Application of CmIAA27 in regulation and control of lateral branch development of chrysanthemum

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of CmIAA27 in regulation and control of lateral branch development of chrysanthemum. According to the invention, phenotypic analysis is carried out on a CmCMT2-RNAi transgenic 'Shenma' plant obtained at an earlier stage, and lateral branches of the CmCMT2-RNAi transgenic 'Shenma' are increased in a vegetative growth period. RNA-seq is carried out on the gene CmIAA27, it is found that the expression quantity of the auxin inhibitory signal key gene CmIAA27 is remarkably increased, McrBC and BSP experiments show that the CHH type methylation level on a CmIAA27 promoter in a transgenic line is remarkably lower than that of WT, and the expression of the plant lateral branch development key inhibitory gene CmBRC1 in the transgenic line is remarkably reduced. Y1H, LUC and EMSA experiments show that the CmCMT2 can be directly combined with a CmIAA27 promoter and regulate and control the CHH type methylation level in the region so as to influence the expression of the CmIAA27. Through transcription analysis of CmCMT2-RNAi transgenic Shenma and WT, a key factor CmIAA27 for downstream regulation and control of chrysanthemum lateral branch development is obtained, functional verification is carried out, and excellent gene reserve and a new strategy are provided for regulation and control of chrysanthemum lateral branches.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving CmIAA27 Application of genes in regulating the development of chrysanthemum lateral branches. Background Technology

[0002] chrysanthemum( Chrysanthemum morifolium Chrysanthemum is one of my country's ten traditional famous flowers, possessing ornamental, edible, tea-making, and medicinal functions, and has extremely high ornamental and economic value. Plant type is an important trait in chrysanthemum cultivation; branching is a major factor determining the morphology of the chrysanthemum and also a key trait determining its yield potential. Therefore, optimizing branching structure is one of the key factors in enhancing the ornamental value of chrysanthemums. Chrysanthemum branching types are diverse, mainly determined by genetics, and can be shaped through cultivation methods. Chrysanthemums are a classic model for studying the core life phenomenon of apical dominance. By studying its lateral branch development, we can gain a deeper understanding of how hormones such as auxin, strigolactone, and cytokinin interact, as well as key genes (such as...). Aux / IAA , TB1 How to precisely regulate the germination and elongation of lateral buds (e.g., [missing information]) deepens our understanding of the principles of overall plant morphology construction. By identifying key genes controlling the number, length, and angle of lateral branches, molecular marker-assisted selection or gene editing technologies can be used to precisely breed new chrysanthemum varieties that meet market demands. For example, cultivating cut chrysanthemums with fewer lateral branches and no need for bud removal can save labor, or cultivating potted chrysanthemums with dense lateral branches and full plant shapes can enhance their ornamental value. In short, research on chrysanthemum lateral branch development perfectly connects basic biological exploration with industrial application innovation. It not only helps us answer the fundamental scientific question of how plants "decide" their growth morphology, but also directly translates into a key technological source for enhancing ornamental value and optimizing agricultural productivity.

[0003] DNA methylation, as an important epigenetic modification, plays a crucial role in plant growth and development. DNA methylation is classified into three types: CG methylation, CHG methylation, and CHH methylation. It is generally believed that CHH methylation can only be maintained by DRM2 via the RdDM pathway. However, in Arabidopsis thaliana, DRM2-mediated CHH methylation accounts for only about 35% of total CHH methylation. CMT2 can maintain approximately 70% of CHH methylation levels, independent of RdDM. CMT2 plays an important role in plant growth and development; current reports indicate that CMT2 participates in regulating multiple growth and developmental processes, including flowering, flower color, secondary metabolism, biosynthesis, and environmental responses. However, reports on CMT2 regulation of lateral branch development are less common. In rice... Osnrpd In the mutant, the styracin receptor gene OsD14 The methylation level of CHH types in downstream MITE transposons is reduced. OsD14Decreased expression levels lead to increased tillering in rice. Therefore, it is speculated that CMT2 may regulate lateral branching by modulating the CHH-type methylation level of gene sequences related to lateral branching development.

[0004] Auxin, one of the earliest discovered and most crucial hormones in plants, plays a vital role throughout almost the entire life cycle of a plant. Its most famous characteristic is that low concentrations promote growth, while high concentrations inhibit it. As one of the core hormonal signaling systems in plants, auxin plays a global regulatory role in plant growth and development. Its most classic function is shaping the basic morphological structure of the plant, one of the most significant manifestations of which is inhibiting lateral branch development and maintaining "apical dominance." This regulation is mainly achieved through a sophisticated "instruction transmission" system: auxin synthesized in the apical bud is actively transported from top to bottom along the stem, forming a concentration gradient. This transport process itself triggers a series of complex signal transductions. When the auxin signal reaches the lateral bud, it does not directly poison the cells, but rather induces the growth of auxin in the lateral bud... BRC1 The expression of key repressor genes and the regulation of the activity of local auxin efflux carriers (such as PIN proteins) lead to the excessive accumulation of auxin in lateral buds, preventing its effective export. This "locally retained" auxin further inhibits cell division and elongation, and interacts with other hormones (such as cytokinins and strigolactones) to jointly suppress the germination and growth of lateral buds. Therefore, auxin determines the "fate" of lateral branches through this systemic signal transduction and fine regulation of the local microenvironment. IAA Genes are crucial "negative regulatory brakes" and "dynamic regulators" in the auxin signaling pathway. They directly inhibit the core response of auxin by encoding a large family of short-lived nuclear proteins, Aux / IAA. IAA Genes, through their encoded repressor proteins, provide a rapidly repressible state for auxin signaling, a timely negative feedback brake, and the molecular basis for signal specificity. This is the core link in auxin's ability to precisely coordinate plant growth and development. Summary of the Invention

[0005] The purpose of this invention is to... CmCMT2 -The phenotypes of RNAi transgenic "Shenma" plants (obtained by the method described in patent 202510225213.2) were observed and statistically analyzed, and samples were collected. CmCMT2- RNAi transgenic 'Shenma' plants and wild-type lateral branch samples were analyzed using RNA-seq to identify key genes influencing chrysanthemum lateral branch development. CmCMT2 Mechanisms and interactions of genes and genes related to lateral branch development regulating the occurrence of lateral branches in chrysanthemum.

[0006] To achieve the above objectives, the present invention utilizes data obtained in the laboratory in the early stages. CmCMT2-Phenotypic analysis was performed on RNAi transgenic 'Shenma' plants (obtained using the method described in patent 202510225213.2), and it was found that... CmCMT2- RNAi transgenic 'Shenma' plants exhibited increased lateral branching during the vegetative growth stage. RNA-seq analysis revealed a key gene for auxin inhibition signaling. CmIAA27 The expression level was significantly increased. McrBC and BSP experiments showed that in transgenic lines... CmIAA27 The CHH methylation level on the promoter was significantly lower than that on the WT, and it was a key repressor gene for plant lateral branch development. CmBRC1 The expression of [specific gene] was significantly reduced in transgenic lines. Y1H, LUC, and EMSA experiments showed that... CmCMT2 Can be directly combined CmIAA27 The promoter regulates the CHH-type methylation level in this region, thereby affecting CmIAA27 The expression. CmIAA27 Functional analysis revealed that, CmIAA27 Overexpression of chrysanthemum resulted in significantly more lateral branches than the root-weighted variety. This invention utilizes... CmCMT2- Transcriptional analysis of RNAi transgenic 'Shenma' and WT revealed key downstream factors regulating chrysanthemum lateral branch development. CmIAA27 Furthermore, functional verification was conducted to provide an excellent gene reserve and new strategies for the regulation of chrysanthemum lateral branches.

[0007] Furthermore, this invention first provides the application of chrysanthemum auxin signal negative regulatory genes in regulating the plant architecture of Asteraceae plants (e.g., chrysanthemum).

[0008] Furthermore, the present invention also provides the application of chrysanthemum auxin signal negative regulatory genes in regulating the occurrence or development of lateral branches in Asteraceae plants (e.g., chrysanthemum).

[0009] Furthermore, the present invention also provides the application of the promoter of the chrysanthemum auxin signal negative regulation gene in regulating the occurrence or development of lateral branches in Asteraceae plants (e.g., chrysanthemum).

[0010] Specifically, the chrysanthemum auxin signal negative regulatory gene is CmIAA27 .

[0011] Specifically, CmIAA27 The nucleotide sequence of the gene is shown in SEQ ID NO.1. CmIAA27 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0012] Specifically, the promoter of the chrysanthemum auxin signal negative regulator gene (i.e. CmIAA27 The nucleotide sequence of the gene promoter is shown in SEQ ID NO.3.

[0013] Specifically, when CmIAA27Increased expression levels of the gene, specifically the chrysanthemum auxin signaling negatively regulated gene. CmIAA27 By expressing the CmIAA27 protein, its ability to inhibit the auxin signaling pathway can be enhanced, thereby blocking the auxin signal transduction process and promoting the development and elongation of chrysanthemum lateral branches.

[0014] Specifically, the chrysanthemum auxin signal negative regulatory gene CmIAA27 By promoting leaf development, inhibiting internode and main stem elongation, and promoting lateral branch development, a compact and vigorously branching plant shape can be created for chrysanthemums.

[0015] Specifically, the chrysanthemum variety mentioned is either 'Shenma' or 'Zhongshan Zigui'.

[0016] Specifically, in application, CmIAA27 The CDS sequence of the gene was ligated into the overexpression vector pORE-R4-2×35SAA (carrying the CaMV 35S enhancer promoter), resulting in... CmIAA27 Gene overexpression vector, and then the constructed CmIAA27 Gene overexpression vectors were transformed into Agrobacterium competent cells, and then leaves of Asteraceae plants (such as chrysanthemums) were transformed using Agrobacterium-mediated transformation to obtain... CmIAA27 Overexpression transgenic chrysanthemum lines (named OX- in this invention) CmIAA27 Genetically modified chrysanthemum), for OX- CmIAA27 Phenotypic observation and measurement were conducted on transgenic chrysanthemums.

[0017] Furthermore, this invention also provides a chrysanthemum methyltransferase gene. CmCMT2 In regulation CmIAA27 Applications related to promoter methylation variation regions.

[0018] Specifically, through the CmCMT2 Transcriptome analysis was performed on the RNAi transgenic lines, and the results were validated using yeast (Y1H) one-hybrid assays and electrophoretic migration assays (EMSA). CmCMT2 and CmIAA27 Interactions in promoter methylation change regions; detection by dual-luciferase assay (LUC) CmCMT2 right CmIAA27 Regulation of the activity of promoter methylation change regions.

[0019] Furthermore, this invention also provides a chrysanthemum methyltransferase gene. CmCMT2 Applications in regulating auxin synthesis and signal transduction during the vegetative growth stage of chrysanthemum.

[0020] Furthermore, the present invention also provides a methyltransferase gene. CmCMT2 Application in regulating the occurrence or development of lateral branches in plants of the Asteraceae family (such as chrysanthemum).

[0021] Specifically, methyltransferase gene CmCMT2 By inhibiting CmIAA27 Expression thereby regulates the development of plant lateral branches.

[0022] Specifically, methyltransferase gene CmCMT2 By direct combination CmIAA27 The promoter regulates its methylation level, thereby inhibiting CmIAA27 The expression.

[0023] Specifically, methyltransferase gene CmCMT2 Through mediation CmIAA27 The level of CHH-type methylation in the promoter region regulates and thus controls CmIAA27 The expression.

[0024] Specifically, methyltransferase gene CmCMT2 Through with CmIAA27 It directly binds to the methylated region of the gene promoter, regulates the methylation state of that region, and thus regulates the occurrence of lateral branches.

[0025] Specifically, methyltransferase gene CmCMT2 Through negative regulation CmIAA27 The expression of auxin enhances the signal transduction of auxin (IAA) in the lateral branches of chrysanthemum, leading to abnormal auxin content in the lateral branches and thus regulating the development of chrysanthemum lateral branches.

[0026] Specifically, methyltransferase gene CmCMT2 The nucleotide sequence is shown in SEQ ID NO.4.

[0027] Specifically, the chrysanthemum variety is either "Shenma" or "Zhongshan Zigui".

[0028] Specifically, in application, it includes the following aspects: (1) To CmCMT2- Determination of hormone content in lateral branches of RNAi transgenic 'Shenma' plants; (2) Changes in the methylation level of candidate gene promoters were detected by McrBC assay, and target genes with changes in the methylation level of promoter regions (i.e., CmIAA27 ); (3) Using BSP experiments to... CmIAA27 Quantitative analysis was performed on the methylation levels of three types of cytosine: CG, CHG, and CHH, in the methylation change region of the gene promoter.

[0029] Compared with the prior art, the advantages of the present invention are: This invention, through the CmCMT2 Phenotypic observation and statistical analysis were conducted on RNAi transgenic 'Shenma' plants, and key genes affecting the development of chrysanthemum lateral branches were identified using RNA-seq. CmIAA27Verification was performed using molecular interaction experiments (Y1H, EMSA, LUC). CmCMT2 right CmIAA27 The regulatory relationship. Obtained via Agrobacterium-mediated leaf disc transformation. CmIAA27 Overexpression of the transgenic 'Shenma', through interaction with wild-type and OX- CmIAA27 Phenotypic observation and analysis of genetically modified 'Shenma' further validates CmIAA27 The regulatory effect on the development of chrysanthemum lateral branches.

[0030] The results of this study indicate that, CmCMT2 direct combination CmIAA27 Promoters regulate their methylation levels, thereby inhibiting CmIAA27 The expression, OX- CmIAA27 The transgenic 'Shenma' variety exhibited significantly longer lateral branches during its vegetative growth stage compared to the WT variety. CmCMT2 Through negative regulation CmIAA27 The expression of IAA enhances the signal transduction of IAA in the lateral branches of chrysanthemum, leading to abnormal auxin content in the lateral branches and thus regulating the development of chrysanthemum lateral branches. Attached Figure Description

[0031] Figure 1 for CmCMT2 Phenotypic analysis of regulation of lateral branching in chrysanthemum, including (A) WT and CmCMT2 -Leaf number analysis of RNAi plants; (B)WT and CmCMT2 - Stem diameter analysis of RNAi plants from the bottom third to the top; (C) WT and CmCMT2 -Internode number analysis of RNAi plants; (D)WT and CmCMT2 -RNAi plant with the eighth fully expanded leaf, target value: 1 cm; (E)WT and CmCMT2 -Statistical analysis of the length and width of the eighth unfolded leaf of RNAi plants; (F)WT and CmCMT2 -RNAi transgenic chrysanthemum plant height phenotype (60 days), target value: 2 cm; (G)WT and CmCMT2 -RNAi plant height analysis (60 days); (H)WT and CmCMT2 -RNAi plant lateral bud phenotype, target value: 2 cm; (I) WT and CmCMT2 -RNAi line lateral bud analysis, each column represents a plant, each layer represents a node in a chrysanthemum, squares indicate the range of bud length, white squares do not represent nodes;* P <0.05 ** P <0.01 (T-test); Figure 2 For WT and CmCMT2 Analysis of IAA content in lateral buds of transgenic plants, with at least 5 plants per combination. P<0.01 (T-test); Figure 3 for CmCMT2 -RNAi transgenic chrysanthemum RNA-Seq analysis, including (A) DEG quantity analysis; (B) KEGG enrichment analysis; (C) lateral branching-related gene heatmap analysis; (D) correlation analysis between lateral branching-related gene transcriptome and RT-qPCR data; (E) expression analysis of lateral branching-related genes in WT and transgenic lines #1 and #5;* p <0.05,** p <0.01; Figure 4 McrBC-PCR for genes related to plant lateral branch development; Figure 5 for CmIAA27 methylation level analysis of promoters; where (A)WT and CmCMT2 #1 CmCMT2 #5 CmIAA27 (A) Location of promoter methylation region (1273-1548 bp) and methylation status of each site in CG, CHG, and CHH types; (B) Pie chart of methylation level in CHH type (changes in methylation status of CHH sites in the red box from 1422-1495 bp); (C) WT and CmCMT2 #1 CmCMT2 #5 CmIAA27 Analysis of methylation levels of promoters of CG, CHG, and CHH types; Figure 6 For CmCMT2 and CmIAA27 Interaction analysis of promoter methylation alteration regions; where, (A) CmCMT2 and CmIAA27pro Experiments on Y1H in regions altered by methylation; (B) CmCMT2 and CmIAA27pro EMSA experiments on methylation-altered regions; (C) mCMT2 regulation CmIAA27pro Fluorescence analysis of methylation altered regions, with color bars indicating fluorescence intensity; (D) CmCMT2 regulation CmIAA27pro Activity analysis of methylation-altered regions, n=3, LUC / REN normalized, * p <0.05,** P <0.01, ns represents no difference; Figure 7 For OX- CmIAA27 Identification of transgenic chrysanthemums; including (A) DNA level identification of OX-CmIAA27 transgenic chrysanthemums; (B) Expression level of CmIAA27 gene in wild-type and overexpression lines; M: DL2000; * P <0.05 ** P<0.01 (T-test); Figure 8 for CmIAA27 Phenotypic analysis of transgenic chrysanthemums; among which, (A) WT and OX- CmIAA27 Leaf count analysis of plants; (B) WT and OX- CmIAA27 Analysis of mean internode length of plants; (C) WT and OX- CmIAA27 Analysis of stem diameter at the base of the plant; (D) WT and OX- CmIAA27 Plant height phenotype (60 days), target value: 2 cm; (E)WT and CmCMT2 -Statistical analysis of the length and width of the eighth unfolded leaf of RNAi plants; (F)WT and OX- CmIAA27 Analysis of the length and width of the eighth fully expanded leaf of the plant; (G)WT and OX- CmIAA27 Plant lateral bud phenotype, target value: 2 cm; (H)WT and CmCMT2 -RNAi line lateral bud analysis, each column represents a plant, each layer represents a node of chrysanthemum, squares indicate the range of bud length, white squares do not represent nodes; (I) WT and OX- CmIAA27 Analysis of total lateral bud length of the plant. P <0.05 ** P <0.01 (T-test); Figure 9 For CmCMT2 through mediation CmIAA27 Methylation of the promoter regulates the development of chrysanthemum lateral branches. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention. Example 1

[0033] 1.1 Experimental Materials 1.1.1 Plant materials The transgenic material used in this study was tissue culture seedlings of the chrysanthemum variety 'Shenma' preserved in our laboratory.

[0034] The material used for transcriptome sequencing in this study was chrysanthemum ( Chrysanthemum morifolium ) 'Shenma' Wild Type (WT) and CmCMT2- The RNAi transgenic 'Shenma' plant, and both are materials that the inventors used in their previous experimental work and have already disclosed, among which... CmCMT2-RNAi transgenic 'Shenma' plants were obtained by the method described in patent 202510225213.2; WT plants, CmCMT2 - Data were recorded and analyzed after 60 days of culture of RNAi transgenic plants (when lateral branching was more obvious), and samples of lateral branch / bud tissue were taken, flash-frozen in liquid nitrogen, and stored at -80°C. These samples were used for RNA extraction in transcriptome sequencing.

[0035] 1.1.2 Main Reagents Kanamycin sulfate (25389-94-0), carbenicillin disodium (4800-94-6), rifampicin (13292-46-1), MS medium (M519), agar powder (9002-18-0), sucrose (57-50-1), etc., were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Maclean); DNA extraction kit (D505, BF FT-Plant DNA kit) was purchased from Baoge Biotechnology Co., Ltd.; KK Fast Plant Total RNA Kit (ZP405K), DNA Marker (ZM404; DL2000 DNA Marker), nucleic acid electrophoresis dye (ZS203, ExRed) and K5 HiFi 2×PCR Master Mix (ZT211) were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd. (Zhuangmeng); restriction endonucleases EcoR Ⅰ (R3101V) Xho I (R0146V) Nde Ⅰ (R0111V) PstⅠ (R3140V) BamHⅠ (R3136V) Sall (R3138V) and SacⅠ(R3156V) was purchased from New England Biotechnology (Beijing) Co., Ltd. (NEB); the DNA Bisulfite Conversion Kit (DP215-02) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the Reverse Transcription Kit (RR047A, PrimeScript™ RT reagent Kit with gDNA Eraser) and the McrBC Enzyme Kit (1234A) were purchased from Takara Biotech (Beijing) Co., Ltd.; the Real-Time PCR Mix (Q711-02, ChamQ Universal SYBR qPCR Master Mix), Plasmid Extraction Kit (DC201, FastPure Plasmid Mini Kit), DNA Purification Kit (DC301, FastPure Gel DNA Extraction Mini Kit), Homologous Recombination Kit (C115, ClonExpress Ultra One Step Cloning Kit), T-Vector Ligation Kit (C603, Ultra-UniversalTOPO Cloning Kit), and Dual Luciferase Kit (DL101) were purchased from New England Biotechnology (Beijing) Co., Ltd. (NEB); the DNA Bisulfite Conversion Kit (DP215-02, DNA Bisulfite Conversion Kit) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the Reverse Transcription Kit (RR047A, PrimeScript™ RT reagent Kit with gDNA Eraser) and the McrBC Enzyme Kit (1234A) were purchased from Takara Biotech (Beijing) Co., Ltd. (Takara Biotech); the PCR kit (R3156V) was purchased from New England Biotechnology (Beijing) Co., Ltd. (NEB); the DNA Bisulfite Conversion Kit (DP215-02, DNA Bisulfite Conversion Kit) was purchased from Tiangen Biotech (Beijing) Co., Ltd. (Takara Biotech); the DNA Bisulfite Conversion Kit (DP215-02, DNA Bisulfite Conversion Kit) was purchased from Tiangen Biotech (Beijing The following reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.: yeast extract, tryptone, NaCl, NaOH, etc., were purchased from Beijing Solarbio Science & Technology Co., Ltd.: methanol and formic acid (HPLC eluent) for primary chromatography were purchased from Comio Chemical Reagent Co., Ltd.: 1 mL syringe, 0.22 μm filter membrane (organic phase), inner tube, and vial were purchased from Solarbio Science & Technology Co., Ltd.: SDS-PAGE gel preparation kit (P0012A), 5×EMSA / Gel-Shif binding buffer (GS005), nylon membrane (FFN10), blocking buffer (GS009B), washing buffer (GS009W), detection equilibration buffer (GS009A), and BeyoGold™ protein ultrafiltration tubes (FUF550) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0036] 1.1.3 Carriers and strains Vectors such as pORE-R4-2×35SAA, pHis2, pGADT7, AD-GUS, pGreenⅡ-0800-LUC, pGreen-62 SK-LUC, and pCold-TF are preserved in this laboratory.

[0037] Agrobacterium EHA105 competent cells (ZC142, Chemically Competent Cell), GV3101 competent cells (ZC141, GV3101 Chemically Competent Cell), GV3101 (pSoup-p19) strain (ZC1407, GV3101-pSoup-p19 Chemically Competent Cell), and prokaryotic expression strain (ZC121, BL21-DE3-Chemically Competent Cell) were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd. (Zhuangmeng); DH5α competent cells (C502, DH5α Chemically Competent Cell) were purchased from Nanjing Novizan Biotechnology Co., Ltd. (Novizan).

[0038] 1.2 Experimental Methods 1.2.1 CmCMT2- Observation of growth phenotype in RNAi transgenic chrysanthemum Select those with consistent growth CmCMT2- RNAi transgenic 'Shenma' plant (hereinafter referred to as...) CmCMT2 RNAi transgenic chrysanthemum and wild-type WT chrysanthemum were propagated by cuttings of terminal buds (peat moss: vermiculite = 2:1). After the cuttings rooted, they were transplanted into large nutrient pots and cultured under natural light. When the seedlings reached 60 days of age, the WT and wild-type WT chrysanthemums were further propagated by cuttings of terminal buds. CmCMT2 The plant height, number of leaves, stem diameter, number of internodes, length and width of the eighth leaf (from top to bottom), number and length of lateral branches of RNAi transgenic chrysanthemums were statistically analyzed.

[0039] 1.2.2 CmCMT2- Determination of hormone content in RNAi transgenic chrysanthemum lateral branches Select those with consistent growth CmCMT2 -RNAi transgenic 'Shenma' plant (abbreviated as...) CmCMT2RNAi transgenic chrysanthemums and wild-type WT terminal buds were propagated by cuttings in a peat moss:vermiculite = 2:1 substrate. After rooting, they were transferred to large nutrient pots and cultured under natural light. When the seedlings reached 60 days of age, the plant height, number of leaves, stem diameter, number of internodes, length and width of the eighth leaf (from top to bottom), number of lateral branches, and length of lateral branches were recorded. Subsequently, lateral branch samples were taken for hormone content determination: hormone extraction solution (methanol:water:formic acid = 80:19:1) was added, mixed on ice, and shaken at 4°C for 3-5 h, then allowed to stand overnight at 4°C; the next day, the sample was centrifuged at 4°C and 10,000 rpm for 15 min, and 800 μL of supernatant was collected; the sample was dried in the dark using a nitrogen blower (about 1.5-2 h), reconstituted with 100 μL of 40% methanol, briefly centrifuged, and filtered through a 0.22 μm organic filter membrane. The filtrate was transferred to a liquid chromatography vial with an inner tube, capped with a pre-cut cap, and used for UPLC-MS / MS (mass spectrometry) analysis.

[0040] 1.2.3 CmCMT2 RNAi transgenic chrysanthemum lateral branch transcriptome sequencing At 60 days of age, WT and CmCMT2-RNAi transgenic chrysanthemum seedlings with uniform growth were selected, and samples were taken from lateral branches. Three biological replicates (at least three plants per replicate) were obtained from each line and sent to Guangzhou GeneDio Biotechnology Co., Ltd. for RNA-seq. The company used the Trizol method to extract total RNA, and Agilent 2100 and agarose gel electrophoresis were used to assess quality. mRNA was enriched using Oligo(dT) magnetic beads, and the purified cDNA underwent end repair, A addition, and ligation with Illumina adapters. After purification with AMPure XPBeads, PCR amplification was performed, and finally sequencing was conducted on the Illumina Novaseq 6000 platform. Raw data were filtered using FASTP to obtain clean reads, aligned to the reference genome using HISAT2, and used for transcript assembly with StringTie. RSEM was used to calculate FPKM as gene expression levels, and principal component analysis was performed using the R package gmodels. Differentially expressed genes (FDR < 0.05 and |fold change| ≥ 2) were screened using DESeq2, and GO functional annotation and KEGG pathway enrichment analysis were performed on the differentially expressed genes.

[0041] 1.2.4 Transcriptome RT-qPCR Validation (1) Extract RNA according to the steps of the RNA extraction kit (ZP405K, Zhuangmeng, Beijing). CmCMT2 Total RNA was collected from RNAi transgenic chrysanthemum and wild-type (WT) chrysanthemum lateral branch samples. RNA was reverse transcribed into cDNA following the reverse transcription kit steps and procedures (RR047A, Takara).

[0042] (2) RT-qPCR verification: Candidate genes were screened from differentially expressed genes in the transcriptome. The corresponding CDS sequences were obtained from the Nanjing Agricultural University Chrysanthemum website (https: / / cgd.njau.edu.cn / asteraceae / homePage). Quantitative primers were designed based on the sequences using Premier.5.0 software. The cDNA obtained from the previous reverse transcription was subjected to real-time PCR (the reaction system and procedure are shown in Tables 1 and 2).

[0043] Table 1. Reaction system.

[0044]

[0045] Table 2 Reaction Procedure.

[0046]

[0047] 1.2.5 Methylation-sensitive restriction endonuclease PCR (McrBC-PCR) (1) DNA extraction: DNA was extracted using a rapid DNA extraction kit (D505, Baoge, Zhengzhou). CmCMT2 Genomic DNA from lateral branch samples of RNAi transgenic chrysanthemum and WT.

[0048] (2) Enzyme digestion: 500 ng of sample gDNA was added to 20 μL of McrBC enzyme digestion system; the digestion was carried out overnight at 37℃. GTP was added as a positive control, and ddH2O was used instead of GTP as a negative control (see Table 3 for the enzyme digestion system). The digested product (1 μL) was used as a template for PCR amplification. The amplification program was 95℃, 3 min; (95℃, 15 s; 58℃, 15 s; 72℃, 15 s), 32 cycles; 72℃, 5 min; 4℃, storage.

[0049] Table 3 McrBC digestion system.

[0050]

[0051] 1.2.6 Bisulfite Sequencing PCR (BSP-PCR) The sample gDNA (500 ng) was processed according to the instructions of the DNA bisulfite conversion kit (DP215, Tiangen, Beijing). Primers for BSP-PCR amplification were designed using the MethPrimer website. 2 μL of the processed gDNA was used as a template for PCR amplification. The amplified product was subjected to 1.0% agarose gel electrophoresis, gel excision, and purification using a gel extraction kit (DC301, Novizan, Nanjing). The purified product was ligated into a T vector (C603, Novizan), and the recombinant vector was then transformed into DH5α competent cells (C603, Novizan). Single clones were picked for PCR. The amplified product was checked for the presence of the target band using 1.0% agarose gel electrophoresis. After verification as positive bacteria, sequencing was performed (Qingke). Fifteen positive single clones from each group were sequenced. CyMATE was used to analyze the cytosine methylation level of the sequencing results.

[0052] 1.2.7 Yeast (Y1H) Single-hybrid Experiment (1) Primers with corresponding vector homologous arm sequences were designed using Premier 5.0 software. Using 'Shenma' as material, PCR amplification was performed on gDNA. CmIAA27 Gene promoter methylation fragments amplified from cDNA CmCMT2 .use Eco RⅠ and enzymes Sac I. The pHIS2 plasmid was digested with enzymes, and then... Eco R Ⅰ and Bam The pGADT7 plasmid was digested with H I and incubated overnight at 37°C. The promoter sequence was then constructed into the pHIS2 linear vector using a homologous recombination kit (C115, Novizan). CmCMT2 The sequence was constructed into the pGADT7 linear vector.

[0053] (2) Y187 yeast strain was placed on YPDA solid medium and cultured at 30℃ for 3 days. Single colonies were picked and cultured in 20 mL of YPDA liquid medium at 28℃ and 200 rpm for 12 h. 30 mL of YPDA liquid medium was added and cultured at 28℃ and 200 rpm for 2-3 h until the cells were activated to OD. 600 =1.0. Transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature; discard the supernatant, add 30 mL of sterile water to the centrifuge tube to suspend the bacterial cells, and centrifuge at 1000 rpm for 5 min at room temperature; discard the supernatant, add 1.5 mL of 1×TE / LiAC to the centrifuge tube to suspend the bacterial cells, transfer the suspension to two 2 mL centrifuge tubes, and centrifuge at 12000 rpm for 15 s at room temperature; discard the supernatant, add 600 μL of 1.1×TE / LiAC to each centrifuge tube, and place the competent yeast cells on ice for later use.

[0054] (3) Add 1000 ng of the constructed recombinant plasmid (control group: pGADT7-GUS+pHIS2-) to a pre-cooled 2 mL centrifuge tube. CmIAA27pro Experimental group: pGADT7- CmCMT2 +pHIS2- CmIAA27pro Add 10 μL of Yeast maker Carrier DNA and 100 μL of Y187 competent cells, then add 500 μL of PEG / LiAc, gently pipette to mix, and incubate at 28℃ and 200 rpm for 30 min; add 20 μL of DMSO, pipette to mix, incubate at 42℃ for 15 min, gently shake to mix every 5 min, centrifuge at 12000 rpm for 15 s at room temperature, discard the supernatant, and add 100 μL of 1×TE to suspend; spread the suspension on SD-Trp / -His / ~Leu solid medium and incubate at 30℃ for 3-5 days.

[0055] (4) Pick a single colony and place it in 1 mL of sterile water. Mix thoroughly by pipetting and adjust to OD. 600 =1.0. Dilute the bacterial suspensions to 10... -1 10 -2 and 10 -3 Diluted bacterial cultures (2 μL each) were taken from the control and experimental groups and cultured on SD-Trp / -His / -Leu solid medium containing 0, 20, 40, 60, 80, and 100 mM 3-AT, respectively, at 30°C for 3–5 days. Once the colonies on the 0 mM 3-AT SD-Trp / -His / -Leu solid medium reached a rounded state, the overall growth of each plate was photographed.

[0056] 1.2.8 Gel Electrophoresis Migration Assay (EMSA) (1) Primers with homologous arm sequences of the pCold-TF vector were designed using Premier 5.0 software, and amplified from 'Shenma' cDNA by PCR. CmCMT2 Gene sequence; use Nde I and Pst I. The pCold-TF plasmid was digested with enzymes overnight at 37°C. The plasmid was then digested using a homologous recombination kit (C115, Novizan). CmCMT2 The plasmid was ligated into the pCold-TF expression vector. The recombinant plasmid was then transformed into the prokaryotic expression strain BL21(DE3).

[0057] (2) Pick a single colony and place it in 5 mL of LB medium containing Amp resistance, and incubate at 37°C and 200 rpm for 12 h; take 2.5 mL of bacterial culture and add it to 250 mL of LB medium containing Amp resistance, and incubate at 37°C and 200 rpm for 4-5 h to activate the strain to OD. 600 =0.6, add IPTG to make the final concentration 1 mM (take 1 mL of bacterial culture before adding IPTG as a pre-induction control), incubate in a 15℃ water bath for 30 min, and then incubate at 15℃ and 100 rpm for 16 h; take 1 mL of the cultured bacterial culture as the induction bacterial culture, centrifuge the remaining bacterial culture at 4℃ and 5000 rpm for 10 min, discard the supernatant, and retain the bacterial precipitate.

[0058] (3) Add 5 mL of sonication buffer to suspend the bacterial precipitate, transfer the bacterial solution to a centrifuge tube, add 250 μL of 100 mM protease inhibitor PMSF to 25 mL of resuspended bacterial solution, and mix by inversion; place the bacterial cells in a 1 L beaker filled with ice and sonicate for 20 min (sonic intensity 220-300 w, continuous sonication for 4 s, stop for 5 s) until the bacterial cells are clear; centrifuge the bacterial solution at 4℃ and 13000 rpm for 40 min, take 1 mL of supernatant as the supernatant after sonication for later use, discard the supernatant, and keep the precipitate as the precipitate after sonication.

[0059] (4) Pipette 2 mL of resuspended HIS-tagged protein agarose gel (NTA-Ni) into the chromatography column, add 10 mL of binding buffer to equilibrate the gel, and repeat twice; close the lower outlet of the chromatography column, add the prepared HIS-tagged CmCMT2 protein into the chromatography column, close the upper outlet of the chromatography column, place it on a shaker at 50 rpm, and incubate at room temperature for 2 h; after incubation, open the inlet and outlet of the chromatography column, and after all the supernatant flows out, collect the supernatant as flow-through and place it in a 4℃ refrigerator for subsequent detection; add 10 mL of washing buffer into the chromatography column, collect the washing buffer, place it in a 4℃ refrigerator for subsequent detection, and repeat washing 4 times; add 1 mL of elution buffer into the chromatography column, and use 5 1.5 mL centrifuge tubes to collect the eluent, collecting about 200 μL of eluent from each tube.

[0060] (5) Prepare an SDS-PAGE gel. Load the pre-induction, post-induction, sonication supernatant, post-sonication precipitate, flow-through, washing buffer, elution buffer, and original sample onto the gel. Perform electrophoresis at 220 V for 30 min. Remove the SDS-PAGE gel and add an appropriate amount of rapid protein staining solution to stain the gel. Place it on a shaker at 100 rpm and stain for 30 min. Observe the purification effect. Rinse the ultrafiltration tube with PBS buffer, add elution buffer to the ultrafiltration tube, centrifuge at 4000 rpm for 15-90 min at room temperature, collect the filtrate, determine the protein concentration, and store it in a -80℃ freezer for subsequent EMSA experiments.

[0061] (6) Use the biotin-labeled and non-biotin-labeled protein binding sequence primers to synthesize the biotin-labeled primers. Use PCR to synthesize double-stranded primers. The reaction system and procedure are shown in Tables 4 and 5.

[0062] Table 4. EMSA double-chain synthesis reaction system.

[0063]

[0064] Table 5. EMSA double-chain synthesis reaction procedure.

[0065]

[0066] (7) Add ultrapure water, labeled probe, protein, and EMSA binding buffer to a 200 μL PCR tube. Do not add cold probe to the cold probe group. The reaction system is shown in Table 6. After instantaneous centrifugation, let the tube stand at room temperature for 30 min. Add labeled probe to the cold probe group, and then add 1 μL of 10× Loading buffer to each group before centrifugation for subsequent non-denaturing PAGE gel electrophoresis.

[0067] Table 6. EMSA reaction system.

[0068]

[0069] (8) Prepare an 8% non-denaturing PAGE gel. Place the prepared gel in an electrophoresis apparatus filled with 1×TBE solution and load the protein marker, control group, experimental group, 500× cold probe group, and 1000× cold probe group in sequence. Electrophore at 100 V for 1 h. Soak the nylon membrane in 1×TBE solution for 10 min. Place the black filter cotton, double-layer filter paper, nylon membrane, PAGE gel, double-layer filter paper, and black filter cotton in the transfer clamp from top to bottom. Clamp the transfer clamp and transfer the membrane into an electrophoresis apparatus filled with 1×TBE solution. Wrap the electrophoresis apparatus with ice. Transfer at 380 mA for 30 min. After the transfer is complete, remove the nylon membrane, blot the surface moisture with qualitative filter paper, place it in a square petri dish, and irradiate it under a UV lamp at a distance of 5-10 cm for 15 min. Add 15 mL of blocking solution to the culture dish and incubate at 75 rpm for 15 min on a shaker. Discard the blocking solution and add 7.5 μL of horseradish peroxidase to a new 15 mL blocking solution. Mix well by pipetting and then add the mixture to the culture dish containing the nylon membrane. Incubate at 75 rpm for 15 min on a shaker.

[0070] (9) Mix 25 mL of 5× washing solution and 100 mL of deionized water to prepare 1× washing solution. Discard the blocking solution in the culture dish, add 15 mL of 1× washing solution, and incubate on a shaker at 75 rpm for 1 min. Discard the washing solution and repeat the washing process three times. After discarding the washing solution, add 20 mL of blocking solution to the culture dish and incubate on a shaker at 75 rpm for 5 min. Under light-protected conditions, mix 5 mL of developer A and 5 mL of developer B, blot dry the surface of the nylon membrane with qualitative filter paper, place it in a square culture dish, pour 10 mL of the mixture into the culture dish, incubate at room temperature for 3 min, and then develop the membrane. Observe the results.

[0071] 1.2.9 Dual-luciferase assay (LUC) Design primers with homologous arms to amplify 'shenma' gDNA. CmIAA27 Promoter methylation changes fragments amplified from cDNA CmCMT2 The sample was recovered and purified by agarose gel electrophoresis. Sal Ⅰ / Bam HⅠ double digestion of pGreenⅡ 0800 vector, using Eco R Ⅰ / Bam The pGreenII 62-SK vector was digested with H I double enzymes, and the linearized vector was recovered after overnight digestion at 37°C. The promoter fragment was cloned into pGreenII 0800 and CmCMT2 was cloned into pGreenII 62-SK via homologous recombination, and then transformed into GV3101 (pSoup-p19) Agrobacterium.

[0072] Agrobacterium containing the recombinant plasmid and Agrobacterium pGreenⅡ 62-SK empty vector (for control) were removed from -80℃ and added to LB liquid medium containing Kan resistance, and cultured at 28℃ and 200 rpm until OD. 600 =0.8, centrifuge to collect bacterial cells, and resuspend in suspension (10 mmol / L MgCl2, 10 mmol / L MES, pH 5.6, containing 200 μmol / L acetylsuccinone). Follow the control group (pGreenⅡ 62-SK empty vector + pGreenⅡ 0800- CmIAA27pro ) and experimental group (pGreenⅡ 62-SK- CmCMT2 + pGreenⅡ 0800- CmIAA27pro Mix the two bacterial cultures in equal proportions, wrap them in aluminum foil, and incubate them in the dark at room temperature for 4 hours.

[0073] Selected 4-6 leaf-age *Nicotiana benthamiana* plants. Marked equal-area areas on both sides of the midrib of fully expanded leaves, and injected bacterial suspension into the control and experimental groups using a 1 mL syringe. After injection, the leaves were incubated in the dark for 1 day, then transferred to a light incubator for 2-3 days. The tobacco leaves were removed, coated with 100 mM sodium salt solution, and exposed to light using a luminometer for 10 min to record the results. The developed leaves were placed in 2 mL centrifuge tubes containing glass beads, frozen in liquid nitrogen, and then pulverized. The activities of LUC and REN enzymes were detected using a dual-luciferase reporter kit, with the LUC / REN ratio as the final result. Each group was repeated three times.

[0074] 1.2.10 OX- CmIAA27 Obtaining and phenotypically observing transgenic chrysanthemums Amplification CmIAA27 Sequence, after recovery Eco R Ⅰ / Xho I. The pORE-R4-2×35SAA vector was double-digested with enzymes, and the pORE-R4-2×35SAA-CmIAA27 overexpression vector was obtained by homologous recombination. This vector was transformed into EHA105 Agrobacterium competent cells and stored at -80℃ for later use.

[0075] The terminal buds of wild-type 'Shenma' tissue culture seedlings were used for propagation. After 35 days of culture, the 3rd to 6th fully expanded leaves were taken and cut into 0.2 × 0.2 cm leaf discs, avoiding the midrib. The discs were placed face up on the pre-culture medium (containing 1.0 mg / L 6-BA and 0.5 mg / L NAA) and cultured for 3 days. On the evening of the second day of pre-culture, the preserved Agrobacterium was activated to OD600≈0.5, centrifuged, and adjusted to OD600≈0.5 with resuspended medium (MS + 30 g / L sucrose, pH 5.8). The leaf discs were immersed in the bacterial solution for 8 min, blotted dry with filter paper, air-dried in a clean bench for 10 min, and then transferred to the original pre-culture medium for dark co-culture for 3 days. After co-culturing, the seedlings were transferred to decarboxylation medium (containing 500 mg / L Carb) and cultured under light for 7 days. Subsequently, they underwent four selection stages: Selection I, Selection II, Selection III, and Selection IV (the medium was changed every two weeks, with kanamycin concentration gradually reduced from 10 mg / L to 8 mg / L and carbenicillin from 350 mg / L to 200 mg / L). For Selection IV, glass bottles were used. When resistant shoots reached 2-3 cm in length, individual plants were cut and transferred to rooting selection medium (containing 8 mg / L Kan and 200 mg / L Carb). Seedlings that rooted normally were retained, and DNA was extracted from leaves for PCR verification (using Agrobacterium as a positive control, wild-type as a negative control, and water as a blank control). Transgenic seedlings were then propagated and grown in medium for 14 days before being transplanted into soil.

[0076] Select transgenic OX- with consistent growth CmIAA27 Wild-type WT terminal bud cuttings were propagated in a peat moss:vermiculite = 2:1 substrate and cultured until 60 days old. Plant height, number of leaves, stem diameter, length and width of the eighth leaf, number and length of lateral branches were recorded for phenotypic comparison.

[0077] 2.1 Results and Analysis 2.1.1 CmCMT2 Phenotypic analysis of regulation of chrysanthemum lateral branching Phenotypic observations of various transgenic lines over two consecutive years revealed that... CmCMT2 RNAi transgenic chrysanthemums exhibited more lateral buds during the vegetative growth stage compared to WT chrysanthemums. To investigate... CmCMT2 The regulatory effect on lateral branching in chrysanthemum was investigated by phenotypic analysis of lines #1 and #5. The results showed no significant differences in plant height, leaf number, stem diameter, and internode number between 60-day-old WT chrysanthemum seedlings and transgenic lines #1 and #5. Figure 1 A, B, C); the leaf length and width of the eighth leaf in transgenic lines #1 and #5 were significantly lower than those in WT (A, B, C). Figure 1 D, E); the number and length of lateral branches in transgenic lines #1 and #5 were significantly higher than those in WT ( Figure 1 H, I).

[0078] Methyltransferase gene CmCMT2 The nucleotide sequence is shown in SEQ ID NO.4.

[0079] 2.1.2 WT and CmCMT2 Analysis of IAA content in RNAi transgenic chrysanthemum lateral buds Previous studies have shown that auxin (IAA) plays an important regulatory role in the development of lateral branches in plants, providing a basis for further research. CmCMT2 The molecular mechanism regulating lateral branching in chrysanthemum CmCMT2 Lateral branches of transgenic lines and WT were sampled, and the content of IAA was detected. The results showed that... CmCMT2 The IAA content in transgenic lines #1 and #5 was significantly higher than that in WT lines by 10.59% and 69.13%, respectively. Figure 2 ).

[0080] 2.1.3 Chrysanthemum WT and CmCMT2 Transcriptome analysis of RNAi transgenic lines For further research CmCMT2 To investigate the molecular mechanisms regulating lateral branch development in chrysanthemum, transcriptome sequencing was performed on the lateral branches of transgenic lines #5 and WT. Transcriptome data quality assessment results showed that the alignment rate between the transcriptome data and the genome data of the chrysanthemum variety 'Zhongshan Zigui' was between 75.12% and 77.36% (Table 7), indicating the reliability of the transcriptome data. Differentially expressed genes (DEGs) analysis revealed 15,302 upregulated DEGs and 6,027 downregulated DEGs in the transgenic lines. Figure 3 A). KEGG enrichment analysis showed that DEGs were concentrated in pathways such as protein biosynthesis, photosynthesis, and secondary metabolism. Plant lateral branch development is closely related to hormones; therefore, the focus was placed on DEGs in secondary metabolic biosynthesis and metabolic pathways. Figure 3 B).

[0081] Analysis of DEGs in secondary metabolic biosynthetic pathways and metabolic pathways identified nine DEGs that may be related to lateral branch development, namely, genes related to the brassinolide synthesis pathway. CmDWF1 , CmDWF4 , CmDET2 , CmCYP92A6 (Metabolic pathway), sucrose synthase gene CmSUS1 (Metabolic pathway), abscisic acid 8' hydroxylase gene CmCYP707A (Metabolic pathway), genes negatively regulating abscisic acid signaling CmWRKY40, Gibberellin synthase gene CmGA20ox1 (Metabolic pathways) and genes negatively regulating auxin signaling CmIAA27 (hormone signal transduction pathway) Figure 3C). To verify the reliability of the transcriptome data, RT-qPCR was performed on nine candidate genes. Linear regression analysis further showed that the FPKM value of the transcript was highly correlated with the RT-qPCR data. Figure 3 D). RT-qPCR results showed that the expression levels of the nine candidate genes were consistent in both transgenic lines. Figure 3 E), indicating that the transcriptome data is reliable.

[0082] Table 7. Transcriptome data quality assessment.

[0083]

[0084] 2.1.4 Analysis of methylation status of promoters of genes related to lateral branch development To further explore CmCMT2 The regulatory mechanism governing lateral branch development in chrysanthemum was investigated by examining the methylation status of promoters of nine candidate genes. The promoters were divided into 2-3 fragments, digested with McrBC, and the digestion products were subjected to PCR. The results showed that... CmDWF1 , CmDWF4 , CmDET2 , CmCYP92A6, CmSUS1, CmCYP707A, CmWRKY40 and Cm GA20ox1 There was no significant difference in the bands of the genes in the transgenic lines and WT, indicating that the methylation level of the promoters of these genes remained unchanged; CmIAA27 fragments of gene promoters CmIAA27pro In the -1 region, the transgenic lines showed higher band brightness than the WT lines, indicating... CmIAA27pro The methylation level in the -1 region was lower than that in WT. Figure 4 ).

[0085] CmIAA27 The nucleotide sequence of the gene is shown in SEQ ID NO.1. CmIAA27 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0086] Chrysanthemum auxin signal negative regulatory gene promoter (i.e. CmIAA27 The nucleotide sequence of the gene promoter is shown in SEQ ID NO.3.

[0087] 2.1.5 CmIAA27 Promoter methylation level analysis For further analysis CmIAA27pro The methylation level of the -1 region was detected using BSP experiments. The results showed that this region contained 6 CG sites, 2 CHG sites, and 37 CHH sites, with significant changes in methylation in the area within the red box. Figure 5A). Fan-shaped analysis of methylation of cytosine in the red-framed region revealed that transgenic lines #1 and #5 had significantly less methylated cytosine compared to WT. Figure 5 B). Methylation level analysis showed that in WT and transgenic lines #1 and #5, the CG type was 95%, 91.67%, and 86.67%, respectively; the CHG type was 5%, 5%, and 10%, respectively; and the CHH type was 21.62%, 4.03%, and 4.03%, respectively. Figure 5 C). Previous studies have shown that... CmCMT2 It primarily maintains CHH-type methylation and mediates CHG methylation to a lesser extent. These results, taken together, indicate that... CmCMT2 Possibly through mediation CmIAA27 The level of CHH-type methylation in the promoter region regulates and thus controls CmIAA27 The expression.

[0088] 2.1.6 CmCMT2 and CmIAA27 Interaction analysis of promoter methylation regions To investigate CmCMT2 Regulation CmIAA27 The molecular mechanism was determined using AD-GUS as a negative control via Y1H experiments. CmCMT2 and CmIAA27 The binding relationship of the promoter methylation region was investigated. Results showed that at a concentration of 40 mM 3-AT, the control group AD-GUS+pHis2- CmIAA27pro Yeast strain growth was inhibited, while AD- CmCMT2 +pHis2- CmIAA27pro The yeast strain can grow normally ( Figure 6 A), indicating that CmCMT2 may be related to CmIAA27 The promoter methylation region binds directly.

[0089] To further verify the relationship between CmCMT2 protein and CmIAA27 The binding of the promoter methylation region sequence was investigated using EMSA experiments. The results showed that the negative control group had no protein binding band, while the conventional group had a protein binding band. The binding affinity gradually weakened with increasing competitive probe concentration. Figure 6 B) indicates that CmCMT2 can specifically bind in vitro. CmIAA27 Promoter methylation region.

[0090] To verify CmCMT2 and CmIAA27 The regulatory relationship of promoter methylation regions was investigated using LUC experiments. The results showed no significant difference in fluorescence signals between the control and experimental groups, and no significant difference in dual-luciferase activity either. Figure 6 (C, D) indicates that CmCMT2 does not directly participate. CmIAA27The regulatory role of the promoter methylation region. The above results indicate that CmCMT2 regulates the methylation region by binding to... CmIAA27 Promoter methylation regions regulate the methylation state of these regions, thereby regulating the occurrence of side branches.

[0091] 2.1.7 Chrysanthemum CmIAA27 Construction of hyperexpression vectors and CmIAA27 Expression analysis To further explore CmIAA27 The branching mechanism regulating the development of lateral branches in chrysanthemums was constructed. CmIAA27 Overexpression vectors were used, and five DNA-level genes were identified via Agrobacterium-mediated leaf disc transformation. CmIAA27 Overexpression of transgenic chrysanthemum ( Figure 7 A). RT-qPCR results showed that these 5 CmIAA27 Compared to WT, genetically modified organisms (GMOs) CmIAA27 Expression levels increased significantly by 0.99-2.70 times. Figure 7 B).

[0092] 2.1.8 CmIAA27 Phenotypic observation of transgenic chrysanthemums with overexpression To clarify CmIAA27 Biological functions in the development of chrysanthemum lateral branches, selection CmIAA27 Two independent transgenic lines (OX-4 and OX-5) with high expression levels were subsequently analyzed for morphological phenotypic differences. The results showed that, compared with the wild-type WT, the transgenic lines OX-4 and OX-5 had significantly higher leaf numbers (14.23% and 8.99%, respectively). Figure 7 A), the average intersegmental length was significantly shortened by 14.37% and 12.01%, respectively. Figure 7 B); the basal stem diameter decreased significantly by 9.57% and 9.33%, respectively ( Figure 7 C). The leaf width of the eighth leaf in transgenic lines OX-4 and OX-5 was significantly shorter than that in WT, and the leaf length of transgenic line OX-4 was significantly shorter than that in WT. The leaf length of transgenic line OX-5 showed no significant difference compared to WT. Figure 7 F); Compared to WT, the transgenic lines OX-4 and OX-5 had significantly higher numbers and lengths of lateral branches than WT. Figure 7 G, H), the total lateral branch length increased by 56.39% and 38.45% respectively. Figure 7 I). In summary, CmIAA27 By promoting leaf development, inhibiting internode and main stem elongation, and significantly promoting lateral branch development, the chrysanthemum is shaped into a compact and vigorously branching plant.

[0093] 2.1.9 CmCMT2 By inhibiting CmIAA27 Expression thereby regulates the development of plant lateral branches In wild-type plants, the DNA methyltransferase CmCMT2 can directly bind to the auxin signaling repressor gene. CmIAA27 The promoter region specifically maintains the CHH-type DNA methylation level in that region. This epigenetic modification persistently suppresses... CmIAA27 The transcription of auxin leads to its expression level remaining low. Consequently, the inhibition of the auxin (IAA) signaling pathway is relieved, and the activity of the signaling pathway is enhanced, ultimately resulting in a significant inhibition of chrysanthemum lateral branch development. Conversely, in CmCMT2 -RNAi plants CmCMT2 Gene expression was effectively knocked down. This led to CmIAA27 The CHH methylation level in the promoter region was significantly reduced, thereby relieving the epigenetic repression of the gene, making... CmIAA27 The expression level of CmIAA27 protein was abnormally elevated. High levels of CmIAA27 protein enhanced its inhibitory ability on the auxin signaling pathway, thereby blocking the auxin signal transduction process and ultimately promoting the development and elongation of chrysanthemum lateral branches. Figure 9 ).

[0094] The primers and probes involved in this invention are as follows: Table 8. Primers related to RT-qPCR.

[0095]

[0096] Table 9. Primers related to McrBC and BSP.

[0097]

[0098] Table 10 Primers related to vector construction.

[0099]

[0100] Table 11 EMSA probe sequences.

[0101]

Claims

1. A gene that negatively regulates chrysanthemum auxin signaling, characterized in that, The gene is CmIAA27 ; CmIAA27 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. CmIAA27 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. The application of the chrysanthemum auxin signal negative regulatory gene as described in claim 1 in regulating the plant architecture of Asteraceae plants, characterized in that, Chrysanthemum is a plant belonging to the Asteraceae family.

3. The application of the chrysanthemum auxin signal negative regulatory gene as described in claim 1 in regulating the occurrence or development of lateral branches in Asteraceae plants, characterized in that, Chrysanthemum is a plant belonging to the Asteraceae family.

4. The application according to claim 2 or 3, characterized in that, The chrysanthemum auxin signal negative regulatory gene, by expressing the CmIAA27 protein, can enhance its ability to inhibit the auxin signaling pathway, thereby blocking the auxin signal transduction process and promoting the development and elongation of chrysanthemum lateral branches.

5. The application according to claim 2 or 3, characterized in that, The chrysanthemum auxin signal negative regulatory gene promotes leaf development, inhibits internode and main stem elongation, and promotes lateral branch development, thus shaping a compact and vigorously branching chrysanthemum plant.

6. The application according to claim 2 or 3, characterized in that, The chrysanthemum variety mentioned is either 'Shenma' or 'Zhongshan Zigui'.

7. Methyltransferase gene CmCMT2 Its application in regulating the occurrence or development of lateral branches in Asteraceae plants is characterized by... Chrysanthemum is a plant belonging to the Asteraceae family.

8. The application according to claim 7, characterized in that, Methyltransferase gene CmCMT2 By inhibiting CmIAA27 Expression thereby regulates the development of plant lateral branches.

9. The application according to claim 7, characterized in that, Methyltransferase gene CmCMT2 By direct combination CmIAA27 The promoter regulates its methylation level, thereby inhibiting CmIAA27 The expression.

10. The application according to claim 7, characterized in that, Methyltransferase gene CmCMT2 Through negative regulation CmIAA27 The expression of auxin enhances the signal transduction of auxin in the lateral branches of chrysanthemum, leading to abnormal auxin content in the lateral branches and thus regulating the development of chrysanthemum lateral branches.

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