Mint McTTG1 gene as well as expression protein and application thereof

Abstract

The invention discloses a mint McTTG1 gene as well as an expression protein and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the mint McTTG1 gene disclosed by the invention is shown as SEQ ID NO.1, the amino acid sequence of the expression protein of the mint McTTG1 gene is shown as SEQ ID NO.2, and the mint McTTG1 gene belongs to WD40 family genes participating in regulation and control of epidermal hair development and synthesis of anthocyanin and procyanidine. Transgenic experiments show that after the McTTG1 gene is over-expressed in the arabidopsis thaliana mutant ttg1-13 with epidermal hair loss and significantly reduced accumulation of anthocyanin and procyanidine, the formation of the epidermal hair on the surface of the mutant can be recovered, and the accumulation of anthocyanin and procyanidine can be promoted. Gene resources are provided for plant variety improvement, and cultivation of new mint varieties is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the mint McTTG1 gene, its expressed protein, and its applications. Background Technology

[0002] Peppermint (Mentha haplocalyx Briq.) is an important spice and medicinal plant, with its essential oil rich in volatile terpenes, widely used in the pharmaceutical, food, and daily chemical industries, demonstrating significant economic value. However, my country's peppermint industry faces problems such as germplasm degradation, low oil yield, and unstable quality, urgently requiring the cultivation of high-yielding and high-quality new varieties through genetic improvement.

[0003] Peppermint essential oil is synthesized and stored in glandular trichomes, with shield-shaped trichomes being the primary site. Plant epidermal trichomes act as physical barriers, playing a crucial role in resisting biotic and abiotic stresses. Anthocyanins and proanthocyanidins, as flavonoid compounds, not only affect plant stress resistance and color but also possess health benefits such as antioxidant, anticancer, and cardiovascular disease prevention properties, making them highly valuable for applications.

[0004] WD40 proteins (WD-repeat proteins) are a conserved superfamily of regulatory proteins in eukaryotes, characterized by the presence of several to dozens of WD40 repeat motifs, each consisting of approximately 40 amino acids, typically beginning with glycine-histidine and ending with tryptophan-aspartic acid (GH…WD). These repeat units fold to form β-helical propeller structures, providing a binding interface for protein-protein interactions, thereby participating in various biological processes such as cell division, signal transduction, and transcriptional regulation. In plants, TTG1 (TRANSPARENT TESTA GLABRA1), an important member of the WD40 family, usually contains multiple WD40 repeat units and participates in regulating life processes such as epidermal and root hair development, anthocyanin and proanthocyanidin accumulation, seed morphology and color, flowering time, and mucilage formation. It also plays a crucial role in plant responses to high salt, drought, and sucrose stress.

[0005] Currently, systematic research on WD40 proteins in medicinal plants remains limited, especially in peppermint, where there are no reports on the function of this gene family. Therefore, cloning and identifying WD40 genes involved in the development of epidermal hairs and the regulation of anthocyanin and proanthocyanidin biosynthesis from peppermint will not only help deepen our theoretical understanding of the regulatory network of epidermal hair formation and flavonoid metabolism in plants, but will also provide key gene resources for the genetic improvement of peppermint at the application level, which is of great value for breeding new peppermint varieties with high yield, high essential oil content, and high stress resistance. Summary of the Invention

[0006] To address the existing technical problems, the first technical problem to be solved in this application is to provide a gene McTTG1 related to the development of peppermint epidermal hairs and the synthesis of anthocyanins and proanthocyanidins, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0008] An expression protein of the peppermint McTTG1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] An isolated polynucleotide encoding the expressed protein of the peppermint McTTG1 gene.

[0010] In some embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO.1.

[0011] A biomaterial, said biomaterial being an expression cassette, recombinant vector, or recombinant cell containing the said polynucleotide.

[0012] The application of the biomaterials in the cultivation of transgenic plants or molecular breeding.

[0013] A method for promoting the development of plant epidermal hairs, comprising overexpressing the expressed protein of the peppermint McTTG1 gene in the plant.

[0014] A method for promoting anthocyanin accumulation in plants includes overexpressing the expressed protein of the peppermint McTTG1 gene in plants.

[0015] A method for promoting the accumulation of proanthocyanidins in plants includes overexpressing the expressed protein of the peppermint McTTG1 gene in plants.

[0016] A method for cultivating transgenic plants includes: introducing a recombinant expression vector containing the polynucleotide into plant cells or tissues, and cultivating transgenic plantlets, wherein the transgenic plantlets have one or more of the following traits: enhanced epidermal hair development, increased anthocyanin content, and increased proanthocyanidin content.

[0017] In some embodiments, the plant is a dicotyledonous plant.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This application is the first to clone a WD40 family gene involved in regulating epidermal trichome development and anthocyanin and proanthocyanidin synthesis from peppermint, named McTTG1, whose nucleotide sequence is shown in SEQ ID NO.1, and whose expressed protein is shown in SEQ ID NO.2. Heterologous overexpression of the McTTG1 gene in Arabidopsis thaliana ttg1-13 (without epidermal trichomes and low anthocyanin and proanthocyanidin content) showed the following effects:

[0020] 1) The leaf epidermal hair density of the Arabidopsis mutant ttg1-13, which was transformed with McTTG1, was significantly restored to near the wild-type level, indicating that this gene can compensate for the epidermal hair development defects caused by the absence of AtTTG1.

[0021] 2) The seedlings and mature leaves of the Arabidopsis mutant ttg1-13, which was transformed into McTTG1, were purplish-red, and the anthocyanin content was significantly increased and close to that of the wild type, indicating that its function was similar to that of AtTTG1.

[0022] 3) The seeds of the Arabidopsis mutant ttg1-13 expressing McTTG1 showed a darker color and deeper DMACA staining, and the proanthocyanidin content was significantly restored to the wild-type level, proving that this gene can rescue the proanthocyanidin synthesis defect caused by the AtTTG1 mutation.

[0023] In summary, this invention is the first to report that the McTTG1 gene is a transcription factor in mint that positively regulates epidermal hair development and anthocyanin and proanthocyanidin synthesis. The McTTG1 gene provided by this invention offers a gene resource for plant variety improvement and has promising application prospects in breeding superior plant varieties. Attached Figure Description

[0024] Figure 1 This is an electrophoretic image of the McTTG1 gene CDS clone. In the image, M: DNA Marker; 1: Full-length McTTG1 gene CDS sequence.

[0025] Figure 2 Construction and identification of McTTG1 gene overexpression vector. In the figure, M: DNA Marker, 1: recombinant plasmid pHellsgate8-McTTG1-GFP, 2: recombinant plasmid pHellsgate8-McTTG1-GFP digested with XhoI enzyme.

[0026] Figure 3 RNA level identification in McTTG1 transgenic Arabidopsis thaliana;

[0027] Figure 4This image shows the development of leaf epidermal trichomes in the Arabidopsis wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2. A shows soil seedlings of each line approximately three weeks old; B shows the leaf surface of each line as captured by scanning electron microscopy; and C shows the number of leaf epidermal trichomes for each line.

[0028] Figure 5 The expression levels of genes related to epidermal hair development in the Arabidopsis wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2;

[0029] Figure 6 The seedling (A), mature seedling phenotype (B), and relative anthocyanin content (C) of Arabidopsis wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 are shown.

[0030] Figure 7 The expression levels of anthocyanin synthesis-related genes in the Arabidopsis wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2;

[0031] Figure 8 Seed color, DMACA staining (A), and proanthocyanidin content (B) of wild-type Arabidopsis thaliana WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2.

[0032] Figure 9 The expression levels of proanthocyanidin synthesis-related genes in the Arabidopsis wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0034] Example 1

[0035] 1. Cloning of the peppermint McTTG1 gene and construction of an overexpression vector

[0036] 1.1 Amplification of the CDS sequence of the peppermint McTTG1 gene

[0037] A suitable amount of leaf tissue samples were taken from the tested material, peppermint variety '68-7', and RNA was extracted from the samples using the plant tissue total RNA extraction kit provided by Nanjing Novizan Biotechnology Co., Ltd. The extracted RNA was then used to synthesize cDNA using Novizan Biotechnology Co., Ltd.'s third-generation high-efficiency cDNA one-strand synthesis kit (genomic removal). Based on the transcriptome sequence of the peppermint McTTG1 gene, the following primers were designed: Forward: 5'-ATGGACAATTCGACCCAGGAATCCC-3'; Reverse: 5'-TCATACTTTGAGCATCTGCATCTTG-3'. The CDS sequence of the McTTG1 gene was obtained by PCR, and the PCR amplification electrophoresis image is shown below. Figure 1 As shown in the figure. The CDS sequence of the McTTG1 gene is shown in SEQ ID NO:1, which is 1002 bp in length and encodes 333 amino acids, as shown in SEQ ID NO:2.

[0038] 1.2 Construction of McTTG1 gene overexpression vector

[0039] Based on the CDS sequence of the McTTG1 gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction enzyme site), amplification primers were designed: Forward: 5'-CATTTGGAGAGGACACGCTCGAGATGGACAATTCGACCCAG-3'; Reverse: 5'-CTTGCTCACCATGAATTCCTCGAGTACTTTGAGCATCTGCATC-3'. The target fragment of the McTTG1 gene was amplified by PCR. The pHellsgate8-GFP vector was digested with XhoI at 37℃ for 3 h. The PCR products and digestion products were recovered from the gel using a Shanghai Sangon Biotech gel extraction kit to obtain the target fragment and the linearized vector fragment. The target fragment was ligated into the pHellsgate8-GFP linear vector using the ClonExpress II One Step Cloning Kit to construct the 35S::McTTG1 overexpression vector. The ligation system was as follows: 2 μl of 5X CEII buffer, approximately 100–200 ng of linearized vector, approximately 50–100 ng of insert fragment, 1 μl of Exnase II, and ddH2O to a final volume of 10 μl. The mixture was incubated at 37°C for 30 min and then immediately transferred to ice. After thawing E. coli DH5α competent cells on ice, the recombinant product was added to the competent cells and mixed thoroughly. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 sec, then cooled on ice for 5 min. 500 μl of LB medium was added, and the cells were incubated at 37°C with shaking for 1 h. The bacterial culture was then plated onto plates containing 100 mg / L spectinomycin and incubated upside down at 37°C for 12–16 h. Single colonies were validated using colony PCR. Positive colonies were picked and inoculated into LB liquid medium containing 100 mg / L spectinomycin, and cultured at 37°C with shaking at 200 rpm for 24 h. Plasmids were extracted and verified by enzyme digestion. Figure 2 After verification, sequencing was performed. If the sequencing was correct, it was identified as the 35S::McTTG1 overexpression vector.

[0040] 2. Screening of positive transgenic Arabidopsis thaliana strains with the McTTG1 gene

[0041] 2.1 Agrobacterium infection in Arabidopsis thaliana

[0042] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

[0043] The 35S::McTTG1 overexpression vector plasmid was electroporated into Agrobacterium GV3101 competent cells. Positive strains were obtained by colony PCR verification. The Arabidopsis thaliana ttg1-13 mutant plants were then transformed using the flower-dipping method. Transformation medium was prepared first, consisting of 1 / 2 MS medium, 0.01 μg / ml BAP, 5% sucrose, and 0.02% Silwet L-77, adjusted to pH 5.7 with KOH. Positive strains of Agrobacterium 35S::McTTG1 were picked and cultured in YEP liquid medium containing 100 mg / L spectinomycin and 50 mg / L rifampin, and incubated at 28°C and 200 rpm for 16 h. 0.5 ml of the bacterial culture was then inoculated into 50 ml of YEP liquid medium containing the aforementioned antibiotics, and cultured at 28°C and 200 rpm until the OD value increased. 600 The value reaches approximately 1.8–2.0. Collect the bacterial culture in a centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the precipitate in conversion medium until the OD value of the resuspended solution reaches approximately 1.8–2.0. 600 Approximately 0.8. Place the Arabidopsis thaliana plants to be transformed horizontally, immerse the flower buds in the transformation medium for 1 minute, then incubate in the dark for 16–24 hours, followed by normal culture.

[0044] 2.2 Screening and identification of positive plants

[0045] After disinfecting the infected seeds with chlorine, they were cultured on MS medium containing 50 mg / L kanamycin. Resistant positive seedlings were selected and transplanted into soil. After seed harvest, the seedlings were screened again on MS medium containing 50 mg / L kanamycin. Transformed seedlings with a 3:1 segregation ratio were selected and transplanted into soil. After seed harvest, the seedlings were screened again on MS medium containing 50 mg / L kanamycin to select transformant lines with 100% kanamycin resistance. The homozygous McTTG1 transgenic lines were named ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2. RNA was extracted from the leaves of the homozygous transgenic positive lines, and cDNA was obtained by reverse transcription. Semi-quantitative primers were designed and semi-quantitative verification was performed. Figure 3 The semi-quantitative primers are as follows: Forward: 5'-GTACTCTGCTCCGCTAACTTCAT-3'; Reverse: 5'-TCACCATGTACCTAAGATCCTGC-3'. The total volume of the PCR reaction system is 20 μL: 1 μL each of the forward and reverse primers. The Robust PCR Master Mix consisted of 10 μL of cDNA and 7 μL of ddH2O. PCR reactions were performed using a PCR instrument with the following program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. A total of 40 cycles of annealing and extension were performed.

[0046] 3. The role of the McTTG1 gene in the development of epidermal trichomes in transgenic Arabidopsis thaliana

[0047] 3.1 Effect of heterologous expression of the McTTG1 gene in the Arabidopsis mutant ttg1-13 on epidermal trichome formation. Arabidopsis seeds (wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2) were sterilized with 75% ethanol and 2% sodium hypochlorite solution, then sown on 1 / 2 MS solid medium and cultured in a light incubator for 7 days. After seedlings were transplanted into soil and grown for approximately 2 weeks, the epidermal trichomes of wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 were observed and photographed using cryo-scanning electron microscopy. Figure 4 It can be seen that the leaves of the mutant ttg1-13 have no epidermal hairs, while the leaves of the ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 lines have obvious epidermal hair formation, and their epidermal hair density is close to that of the wild type. Figure 4 ).

[0048] 3.2 The mechanism by which the McTTG1 gene positively regulates the formation of epidermal trichomes in transgenic Arabidopsis thaliana. To further understand...

[0049] The McTTG1 gene regulates the formation of leaf epidermal trichomes. This invention used qRT-PCR to detect the gene expression levels of regulatory factors (such as AtMYB23, AtMYB5, AtGL3, AtEGL3, AtGL2, and AtTTG2) related to epidermal trichome formation in Arabidopsis wild-type WT, the mutant ttg1-13, and the McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2) using epidermal trichomes. Upregulation of these genes promoted epidermal trichome formation, while downregulation reduced the number of epidermal trichomes. Figure 5 It is evident that, compared to the mutant ttg1-13, most of these genes were significantly upregulated in the ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 lines. This suggests that McTTG1 can promote trichome formation in transgenic Arabidopsis thaliana at the molecular level by upregulating the expression of AtMYB23, AtMYB5, AtGL3, AtGL2, and AtTTG2.

[0050] 4. The role of the McTTG1 gene in anthocyanin accumulation in transgenic Arabidopsis thaliana

[0051] 4.1 Effect of heterologous expression of the McTTG1 gene in the Arabidopsis mutant ttg1-13 on anthocyanin accumulation. Arabidopsis seeds (wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2) were sterilized and planted on 1 / 2 MS solid medium. After being placed at 4°C for two days, they were cultured in a light incubator for four days. The color of the cotyledons, cotyledon margins, and hypocotyls of the seedlings were observed and photographed under a stereomicroscope. Seedlings were further cultured for 7 days and then transplanted into soil for approximately 4 weeks of growth. Anthocyanins were extracted from rosette leaves. The anthocyanin extract consisted of n-propanol, concentrated hydrochloric acid, and water (n-propanol:concentrated hydrochloric acid:water / 18:1:81, v:v:v). Weigh approximately 0.1g of plant leaf tissue, quick-freeze and grind it in liquid nitrogen, add 1ml of anthocyanin extraction solution, place at room temperature in the dark, extract overnight, centrifuge at 13000g for 20min, transfer the supernatant to a clean centrifuge tube, and dilute 4 times for determination. Measure the absorbance at 535nm and 650nm using a spectrophotometer, and calculate according to the formula 4x(A). 535 -A 650 The relative anthocyanin content per gram of fresh weight of plant tissue is calculated using this method. Figure 6 It can be seen that the seedlings of mutant ttg1-13 have light green cotyledon petioles, cotyledon margins, and hypocotyls, while the seedlings of ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 have purplish-red cotyledon petioles, cotyledon margins, and hypocotyls, similar to WT. The mature leaves of mutant ttg1-13 have green petioles, while the mature leaves of ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 have purplish-red petioles, similar to WT. These differences stem from the lower anthocyanin content in mutant ttg1-13 compared to WT, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2 lines. The relative anthocyanin content in the leaves was measured... Figure 6 The results showed that the relative anthocyanin content in the leaves of the mutant ttg1-13 was 0.028. Overexpression of the McTTG1 gene in the mutant ttg1-13 significantly increased the relative anthocyanin content in the leaves (1.369 for ttg1-13 / 35S::McTTG1-1 and 1.309 for ttg1-13 / 35S::McTTG1-2), showing no significant difference from the relative anthocyanin content in the leaves of the wild-type WT (1.531). This result indicates that overexpression of the McTTG1 gene can promote the accumulation of anthocyanins in plants.

[0052] 4.2 The Mechanism of McTTG1 Gene in Positively Regulating Anthocyanin Accumulation in Plants. To further understand the regulatory mechanism of the McTTG1 gene on anthocyanin accumulation in plants, this invention used qRT-PCR technology to detect the expression levels of anthocyanin synthesis genes in Arabidopsis thaliana WT, ttg1-13, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2 lines. The proteins encoded by the AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX, and AtUF3GT genes are important proteases in the anthocyanin synthesis pathway. Upregulation of these genes promotes anthocyanin synthesis, while downregulation leads to reduced anthocyanin synthesis. The results are as follows: Figure 7 As shown, compared with ttg1-13, the expression levels of AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX, and AtUF3GT were all significantly upregulated in the ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 lines. This indicates that overexpression of McTTG1 in ttg1-13 can increase the expression of anthocyanin synthesis genes, thereby promoting anthocyanin accumulation in transgenic Arabidopsis. Therefore, McTTG1 is a positive regulator of anthocyanin synthesis.

[0053] 5. The role of the McTTG1 gene in proanthocyanidin accumulation in transgenic Arabidopsis seeds

[0054] 5.1 Effect of heterologous expression of the McTTG1 gene in the Arabidopsis mutant ttg1-13 on proanthocyanidin accumulation in seeds. Arabidopsis seeds (wild-type WT, mutant ttg1-13, and McTTG1 heterologous replacement transgenic lines ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2) were collected, observed, and photographed under a stereomicroscope. Seeds from the four lines were also used for DMACA staining. A 0.1% (w / v) DMACA solution was prepared by dissolving an appropriate amount of DMACA powder in 0.12N HCl-MeOH solution. An appropriate amount of Arabidopsis seeds were immersed in the DMACA solution and stained at room temperature for approximately 2 hours. After rinsing with deionized water, the seeds were observed and photographed under a stereomicroscope. The proanthocyanidin content in the seeds was further determined using the sulfuric acid-vanillin method. Prepare a 1 mg / ml proanthocyanidin standard solution and dilute it to concentrations of 0, 0.025, 0.05, 0.125, 0.25, 0.5, and 1 mg / ml to construct a standard curve. Proanthocyanidins were extracted with hydrochloric acid-methanol (0.1% HCl, v / v). Weigh 1 g of seeds, add 800 μL of extraction buffer, grind thoroughly, centrifuge at 8000 rpm for 15 min, transfer the supernatant, and continue extraction twice with the extraction buffer. Centrifuge again, collect the supernatant, combine the supernatants, dry under nitrogen, and redissolve in methanol. Take 200 μL of proanthocyanidin extract, add 500 μL of 1% (w / v) vanillin-methanol and 500 μL of 25% (v / v) H2SO4-methanol solution, incubate in a 30℃ water bath for 15 min, and determine the A value using a spectrophotometer. 500 The value is used to calculate the proanthocyanidin content. (From...) Figure 8 It can be seen that, without DMACA staining, the seed coat color of mutant ttg1-13 is lighter than that of WT, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2. After DMACA staining, the seed coat coloring of WT, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2 lines is significantly higher than that of mutant ttg1-13. These differences are due to the lower proanthocyanidin content in the seeds of mutant ttg1-13 compared to WT, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2 lines. The proanthocyanidin content in the seeds was determined by... Figure 8The results showed that the proanthocyanidin content in wild-type WT seeds was approximately 0.566 mg / g seed, while that in the mutant ttg1-13 was approximately 0.209 mg / g seed. Overexpression of the McTTG1 gene in the ttg1-13 mutant significantly increased the proanthocyanidin content (0.649 mg / g seed in ttg1-13 / 35S::McTTG1-1 and 0.703 mg / g seed in ttg1-13 / 35S::McTTG1-2). This result indicates that overexpression of the McTTG1 gene can promote proanthocyanidin accumulation.

[0055] 5.2 The Mechanism of McTTG1 Gene in Positively Regulating Proanthocyanidin Accumulation in Seeds. To further understand the regulatory mechanism of the McTTG1 gene on proanthocyanidin accumulation in seeds, this invention used qRT-PCR technology to detect the expression levels of proanthocyanidin synthesis genes and regulatory genes in the seeds of Arabidopsis thaliana WT, ttg1-13, ttg1-13 / 35S::McTTG1-1, and ttg1-13 / 35S::McTTG1-2 lines. AtANR, AtTT19, AtTT13, AtTT12, and AtTT10 genes are proanthocyanidin synthesis genes in Arabidopsis thaliana seeds, while AtTT8 and AtTTG2 genes are regulatory genes for proanthocyanidin synthesis in Arabidopsis thaliana seeds, playing an important role in proanthocyanidin accumulation in Arabidopsis thaliana seeds. Figure 9 It is evident that, compared to the mutant ttg1-13, the AtANR, AtTT19, AtTT13, AtTT12, AtTT8, and AtTTG2 genes were all significantly upregulated in the ttg1-13 / 35S::McTTG1-1 and ttg1-13 / 35S::McTTG1-2 lines. This indicates that overexpression of McTTG1 in ttg1-13 can enhance the expression of proanthocyanidin synthesis genes and regulatory genes, thereby promoting the accumulation of proanthocyanidins in transgenic Arabidopsis seeds. Therefore, McTTG1 is a positive regulator of proanthocyanidin synthesis.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An expression protein of the peppermint McTTG1 gene, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the expression protein of the peppermint McTTG1 gene as described in claim 1.

3. The polynucleotide according to claim 2, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

1.

4. A biomaterial, characterized in that, The biomaterial is an expression cassette, recombinant vector, or recombinant cell containing the polynucleotides described in claim 2 or 3.

5. The application of the biomaterial described in claim 4 in the cultivation of transgenic plants or molecular breeding.

6. A method for promoting the development of plant epidermal hairs, characterized in that, This includes overexpression of the peppermint McTTG1 gene protein as described in claim 1 in plants.

7. A method for promoting anthocyanin accumulation in plants, characterized in that, This includes overexpression of the peppermint McTTG1 gene protein as described in claim 1 in plants.

8. A method for promoting the accumulation of proanthocyanidins in plants, characterized in that, This includes overexpression of the peppermint McTTG1 gene protein as described in claim 1 in plants.

9. A method for cultivating transgenic plants, characterized in that, include: A recombinant expression vector containing the polynucleotides described in claim 2 or 3 is introduced into plant cells or tissues to cultivate transgenic plant plants, wherein the transgenic plant plants have one or more of the following traits: enhanced epidermal hair development, increased anthocyanin content, and increased proanthocyanidin content.

10. The method according to claim 9, characterized in that, The plant in question is a dicotyledonous plant.

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