A pimarerene reductase gene promoter and its use in driving specific expression of a gene of interest in the glandular head of a plant trichome

Abstract

The application discloses a pulegone reductase gene promoter and application thereof in driving specific expression of a target gene in a plant glandular hair gland head, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the promoter is shown as SEQ ID NO. 1. The promoter is cloned from mint leaves and constructed into a plant expression vector pMcPR::GUS. GUS staining results of transgenic tobacco show that the promoter can drive specific expression of a downstream gene in long-stalk glandular hair gland head cells of tobacco leaves, stems, petals, sepals and anther stalks, and hardly expresses in gland stalks and other tissues. The promoter provides an important tool for research on glandular hair development regulation, synthesis of secondary metabolites in the glandular hair, functions of genes related to the secondary metabolites and transcriptional regulation of the secondary metabolism.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, it relates to a menthol reductase gene promoter and its application in driving the specific expression of the target gene in plant glandular heads. Background Technology

[0002] Menthone, with the molecular formula C 10 H 18 O,2-Isopropyl-5-methylcyclohexanone is a natural cyclic terpene ketone compound found in peppermint and other mint species, possessing antibacterial, anti-inflammatory, and antioxidant pharmacological effects. Menthone is an upstream volatile substance in menthol metabolism. It is formed by the condensation of isopentenyl pyrophosphate (IPP) and dimethylbenzyl pyrophosphate (DMAPP) under the catalysis of geranyl pyrophosphate synthase (GPPS), generating GPP, the only precursor in monoterpene synthesis. GPP is then catalyzed by monoterpene synthase (mTPS) to form various monoterpene skeletal structures. The final skeletal structure undergoes structural modifications such as methylation and isomerization to form the final structure. Menthone reductase is the final step in menthone synthesis. Menthone reductase genes have been isolated and identified from various plants, but the promoter sequence of menthone reductase has not yet been reported.

[0003] Tissue-specific promoters are a class of promoters that drive gene expression only in specific tissues, organs, or cell types, while exhibiting very low activity or complete silence in other tissues. They are core components of spatiotemporal gene expression regulation and important tools for plant genetic improvement and functional research. By utilizing glandular trichome-specific promoters to drive gene expression, the accumulation of target products in glandular trichomes can be directionally increased without affecting the development of other plant tissues, achieving precise regulation of essential oil quality. The glandular trichome tissue structure can be divided into basal cells, stalk cells, and glandular heads, each with completely different functions. The glandular cells that synthesize secondary metabolites constitute the glandular head. However, most glandular trichome-specific promoters drive the expression of target proteins throughout the glandular trichome, not just within glandular cells. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a promoter for the menthone reductase gene. Another problem to be solved by the present invention is to provide the application of the aforementioned menthone reductase gene promoter.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A promoter for a menthone reductase gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Expression cassettes containing the promoter, recombinant vectors, transgenic cell lines, recombinant bacteria, or recombinant viruses.

[0008] In some embodiments, the expression cassette consists of the promoter, the target gene transcribed by the promoter, and a terminator.

[0009] In some embodiments, the recombinant bacteria is Agrobacterium.

[0010] In some embodiments, the recombinant vector is a recombinant plant expression vector containing a target gene transcribed by the promoter.

[0011] In some embodiments, the target gene is a GUS reporter gene.

[0012] The application of the described menthol reductase gene promoter in driving the specific expression of the target gene in the glandular heads of plant trichomes.

[0013] The application includes the following steps: operatively linking the target gene downstream of the promoter described in claim 1 to construct a recombinant plant expression vector, then introducing the recombinant plant expression vector into plant cells, tissues or organs, and then cultivating the transformed plant cells, tissues or organs into plants to obtain transgenic plants.

[0014] In some embodiments, the plant is a plant with glandular hairs.

[0015] In some embodiments, the plant is a plant of the Lamiaceae family or the Solanaceae family.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) This invention clones and provides the promoter sequence of the menthone reductase gene for the first time, filling the gap in the promoter sequence of this gene.

[0018] (2) The promoter recombinant expression vector constructed in this invention can drive the downstream gene to be specifically and efficiently expressed in the glandular head of the secretory long-stalked glandular hair in the leaves of transgenic tobacco, while it is hardly expressed in the glandular stalk, basal cells and other tissues or developmental stages of the plant, showing extremely high tissue specificity.

[0019] (3) The promoter is derived from peppermint. Its specific expression in the glandular hairs of tobacco leaves indicates that the promoter has a conserved regulatory function across species and can be used to target the expression of the target gene in peppermint and other plants with glandular hairs.

[0020] (4) This invention provides an important tool for gene function research and metabolite production using the plant glandular trichome system, and has broad application prospects in the field of plant metabolic engineering. Attached Figure Description

[0021] Figure 1 This is an electrophoresis image of the PCR amplification of the menthone reductase promoter, where the lane bands represent the McPR promoter.

[0022] Figure 2 Schematic diagram of the cis-acting element of the McPR promoter of menthone reductase;

[0023] Figure 3 This is a PCR identification diagram of transgenic tobacco (lanes 2-6 show bands indicating positive plants, lane 1 shows wild-type plants without bands, M: DL2000 molecular weight standard).

[0024] Figure 4 GUS staining images of young leaves of pMcPR::GUS transgenic tobacco (A), long-stalked glandular hairs of stem (B), long-stalked glandular hairs of tobacco leaves (C), glandular hairs of tobacco petals (D), glandular hairs of tobacco calyx (E), and glandular hairs of tobacco calyx stalk (F).

[0025] Figure 5 The image shows the specific expression of the glandular heads in the long-stalked glandular hairs of tobacco leaves (red arrow) and the absence of color on the stalked cells of the glandular hairs (blue arrow). Detailed Implementation

[0026] 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 specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The plant material used in this invention is *Mentha canadensis* Linn., a plant of the genus *Mentha* in the family Lamiaceae, cultivated in the nursery of the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province. The tobacco variety used in this invention is "K326," cultivated in the greenhouse of the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province.

[0027] Example 1

[0028] 1. Extracting peppermint genomic DNA

[0029] Place approximately 300 mg of fresh peppermint leaves into a 1.5 mL centrifuge tube, along with 3 stainless steel beads (2-3 mm in diameter). Place the centrifuge tube symmetrically into a tissue homogenizer adapter and perform vertical homogenization at a frequency of 50-60 Hz for 90-120 seconds. Immediately add 600 μL of 2×CTAB (hexadecyltrimethylammonium bromide) nucleic acid extraction buffer. Invert or vortex for 5-10 seconds to thoroughly mix the powder with the buffer, ensuring all tissue fragments are wetted by the lysis buffer. Incubate the centrifuge tube in a 65°C water bath for 1-1.5 hours, then cool to room temperature. Add an equal volume (600 μL) of chloroform-isoamyl alcohol (24:1 volume ratio) to the cooled homogenate. Tightly cap the tube and vortex vigorously for 15-30 seconds until a uniform milky white emulsion forms, ensuring complete separation of proteins and nucleic acids. Then, incubate the centrifuge tube at 4°C for 10-15 minutes. Centrifuge at 12000 rpm for 10 min, discard the supernatant; vortex to mix, incubate at 4℃ for 30 min, centrifuge at 4℃ and 12000 rpm for 10-12 min to obtain the supernatant; add 1 / 10 volume of 3M NaAc (pH 4.8) and an equal volume of isopropanol (pre-cooled at 4℃), invert to mix; centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant; rinse with 600 μL of 70% ethanol (pre-cooled at 4℃), centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant, invert the centrifuge tube on a spread paper towel, and after a few minutes, upright the centrifuge tube to air dry at room temperature; dissolve in 40-100 μL of 1×TE buffer or ddH2O and store at -20℃ for later use.

[0030] 2. Amplification of the Menthol Reductase McPR Promoter

[0031] Based on the previously obtained McPR sequence of the menthol reductase gene (Genebank ID MT447842.1) in our laboratory, a search was conducted in the Mentha Root Genome Database (http: / / langelabtools.wsu.edu / mgr / ) to find the homologous gene. Primers for amplifying the McPR promoter of the menthol reductase gene were designed based on the upstream promoter sequence of this gene. The primer sequences are shown below:

[0032] pMcPR-F: 5'-TAATGCTACTTTGCCTATTGC-3' (SEQ ID NO. 2);

[0033] pMcPR-R: 5'-GGATATCGAGTTTTCCATGCG-3' (SEQ ID NO. 3).

[0034] Using peppermint genomic DNA as a template, the promoter of the peppermint reductase gene McPR was amplified by PCR using pMcPR-F and pMcPR-R. The PCR reaction system followed the instructions of Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing). The PCR program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, for 35 cycles; 72℃ extension for 5 min.

[0035] The PCR products were subjected to agarose gel electrophoresis. Figure 1 The sequence was then sequenced, and the nucleotide sequence of the promoter of the menthone reductase gene McPR was obtained as shown in SEQ ID NO.1.

[0036] Core functional element analysis was performed on the promoter sequence of the menthone reductase gene McPR obtained by sequencing. This promoter contains the basic conserved elements of eukaryotic promoters, namely the CAAT-box and TATA-box, as well as transcription factor binding sites and light-responsive elements. Figure 2 ).

[0037] The PCR product was ligated into the pCE2-TAvector vector from Vazyme to obtain the proMcPR-pCE2-TA recombinant plasmid containing the McPR promoter sequence of the menthone reductase gene.

[0038] Example 2

[0039] 1. Vector double enzyme digestion

[0040] The pGC-GUS(G5) vector was double-digested using KpnI enzyme. The resulting double-digested vector was subjected to agarose gel electrophoresis and then recovered using a kit.

[0041] 2. Construction of expression vectors containing the GUS gene

[0042] Design primers to introduce the restriction enzyme site KpnⅠ. The primer sequences are shown below:

[0043] pMcPR-G5-F:

[0044] 5'-CGCGTTGGGAGTCCCTCGAGGGTACCTAATGCTACTTTGCCTATTGC-3' (SEQ ID NO. 4);

[0045] pMcPR-G5-R:

[0046] 5'-ACTCATTCTAGAGAATTCAAGCTTGGGTACCTCTGAGAACTATGTGTTG-3' (SEQ ID NO. 5);

[0047] PCR amplification was performed using the recombinant plasmid pMcPR-pCE2-TA as a cloning template. The system is shown in Table 1 below:

[0048] Table 1 Amplification System

[0049]

[0050] The PCR product was transformed into E. coli, and the specific steps are as follows:

[0051] 100 μL of competent Escherichia coli DH5α cells (TSINGKE, Beijing) were thawed on ice, and the PCR product was added. The cells were then incubated on ice for 30 min. After heat shock in a 42°C water bath for 90 s, the cells were quickly transferred to ice and placed for 2 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were incubated at 200 rpm in a shaker at 37°C for 1 h. After centrifugation at 5000 g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid medium containing 50 mg / mL Kanabolic acid. The plate was spread evenly and inverted in a 37°C incubator for 12-16 h. 100 μL of Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd., commercial strain) were removed from the -80℃ freezer and slowly thawed on ice. Once the cells were completely thawed, the PCR product was added immediately, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 90 s and quickly transferred to ice for 2 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were cultured at 200 rpm in a shaker at 37℃ for 1 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid medium containing 50 mg / mL Kana antibiotic. The plate was spread evenly and inverted in a 37℃ incubator for 12-16 h.

[0052] Single clones were selected for bacterial culture PCR detection using primers (5'-GAATTATTATTCATGTCA-3' (SEQ ID NO.6)) and (5'-AAGGAAAGATTAATCATG-3' (SEQ ID NO.7)). Positive clones were identified as expression vectors containing the GUS gene, pMcPR::GUS.

[0053] 3. Transformation of Agrobacterium

[0054] The obtained peppermint McPR promoter plant expression vector pMcPR::GUS was transformed into Agrobacterium. The specific steps are as follows: 100 μL of competent Agrobacterium cells EHA105 (TSINGKE, Beijing) were thawed on ice, 5 μL of recombinant plasmid was added, and the cells were incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and on ice for 5 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were cultured at 200 rpm and 28℃ on a shaker for 2 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving about 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid medium containing the specified antibiotic. The plate was spread evenly and incubated upside down in a 37℃ incubator for 2-3 days. Single colonies were picked for PCR identification.

[0055] 4. Tobacco Conversion Method

[0056] Take an appropriate amount of mature tobacco K326 seeds and pour them into a 1.5 mL centrifuge tube. Add 1 mL of 75% ethanol and soak for 1 min. Use a pipette to remove the 75% ethanol, avoiding the seeds, and wash the seeds once with sterile water. Add 1 mL of 2% sodium hypochlorite and soak for 10 min, then wash three times with sterile water. Transfer the seeds to MS solid medium, about 30 seeds per plate. Seal the plates and culture them in a culture room at 25℃, 16 h light / 8 h dark, and 1500 Lux light intensity for about 1 week until the tobacco seeds germinate. Transfer the germinated tobacco seedlings to tissue culture bottles containing MS solid medium and culture for about 3 weeks. They can be used for transformation. Pick Agrobacterium containing recombinant plasmids in a clean bench and transfer them to LB liquid medium containing 50 mg / L LRFP and 100 mg / L Spec. Culture overnight in a shaker at 200 rpm and 28℃. Transfer 1 mL of the overnight cultured Agrobacterium to 25 mL of medium containing the same antibiotics. In LB liquid medium, incubate at 200 rpm and 28°C on a shaker for 3-4 hours until OD reaches zero. 600 The concentration was 0.4-0.6; after centrifugation at 5000g for 5 min, Agrobacterium cells were collected and resuspended in MS salt infection medium containing 100 μM / s. The final OD value was calculated. 600The concentration of the bacterial culture medium was 0.4-0.6. The medium was placed at room temperature in the dark for 2-4 hours. Healthy tobacco leaves were taken, and the leaf margins and veins were removed in a clean bench and the leaves were cut into small pieces. These pieces were then placed in the infection solution and shaken at 200 rpm and 28°C for 15-20 minutes. Excess bacterial culture solution was absorbed with absorbent paper, and the leaves were placed on a tobacco co-culture medium with filter paper on the surface. The leaves were co-cultured at 25°C in the dark for 4-6 days. After 4-6 days of co-culture, the leaves were transferred to a tobacco selection and differentiation medium containing 500 mg / L Cef and 100 mg / L Spec. The medium was cultured at 25°C in a culture chamber with 16 hours of light / 8 hours of darkness and a light intensity of 1500 Lux until buds differentiated. The buds were then cut off and placed on a selection and rooting medium containing the same 500 mg / L Cef and 100 mg / L Spec, and allowed to root.

[0057] The results are as follows Figure 3 As shown, PCR was used to identify the transgenic positive plants, and five pMcPR::GUS transgenic tobacco lines were successfully obtained.

[0058] 5. GUS expression activity in transgenic positive plants

[0059] Wild-type tobacco was used as the negative control, and tobacco with the 35S promoter was used as the positive control. GUS staining was performed on various tissue parts of the peppermint McPR promoter transgenic tobacco using a GUS staining kit (Coolaber, Beijing). The steps were as follows: Tissue parts of the McPR promoter transgenic tobacco and the negative control wild-type tobacco were immersed in GUS staining solution and placed overnight in a 37 ℃ incubator in the dark. Except for the parts without chlorophyll, the remaining samples were destained in 75% ethanol 2-3 times until the negative control material turned white. The presence of blue areas was observed under a stereomicroscope, and the blue areas indicated GUS gene expression.

[0060] The results are as follows Figure 4 , 5 As shown, the GUS gene is specifically expressed only in the glandular head cells of the long-stalked glandular hairs of tobacco leaves, and does not show color in other leaf tissues or glandular hair stalk cells, exhibiting high tissue specificity.

[0061] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A promoter for a menthone reductase gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An expression cassette, recombinant vector, recombinant bacteria, or recombinant virus containing the promoter of claim 1.

3. The expression box according to claim 2, characterized in that, The expression cassette consists of the promoter as described in claim 1, the target gene transcribed by the promoter, and a terminator.

4. The recombinant bacteria according to claim 2, characterized in that, The recombinant bacteria is Agrobacterium.

5. The recombinant vector according to claim 2, characterized in that, The recombinant vector is a recombinant plant expression vector.

6. The recombinant vector according to claim 5, characterized in that, The target gene is the GUS reporter gene.

7. The application of the menthone reductase gene promoter according to claim 1 in driving the specific expression of the target gene in the glandular heads of plant trichomes, characterized in that, The plant in question is either mint or tobacco.

8. The application according to claim 7, characterized in that, Includes the following steps: The target gene is operatively linked downstream of the promoter described in claim 1 to construct a recombinant plant expression vector. The recombinant plant expression vector is then introduced into plant cells, tissues, or organs, and the transformed plant cells, tissues, or organs are then cultured into plants to obtain transgenic plants.

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