Mint McTTG2 gene, its expressed protein and application

Abstract

The present invention discloses a mint McTTG2 gene, its expression protein and application, and relates to the technical field of plant genetic engineering. The mint McTTG2 gene disclosed by the present invention has a nucleotide sequence as shown in SEQ ID NO.1, and an amino acid sequence of its expression protein as shown in SEQ ID NO.2. The present invention constructs an expression vector of the mint McTTG2 gene and transforms it into an Arabidopsis mutant; cultivates, screens and obtains transgenic Arabidopsis plants with significantly increased proanthocyanidin content. Transgenic experiments show that after overexpressing the McTTG2 gene in the mutant ttg2-7, the proanthocyanidin content is significantly increased; compared with the mutant ttg2-7, the epidermal hairs on the leaves of the ttg2-7 / 35S::McTTG2-1 and -2 strains are significantly increased, close to the wild type. It provides genetic resources for plant variety improvement and helps to cultivate new mint varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically relates to a mint McTTG2 gene, an expression protein thereof and an application thereof. Background Art

[0002] Mint (Mentha haplocalyx Briq.) is a perennial herb in the genus Mentha (Mentha L.) of the Lamiaceae family. It is widely cultivated as both a food and medicine and a spice. Mint is a world-renowned spice and an important food and medicine plant, cultivated worldwide. The entire mint plant is used medicinally and contains chemical components such as volatile oils, flavonoids, terpenes, phenolic acids, quinones, and phenylpropanoids. Mint essential oil, a key secondary metabolite synthesized by mint, is primarily composed of volatile monoterpenoids. These compounds exhibit antibacterial, antimicrobial, and preservative properties. It is used in pharmaceutical development and is also widely used as a flavoring and fragrance in food and household chemicals, possessing significant economic value. Domestic demand for mint and mint essential oil is growing, but due to the limited availability of mint germplasm resources, degraded varieties, and limited planting area in my country, mint production and oil yields are low, and the quality of the essential oil is unstable. This makes it impossible to meet domestic demand and necessitates imports. Therefore, carrying out genetic improvement and innovation of mint germplasm resources, cultivating high-quality and high-yield mint varieties, and improving the yield and quality of essential oils will be beneficial to promoting the development of my country's mint industry.

[0003] Plant trichomes are hair-like structures formed by specialized differentiation of epidermal cells on the surface of aerial plant parts, such as leaves and stems. These trichomes form an important physical barrier between the plant epidermis and the environment, helping plants adapt to changes in their surroundings. They not only defend against biotic stresses such as herbivory and pathogen infection, but also help plants withstand abiotic stresses such as high temperature, drought, high salinity, and ultraviolet light. Plant trichomes are generally classified as non-glandular or glandular, depending on whether they have synthetic or secretory functions. Many plant-derived secondary metabolites are synthesized, stored, or secreted within these glandular trichomes. Peppermint essential oil, a crucial plant secondary metabolite, is synthesized and stored within these glandular trichomes. Peppermint trichomes generally come in three types: non-glandular, capitate, and peltate. Both capitate and peltate trichomes are capable of essential oil synthesis, while peltate trichomes are larger than capitate trichomes and serve as the primary site for essential oil synthesis and storage.

[0004] Proanthocyanidins are a mixture of flavonoids, whose basic units are flavan-3-ol compounds. They are widely found in plant stems, bark, flowers, fruit peels, and seeds. They have various biological functions and are widely used in the pharmaceutical, food, and cosmetics markets. Based on the degree of polymerization, anthocyanidins are generally divided into oligomeric proanthocyanidins and polymeric proanthocyanidins. The former usually contain 2-5 monomers and are soluble, while the latter contain more than 5 monomers and are insoluble. Plants use proanthocyanidins to regulate seed dormancy and germination and to resist biotic and abiotic stresses, such as UV protection, resistance to pathogens, and predation. For humans, proanthocyanidins are natural antioxidants, nutritional supplements, and DNA protectants. They can effectively scavenge oxidative free radicals, lower blood cholesterol and blood sugar levels, protect the cardiovascular system, and prevent cardiovascular disease. In addition, proanthocyanidins affect the taste of some fruits and processed foods, such as persimmons, grapes, strawberries, and wine.

[0005] WRKY transcription factors are a class of transcription factors unique to plants. They contain conserved WRKY domains and C2H2 or C2HC zinc finger domains at their N- and C-termini, respectively. They can specifically bind to promoters containing the W-box (C / TTGACT / C), SURE (sugar-responsive cis-element, an AT-rich sequence), and other related elements, thereby regulating the expression of target genes. WRKYs not only play a crucial role in plant responses to biotic and abiotic stresses, but also in plant development and the synthesis of secondary metabolites.

[0006] At present, WRKY transcription factors have been identified in other medicinal plants, but there are few reports on WRKY transcription factors in mint, especially mint WRKY transcription factors that regulate epidermal hair development and proanthocyanidin synthesis. Summary of the Invention

[0007] In response to the aforementioned problems in the prior art, the present invention aims to provide a mint McTTG2 gene. Another technical problem addressed by the present invention is to provide an expression protein of the mint McTTG2 gene. A further technical problem addressed by the present invention is to provide the use of the mint McTTG2 gene for regulating plant epidermal hair formation and proanthocyanidin accumulation.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0009] A mint McTTG2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0010] The amino acid sequence of the mint McTTG2 gene protein is shown in SEQ ID NO. 2.

[0011] A vector, recombinant bacteria or host cell containing the mint McTTG2 gene.

[0012] The application of the mint McTTG2 gene in regulating the content of proanthocyanidins in plants includes:

[0013] 1) Constructing an expression vector for the mint McTTG2 gene;

[0014] 2) Transforming the constructed mint McTTG2 gene expression vector into Arabidopsis mutants;

[0015] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with significantly increased proanthocyanidin content.

[0016] The application of the mint McTTG2 gene in regulating the content of proanthocyanidins in plants can regulate the content of proanthocyanidins in plants by regulating the expression of key genes for proanthocyanidin accumulation.

[0017] The key genes for proanthocyanidin accumulation are AtTT12 and AtTT13 genes.

[0018] The application of the mint McTTG2 gene in regulating the development of plant epidermal hairs can regulate the development of plant epidermal hairs by regulating the expression of regulatory factors related to the formation and branching of epidermal hairs.

[0019] The regulatory factors related to epidermal hair formation and branching are AtGL3, AtEGL3, AtGL2, AtMYB23, AtBRK1, AtARP2 and AtWAVE2.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) An expression vector for the mint McTTG2 gene constructed in this study was transformed into Arabidopsis thaliana lines. Before staining, the seed coat color of the transgenic Arabidopsis mutant ttg2-7 was lighter than that of the WT, ttg2-7 / 35S::McTTG2-1, and ttg2-7 / 35S::McTTG2-2 lines. After DMACA staining, the seed coat color of the WT, ttg2-7 / 35S::McTTG2-1, and ttg2-7 / 35S::McTTG2-2 lines was significantly higher than that of the mutant ttg2-7. Overexpression of the McTTG2 gene in the mutant ttg2-7 significantly increased the proanthocyanidin content. These results indicate that overexpression of the McTTG2 gene promotes proanthocyanidin accumulation.

[0022] 2) Compared to the mutant ttg2-7, AtTT12 and AtTT13 genes were significantly upregulated in both ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2, approaching their wild-type expression levels. These results suggest that McTTG2 positively regulates proanthocyanidin accumulation in transgenic Arabidopsis seeds at the molecular level by upregulating the expression of AtTT12 and AtTT13 genes.

[0023] 3) The expression vector of the mint McTTG2 gene constructed in the present invention was transformed into Arabidopsis strains. Compared with the mutant ttg2-7, the ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 strains had significantly more epidermal hairs on the leaves, close to the wild type.

[0024] 4) Compared to the ttg2-7 mutant, the expression levels of AtGL3, AtEGL3, AtGL2, AtMYB23, AtBRK1, AtARP2, and AtWAVE2 genes were increased in the ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 lines. These results suggest that McTTG2 promotes epidermal trichome formation in transgenic Arabidopsis by upregulating the expression of these genes at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the electrophoresis detection diagram of McTTG2 gene CDS cloning (M is DNA Marker; 1 is the full-length sequence of McTTG2 gene CDS);

[0026] Figure 2 Figure 2 shows the construction and identification of the McTTG2 gene overexpression vector (M is a DNA marker, 1 is the recombinant plasmid pHellsgate8-McTTG2-GFP, and 2 is the recombinant plasmid pHellsgate8-McTTG2-GFP digested with XhoI);

[0027] Figure 3 Figure 1 is the RNA level identification diagram of McTTG2 transgenic Arabidopsis;

[0028] Figure 4 Diagrams showing the development of leaf epidermal hairs in the Arabidopsis wild type WT, the mutant ttg2-7, and the McTTG2-complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 (A is a scanning electron microscope image of the leaf surface of each line; B is a statistical analysis of the number of epidermal hairs on the leaves of each line; C is a statistical analysis of the proportion of epidermal hairs on different branches of the leaves of each line);

[0029] Figure 5The expression levels of genes related to epidermal hair development in Arabidopsis wild type WT, mutant ttg2-7, and McTTG2 heterologous complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2;

[0030] Figure 6 The seed colors and DMACA staining results of Arabidopsis thaliana wild type WT, mutant ttg2-7, and McTTG2 heterologous complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 are shown;

[0031] Figure 7 The graph shows the proanthocyanidin content in Arabidopsis wild type WT, mutant ttg2-7, and McTTG2 heterologous complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2;

[0032] Figure 8 The expression levels of genes related to proanthocyanidin synthesis in Arabidopsis wild type WT, mutant ttg2-7 and McTTG2 heterologous complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0034] The plant material used in this application, the mint variety "68-7", was collected in the nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, and provided by Liang Chengyuan's research group; the Arabidopsis ttg2-7 mutant (with sparse epidermal hairs, abnormal morphology, and low proanthocyanidin content) was purchased from the Arabidopsis germplasm resource bank TAIR (The Arabidopsis Information Resource).

[0035] Example 1

[0036] 1. Total RNA extraction and cDNA acquisition

[0037] Total RNA from mint was extracted using a plant tissue total RNA extraction kit (Nanjing Novozymes Biotech Co., Ltd.). cDNA was synthesized using the third-generation high-efficiency cDNA first-strand synthesis kit (genomic-free) (Nanjing Novozymes Biotech Co., Ltd.) using the extracted total RNA as a template.

[0038] 2. Amplification of the CDS sequence of the mint McTTG2 gene

[0039] Based on the transcriptome sequence of the mint McTTG2 gene, the following primers were designed:

[0040] Forward: 5'-ATGATTGAAGTAGAGGAGAAAGAAAAAATC-3',

[0041] Reverse: 5'-TCAGTTCTTAGTACTCGTCTTGGATGCATC-3'.

[0042] The CDS sequence of McTTG2 gene was obtained by PCR. The electrophoresis diagram of PCR amplification is shown in Figure 2. Figure 1 shown.

[0043] The CDS sequence of the McTTG2 gene obtained by final sequencing is shown in SEQ ID NO.1, totaling 1248 bp; it encodes a total of 415 amino acids, as shown in SEQ ID NO.2 in the sequence listing.

[0044] Example 2

[0045] 1. Construction of mint McTTG2 gene overexpression vector

[0046] Based on the CDS sequence of the McTTG2 gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction site), amplification primers were designed. The primer sequences are as follows:

[0047] Forward:

[0048] 5'-CATTTGGAGAGGACACGCTCGAGATGATTGAAGTAGAGGAG-3';

[0049] Reverse:

[0050] 5'-CTTGCTCACCATGAATTCCTCGAGGTTCTTAGTACTCGTCTTG-3'.

[0051] PCR amplify the target fragment of the McTTG2 gene. Digest the pHellsgate8-GFP vector with XhoI at 37°C for 3 h. Recover the PCR and digestion products using a Shanghai Biotech Gel Extraction Kit to obtain the target fragment and linearized vector fragment. Ligate the target fragment with the pHellsgate8-GFP linearized vector using the ClonExpress II One-Step Cloning Kit for homologous recombination to construct a 35S::McTTG2 overexpression vector. The ligation system is as follows: 2 μL of 5× CE II buffer, approximately 100–200 ng of linearized vector, approximately 50–100 ng of insert, 1 μL of Exnase II, and add ddH2O to 10 μL. Incubate at 37°C for 30 min, then immediately transfer to ice. After the E. coli DH5α competent cells were thawed on ice, the recombinant product was added to the competent cells and mixed. The cells were incubated on ice for 30 minutes, followed by heat shock at 42°C for 45 seconds, and then cooled on ice for 5 minutes. 500 μL of LB medium was added and the cells were shaken at 37°C for 1 hour. The bacterial liquid was spread on a plate containing 100 mg / L spectinomycin and incubated in an inverted manner at 37°C for 12-16 hours. Single colonies were verified by colony PCR. Positive colonies were picked and inoculated into LB liquid medium containing 100 mg / L spectinomycin and shaken at 200 rpm at 37°C for 24 hours. Plasmid enzyme digestion verification ( Figure 2 ), and then sequenced after verification. The correct sequence is the 35S::McTTG2 overexpression vector.

[0052] 2. Agrobacterium infection of Arabidopsis

[0053] The 35S::McTTG2 overexpression vector plasmid was electroporated into Agrobacterium GV3101 competent cells, and the positive strains were obtained by colony PCR verification. The Arabidopsis thaliana ttg2-7 mutant plants were transformed using the flower dipping method. First, the transformation medium was prepared. The components included 1 / 2MS culture medium, 0.01μg / mL BAP, 5% sucrose, 0.02% silwetL-77, and the pH was adjusted to 5.7 with KOH. The 35S::McTTG2 Agrobacterium positive strain was picked and added to the YEP liquid medium containing 100mg / L spectinomycin and 50mg / L rifampicin, and shaken at 28℃, 200rpm, for 16h. Take 0.5mL of the above bacterial solution and add it to 50mL of YEP liquid medium containing the above antibiotics, and shake at 28℃, 200rpm to make the OD 600 The value reaches about 1.8~2. Collect the bacterial solution in a centrifuge tube, centrifuge at 5000rpm for 5 minutes, discard the supernatant, and resuspend the pellet with transformation medium until the resuspension OD 600Place the Arabidopsis plants to be transformed flat, immerse the flower buds in the transformation medium for 1 minute, and then incubate in the dark for 16-24 hours before resuming normal culture.

[0054] 3. Screening and identification of positive plants

[0055] The infected seeds were disinfected with chlorine and cultured on MS medium containing 50 mg / L kanamycin. The resistant seedlings were screened and transplanted into the soil. After the seeds were harvested, they were screened on MS medium containing 50 mg / L kanamycin. Transformed seedlings with a 3:1 segregation ratio were selected and transplanted into the soil. After the seeds were harvested, they were screened on MS medium containing 50 mg / L kanamycin. Transformed seedlings with 100% kanamycin resistance were selected. The McTTG2 homozygous transgenic lines were named ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2. RNA was extracted from the leaves of the homozygous transgenic positive lines, and its cDNA was obtained by reverse transcription. Quantitative primers were designed and semi-quantitative verification was performed ( Figure 3 ). The quantitative primers are as follows:

[0056] Forward: 5'-GGACAGAAACAGGTTAAAGGAAGTG-3';

[0057] Reverse: 5'-TCTTCAAACGTTCTCTCCACATACT-3'.

[0058] The total volume of the PCR reaction system was 20 μL: 1 μL each of the upstream and downstream primers, 2×Hieff ® Robust PCR MasterMix 10 μL, cDNA 1μL, ddH2O 7μL.

[0059] The PCR reaction was performed using a PCR instrument with the following program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, with a total of 40 cycles of annealing and extension.

[0060] Example 3

[0061] 1. Effect of overexpression of the McTTG2 gene on Arabidopsis epidermal hair formation

[0062] Arabidopsis seeds (WT, wild-type WT, mutant ttg2-7, and McTTG2 heterologous complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2) were disinfected with 75% ethanol and 2% sodium hypochlorite solution, planted on 1 / 2MS solid culture medium, and cultured in a light incubator for 7 days. After the seedlings were transferred to soil and grown for 2-3 weeks, the epidermal hairs of each line were observed using a cryo-scanning electron microscope.

[0063] The results are as follows Figure 4 As shown, compared with the mutant ttg2-7, the ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 lines had significantly more hairs on the leaf epidermis, close to that of the wild type.

[0064] qRT-PCR was used to examine the gene expression levels of regulatory factors involved in trichome formation and branching, such as AtGL3, AtEGL3, AtGL2, AtMYB23, AtBRK1, AtARP2, and AtWAVE2, in each strain. Upregulation of these genes promotes trichome formation and normal branching, while downregulation reduces trichome number and branching.

[0065] The results are as follows Figure 5 As shown in the figure, compared with the mutant ttg2-7, the expression levels of these genes were increased in the ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 lines. The results showed that McTTG2 promoted epidermal hair formation in transgenic Arabidopsis by upregulating the expression of AtGL3, AtEGL3, AtGL2, AtMYB23, AtBRK1, AtARP2, and AtWAVE2 at the molecular level.

[0066] 2. Effect of overexpression of the McTTG2 gene on proanthocyanidin accumulation in Arabidopsis seeds

[0067] Arabidopsis seeds (WT, wild-type WT, mutant ttg2-7, and McTTG2-complemented transgenic lines ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2) were collected, observed under a stereomicroscope, and photographed. Seeds from the four aforementioned lines were also collected for DMACA staining. A 0.1% (w / v) DMACA solution was prepared by weighing an appropriate amount of DMACA powder and dissolving it in a 0.12N HCl-MeOH solution. An appropriate amount of Arabidopsis seeds were immersed in the DMACA solution and stained at room temperature for approximately 1.5 hours. After rinsing with deionized water, the seeds were observed under a stereomicroscope and photographed. The proanthocyanidin content of the seeds was further determined using the sulfuric acid-vanillin method. Prepare a 1 mg / mL proanthocyanidin standard solution and dilute it into proanthocyanidin solutions with concentrations of 0, 0.025, 0.05, 0.125, 0.25, 0.5, and 1 mg / mL to prepare a standard curve. Proanthocyanidins were extracted with hydrochloric acid-methanol (0.1% HCl, v / v). Weigh 1 g of seeds, add the extract and grind thoroughly, centrifuge at 8000 rpm for 15 minutes, transfer the supernatant, continue to extract with the extract twice, centrifuge and take the supernatant, combine the supernatants, blow dry with nitrogen, and re-dissolve in methanol. Take 200 μL of proanthocyanidin extract, add 500 μL of 1% (w / v) vanillin-methanol and 500 μL of 25% (v / v) H2SO 4- Methanol solution, 30℃ water bath, reaction for 15min, spectrophotometer to measure A 500 The proanthocyanidin content was calculated.

[0068] The results are as follows Figure 6 As shown in the figure, before staining, the seed coat color of the mutant ttg2-7 was lighter than that of the WT, ttg2-7 / 35S::McTTG2-1, and ttg2-7 / 35S::McTTG2-2. After DMACA staining, the seed coat coloration of the WT, ttg2-7 / 35S::McTTG2-1, and ttg2-7 / 35S::McTTG2-2 lines was significantly higher than that of the mutant ttg2-7. These differences are attributed to the lower proanthocyanidin content in the seeds of the mutant ttg2-7 compared with the WT, ttg2-7 / 35S::McTTG2-1, and ttg2-7 / 35S::McTTG2-2 lines.

[0069] The results are as follows Figure 7As shown, the proanthocyanidin content of the wild-type WT seeds was approximately 0.292 mg / g, and the proanthocyanidin content of the mutant ttg2-7 seeds was approximately 0.095 mg / g. However, overexpression of the McTTG2 gene in the mutant ttg2-7 significantly increased the proanthocyanidin content (ttg2-7 / 35S::McTTG2-1 was 0.176 mg / g, and ttg2-7 / 35S::McTTG2-2 was 0.314 mg / g). The results indicate that overexpression of the McTTG2 gene promotes the accumulation of proanthocyanidins.

[0070] qRT-PCR was used to detect the expression levels of key genes (AtTT12 and AtTT13) that regulate seed proanthocyanidin accumulation in each strain. AtTT12 and AtTT13 are positive regulators of proanthocyanidin synthesis in Arabidopsis seeds.

[0071] The results are as follows Figure 8 As shown, compared with the mutant ttg2-7, AtTT12 and AtTT13 genes were significantly upregulated in the ttg2-7 / 35S::McTTG2-1 and ttg2-7 / 35S::McTTG2-2 lines, approaching their expression levels in the wild type. These results suggest that McTTG2 positively regulates proanthocyanidin accumulation in transgenic Arabidopsis seeds at the molecular level by upregulating the expression of AtTT12 and AtTT13 genes.

[0072] The above description is only illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of them will fall within the scope of protection of the present invention.

Claims

1. A mint McTTG2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. mint according to claim 1 McTTG2 The amino acid sequence of the expressed protein of the gene is shown in SEQ ID NO.

2.

3. Containing the mint according to claim 1 McTTG2 gene vector or host cell.

4. mint according to claim 1 McTTG2 The application of genes in regulating the content of proanthocyanidins in plants is characterized in that: The content of proanthocyanidins in a plant is regulated by regulating the expression of key genes for proanthocyanidin accumulation; the plant is Arabidopsis thaliana or mint.

5. The use according to claim 4, characterized in that include: 1) Build Mint McTTG2 Gene expression vector; 2) The mint that will be built McTTG2 Arabidopsis thaliana ttg2-7 in mutants or mint plants; 3) Cultivate, screen and obtain transgenic Arabidopsis or mint plants with significantly increased proanthocyanidin content.

6. mint according to claim 1 McTTG2 The application of a gene in regulating the development of plant epidermal hairs is characterized in that: The development of plant epidermal hairs is regulated by regulating the expression of regulatory factors related to the formation and branching of epidermal hairs; the plant is Arabidopsis or mint.

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