Peppermint transcription factor McMYC2 and its application in regulating essential oil synthesis

Abstract

The present application belongs to the technical field of genetic engineering breeding, and discloses a mint transcription factor McMYC2 and application thereof in regulating synthesis of essential oil. A mint MYC2 transcription factor, namely McMYC2, is screened, and a coding gene (namely McMYC2 gene) of the McMYC2 transcription factor is overexpressed in a mint plant. The transcription factor regulates transcription levels of relevant genes such as McGPPS-LSU and McGPPS-SSU in a mint essential oil synthesis pathway, and thus plays a role in regulating synthesis of mint essential oil. It is clarified that the McMYC2 has a function of regulating synthesis of mint essential oil, and valuable gene resources are provided for breeding of a new mint variety with high essential oil content, and the present application has important application value in improvement of the mint.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering breeding technology and relates to the peppermint transcription factor McMYC2 and its application in regulating essential oil synthesis. Background Technology

[0002] Peppermint (Mentha canadensis L.) is a perennial herb belonging to the genus Mentha in the family Lamiaceae. It is an important spice plant and a commonly used traditional Chinese medicine. The main medicinal components of peppermint are volatile monoterpenoids from its aerial parts, commonly known as "peppermint essential oil," which possesses antibacterial, antioxidant, anti-inflammatory, analgesic, antitumor, and central nervous system stimulant activities. It is currently widely used in medicine, food, fragrances, and cosmetics. In addition, peppermint essential oil has insecticidal and bactericidal activities, making it a plant-derived "green pesticide" for pest control and biological defense in storage, playing an important ecological role in reducing the use of harmful pesticides and promoting green agricultural production. Therefore, essential oil content is a key indicator for evaluating the quality and industrial value of peppermint.

[0003] The activity of peppermint essential oil-related synthases is crucial for the synthesis and accumulation of peppermint essential oil. These synthases are regulated by various transcription factors (TFs) or other genes. Identifying transcription factors related to the regulation of secondary metabolite synthesis in medicinal plants and elucidating their regulatory mechanisms is of great significance for future genetic engineering improvements of medicinal plants using transcription factors.

[0004] However, research on the regulatory genes of peppermint essential oil synthesis is relatively limited, with only a few reports in the genus *Mentha*. Furthermore, the molecular mechanisms by which transcription factors regulate essential oil synthesis are still unclear, and very few transcription factors positively regulating essential oil synthesis have been identified in peppermint. This significantly restricts the progress of molecular breeding in peppermint. Summary of the Invention

[0005] In order to cultivate new peppermint varieties with high quality and strong adaptability and increase the content of peppermint essential oil, this invention uses genetic engineering to clarify the important function of peppermint transcription factor McMYC2 in essential oil synthesis.

[0006] First, this invention seeks protection for a peppermint transcription factor McMYC2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Secondly, the present invention seeks protection for the encoding gene of the above-mentioned peppermint transcription factor McMYC2, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] Thirdly, the present invention provides the application of the above-mentioned peppermint transcription factor McMYC2: the peppermint transcription factor McMYC2 is used to regulate the transcription level of related genes in the peppermint essential oil synthesis pathway, thereby increasing the content of peppermint essential oil.

[0009] Specifically, in the above applications, the relevant genes include: McGPPS-LSU, McGPPS-SSU, McLS, and McPR.

[0010] Fourthly, the present invention provides a recombinant vector comprising the above-mentioned coding gene.

[0011] Fifthly, the present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium comprises the above-mentioned recombinant vector.

[0012] In a sixth aspect, the present invention provides a method for cultivating peppermint varieties that promote essential oil synthesis, comprising: overexpressing the McMYC2 gene in peppermint plants; the nucleotide sequence of the McMYC2 gene is shown in SEQ ID NO:2.

[0013] Compared with existing technologies, the present invention, "Peppermint Transcription Factor McMYC2 and Its Application in Regulating Essential Oil Synthesis," has the following beneficial effects:

[0014] This invention screened out a peppermint MYC2 transcription factor (i.e., McMYC2), and overexpressed the gene encoding the McMYC2 transcription factor (i.e., the McMYC2 gene) in peppermint plants. This transcription factor regulates the transcription level of related genes such as McGPS-LSU, McGPS-SSU, McLS, and McPR in the peppermint essential oil synthesis pathway, thereby regulating the synthesis of peppermint essential oil.

[0015] This invention clarifies that McMYC2 has the function of regulating essential oil synthesis, providing valuable genetic resources for cultivating new peppermint varieties with high essential oil content, and has important application value in peppermint improvement. Attached Figure Description

[0016] Figure 1 This is a clone of the peppermint McMYC2 gene; the red arrow indicates the target gene band.

[0017] Figure 2 Phylogenetic analysis of the peppermint McMYC2 protein with MYC2 proteins from other species.

[0018] Figure 3 Analysis of the expression of the peppermint McMYC2 gene in different tissue sites.

[0019] Figure 4This study analyzed the expression of the mint McMYC2 gene in different leaf arrangements. L1–L8 represent the 1st to 8th leaves from top to bottom in terms of morphology.

[0020] Figure 5 Expression patterns of the McMYC2 gene in peppermint leaves (A) and roots (B) under MeJA treatment.

[0021] Figure 6 This study analyzed the co-expression of the McMYC2 gene and the essential oil synthase gene under MeJA treatment. CK1 and CK2 served as control groups; Mock represented the expression of McMYC2 at different time points under 0.1% ethanol aqueous solution treatment.

[0022] Figure 7 Subcellular localization analysis of McMYC2 and AtMYC2 proteins. Figure 7 In this context, A represents the expression of McMYC2 and AtMYC2 in tobacco leaves; Figure 7 In this context, B represents McMYC2 expression in peppermint leaves. GFP: Green fluorescent protein; mCherry: Red fluorescent protein.

[0023] Figure 8 Analysis of transcriptional autoactivation and transcriptional activation functional regions of peppermint McMYC2. Figure 8 A in the diagram represents a vector containing the full-length McMYC2 gene sequence and some of its domains. Figure 8 In B, the growth of yeast AH109 after transformation by each vector in A is shown on the auxotrophic media SD / -Trp, SD / -Trp / -His, and SD / -Trp / -His / -Ade.

[0024] Figure 9 This is a schematic diagram of the specific primer activation sites during transient overexpression of McMYC2.

[0025] Figure 10 The expression of McMYC2-GFP was detected by RT-PCR. VC served as the empty vector (pCAMBIA2300-GFP) control.

[0026] Figure 11 This shows the transient overexpression of the McMYC2 gene in leaves. VC is the empty vector (pCAMBIA2300-GFP) control.

[0027] Figure 12 The expression of the McGPS-LSU, McGPS-SSU, McLS, and McPR genes in leaves is shown. VC is the empty vector control.

[0028] Figure 13The integration status of the McMYC2 gene was detected using genomic-PCR technology.

[0029] Figure 14 The expression level of McMYC2 in transgenic peppermint. WT represents wild type.

[0030] Figure 15 Plant height, leaf fresh weight, leaf length, leaf area, and leaf width were measured for WT peppermint and genetically modified peppermint.

[0031] Figure 16 Microscopic observation of secretory glandular trichomes in WT peppermint and transgenic peppermint.

[0032] Figure 17 Statistical analysis of secretory glandular trichomes in WT peppermint and transgenic peppermint. Detailed Implementation

[0033] The present invention will now be described in conjunction with embodiments, providing a clear and complete description of the technical solutions in these embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] The peppermint (M. canadensis L.) and peppermint (Mentrapiperita) used in the experiment were grown in an artificial climate greenhouse under 16 hours of light / 8 hours of darkness. The tobacco used in the experiment was Nicotiana benthamiana, grown in a constant temperature and light incubator, and grown for 4-5 weeks under normal conditions before use.

[0035] Example 1

[0036] This embodiment provides screening for the peppermint McMYC2 gene.

[0037] Studies have shown that MYC2, a core bHLH transcription factor in the JA signaling pathway, plays a crucial role in regulating plant secondary metabolism. Therefore, this invention first screened peppermint bHLH genes. Using GO annotation of previous peppermint MeJA-treated transcriptome data and Blast alignment of local homologous proteins with Arabidopsis bHLH family members, a total of 123 bHLH transcription factor unigenes were screened. The RPKM values ​​of these bHLH transcription factors under normal conditions (CK) and MeJA treatment were obtained using transcriptome data. The Log2(MeJA / CK) value of the RPKM was then calculated. The results showed that 61 bHLH genes were upregulated under MeJA induction, while 62 were downregulated.

[0038] Subsequently, four MYCs-like genes that were significantly upregulated by jasmonic acid were identified from the upregulated bHLH transcription factors, with Unigene0042863 showing the most significant upregulation. Based on the expresssequence tag (EST) sequences obtained from transcriptome sequencing, partial cDNA sequences of the above four MYCs genes were spliced ​​together. Further annotation and sequence analysis using the NR (non-redundant protein) database showed that Unigene0042863 is homologous to MYC2, a MYCs gene involved in the regulation of secondary metabolism in plants; therefore, it was named the McMYC2 gene.

[0039] Example 2

[0040] This embodiment provides a clone of the peppermint McMYC2 gene.

[0041] 1. Total RNA extraction and first-strand cDNA synthesis from peppermint

[0042] Two-week-old young mint leaves were collected under normal culture conditions, frozen in liquid nitrogen, and ground into powder. Total RNA was extracted from the leaves according to the steps described in the Tiangen RNAprepPure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441). The RNA integrity was assessed by 1% agarose gel electrophoresis, and the RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer. The first strand of cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNAwiper) (R212, vazyme) according to the manufacturer's instructions. The reaction system and RT-PCR reaction procedure are shown in Table 1.

[0043] Table 1. Reverse transcription reaction system and RT-PCR reaction procedure

[0044] Components Dosage 4×gDNAwiperMix 4μL Oligo(dT)23VN(50μM) 1μL Randomhexamers (50 ng / μl) 1μL TotalRNA 1μg <![CDATA[RNasefreeddH2O]]> To 16μL 42℃, 2min / 10×RTMix 2μL HiScriptIIEnzymeMix 2μL 50℃, 15min; 85℃, 15s /

[0045] 2. McMYC2 gene amplification and DNA purification

[0046] Based on the McMYC2 gene sequence obtained above, primers for full-length amplification of the McMYC2 gene were designed using DNAMAN software: McMYC2-F: 5'-ATGATTGATTACCGTCTGCCG-3' (SEQ ID NO:3); McMYC2-R: 5'-CTAGTTTATTTCAGCAGCAAGTC-3' (SEQ ID NO:4).

[0047] The McMYC2 gene was amplified using peppermint leaf cDNA or DNA as a template. The PCR amplification system is shown in Table 2, and the amplification program is shown in Table 3. The PCR products were identified and separated by 1% agarose gel electrophoresis. The PCR products of the McMYC2 gene were recovered using a Biomed DNA gel recovery kit (DH103-01) according to the instructions. The concentration of the PCR products was determined using a Nanodrop 2000 spectrophotometer for later use.

[0048] Table 2. PCR amplification system for McMYC2 gene

[0049]

[0050]

[0051] Table 3. PCR amplification program for the McMYC2 gene

[0052]

[0053] The PCR product was sequenced and aligned to obtain the correct CDS sequence. Sequence analysis showed that the full-length McMYC2 gene is 1854 bp (nucleotide sequence as shown in SEQ ID NO:2), encoding 617 amino acids (amino acid sequence as shown in SEQ ID NO:1), with a theoretical molecular weight of 67.728 kDa and a theoretical isoelectric point of 5.64. The McMYC2 gene DNA sequence was subsequently cloned by PCR. Figure 1 (As indicated by the arrow). Post-sequencing structural analysis showed that the McMYC2 gene lacks intron structure, which is similar to the structure of the MYC2 genes in Arabidopsis thaliana, Artemisia annua, and Rubber tree.

[0054] 3. Ligation of the McMYC2 gene with the vector, transformation, and identification of recombinant bacteria

[0055] Following the steps described in the pClone007 BluntVectorKit (TSV-007B) gene cloning kit from Beijing Qingke Biotechnology Co., Ltd., the PCR product of the McMYC2 gene was ligated into the intermediate vector pClone007 BluntVector. The reaction system is shown in Table 4.

[0056] Table 4. McMYC2 gene cloning reaction system

[0057] Components Dosage Target fragment 1~8μL pClone007BluntVector 1μL 10×Topo 1μL <![CDATA[ddH2O]]> up to 10 μL

[0058] The above reaction system was placed in a metal bath at 25°C for 15 min. After the reaction, the reaction solution was transformed into *E. coli* DH5α competent cells. The steps are as follows: Take 50 μL of *E. coli* DH5α competent cells thawed on ice, add 10 μL of the reaction product, mix gently, and incubate on ice for 30 min; heat shock in a 42°C water bath for 45–60 s, quickly transfer to an ice bath, and incubate for 2 min; add 200 μL of antibiotic-free LB medium to a centrifuge tube, mix well, and place in a 37°C shaker at 200 rpm for 1 h; evenly spread 200 μL of the recovery solution onto an LB medium plate containing Amp resistance, and incubate the plate upside down in a 37°C incubator overnight. Single colonies were picked and cultured overnight in LB liquid medium containing 50 mg / mL Amp. Recombinant cells were identified by bacterial PCR, and positive bacterial cultures containing the target fragment were sequenced (Beijing Qingke Biotechnology Co., Ltd.), confirming successful transformation of the McMYC2 gene sequence. After mixing the positive bacterial solution with 50% glycerol at a volume ratio of 1:1, store at -20℃ for later use.

[0059] Example 3

[0060] This embodiment provides a bioinformatics analysis of the peppermint McMYC2 gene.

[0061] 1. McMYC2 protein structure analysis

[0062] To gain a preliminary understanding of the basic structure of the McMYC2 protein, this invention analyzed its secondary structure using the SOPMA online tool. The results showed that the protein contains classic secondary structures such as α-helices, β-sheets, extended strands, and random coils, accounting for 36.47%, 1.94%, 15.72%, and 45.87% of the total protein, respectively. The tertiary structure of the McMYC2 protein was predicted using the SWISS-MODEL online tool. The prediction model showed a Global Model Quality Estimate (GMQE) of 0.61, indicating a certain degree of reliability in the prediction results. The entire protein model mainly consists of extended strands and random coils, with α-helices and β-sheets distributed on both sides of the model, and the β-sheets being wrapped by α-helices.

[0063] 2. Analysis of conserved domains of the McMYC2 protein

[0064] To clarify the functional similarities and differences between the peppermint McMYC2 protein and MYC2 proteins from other species, this invention systematically analyzed its amino acid sequence and evolution. Amino acid sequence alignment revealed that the McMYC2 protein shares certain similarities with MYC2 proteins from other species (Artemisia annua, Aquilaria sinensis, Arabidopsis thaliana, Vinca minor, and Salvia miltiorrhiza), all possessing highly conserved JID (JAZ Interaction Domain), transcriptional activation domain (AD), HLH domain, and nuclear localization signaling (NLS). Studies showed that the JID domain is responsible for interaction with the JA signaling repressor JAZ protein, while the transcriptional activation domain is responsible for regulating its downstream target genes, and the NLS domain is responsible for its localization to the cell nucleus. This suggests that the McMYC2 protein is highly likely to regulate the peppermint essential oil synthase gene in the cell nucleus in response to JA signaling.

[0065] 3. McMYC2 protein phylogenetic analysis

[0066] Download the following species from the NCBI online website: Arabidopsis thaliana, Hedysarum brasiliensis, Vitis vinifera, P. trifoliata, M. domestica, Aquilaria sinensis, Tripterygium wilfordii, M. truncatula, Artemisia annua, T. cinerariifolium, G. rigescens, and Gracilaria stenoptera. The amino acid sequences of MYC2 proteins from various plants, including tobacco (N. attenuata), tobacco (N. tabacum), night sorrel (W. somnifera), jalapeno (C. annuum), potato (S. tuberosum), tomato (S. lycopersicum; L. esculentum), salvia (S. miltiorrhiza), peppermint (M. longifolia), rice (O. sativa), freesia (F. hybrida), dendrobium officinale, lily (Lilium hybrid 'Sorbonne'), Chinese cabbage (B. rapa), yew (T. cuspidata), and yew (T. chinensis), were obtained. Considering that peppermint is an allopolyploid, the amino acid sequence of the McMYC2 protein was used to perform local amino acid alignment of the three generations of peppermint transcriptome data, resulting in a candidate gene with high homology to McMYC2, named McMYC2-like. Subsequently, a phylogenetic tree of the MYC2 gene among the aforementioned different species was constructed using MEGA 5.0 software. The results showed that McMYC2 clustered with the diploid peppermint MlMYC2, exhibiting the highest homology and closest phylogenetic relationship, followed by *Salvia miltiorrhiza*, also belonging to the Lamiaceae family. Figure 2 Furthermore, research revealed that the MYC2 protein is involved in regulating the synthesis of secondary metabolites in various plants, including Arabidopsis thaliana, further suggesting that McMYC2 may serve as a core transcription factor in the peppermint JA signaling pathway and has a potential role in regulating peppermint essential oil synthesis.

[0067] Example 4

[0068] This embodiment provides an analysis of the expression characteristics of the peppermint McMYC2 gene.

[0069] 1. Analysis of McMYC2 gene expression in peppermint tissue and foliage

[0070] Two-month-old mint plants with uniform growth were selected, and samples were taken from different tissues, including roots, stems, leaves, flowers, buds, and stem tips. At the same time, five-week-old mint plants with uniform growth were selected, and samples were taken from different leaf indices (morphologically, the 1st to 8th leaves from top to bottom (marked as L1 to L8)). Each group of samples was replicated in triplicate, and after being flash-frozen in liquid nitrogen, they were stored in an ultra-low temperature freezer at -80°C.

[0071] qRT-PCR primers (qMcMYC2-F and qMcMYC2-R, see Table 5) were designed based on the McMYC2 gene sequence to analyze McMYC2 gene expression in different tissues and leaf orders, using β-actin as an internal reference gene. Quantitative PCR was performed using a BIO-RAD qPCR instrument. The qRT-PCR reaction system is shown in Table 6, and the qRT-PCR program is shown in Table 7. Each sample was tested in triplicate. -ΔΔCT The relative mRNA expression level of the McMYC2 gene was calculated using a method, and significance analysis was performed using IBM SPSS Statistics 26 software.

[0072] Table 5. qRT-PCR specific primer design

[0073]

[0074]

[0075] Table 6. qRT-PCR reaction system

[0076] Components Dosage 2×SYBRGreenProTaqHSPremix 5μL F primer 0.5μL R primer 0.5μL cDNA 2μL <![CDATA[ddH2O]]> up to 10 μL

[0077] Table 7. qRT-PCR program

[0078]

[0079] The expression of the McMYC2 gene in different tissues of mint is shown in the figure. Figure 3 , Figure 3 The results showed that McMYC2 was expressed in different tissues of mint, with relatively high expression levels in leaves, the main site of mint essential oil synthesis, and even higher expression levels in older leaves than in young leaves. Expression was also observed in flowers and buds, with relatively low levels in roots, stems, and stem tips. This indicates that McMYC2 is a widely expressed gene in mint, and its function may differ in different tissues.

[0080] The expression of the McMYC2 gene in different leaf arrangements of mint is shown in the figure. Figure 4 , Figure 4The results showed that the expression of the McMYC2 gene fluctuated in different leaves as the leaves aged, generally exhibiting a trend of gradually increasing expression with leaf senescence. These results suggest that McMYC2 may not only be involved in the synthesis of peppermint essential oil but also in the development or senescence process of peppermint leaves.

[0081] 2. Response analysis of peppermint McMYC2 gene and essential oil synthesis gene to MeJA

[0082] Select uniformly growing mint seedlings, harvest the above-ground parts for hydroponics, and when the root system is vigorous (about 3 weeks), select uniformly growing mint plants again, and induction treatment is performed by spraying the leaves with 100 μmol / L MeJA solution, referring to the MeJA treatment method of Ma et al. At 0, 1, 3, 6, 12 and 24 h after treatment, the leaves and roots are sampled separately, flash-frozen in liquid nitrogen at -80℃ and stored for later use. Each treatment is set up with 3 biological replicates.

[0083] Using qRT-PCR primers for the McMYC2 gene, specific qRT-PCR primers for essential oil synthase genes (McGPPS-LSU, McGPPS-SSU, McLS, McL3OH, MciPD, MciPR, McPR, McMMR, McMFS) were designed (see Table 5). The experimental conditions, including the internal reference gene, qRT-PCR reaction system, and procedure, were the same as those in the "Analysis of Mint McMYC2 Gene Tissue and Leaf Arrangement Expression".

[0084] Figure 5 The expression patterns of the McMYC2 gene in peppermint leaves (A) and roots (B) under MeJA treatment were shown. The results indicated that MeJA treatment significantly induced the expression of the McMYC2 gene in both leaves and roots, with a higher expression level in leaves (A) and roots (B). Figure 5 In A), the McMYC2 gene expression peaked 1 hour after MeJA treatment, followed by a slight decrease, a second peak at 12 hours, and then decreased to a level insignificantly different from the control; in root ( Figure 5 In B), the expression of the McMYC2 gene showed a gradually increasing trend, indicating that MeJA activates the expression of peppermint McMYC2 at the transcriptional level.

[0085] Figure 6This study analyzed the co-expression of the McMYC2 gene and essential oil synthase genes under MeJA treatment. CK1 and CK2 served as control groups; Mock represented the expression of McMYC2 at different time points under 0.1% ethanol aqueous solution treatment. Results showed that after 3 hours of MeJA treatment, the expression of peppermint essential oil synthase genes McCPPS-LSU, McL3OH, MciPD, MciPR, McPR, and McMFS gradually increased, reaching its peak at 24 hours. The expression of McCPPS-SSU and McLS reached its highest level at 6 hours of MeJA treatment and maintained a high expression level with increasing treatment time. The expression of McMMR showed a slight increase at 6 and 24 hours. McMYC2 expression was significantly upregulated and reached its peak at 1 hour of MeJA treatment. These results indicate that MeJA treatment rapidly induces McMYC2 expression, and changes in McMYC2 transcriptional levels precede those of essential oil synthase genes. In summary, McMYC2 is an early response gene to MeJA signaling and is co-induced by MeJA treatment along with the peppermint essential oil synthase gene, exhibiting a co-induced expression pattern. It is speculated that McMYC2 may participate in the regulation of essential oil synthesis.

[0086] Example 5

[0087] This embodiment provides a characterization analysis of the McMYC2 protein.

[0088] 1. Subcellular localization of McMYC2 protein

[0089] To further clarify the subcellular localization of McMYC2, this invention constructed the McMYC2 gene (with the stop codon removed) and the Arabidopsis thaliana AtMYC2 gene into the pCAMBIA2300-GFP vector, and performed transient expression via Agrobacterium-mediated transient transformation. The specific procedures are as follows:

[0090] Homologous recombination PCR primers for the McMYC2 and Arabidopsis thaliana AtMYC2 gene sequences were designed (see SEQ ID NO: 27-30 in Table 8). PCR amplification was performed using pClone007-McMYC2 plasmid or Arabidopsis thaliana leaf cDNA as templates. The reaction system consisted of 12.5 μL of 2×ApexHF FS PCR Master Mix, 0.5 μL each of F and R primers, 1 μL of template plasmid / cDNA, and 10.5 μL of ddH2O. The PCR reaction program was as follows: 94℃ for 5 min; 95℃ for 15 s, 50℃ for 20 s, 72℃ for 45 s (1 kb / 5 s), 34 cycles; 72℃ for 10 min, 4℃ for 1 min.

[0091] Table 8. Primers for homologous recombination PCR amplification

[0092]

[0093]

[0094] Table 9. Double Enzyme Digestion System

[0095] Components Dosage <![CDATA[10×Cutone TM buffer]]> 5μL Fast cleavage enzyme 1 5μL Fast cleavage enzyme 2 5μL plasmid 5μg <![CDATA[ddH2O]]> up to 50μL

[0096] Table 10. Homologous recombination reaction systems

[0097] Components Dosage Exnase II 1μL 5×CEIIBuffer 2μL PCR products 5~100ng vector 25~100ng <![CDATA[RNasefreeddH2O]]> up to 10 μL

[0098] Table 11. Subcellular localization infection fluid system

[0099] Components Dosage 0.5MMES 2mL <![CDATA[100mMMgCl2]]> 10mL 150mMAS 75μL <![CDATA[ddH2O]]> up to 50mL

[0100] The pCAMBIA2300-GFP(VC) vector was double-digested with BamHI and SalI, as shown in Table 9. The PCR products and VC vector digestion products were separated by agarose gel electrophoresis and the gels were recovered for later use. Following the instructions of the IIOneStep Cloning Kit (C112, vazyme), homologous recombination was performed. The reaction system is shown in Table 10. The PCR product was ligated into the VC vector via in vitro homologous recombination. The ligation, transformation, and identification methods were the same as in Example 2, and the sequence correctness was confirmed.

[0101] The correct VC vector, nuclear localization vector PHB-SV40-mCherry, and fusion expression vector pCAMB IA2300-McMYC2 / AtMYC2-GFP were transformed into Agrobacterium GV3101 and EHA105 by freeze-thaw method, respectively.

[0102] 1) Subcellular localization in tobacco leaves: Agrobacterium GV3101 carrying the above four plasmids was propagated, centrifuged, and resuspended in infection solution (infection solution system is shown in Table 11). The OD of the resuspended solution was... 600 The value was adjusted to 0.6. Then, the Agrobacterium GV3101 resuspension carrying the fusion expression vector and the VC vector was mixed with the Agrobacterium GV3101 resuspension containing the nuclear localization vector at a 1:1 volume ratio. After standing at room temperature for 1 hour, the mixture was injected into the leaves of 5-week-old Nicotiana benthamiana and placed in a light incubator at 23°C in darkness for 16 hours. After normal culture for 2 days, the green fluorescent protein (GFP) or red fluorescent protein (mCherry) signals were detected using a laser confocal microscope. Each experiment was repeated three times.

[0103] 2) Subcellular localization in peppermint leaves: Agrobacterium EHA105 carrying the fusion expression vector pCAMBIA2300-McMYC2-GFP was propagated, centrifuged, and resuspended in the infection solution (the infection solution system is shown in Table 11). The OD of the resuspended solution was...600 The concentration was adjusted to 0.7. Similarly, Agrobacterium EHA105 resuspension carrying the fusion expression vector and VC vector was mixed with Agrobacterium EHA105 resuspension containing the nuclear localization vector at a 1:1 volume ratio. After standing at room temperature for 1 hour, the mixture was injected into the middle leaves of mint seedlings (about 6 cm tall). After incubation at 23°C in the dark for 16 hours, the seedlings were transferred to normal conditions for 2 days. The fluorescence signal was detected using a laser confocal microscope. Each experimental combination was repeated 3 times.

[0104] Subcellular localization analysis results are shown in Figure 7 . Figure 7 In the figure, A represents the expression of McMYC2 and AtMYC2 in tobacco leaves. The results showed that the green fluorescence signal of the empty vector VC group was widely distributed in the cytoplasm, cell membrane, and nucleus. In tobacco leaves containing the fusion expression vector pCAMBIA2300-McMYC2-GFP, the green fluorescence signal could only be observed in the nucleus, which overlapped with the red fluorescence signal, indicating that McMYC2, like Arabidopsis thaliana AtMYC2, is located in the nucleus of tobacco cells. Figure 7 In the figure, B represents the expression of McMYC2 in peppermint leaves. The results show that, consistent with those in tobacco, McMYC2 is also localized in the nucleus of peppermint leaf cells. In summary, McMYC2 is located in the nucleus and may have a function in regulating the transcriptional levels of downstream genes.

[0105] 2. McMYC2 protein transcriptional autoactivation analysis

[0106] To analyze the transcriptional activation activity and self-activation functional regions of the McMYC2 protein, this invention constructed the full-length McMYC2 gene and truncated nucleic acid sequences encoding various structural regions into the pGBKT7(BD) vector. These were then transformed into yeast AH109, ​​and their growth on the selection medium was observed after 3 days to analyze their transcriptional self-activation ability. Details are as follows:

[0107] Design the complete McMYC2 encoding sequence ( Figure 8 The homologous recombination PCR amplification primers for A (see label 1) and the N-terminus encoding the McMYC2 protein (containing the PRD+JID+AD domain) Figure 8 In the text, A (see label 2) and C (HLH structural domain) Figure 8 A in the text, see note 6), PRD+JID structural field ( Figure 8 A in the text, see note 3), JID+AD structural domain ( Figure 8 A in the text, see label 4), AD structural domain ( Figure 8The homologous recombination PCR amplification primers for gene sequence A (see label 5) (see SEQ ID NO: 31-39 in Table 8) were used for PCR amplification with pClone007-McMYC2 plasmid as template. The pGBKT7(BD) vector was double-digested with NdeI and EcoRI, with the digestion system as shown in Table 9. The PCR amplification product and the vector double-digestion product were separated by gel electrophoresis and purified using the steps described in the Biomed DNA Gel Recovery Kit (DH103-01). The instructions for the IIOneStep Cloning (C112, vazyme) kit state that the PCR amplification product and the vector double-digestion product should be subjected to in vitro homologous recombination to obtain the recombinant vector. Ligation, transformation, and identification methods are the same as in Example 2 to confirm sequence correctness. The pGBKT7 (BD) vector was used as a control. Figure 8 (See label 7 for A in the text).

[0108] Yeast was transformed using the PEG / LiAC method: 1 μg of pGBKT7(BD) vector and the above recombinant vector were mixed with 50 μL of AH109 competent yeast cells, respectively. 346 μL of PEG / LiAC mixture was added, and the mixture was shaken at 30℃ for 30 min, then at 42℃ for 20 min. After recovery culture in 500 μL of YPDA liquid medium for 90 min, the cells were centrifuged, washed with ddH2O, and resuspended in 200 μL of 0.9% NaCl. The resuspended cells were evenly spread on SD / -Trp auxotrophic medium and cultured at 30℃ for 3 days. After the culture, an appropriate amount of cells was resuspended in 100 μL of 0.9% NaCl solution. 5 μL of the yeast cell resuspended cells was spotted onto auxotrophic medium SD / -Trp, SD / -Trp / -His, and SD / -Trp / -His / -Ade. The cells were cultured at 30℃ for 2 days. The growth of yeast cells was observed, and the transcriptional activation activity and transcriptional activation functional region of McMYC2 were determined. Five replicates were set up for each experimental group.

[0109] The results showed that all combinations grew normally on SD / -Trp medium, indicating that all combinations were successfully transformed. Further analysis revealed that, similar to Arabidopsis thaliana AtMYC2, yeast transfected with the full-length McMYC2 gene grew normally on selective media SD / -Trp / -His and SD / -Trp / -His / -Ade, indicating that peppermint McMYC2 can activate the expression of reporter genes His and Ade in yeast, possessing transcriptional activation activity. Figure 8(B) Sequence truncation experiments showed that yeast AH109 transformed with PRD and JID alone, and HLH alone, could not grow on selective medium. However, yeast transformed with fragments containing the McMYC2AD structure and fragments containing only the AD structure grew normally on SD / -Trp / -His medium, indicating that the AD structure is a key region for McMYC2 transcriptional activation. Furthermore, when McMYC2 retained the PRD, JID, and AD structures simultaneously, the transformed yeast grew normally on the more stringent SD / -Trp / -His / -Ade medium, indicating that the integrity of the N-terminus of the McMYC2 protein has a certain influence on its transcriptional activation ability. These results suggest that the McMYC2 protein possesses transcriptional self-activation activity and can function as a transcription factor to regulate downstream gene expression in the cell nucleus.

[0110] Example 6

[0111] This embodiment provides the effect of transient overexpression of the McMYC2 gene on genes related to peppermint essential oil synthesis.

[0112] The fusion expression vector pCAMBIA2300-McMYC2-GFP (experiment) and the empty vector pCAMBIA 2300-GFP (control) were transformed into Agrobacterium tumefaciens EHA105 using the freeze-thaw method. Following the method in Example 5, Agrobacterium tumefaciens containing these two vectors were injected into the lower epidermis of opposite leaves in the middle of mint seedlings (approximately 6 cm in height). After culturing at 23°C for 3 days, the leaves were collected, RNA was extracted, and reverse transcribed into cDNA, as described in Example 2. Specific primers were designed as follows: sMcMYC2-F: 5'-AGTAGTGAGTTGGAGTTGC T-3' (SEQ ID NO:40); sGFP-R: 5'-GTCGTCCTTGAAGAAGATGG-3' (SEQ ID NO:41).

[0113] The expression of McMYC2 was detected by RT-PCR, with six biological replicates. Following the method described in Example 4, qRT-PCR (primers shown in Table 5) was used to analyze the changes in the expression levels of McMYC2 and essential oil synthase genes in the leaves of the control and experimental groups.

[0114] RT-PCR results showed that the effective primers sMcMYC2-F on the McMYC2 gene and sGFP-R on the GFP gene (the effective sites are shown in the figure) were successfully applied. Figure 9 A PCR fragment of the predicted size was successfully detected, while the target fragment was not amplified in the control group (VC). Figure 10qRT-PCR analysis showed that, compared with the control group, the expression of McMYC2 was significantly upregulated in peppermint leaves with transient overexpression. Figure 11 This indicates that McMYC2 was successfully overexpressed in peppermint leaves.

[0115] Figure 12 The expression of essential oil synthesis-related genes McGPPS-LSU, McGPPS-SSU, McLS, and McPR in leaves. Figure 12 The results showed that the expression levels of McGPS-LSU, McGPS-SSU, McLS, and McPR were all upregulated to varying degrees. These results indicate that McMYC2 may affect the synthesis of essential oil-related metabolites by regulating the transcriptional levels of related enzyme genes in the peppermint essential oil synthesis pathway.

[0116] Example 7

[0117] This embodiment provides the effect of stable overexpression of the McMYC2 gene on the growth and glandular hair count of peppermint. Homologous recombination primers were designed to ligate the McMYC2 gene into the overexpression vector pCAMBIA1300-GFP:

[0118] pCAMBIA1300-GFP-McMYC2-F: 5'-GGGGCCCGGGGTCGACATGATTGAT TACCGTCTGCCG-3' (SEQ ID NO: 42); pCAMBIA1300-GFP-McMYC2-R: 5'-TACCGGATCCACTAGTGTTTATTTCAGCAGCAAGTG-3' (SEQ ID NO: 43).

[0119] Following the method used by Ma et al. for constructing transgenic vectors, a McMYC2 overexpression vector was constructed. The McMYC2 overexpression vector was transformed into EHA105 cells using a freeze-thaw method. Stably transformed peppermint was obtained through Agrobacterium infection and resistant shoot differentiation screening. Total DNA from wild-type and transgenic peppermint was obtained using the method described in the Bomeide CTAB Plant Genomic DNA Rapid Extraction Kit (DL114-01). Specific primers were designed: McMYC2-T1F-E06: 5'-AAAGCCTAGGAAACGGGGGA-3' (SEQ ID NO:44); p1300-GFP-R: 5'-GCTGAACTTGTGGCCGTTTAC-3' (SEQ ID NO:45).

[0120] DNA levels were identified by PCR and gel electrophoresis. The PCR amplification procedure is shown in Table 3. RNA levels were identified according to the method of Ma et al. (primers are shown in Table 5), and positive plants were obtained. The following analyses were performed:

[0121] 1. Gene expression: Wild-type and transgenic peppermint plants with similar growth were used in triplicate, and the expression of the McMYC2 gene in wild-type and transgenic peppermint plants was analyzed by qRT-PCR.

[0122] 2. Agronomic traits: In addition, 8cm of the above-ground part of wild-type and transgenic peppermint plants with uniform growth were cut and propagated. After three weeks of cultivation at 25℃ under 16h light / 8h dark conditions, the plant height and morphological characteristics of the third leaf from the top were observed and statistically analyzed. The results were repeated six times.

[0123] 3. Number of glandular hairs: Take three more replicate samples and divide the peppermint leaves into three regions: upper leaf, middle leaf, and lower leaf. Observe the growth of glandular hairs using a stereomicroscope.

[0124] Fourteen *Mentha haplocalyx* strains were obtained through *Agrobacterium* infection, differentiation, and resistance screening of regenerated seedlings, and named McMYC2OE-1, McMYC2OE-2, ..., McMYC2OE-14, respectively. This invention extracted leaf DNA from transgenic *Mentha haplocalyx* seedlings and wild-type (WT) seedlings. Using genomic-PCR technology, the integration of the *Mentha haplocalyx* gene was detected using the forward primer McMYC2-T1F-E06 on the *Mentha haplocalyx* gene and the reverse primer p1300-GFP-R on the *GFP* gene. Figure 13 The integration of the McMYC2 gene was detected using genomic-PCR. The results showed that, compared to the wild type, except for the McMYC2OE-5 and McMYC2OE-6 lines, the target fragment McMYC2-GFP was detectable at the DNA level in all other transgenic progeny plants, indicating that this invention yielded transgenic positive pepper-like mint plants that stably overexpress the McMYC2 gene.

[0125] In addition, using qRT-PCR technology, the expression level of McMYC2 was identified in two transgenic peppermint plants, McMYC2OE-7 and McMYC2OE-14. Figure 14 The results showed that the transcriptional level of McMYC2 in McMYC2OE-7 and McMYC2OE-14 was higher than that in WT peppermint, indicating that McMYC2 was successfully transferred into peppermint and could be stably overexpressed.

[0126] Figure 15Plant height, leaf fresh weight, leaf length, leaf area, and leaf width were compared between WT peppermint and two transgenic peppermint plants. No significant phenotypic differences were found between McMYC2OE-7 and McMYC2OE-14 and WT peppermint in these parameters, indicating that overexpression of the McMYC2 gene does not affect the agronomic traits of peppermint.

[0127] Figure 16 Microscopic observations of secretory glandular trichomes in WT peppermint and two transgenic peppermint plants. Results showed no significant difference in secretory glandular trichome density among WT, McMYC2OE-7, and McMYC2OE-14 plants in the upper, middle, and lower leaf regions. Further statistical analysis (…) Figure 17 It was found that the number of secretory glandular hairs did not differ significantly among the upper, middle, and lower parts of the leaves of the above materials, indicating that McMYC2 does not affect the development of secretory glandular hairs in peppermint leaves.

[0128] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A peppermint transcription factor McMYC2, characterized in that, The amino acid sequence of McMYC2 is shown in SEQ ID NO:

1.

2. The gene encoding the peppermint transcription factor McMYC2 according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. The application of the peppermint transcription factor McMYC2 according to claim 1, characterized in that, The peppermint transcription factor McMYC2 is used to positively regulate the transcriptional level of related genes in the peppermint essential oil synthesis pathway, thereby increasing the content of peppermint essential oil.

4. The application according to claim 3, characterized in that, The relevant genes are those identified in peppermint that are involved in the synthesis pathway of peppermint essential oil, specifically: McGPPS-LSU , McGPPS-SSU , McLS , McPR ; The nucleotide sequences of the relevant genes were obtained by amplification using specific primers for qRT-PCR with mint leaf cDNA as a template. The McGPPS-LSU The specific primers for qRT-PCR of the gene are: SEQ ID NO:7 and SEQ ID NO:8; The McGPPS-SSU The specific primers for qRT-PCR of the gene are: SEQ ID NO:9 and SEQ ID NO:10; The McLS The specific primers for qRT-PCR of the gene are: SEQ ID NO:11 and SEQ ID NO:12; The McPR The specific primers for qRT-PCR of the gene are: SEQ ID NO:19 and SEQ ID NO:

20.

5. A recombinant vector, characterized in that, The recombinant vector contains the coding gene as described in claim 2.

6. A recombinant genetically engineered bacterium, characterized in that, The recombinant genetically engineered bacteria comprises the recombinant vector as described in claim 5.

7. A method for cultivating peppermint varieties that promote essential oil synthesis, characterized in that, include: Overexpression in mint plants McMYC2 Gene; The McMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

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