Mint transcription factor gene McHD-Zip1 as well as expression protein and application thereof

Abstract

The invention discloses a mint transcription factor gene McHD-Zip1 as well as an expression protein and application thereof, and relates to the technical field of gene engineering. The full-length coding sequence of the mint transcription factor gene McHD-Zip1 disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the mint transcription factor gene McHD-Zip1 is as shown in SEQ ID NO.2. An overexpression vector of the mint transcription factor gene McHD-Zip1 is constructed and transformed into peppermint, and the result of the embodiment shows that the McHD-Zip1 can regulate and control the development of glandular hair of mint leaves, and overexpression of the gene can remarkably increase the glandular hair density of the leaves and regulate and control the content of total volatile oil, menthone and limonene in mint bodies. The invention provides a new gene resource for research on regulation and control of plant glandular hair development, and also provides theoretical support and gene resources for improvement of mint germplasm and improvement of the yield and quality of mint essential oil.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and more specifically, relates to a peppermint transcription factor gene McHD-Zip1, its expression protein, and its applications. Background Technology

[0002] Plants of the genus *Mentha* (L.) are perennial or rarely annual herbs belonging to the family Lamiaceae. They are aromatic and comprise over 25 species, 12 of which are found in my country. Among them, *Mentha canadensis* L., *Mentha × pipeita*, and *Mentha spicata* L. are important aromatic plants widely used both domestically and internationally. The aroma of *Mentha* plants comes from the abundant essential oils in their stems and leaves, primarily composed of volatile monoterpenes. The essential oils of *Mentha* and *Mentha × pipeita* are quite similar, mainly consisting of menthol, menthone, and limonene. Peppermint essential oils are used in various industries, including food, biomedicine, and cosmetics, and have significant economic and medicinal value. Therefore, improving the yield and quality of essential oils is of great importance for improving peppermint germplasm and promoting the development of my country's peppermint industry.

[0003] Glandular trichomes are a type of epidermal hair in plants. Based on structure and type, they can be divided into secretory and non-secretive glandular trichomes. Secretory glandular trichomes include capitulum and peltate glandular trichomes. The stems and leaves of peppermint plants possess both capitulum and peltate glandular trichomes. The peltate glandular trichomes are the sites of essential oil synthesis, secretion, transport, and storage; they are the "biofactories" for essential oil production. Glandular trichome density is positively correlated with essential oil yield, and the morphology and number of glandular trichomes determine the essential oil production. Therefore, increasing the density of peppermint glandular trichomes is an effective means to increase peppermint essential oil yield. The development of plant glandular trichomes involves multiple genes, and has been extensively studied in plants such as Arabidopsis thaliana, tomato, and Artemisia annua. However, the molecular mechanism of peppermint glandular trichome development remains unclear.

[0004] Because peppermint is a polyploid plant and reproduces asexually in production, germplasm innovation is slow, varieties are limited and degenerate, and domestic peppermint essential oil supply cannot meet demand, leading to increasing reliance on imports. Therefore, identifying key genes regulating the development of peppermint glandular trichomes can not only enrich the molecular network regulating glandular trichome development but also provide excellent genetic resources and theoretical support for improving peppermint germplasm resources and increasing peppermint essential oil yield through molecular biotechnology. 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 transcription factor gene McHD-Zip1. Another technical problem to be solved by this invention is to provide the expression protein of the peppermint transcription factor gene McHD-Zip1. A further technical problem to be solved by this invention is to provide the application of the peppermint transcription factor gene McHD-Zip1 for regulating the development of peppermint glandular trichomes.

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

[0007] A peppermint transcription factor gene, McHD-Zip1, has its full-length coding sequence as shown in SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the peppermint transcription factor gene McHD-Zip1 is shown in SEQ ID NO.2.

[0009] Primers used to amplify the peppermint transcription factor gene McHD-Zip1, the primer sequences of which are shown below:

[0010] McHD-Zip1-F: 5'-ATGTATGGAGATTGTCAGGTGC-3',

[0011] McHD-Zip1-R: 5'-TTAAGCATCAACAGCTGCCT-3'.

[0012] Vectors containing the peppermint transcription factor gene McHD-Zip1.

[0013] A method for constructing a vector of the peppermint transcription factor gene McHD-Zip1 includes the following steps:

[0014] 1) The overexpression vector was double-digested with BamHI and SalI and the linearized vector was recovered;

[0015] 2) Design primers for the McHD-Zip1 overexpression vector, introduce the BamHI restriction sequence into the upstream primer and the SalI restriction sequence into the downstream primer;

[0016] 3) The mint transcription factor gene McHD-Zip1 was ligated to the linearized vector using homologous recombination primers for the McHD-Zip1 overexpression vector.

[0017] The primer sequences for the McHD-Zip1 overexpression vector are shown below:

[0018] McHD-Zip1-OE-F:

[0019] 5'-GGGTACCCGGGGATCCATGTATGGAGATTGTCAGGTGC-3',

[0020] McHD-Zip1-OE-R:

[0021] 5'-ATTCCTGCAGGTCGACTTAAGCATCAACAGCTGCCT-3'.

[0022] Application of the peppermint transcription factor gene McHD-Zip1 in regulating the development of glandular trichomes in peppermint leaves.

[0023] Application of the peppermint transcription factor gene McHD-Zip1 in regulating the total volatile oil content in peppermint.

[0024] This paper describes the application of the peppermint transcription factor gene McHD-Zip1 in regulating the menthone content in peppermint.

[0025] Application of the peppermint transcription factor gene McHD-Zip1 in regulating limonene content in peppermint.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1) This invention discloses for the first time the nucleotide sequence (SEQ ID NO.1) of the peppermint transcription factor gene McHD-Zip1 and the amino acid sequence of its expressed protein (SEQ ID NO.2), providing new gene resources for the study of the regulation of plant glandular hair development.

[0028] 2) This invention constructs an overexpression vector for the peppermint transcription factor gene McHD-Zip1 and transforms it into peppermint. The results of the examples show that the density of glandular hairs on the leaf surface of peppermint strains overexpressing the McHD-Zip1 gene is significantly higher than that of wild-type strains. This gene increases the number of glandular hairs by regulating the development process of glandular hairs, thus laying a structural basis for improving essential oil synthesis.

[0029] 3) This invention constructs an overexpression vector for the peppermint transcription factor gene McHD-Zip1 and transforms it into peppermint. Headspace gas chromatography-mass spectrometry (HS-GC-MS) was used for detection. The results showed that the total volatile oil content of the transgenic peppermint plants was significantly higher than that of the wild type; the increases in menthone and limonene were most significant in the transgenic plants. This confirms that the McHD-Zip1 gene promotes the overall synthesis and accumulation of volatile oils by enhancing the secretory function of glandular trichomes or related metabolic pathways. This gene not only increases the total amount of volatile oils but also specifically regulates the synthesis of key aroma components (such as menthone) and functional components (such as limonene), which is of great significance for improving the quality of peppermint essential oil. Attached Figure Description

[0030] Figure 1 The graph shows the changes in expression levels of the peppermint transcription factor gene McHD-Zip1 in the strain.

[0031] Figure 2 Image of glandular trichomes on leaf surface of a strain overexpressing the peppermint transcription factor gene McHD-Zip1 under a scanning electron microscope;

[0032] Figure 3 The images show the observation and statistical diagrams of glandular trichomes on the leaf surface of the McHD-Zip1 strain overexpressing the peppermint transcription factor gene under a stereofluorescence microscope (A is a three-field diagram of glandular trichome density; B is a three-field diagram of glandular trichomes observed in the wild-type and control strains under a stereofluorescence microscope; C is a statistical diagram of glandular trichome density in the three-field diagram).

[0033] Figure 4 Figure showing the essential oil content analysis in the McHD-Zip1 strain overexpressing the peppermint transcription factor gene. Detailed Implementation

[0034] 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 described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0035] The plant material used in the following examples is mint, which is preserved in the germplasm resource nursery of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province.

[0036] Example 1

[0037] 1. Cloning the peppermint transcription factor gene McHD-Zip1

[0038] Total RNA was extracted from peppermint leaves using a plant tissue total RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). The extracted total RNA was then used as a template to synthesize cDNA using reverse transcriptase. Based on the peppermint transcriptome data, McHD-Zip1 cloning primers were designed.

[0039] McHD-Zip1-F: 5'-ATGTATGGAGATTGTCAGGTGC-3',

[0040] McHD-Zip1-R: 5'-TTAAGCATCAACAGCTGCCT-3'.

[0041] After PCR reaction, the cloned fragment was ligated into the T vector, and after sequencing, the full-length coding sequence of the peppermint transcription factor gene McHD-Zip1 was obtained as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein was shown in SEQ ID NO.2.

[0042] 2. Construction of the overexpression vector for the peppermint transcription factor gene McHD-Zip1

[0043] Primers for the McHD-Zip1 overexpression vector were designed, with a BamHI restriction sequence introduced into the upstream primer and a SalI restriction sequence introduced into the downstream primer. The primer sequences are shown below:

[0044] McHD-Zip1-OE-F:

[0045] 5'-GGGTACCCGGGGATCCATGTATGGAGATTGTCAGGTGC-3',

[0046] McHD-Zip1-OE-R:

[0047] 5'-ATTCCTGCAGGTCGACTTAAGCATCAACAGCTGCCT-3'.

[0048] The McHD-Zip1 gene fragment was amplified using Phanta Max Super-Fidelity DNA Polymerase. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 3 min, repeated 35 times; 72℃ extension for 5 min. The amplified fragment was recovered using a Shanghai Sangon DNA fragment gel recovery kit. The plant expression vector p35SGK was double-digested with BamHI and SalI at 37℃ for 3 h, and the digestion products were recovered using a DNA fragment recovery kit. Homologous recombination of the gene fragment and vector fragment was performed using the ClonExpress II One Step Cloning Kit. The reaction system was: 1 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 ddH2O to a final volume of 5 μL. The reaction was carried out at 37℃ for 30 min, and then immediately transferred to ice. The recombinant product was added to *E. coli* DH5α thawed on ice, incubated on ice for 30 min, heat-shocked in a 42°C water bath for 90 s, quickly transferred to ice for 2 min, and then 1 mL of LB medium was added. The mixture was incubated at 37°C and 200 rpm for 1 h in a shaker. An appropriate amount of the bacterial culture was spread onto a solid medium containing 50 mg / mL Kana antibiotic and incubated upside down at 37°C for approximately 12 h. Single colonies were picked for PCR detection. Positive colonies underwent plasmid extraction and sequencing. Correct sequencing confirmed the presence of the McHD-Zip1 gene overexpression recombinant vector.

[0049] 3. Transformation of peppermint with the peppermint transcription factor gene McHD-Zip1

[0050] The constructed overexpression vector was transformed into Agrobacterium EHA105, and after positive identification, positive clones were selected and cultured in YEB medium until OD600. 600 =0.6, after centrifugation, the supernatant was discarded, and the mixture was resuspended in a resuspension solution (MS solution containing 200 μmol / L acetylsyringone, pH 5.4) to obtain the infection solution; the cut peppermint stem segments were cultured on pre-culture medium for 3 days and then transferred to the prepared infection solution. The mixture was incubated at 28°C for 0.5 hours. After aspirating the bacterial suspension, it was placed in a co-culture medium (MS + 30 g / L sucrose + 5 mg / L TDZ + 0.2 mg / L IAA + 20% coconut milk + 200 μmol / L acetylsyringone + 7 g / L agar, pH 5.4) and co-cultured at 25°C in the dark for 3 days. The culture was then transferred to a selection and differentiation medium (MS + 30 g / L sucrose + 5 mg / L TDZ + 0.2 mg / L IAA + 20% coconut milk + 7 g / L agar + 250 mg / L cephalosporin + 50 mg / L kanamycin, pH 5.4). 5.8) Culture under normal light. After the resistant buds have grown, transfer them to the screening rooting medium (MS + sucrose 30g / L + cephalosporin 250mg / L + kanamycin 50mg / L, pH 5.8) for rooting culture. When the roots grow to 3-5cm, transfer them to a plant light incubator.

[0051] 4. Identification of transgenic positive plants

[0052] After antibiotic screening and GUS staining identification, wild-type and overexpressing plants were transferred to a substrate for culture. Peppermint leaves from the same growth stage and location of both wild-type and overexpressing plants were selected, and RNA was extracted and reverse transcribed into cDNA. Real-time quantitative PCR primers were designed based on the McHD-Zip1 gene sequence, and the primer sequences are shown below.

[0053] qMcHD-Zip1-F: 5'-TGGTGAGATGGGCTATGA-3',

[0054] qMcHD-Zip1-R: 5'-TCCCGTTGTATTGCTGTGA-3'.

[0055] The results are as follows Figure 1 As shown, the expression level of McHD-Zip1 was significantly increased in all overexpression lines, with the most significant increase observed in the McHD-Zip1-1OE and McHD-Zip1-3OE lines.

[0056] 5. Statistics on glandular trichomes on leaves of transgenic positive plants

[0057] Two overexpression lines, McHD-Zip1-1 OE and McHD-Zip1-3 OE, with the most significant increase in expression levels, were selected for scanning electron microscopy to observe glandular trichome density. To further analyze changes in glandular trichome density, three fields of view on the leaves were observed under a stereofluorescence microscope. Figure 3 (A).

[0058] The results are as follows Figure 2 As shown, the glandular trichome density of both overexpression lines was significantly higher than that of the wild-type line.

[0059] The results are as follows Figure 3 As shown, the glandular trichome density of the two overexpression lines, McHD-Zip1-1 OE and McHD-Zip1-3 OE, was significantly higher than that of the wild type. Figure 3 Density statistics were performed on the B-type strains, and it was found that the glandular trichome density of both overexpression lines was significantly higher than that of the wild-type line. Figure 3 The results showed that McHD-Zip1 promotes glandular hair development.

[0060] 6. Determination of volatile oil content in transgenic positive plants

[0061] The essential oil content of wild-type and transgenic peppermint was determined using headspace gas chromatography-mass spectrometry (HS-GC-MS). The procedure was as follows: Leaves from the same growth location were selected and air-dried in a well-ventilated area. 0.5 g of each dried leaf was placed in a headspace vial, with 0.3 mg of camphor added as an internal standard. Extraction was performed using a fiber extractor at 40℃ for 40 min, with an injection port temperature of 250℃ and a flow rate of 50 mL / min, followed by desorption for 3 min. Based on the proportion of each component, and using 0.3 mg of camphor as an internal standard, the total volatile oil and the content of each component were calculated.

[0062] The results are as follows Figure 4 As shown, compared with the wild type, the total volatile oil content of the transgenic plants was significantly increased, with the most significant increases in menthone and limonene content.

[0063] 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 transcription factor gene, McHD-Zip1, the full-length coding sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the peppermint transcription factor gene McHD-Zip1 as described in claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. Primers for amplifying the peppermint transcription factor gene McHD-Zip1 as described in claim 1, characterized in that, The primer sequences are shown below: McHD-Zip1-F: 5'-ATGTATGGAGATTGTCAGGTGC-3', McHD-Zip1-R: 5'-TTAAGCATCAACAGCTGCCT-3'.

4. A vector containing the peppermint transcription factor gene McHD-Zip1 as described in claim 1.

5. A method for constructing the carrier according to claim 4, characterized in that, Includes the following steps: 1) The overexpression vector was double-digested with BamHI and SalI and the linearized vector was recovered; 2) Design primers for the McHD-Zip1 overexpression vector, introduce the BamHI restriction sequence into the upstream primer and the SalI restriction sequence into the downstream primer; 3) The mint transcription factor gene McHD-Zip1 was ligated to the linearized vector using homologous recombination primers for the McHD-Zip1 overexpression vector.

6. The application according to claim 5, characterized in that, The primer sequences for the McHD-Zip1 overexpression vector are shown below: McHD-Zip1-OE-F: 5'-GGGTACCCGGGGATCCATGTATGGAGATTGTCAGGTGC-3', McHD-Zip1-OE-R: 5'-ATTCCTGCAGGTCGACTTAAGCATCAACAGCTGCCT-3'.

7. The application of the peppermint transcription factor gene McHD-Zip1 as described in claim 1 in regulating the development of glandular trichomes in peppermint leaves.

8. The application of the peppermint transcription factor gene McHD-Zip1 as described in claim 1 in regulating the total volatile oil content in peppermint.

9. The application of the peppermint transcription factor gene McHD-Zip1 as described in claim 1 in regulating the content of menthone in peppermint.

10. The application of the peppermint transcription factor gene McHD-Zip1 as described in claim 1 in regulating the limonene content in peppermint.

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