Chrysanthemum G protein coupled receptor GCR or related factors thereof and application thereof in flower development regulation

Abstract

The invention relates to the technical field of bioengineering, and provides a chrysanthemum G protein coupled receptor GCR or related factors thereof and application thereof in flower development regulation. Specifically, the invention provides a method for regulating chrysanthemum flower development, the method is carried out by regulating expression of CmGCR and / or CmGCR related factors, and the CmGCR related factors are downstream factors regulated by CmGCR in a photoperiod pathway, downstream factors regulated by CmGCR in a hormone signal pathway or downstream factors regulated by CmGCR in floral organ development. The invention also provides an application of the CmGCR and / or the CmGCR related factor in regulating and controlling the flowering time of the plant. New functions of the CmGCR and the CmGCR related factors are applied to flower development regulation and control, and the inflorescence development process and the flowering time can be accurately regulated, so that the CmGCR is particularly suitable for improving germplasm in the flowering phase and creating early-flowering or late-flowering germplasm.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the application of chrysanthemum G protein-coupled receptor GCR and its related factors in regulating flower development. Background Technology

[0002] Flower development is a crucial step in the transformation of angiosperms from vegetative to reproductive growth. Abnormal development of floral organs can severely impact the economic value of plants, such as ornamental plants. Flowering in higher plants is precisely regulated by photoperiod, ambient temperature, vernalization, gibberellin, age, and autonomous pathways. Ultimately, flowering integrators (such as SOC1) activate floral meristem characteristic genes (such as LFY / AP1), which in turn regulate floral organ formation through the ABCDE and tetramer models.

[0003] G protein-coupled receptors (GPCRs) are the most numerous class of membrane protein receptors, regulating a variety of biological processes. However, research on GPCRs in plants is very limited, and their role in flower development remains unclear.

[0004] Currently, thousands of GPCRs have been discovered in animals; however, in plants, such as plant cells, GPCRs have only been studied in preliminary stages and progress has been relatively slow.

[0005] Because GPCR sequences are less conserved, their signaling mechanisms may differ significantly from ion channel regulation, phospholipase C (PLC) / IP3 / DAG / Ca2+. 2+ The classic GPCR signal transduction pathways in three animal species: / PKC, cAMP / PKA, etc.

[0006] Based on studies of model plants such as Arabidopsis thaliana and rice, GPCRs are considered to play an important role in regulating plant growth and development. By constructing loss-of-function mutants and overexpression lines of key components of the G protein signaling pathway, it was found that plant GPCRs not only participate in the regulation of various plant hormone signals but also play important regulatory roles in abiotic stress responses and multiple developmental stages. However, research gaps remain regarding the regulatory functions of GPCRs in plant development: the functions of GPCRs in plants are currently limited to seed germination and seedling development. For example, in Arabidopsis thaliana, two novel GPCR-like G protein members, GTG1 and GTG2, mediate the ABA signaling pathway to regulate seed germination; while Arabidopsis thaliana GCR1 may respond to cytokinin signal transduction to participate in seedling development.

[0007] Whether GPCRs are involved in flowering regulation pathways, especially photoperiod / hormone signaling pathways, remains unknown.

[0008] Chrysanthemums are important ornamental and economic crops. Floral organs are crucial quality traits of chrysanthemums. Abnormal development of floral organs directly affects the ornamental value and economic benefits of chrysanthemums. This invention has discovered that the GCR (CmGCR) gene plays a vital role in chrysanthemum flower development and proposes a novel chrysanthemum flower development regulation technology based on CmGCR and related factors. Furthermore, this invention elucidates the spatiotemporal expression patterns of chrysanthemum GCR (CmGCR) genes during flower development, analyzes their biological functions in the flower development process, and reveals the signaling pathway networks they regulate and key downstream target genes. This is of great significance for a deeper understanding of the molecular mechanisms by which GPCR-mediated signal transduction regulates chrysanthemum flower development and for providing new targets and theoretical basis for molecular breeding to improve important traits related to chrysanthemum flowering time. Summary of the Invention

[0009] In order to solve one or more of the problems mentioned above in the prior art, the inventors, through long-term and in-depth research, discovered that... CmGCR, A chrysanthemum G protein-coupled receptor gene was upregulated by short-day induction, with the highest expression levels observed during the inflorescence meristem swelling stage and the floret primordium arrangement stage. Its expression primarily modulates gene expression. CmSAUR50 , CmGRAS17 , CmGI , CmAP1 and flowering integrators CmSOC1 and optical signal transmission factor CmCO The expression of [something] delays the arrangement of floret primordia and the flowering process, thus completing this invention.

[0010] This application provides, in a first aspect, a method for regulating the development of chrysanthemum flowers, the method being achieved by regulating... CmGCR and / or CmGCR The expression of relevant factors is used to determine the nature of the problem. CmGCR Related factors include downstream factors regulated by CmGCR in the photoperiodic pathway, downstream factors regulated by CmGCR in hormone signaling pathways (such as downstream factors regulated by CmGCR in the gibberellin pathway), or factors regulated by CmGCR in floral organ development. CmGCR Downstream factors that regulate.

[0011] Preferably, the regulation of chrysanthemum flower development is: (1) delaying or advancing the flowering process; (2) regulating flower bud differentiation and interfering with the development rate of the inflorescence meristem swelling period or the floret primordium arrangement period.

[0012] Preferably, the photoperiodic path is affected by... CmGCR Downstream factors that are regulated include chrysanthemum flowering integrators. CmSOC1 Timing factor CmGI Optical signal transmission factor CmCO .

[0013] Preferably or more preferably, the downstream factors regulated by CmGCR in the hormone signaling pathway include C mGRAS17, CmSAUR50.

[0014] Preferably or more preferably, the downstream factors regulated during the development of floral organs by CmGCR include C mAP1.

[0015] Preferably, the regulation of chrysanthemum flower development inhibits the arrangement process of floret primordia, delays flowering, and is achieved by overexpressing the CmGCR and / or reducing the expression of the CmGCR related factors.

[0016] Preferably, the regulation of chrysanthemum flower development accelerates the arrangement process of floret primordia, advances flowering, and is achieved by reducing the expression of CmGCR and / or increasing the expression of the CmGCR related factors.

[0017] More preferably, the method is carried out by at least one of the following: (1) knocking out the gene encoding the CmGCR or overexpressing the gene encoding the CmGCR related factor; (2) RNA interfering with the gene encoding the CmGCR or overexpressing the expression of the gene encoding the CmGCR related factor.

[0018] Preferably, the chrysanthemum is a ground-cover chrysanthemum. More preferably, the chrysanthemum is the ground-cover chrysanthemum cultivar 'Qiuyan' ( Chrysanthemum morifolium , cv. Fall Color).

[0019] It should be understood that those skilled in the art can also, without creative efforts, extend the new functions of the CmGCR and / or CmGCR related factors discovered by the present inventors to other plants, especially Compositae plants, as long as the plants targeted have a need for regulating flower development.

[0020] The present application provides in a second aspect the use of CmGCR and / or CmGCR related factors in regulating the flowering time of plants, wherein the CmGCR related factors are downstream factors regulated by CmGCR in the photoperiod pathway, downstream factors regulated by CmGCR in the hormone signaling pathway, or downstream factors regulated during the development of floral organs by CmGCR regulation.

[0021] Preferably, the downstream factors regulated in the photoperiod pathway by CmGCRThe regulated downstream factors include flowering integrators SOC1 , timekeeping factors GI , light signal transduction factors CO .

[0022] Preferably, the downstream factors regulated by CmGCR in the hormone signaling pathway include GRAS17 SAUR50.

[0023] Preferably, the downstream factors regulated by CmGCR in floral organ development include AP1.

[0024] Preferably, the plant is a Compositae plant. More preferably, the plant is a chrysanthemum, further preferably a ground-cover chrysanthemum, and even more preferably the ground-cover chrysanthemum cultivar 'Qiuyan' ( Chrysanthemum morifolium , cv. Fall Color).

[0025] Similar to the first aspect of the present invention: The regulation of plant flowering time can be: (1) delaying or advancing the flowering process; (2) regulating flower bud differentiation and intervening in the development rate of the inflorescence meristem swelling stage or the floret primordium arrangement stage; the downstream factors regulated by CmGCR in the photoperiod pathway can include or be chrysanthemum flowering integrators CmSOC1 , timekeeping factors CmGI , light signal transduction factors CmCO ; the downstream factors regulated by CmGCR in the hormone signaling pathway can include or be C mGRAS17, CmSAUR50; The downstream factors regulated by CmGCR in floral organ development can include or be C mAP1.

[0026] Similarly, the regulation of plant flowering time can be achieved by inhibiting the arrangement process of floret primordia, delaying flowering, and by overexpressing the CmGCR and / or reducing the expression of the CmGCR related factors; or, the regulation of plant flowering time can be to accelerate the arrangement process of floret primordia, advance flowering, and by reducing the expression of CmGCR and / or increasing the expression of the CmGCR related factors.

[0027] Similarly, the application is carried out by at least one of the following methods: (1) knocking out the gene encoding the CmGCR or overexpressing the gene encoding the CmGCR related factors; (2) RNA interfering with the gene encoding the CmGCR or overexpressing the gene encoding the CmGCRThe expression of genes related to these factors.

[0028] Compared with the prior art, the present invention has the following technical effects: (1) This invention discovered CmGCR It plays a crucial role in chrysanthemum flower development, especially during the swelling stage of inflorescence meristem and the arrangement stage of floret primordia. Therefore, by regulating its expression level in chrysanthemum (such as overexpression or RNAi silencing), the inflorescence development process and flowering time can be precisely regulated.

[0029] (2) This invention discovered that CmGCR mainly inhibits the expression of the flowering integrator CmSOC1 through the photoperiodic pathway and affects the auxin signal transduction pathway and the expression network of MADS-box genes in flower development. Therefore, it is possible to intervene in the CmGCR-CmSOC1 signaling axis (such as regulating its expression or interaction) or downstream key genes (such as... CmSAUR50 , CmGRAS17 , CmGI , CmAP1 (etc.) to affect the development of floral organs and the flowering process.

[0030] (3) This invention can also be applied to intervene in the flowering process of plants, such as advancing or delaying the flowering time. This invention is also particularly suitable for improving germplasm in terms of plant flower development, especially flowering time, such as for creating early-flowering or late-flowering germplasm. Attached Figure Description

[0031] Figure 1 For the chrysanthemum variety 'Shenma' ( Chrysanthemum morifolium Transcriptome data analysis of *C. Jinba* at different stages of transition to short-day conditions showed... CmGCR FPKM values ​​under different optical periods.

[0032] Figure 2 Sequence alignment of the CmGCR protein with GCRs from other species.

[0033] Figure 3 The N-terminal signal peptide of the CmGCR protein was analyzed using the SignalP6.0 website.

[0034] Figure 4 The transmembrane domain analysis of the CmGCR protein was performed using the TMHMM website.

[0035] Figure 5 The CmGCR protein structure prediction was completed on the Alphafold official website.

[0036] Figure 6 Subcellular localization analysis of CmGCR in tobacco leaves. Scale bar = 100 μm; excitation wavelengths were 488 nm and 561 nm, respectively.

[0037] Figure 7 for CmGCR The relative expression levels of genes in different tissues of chrysanthemum were determined. Quantitative real-time PCR (QRT) analysis was performed to assess the expression of each gene. CmUBI This is an internal reference gene. Three independent replicates were performed. Error bars represent standard deviations, and asterisks indicate significant differences according to t-tests (n ≥ 3, *P < 0.05, **P < 0.01).

[0038] Figure 8 for CmGCR The relative expression levels of genes in the apical buds at different growth and development stages.

[0039] Figure 9 for CmGCR Relative gene expression levels during chrysanthemum apical bud development. The top image shows gene expression levels, while the bottom images show the corresponding phenotypic diagrams, stereomicrographs, and scanning electron microscope images at each stage.

[0040] Figure 10 for CmGCR Detection of expression levels in overexpression lines.

[0041] Figure 11 For RNAi strains CmGCR Expression level detection.

[0042] Figure 12 for CmGCR Flowering time statistics of transgenic lines. Three independent replicates were performed. Error bars represent standard deviations, and asterisks indicate significant differences according to t-tests (n≧3, *P<0.05, **P<0.01).

[0043] Figure 13 for CmGCR Phenotypic characteristics of transgenic lines at different stages of floral development. Scale bar = 0.5 cm.

[0044] Figure 14 for CmGCR Statistical analysis of the time of each stage of flower development in transgenic lines. Error bars represent standard deviation.

[0045] Figure 15 for CmGCR Microscopic phenotype of floral organ development in transgenic lines. Scale bar = 250 μm.

[0046] Figure 16 for CmGCR Electron microscopic phenotype of floral organ development in transgenic lines. Scale bar = 250 μm.

[0047] Figure 17For the statistical analysis of the number of differentially expressed genes. Among them, blue represents the total number of differentially expressed genes, green represents the number of down-regulated genes, and orange represents the number of up-regulated genes.

[0048] Figure 18 For the Venn diagram of the number of differentially expressed genes.

[0049] Figure 19 For the expression analysis of candidate differentially expressed genes.

[0050] Figure 20 For CmGCR The expression analysis of flowering-related genes in transgenic lines. Specific implementation manners

[0051] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] Embodiment The present invention will be further described below through embodiments, but it should be understood that the protection scope of the present invention is not limited to these embodiments.

[0053] Example 1: Plant materials and treatment The chrysanthemum used in the embodiment is the ground-cover chrysanthemum variety 'Qiuyan' ( Chrysanthemum morifolium , cv.Fall Color). The tissue-cultured seedlings with a seedling age of 40 days (d) were transplanted into pots with a diameter of 9 cm containing a 1:1 (v / v) mixture of peat:vermiculite, and grown in a temperature-controlled culture room at a temperature of 23 ± 1 °C, a relative humidity of 40%, and a fluorescent lamp illumination of 100 μmol / m −2 s −1 , and a photoperiod of 16 h light / 8 h darkness. After the chrysanthemum plants were cultured under long-day (16 h light / 8 h darkness) conditions for 21 days, the plants were transferred to a short-day (10 h light / 14 h darkness) culture room to induce flowering.

[0054] Chrysanthemum seedlings cultured under long-day conditions for 21 days were sampled after short-day induction (three biological replicates were set up for each sample). Tissue-specific sampling sites included leaves, roots, stems, and terminal buds during the vegetative growth stage. Sampling sites for different developmental stages included terminal buds / flowers at 0 days of short-day induction (control), 2mm bud stage, 6mm bud stage, color-showing stage, and opening stage. Time-series expression sampling sites included terminal buds at 13 time points (0, 7, 11, 14, 18, 21, 24, 26, 28, 30, 35, 38, and 42 days) within 0–42 days of short-day induction. All samples were flash-frozen in liquid nitrogen and then stored at -80°C.

[0055] Example 2: CmGCR Genes and protein bioinformatics analysis Nucleic acid / amino acid sequences were aligned using NCBI BLAST (https: / / www.ncbi.nlm.nih.gov / ). A phylogenetic tree of GCR proteins from homologous species was constructed using the neighbor-joining method based on MEGA11. The tertiary structure of the proteins was predicted using AlphaFold (https: / / alphafoldserver.com / ). The signal peptide and transmembrane domain were analyzed using SignalP 6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ) and TMHMM 2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ), respectively.

[0056] Example 3: RNA extraction and qRT-PCR Total RNA was extracted from chrysanthemum tissue using a modified TRIzol method. After treatment with DNase I (Beijing Polymer Biotechnology), cDNA was synthesized using the M5 Super plus Mix kit. The chrysanthemum ubiquitin gene (GenBank accession number: EU862325) was used as an internal control, and qRT-PCR was performed on a StepOne Plus system using 2×M5 HiPer SYBR mix. The reaction program was: 95℃ for 3 min; 40 cycles (95℃ for 3 seconds, 60℃ for 30 seconds); melting curve analysis (65–95℃, 0.2℃ / step). -△△Ct The relative expression levels of the gene were calculated using a method. Quantitative primers are listed in SEQ ID No: 12-35.

[0057] Example 4: CmGCR Cloning of genes Total RNA was extracted from chrysanthemum tissue using a modified TRIzol method. After gDNA removal and reverse transcription to synthesize cDNA, high-fidelity PCR amplification was performed using cloning primers. The product was recovered from the gel and ligated into a T vector, transformed into *E. coli* DH5α, and positive clones were screened and sequenced for verification. CmGCR Full-field CDS sequence of the gene.

[0058] The primer sequences used for cloning are as follows: CmGCR -FULL-F:5′-TGCAACGATCCTTGAGTCATAAAC-3′ (SEQ ID No: 1); CmGCR -FULL-R:5′-TAGGATCCATTCCCTCTTCGAGT-3′ (SEQ ID No: 2); The CDS sequence is shown in SEQ ID No: 3.

[0059] Example 5: Subcellular localization Will CmGCR The gene ORF sequence was ligated into the pSuper1300-GFP vector (restriction site: XbaI / KpnI) via homologous recombination to construct the pSuper::CmGCR-GFP recombinant vector. After transforming Agrobacterium GV3101 with this vector, the cells were resuspended in infection solution (containing 10 mM MES, 10 mM MgCl2, and 200 μM AS) to OD200. 600 =0.6-0.8. Using pSuper1300-GFP as a control, a bacterial suspension was mixed at a ratio of pSuper1300:nuclear marker:P19 = 3:2:2 and incubated in the dark for 2-3 hours before injection into *Nicotiana benthamiana* leaves. Three days after injection, leaf tissue was collected and observed using an OLYMPUS FV1000 confocal laser scanning microscope. GFP images were obtained under confocal microscopy with excitation at 488 nm and emission at 525 nm. The primer sequences are as follows: Supper1300-XbaI- CmGCR -F:ccaaatcgactctagtctagaATGAACCACAGGATCAGTTTATTGC (SEQ ID No: 4); Supper1300-KpnI- CmGCR -R:gcccttgctcaccatggtaccGGTCCCGTATCTTCTCTCACTTGT (SEQ ID No: 5); The lowercase part is designed for the vector sequence, and the uppercase part is designed for the target gene sequence. The same applies throughout the text.

[0060] Example 6: Genetic transformation of chrysanthemum Using leaves from tissue-cultured seedlings of the chrysanthemum variety 'Qiuyan' as explants, a system was constructed. CmGCR Overexpression vector (using the method described with subcellular localization) and CmGCR- RNAi vectors. The construction process for RNAi vectors is as follows: cloning. CmGCR The 3' UTR fragment (approximately 300 bp) was inserted into the BamHI / PacHI site of the pFGC1008 vector via homologous recombination, with the sense fragment inserted into the AscHI / SwaHI site, forming an intron containing hairpin RNA (ihpRNA). The recombinant vector was transformed into Agrobacterium EHA105, and chrysanthemum leaf explants were infected using the leaf disc method. Infection conditions included OD... 600 =0.1~0.2% bacterial suspension for 30 minutes. Induction medium (IM: containing 1.5 mg / L 2,4-D + 0.5 mg / L 6-BA) was used for dark culture for 3 days; subculture was performed every 14 days using conidial medium (RM: containing 50 μg / mL hygromycin); positive seedlings were selected using rooting medium (RTM: containing 15 μg / mL hygromycin); and transgenic plants were detected by qRT-PCR. CmGCR The amount of expression, ultimately obtained CmGCR -OX and CmGCR -RNAi stable lines.

[0061] The cloning CmGCR The primers for the 3'UTR fragment are as follows: CmGCR -3'UTR-F: 5'-CCTATTATGCCCTCAAAGC-3' (SEQ ID NO: 6); CmGCR -3'UTR-R: 5'-TCCAGAGTCTCCCATTCCT-3' (SEQ ID NO: 7).

[0062] The primers used to construct the RNAi vector are as follows: sen- CmGCR -BamHI-F:cgatctctttgatggggatccCAGTGAGTGGTGTTGGAGGAGG (SEQ IDNO: 8); sen- CmGCR -PacI-R:gactctagggactagttaattaaCTTTGAGGGCATAATAGGAAAAAAG (SEQID NO: 9); Anti- CmGCR-AscI-F:ttacaattaccatggggcgcgccCTTTGAGGGCATAATAGGAAAAAAG (SEQ ID NO: 10); Anti- CmGCR -SwaI-R:catgttcatctggggatttaaatCAGTGAGTGGTGTTGGAGGAGG (SEQ ID NO: 11).

[0063] Table 1. Quantitative primers used in the examples The results obtained in each embodiment will be analyzed from different aspects below.

[0064] 1. CmGCR is an atypical G protein-coupled receptor. In their previous research, the inventors analyzed transcriptome data of the chrysanthemum variety 'Shenma' under different periods of short-day treatment. Figure 1 Differentially expressed genes were obtained through screening. CmGCR In order to investigate CmGCR The inventor cloned the gene's function from the ground cover chrysanthemum variety 'Qiuyan'. CmGCR The full-length cDNA sequence is 1359 bp, of which the cds sequence is 1212 bp. Figure 2 It encodes 403 amino acids. Phylogenetic analysis revealed its phylogenetic relationship with the gene AT4G36440 on Arabidopsis thaliana chromosome 4, belonging to the G protein-coupled receptor family, hence it was named the chrysanthemum G protein-coupled receptor GCR. The inventors used the SignalP 6.0 website (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ) to analyze the localization signal sequence of the CmGCR protein, finding that the CmGCR protein contains a signal peptide, which is amino acids 1-24 of the N-terminus (…). Figure 3 The signal peptide consists of amino acids 1-4 (n-region), 5-19 (h-region), and 20-24 (c-region). The transmembrane domain of the CmGCR protein was then analyzed using the TMHMM website (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). The CmGCR protein possesses one transmembrane domain, with the N-terminus located on the outer side of the cell membrane and the C-terminus on the inner side. Of the 403 amino acids in the CmGCR protein, amino acids 1-309 are located outside the membrane, amino acids 310-332 form the transmembrane domain, and amino acids 333-403 are located inside the membrane. Figure 4To investigate the tertiary structure of the CmGCR protein, the inventors used the online prediction function of the Alphafold official website (https: / / alphafoldserver.com / ) to predict the tertiary structure of the CmGCR protein. The results showed that CmGCR may contain 3 α-helices and 19 β-sheets. Figure 5 Based on its structure, CmGCR is speculated to be a membrane receptor protein.

[0065] To investigate the intracellular localization characteristics of CmGCR protein, the expression vector Supper1300 was successfully constructed and transformed into Agrobacterium GV3101. Subsequently, tobacco leaves were injected and infected with the transformed plasmid. Observation under a fluorescence confocal microscope revealed green fluorescence signals in both the cell membrane and endoplasmic reticulum of the tobacco leaf cells. Figure 6 This indicates that the CmGCR protein is mainly located in the membrane system, including the cell membrane and endoplasmic reticulum. Therefore, the CmGCR protein is a membrane protein, consistent with the prediction of CmGCR based on its transmembrane domains and tertiary structure.

[0066] 2. CmGCR High expression in the apical meristem during flower differentiation and arrangement of floret primordia The inventor tested CmGCR The expression of genes in different tissues of chrysanthemum plants was investigated. The results showed that... CmGCR The gene was expressed in chrysanthemum leaves, stems, and terminal buds, with the expression level in terminal buds being significantly higher than that in leaves and stems, approximately twice that in leaves and stems. Figure 7 This indicates CmGCR It plays an important role in the physiological processes related to the apical bud. To investigate... CmGCR Expression patterns at different stages of chrysanthemum apical bud development: The inventors examined gene expression levels in the apical buds of chrysanthemum plants at different developmental stages. The results showed that... CmGCR The expression level of CmGCR was significantly higher during the budding stage than during the vegetative growth stage, and reached its highest level at the 2mm budding stage. During subsequent development, the expression of CmGCR gradually decreased and returned to the level of the vegetative growth stage. Figure 8 These results indicate that CmGCR It plays an important role in the development of chrysanthemum flowers and is closely related to the development of flower buds. To more accurately determine... CmGCRTo investigate the role of CmGCR in the development of chrysanthemum apical buds, the inventors selected apical buds from chrysanthemum plants that had been subjected to short-day conditions for 0, 7, 11, 14, 18, 21, 24, 26, 28, 30, 35, 38, and 42 days as samples for expression analysis. Phenotypic observation was also conducted using stereomicroscopy and electron microscopy. The results showed that the expression level of CmGCR significantly increased after being subjected to short-day conditions, remaining high from day 7 to day 26, and subsequently returning to the level of the vegetative growth period. Figure 9 Phenotypic observations using both stereomicroscopy and electron microscopy revealed that high CmGCR expression occurred during the inflorescence meristem swelling stage and the floret primordium arrangement stage. Once the floret primordium arrangement was complete, CmGCR expression returned to normal levels. These results indicate that CmGCR plays a role in the inflorescence meristem swelling stage and the floret primordium arrangement stage of chrysanthemum flower development, and is closely related to the physiological processes of flower development during these two stages.

[0067] 3. CmGCR Acting at the inflorescence meristem stage and at the stage of primordia arrangement to regulate the process of flower development To obtain stably heritable CmGCR overexpression lines and RNAi lines, the inventors successfully constructed the CmGCR overexpression vector pSupper1300 and the RNAi silencing vector pFGC1008. Through screening on hygromycin HYP selection medium, detection of relative CmGCR expression levels at the RNA level, and preliminary observation of chrysanthemum plant phenotypes, 14 stably heritable CmGCR overexpression lines and 9 CmGCR RNAi silencing lines were successfully obtained. Figure 10 The inventors selected the overexpression lines OX-4 and OX-5, as well as the silenced lines RNAi-1 and RNAi-5, as representatives for in-depth research. Figure 11 ).

[0068] Through the CmGCR Phenotypic observations of various transgenic lines revealed that... CmGCR- The flowering time of the OX strain was significantly later than that of the WT strain, and CmGCR- The flowering time of the RNAi line was significantly earlier than that of the WT line. Figure 12 ). CmGCR- RNAi lines showed obvious flower buds as early as day 21 after being introduced into short-day conditions, while CmGCR- For the OX and WT strains, it takes until the 28th day to observe obvious flower buds. Figure 13 The timing of each stage of flower development in the transgenic lines was statistically analyzed. The results showed that the differences in flowering time among the lines were mainly due to differences in the flower bud differentiation period and the budding stage. Figure 14 There is no difference in the developmental process between the color-revealing stage and the opening stage.

[0069] Therefore, the inventors conducted further observations on the developmental processes of flower bud differentiation and bud formation in various strains. The observations under a stereomicroscope showed that ( Figure 15 ), CmGCR- The arrangement of floret primordia in the OX strain was later than that in the WT strain, and CmGCR- The floret primordium arrangement process in the RNAi strain is earlier than that in the WT strain. On day 14 after transition to short-day conditions, CmGCR- In the OX strain, the floret primordia were not yet arranged, while in the WT strain, only half were arranged. CmGCR- The RNAi strains have been largely arranged. There was no significant difference in the rate of bud development among the strains. Electron microscopic observations were largely consistent with stereomicroscopic findings. Figure 16 ).show CmGCR The gene mainly functions during the swelling stage of the inflorescence meristem and the arrangement stage of the floret primordia, and may be involved in regulating the flower development process during these two stages.

[0070] 4. CmGCR Regulation of flowering time by suppression of the expression of the flowering integrator SOC1 through the photoperiod pathway To verify CmGCR To investigate the function of chrysanthemum flowers in development, the inventors sampled apical buds from various transgenic lines before and after flower bud differentiation and performed transcriptome sequencing. The number of differentially expressed genes was statistically analyzed. The results showed that in RNAi lines, the number of upregulated genes was significantly greater than the number of downregulated genes, and in overexpression lines, the number of downregulated genes was significantly greater than the number of upregulated genes. Figure 17 , 18 ).

[0071] GO classification and annotation of differentially expressed genes revealed that they were mainly enriched in biological processes such as cellular processes, metabolic processes, biological regulation, response to stimulus, signaling, developmental processes, and localization. KEGG classification and annotation showed that differentially expressed genes are mainly involved in signaling pathways such as plant hormone signal transduction, MAPK signaling pathway, carbon metabolism, amino sugar and nucleotide sugar metabolism, and plant-pathogen interactions.

[0072] Table 2 shows the log2FC values ​​and annotations of key differentially expressed genes. The log2FC value represents the base-2 logarithm of the fold change, measuring the degree of difference in gene expression levels among different groups. Differentially expressed genes related to flower development were screened, and seven key candidate genes related to flower development were identified. These genes encode auxin-responsive proteins SAUR, DELLA, GIGANTEA, and several MADS-box transcription factors that regulate flower organ development (Table 2).

[0073] Table 2. Log2FC values ​​and annotations of key differentially expressed genes.

[0074]

[0075] Further screening of the above genes using qPCR revealed the following four key genes: members of the auxin-responsive gene family. CmSAUR50 Expression level CmGCR Significantly increased in -RNAi lines and CmGCR Significantly reduced in -OX strains; a member of the DELLA protein family, a key inhibitor of the gibberellin pathway. CmGRAS17 exist CmGCR The expression level of -RNAi in the strain was significantly lower than that in the WT strain. CmGCR -OX strains were higher than WT strains; CmGI It is a key timing factor in the photoperiodic pathway. CmAP1 These are key A-type genes in the ABCDE model of flower development; they play a crucial role in... CmGCR The expression levels of RNAi in the RNAi lines were all higher than those in the WT lines. Figure 19 Based on the above results, therefore, CmGCR It may exert its effects through the gibberellin pathway and the photoperiod pathway, and participate in the development of inflorescence meristems by regulating the auxin signal transduction pathway and MADS-box genes in flower development.

[0076] To further determine CmGCR This invention involves the regulation of specific pathways in flowering and flower development. The inventors sampled apical buds from CmGCR transgenic lines during their vegetative growth and flower bud differentiation stages to detect the expression levels of several flowering-related genes, including key genes involved in various major flowering pathways. GI , GA3ox , GA20ox , FLC , SPL3 , SPL9 , CO And some flowering integrators including SOC1 , AFT The results showed that key genes in the photoperiodic pathway... CmCO , CmGI During the flower bud differentiation period CmGCR -OX expression levels were significantly lower in the WT strain than in the WT strain. CmGI exist CmGCR -RNAi strains showed higher expression levels than WT; key genes in the gibberellin pathway CmGA3ox , CmGA20ox During the vegetative growth period CmGCR -OX expression levels were significantly lower than WT in the OX lines, but there were no significant differences among the lines during flower bud differentiation; in the temperature pathway, key genes CmFLC transgenic There were no significant differences among strains; in the age pathway... CmSPL3 , CmSPL9 During the flower bud differentiation period CmGCR -OX expression levels were lower than WT in the OX strain, but CmGCR No significant differences were observed in RNAi lines. Flowering integrative factors CmSOC1 exist CmGCR -OX expression levels were significantly lower in the OX strain than in the WT strain. CmGCR The RNAi strain was significantly higher than the WT strain; CmAFT There were no significant differences among the lines during the flower bud differentiation period. Figure 20 The above results indicate that... CmGCR It may primarily function through the photoperiodic pathway, by inhibiting flowering integrators. CmSOC1 The expression of these substances participates in the regulation of the development process of inflorescence meristems.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for regulating chrysanthemum flower development, characterized in that: The method involves regulation CmGCR and / or CmGCR The expression of relevant factors is used to determine the nature of the problem. CmGCR The relevant factors are downstream factors regulated by CmGCR in the photoperiodic pathway, downstream factors regulated by CmGCR in the hormone signaling pathway, or factors regulated by CmGCR in floral organ development. CmGCR Downstream factors that regulate.

2. The method according to claim 1, characterized in that, The regulation of chrysanthemum flower development is as follows: (1) Delay or advance the flowering process; (2) Regulate flower bud differentiation and intervene in the development rate of the inflorescence meristem swelling period or the floret primordium arrangement period.

3. The method according to claim 1, characterized in that: The photoperiodic path is affected by CmGCR Downstream factors that are regulated include chrysanthemum flowering integrators. CmSOC1 Timing factor CmGI Optical signal transmission factor CmCO ; The downstream factors regulated by CmGCR in the hormone signaling pathway include C mGRAS17, CmSAUR50; Or the development of the flower organs is affected CmGCR Downstream factors that regulate include C mAP1.

4. The method according to claim 1, characterized in that: The regulation of chrysanthemum flower development involves inhibiting the arrangement of floret primordia, delaying flowering, and is achieved by overexpressing the aforementioned... CmGCR and / or reduce the CmGCR It is done by analyzing the expression of relevant factors.

5. The method according to claim 1, characterized in that: The regulation of chrysanthemum flower development accelerates the arrangement of floret primordia, promotes earlier flowering, and reduces... CmGCR The expression and / or enhancement of the CmGCR It is done by analyzing the expression of relevant factors.

6. The method according to claim 5, characterized in that, The method is performed in at least one of the following ways: (1) Knockout encoding CmGCR The gene or overexpression encoding the CmGCR Genes of related factors; (2) RNA interference encoding the above CmGCR The gene or overexpression encoding the CmGCR The expression of genes related to these factors.

7. The method according to any one of claims 1 to 6, characterized in that, The chrysanthemum mentioned is a ground cover chrysanthemum.

8. CmGCR and / or CmGCR The application of relevant factors in regulating plant flowering time, wherein CmGCR The relevant factors are downstream factors regulated by CmGCR in the photoperiodic pathway, downstream factors regulated by CmGCR in the hormone signaling pathway, or factors regulated by CmGCR in floral organ development. CmGCR Downstream factors that regulate.

9. The application according to any one of claims 8, characterized in that: The photoperiodic path is affected by CmGCR Downstream factors that regulate this include flowering integrators. SOC1 Timing factor GI Optical signal transmission factor CO ; The downstream factors regulated by CmGCR in the hormone signaling pathway include GRAS17, SAUR50; or The development of the floral organs is affected by CmGCR Downstream factors that are regulated include AP1.

10. The application according to claim 9, characterized in that: The plant is a member of the Asteraceae family; and / or The photoperiodic path is affected by CmGCR Downstream factors that are regulated include chrysanthemum flowering integrators. CmSOC1 Timing factor CmGI Optical signal transmission factor CmCO The downstream factors regulated by CmGCR in the hormone signaling pathway include C... mGRAS17 CmSAUR50; and / or the development of the floral organs affected by CmGCR Downstream factors that regulate include C mAP1.

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