanthemum in open fields.

3. The application of a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.3 in delayed flowering of *Chrysanthemum indicum*, characterized in that, The starting vector for the recombinant vector is the pCAMBIA1305 vector.

4. Application of recombinant microbial cells containing the nucleotide sequence shown in SEQ ID NO.3 in delayed flowering of Chrysanthemum in open field.

5. The application according to claim 4, characterized in that, The recombinant microbial cells are eukaryotic or prokaryotic microbial cells.

6. A method for delaying the flowering of chrysanthemums in open fields, characterized in that, The steps of the method are as follows: Step 1: The nucleotide sequence shown in SEQ ID NO.3 is ligated into the pCAMBIA1305 vector to obtain the recombinant vector; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfer the recombinant Agrobacterium obtained in Step 2 into Chrysanthemum indicum to obtain transgenic Chrysanthemum indicum.

7. A method for advancing the flowering of chrysanthemums in open fields, characterized in that, The steps of the method are as follows: Step 1: The nucleotide sequence shown in SEQ ID NO.3 is ligated into the pFGC5941 vector to obtain the recombinant vector; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfer the recombinant Agrobacterium obtained in Step 2 into Chrysanthemum indicum to obtain transgenic Chrysanthemum indicum.

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