Application of methyltransferase genes CmCMT2 and CmDRM2 in regulation and control of flowering of chrysanthemum

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of methyltransferase genes CmCMT2 and CmDRM2 in regulation and control of flowering of chrysanthemum. According to the invention, CmCMT2 and CmDRM2 genes in a chrysanthemum variety 'Shenma' are separated, an RNAi silent expression vector is constructed, CmCMT2-RNAi and CmDRM2-RNAi transgenic 'Shenma' plants are obtained by using a genetic transformation method, then phenotype observation and statistics are carried out on the transgenic plants, key genes influencing chrysanthemum flowering are excavated by using an RNA-seq method, and a mechanism of the CmCMT2 and CmDRM2 genes influencing chrysanthemum early flowering is explored. The CmCMT2 gene and the CmDRM2 gene are cloned from a chrysanthemum variety 'Shenma', and spatio-temporal expression analysis shows that the CmCMT2 gene and the CmDRM2 gene are highly expressed in the process from bud development to coloring of the 'Shenma'; moreover, in the growth and development process of the'miraculous horse ', the expression levels of the CmCMT2 and the CmDRM2 in leaves are gradually reduced. Research results show that the CmCMT2 and the CmDRM2 change the content of the GA1 by negatively regulating the expression of the CmG20ox2a and the CmG20ox2b, so that the florescence of the chrysanthemum is respectively advanced by 15 days and 8 days.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of methyltransferase genes CmCMT2 and CmDRM2 in regulating the flowering of chrysanthemum. Background Art

[0002] DNA methylation is crucial for regulating gene transcription, transposon silencing, and genomic imprinting in eukaryotic genomes. It also plays a crucial role in regulating plant growth and development. In addition to CG methylation, plant DNA also contains CHG and CHH methylation types. It is generally believed that CHH methylation is maintained exclusively by DRM2 through the RdDM pathway. However, in Arabidopsis, DRM2-mediated CHH methylation accounts for only approximately 35% of total CHH methylation. CMT2 maintains approximately 70% of CHH methylation levels independently of RdDM. Furthermore, the DRM2-mediated RdDM pathway primarily methylates CHH at more homochromatic regions, including short transposable elements (TEs) within chromosome arms and at the edges of long TEs within pericentromeric regions. At more heterochromatic sites, such as long TEs, CHH methylation is mediated by CMT2. Studies have shown that in Arabidopsis, DRM2 is strongly expressed in petals, anthers, and stigmas. In kiwifruit, CMT2 is prominently expressed in flowers, with DRM2 expression more pronounced in stems and flowers than in early developmental stages. In Xanthoceras sorbifolia, XsCMT2 expression decreases during flower development. In summary, the distinct expression patterns of CMT2 and DRM2 genes in flowers suggest that they may play important regulatory roles in flower development and flowering.

[0003] Gibberellic acid (GA) is an important hormone involved in cell growth and differentiation and has been shown to promote flowering in many plants, including Arabidopsis thaliana. GA comprises a large class of compounds and intermediates, with over 130 GA species currently identified. Of these, only GA1, GA3, GA4, and GA7 are considered biologically active, with GA1 and GA4 being the primary active GAs promoting plant growth and development. In Arabidopsis thaliana, under LD conditions, GA acts simultaneously with CO to induce flowering by promoting the expression of FT and TSF in leaves. Under SD conditions, GA is required to promote expression of the LFY promoter. Consequently, the GA-deficient ga1-3 mutant fails to express LFY and SOC1, resulting in a failure to flower under SD conditions. Although extensive research has examined how GA influences the flowering pathway, the mechanisms by which altered methylation levels, particularly those of non-CG methylation, influence GA content and flowering time remain unclear. Summary of the Invention

[0004] The purpose of the present invention is to isolate the CmCMT2 and CmDRM2 genes in the chrysanthemum variety "Shenma", construct an RNAi silencing expression vector, obtain CmCMT2-RNAi and CmDRM2-RNAi transgenic "Shenma" plants by genetic transformation, then observe and count the phenotypes of the transgenic plants, and use RNA-seq to explore the key genes that affect chrysanthemum flowering, and explore the mechanism by which the CmCMT2 and CmDRM2 genes affect the early flowering of chrysanthemum.

[0005] To achieve the above objectives, the present invention first cloned the CmCMT2 and CmDRM2 genes from the chrysanthemum variety "Shenma" through bioinformatics methods, and identified them through fluorescence quantitative PCR, subcellular localization, transgenic verification, RNA-seq and other analytical methods.

[0006] Furthermore, the present invention provides the use of the chrysanthemum methyltransferase gene CmCMT2 and / or CmDRM2 in regulating the growth and development, flowering timing or flowering period of Asteraceae plants (e.g., chrysanthemum). Specifically, the chrysanthemum variety is "Shenma" or "Zhongshan Zigui".

[0007] Specifically, the present invention is based on the chrysanthemum methyltransferase genes CmCMT2 and CmDRM2, by constructing an RNAi gene silencing expression vector, transforming Agrobacterium competent cells, and transforming chrysanthemum leaves through Agrobacterium-mediated method, thereby studying the effects of CmCMT2 and / or CmDRM2 on the growth and development, flowering timing or flowering period of chrysanthemum.

[0008] Specifically, the decreased expression levels of the chrysanthemum methyltransferase genes CmCMT2 and CmDRM2 can lead to varying degrees of early flowering, that is, the early flowering period.

[0009] Furthermore, the present invention also provides the use of the methyltransferase gene CmCMT2 and / or CmDRM2 in regulating flower bud development, regulating flower color or improving flowering period of Asteraceae plants (such as chrysanthemum).

[0010] Furthermore, the present invention also provides the use of chrysanthemum methyltransferase genes CmCMT2 and / or CmDRM2 in regulating gibberellin synthesis during the flowering period.

[0011] Furthermore, the present invention also provides the use of an RNAi gene silencing expression vector in regulating the growth and development, flowering timing or flowering period of Asteraceae plants (such as chrysanthemum).

[0012] Specifically, during application, the specific sequences of the CmCMT2 gene and the CmDRM2 gene are connected to a composite vector constructed by plasmids pART27 and pKANNIBAL to obtain an RNAi gene silencing expression vector, and then the constructed RNAi gene silencing expression vector is transformed into Agrobacterium competent cells, and then transformed into leaves of Asteraceae plants (such as chrysanthemum) through Agrobacterium-mediated method.

[0013] Furthermore, the present invention also provides an RNAi gene silencing expression vector constructed using the CmCMT2 gene and the CmDRM2 gene, and the plasmids pART27 and pKANNIBAL.

[0014] Specifically, the RNAi gene silencing expression vector is constructed using the following method:

[0015] 1) Plasmids pART27 and pKANNIBAL were digested with Not I, and the intron-containing portion of plasmid pKANNIBAL was ligated into plasmid pART27 using T4 DNA ligase to construct a composite expression vector;

[0016] 2) Based on the conserved sequences of the CmCMT2 gene and the CmDRM2 gene, a 200-500 bp RNAi fragment was designed, and primers were designed using Premier 5.0 software. The primers were then ligated with the composite vector prepared in step 1).

[0017] Specifically, the RNAi fragment sequence of the CmCMT2 gene is shown as SEQ ID NO.1, and the RNAi fragment sequence of the CmDRM2 gene is shown as SEQ ID NO.2.

[0018] Specifically, during the construction of the RNAi gene silencing expression vector, some primer sequences used are as follows:

[0019] Positive CMT2-F-XhoⅠ:TTGGAGAGGACACGCTCGAG;

[0020] CTTACCTCGTCCGGGTGATGTTGAT;

[0021] Positive CMT2-R-KpnⅠ:ATTTCCTTACCAAGCTGGGGTACC;

[0022] ATTGAGGAAGACCATAACAGCCAGC;

[0023] Anti-CMT2-F-XbaⅠ:TCATTAAAGCAGGACTCTAGA;

[0024] CTTACCTCGTCCGGGTGATGTTGAT;

[0025] Anti-CMT2-R-HindIII:GGTTCGAAATCGATAAGCTT;

[0026] ATTGAGGAAGACCATAACAGCCAGC;

[0027] Positive DRM2-F-XhoⅠ:TTGGAGAGGACACGCTCGAG;

[0028] TATGACGAGAGTCTTCTTGATGG;

[0029] Positive DRM2-R-KpnⅠ:ATTTCCTTACCAAGCTGGGGTACC;

[0030] GTCCCTTCTGCTTCTTTCTTTTC;

[0031] Anti-DRM2-F-XbaⅠ:TCATTAAAGCAGGACTCTAGA;

[0032] TATGACGAGAGTCTTCTTGATGG;

[0033] Anti-DRM2-R-HindⅢ:GGTTCGAAATCGATAAGCTT;

[0034] GTCCCTTCTGCTTCTTTCTTTTC

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This study isolated the CmCMT2 and CmDRM2 genes from the chrysanthemum variety "Shenma," constructed RNAi silencing expression vectors, and used genetic transformation to generate CmCMT2-RNAi and CmDRM2-RNAi transgenic "Shenma" plants. Phenotypic observation and statistical analysis of the transgenic plants were then performed. RNA-seq was used to identify key genes influencing chrysanthemum flowering and to investigate the mechanisms by which CmCMT2 and CmDRM2 genes influence early flowering. Spatiotemporal expression analysis of the cloned CmCMT2 and CmDRM2 genes from "Shenma" revealed that they are highly expressed from bud development to color change in "Shenma" plants. Furthermore, their expression levels in leaves gradually decrease during growth and development. These results suggest that CmCMT2 and CmDRM2 regulate chrysanthemum flowering by negatively regulating the expression of CmG20ox2a and CmG20ox2b, thereby altering GA1 levels and thereby regulating flowering time. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the construction principle of the RNAi vector of the present invention;

[0038] Figure 2 Phylogenetic tree analysis of CmCMT2 and CmDRM2; A is CmCMT2, B is CmDRM2;

[0039] Figure 3 Models of RNAi fragments and subcellular localization of CmCMT2 and CmDRM2; (A) Positional model of RNAi fragments in CmCMT2 and CmDRM2 gene silencing expression vectors; (B) Subcellular localization of CmCMT2 and CmDRM2 proteins;

[0040] Figure 4 The spatiotemporal expression analysis of CmCMT2 (A) and CmDRM2 (B) genes; EFα was used as an internal reference, and 2 -ΔΔCt Determined by the method; Values ​​represent the mean ± SE of three repeated measurements (P < 0.05); SS: seedling stage; VS: vegetative growth stage; RS: reproductive growth stage; Root: root; Stem: stem; Leaf: leaf; FBD, flower bud development stage; VC, color change stage; EO, early opening stage; OF, open flower stage;

[0041] Figure 5 Phenotypic identification of CmCMT2-RNAi and CmDRM2-RNAi transgenic Shenma plants; (A) Expression analysis of CmCMT2 and CmDRM2 in WT, CmCMT2-RNAi, and CmDRM2-RNAi plants; (B) Flower bud development in wild-type and transgenic plants; FBD, flower bud development stage; VC, color change stage; EO, early opening stage; OF, open flower stage; SF, senescent flower stage; target size: 1 cm; Phenotypic photos at 90 days (C) and 110 days (D); target size: 2 cm;

[0042] Figure 6 Principal component analysis (A) and sample correlation heat map (B) of RNA-seq of CmCMT2 and CmDRM2 transgenic chrysanthemum.

[0043] Figure 7 RNA-seq analysis of CmCMT2 and CmDRM2 transgenic chrysanthemums; (A) Statistics of the number of differentially expressed genes (DEGs) in CmCMT2 and CmDRM2 transgenic chrysanthemums; KEGG enrichment analysis of differentially expressed genes between WT vs RNAi-4 (B) and WT vs RNAi-3 (C);

[0044] Figure 8 Expression analysis of genes involved in flowering and GA content regulation pathways in WT vs RNAi-4 (A) and WT vs RNAi-3 (B);

[0045] Figure 9 Analysis of gibberellin content in CmCMT2 and CmDRM2 transgenic chrysanthemum. DETAILED DESCRIPTION

[0046] Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.Unless otherwise stated, the reagent, method and equipment adopted in the present invention are conventional reagents, methods and equipment in the art.Unless otherwise defined, all professional and scientific terms used in the text are identical in meaning with those skilled in the art.In addition, any method and material similar to or equivalent to the described content all can be applied to the inventive method.

[0047] Example 1

[0048] 1.1 Experimental Materials

[0049] 1.1.1 Plant materials

[0050] The material used in this study was the chrysanthemum variety 'Shenma'. Tissue culture seedlings of the chrysanthemum variety 'Shenma' were cultured in an incubator, and samples were collected from roots (R), stems (S), and leaves (L) at the seedling stage (SS), vegetative growth stage (VS), and reproductive growth stage (RS), as well as from flower bud development (FBD), color change stage (VC), early flowering stage (EO), and flowering stage (OF) during the reproductive growth stage. Each sample contained at least three biological replicates, was quick-frozen in liquid nitrogen, and then stored in a -80°C refrigerator for RNA extraction in subsequent spatiotemporal expression analysis experiments.

[0051] The transgenic material used in this study was the tissue culture seedlings of the chrysanthemum variety 'Shenma' preserved in our laboratory.

[0052] The materials used for transcriptome sequencing in this study were transgenic and wild-type (WT) chrysanthemum cultivar 'Shenma'. WT, CmCMT2-RNAi, and CmDRM2-RNAi materials were cultured until bud formation (the earliest observed bud formation among all materials). Three to five leaves (fully expanded leaves) were sampled from top to bottom, snap-frozen in liquid nitrogen, and stored at -80°C. This was used for RNA extraction for transcriptome sequencing.

[0053] 1.1.2 Main reagents

[0054] Spectinomycin hydrochloride (22189-32-8), kanamycin sulfate (25389-94-0), rifampicin (13292-46-1), MS medium (M519), agar powder (9002-18-0), sucrose (57-50-1) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (MacLean); polysaccharide and polyphenol plant DNA extraction kit (ND-26, Plant DNA Mini Syetem) was purchased from Beijing Nolay Biotechnology Co., Ltd.; KK ultrafast plant total RNA extraction kit (ZP405K, KK Fast Plant Total RNA Kit), microcolumn concentrated DNA gel recovery kit (ZPV202, V-ELUTE Gel Mini Purification Kit), mini DNA product purification kit (ZP201, Miniquick Purification Kit), plasmid miniprep kit (ZP101, DNA Marker (ZM404; DL2000 DNA Marker), nucleic acid electrophoresis dye (ZS203, ExRed) and SE Seamless Cloning and Assembly Kit (ZC231, SE Seamless Cloning and Assembly Kit) were purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd. (Zhuangmeng); restriction endonucleases Kpn I (R3142V), Xho I (R0146V) and specific endonuclease McrBC (M0272) were purchased from New England Biotechnology (Beijing) Co., Ltd. (NEB); sulfite conversion kit (DP215-02, DNA Bisulfite Conversion Kit) and common mix (KT201, 2× Taq PCR Mix) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; high-fidelity mix (R045A, Max DNA Polymerase) and cDNA synthesis kit (RR047A, PrimeScript TMRT reagent Kit with gDNA Eraser) was purchased from Takara Biotechnology (Beijing) Co., Ltd. (Takara); fluorescent quantitative mix (Q711-02, ChamQ Universal SYBR qPCR Master Mix) was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; yeast extract, sorbitol, spermidine, CaCl2, tryptone, NaCl, NaOH and surfactant (Silwet-L77) were purchased from Beijing Solebold Technology Co., Ltd.; primary chromatography methanol and formic acid (high-performance liquid chromatography eluent HPLC) were purchased from Komiou Chemical Reagent Co., Ltd.; 1 mL syringe, 0.22 μm filter membrane (organic phase), inner cannula, injection vial and inner cannula were purchased from Solebold Co., Ltd.

[0055] 1.1.3 Vectors and strains

[0056] DH5α competent cells (ZC101, DH5α Chemically Competent Cell) and GV3101 competent cells (ZC141, GV3101 Chemically Competent Cell) were purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd. (Zhuangmeng); the overexpression vectors pSAK277 and pSAK277-GFP (carrying the CaMV 35S enhanced promoter) and the RNAi silencing expression pART27 vector (commercially available) and pKANNIBAL vector (commercially available) were stored in our laboratory.

[0057] 1.2 Experimental methods

[0058] 1.2.1 Identification and phylogenetic tree construction of CmCMT2 and CmDRM2 genes in Chrysanthemum ‘Shenma’

[0059] Based on the published transcriptome data of the chrysanthemum variety 'Shenma' (Zhao W, Ding L, Liu J, et al. Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum[J]. Journal of Experimental Botany, 2022, 73(8): 2403-19), a local Blast database of 'Shenma' was constructed. Since our laboratory has previously identified CMT2 and DRM2 in the wild chrysanthemum species Chamomile, Juhuanao, Ganyeju Gojo-0 and the cultivated chrysanthemum species 'Zhongshan Zigui', CMT2 and DRM2 in these wild and cultivated chrysanthemum species as well as Arabidopsis, tomato, rice, corn and poplar were searched in the local Blast database of the chrysanthemum variety 'Shenma' to obtain CMT2 and DRM2 in the chrysanthemum variety 'Shenma' and construct a phylogenetic tree.

[0060] The alignment was performed using the Alignment and Align by ClustaIW (default parameters) options in MAGA 7.0 software (Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Molecular biology and evolution, 2016, 33(7): 1870-1874). After the alignment was completed, Data-Export Alignment was selected to save the alignment as a .meg file. The .meg file was then used to construct a phylogenetic tree using MAGA 7.0 software. The parameters were set to: Neighbor-joining, p-distance, complete deletion, bootstrap (replicates = 1000), and the default values ​​were used for the parameters.

[0061] Nucleotide sequence of the CmCMT2 gene:

[0062]

[0063] Nucleotide sequence of the CmDRM2 gene:

[0064]

[0065] Amino acid sequence encoded by the CmCMT2 gene:

[0066] MVADNGEAPTSQPPPLPPLPPPSSPSQPTPTQPTPQLHSIRRRSPRLVECGNSVEVPAKKKAKVTTSLVGVEFNDGGERVSFLIGDPVNDEEARRRWPWRYDIEELKRKGRYPTTKINSEDDDDELILNVKCHYLQAKILKHVFDLGDCAIVKGEEEGHDHVGRILEFFKTNTNEEYFRVQWFFRAEDTVIKDAATSNDKKRVFYSTLMNDNPLDCIVSKVKVLKVAPGINLKSIPKCDFYYDMEYNINYSTFSTIKDDVSSNNCCLSSSDKKEEIHSNGSIGKEEIHSNGSIGKEEIHSNGSIGKEDIHSNGSIGKEEIHSNGSIGKEEIHSNGSIGTSINGTLTRCKSHKSELTLLDIYSGCGGMSTGLCFGAKLSGVDLSTKWAVDANKCACESLRLNHPETQIRNEYAEDFLDLIKEWDKLCKKYKVKEEKTDGHDPMLSGSQDDVTPSKADNIVAKDEYEVEKLVDICYSDLDGTNKRELKFKVRWAGYGPSDDTWESIQGLRKCQDKIREFVHKGVKAKILPRPGDVDIICGGPPCQGISGHNRHRNFESPLEDERNYQIVVFMEIISFLKPRYVLMENVGDILRYANGSLARYAISCLVREYYQVRLGIMAAGCYGLPQFRLRVFLWGAHHDEHLPPFPLPTHDVVFKYGSPTGFERNVVAYDKGKLQNIEKCVLLKDAISDLPPVSNSESRDEMNYTNAPETEFQKFIRATKSDMLGNASLSSCDKKKPLLYDHRPFSLNEDDCLRVSRVPKRKGASFRDLPGIVFGDDNVVTRAPEPELMPSGKHWVPNYAINFNDGKNMKSFARVWWDETVSTVLCAPNYRTEAIIHPEQDRVLTIRECARLQGFPDFYALSGTVKERYRQVGNAVAVPVGRALGYTLGMALQKLSGDEPLVTLPPKFAHSTTIDLLQPSLAIEP

[0067] Amino acid sequence encoded by the CmDRM2 gene:

[0068] MDVHASVEENNAIDWDTEDELEIENIAMTSNAASTSAGPSNVSLIQHFIGMGFPENFVTKAIKKNGESDSDRLLETILAYMDGHVSDEENVDIDSDTKDDLEIQNIASTSCSTPVTSNNGEASSSGGPSNSNSVQHFIDMGFPAELVTRAIKENEENDTEKILES LLTYSQMAPEELDTCHASSSQQQCVTYDELSSDYDESLLDGFSEDDSLSESEITDNKSLPENEQMLLSLASMGYTVEEASAAVARCGPDASIAELTDFISAAQMAKTEAAFFEEEKPKVFLNPKLKRKMYQEALKRKKQKGPLTEEEEVIRIPKPMIGFGVPSES SIVTHRTLPDAAIGRPFFYYENVALAPKGVWDTISRFLFDVAPEFVDSKHFCAAARKRGYVHNLPIDNRFPLLPLQPRTIHGALPLTRRWWPAWDKRTNLNCLQTAYGSAKLTDRLRKALEKWGDEPPDHVKRYVLDECRRWNLVWVGKNKLATLEPDEVEMLLG FPRNHTRGGGISRTDRYKSLGNSFQVDTVAYHLSVLKKIFPNGINVLSLFSGIGGAEVALHRLGVPLKTVVSVELSEANRDIVRSWWEQTNQKGNLVHLDDVQKLHGDKLEELIKLYGRFDLVVGGSPCNNLAGGNRVSRDGLEGEQSSLFYDYFRILDTVKAIMK

[0069] 1.2.2 Construction of RNAi vector

[0070] The vector construction primers are shown in Table 1. The specific construction method is as follows:

[0071] First, pART27 and pKANNIBAL vectors were digested with Not I. The intron-containing portion of the pKANNIBAL vector was then ligated into pART27 using T4 DNA tau ligase to create a composite vector. Next, 200-500 bp RNAi fragments were designed based on the conserved sequences of the CmCMT2 and CmDRM2 genes. Primers were designed using Premier 5.0 software (primers are shown in Table 1) and then ligated into the composite vector. A schematic diagram and principle of RNAi vector construction are shown below. Figure 1 The sequence of the RNAi fragment of the CmCMT2 gene is shown in SEQ ID NO. 1, and the sequence of the RNAi fragment of the CmDRM2 gene is shown in SEQ ID NO. 2.

[0072] Table 1 Primer list

[0073]

[0074]

[0075] 1.2.3 Analysis of the spatiotemporal expression of CmCMT2 and CmDRM2 genes in Chrysanthemum ‘Shenma’

[0076] Total RNA was extracted from the preserved materials in step 1.1 (roots (R), stems (S) and leaves (L) in the seedling stage (SS), vegetative growth stage (VS) and reproductive growth stage (RS), and flower buds and flowers in the flower bud development stage (FBD), color change stage (VC), early flowering stage (EO) and flowering stage (OF) in the reproductive growth stage) using the KK Ultrafast Plant Total RNA Extraction Kit (Zangmeng) (refer to the instructions for the extraction steps). The total RNA of each extracted sample was detected by 1% agarose gel electrophoresis, and the RNA samples were detected using a NanoDrop2000 microspectrophotometer (Thermo Fisher). RNA samples with A260 / A280 values ​​between 1.8 and 2.2 were reverse transcribed, and the reverse transcribed cDNA was diluted to 200 ng / μL for subsequent experiments. Quantitative primers were designed using Premier 5.0 software. The primers (Table 1) were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. Fluorescence quantitative Mix (SYBR qPCR Master Mix) was used in Fluorescence quantitative PCR reaction was performed on a 480II real-time fluorescence quantitative PCR instrument (Roche) (reaction system shown in Table 2), using the CmEFα gene as an internal reference, and each sample was repeated three times.

[0077] According to the calculation method of relative expression in the literature (Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method [J]. Methods, 2001, 25 (4): 402-408), the Cp value obtained from the qRT-PCR data was 2-fold modified using Excel software. -△△CT The relative expression levels were calculated, and the calculated values ​​were analyzed for significance of difference using SPSS 19.0 software (Duncan test, P < 0.05).

[0078] Table 2 Fluorescence quantitative PCR reaction system

[0079]

[0080]

[0081] 1.2.4 Transformation of Chrysanthemum “Shenma” with CmCMT2 and CmDRM2 Gene Silencing Expression Vectors

[0082] The constructed RNAi vector was transformed into Agrobacterium competent cells, and then transformed into the leaves of 'Shenma' by Agrobacterium-mediated method. The specific steps are as follows:

[0083] (1) The wild type 'Shenma' tissue culture seedlings were propagated on MS solid medium (the terminal buds were taken). When the culture reached 35-45 days, the third and fourth expanded leaves of the tissue culture seedlings were taken and cut into 0.3×0.3 cm pieces using a scalpel in a clean bench. 2 The squares were cultured with the leaves facing upward in pre-culture medium (MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L, pH 5.8) for 3 days.

[0084] (2) On the second day of leaf disc culture, take out the preserved glycerol Agrobacterium from the -80℃ freezer, thaw naturally, take 500-1000 μL and add it to 40-50 mL of YEB liquid culture medium containing rifampicin (Rif) and spectinomycin (Spec) resistance (100 mL conical flask), and culture overnight at 28℃ in a shaking incubator.

[0085] (3) The next day, wait for the bacterial solution to be activated to OD 600When the value is about 0.5, centrifuge the cells in a centrifuge (room temperature, 5000 rpm, 10 min), discard the supernatant in a clean bench, and reselect with a gravity solution (MS 4.43 / L + sucrose 30 g / L, pH 5.8) to make the final resuspended cells OD 600 It is about 0.5 (the ratio of gravity separation liquid to bacteria is generally 1:1).

[0086] (4) Immerse the leaf disc after pre-culture in the prepared re-selection bacterial solution and infect it with slow shaking for 10 minutes. Then use filter paper to absorb the bacterial solution and finally place it in the co-culture medium (same as the pre-culture medium) for dark culture for 3 days.

[0087] (5) After the co-cultivation, the leaf disc was transferred to decarboxylation medium (MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L + Carb 500 mg / L) and cultured under light for 5-7 days.

[0088] (6) After decarboxylation, the culture medium is changed every two weeks until the seedlings reach stage IV, at which point the culture medium is replaced with a glass bottle to increase the culture space. If seedlings have not yet emerged by stage IV, the culture medium is continued to be changed every two weeks with stage IV medium until seedlings emerge.

[0089] Option I: MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L + Carb 350 mg / L + Kan 10 mg / L;

[0090] Option II: MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L + Carb 300 mg / L + Kan 9 mg / L;

[0091] Option III: MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L + Carb 250 mg / L + Kan 8 mg / L;

[0092] Option IV: MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + 6-BA 1.0 mg / L + NAA 0.5 mg / L + Carb 200 mg / L + Kan 7 mg / L.

[0093] (7) After the resistant buds are differentiated, when they grow to 2-3 cm, they are transferred to the rooting screening medium (Kana resistance: MS 4.43 g / L + sucrose 30 g / L + agar 7 g / L + Carb 200 mg / L + Kan 7 mg / L) for screening. The seedlings that can normally root in the rooting screening medium are retained, and then their leaves are taken for PCR verification. The verification primers are shown in Table 1.

[0094] (8) Finally, the transgenic seedlings are obtained for propagation, and then the propagated transgenic seedlings are hardened. Finally, they are grown in an incubator for about 30 days and then transplanted into the field. The phenotypic observations and data statistics are performed more than twice.

[0095] 1.2.5 Subcellular localization

[0096] According to the method described in the literature (Von A A. Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells [J]. CSH protocols, 2007, 2007(2): pdb.prot4689), 35:GFP-ClROS1a and 35:GFP plasmids were transiently introduced into onion epidermal cells using high-pressure gas gene gun particle bombardment. The transformed cells were cultured in MS medium at 22°C in the dark for 16 hours and then observed using a Zeiss LSM780 confocal microscope. mCherry was used as a nuclear marker and served as a control. The specific method is as follows:

[0097] (1) Microprojectile preparation

[0098] Weigh 1.0 μm gold powder into a 2 mL sterile centrifuge tube; then slowly add 1 mL of anhydrous ethanol to the centrifuge tube and shake thoroughly to mix; let it stand for 10 minutes; centrifuge at 10,000 rpm for 1 minute; discard the supernatant, add 1 mL of sterile water again, shake thoroughly to mix; centrifuge at 10,000 rpm for 1 minute, repeat 3-4 times; discard the supernatant, prepare a 100 mg / mL gold powder solution, and store in a -20°C refrigerator for later use.

[0099] (2) Prepare culture medium

[0100] Hypertonic medium: MS 4.43g / L + sucrose 30g / L + agar 10g / L + sorbitol 72.8g / L

[0101] MS medium: MS 4.43 g / L + sucrose 30 g / L + agar 10 g / L

[0102] (3) Prepare onions

[0103] Choose fresh, yellow, large onions and cut them between 7:00 and 9:00 in the evening. Cut the third to fifth layers into 2-3cm pieces. 2 The onion pieces were cut into squares of different sizes and spread onto hypertonic culture medium (the side not close to the onion pulp was in contact with the culture medium) and cultured overnight in the dark in a tissue culture room.

[0104] (4) Extraction of plasmid

[0105] The operation method of the plasmid extraction kit of different companies shall prevail, but the plasmid concentration must be high and free of impurities.

[0106] (5) Reagent preparation

[0107] 2.5M CaCl2: Dissolve 2.7745 g of CaCl2 in 10 mL of sterile distilled water. Filter the solution through a water filter membrane in a laminar flow hood into a sterile centrifuge tube and store in a refrigerator at 4°C until use.

[0108] 0.1 M spermidine: Dissolve 15.7 μL spermidine in 984.3 μL sterile water and filter through a water filter (this reagent should be prepared immediately before use).

[0109] (6) Mixing plasmid and gold powder

[0110] Mix the preserved gold powder thoroughly, take 30 μL of gold powder into a sterile centrifuge tube, add 5 μg of plasmid, and mix thoroughly; then add 20 μL of 2.5 M CaCl2, and mix thoroughly; then add 8 μL of 0.1 M spermidine, and mix thoroughly; place on ice for 20 minutes, vortexing 4-5 times during the period.

[0111] (7) Cleaning plasmids and gold powder

[0112] Add 80 μL of anhydrous ethanol, centrifuge at 13,000 g for 20 seconds, and discard the supernatant (retain about 20 μL); add 20-30 μL of anhydrous ethanol, vortex vigorously for 3-5 seconds, add anhydrous ethanol to 20 μL, centrifuge at 13,000 g, discard the supernatant, and repeat 3 times; add 80 μL of anhydrous ethanol to resuspend and mix thoroughly by pipetting.

[0113] (8) Using a gene gun

[0114] Transfer the onion epidermis from high-density culture medium to MS culture medium for gene gun use. Soak the yellow flight membrane, steel mesh, and holder in 75% alcohol, allow to dry in a clean bench, and then perform the procedure. Place gold powder on the yellow flight membrane (located at the center hole), allow to dry, and then install it in the holder. The small disc is placed at the top of the gene gun and must be replaced each time.

[0115] (9) Cultivation

[0116] The onion surfaces that have been injected with the gene gun are cultured in a tissue culture room under dark conditions for 16-24 hours.

[0117] (10) Observe GFP signal

[0118] Fluorescence signals were observed and photographed using an LSM710 laser confocal microscope.

[0119] 1.2.6 RNA extraction and transcriptome sequencing and analysis

[0120] The top buds of transgenic CmCMT2-RNAi, CmDRM2-RNAi, and wild-type WT plants with consistent growth were selected for cuttings around mid-July of the same year. After the cuttings grew and took root (around early August), they were transferred to large nutrient pots and cultured in an incubator. When the number of fully expanded leaves of the transgenic and wild-type 'Shenma' plants reached 15, they were transferred outdoors for natural dark treatment (around early September). The dark treatment lasted for about a month. When the earliest buds appeared, the leaves of the transgenic and wild-type 'Shenma' plants were sampled (the 3rd to 5th fully expanded leaves from top to bottom). Three biological replicates were performed for each sample, with at least three plants in each replicate. Guangzhou Kidio Biotechnology Co., Ltd. extracted RNA, constructed libraries, and then sequenced them using Illumina Novaseq6000. The specific steps are as follows:

[0121] (1) RNA extraction, library construction, and sequencing

[0122] Total RNA was extracted according to the Trizol kit instructions. RNA quality was monitored using an Agilent 2100 and agarose gel electrophoresis. mRNA was then enriched using Oligo(dT) beads. After purification, the cDNA fragments were end-repaired, A bases were added, and ligated with Illumina sequencing adapters. The ligation reaction was purified using AMPure XP Beads (1.0X) and amplified by PCR. The resulting cDNA library was sequenced by Kidio Biotechnology Co., Ltd. (Guangzhou, China) using the Illumina Novasek 6000 platform.

[0123] (2) Bioinformatics analysis

[0124] The raw reads were filtered using fastp (version 0.18.0) to obtain clean reads. A reference genome index was established, and paired-end clean reads were mapped to the reference genome using HISAT2.2.4. StringTiev1.3.1 was used to assemble the mapped reads of each sample based on the reference genome. RSEM software was then used to calculate FPKM as the expression level of gene i, where C was the number of fragments mapped to gene i, N was the total number of fragments mapped to the reference gene, and L was the number of bases in gene i. Therefore, the calculated gene expression levels can be directly used to compare the differences in gene expression levels between different samples. Finally, principal component analysis (PCA) was performed using the R software package gmodels (http: / / www.r-project.org / ).

[0125] (3) Differentially expressed genes (DEGs) and enrichment analysis

[0126] DESeq2 software was used to analyze differential RNA expression between the two groups of samples. Genes with a false discovery rate (FDR) less than 0.05 and an absolute fold change ≥ 2 were considered differentially expressed. These differentially expressed genes were then subjected to Gene Ontology (GO) functional classification and enrichment analysis, as well as KEGG biological pathway classification and enrichment analysis.

[0127] 1.2.7 Hormone determination

[0128] For hormone determination, follow the same procedures as in step 1.2.6. Grind the tissue sample thoroughly in liquid nitrogen, place 0.2 g of sample in a 2.0 mL centrifuge tube, and store in liquid nitrogen. Add the prepared hormone extract (methanol: water: formic acid = 80%: 19%: 1%) to the centrifuge tube containing the sample, mix thoroughly, and place on ice. Then, shake it on an oscillator at 4°C for 3-5 hours and place it in a 4°C refrigerator overnight. After overnight low-temperature extraction, centrifuge in an ultra-high-speed low-temperature centrifuge at 4°C, 10,000 rpm for 15 minutes, and collect 800 μL of the supernatant into a 1.5 mL centrifuge tube. Blow nitrogen in a nitrogen blower in the dark until dry (approximately 1.5-2 hours), and finally redissolve with 100 μL of 40% methanol, mix thoroughly and centrifuge briefly, then filter with a 0.22 μm organic filter membrane, add the filtered liquid to a liquid phase vial with an inner tube, and cover with a pre-cut cap for UPLC-MS / MS analysis (mass spectrometry).

[0129] 2.1 Results and Analysis

[0130] 2.1.1 Identification and phylogenetic tree analysis of CmCMT2 and CmDRM2

[0131] Previous studies have found that CMT2 and DRM2 genes are highly expressed during the flowering period, so the transcriptome data of 'Shenma' were subjected to local blasting using two methyltransferases of Arabidopsis, AtCMT2 and AtDRM2, to obtain the CmCMT2 and CmDRM2 genes in 'Shenma'. The nucleotide lengths of CmCMT2 and CmDRM2 genes are 2778 and 1986 bp, respectively. Phylogenetic trees were constructed for CmCMT2 and CmDRM2, respectively, in relation to CMT2 and DRM2 in wild and cultivated chrysanthemum species, as well as model plants Arabidopsis, rice, tomato, corn, and poplar. The results showed that CmCMT2 was most closely related to ClCMT2a / c in chamomile and CsGCMT2a in Gojo-0, followed by CmoCMT2a / b in 'Zhongshan Zigui' ( Figure 2 A). CmDRM2 is closely related to ClDRM2a in chamomile, CnDRM2a in chrysanthemum, and CmoDRM2a / b / c in ‘Zhongshan Zigui’, and they are clustered in the same subgroup ( Figure 2 B).

[0132] 2.1.2 Construction of CmCMT2 and CmDRM2 gene silencing vectors and protein localization analysis

[0133] Previous studies have shown that the methylation level of early-flowering varieties of chrysanthemum is lower than that of late-flowering varieties, and the expression levels of ClCMT2 and ClDRM2 are higher during the flowering period than at other times. In order to explore the functions of CmCMT2 and CmDRM2 in 'Shenma'. This study first constructed silencing expression vectors for these two genes. The gene fragments used to construct the silencing expression vectors are located in the conserved regions of the CmCMT2 and CmDRM2 genes, respectively. In the CmCMT2 gene, the sequence of the 1578-1879bp region was selected for constructing the RNAi vector, and in the CmDRM2 gene, the sequence of the 595-849bp region was selected for constructing the RNAi vector ( Figure 1 and Figure 3 A).

[0134] In order to further explore the function of CmCMT2 and CmDRM2 proteins, the full-length CmCMT2 and CmDRM2 gene sequences were isolated from Shenma, excluding the stop codon, and linked to an overexpression vector containing a GFP tag. pro :GFP-CmCMT2 and 35S pro :GFP-CmDRM2 fusion vectors were injected into onion epidermal cells and the 35S pro :GFP-CmCMT2 and 35S pro: The fluorescence position of GFP-CmDRM2 fusion protein was evaluated. The results showed that 35S pro :GFP-CmCMT2 and 35S pro :GFP-CmDRM2 fusion protein has green fluorescence signal in the cell nucleus, while 35S pro :GFP protein has green fluorescence signals in the cell membrane and nucleus, and the nuclear localization marker mCherry protein shows green fluorescence at the same position as 35S pro :GFP-CmCMT2 and 35S pro :GFP-CmDRM2 fusion protein is located at the same position ( Figure 3 B) In summary, CmCMT2 and CmDRM2 are localized in the cell nucleus and their functions are mainly performed in the cell nucleus.

[0135] 2.1.3 Spatiotemporal expression analysis of CmCMT2 and CmDRM2 genes

[0136] To determine the expression patterns of CmCMT2 and CmDRM2 genes in 'Shenma', qRT-PCR assays were performed using RNA extracted from different organs at different developmental stages ( Figure 4 ). The results showed that the expression levels of CmCMT2 and CmDRM2 during the development of flower buds until color change were significantly higher than those in other organs at other stages. The expression level of CmCMT2 gradually decreased from flower buds to full flower opening, while CmDRM2 began to decrease significantly after the flower changed color. In addition, in leaves, the expression levels of CmCMT2 and CmDRM2 showed a gradually decreasing trend throughout the growth process. In stems, the expression levels of CmCMT2 and CmDRM2 were relatively stable, and there was no significant difference between different stages. In roots, CmCMT2 and CmDRM2 showed different expression patterns. CmCMT2 decreased significantly in roots from the seedling stage to the reproductive stage, while the expression level of CmDRM2 remained almost unchanged during the seedling stage and vegetative growth period, but increased significantly during the reproductive growth period. In conclusion, the expression patterns of CmCMT2 and CmDRM2 in Shenma's organs except roots were almost identical at different developmental stages, indicating that CmCMT2 and CmDRM2 may have similar functions during the growth and development of Shenma'.

[0137] 2.1.4 Reduced expression of CmCMT2 and CmDRM2 leads to premature flowering in 'Shenma'

[0138] Given that the expression levels of CmCMT2 and CmDRM2 genes in the leaves of Shenma gradually decrease during its growth and development, and their expression levels decrease significantly from the bud to the full flowering stage, we aimed to investigate whether CmCMT2 and CmDRM2 are involved in regulating the flowering period of Shenma. First, we transformed the silencing expression vectors of CmCMT2 and CmDRM2 genes into Shenma, and obtained transgenic chrysanthemums with silenced expression of CmCMT2 and CmDRM2 ( Figure 5 A). The results showed that the expression levels of CmCMT2 and CmDRM2 in CmCMT2-RNAi and CmDRM2-RNAi transgenic plants were reduced by 27.1% and 28.7% and by 22.1% and 29.4%, respectively.

[0139] Secondly, to explore the effects of CmCMT2 and CmDRM2 on the flowering period of Shenma, two CmCMT2-RNAi lines (CmCMT2-RNAi-4, CmCMT2-RNAi-17) and two CmDRM2-RNAi lines (CmDRM2-RNAi-3, CmDRM2-RNAi-4) were cultured. The results showed that both CmCMT2 and CmDRM2 could advance the flowering period of Shenma, but the flowering time showed differences. The two CmCMT2-RNAi lines were 15 days earlier than the WT in the bud and early flower opening stages ( Figure 5 B), and the flowering period was significantly earlier than that of WT on days 90 and 110 ( Figure 5 C and D). The two CmDRM2-RNAi lines were 8 days earlier than the WT in the budding and early flower opening stages ( Figure 5 B), also showed an early flowering phenotype compared to WT on days 90 and 110 ( Figure 5 C and D).

[0140] 2.1.5 Transcriptome Analysis of CmCMT2 and CmDRM2 Transgenic Chrysanthemum

[0141] To further elucidate the molecular mechanisms by which CmCMT2 and CmDRM2 regulate flowering, transcriptome sequencing was performed on leaves (3–5 leaves from top to bottom, approximately 80 days old) from wild-type 'Shenma', CmCMT2-RNAi-4 (RNAi-4), and CmDRM2-RNAi-3 (RNAi-3) transgenic lines. Three natural replicates were used for each sample from the wild-type and transgenic lines, for a total of nine samples. The genome of the chrysanthemum cultivar 'Zhongshan Zigui' was used as the reference genome. Across the nine samples, the lengths of raw and clean reads ranged from 5,455,489,800 bp to 5,292,205,534 bp, respectively. After filtering, the data had Q20 values ​​greater than 95.83%, Q30 values ​​greater than 89.39%, and a GC ratio greater than 43.16% (Table 3). Clean reads were then aligned to the 'Zhongshan Zigui' genome. Across the nine samples, an average of 44,828,400 reads were obtained, with less than 25.08% of reads unaligned. The single alignment rate exceeded 51.59%, the multiple alignment rate was less than 26.37%, and the total alignment rate exceeded 74.92% (Table 4). Finally, the distribution of reads that could be mapped to the genome was calculated, and the mapped regions were divided into exons, introns, and intergenic regions. The highest alignment rate was found in exons, with an average alignment rate of 86.58%. Alignment rates were lower in introns and intergenic regions, with introns and intergenic regions having an alignment rate of less than 5.78% and 8.08% respectively (Table 5).

[0142] In order to detect the correlation between transcriptome samples, principal component analysis (PCA) and correlation analysis between samples were performed on 9 samples. PCA results showed that the dispersion between the replicates of WT, RNAi-4 and RNAi-3 groups was small, among which WT had the smallest dispersion ( Figure 6 A). The correlation heat map between samples showed that the three biological repeatability of WT, RNAi-4 and RNAi-3 were good, with correlation coefficients above 0.996 ( Figure 6 B) In summary, the repeats of WT, RNAi-4, and RNAi-3 are well correlated, and the sequencing data are highly reliable.

[0143] Table 3 Transcriptome read statistics

[0144]

[0145] Table 4 Comparative analysis with the genome of 'Zhongshan Zigui'

[0146]

[0147]

[0148] Table 5 Statistical analysis of genomic regions compared with 'Zhongshan Zigui'

[0149]

[0150]

[0151] By comparing the wild type with RNAi-4 and RNAi-3, differentially expressed genes (EDGs) were screened out. The criteria for screening differentially expressed genes were: FDR < 0.05; |log2 FoldChange |>log2(2). The result is as follows Figure 7 As shown, 25,002 and 22,172 EDGs were screened in WT vs RNAi-4 and WT vs RNAi-3, respectively. Figure 7 A). Among them, 8083 genes were up-regulated and 16919 genes were down-regulated in WT vs RNAi-4; 7186 genes were up-regulated and 14986 genes were down-regulated in WT vs RNAi-3 ( Figure 7 A).

[0152] To explore the cause of premature flowering caused by silencing expression of CmCMT2 and CmDRM2 in chrysanthemum, KEGG enrichment analysis was performed on EDGs in the transcriptome. In WT vs RNAi-4, differentially expressed genes were enriched in pathways such as ribosome, biosynthesis of secondary metabolites, photosynthesis-antennary protein, metabolic pathway, biosynthesis of phenylalanine, photosynthesis, fatty acid elongation, plant-pathogen interaction, MAPK signaling pathway-plant and biotin metabolism ( Figure 7 B); In WT vs RNAi-3, differentially expressed genes were enriched in ribosome, photosynthesis-antennary protein, photosynthesis, biosynthesis of secondary metabolites, metabolic pathways, biosynthesis of phenylalanine, plant-pathogen interaction, fatty acid elongation, MAPK signaling pathway-plant, and biotin metabolism ( Figure 7 C). In summary, most pathways were co-enriched between WT vs RNAi-4 and WT vs RNAi-3, and flowering was primarily due to the influence of photoperiod and hormones. The photoperiod pathway was not significantly enriched, while gibberellin (GA) has been shown to affect flowering time. Therefore, the present invention focuses on hormone regulation in metabolic pathways (biosynthesis and metabolic pathways of secondary metabolites), which may be the reason why silencing CmCMT2 and CmDRM2 expression in chrysanthemum leads to changes in flowering time.

[0153] 2.1.6 Analysis of differentially expressed genes in GA synthesis and flowering regulation

[0154] Further screening of differentially expressed genes regulating flowering and gibberellin (GA) content in the photoperiod pathway was conducted, and genes that may regulate flowering were screened in WT-vs-RNAi-4 and WT-vs-RNAi-3. The results showed that they had similar differentially expressed genes. In the flowering regulation pathway, multiple genes with relatively significant expression differences were screened in WT-vs-RNAi-4 and WT-vs-RNAi-3, such as GIs, COs, FTs, and AP1s ( Figure 8 In addition, the differentially expressed genes regulating GA were found to be the same in WT-vs-RNAi-4 and WT-vs-RNAi-3, namely GA20ox1a, GA20ox1 (evm.TU.scaffold_1806.761 and evm.TU.scaffold_9272.33 were down-regulated) and GA20ox2a, GA20ox2b (evm.TU.scaffold_166.251 and evm.TU.scaffold_12101.1 were up-regulated) ( Figure 8 Since GA20ox2a and GA20ox2b positively regulate GA content, it is speculated that the early flowering of CmCMT2 and CmDRM2 transgenic chrysanthemums is due to the up-regulated expression of GA20ox2a and GA20ox2b, which promotes the increase in GA content, leading to changes in the expression of flowering regulatory genes, and thus resulting in changes in the flowering period of CmCMT2 and CmDRM2 transgenic chrysanthemums.

[0155] 2.1.7 Analysis of gibberellin content in CmCMT2 and CmDRM2 transgenic chrysanthemums

[0156] To investigate whether changes in GA content lead to changes in flowering period in CmCMT2 and CmDRM2 transgenic chrysanthemums, UPLC-MS / MS was used to analyze the GA content in CmCMT2 and CmDRM2 transgenic chrysanthemums. The results showed that only GA1 was detected in CmCMT2 and CmDRM2 transgenic chrysanthemums, while the other active GA3, GA4, and GA7 were likely present at low levels and could not be detected. The GA1 content in both CmCMT2 transgenic chrysanthemum lines was significantly higher than that in the WT, and the GA1 content was approximately twice that of the WT ( Figure 9 The GA1 content in the two lines of CmDRM2 transgenic chrysanthemum was also significantly higher than that in WT, but the increase in GA1 content was relatively small compared with that in WT ( Figure 9 ).

[0157] 2.2 Discussion

[0158] Gibberellic acid (GA) is a key hormone involved in cell growth and differentiation. It plays a crucial role in regulating the elongation or expansion of various tissues, including roots, hypocotyls, leaves, stems, stamens, and pistils. It has been shown to promote flowering in many plants, including Arabidopsis thaliana. In Arabidopsis thaliana, GA4 is the primary bioactive GA involved in growth and flowering induction. The activity of the gibberellin biosynthetic enzymes GA20-oxidases (GA20ox) regulates GA content in various plants. In Arabidopsis thaliana, these enzymes are encoded by five gene families: AtGA20ox1-AtGA20ox5. AtGA20ox1 and AtGA20ox2 play redundant roles in promoting hypocotyl and internode elongation, flowering time, anther filament elongation, seed number per pod, and pod elongation. AtGA20ox1 contributes more to internode and filament elongation, while AtGA20ox2 contributes more to flowering time and pod length.

[0159] Most cultivated chrysanthemums require short-day induction to flower, but in the model plant Arabidopsis thaliana, GA treatment can accelerate flowering. Studies have found that exogenous GA3 application can advance flowering by 5 days and extend the flowering period by 7 days. A mutant of the long-day flowering chrysanthemum cultivar 'Shenma' shows upregulation of circadian clock genes and CmFTL3 under short-day conditions, inducing flower bud formation and flowering. Under long-day conditions, GA20ox and GID, genes involved in the GA signaling pathway, are upregulated, while GA2ox and GAI are downregulated, inducing transcription of SOC1 and LFY. Furthermore, the mutant's leaves have higher GA content than the wild-type under both LD and SD conditions, and exhibit more branching. Furthermore, in the chrysanthemum mutant CmBBX24-RNAi, genes involved in the GA biosynthesis pathway are upregulated, exhibiting an early-flowering phenotype. Furthermore, the active GA1 content in both the mutant and the WT was significantly higher than that in the WT under both LD and SD conditions. Under LD conditions, the GA1 and GA4 content in leaves of CmBBX24-RNAi plants was 2.3 and 1.8 times higher than that of the WT, respectively, and GA1 content was at least five times higher than that of the other active GAs. In chrysanthemum, CmBBX24 regulates flowering by affecting gibberellin synthesis. Overexpression of CmMYB2 resulted in early flowering, while CmMYB2-RNAi plants flowered later. Interaction experiments revealed that CmMYB2 interacts with CmBBX24, suggesting that CmMYB2 regulates flowering by affecting gibberellin synthesis.

[0160] In this study, the present invention constructed chrysanthemum CmCMT2 and CmDRM2 silenced expression vectors and found that in 'Shenma', the decrease in CmCMT2 and CmDRM2 expression levels led to different degrees of early flowering ( Figure 5), and then RNA-seq sequencing was performed, and KEGG analysis found that EDGs were mainly enriched in metabolic pathways. It is reported that gibberellins (GA) promote plant flowering, so the EDGs involved in gibberellin regulation in the metabolic pathway were analyzed, and it was found that the GA synthesis genes CmG20ox2a and CmG20ox2b were significantly upregulated in CmCMT2 and CmDRM2 transgenic chrysanthemums. Then, for active GA (GA1, GA3, GA4 and GA7), it was found that GA1 increased in CmCMT2 and CmDRM2 transgenic chrysanthemums. This is inconsistent with the study in Arabidopsis, where the active gibberellins involved in flowering regulation are mainly GA4. However, in chrysanthemum, CmBBX24-RNAi mutant chrysanthemums showed an early flowering phenotype, and their GA1 content was at least 5 times higher than that of other active GAs, which is consistent with the high GA1 content in this study ( Figure 9 ), and these differences may be due to species specificity and different sampling sites. Therefore, the changes in GA1 content in CmCMT2 and CmDRM2 transgenic chrysanthemums in this study may be the main cause of early flowering, and GA1 content may have a dosage effect on early flowering. In summary, CmCMT2 and CmDRM2 genes may alter the methylation of the promoters of the CmG20ox2a and CmG20ox2b genes in chrysanthemum, thereby changing the expression levels of CmG20ox2a and CmG20ox2b and thus altering flowering time.

[0161] 2.3 Summary

[0162] This study used bioinformatics methods to identify the CmCMT2 and CmDRM2 genes from Shenma, and obtained the following conclusions through quantitative PCR, subcellular localization, transgenic verification, and RNA-seq analysis:

[0163] (1) The CmCMT2 and CmDRM2 genes were cloned from Shenma. Spatiotemporal expression analysis showed that CmCMT2 and CmDRM2 were highly expressed during the development of flower buds and color change in Shenma. Furthermore, the expression levels of CmCMT2 and CmDRM2 in leaves gradually decreased during the growth and development of Shenma. (2) The expression levels of CmCMT2-RNAi and CmDRM2-RNAi transgenic Shenma decreased by 27.1% and 28.7% and by 22.1% and 29.4%, respectively. Phenotypic analysis showed that the flowering date of CmCMT2-RNAi-4, CmCMT2-RNAi-17, and CmDRM2-RNAi-3, CmDRM2-RNAi-4 transgenic Shenma was advanced by 15 and 15 days, and by 8 and 8 days, respectively. (3) RNA-seq analysis of CmCMT2-RNAi and CmDRM2-RNAi transgenic 'Shenma' showed that differentially expressed genes (DEGs) in metabolic pathways were significantly enriched, and the expression of GA biosynthesis genes CmG20ox2a and CmG20ox2b was significantly increased. (4) Hormone content analysis showed that the GA1 content in CmCMT2-RNAi-4, CmCMT2-RNAi-17, CmDRM2-RNAi-3, and CmDRM2-RNAi-4 was 2.05 and 1.73 times that of WT, and 1.12 and 1.24 times that of WT, respectively, and was significantly different from WT.

[0164] In conclusion, our results indicate that CmCMT2 and CmDRM2 negatively regulate the expression of CmG20ox2a and CmG20ox2b to alter the content of GA1, thereby regulating the flowering period of chrysanthemum.

Claims

1. Chrysanthemum methyltransferase gene CmCMT2 and / or CmDRM2 Application in regulating the growth and development, flowering timing or flowering period of Asteraceae plants.

2. The use according to claim 1, characterized in that: The chrysanthemum variety is "Shenma" or "Zhongshan Zigui".

3. The use according to claim 1 or 2, characterized in that: Methyltransferase gene CmCMT2 and / or CmDRM2 Application in regulating the development of flower buds of Asteraceae plants, regulating flower color or improving flowering period.

4. The use according to claim 1 or 2, characterized in that: Methyltransferase gene CmCMT2 and / or CmDRM2 Application in regulating gibberellin synthesis during flowering period.

5. The use according to claim 1 or 2, characterized in that: Application of RNAi gene silencing expression vector in regulating the growth and development, flowering timing or flowering period of Asteraceae plants.

6. The use according to claim 5, characterized in that: When applied, CmCMT2 Genes and CmDRM2 The specific sequence of the gene was connected to a composite vector constructed by plasmids pART27 and pKANNIBAL to obtain an RNAi gene silencing expression vector, which was then transformed into Agrobacterium competent cells and then transformed into leaves of Compositae plants through Agrobacterium-mediated method.

7. The use according to claim 6, characterized in that: The RNAi gene silencing expression vector is constructed by the following method: 1) The plasmids pART27 and pKANNIBAL were digested with Not I, and then the intron-containing part of the plasmid pKANNIBAL was ligated into the plasmid pART27 using T4 DNA ligase to construct a composite vector; 2) According to CmCMT2 Genes and CmDRM2 Gene conserved sequence, design 200-500 bp RNAi fragments, and use Premier 5.0 software to design primers, and then connect with the composite vector in step 1).

8. The use according to claim 7, characterized in that: CmCMT2 The RNAi fragment sequence of the gene is shown in SEQ ID NO.1, CmDRM2 The RNAi fragment sequence of the gene is shown in SEQ ID NO.

2.

9. Utilize CmCMT2 Genes and CmDRM2 Gene, and RNAi gene silencing expression vector constructed by plasmids pART27 and pKANNIBAL.

Images