Application of protein CmMYC2 in regulating flowering stage, flowering amount and natural plant type roundness of garden chrysanthemum
By suppressing or silencing the CmMYC2 gene in garden chrysanthemums, new varieties with early flowering, more branching, and rounder shape were bred. This solved the problems of garden chrysanthemums having unique flower shapes and bright colors but tall, thin plants that are prone to lodging and have few flowers. It achieved earlier flowering, more branching, and more flowers, thus enhancing the ornamental value and landscape effect of garden applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING FORESTRY UNIVERSITY
- Filing Date
- 2026-01-22
- Publication Date
- 2026-06-05
AI Technical Summary
Existing garden chrysanthemum varieties have unique flower shapes and vibrant colors, but their tall and slender plants are prone to lodging, produce few flowers, and bloom late, making it difficult to meet the demands of major festivals and to create a concentrated landscape when paired with early-flowering plants. Traditional breeding methods are inefficient, and there are few cases of improvement through genetic modification.
By inhibiting or reducing the activity or expression of the protein CmMYC2 in garden chrysanthemums, the CmMYC2 gene is silenced using RNAi technology, and new varieties with early flowering, many branches, naturally round plant shape, and large number of flowers are bred. Gene knockout or gene silencing methods are used to reduce the expression of the CmMYC2 encoding gene.
It achieves earlier flowering period, increased lateral branch length, more branches, rounder plant shape, and increased flowering volume for garden chrysanthemums, while reducing production costs, meeting the needs of garden applications, and enhancing ornamental value and landscape effect.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of protein CmMYC2 in regulating the flowering period, flowering quantity, and natural plant shape of garden chrysanthemums. Background Technology
[0002] chrysanthemum( Chrysanthemum × morifolium Ramat. is a perennial herbaceous plant belonging to the genus Chrysanthemum in the family Asteraceae. It is also one of the traditional famous flowers, possessing high ornamental and economic value. Garden chrysanthemums are an important branch among the many application categories of cultivated chrysanthemums. In recent years, with policy support and rising market demand, the garden chrysanthemum industry has developed rapidly and occupies an important position in flower production and landscaping applications.
[0003] Unlike cut chrysanthemums, which prioritize a single outstanding bloom in production and breeding, garden chrysanthemums, due to their different application, are best characterized by early flowering, abundant branching, good ball-forming ability, rounded plant shape, and profuse flowering. However, there are currently few highly ornamental varieties that meet these requirements. Some varieties have unique flower shapes, vibrant colors, and high ornamental value, but they are tall and slender, prone to lodging, and produce few flowers. During production, they require constant manual pruning to achieve a more rounded plant shape, which increases costs. In addition, chrysanthemums are typical short-day plants with a late flowering period, which cannot adequately meet the needs of major festivals. Furthermore, they are difficult to combine with other early-flowering plants to create a concentrated landscape. For these reasons, some excellent varieties cannot be effectively promoted and applied.
[0004] With the rapid development of molecular biology, Agrobacterium-mediated genetic transformation technology has gradually become an important means of modern breeding to alter the horticultural traits of ornamental plants. Compared with traditional breeding methods that mainly rely on hybridization, this method is more targeted and precise, greatly improving breeding efficiency and shortening breeding time. It provides new means and pathways for improving chrysanthemum varieties. However, there are currently few reports of cases using transgenic methods to improve chrysanthemum plant shape and flowering quantity. Therefore, genetically modifying existing highly ornamental garden chrysanthemums to cultivate new varieties with early flowering, abundant branching, naturally rounded plant shape, and high flowering quantity has significant practical value and economic significance for promoting variety improvement and landscaping applications. Summary of the Invention
[0005] The technical problem to be solved by this invention is how to regulate the flowering period, number of branches, and amount of flowers of garden chrysanthemums.
[0006] To address the above technical problems, this invention provides the application of substances that reduce the activity or content of protein CmMYC2, or substances that inhibit or reduce the expression of the gene encoding said protein CmMYC2, wherein the application is any one of Y1-Y7: Y1. Application in advancing the flowering period of chrysanthemums in gardens; Y2. Application in increasing the natural roundness of the plant shape of garden chrysanthemums; Y3. Application in increasing the length of lateral branches of garden chrysanthemums; Y4. Application in increasing the number of branches of garden chrysanthemums; Y5. Application in increasing the number of chrysanthemums in gardens; Y6. Application in increasing the crown width of garden chrysanthemums; Y7. Application in the breeding of garden chrysanthemums; The protein CmMYC2 is a protein that is, as shown in A1), A2), or A3), the following: A1) The amino acid sequence is that of the protein listed as SEQ ID No:2; A2) A protein that has more than 80% identity with and is functionally similar to the protein shown in A1, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No:2 in the sequence listing. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).
[0007] The breeding program aims to select early-flowering and / or naturally round plant shape and / or many branches and / or large number of flowers and / or large crown size garden chrysanthemums.
[0008] The garden chrysanthemums mentioned are multi-flowered chrysanthemums.
[0009] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.
[0010] In the above applications, the protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.
[0011] In the above applications, the gene encoding the protein CmMYC2 can be a DNA molecule as shown in a1), a2), or a3) below: a1) The coding sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing; a2) has 90% or more identity with the nucleotide sequence defined by a1) and encodes a DNA molecule that encodes the protein CmMYC2 described above; a3) hybridizes under strict conditions with the nucleotide sequence defined by a1) or a2) and encodes a DNA molecule that encodes the protein CmMYC2 described above.
[0012] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Having 90% or more identity can mean at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
[0013] In the above applications, inhibiting or reducing the expression of the gene encoding the protein CmMYC2 can be achieved by gene knockout or gene silencing.
[0014] Gene knockout refers to the process of making a specific target gene lose its function by altering its DNA sequence.
[0015] Gene silencing refers to the phenomenon of preventing or reducing the expression of a gene without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes RNA interference (RNAi), antisense RNA, co-suppression, gene quelling, and microRNA-mediated translational repression.
[0016] In the above applications, the substance that inhibits or reduces the expression of the gene encoding the protein CmMYC2 can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.
[0017] In the above applications, the substance that inhibits or reduces the expression of the gene encoding the protein CmMYC2 can be any one of the following c1)-c4): c1) Nucleic acid molecules that inhibit or reduce the expression of the protein CmMYC2 encoding gene; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).
[0018] In the above application, c1) the nucleic acid molecule is a nucleic acid molecule that targets the gene encoding the protein CmMYC2, and the nucleic acid molecule includes a sense fragment with the sequence shown as positions 1228-1567 of SEQ ID No. 1 and an antisense fragment that is inversely complementary to it.
[0019] To address the aforementioned technical problems, the present invention also provides a method for breeding early-blooming garden chrysanthemums, comprising obtaining early-blooming garden chrysanthemums by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient garden chrysanthemum, wherein the flowering period of the early-blooming garden chrysanthemum is earlier than that of the recipient garden chrysanthemum.
[0020] To address the aforementioned technical problems, the present invention also provides a method for breeding naturally round-shaped garden chrysanthemums, comprising obtaining naturally round-shaped garden chrysanthemums by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient garden chrysanthemum, wherein the natural plant shape of the naturally round-shaped garden chrysanthemum is more rounded than that of the recipient garden chrysanthemum.
[0021] To address the aforementioned technical problems, the present invention also provides a method for increasing the length of lateral branches and / or increasing the number of branches of garden chrysanthemums, comprising the step of obtaining a target garden chrysanthemum by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient garden chrysanthemum, wherein the lateral branch length of the target garden chrysanthemum is longer than and / or the number of branches is greater than that of the recipient garden chrysanthemum.
[0022] To address the aforementioned technical problems, the present invention also provides a method for breeding garden chrysanthemums with high flowering volume, comprising obtaining garden chrysanthemums with high flowering volume by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient garden chrysanthemum, wherein the flowering volume of the garden chrysanthemum with high flowering volume is greater than that of the recipient garden chrysanthemum.
[0023] To address the aforementioned technical problems, the present invention also provides a method for breeding large-crown garden chrysanthemums, comprising obtaining large-crown garden chrysanthemums by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient garden chrysanthemum, wherein the crown width of the large-crown garden chrysanthemum is greater than that of the recipient garden chrysanthemum.
[0024] The above-mentioned inhibition or reduction of the expression level of the gene encoding the protein CmMYC2 in the genome of the receptor Chrysanthemum in the garden can be achieved by any method in the prior art, so as to induce deletion mutations, insertion mutations or base transformation mutations in the gene, thereby reducing or losing gene function. Specifically, this can be achieved by chemical mutagenesis, physical mutagenesis, RNAi, site-directed genome editing or homologous recombination, etc.
[0025] The method described above may include introducing a substance into the recipient *Chrysanthemum indicum* that inhibits or reduces the expression level of the gene encoding the protein CmMYC2 in the genome of the recipient *Chrysanthemum indicum*, or introducing a substance that reduces or inhibits the expression of the gene encoding the protein CmMYC2. The substance that reduces or inhibits the activity of the protein CmMYC2, or the substance that reduces or inhibits the expression of the gene encoding the protein CmMYC2, may be any one of the following c1)-c4): The substance that inhibits or reduces the expression of the gene encoding the protein CmMYC2 can be any one of the following c1)-c4): c1) Nucleic acid molecules that inhibit or reduce the expression of the protein CmMYC2 encoding gene; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).
[0026] In the above method, c1) refers to a nucleic acid molecule that targets the gene encoding the protein CmMYC2. The nucleic acid molecule includes a sense fragment with a sequence as shown in positions 1228-1567 of SEQ ID No. 1 and an antisense fragment that is inversely complementary to it.
[0027] The present invention also provides the protein CmMYC2.
[0028] The biomaterials related to the protein CmMYC2 are also within the scope of protection of this invention; the biomaterials are any one of B1 to B5 below: B1. The nucleic acid molecule encoding the protein CmMYC2; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4. Recombinant microorganisms containing the nucleic acid molecules described in B1, or recombinant microorganisms containing the expression cassette described in B2, or recombinant microorganisms containing the recombinant vector described in B3; B5. Transgenic plant cell lines, transgenic plant tissues, transgenic plant organs, or transgenic plants containing the nucleic acid molecules described in B1.
[0029] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
[0030] This invention utilizes RNAi technology to silence chrysanthemums in gardens. CmMYC2 Genetic analysis and phenotypic observation of transgenic plants revealed that they flowered earlier, had significantly increased lateral branch length, and exhibited more branching, resulting in a more rounded plant shape and a significantly increased number of flowers. This invention can be widely applied to the cultivation of garden chrysanthemums, providing a novel and practical method for improving important horticultural traits of flowers using genetic engineering technology and reducing labor costs in the production process. It has high application value. Attached Figure Description
[0031] Figure 1 In Embodiment 1 of the present invention CmMYC2 -RNAi expression vector CmMYC2 A diagram illustrating the insertion positions of forward and reverse segments.
[0032] Figure 2 Wild type and in embodiment 1 of the present invention CmMYC2 -RNAi transgenic plants CmMYC2 Gene expression analysis.
[0033] Figure 3 Wild type and in embodiment 1 of the present invention CmMYC2 -Statistics on plant type and lateral branch length of RNAi transgenic plants. Figure 3 A represents the plant type. Figure 3 B represents the lateral branch length statistics, where 1-20 indicates the lateral branch number from the apex to the base. **** in the figure represents a significance analysis result of P<0.0001.
[0034] Figure 4 Wild type and in embodiment 1 of the present invention CmMYC2 - Flowering status of RNAi transgenic plants. Figure 4 Photo A shows photos taken 120 days (top) and 160 days (bottom) after transplanting. Figure 4 B represents the number of flower buds and flower branches counted 120 days after planting. Figure 4 C represents the crown width measured 160 days after planting. **** in the figure indicates a significance analysis result of P < 0.0001.
[0035] Figure 5 Wild type and in embodiment 1 of the present invention CmMYC2 -Statistics on flowering period of RNAi transgenic plants. Figure 5 A represents the time period corresponding to each period, with the planting day as 0d; Figure 5 B is a photograph taken at 150 days. Among them, FBD stage is the flower bud development stage, VC stage is the visible color stage, and OF stage is the opened flower stage. Detailed Implementation
[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0038] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.
[0039] The garden chrysanthemum '331' in the following examples has been disclosed in the master's thesis "Huang Jialu. Expression analysis and transgenic study of CmCYC2d gene in the flower shape formation of 'Tuogui' type chrysanthemum. 2023", and is available to the public from the applicant.
[0040] The pART27 vector in the following examples has been disclosed in the master's thesis "Huang Jialu. Expression analysis and transgenic study of CmCYC2d gene in the formation of Togui-type small chrysanthemum. 2023", and is available to the public from the applicant.
[0041] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.
[0042] Example 1 one, CmMYC2 Gene sequence cloning Cloning in chrysanthemum '331' CmMYC2 The nucleotide sequence (CDS) of the gene is SEQ ID No. 1, and the amino acid sequence of the protein CmMYC2 encoded by it is SEQ ID No. 2.
[0043] SEQ ID No.1 SEQ ID No.2 MKCTIFLTTSLFILHHLLLYIIIIRCNKIQSIRYKLREMDDLIVSSSSSSSIVSIPSTAINPNQCDTLQQKLQTLLQNQPQPWAYAIFWQTFNDDSNGCVSLSWGDGHFLSSNDTLPDSFLPDSDLDCRKS VVREIKALLGPDNREDAEWFYVISLTRSFMPGDGSVPGTALGSNSMIWLSGVDQLQSFSCERAKEAQVHGLETMVCIPTCNGVVEMGSYHFIEETWNLAHQAQSLFGFGGGSTKFNELNDGHHNIVSFADMV IMASGLHDEQDEGITPDDEMSKNGGMLCTNMNNAVVTNTYIETGSEQSDSDCQLVLATTENRFQKKKGKHTRGRCPPVNHVEAERQRREKLNQRFYALRSVVPNVSRMDKASLLADAVCYISELKQKVECL ESQLQHRNNNQGKIKKVKTELPHTTDNTCNLYVSTKPTLKNNNKANMNKMTSGFGEIEVKIVGEDAMIRVQSGNSDLPSAKLTDALREMKGQIQHASMSCVNDIMLQDVVVRIPGATDSDELKSDLIRILDL II. pART27- CmMYC2 -RNAi vector construction exist CmMYC2 A 340 bp fragment (positions 1228-1567 of SEQ ID No. 1) from the non-structural domain region of the gene was selected as the interference sequence. Using the CDS of CmMYC2 as a template, PCR amplification of the forward and reverse complementary sequences of this fragment was performed using KOD-Plus series high-fidelity polymerases, respectively. The primer sequences for amplifying the forward and reverse complementary sequences are as follows: Primers for amplifying the forward fragment: Ri-ZF-MYC2:5′ TTTGGAGAGGACACGCTCGAGAAAGTGAAGACAGAA 3′ (bits 22-36 are the same as bits 1228-1242 of SEQ ID No. 1); Ri-ZR-MYC2:5′ TTCCTTACCAATTGGGGTAACCTCCTAATGAGATCGG 3′ (bits 22-36 and bits 1553-1567 of SEQ ID No. 1 are opposite complements).
[0044] Primers for amplifying the reverse fragment: Ri-FF-MYC2:5′ GGGTTCGAAATCGATAAGCTTTCCTAATGAGATCGG 3′ (bits 22-36 and bits 1553-1567 of SEQ ID No. 1 are opposite complements); Ri-FR-MYC2:5′ TCATTAAAGCAGGACTCTAGAAAAGTGAAGACAGAA 3′ (bits 22-36 are the same as bits 1228-1242 of SEQ ID No. 1).
[0045] The reaction procedure was as follows: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, for 35 cycles, followed by a final extension at 72℃ for 10 min. Agarose gel electrophoresis was performed to detect the target band. The target band was excised and recovered using a gel recovery kit (see kit instructions for details). The recovered PCR product and double-digested enzymes (forward fragment: Xho I. Kpn I; Reverse segment: Hind III. Xba I) pART27 vector ligation, transformation DH5 Alpha competent cells (Coolaber cc501) were used to select single clones of the strain. Positive strains were identified by PCR and named... DH5 α-pART27- CmMYC2 -RNAi; plasmid extracted and named pART27- CmMYC2 -See the diagram showing the insertion sites of the forward and reverse fragments on the RNAi expression vector. Figure 1 .
[0046] III. Experimental Procedure for Agrobacterium GV3101-Mediated Leaf Disc Transformation of Chrysanthemum ① Preparation of infection solution and culture medium a. Save DH5 α-pART27- CmMYC2RNAi bacterial culture was activated by streaking on LB solid medium and incubated statically at 28°C for two days. Colonies were then picked using a pipette tip and added to 10 ml of LB liquid medium. The culture was incubated at 28°C with shaking at 200 rpm for 12 h. 1000 μl of the bacterial culture was then added to 100 ml of LB liquid medium for further culture for 12 h. Both media contained 50 mg / L Kana and 30 mg / L Rif.
[0047] b. Centrifuge 100 ml of bacterial culture at 5000 rpm for 5 min at 4°C, discard the supernatant, resuspend the Agrobacterium cells in MS liquid medium (containing 30 g / L sucrose), and then... 600 Adjust the value to 0.4-0.45.
[0048] c. Prepare three types of culture media.
[0049] Co-culture medium: MS + 6-BA 2.0 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + agar 7 g / L; Differentiation induction medium: MS + 6-BA 2.0 mg / L + NAA 1.0 mg / L + Kanamycin 11 mg / L + Carb 400 mg / L + Sucrose 30 g / L + Agar 7 g / L; Rooting medium: MS + NAA 0.3 mg / L + Kana 8 mg / L + Carb 400 mg / L + Sucrose 30 g / L + Agar 7 g / L.
[0050] ② Transformation of chrysanthemum leaves using the leaf disc infection method a. Use robust, sterile '331' garden chrysanthemum seedlings (approximately 40 days after subculturing) as recipient material. Cut leaves into 1 cm × 1 cm pieces and gently score the leaf surface with a scalpel. Place the cut leaf discs in a co-culture medium to prevent them from drying out.
[0051] b. Transfer the leaf disc to a wide-mouthed bottle (cover the mouth of the bottle with clean gauze), pour in the resuspended bacterial solution, and shake continuously to ensure the bacterial solution fully wets the leaf disc. After 10 minutes, pour the bacterial solution into an empty bottle, remove the leaf disc from the wide-mouthed bottle, place it on filter paper to absorb the liquid, and then lay the leaf disc face up on the co-culture medium. Incubate in the dark for 2-3 days. During the co-culture period, there should be no obvious Agrobacterium colony formation.
[0052] c. Remove the leaf discs from the co-culture medium and place them in a wide-mouth bottle. Pour in 450 mg / L Carb sterile water, shake for 10 minutes, and then rinse five times with sterile water for 2 minutes, 2 minutes, 4 minutes, 4 minutes, and 4 minutes respectively. After draining, place the leaf discs on filter paper to absorb excess water, and then lay them flat on the differentiation induction medium with the upper surface facing up. The culture temperature is 25±2℃, the light intensity is 1500-2000 lx, and the photoperiod is 12 h / d. Change the culture medium approximately every 15 days during the culture period.
[0053] d. When the differentiated seedlings grow to 1 cm, the callus tissue is peeled off and inserted into the rooting medium for rooting induction. After rooting, the seedlings are transplanted into the soil substrate and managed in a normal greenhouse.
[0054] IV. Expression Analysis of Transgenic Plants From the leaves of plants resistant to kanamycin (i.e. CmMYC2 Total RNA was extracted from the ray florets of RNAi transgenic plants and wild-type plants (WT), reverse transcribed into cDNA, and subjected to real-time quantitative RT-PCR. PCR testing, with SAND As an internal reference gene, the ΔCT value of each sample was obtained based on data analysis. Using the expression level of wild-type plants as the baseline, the expression level in the transgenic lines was calculated. CmMYC2 The relative expression of genes.
[0055] CmMYC2 The gene-specific amplification fragment is 103 bp, and the primer sequences are as follows: CmMYC2 qRT F: 5' TCGTGGGTGAGGACGCAATGATAAGG 3′ (identical to the sequence of bits 1364-1389 of SEQ ID No. 1); CmMYC2 qRT R: 5' TGCTGGATTTGTCCCTTCATTTCTCG 3′ (reverse complementary to the sequence of SEQ ID No. 1, positions 1441-1466).
[0056] See results Figure 2 , CmMYC2 -RNAi transgenic plants CmMYC2 The expression level was significantly reduced, downregulated by about 98%, indicating a high silencing efficiency.
[0057] V. Phenotypic Observation of Transgenic Plants Plant type observation: Plants planted for 160 days were selected as the observation objects. The plant type was observed and the length of lateral branches was counted. Five replicates were made for each plant.
[0058] See results Figure 2 The wild type (WT) has only some elongated terminal branches, with very short basal lateral branches, resulting in a tall and slender overall shape that is top-heavy; while CmMYC2 -RNAi transgenic plants exhibit extremely significant lateral branch elongation, particularly near the base of the plant, resulting in a significantly increased overall crown width. Furthermore, the length of the lateral branches gradually increases from the tip to the base, leading to... CmMYC2 -RNAi transgenic plants have a more rounded and aesthetically pleasing natural plant shape, effectively reducing the labor cost of topping operations in the application of garden chrysanthemums.
[0059] Flowering quantity: The number of flower buds and flowering branches were counted 120 days and 160 days after planting, and the crown width was measured. The results are shown in the table below. Figure 4 .
[0060] because CmMYC2 -RNAi transgenic plants exhibit significant elongation of basal lateral branches, extending the vegetative growth period and enabling the production of secondary branches. These lateral branches and some secondary branches produce flower buds, resulting in a significant increase in both the total number of flower buds and flowering branches. Compared to the wild type, which has 40-50 flower buds per plant, CmMYC2 -RNAi transgenic plants have up to 100 flower buds, twice that of wild types, greatly enhancing their ornamental value.
[0061] Regarding flowering period: Flowering period was recorded, and the statistical results are shown below. Figure 5 .
[0062] Compared to the wild type, CmMYC2 -RNAi transgenic plants bud about a week earlier and bloom about two weeks earlier, providing more options for flowering period control and landscape design in the production and application of garden chrysanthemums.
[0063] In summary, compared to wild-type plants, CmMYC2 The increased branching, more rounded plant shape, and denser flowering of RNAi transgenic plants significantly enhance their ornamental value in landscaping applications. The increased crown width and naturally rounded plant shape significantly reduce the number of seedlings per unit area and the labor costs of maintenance operations such as topping. In addition, the earlier flowering period effectively solves the problem that chrysanthemums in landscaping tend to have a late flowering period, which makes them unsuitable for major events such as National Day and difficult to form a concentrated landscape with other early-flowering plants. Therefore, they have high application value.
[0064] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. The application of a substance that reduces the activity or content of protein CmMYC2, or a substance that inhibits or reduces the expression of the gene encoding said protein CmMYC2, characterized in that, The application is any one of Y1-Y7: Y1. Application in advancing the flowering period of chrysanthemums in gardens; Y2. Application in increasing the natural roundness of the plant shape of garden chrysanthemums; Y3. Application in increasing the length of lateral branches of garden chrysanthemums; Y4. Application in increasing the number of branches of garden chrysanthemums; Y5. Application in increasing the number of chrysanthemums in gardens; Y6. Application in increasing the crown width of garden chrysanthemums; Y7. Application in the breeding of garden chrysanthemums; the breeding refers to the selection of garden chrysanthemums that bloom early and / or have a naturally round plant shape and / or have many branches and / or have a large number of flowers and / or have a large crown. The protein CmMYC2 is a protein that is, as shown in A1), A2), or A3), the following: A1) The amino acid sequence is that of the protein SEQ ID No:2 in the sequence listing; A2) A protein that has more than 80% identity with and is functionally similar to the protein shown in A1, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No:2 in the sequence listing. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).
2. The application according to claim 1, characterized in that, The gene encoding the protein CmMYC2 is a DNA molecule as shown in a1), a2), or a3) below: a1) The coding sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing; a2) has 90% or more identity with the nucleotide sequence defined by a1) and encodes a DNA molecule that encodes the protein CmMYC2 described above; a3) hybridizes under strict conditions with the nucleotide sequence defined by a1) or a2) and encodes a DNA molecule that encodes the protein CmMYC2 described above.
3. A method for selecting and breeding early-blooming garden chrysanthemums, characterized in that, This includes obtaining early-flowering garden chrysanthemums by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 of claim 1 in the genome of the recipient garden chrysanthemum, wherein the flowering period of the early-flowering garden chrysanthemum is earlier than that of the recipient garden chrysanthemum.
4. A method for selecting and breeding garden chrysanthemums with naturally rounded plant shapes, characterized in that, This includes obtaining a naturally rounded garden chrysanthemum by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 described in claim 1 in the genome of the recipient garden chrysanthemum, wherein the natural plant shape of the naturally rounded garden chrysanthemum is more rounded than that of the recipient garden chrysanthemum.
5. A method for increasing the length of lateral branches and / or increasing the number of branches of garden chrysanthemums, characterized in that, The method includes obtaining a target garden chrysanthemum by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 of claim 1 in the genome of the recipient garden chrysanthemum, wherein the target garden chrysanthemum has lateral branches longer than and / or more branches than the recipient garden chrysanthemum.
6. A method for breeding garden chrysanthemums with high flowering output, characterized in that, This includes obtaining a high-flowering-volume garden chrysanthemum by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 of claim 1 in the genome of the recipient garden chrysanthemum, wherein the high-flowering-volume garden chrysanthemum has a greater flowering volume than the recipient garden chrysanthemum.
7. A method for selecting and breeding large-crowned garden chrysanthemums, characterized in that, This includes obtaining a large-crown garden chrysanthemum by inhibiting or reducing the expression level of the gene encoding the protein CmMYC2 of claim 1 in the genome of the recipient garden chrysanthemum, wherein the crown width of the large-crown garden chrysanthemum is greater than that of the recipient garden chrysanthemum.
8. The method according to any one of claims 3-7, characterized in that, The method includes introducing a substance into the recipient *Chrysanthemum indicum* that inhibits or reduces the expression level of the gene encoding the protein CmMYC2 of claim 1 in the genome of the recipient *Chrysanthemum indicum*, or introducing a substance that reduces or inhibits the expression of the gene encoding the protein CmMYC2; the substance that reduces or inhibits the activity of the protein CmMYC2 of claim 1, or the substance that reduces or inhibits the expression of the gene encoding the protein CmMYC2 of claim 1, is any one of the following c1)-c4): The substance that inhibits or reduces the expression of the gene encoding the protein CmMYC2 can be any one of the following c1)-c4): c1) Nucleic acid molecules that inhibit or reduce the expression of the protein CmMYC2 encoding gene; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c1) The nucleic acid molecule described is a nucleic acid molecule that targets the gene encoding the protein CmMYC2. The nucleic acid molecule includes a sense segment with the sequence shown in positions 1228-1567 of SEQ ID No. 1 and an antisense segment that is inversely complementary to it.
9. The protein CmMYC2 as described in claim 1.
10. A biomaterial relating to the protein CmMYC2 of claim 1, wherein the biomaterial is any one of B1 to B5 below: B1. The nucleic acid molecule encoding the protein CmMYC2; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4. Recombinant microorganisms containing the nucleic acid molecules described in B1, or recombinant microorganisms containing the expression cassette described in B2, or recombinant microorganisms containing the recombinant vector described in B3; B5. Transgenic plant cell lines, transgenic plant tissues, transgenic plant organs, or transgenic plants containing the nucleic acid molecules described in B1.