Mentha mcgata1 gene, expression protein and application thereof

Abstract

The application discloses a Mentha cardiaca McGATA1 gene, an expression protein thereof and application, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the McGATA1 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The McGATA1 gene is cloned from Mentha cardiaca, a plant overexpression vector thereof is constructed, and Mentha cardiaca is transformed to obtain a transgenic plant. Experiments show that overexpression of the McGATA1 gene can significantly up-regulate the expression of key enzyme genes in a menthol synthesis pathway and improve the accumulation amount of menthol in leaves of the transgenic plant. Gel migration retardation experiments further prove that the McGATA1 protein can specifically bind to a DNA probe containing a GATC element. The application provides a key gene and technical basis for cultivating a Mentha cardiaca new variety with high menthol content through transcription factor engineering.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a peppermint McGATA1 gene, its expressed protein, and its applications. Background Technology

[0002] Peppermint, a perennial plant belonging to the genus *Mentha* in the family Lamiaceae, is a widely used traditional Chinese medicine. Its essential oils have antibacterial and preservative properties, and therefore it is also used in the food and daily chemical industries.

[0003] The entire process of monoterpene formation in peppermint volatile oil involves the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) under the catalysis of geranyl pyrophosphate synthase (GPPS) to generate GPP, the only precursor for monoterpene synthesis. GPP is then catalyzed by monoterpene synthase (mTPS) to form various monoterpene skeletal structures. These skeletal structures undergo structural modifications such as methylation and isomerization to ultimately form monoterpene compounds, such as menthol and menthone. While the entire process of peppermint volatile oil synthesis and the enzymes required in the catalytic pathway are relatively well understood, research on the transcriptional regulation of various monoterpene synthases in volatile oil is scarce.

[0004] Plant transcription factors can precisely regulate the initiation and efficiency of gene transcription by recognizing cis-acting elements on target gene promoters and binding to specific sequences. This allows them to regulate plant cell differentiation, tissue and organ development, metabolic networks, and responses to plant hormones and environmental factors. GATA transcription factors play a crucial role in regulating nitrogen metabolism and chlorophyll synthesis in plants, as well as in plant secondary metabolic biology. While GATA family transcription factors have been identified in Arabidopsis thaliana, functional studies of GATA transcription factors and their expressed proteins in peppermint have not yet been reported. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by this invention is to provide the peppermint McGAT1 gene. Another technical problem to be solved by this invention is to provide the expression protein of the peppermint McGAT1 gene. A further technical problem to be solved by this invention is to provide the application of the peppermint McGAT1 gene for regulating menthol accumulation in plants.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A peppermint McGATA1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A peppermint McGATA1 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] A recombinant expression vector containing the nucleic acid sequence of the peppermint McGATA1 protein.

[0010] A host cell containing the peppermint McGATA1 gene or the recombinant expression vector; the host cell is a prokaryotic cell or Agrobacterium.

[0011] A cell or tissue of a transgenic peppermint plant, stably integrated with a nucleic acid sequence encoding the peppermint McGATA1 protein.

[0012] A method for increasing the menthol content in peppermint, comprising the step of increasing the expression level or activity of the peppermint McGATA1 protein in peppermint cells or tissues.

[0013] In some embodiments, the step of increasing the expression level of the peppermint McGATA1 protein includes: constructing an expression vector containing a nucleic acid sequence encoding the peppermint McGATA1 protein, and introducing the expression vector into plant cells or tissues to obtain transgenic plants with increased menthol content.

[0014] The application of the described peppermint McGATA1 protein in the preparation of transgenic peppermint with increased menthol content.

[0015] In some embodiments, the application is achieved by upregulating the expression of key enzyme genes in the menthol synthesis pathway; the key enzyme genes are one or more of the following: gerany pyrophosphate synthase gene, limonene synthase gene, isopistinolide reductase gene, menthene-3-ol dehydrogenase gene, and isopistinolide dehydrogenase gene.

[0016] In some embodiments, the peppermint McGATA1 protein upregulates its expression by binding to and activating the GATC cis-acting element in the promoter of the key enzyme gene.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1) This invention constructs a plant expression vector of the peppermint McGAT1 gene and transforms it into peppermint. After overexpressing the McGAT1 gene, the transgenic peppermint significantly increases the expression of key enzyme genes McGPPS, McLS, McPR, L3OH and IPR in the menthol synthase pathway, elucidating its regulatory mechanism at the transcriptional level.

[0019] 2) This invention constructs a protein expression vector for the peppermint McGATA1 gene, transforms it into competent E. coli BL21 cells, and after induction of expression and purification, obtains the His-tagged McGATA1 protein. Gel migration arrest experiments demonstrate that the McGATA1 protein can bind to the GATC cis-acting element with a biotin-labeled probe. This directly reveals the molecular mechanism by which McGATA1, as a transcription factor, regulates downstream target genes at the molecular interaction level.

[0020] 3) This invention, through genetic transformation technology, obtained peppermint plants overexpressing McGATA1, whose leaves contained significantly higher menthol content than the wild-type control. This demonstrates that by regulating a single key transcription factor, the accumulation of the target metabolite (menthol) can be effectively increased. This provides key genes and core technologies for the targeted improvement of peppermint varieties and the cultivation of new high-yield menthol-producing industrial raw material varieties through molecular breeding, and has excellent prospects for industrial application. Attached Figure Description

[0021] Figure 1 Electrophoretic detection image of the McGATA1 gene CDS clone (M is DNA Marker; 1 is the full-length McGATA1 gene CDS sequence).

[0022] Figure 2 The expression levels of the McGATA1 gene in different positive plants are shown in the figure.

[0023] Figure 3 Figure showing increased expression levels of enzyme genes related to the menthol synthesis pathway in McGATA1-overexpressing peppermint strains;

[0024] Figure 4 The graph shows the changes in menthol content in the McGATA1-overexpressing peppermint strain OE7; where A is a statistical comparison of menthol content between the wild type and the overexpressing strain OE7; and B is a chromatographic graph of menthol detection between the wild type and the overexpressing strain OE7.

[0025] Figure 5 The image shows the results of Western blot analysis of McGATA1 protein expression (M: 10-250 kDa protein marker; 1 is before induction, 2 is after induction).

[0026] Figure 6 The diagram shows the corresponding arrest bands generated when His-McGATA1 expressed protein binds to the promoter of GPPS, a key enzyme gene in the menthol synthesis pathway. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0028] The plant material used in this invention is peppermint (Mentha canadensis Linn.) of the genus Mentha in the family Lamiaceae, which was planted in the nursery of the Jiangsu Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province.

[0029] Example 1

[0030] (1) Total RNA extraction and cDNA acquisition

[0031] Total RNA was extracted from peppermint using a plant tissue total RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). Using the extracted total RNA as a template, cDNA was synthesized using a third-generation high-efficiency cDNA one-strand synthesis kit (genomically degenerated) (Nanjing Novizan Biotechnology Co., Ltd.).

[0032] (2) Amplification of the CDS sequence of the peppermint McGATA1 gene

[0033] Based on the transcriptome sequence of the peppermint McGATA1 gene, the following primers were designed:

[0034] GATA1 Forward: 5'-ATGAACTTAAATTCTCCCCTCTCAC-3' (SEQ ID NO.3);

[0035] GATA1 Reverse: 5'-TCAACCATTAACTAATCCA-3' (SEQ ID NO. 4).

[0036] The CDS sequence of the McGATA1 gene was obtained by PCR, and the PCR amplification electrophoresis image is shown below. Figure 1 As shown.

[0037] The final sequencing yielded the CDS sequence of the McGATA1 gene, as shown in SEQ ID NO.1, which is 879 bp in length and encodes 292 amino acids, as shown in SEQ ID NO.2.

[0038] Example 2

[0039] 1. Construction of a peppermint McGata1 gene overexpression vector

[0040] Based on the CDS sequence of the McGATA1 gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction site), amplification primers were designed, and the primer sequences are shown below:

[0041] Forward

[0042] 5'-CATTTGGAGAGGACACGCTCGAGATGAACTTAAATTCTCCCCCTTCAC-3' (SEQ ID NO. 5);

[0043] Reverse

[0044] : 5'-CTTGCTCACCATGAATTCCTCGAGACCATTAACTAATCCA-3' (SEQ ID NO. 6).

[0045] The target fragment of the McGATA1 gene was amplified by PCR. The pHellsgate8-GFP vector was digested with XhoI at 37°C for 3 h. The PCR products and digestion products were recovered from the DNA 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 a 35S::McGATA1 overexpression vector. The ligation system is as follows:

[0046] 2 μL of 5× CE II buffer, approximately 100-200 ng of linearized vector, approximately 50-100 ng of insert fragment, 1 μL of Exnase II, and bring the ddH2O to a final volume of 10 μL. Incubate at 37°C for 30 min and then immediately transfer to ice.

[0047] After thawing *E. coli* DH5α competent cells on ice, the recombinant product was added to the competent cells and mixed well. The mixture was incubated on ice for 30 min, followed by heat shock at 42°C for 45 sec, and then cooled on ice for 5 min. 500 μL of LB medium was added, and the mixture was incubated at 37°C with shaking for 1 h. The bacterial culture was then spread 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 incubated with shaking at 37°C for 24 h at 200 rpm. Plasmid extraction and enzyme digestion were performed for verification. Sequencing was performed after successful verification; correctly sequenced colonies were identified as the 35S:: McGATA1 overexpression vector.

[0048] 2. Agrobacterium-mediated stable genetic transformation of peppermint

[0049] Take 2-month-old sterile seedlings from tissue culture bottles, and cut their stems into 1-2 mm segments in a clean bench, about 1000 segments in total. Place the cross-section of each stem segment in contact with the culture medium and then place them on pepper-like pre-med medium (MS + 6-BA 2 mg·L⁻¹). -1 +NAA 0.2 mg·L -1 + 30g / L of sucrose -1 +3.8g / L agar -1 Incubate in the dark for 5-7 days. Add Agrobacterium EHA105 containing the 35S:: McGATA1 recombinant plasmid to 30 ml of solution containing 50 mg / L of [unclear text - likely a specific culture medium or solution]. -1 Spectinomycin and 50 mg·L -1 Rifampicin LB liquid medium was cultured on a shaker at 28 °C until the bacterial culture reached OD. 600 The concentration was 0.6-0.8. The bacterial culture was centrifuged at 5000 rpm, the supernatant was discarded, and the culture was resuspended in an equal volume of MS salt containing AS. The pre-cultured peppermint stem segments were immersed in the solution and placed in a live-cell conversion apparatus. Vacuum treatment was performed at 6 kPa for 180 s, repeated three times. The bacterial culture was discarded, the peppermint stem segments were washed with sterile water, and the surface moisture was absorbed on filter paper. The stem segments were then placed in peppermint co-culture medium (MS + 30 g / L sucrose). -1 +3.8 g·L agar -1 The stem segments were cultured in the dark for 3-4 days, after which they were transferred to pepper-like selection medium (MS + 3.0 mg·L⁻¹). -1 TDZ+ 0.2 mg·L -1 The samples were screened and cultured on IAA (25% coconut juice + cephalosporin 250mg / L + kanamycin 50mg / L) for 18-20 days until they sprouted.

[0050] 3. Screening and identification of positive plants

[0051] The shoots differentiated from the selection medium were transferred to selection rooting medium (MS + 30 g / L sucrose + 250 mg / L cephalosporin + 50 mg / L kanamycin, pH 5.8) for further selection. If the shoots could grow normally and develop fibrous roots in the selection rooting medium, they could be preliminarily identified as transgenic positive plants. Using wild-type peppermint as a negative control and Actin as an internal reference gene, the expression level of McGATA1 in transgenic peppermint was analyzed by RT-qPCR. Figure 2 As shown, the overexpression line OE7 (GATA1-OE7) had the highest expression level and was used for subsequent experiments.

[0052] 4. Effect of McGATA1 gene overexpression on the content of volatile components in peppermint plants

[0053] In McGATA1 overexpression lines, the relative expression levels of five candidate volatile oil synthesis pathway-related enzyme genes were analyzed using qRT-PCR. The results showed that ( Figure 3 In the overexpression lines, the expression levels of volatile oil-related synthase genes, namely gerany pyrophosphate synthase (GPPS), limonene synthase (LS), menthene-3-ol dehydrogenase (L3OH), isoprene dehydrogenase (IPR), and isoprene reductase (PR), were significantly increased compared to the wild type. This indicates that McGAT1 overexpression may participate in the regulation of volatile oil synthesis by modulating volatile oil synthase genes.

[0054] The overexpression line OE7, which had the highest McGATA1 gene expression level, was selected for the determination of volatile components. Three biological replicates were taken for each sample, and the results were compared with camphor as a standard. Peak times and mass spectrometry ion fragments were compared. The results showed that the menthol content in the overexpressing plants was significantly higher than that in the wild type. Figure 4 This indicates that McGATA1 overexpression can promote the accumulation of peppermint volatile oil components, demonstrating the positive regulatory effect of McGATA1 on peppermint volatile oil synthesis.

[0055] Example 3

[0056] The McGAT1 gene sequence was constructed into a His-tagged pCold(EcoRI) vector using homologous recombination to obtain the His-McGATA1 recombinant vector. This recombinant vector was transformed into competent E. coli BL21(DE3) cells, and positive clones were screened after sequencing. Positive clones were picked and cultured overnight at 37°C in 10 ml of LB broth containing 100 mg / L ampicillin on a shaker to obtain a saturated culture. In a 1000 ml Erlenmeyer flask, 400 ml of the same antibiotic-containing LB broth was added, and 4 ml of the saturated culture was added at a volume ratio of 1:100. The mixture was then shaken at 37°C until OD (dose elapsed). 600The induction concentration was 0.4-0.6. 2 ml of the induction sample was collected. 400 μl of 1M IPTG was added to the bacterial culture, and the culture was incubated at 16 °C and 100 rpm for 18-20 h. 2 ml of the induction sample was collected. The culture was centrifuged at 8000 rpm and 4 °C for 10 min, and the bacterial culture was collected. The cells were resuspended in 1 / 5 volume of PBS, centrifuged at 8000 rpm and 4 °C for 10 min, and the supernatant was discarded. The cells were resuspended in 80 ml of 0 mM imidazole solution, and 100 mM MMSF (working concentration 1 mM) was added. The mixture was shaken thoroughly. The cells were disrupted using an ultrasonic disruptor at 35% power for 45 min until clear. The culture was then centrifuged at 12000 rpm and 4 °C for 20 h. After min, discard the precipitate and add two column volumes of 10 mM imidazole buffer to the Ni-NTA purification column to equilibrate the column; add the supernatant to the equilibrated column, adjust the flow rate, and collect the flow-through; add two column volumes of 10 mM imidazole buffer to the chromatography column to wash away most of the contaminating proteins; elute with imidazole buffer at concentrations of 20 mM, 50 mM, 70 mM, 100 mM, and 250 mM sequentially, adding 14 ml of each concentration, and collect the eluent using a 2 ml centrifuge tube; detect McGATA1 protein (WB) Figure 5 EMSA gel migration assays were used to detect the interaction between McGAT1 (GATA1) and proMcGPPS (proGPPS). The results showed that the probe could bind to His-McGATA1 protein and produce a corresponding blocking band, indicating that McGAT1 can bind to proMcGPPS in vitro. Figure 6 This confirms at the molecular level that the upregulation of the key gene McGPPS for volatile oil synthesis in transgenic lines is directly regulated by the increased level of McGATA1 protein.

[0057] 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.

Claims

1. A peppermint McGata1 gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A peppermint McGata1 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It contains a nucleic acid sequence encoding the peppermint McGATA1 protein of claim 2.

4. A host cell, characterized in that, The present invention comprises the peppermint McGATA1 gene of claim 1 or the recombinant expression vector of claim 3; the host cell is a prokaryotic cell.

5. A cell or tissue of a transgenic peppermint plant, characterized in that, It is stably integrated with a nucleic acid sequence encoding the peppermint McGATA1 protein of claim 2.

6. A method for increasing the menthol content in peppermint, characterized in that, The step includes increasing the expression level of the peppermint McGATA1 protein of claim 2 in peppermint cells or tissues.

7. The method according to claim 6, characterized in that, The steps for increasing the expression level of the peppermint McGATA1 protein include: constructing an expression vector containing a nucleic acid sequence encoding the peppermint McGATA1 protein, and introducing the expression vector into plant cells or tissues to obtain transgenic plants with increased menthol content.

8. The application of the peppermint McGATA1 gene as described in claim 1 in the preparation of transgenic peppermint with increased menthol content.

9. The application according to claim 8, characterized in that, The application is achieved by upregulating the expression of key enzyme genes in the menthol synthesis pathway; the key enzyme genes are one or more of the following: gerany pyrophosphate synthase gene, limonene synthase gene, isopistinolide reductase gene, menthene-3-ol dehydrogenase gene, and isopistinolide dehydrogenase gene.

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