Application of Chimonanthus praecox CpMADS1 gene in regulating plant anthocyanin biosynthesis

Abstract

The application belongs to the field of biotechnology, and particularly relates to application of a wax plant CpMADS1 gene in regulating biosynthesis of plant anthocyanins. The wax plant CpMADS1 gene belongs to the MADS-box family, and its expression is closely related to flower color formation of the wax plant, but its function in heterologous plants such as tobacco has not been revealed. In the application, the wax plant CpMADS1 gene is introduced into tobacco, and it is found that the gene significantly inhibits synthesis of anthocyanins, and is accompanied by slight down-regulation of flavonoid content. Research shows that CpMADS1 may affect downstream metabolic flux distribution by regulating expression of key enzyme genes in the anthocyanin synthesis pathway, so as to change the flower color phenotype. The application provides a new strategy for regulating plant anthocyanin synthesis by using MADS-box genes, and has important application value for flower breeding and pigment metabolism engineering.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CpMADS1 gene of wintersweet in regulating anthocyanin biosynthesis in plants. Background Technology

[0002] Anthocyanins, important flavonoid compounds in plant secondary metabolites, are key pigments determining the color of petals, fruits, and other organs. Their biosynthetic pathway involves the coordinated expression of multiple structural genes, primarily including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDP-glucosyltransferase (UGT). These genes encode enzymes that sequentially catalyze the conversion of tyrosine metabolism towards anthocyanins, ultimately producing stable anthocyanin glycosides.

[0003] Studies have shown that anthocyanin synthesis is precisely regulated by internal and external environmental factors (such as light, temperature, and sugar signals) and developmental signals, with transcription factors playing a core regulatory role. Known transcription factors involved in anthocyanin synthesis mainly include MYB, bHLH, WD40, and MADS-box types. For example, the MYB-bHLH-WD40 ternary complex in Arabidopsis thaliana can activate the expression of downstream structural genes; while MADS-box transcription factors indirectly affect flower color phenotype by regulating floral organ development.

[0004] MADS-box genes are a highly conserved family of transcription factors in plants. Their name originates from the acronym of four model proteins: yeast MCM1, Arabidopsis AGAMOUS, snapdragon DEFICIENS, and human SRF. Based on sequence characteristics, MADS-box genes can be divided into two main groups: Type I and Type II. The Type II subfamily (also known as the MIKC type) contains four domains: MADS, I, K, and C. The MADS domain is responsible for DNA binding, the I domain regulates protein-protein interactions, the K domain forms a helical-loop-helical structure, and the C domain mediates transcriptional activation or repression.

[0005] During plant development, MADS-box genes (such as...) Figure 1 (As shown) it regulates target gene expression by forming homodimers or heterodimers. For example:

[0006] Class A genes (such as AP1 and AP2): control the initiation of differentiation of sepals and petals;

[0007] Class B genes (such as AP3 and PI): determine the identity of petals and stamens;

[0008] Class C genes (such as AG): regulate stamen and carpel development;

[0009] E-class genes (such as SEPALLATA): involved in the coordinated development of four-flowered organ systems.

[0010] Recent studies have found that some MADS-box genes can also affect flower color by regulating flavonoid metabolism. For example, the PhAN2 gene in petunias promotes the accumulation of red anthocyanins by activating downstream structural genes; the MdMADS1 gene in apples indirectly affects anthocyanin synthesis by regulating the MYB transcription factor. However, the molecular mechanisms by which MADS-box genes directly regulate anthocyanin biosynthesis remain unclear, especially in woody plants where there are few reports.

[0011] Winter sweet (Chimonanthus praecox) is a traditional winter flower in my country, with its flowers primarily in the yellow hue, and a few varieties exhibiting yellowish-green or yellowish-white. Research indicates that the flower color phenotype of winter sweet is mainly determined by flavonoids (flavonols, flavones) and a small amount of anthocyanins. Flavonols (such as kaempferol and quercetin) contribute to the yellow color by chelating metal ions or adjusting pH; while the absence or low accumulation of anthocyanins prevents the flower from displaying red hues.

[0012] Transcriptomic analysis revealed abundant expression of flavonoid metabolism-related genes in wintersweet petals, including structural genes such as CHS, CHI, F3H, and FLS (flavonol synthase), as well as regulatory factors such as MYB and bHLH. Notably, the wintersweet genome contains multiple MADS-box gene family members, among which the CpMADS1 gene is predicted to be a Type II subfamily member, but its biological function has not yet been reported. Previous experiments showed that the CpMADS1 gene exhibits specific high expression during the flower bud differentiation and full bloom stages of wintersweet, suggesting a possible association with floral organ development and pigment metabolism.

[0013] Despite significant progress in the study of anthocyanin synthesis regulation, the following technical bottlenecks still exist in practical applications:

[0014] (1) Limitations of regulatory targets: Existing studies have focused on transcription factors such as MYB and bHLH, while the direct role of MADS-box genes in anthocyanin synthesis has not been explored in depth;

[0015] (2) Poor species adaptability: Most of the developed regulatory genes are derived from model plants (such as Arabidopsis thaliana and petunia), and have problems such as low expression efficiency and poor compatibility in woody horticultural plants;

[0016] (3) Functional redundancy interference: Members of the MADS-box gene family usually have functional redundancy, and it is difficult to achieve significant phenotypic changes by manipulating a single gene;

[0017] (4) Insufficient flower color diversity: Traditional breeding methods are difficult to break through the limitations of yellow wax plum flower color, and it is urgent to explore new regulatory genes to enrich flower color phenotype.

[0018] Based on the spatiotemporal expression characteristics of the CpMADS1 gene in wintersweet and the regulatory characteristics of the MADS-box family, it is speculated that it may participate in the regulation of anthocyanin synthesis through the following mechanisms:

[0019] (1) Direct regulation of structural genes: CpMADS1 may activate or inhibit the expression of key enzyme genes (such as DFR and ANS) in the anthocyanin synthesis pathway by binding to their promoters.

[0020] (2) Co-regulatory network: CpMADS1 may form a complex with transcription factors such as MYB and bHLH, forming a multi-level regulatory network;

[0021] (3) Developmental stage-specific regulation: Its specific expression during the flower bud differentiation period suggests that it may be involved in the synergistic regulation of flower organ morphogenesis and pigment deposition.

[0022] If the function of the CpMADS1 gene in anthocyanin synthesis can be elucidated and heterologous expression can be achieved, it will provide a novel regulatory element for improving the flower color of woody plants, which has important scientific value and economic significance. Summary of the Invention

[0023] This invention first provides the application of the CpMADS1 gene of wintersweet in regulating the biosynthesis of anthocyanins in plants, and the nucleotide sequence of the CpMADS1 gene is shown in SEQ ID NO:1.

[0024] This invention also provides the application of the CpMADS1 protein of wintersweet in inhibiting the biosynthesis of anthocyanins in plants, the amino acid sequence of which is shown in SEQ ID NO:2.

[0025] The present invention also provides a CpMADS1 gene for wintersweet, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0026] The present invention also provides a CpMADS1 protein from wintersweet, the amino acid sequence of which is shown in SEQ ID NO:2.

[0027] The present invention also provides biological materials related to the above-mentioned CpMADS1 protein, which are any one of B1) to B8) below: B1) a nucleic acid molecule encoding the above-mentioned CpMADS1 protein;

[0028] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0029] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0030] B4) A recombinant vector containing the expression cassette described in B2);

[0031] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0032] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0033] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0034] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0035] The present invention also provides a method for preparing transgenic plants, wherein the above-mentioned recombinant vector is introduced into plant cells to obtain transgenic plants that overexpress the CpMADS1 gene.

[0036] In some embodiments, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0037] In some embodiments, the plant is tobacco, Arabidopsis thaliana, chrysanthemum, or rose.

[0038] Finally, this invention also provides a method for improving the flower color of plants by overexpressing the CpMADS1 gene of wintersweet to reduce the anthocyanin content, so that the flower color of the plant is light pink or white.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] (1) Precise regulation of anthocyanin synthesis: By overexpressing the CpMADS1 gene of wintersweet, the anthocyanin content of plants can be significantly reduced (e.g., the anthocyanin content of tobacco OE-5 strain is reduced by 47%), providing an efficient regulatory means for cultivating low anthocyanin or white flowers.

[0041] (2) Expanding the breeding pathways for flower color: The discovery of the CpMADS1 gene provides a new perspective for the study of the function of the MADS-box family in anthocyanin metabolism. Combined with other regulatory genes (such as CHS and F3H), more complex pigment combination regulation can be achieved, promoting the diversification of flower color.

[0042] (3) Enhance the controllability of metabolic pathways: CpMADS1 may optimize the distribution of metabolic flux by regulating the expression of key enzyme genes in the anthocyanin synthesis pathway, providing a theoretical basis for the analysis of the anthocyanin biosynthesis network and metabolic engineering.

[0043] (4) Wide range of applications: This invention is applicable to a variety of plants such as tobacco and flowers, and can provide key technical support for improving the color of horticultural crops, producing raw materials for pigment extraction, and developing natural antioxidants. Attached Figure Description

[0044] Figure 1 The structure of the MADS-box gene;

[0045] Figure 2 Morphological observation of 35S-CpMADS1 transgenic tobacco; Note: A, B: growth of OE-CpMADS1 transgenic tobacco; C: WT, OE-5, and OE-9 tobacco;

[0046] Figure 3 1. OE-CpMADS1 transgenic tobacco RNA electrophoresis bands; Note: M: DL2000 + DNA Marker; 1: OE-CpMADS1-1; 2: OE-CpMADS1-5; 3: OE-CpMADS1-2; 4: OE-CpMADS1-6; 5: OE-CpMADS1-9; 6: OE-CpMADS1-11; 7: OE-CpMADS1-12; 8: OE-CpMADS1-15; 9: OE-CpMADS1-13;

[0047] Figure 4 35S-CpMADS1 real-time PCR detection; Note: ***: p value < 0.001 is highly significant;

[0048] Figure 5 Phenotypic observation of T0 generation 35S-CpMADS1 transgenic tobacco;

[0049] Figure 6 Phenotypic observation of T1 generation tobacco transgenic 35S-CpMADS1 gene;

[0050] Figure 7 Detection of anthocyanin content in T1 generation of 35S-CpMADS1 transgenic tobacco; Note: *: **: significant, p value < 0.01; ***: p value < 0.001, highly significant;

[0051] Figure 8 Detection of flavonoid content in T1 generation of 35S-CpMADS1 transgenic tobacco; Note: ns: not significant. Detailed Implementation

[0052] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0053] Material

[0054] Plant materials: wintersweet HLT015 from Heilongtan; tobacco K326.

[0055] RNA Extraction Kit (Adley Biotech Co., Ltd.), DNA Kit (Bomaide Biotech Co., Ltd.), PCR Kit, cDNA First-Strand Synthesis Kit, PerfectStart Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.), Plant Anthocyanin Kit (Suzhou Keming Biotechnology Co., Ltd.), Plant Flavonoid Kit, Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.)

[0056] Instruments and equipment: ultra-clean workbench, constant temperature water bath, constant temperature incubator shaker, autoclave, DYY-12C electrophoresis apparatus (Beijing Liuyi Instrument Factory); Dongshenglong eQ9600 series 96-well fluorescence quantitative PCR instrument (Suzhou Dongsheng Biotechnology Co., Ltd.); nucleic acid protein detector (Nano Drop 2000C) (Thermo Fisher Scientific); ultraviolet spectrophotometer (Shanghai Meipuda).

[0057] SEQ ID NO:1:

[0058] SEQ ID NO:2: MGRGRVELKRIENKINRQVTFSKRRNGLLKKAYELSVLCDAEVALIIFSTRGKLYEFGSVGTTKTLERYQRCCHDPQDANLSDCQTQSWYQEMSKLKAKYESLQRSQRHLLGDDLGSL NVKELHLLERQLEAALSQTRQRKTQIVMEHIEELRRKERQLGDINKQLRTKLEEEGQGDIRGIRGSWESGAGTNNFSLHPSQSAPNDCEPTLQIGYQTFVPPEAAIPRSTAGEGNFIQGWVL-.

[0059] Example 1: Transgenic Tobacco Planting, Maintenance and Management

[0060] Tobacco seeds of the transgenic 35S-CpMADS1 (obtained through cloning in previous laboratory studies) were used. Before sowing, loose, well-aerated, and absorbent soil that had been autoclaved was prepared. The soil was mixed with a small amount of perlite and then the seeds were sown. A layer of plastic wrap was placed on top to retain moisture, and the soil was placed in an incubator to germinate. For transplanting, a petri dish filled with substrate was selected, a small hole was dug in the center, and a small amount of tobacco seedling with soil was placed in the dish with tweezers. The seedling was then thoroughly watered. Afterward, the soil moisture was checked before each watering to ensure it was neither too wet nor too dry. Suitable fertilizers for tobacco, such as Bilu manure, were used, and the frequency of fertilization was adjusted according to the growth of the tobacco. The greenhouse temperature was controlled at around 25°C, avoiding excessively high or low temperatures to prevent affecting the tobacco's growth. As the tobacco plants grew, their placement was adjusted to prevent leaves from shading each other. At this stage, the transgenic tobacco required good light conditions. To ensure sufficient light and adequate daily sunlight exposure, the tobacco plants were placed in a sunny location in the wintersweet field behind the school.

[0061] While genetically modified tobacco exhibits some resistance to pests, it cannot completely eliminate pests. It is essential to stockpile pesticides, regularly inspect tobacco plants, and promptly remove any pests found. Sterilization can be performed using carbendazim or 75% alcohol, but care should be taken not to spray directly onto the tobacco leaves. Subsequently, as the tobacco grows, flowers, and bears fruit, experimental materials can be obtained.

[0062] Results: This study successfully introduced the CpMADS1 gene, unique to wintersweet, into tobacco, achieving genetic transformation. After continuous observation of the growth of the 35S-CpMADS1 transgenic tobacco, no significant differences were found in the OE-1, OE-6, OE-12, OE-13, and OE-15 varieties. However, the flower color of OE-5 and OE-9 was lighter and whiter than that of the WT strains. Figure 2 ). Example 2

[0063] (1) Extraction of RNA from tobacco flowers and synthesis of cDNA

[0064] Total RNA was extracted from the flowers of wintersweet HLT015 during its full bloom period. The RNA concentration was first detected by 1% agarose gel electrophoresis, then by Nanodrop2000, and finally, the RNA was reverse transcribed into cDNA according to the instructions of the Total Gold Reverse Transcription Kit (cDNA First Strand Synthesis Kit).

[0065] Table 1. RNA Reverse Transcription Reaction System

[0066] Components Dosage Total RNA / mRNA 50 ng - 5 ug / 5 - 500 ng Anchored Oligo(dT)20(0.5 ug / μL) 1 μL or Random primer(N9) (0.1 ug / μL) 1 μL or GSP 2 pmol 5×RTMix 4 μL M-MLV4 1 μL H2O(RNase free) to final volume 20 μL

[0067] (2) Quantitative analysis of tobacco flowers by qRT-PCR

[0068] Flower samples from different strains of genetically modified tobacco were collected, and total RNA was extracted and reverse transcribed into cDNA. Real-time quantitative PCR (qRT-PCR) was used to accurately analyze gene expression levels in the transgenic tobacco. The following primers were designed and synthesized using Premier 3.0 software:

[0069] CpMADS1qF: GAGGGTCATGGGAATCTGGAG (SEQ ID NO: 3);

[0070] CpMADS1qR: CTTCACCAGCAGTGCCTCCTA (SEQ ID NO:4);

[0071] NtRNR2 was used as an internal reference gene. cDNA synthesis was performed using the EasyScript one-step gDNA removal and cDNA synthesis kit. qRT-PCR was performed on an eQ9600. The reaction procedure was as follows: initial denaturation at 94℃ for 10 seconds, followed by denaturation at 94℃ for 5 seconds, annealing at 60℃ for 15 seconds, and extension at 72℃ for 30 seconds. Three biological replicates and three technical replicates were used in the quantitative PCR experiments. −ΔΔ The relative expression levels of each gene were calculated using the CT method. All results were calculated and plotted using Graphpad Prism9.

[0072] Results: RNA expressing pBWA(V)HS-CpMADS1-GLOSCf was extracted from T1 generation positive seedlings using RNA extraction methods. Agarose gel electrophoresis showed that the RNA quality was good. Figure 3 The expression of 35S-CpMADS1 in tobacco was quantified using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Figure 4As shown, the expression level of CpMADS1 in all 10 transgenic lines was significantly higher than that in the wild type. Among them, the OE-9 line had the highest relative expression level of CpMADS1, but there was no significant difference in the relative expression level of CpMADS1 between the OE-12 and OE-13 empty vector lines. Compared with other transgenic lines, the OE-12 line had the lowest relative expression level.

[0073] Example 3: Phenotypic Observation and Analysis of CpMADS1 Transgenic Tobacco

[0074] During the plant's growth period, tobacco flowers were periodically photographed using a high-definition camera. Frontal views of the flowers were captured at specific times, documenting the gradual color change. At peak bloom, the flowers were collected, and intact tobacco flowers were cut off with scissors and immediately placed into pre-prepared sterile cryovials. The time, variety, and characteristics were recorded. The sample tubes were then flash-frozen in liquid nitrogen and transferred to an ultra-low temperature freezer at -80°C for storage.

[0075] Results: Observation of the growth status of OE-CpMADS1 transgenic tobacco revealed no significant differences among the five lines OE-1, OE-6, OE-12, OE-13, and OE-15. However, OE-5 and OE-9 differed significantly from WT (wild type); the flower color of these two plants was lighter than that of WT, approaching white. Figures 5-6 ).

[0076] Example 4: Determination of anthocyanin content

[0077] An anthocyanin content in the OE-CpMADS1 strain was determined by single-wavelength colorimetry using acidic ethanol extraction and the maximum absorbance of anthocyanins at 520 nm. Before the experiment, a visible spectrophotometer and other instruments were prepared, and flowers from different OE-CpMADS1 transgenic tobacco strains were aliquoted into sampling tubes. The specific operating steps are as follows: First, the reagents were prepared. The extraction solution (60 mL) was stored in one bottle at 4℃; reagent one (50 mL) was also stored at 4℃. Next, anthocyanin extraction was performed. The sample mass to extraction solution volume was 1:5 to 10 (recommended ratio: 0.1 g sample to 1 mL extraction solution). The mixture was homogenized, transferred to EP tubes, and brought to a final volume of 1 mL. Extraction was carried out at 75℃ for 20 minutes, followed by centrifugation at 8000 g at room temperature for 10 minutes. The supernatant was then collected for later use. Finally, the determination procedure is as follows: First, preheat the spectrophotometer for at least 30 minutes, and preheat Reagent 1 at 25℃ for at least 10 minutes. Take 50 μL of the supernatant and 50 μL of the extract, and add each to 950 μL of Reagent 1 in a cuvette. Measure the absorbance at 520 nm, and record them as A (determination) and A (blank), respectively. Finally, the formula for calculating anthocyanin content is given; calculate the content based on the determination data.

[0078] Please note that the anthocyanin content (expressed in μg / g fresh weight) is calculated as follows:

[0079] [△A×V÷(ε×d)]×MxFx10%÷W

[0080] =16.7×△A×F÷W

[0081] V represents the volume of the extract, in units of 1 × 10³ L; ε is the molar extinction coefficient of anthocyanins, with a value of 2.69 × 10⁻⁶. 4 L / mol / cm; d is the optical path length of the cuvette, which is 1 cm; M is the relative molecular mass of anthocyanin, which is 449.2 g / mol; F is the dilution factor, which is 20; 10 6 This means 1g equals 10 6 μg; W represents the dry weight of the sample, in g.

[0082] Results: The anthocyanin content in the corolla of several transgenic tobacco plants OE-CpMADS1 with flowers whiter than those of the empty-gene tobacco strain was determined using a single-wavelength colorimetric method. The anthocyanin content was measured experimentally as follows: Figure 3-6 As shown in the analysis results, we found that the anthocyanin content of the OE strains was generally lower than that of the WT strains. Specifically, the anthocyanin content of the OE-5 sample was only 0.53 times that of the WT sample, showing a significant difference, while the anthocyanin content of the OE-9 sample was 0.26 times that of the WT sample, showing a highly significant difference. Figure 7 ).

[0083] Example 5: Determination of flavonoid content

[0084] In phytochemistry, flavonoids are secondary metabolites in plants, and their content determination plays an important role in studying plant physiological characteristics and quality evaluation. The method used in this experiment is as follows: After treating the extracted sample with an alkaline nitrite solution, a red complex is formed with aluminum ions. This complex exhibits characteristic absorption at a wavelength of 510 nm. The flavonoid content is obtained by measuring the absorbance of the sample extract at 510 nm. Before the determination, experimental preparation is necessary. The experimental equipment required includes: a balance (for accurate sample weighing); an oven (for drying the sample to constant weight); a grinder and sieve (for grinding and sieving the sample to ensure homogeneity); an ultrasonic grinder (to assist extraction); a centrifuge (for separating the extract); a visible spectrophotometer; and 1 mL glass cuvettes (for absorbance measurement). Reagents: 60% ethanol (prepare yourself); Reagent 1: 2mL x 1 bottle, store at 4℃; Reagent 2: 2mL x 1 bottle, store at 4℃; Reagent 3: 20mL x 1 bottle, store at 4℃; Distilled water: as needed. Additionally, flowers from different tobacco strains of OE-CpMADS1 should be aliquoted using sampling tubes. Specific experimental steps are as follows:

[0085] Dry sample processing:

[0086] Dry the sample in an oven to constant weight. Grind the sample through a 40-mesh sieve using a pulverizer. Weigh approximately 0.02 g of the sample and place it in a container, then add 2 mL of 60% ethanol extraction solution. Place the container in a shaking metal bath or water bath and extract at 60°C for 2 hours. Transfer the sample solution to a centrifuge tube and centrifuge at 10000 g for 10 minutes at 25°C. Collect the supernatant as the test solution.

[0087] Fresh sample processing:

[0088] Take about 0.1g of tobacco flower tissue, add 2mL of 60% ethanol extract, grind into a homogenate, and then extract with shaking at 60℃ for 2h, just like the dry sample. Centrifuge at 10000g and 25℃ for 10min, and take the supernatant for testing. Note that when extracting in a water bath, you should manually mix it every once in a while.

[0089] Measurement procedure:

[0090] Next, turn on the visible spectrophotometer and zero it using distilled water to ensure accuracy. Carefully add reagents to the blank and test tubes according to the procedure: first add 540 μL of distilled water to the blank tube, then add 30 μL of reagent one, and after complete mixing, let it stand at room temperature for 6 minutes. Next, add 30 μL of reagent two, mix well, and let it stand at room temperature for 5 minutes. Finally, add 400 μL of reagent three, mix thoroughly, and let it stand at 25°C for 15 minutes. Add 540 μL of the sample solution to the test tube, following the same procedure as the blank tube. After the above steps, transfer the prepared blank and test tubes to 1 mL glass cuvettes. Measure the absorbance at 510 nm using the visible spectrophotometer, recording the values ​​as Ablank and Atest, respectively. Calculate the absorbance difference using the formula ΔA = Atest - Ablank. This method only requires preparing one blank tube to meet the experimental requirements. Finally, based on the measured data, the content value was obtained using the formula for calculating flavonoid content.

[0091] Formula for calculating flavonoid content

[0092] Standard curve: y = 5.02x + 0.0007, R² = 0.9996

[0093] The flavonoid content (mg / g dry weight) is calculated as follows: (△A - 0.0007) plus 5.02 divided by (W divided by V total sample) = 0.398 × (AA - 0.0007) ÷ W

[0094] Vtotal: Volume of extract added, 2 mL; W: Sample mass, g

[0095] The limit of detection is 10 μg / g

[0096] Results: The measurement results are as follows Figure 8 Calculations showed that the flavonoid content of OE-5 was 1.08 times that of WT, with no significant difference, while the flavonoid content of OE-9 was 0.82 times that of WT, with no significant difference.

[0097] This invention utilizes transgenic technology to introduce the MADS1-box gene into tobacco and focuses on its effects on flower color and other traits, finding no significant phenotypic differences. The expression level of the CpMADS1 gene differs among transgenic lines. In the floral parts of transgenic tobacco, only OE-5 and OE-9 showed significant changes in petal color, exhibiting varying degrees of white or light yellow, while the stamens and pistils showed relatively small color changes. This suggests that the expression of the CpMADS1 gene may influence tobacco flower color, primarily affecting petal color.

[0098] To further clarify the influence of the CpMADS1 gene on tobacco flower color, we used qRT-PCR to detect the relative expression level of the CpMADS1 gene in transgenic tobacco. We found that the expression level of the CpMADS1 gene in other transgenic lines was significantly different from that in OE-5, OE-9 transgenic lines and wild-type WT, further confirming the conclusion that the high expression of the CpMADS1 gene leads to lighter tobacco flower color.

[0099] Flower color is primarily influenced by the types and amounts of flavonoids and carotenoids in the petals. Furthermore, we measured the anthocyanin and flavonoid content in transgenic tobacco, both important pigments in plants that significantly affect flower color. The results showed that the anthocyanin and flavonoid contents of OE-5 and OE-9 were significantly lower than those of wild-type WT and other transgenic lines. This result further supports our hypothesis that the expression of the CpMADS1 gene likely affects tobacco flower color through its involvement in anthocyanin and flavonoid synthesis. Anthocyanin content analysis showed that the anthocyanin content of the CpMADS1-T1 generation was lower than that of the WT line, with OE-5 having 0.53 times the anthocyanin content of WT. During growth, the flowers of OE-5 were observed to be whiter. The anthocyanin content of OE-9 was 0.26 times that of WT. Flavonoid content analysis showed that the flavonoid content of OE-5 was 1.08 times that of WT, while the flavonoid content of OE-5 was 0.82 times that of WT. This suggests that CpMADS1 inhibits anthocyanin synthesis in tobacco flowers, thus affecting tobacco flower color, but has little effect on flavonoid synthesis.

[0100] This invention has led to a deeper understanding of the role of CpMADS1 in flower color regulation in wintersweet, pointing the way for research on flower color breeding using plant genetic engineering.

[0101] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the CpMADS1 gene of wintersweet in inhibiting anthocyanin biosynthesis in tobacco, characterized by: The nucleotide sequence of the CpMADS1 gene is shown in SEQ ID NO:

1.

2. The application of CpMADS1 protein from wintersweet in inhibiting anthocyanin biosynthesis in tobacco, characterized by: The amino acid sequence of the CpMADS1 protein is shown in SEQ ID NO:

2.

3. A CpMADS1 gene of wintersweet, characterized by: The nucleotide sequence of the CpMADS1 gene is shown in SEQ ID NO:

1.

4. A CpMADS1 protein from wintersweet, characterized in that: The amino acid sequence of the CpMADS1 protein is shown in SEQ ID NO:

2.

5. The biological material associated with the CpMADS1 protein of claim 4 is any one of B1) to B8) below: B1) A nucleic acid molecule encoding the CpMADS1 protein of claim 4; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4).

6. A method for preparing genetically modified tobacco, characterized in that: The recombinant vector described in claim 5 was introduced into tobacco cells to obtain transgenic tobacco plants that overexpress the CpMADS1 gene.

7. A method for improving the color of tobacco, characterized in that: By overexpressing the CpMADS1 gene of wintersweet, the anthocyanin content is reduced, resulting in a light pink or white color for tobacco flowers; the nucleotide sequence of the CpMADS1 gene is shown in SEQ ID NO:1.

Images