Application of g-box tandem cis-regulatory elements, expression cassette, recombinant vector in regulating petal timing color change after flowering in plants

Abstract

This invention discloses the application of a G-box tandem cis-regulatory element, expression cassette, and recombinant vector in regulating the temporal color change of petals after flowering in plants, belonging to the fields of plant genetic engineering and molecular breeding technology. The element is derived from the rose. RhMYB114a The gene promoter region, composed of G-box3 and G-box4 tandemly, can be constructed as an enhanced 4×G3+4×G4, with each element repeated four times. Existing MYB transcription factors only statically deepen flower color. This invention demonstrates that this tandem module, as a light-responsive enhancer, can sustainably drive downstream gene expression and promote the temporal accumulation of anthocyanins, stably mediating post-flowering color change in both roses and tobacco. This element can be used to create ornamental flowers with temporally changing color, and can also serve as a universal light-responsive enhancer to improve crop light-responsive traits, providing a novel core regulatory element for molecular breeding of flowers.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, specifically to the application of a G-box tandem cis-regulatory element, expression cassette, and recombinant vector in regulating the temporal color change of petals after flowering in plants. Background Technology

[0002] Flower color is one of the most important economic traits of ornamental flowers. Most of the existing flower color improvements have yielded constant color phenotypes: these color phenotypes are static traits, and the flower color is basically determined when the flower opens; from opening to senescence, the flower color does not undergo orderly and continuous dynamic changes with the development time after flowering, and the flower color of all open flowers on the same plant is relatively uniform.

[0003] In contrast, petal color change is a dynamic process: the flower initially displays a basic color, and after opening, the petal color gradually changes over time as the flower develops and light signals accumulate. On the same plant, flowers that open at different times display different colors, achieving a unique ornamental effect of "multiple colors on one plant, changing color day by day," which has extremely high horticultural value and commercial potential.

[0004] However, there are very few existing flower varieties with significant temporal color-changing characteristics. Traditional hybridization breeding is time-consuming, inefficient, and difficult to precisely target and improve. Therefore, it is urgent to discover the key genes or regulatory modules that regulate this trait and to use genetic engineering technology to create new color-changing flower varieties.

[0005] Although there are reports on anthocyanin content regulating genes in existing technologies, overexpression or silencing of flower color regulating genes through genetic engineering can only deepen or lighten the petal color overall, and cannot endow plants with the ability to change the color of petals over time after flowering. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of G-box tandem cis-regulatory element, expression cassette, and recombinant vector in regulating the temporal color change of petals after flowering in plants.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an application of a G-box tandem cis-regulatory element in regulating the temporal color change of petals after flowering in plants; the G-box tandem cis-regulatory element comprises at least one G-box3 and at least one G-box4; The nucleotide sequence of G-box3 is CACGTT; the nucleotide sequence of G-box4 is CACGTC.

[0008] In the above applications, the G-box tandem cis-regulatory element specifically drives the temporal high expression of downstream anthocyanin regulatory genes under light conditions, continuously activating the anthocyanin synthesis pathway in petals, thereby enabling the petals to gradually change color as the flower develops after opening.

[0009] Preferably, the downstream anthocyanin regulatory gene is RhMYB114a The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0010] Preferably, the G-box series sequential control element is an enhanced control element (4×G3+4×G4) in which G-box3 and G-box4 are each repeated in series 4 times.

[0011] The G-box series cis-regulator element of this invention is derived from the ancient rose variety 'Butterfly' ( Rosa chinensis 'Mutabilis'). 'Butterfly' is a typical color-changing flower, exhibiting a significant temporal color change characteristic from yellow to pink after flowering. This invention has conducted an in-depth study of its color-changing mechanism, discovering that the RhHY5-RhMYB114a molecular module is a key regulatory pathway driving this complex color-changing process. Further research shows that... RhMYB114a The G-box3 and G-box4 tandem cis-regulatory elements, unique to the gene promoter region, are the core genetic basis for responding to light signals and determining this color-changing phenotype.

[0012] RhMYB114a The CDS sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. RhMYB114a The promoter region sequence of the gene is shown in SEQ ID NO.3; G-box3 is located in the nucleotide region from nucleotides 676 to 681 of the sequence shown in SEQ ID NO.3, and G-box4 is located in the nucleotide region from nucleotides 519 to 524 of the sequence shown in SEQ ID NO.3. Details are as follows: RhMYB114a Gene: ATGGAGGGTTTCGGCGTGAGAAAAGGTGCATGGACTAAAGAGGAAGATGAACTTCTGAGACAGGTCATCGAAAAgcatggagaaggaaaatggcaTCAGGTTCCTTTCAAAGCAGGCTTAAACAGATGCAGGAAGAGCTGTAGACTGAGGTGGCTAAATTATTTGAAGCCAAATATCAAGAGAGGAGAGTTTACAGTTGATGAAGTTGATATGATCATCAGACTTCATAAGCTTCTAGGAAACAGGTGGTCCTTAATTTCTGGAAGACTACCGGGAAGAACAGCCAACGATGTAAAGAACTATTGGAATACTTATCAACGGAAAAAGAATCAAAAGATGACTTCAGGcgcaaaaaaaatgaaagataaaTCCCAAAAAAACACAATCGCCCCTTTGGTTGTAAGACCTCGACCACGAACCTTCATCaaaagtttgaattttttggAAAGAGATGCCAATTTAGAGCATATTCATTCAGAAGAGAATTCTTCCACTTCTTTACCAACAGCACCACCACAAACTCTAGAATTAGAGAATGTAATTGATTGGTGGAAAGTTGTATCTGAAGACAGTACAGGAAGCATTGATAGAACAACATGTTCTAGTCTTGGTTTAGAGGACGACTTCTTCACAAACTTCTGGGTTGAAGATATGGTACAATTGTCAACTATAGATGGCCATGATCTAGTCAACAACTTCTACGCATGA。

[0013] RhMYB114a protein: MEGFGVRKGAWTKEEDELLRQVIEKHGEGKWHQVPFKAGLNRCRKSCRLRWLNYLKPNIKRGEFTVDEVDMIIRLHKLLGNRWSLISGRLPGRTANDVKNYWNTYQRKKNQKMTSGAKKMKDKSQKNTIAPLVVRPRPRTFIKSLNFLERDANLEHIHSEENSSTSLPTAPPQTLELENVIDWWKVVSEDSTGSIDRTTCSSLGLEDDFFTNFWVEDMVQLSTIDGHDLVNNFYA。

[0014] RhMYB114a Gene promoter sequence: CAGCAACACAAGTTATGGCTGATGGCAATCAAGAACTATCAATTCCAGAGTCTAACAGTGCATTCATGTGAACTGTCAGCAAAGAAAAATAAACTTGATAGTGACCTTCACCTAATTAACGTACGAATTGCCCAGCGATCATATGCATATACACCTCAAAACATGACAAACAGCATCAGTATCTCTTTTTCCAGTTTATTATAATTTTTTCTTCTGGAGATGAGTAATTAAAACAGTTTGTCCCCGTGTATTGAAGAAGGAGAAAGAAGTCTAGTAGGGCTTGGTCAGTGCTACAGACTATAATCCACGCACGGTCCCAAGGGATCACCAGATTTTTAAATCTTTTGTTCTTCTGTGCGCTTTTCTAGAAGTGGAATTGAATTTAAGCATGCGGTTTGACTAGTTATTAGTTAAGCATCTTTGATCATCAAATTTGATTCCAACTTAGAGTTGTTTTCACTCGTCGTCCCAGCTAACCTGCTTTCTCTGATTTTCTCTTCGCGTGGATACCACAGATG CACGTC

[0015] Note: The bolded area in the promoter sequence is G-box3; the italicized area is G-box4.

[0016] Preferably, the plant is an ornamental plant; more preferably, it is a rose or tobacco.

[0017] The regulatory mechanism of the G-box tandem cis-regulatory element of this invention is as follows: Under light conditions, endogenous HY5 protein accumulates in the plant and enters the nucleus, specifically binding to the core sequences of G-box3 and G-box4. It recruits the transcription complex to synergistically enhance the transcriptional efficiency of downstream anthocyanin-regulating genes. As the duration of light exposure accumulates after flowering, the expression level of downstream genes gradually increases, continuously driving the accumulation of anthocyanins in the petals, ultimately resulting in a temporal color change phenotype from light to dark after the flower opens. The downstream anthocyanin-regulating gene is the rose. RhMYB114a The gene can also be replaced with other anthocyanin synthesis MYB transcription factor genes with similar functions.

[0018] A second aspect of the present invention provides an application of an expression box in regulating the temporal color change of petals after flowering in plants; the expression box includes the above-mentioned G-box tandem cis-regulatory element.

[0019] Furthermore, the expression cassette also contains an anthocyanin synthesis regulatory gene operatively linked to a G-box tandem cis-regulatory element.

[0020] Preferably, the anthocyanin synthesis regulatory gene is RhMYB114a The gene encodes a product that can activate the anthocyanin synthesis pathway in plants.

[0021] A third aspect of the present invention provides an application of a recombinant vector in regulating the temporal color change of petals after flowering in plants; the recombinant vector comprises the above-mentioned G-box tandem cis-regulatory element or expression cassette.

[0022] In some preferred embodiments, the recombinant vector uses a plant binary vector as its backbone and carries selection marker genes such as hygromycin and kanamycin, making it suitable for Agrobacterium-mediated transient and stable genetic transformation of plants.

[0023] In the above application, the method for regulating the temporal color change of petals after flowering in plants is as follows: the recombinant vector is transformed into the target plant, positive transformants are screened, and transgenic plants in which petals exhibit temporal color change after flowering are obtained.

[0024] Preferably, the recombinant vector is transformed into the target plant through transient or stable genetic transformation.

[0025] Preferably, the target plant is an ornamental plant; more preferably, it is a rose or tobacco.

[0026] The aforementioned tandem repeat G-box cis-regulatory elements, expression cassettes, and recombinant expression vectors have multiple applications: first, they are used to create transgenic ornamental plants with sequential petal color changes after flowering, cultivating new high-value-added flower varieties with "multiple colors per plant"; second, they are used for molecular improvement breeding of plant flower color, quantitatively regulating the intensity and rhythm of color change by adjusting the copy number of the elements. The beneficial effects of this invention are: (1) This invention reveals for the first time that the G-box3-G-box4 tandem cis-regulatory element is the core genetic basis for determining the temporal color change of roses after flowering. This invention relies on two systems—promoter segmental mutagenesis, transient transformation in roses, and stable transformation in tobacco—to complete dual functional verification, clarifying that the G-box3-G-box4 tandem module is the essential regulatory unit driving the gradual change of petals day by day. Mutating any element alone or simultaneously at two sites will completely eliminate the ability to change color temporally, effectively solving the technical bottleneck of existing breeding methods that can only deepen flower color but cannot achieve temporal color change. This tandem regulatory module has cross-species functional conservation characteristics, which can not only activate the temporal anthocyanin synthesis pathway in the original petals of roses, but also completely replicate the color change phenotype from light to dark after flower opening in the tobacco heterologous system. At the same time, by constructing enhanced elements with G-box3 and G-box4 tandemly four times each, the intensity of color change can be quantitatively controlled. The natural single-copy module can meet the basic color change breeding needs, and the four-fold tandem enhanced element can further enhance the color change intensity. RhMYB114a The transcriptional level and anthocyanin accumulation in petals accelerate the color change rate and enhance color saturation. The element copy number can be flexibly adjusted according to the breeding goals of different ornamental flowers to meet the needs of differentiated flower color improvement. Moreover, the entire experimental process is standardized and the element sequence is clear and well-defined. It can be adapted to precision molecular breeding technologies such as CRISPR promoter editing and site-directed gene knock-in, and can quickly cultivate new high-value-added flower varieties with the characteristics of "multiple colors on one plant and changing colors every day". It has strong feasibility for industrialization.

[0027] (2) The tandem G-box module disclosed in this invention is essentially a high-efficiency light signal transcription enhancement converter. Its application scenarios are not limited to the improvement of flower color in ornamental plants. It has broad-spectrum breeding value covering a wide range of crops. After integrating this element into the upstream regulatory region of related genes for fruit coloring, key photosynthetic enzymes, and photomorphology, it can specifically enhance the expression level of downstream target genes under light conditions. It can promote the accumulation of anthocyanins in the peel of horticultural crops such as tomatoes, improve the uniformity and color depth of fruit coloring, enhance the light-induced expression efficiency of crop photosynthetic pathway genes, increase the overall biomass of plants, and regulate the shade avoidance response genes of plants to alleviate the problem of excessive growth under dense planting. In addition, this light-responsive controllable enhancement element can also be used to build a synthetic biology light-induced gene regulation circuit, expanding the development and application space of light-responsive cis elements in the fields of basic molecular biology and crop genetic improvement. Attached Figure Description

[0028] Figure 1 The color phenotypes of petals at different developmental stages (S1~S6) of the 'Butterfly' rose demonstrate the sequential color change process of the petals from yellow to pink.

[0029] Figure 2 Comparison of petal colors of the 'Butterfly' rose in S3 stage after 24 hours of darkness and light treatment.

[0030] Figure 3 : Different developmental stages of the 'Butterfly' rose and the petals under 24 hours of darkness and light treatment RhMYB114a Expression level (FPKM value); in the figure, a represents the expression level in the petals of stages S1 to S6. RhMYB114a Expression level; b represents the petals in the dark and light treatments for 24 hours. RhMYB114a Level of expression.

[0031] Figure 4 Based on the 'Butterfly' rose RhMYB114a Schematic diagram of the structure of a series of expression vectors constructed by promoters.

[0032] Figure 5 : Sole overexpression RhMYB114a The color phenotype of rose petals.

[0033] Figure 6 : Sole overexpression RhMYB114a The color phenotypes of tobacco petals and the color phenotypes of petals at different times after flowering; in the figure, 'a' represents overexpression alone. RhMYB114a The color phenotype of tobacco petals, with #1, #2, #3, etc. representing different transgenic lines; b represents overexpression. RhMYB114a The petal color phenotype of the transgenic tobacco lines at 1, 2 and 3 days after flowering.

[0034] Figure 7 Instant transformation based on the 'Butterfly' rose RhMYB114a After constructing a series of expression vectors using promoters, the flower color phenotype of roses was observed.

[0035] Figure 8 Instant transformation based on the 'Butterfly' rose RhMYB114a After constructing a series of expression vectors using promoters, the rose petals... RhMYB114a Relative gene expression levels and anthocyanin content; in the figure, a represents... RhMYB114a b represents the relative expression levels of genes; b represents the anthocyanin content determination results.

[0036] Figure 9 Stable conversion in tobacco based on 'Butterfly' rose RhMYB114a Flower phenotypes after a series of expression vectors constructed using promoters.

[0037] Figure 10 Stable conversion in tobacco based on 'Butterfly' rose RhMYB114a A series of expression vectors constructed from promoters RhMYB114a Relative gene expression levels and anthocyanin content; in the figure, a represents... RhMYB114a b represents the relative expression levels of genes; b represents the anthocyanin content determination results. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0040] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The Agrobacterium was identified as GV3101, a member of the tumefaciens family, and was described in the journal article "Han ZF, Hunter DM, Sibbald S, et al. Biological activity of the tzs gene of nopaline Agrobacterium tumefaciens GV3101 in plant regeneration and genetic transformation[J].Molecular Plant-microbe Interactions:MPMI, 2013, 26(11): 1359-1365."

[0041] The pCY-H05252 vector is described in the journal article “Li X, Wu Q, Wang J, et al. 2025. Analysis of volatile organic compound diversity revealed the differentiation of scent traits and candidate genes for sweet-aroma formation rose petals. Ornamental Plant Research, 5:e042.”

[0042] The pink-flowered tobacco variety used was the "(Shanxi) nc strain" variety. Nicotiana tabacum cv.Xanthi nc).

[0043] Example 1: RhMYB114a Analysis of gene expression patterns in the 'Butterfly' rose 1. Test method: According to the developmental sequence of the flower from bud to full bloom, the 'Butterfly' rose is divided into six stages, S1 to S6, forming a continuous time development line: S1 (small bud) → S2 (large bud) → S3 (flowers first open, pale yellow) → S4 (yellow fades, pink appears) → S5 (turns into pink) → S6 (deep pink).

[0044] Petal samples were collected as experimental materials at stages S1 (8:00 AM on day 1), S2 (8:00 AM on day 1), S3 (8:00 AM on day 1), S4 (2:00 PM on day 1), S5 (6:30 PM on day 1), and S6 (8:00 AM on day 2).

[0045] In addition, the petal material of the S3 stage was placed in a light incubator and subjected to darkness treatment for 24 hours and light treatment for 24 hours, respectively.

[0046] Based on transcriptome data RhMYB114a Primers for real-time quantitative PCR were designed for the CDS region of the gene, and their sequences are shown below: F: TTACCAACAGCACCACCACAA; (SEQ ID NO.4) R: GTGAAGAAGTCGTCCTCTAAAACCA. (SEQ ID NO.5) Using the SYBR Green Pro Taq HS premixed qPCR kit, quantitative real-time PCR was performed according to the PCR system and procedure specified in the kit instructions to obtain the cycle number at which the fluorescence threshold was reached. Based on 2... -ΔΔCt The method calculates RhMYB114a Relative expression levels in petals of the 'Butterfly' rose during S1-S6 stages and 24 hours after dark and light treatments.

[0047] 2. Test Results The petal color phenotype of the 'Butterfly' rose at different developmental stages is as follows: Figure 1 As shown; the petal color phenotypes of the material in stage S3 after 24 hours of darkness and light treatment are as follows. Figure 2 As shown.

[0048] RhMYB114a Gene expression level measurement results as follows Figure 3 As shown, RhMYB114a The expression level was extremely low in stage S1, increased significantly in stage S2, continued to rise sharply in stages S3 and S4, decreased sharply in stage S5, and then rose back to a high level in stage S6. Furthermore, dark treatment significantly suppressed [the expression level]. RhMYB114a Expression. The above results indicate that, RhMYB114a Gene expression is induced by light and exhibits stage-specific high expression characteristics after flowering.

[0049] Example 2: Construction of a series of recombinant vectors Using homologous recombination method RhMYB114a The gene CDS sequence (SEQ ID NO.1) was ligated into the multiple cloning site after the Ps promoter in the pCY-H05252 vector to construct the gene CDS sequence. RhMYB114a Gene overexpression vector (containing only) RhMYB114a Gene CDS sequence, without RhMYB114a (Self-promoter sequence).

[0050] based on RhMYB114a Self-promoter constructs a series of expression vectors, such as Figure 4 As shown, the construction process is as follows: The one shown in SEQ ID NO.3 RhMYB114a The promoter region sequence of the gene was replaced with the Ps promoter in the pCY-H05252 vector, and then ligated into the multiple cloning site following the promoter. RhMYB114a Gene CDS sequences were used to construct a gene containing wild-type G-box3 and G-box4. RhMYB114a Gene overexpression vector (G3+G4); The one shown in SEQ ID NO.3 RhMYB114a The G-box3 element in the promoter region of the gene was mutated to TTTTTT, and then the Ps promoter in the pCY-H05252 vector was replaced. The gene was then ligated into a multiple cloning site following the promoter. RhMYB114a Gene CDS sequence was used to construct a single G-box3 mutant. RhMYB114a Gene overexpression vector (G3-mut); The one shown in SEQ ID NO.3 RhMYB114a The G-box4 element in the promoter region of the gene was mutated to TTTTTT, and then the Ps promoter in the pCY-H05252 vector was replaced. The gene was then ligated into a multiple cloning site following the promoter. RhMYB114a Gene CDS sequence was used to construct a G-box4 single mutant. RhMYB114a Gene overexpression vector (G4-mut); The one shown in SEQ ID NO.3 RhMYB114aThe G-box3 and G-box4 elements in the promoter region of the gene were simultaneously mutated to TTTTTT, and then the Ps promoter in the pCY-H05252 vector was replaced. The gene was then ligated into a multiple cloning site following the promoter. RhMYB114a Gene CDS sequences were used to construct G-box3 and G-box4 double mutants. RhMYB114a Gene overexpression vector (G3-mut + G4-mut); The one shown in SEQ ID NO.3 RhMYB114a The G-box3 and G-box4 elements in the promoter region of the gene are each repeated tandemly four times. Then, the Ps promoter in the pCY-H05252 vector is replaced, and the gene is ligated into a multiple cloning site following the promoter. RhMYB114a Gene CDS sequences were used to construct G-box3 and G-box4 enhanced genotypes. RhMYB114a Gene overexpression vector (4×G3 + 4×G4).

[0051] Example 3: RhMYB114a Gene function identification 1. Test method: (1) Preparation of the inoculation solution: The construction in Example 2 RhMYB114a Gene overexpression vector (containing only) RhMYB114a Gene CDS sequence, without RhMYB114a Agrobacterium was transformed with its own promoter sequence to obtain recombinant Agrobacterium.

[0052] The pCY-H05252 vector was transformed into Agrobacterium as an empty vector control using the same method.

[0053] Inoculate 2 mL of recombinant Agrobacterium bacterial suspension into 20 mL of YEB liquid medium (containing 100 mg / L rifampin and 50 mg / L kanamycin); incubate in the dark at 28°C and 200 rpm with shaking for 1-2 h until OD reaches zero. 600 Centrifuge at 4℃, 5000 rpm for 10 min, collect the bacterial cells, resuspend the cells in pre-cooled infection buffer, and adjust the OD value to 0.6-0.8. 600 =0.6, and the solution was prepared by standing at 24°C for 2 h in the dark.

[0054] (2) Instantly transforming rose petals: Take a 'Old Blush' rose stem (approximately 10 cm in length) with half-open flowers, cut it off, and place it in water before bringing it back to the laboratory. Inject the infection solution into the petals, then place the stem in water and incubate it in a light incubator in the dark for 24 hours. Subsequently, transfer it to a light intensity of 4000 lux and continue incubation for 48 hours. Finally, take photos and samples.

[0055] (3) Stabilizing the conversion of tobacco: Stable genetic transformation of pink-flowering tobacco was performed using the Agrobacterium-mediated leaf disc method. Sterile tobacco leaf discs were infected with an inoculum solution and co-cultured before being transferred to a selection medium containing hygromycin (MS + 30 g / L sucrose + 1 mg / L 6-BA + 0.1 mg / L NAA + 0.002 mg / L TDZ + 8 g / L agar + 25 mg / L hygromycin + 300 mg / L cephalosporin) for positive transformant selection. Regenerated resistant shoots were rooted and hardened off before being transplanted into a substrate (peat:vermiculite = 7:1, volume ratio) for further growth. Positive transgenic lines were identified using RT-qPCR, and phenotypic and flower color changes between the transgenic lines and wild-type plants were continuously observed and recorded.

[0056] 2. Experimental Results: The CDS sequence (excluding the promoter sequence) will be used. RhMYB114a Genes are transiently overexpressed in the petals of the 'Old Blush' rose, such as Figure 5 As shown, compared with the empty vector control (EV-OE), overexpression RhMYB114a The petals are significantly darker in color.

[0057] The CDS sequence (excluding the promoter sequence) will be used. RhMYB114a After the gene was stably overexpressed in pink-flowered tobacco, the petal color of the transgenic tobacco was significantly darker than that of the wild type, but it did not exhibit the phenotype of color change after flowering. Figure 6 ).

[0058] The above results indicate that overexpression alone RhMYB114a Genes can only produce materials with deeper flower color, but are insufficient to create new varieties with time-varying color characteristics.

[0059] Example 4: RhMYB114a Functional verification of G-box3-G-box4 modules in gene promoter region 1. Test method: The experimental method is the same as in Example 3, but the method based on Example 2 is used instead. RhMYB114a A series of expression vectors were constructed using the self-promoter and transiently transformed into rose petals; and tobacco was stably genetically transformed to obtain T0 generation transgenic plants. The petal color phenotype was observed, and the concentration of certain nutrients in the petals was measured. RhMYB114a The relative expression levels of genes and anthocyanin content.

[0060] 2. Experimental Results: The instantaneous transformation result of rose petals is as follows: Figures 7-8 As shown, transient transformation of G-box3 and G-box4 double mutations RhMYB114aGene overexpression vector (G3-mut + G4-mut), in RhMYB114a There were no significant differences in expression levels, anthocyanin content, and flower color phenotype compared to the control (EV).

[0061] All other carriers can improve to varying degrees. RhMYB114a Anthocyanin expression levels and accumulation lead to deeper flower color, with the enhancing effect in the following order: 4×G3+4×G4>G3+G4>G4-mut>G3-mut. This result indicates that mutations or doubling of G-box3 and G-box4 elements can effectively regulate... RhMYB114a Expression, and consequently anthocyanin synthesis, is affected. The G-box3 / 4 driven expression system is a controllable and tunable intensity switch.

[0062] Experimental results in stable conversion tobacco are as follows: Figures 9-10 As shown, transformation of G-box3 and G-box4 double mutants RhMYB114a Plants with gene overexpression vectors (G3-mut + G4-mut) have... RhMYB114a Expression levels, anthocyanin content, and flower color phenotype were not significantly different from the control (EV). Other transgenic plants showed significantly enhanced corresponding indicators, and the order of increase was consistent with the transient transformation results.

[0063] It is worth noting that the conversion of wild-type G-box3 and G-box4... RhMYB114a Plants overexpressing the gene vector (G3+G4) and transformed with G-box3 and G-box4 enhanced genes RhMYB114a Plants overexpressing the gene vector (4×G3 + 4×G4) all exhibited a temporal color change phenotype after flowering, with the latter showing a more pronounced color change; however, plants transformed with each mutant vector (G3-mut, G4-mut, G3-mut+ G4-mut) did not exhibit this color change phenotype.

[0064] The above results demonstrate that the G-box3-G-box4 module significantly enhances performance compared to a single G-box element. RhMYB114a Expression and successful induction of post-flowering temporal color change phenotype in heterologous systems are key genetic bases for determining petal color change phenotype.

[0065] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of a G-box tandem cis-regulatory element in regulating the temporal color change of petals after flowering in plants, characterized in that, The G-box series cis-control element consists of at least one G-box3 and at least one G-box4; The nucleotide sequence of G-box3 is CACGTT; the nucleotide sequence of G-box4 is CACGTC.

2. The application according to claim 1, characterized in that, The G-box series cis-control element is an enhanced control element in which G-box3 and G-box4 are each repeated in series 4 times.

3. The application according to claim 1 or 2, characterized in that, The G-box tandem cis-regulatory element specifically drives the temporal high expression of downstream anthocyanin regulatory genes under light conditions, continuously activating the anthocyanin synthesis pathway in petals, thus enabling the petals to gradually change color as the flower develops after opening.

4. The application according to claim 3, characterized in that, The downstream anthocyanin regulatory gene is RhMYB114a The gene, whose nucleotide sequence is shown in SEQ ID NO.

1.

5. The application of an expression cassette in regulating the temporal color change of plant petals after flowering, characterized in that, The expression box includes the G-box cascaded cis-regulatory element of claim 1.

6. The application according to claim 5, characterized in that, The expression cassette also contains an anthocyanin synthesis regulatory gene that is operatively linked to a G-box tandem cis-regulatory element.

7. The application of a recombinant vector in regulating the temporal color change of plant petals after flowering, characterized in that, The recombinant vector comprises the G-box tandem cis-regulatory element of claim 1 or the expression cassette of claim 5.

8. The application according to claim 7, characterized in that, The method for regulating the temporal color change of petals after flowering in plants is as follows: the recombinant vector is transformed into the target plant, positive transformants are screened, and transgenic plants in which petals exhibit temporal color change after flowering are obtained.

9. The application according to claim 8, characterized in that, The recombinant vector is transformed into the target plant through transient or stable genetic transformation.

10. The application according to claim 8, characterized in that, The target plant is an ornamental plant.

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