Mint transcription factor McMYC2 and application thereof in regulation and control of essential oil synthesis

Abstract

The invention belongs to the technical field of genetic engineering breeding, and discloses a mint transcription factor McMYC2 and application thereof in regulation and control of essential oil synthesis. A mint MYC2 transcription factor, namely McMYC2, is screened, an encoding gene (namely McMYC2 gene) of the McMYC2 transcription factor is over-expressed in a mint plant, and the transcription factor plays a role in regulating synthesis of mint essential oil by regulating and controlling transcriptional levels of related genes such as McGPPS-LSU and McGPPS-SSU in a mint essential oil synthesis route. The McMYC2 has the function of regulating and controlling synthesis of the peppermint essential oil, a valuable gene resource is provided for cultivating a new variety of peppermint with high essential oil content, and the McMYC2 has an important application value in the aspect of peppermint improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering breeding, and relates to a mint transcription factor McMYC2 and an application thereof in regulating the synthesis of essential oils. Background Art

[0002] Mint (Mentha canadensis L.) is a perennial herbaceous plant of the genus Mentha in the family Lamiaceae. It is an important spice plant and also a commonly used Chinese herbal medicine. The main medicinal ingredients of mint are volatile monoterpenoid compounds in its aerial parts, commonly known as "mint essential oil", which have biological activities such as antibacterial, antioxidant, anti-inflammatory, analgesic, anti-tumor, and central nervous system excitation. It is currently widely used in the fields of medicine, food, spices, cosmetics, etc. In addition, mint essential oil has insecticidal and bactericidal activities. It is a plant-derived "green pesticide" in terms of storage pest control and biological defense. It plays an important ecological role in reducing the use of harmful pesticides and promoting green agricultural production. Therefore, the essential oil content is the main indicator for evaluating the quality of mint medicinal materials and industrial output value.

[0003] The activity of mint essential oil-related synthases is the key to the synthesis and accumulation of mint essential oil. Mint essential oil synthases are regulated by various transcription factors (TFs) or other genes. It is of great significance to explore the transcription factors related to the regulation of the synthesis of secondary metabolites of medicinal plants and analyze their regulatory mechanisms for the future use of transcription factors for genetic engineering to improve the quality of medicinal plants.

[0004] However, there are relatively few studies on the regulatory genes for mint essential oil synthesis, with only a few reports in plants of the genus Mentha. In addition, the molecular mechanism of transcription factor regulation of essential oil synthesis is still unclear, and very few transcription factors that positively regulate essential oil synthesis have been identified in mint, which greatly limits the molecular breeding process of mint. Summary of the invention

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

[0006] First, the present invention seeks to protect a mint transcription factor McMYC2, whose amino acid sequence is shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention seeks to protect the gene encoding the mint transcription factor McMYC2, the nucleotide sequence of which is shown in SEQ ID NO:2.

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

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

[0010] In a fourth aspect, the present invention provides a recombinant vector comprising the above-mentioned encoding gene.

[0011] In a fifth aspect, 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 breeding mint varieties that promote essential oil synthesis, comprising: overexpressing the McMYC2 gene in a mint plant; the nucleotide sequence of the McMYC2 gene is shown in SEQ ID NO:2.

[0013] Compared with the prior art, the present invention "mint transcription factor McMYC2 and its application in regulating essential oil synthesis" has the following beneficial effects:

[0014] The present invention screens out a mint MYC2 transcription factor (i.e., McMYC2), and overexpresses the coding gene of the McMYC2 transcription factor (i.e., the McMYC2 gene) in a mint plant. The transcription factor regulates the transcription levels of related genes such as McGPPS-LSU, McGPPS-SSU, McLS, and McPR in the mint essential oil synthesis pathway, thereby regulating the synthesis of mint essential oil.

[0015] The present invention clarifies that McMYC2 has the function of regulating the synthesis of essential oils, provides valuable gene resources for breeding new mint varieties with high essential oil content, and has important application value in mint improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the mint McMYC2 gene clone, and the red arrow points to the target gene band.

[0017] Figure 2 Phylogenetic tree analysis of mint McMYC2 protein and MYC2 proteins from other species.

[0018] Figure 3 This is the expression analysis of the mint McMYC2 gene in different tissues.

[0019] Figure 4This is the expression analysis of the McMYC2 gene in different leaf sequences of mint. Among them, L1~L8 are the 1st to 8th leaves from top to bottom in morphology.

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

[0021] Figure 6 This is the co-expression analysis of McMYC2 gene and essential oil synthase gene under MeJA treatment. CK1 and CK2 are the control groups; Mock is the expression of McMYC2 at different time points under 0.1% alcohol aqueous solution treatment.

[0022] Figure 7 Subcellular localization analysis of McMYC2 protein and AtMYC2 protein. Figure 7 A in the figure indicates the expression of McMYC2 and AtMYC2 in tobacco leaves; Figure 7 B in the figure shows the expression of McMYC2 in mint leaves. GFP: green fluorescent protein; mCherry: red fluorescent protein.

[0023] Figure 8 Analysis of the transcriptional autoactivation and transcriptional activation functional regions of mint McMYC2. Figure 8 A in the figure is a schematic diagram of a vector containing the full-length and partial domain gene sequences of McMYC2; Figure 8 B in the figure shows the growth of yeast AH109 transformed with each vector in A on the nutrient-deficient medium SD / -Trp, SD / -Trp / -His, and SD / -Trp / -His / -Ade.

[0024] Fig. 9 Schematic diagram of specific primer action sites in McMYC2 transient overexpression.

[0025] Fig.10 RT-PCR was used to detect the expression of McMYC2-GFP. VC was the empty vector (pCAMBIA2300-GFP) control.

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

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

[0028] Fig.13The integration status of McMYC2 gene detected by genomic-PCR technology.

[0029] Fig.14 is the expression level of McMYC2 in transgenic peppermint. WT indicates wild type.

[0030] Fig.15 Plant height, leaf fresh weight, leaf length, leaf area and leaf width of WT peppermint and transgenic peppermint.

[0031] Fig.16 The results are shown in the microscope observation of the secretory glandular hairs of WT peppermint and transgenic peppermint.

[0032] Fig.17 Statistical analysis of secretory glandular trichomes of WT peppermint and transgenic peppermint. DETAILED DESCRIPTION

[0033] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0035] Example 1

[0036] This example provides the screening of the mint McMYC2 gene.

[0037] Studies have shown that MYC2, a core bHLH transcription factor in the JA signaling pathway, plays an important role in regulating plant secondary metabolism. Therefore, the present invention first screened mint bHLH genes. Using the GO annotation of the previous mint MeJA-treated transcriptome data and the 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-treated conditions were obtained using transcriptome data. The Logarithm of RPKM was taken. 2 The (MeJA / CK) values ​​showed that 61 bHLH genes were upregulated and 62 were downregulated under MeJA induction.

[0038] Subsequently, four MYCs genes were identified from the up-regulated bHLH transcription factors that were significantly up-regulated by jasmonic acid, among which Unigene0042863 was the most significantly up-regulated. Based on the expresssequence tag (EST) sequence obtained by transcriptome sequencing, the partial cDNA sequences of the above four MYCs genes were spliced. Further NR (non-redundant protein) database annotation and sequence analysis showed that Unigene0042863 was homologous to the MYCs gene MYC2 related to the regulation of secondary metabolism in plants, so it was named McMYC2 gene.

[0039] Example 2

[0040] This example provides the mint McMYC2 gene cloning.

[0041] 1. Extraction of total RNA from mint and synthesis of first-strand cDNA

[0042] Two-week-old young leaves of peppermint were collected and frozen in liquid nitrogen and then ground into powder. Total RNA from the leaves was extracted according to the steps of Tiangen RNAprepPure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441). The obtained RNA was subjected to 1% agarose gel electrophoresis to determine RNA integrity, and the RNA concentration and purity were determined by Nanodrop 2000 spectrophotometer. The first strand of cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit (+gDNAwiper) (R212, vazyme) according to the 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 Random hexamers (50ng / μl) 1μL Total RNA 1 μg <![CDATA[RNasefreeddH 2 The]]> To16μL 42℃,2min / 10×RTMix 2μL HiScriptIIEnzymeMix 2μL 50℃, 15min; 85℃, 15s /

[0045] 2. McMYC2 gene amplification and DNA purification

[0046] According to the McMYC2 gene sequence obtained above, the full-length amplification primers of 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 mint 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 product was identified and separated by 1% agarose gel electrophoresis, and the PCR product of the McMYC2 gene was recovered using the Biomed DNA gel recovery kit (DH103-01) according to the instructions. The concentration of the PCR product was determined by Nanodrop 2000 spectrophotometer and set aside.

[0048] Table 2. McMYC2 gene PCR amplification system

[0049]

[0050]

[0051] Table 3. McMYC2 gene PCR amplification procedure

[0052]

[0053] The PCR product was sequenced and aligned to obtain the correct CDS sequence. Sequence analysis showed that the full length of the McMYC2 gene was 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 DNA sequence of the McMYC2 gene was further cloned by PCR ( Figure 1 , as indicated by the arrow). Structural analysis after sequencing showed that the McMYC2 gene had no intron structure, which was similar to the structure of the MYC2 genes of Arabidopsis thaliana, Artemisia annua and Hevea brasiliensis.

[0054] 3. McMYC2 gene and vector connection, transformation and identification of recombinant bacteria

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

[0056] Table 4. McMYC2 gene cloning reaction system

[0057] Components Dosage Purpose fragment 1~8μL pClone007BluntVector 1μL 10×Topo 1μL <![CDATA[ddH 2 The]]> up to 10 μL

[0058] The above reaction system was placed in a metal bath at 25°C for 15 minutes. 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 melted in an ice bath, add 10 μL of the reaction product, mix gently, and stand on ice for 30 minutes; heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and stand for 2 minutes; add 200 μL of non-resistant LB culture medium to the centrifuge tube, mix well, place in a 37°C shaker, and recover at 200rpm for 1 hour; absorb 200 μL of the recovery solution and evenly spread it on the LB medium plate containing Amp resistance, and invert the plate in a 37°C incubator for overnight culture. Pick a monoclonal colony and place it in LB liquid culture medium containing 50 mg / mL Amp for overnight culture. Use bacterial liquid PCR to identify the recombinant, and sequence the positive bacterial liquid containing the target fragment (Beijing Qingke Biotechnology Co., Ltd.) to confirm that the McMYC2 gene sequence has been successfully transformed. The positive bacterial solution was mixed evenly with 50% glycerol at a volume ratio of 1:1 and stored at -20°C for later use.

[0059] Example 3

[0060] This example provides bioinformatics analysis of the mint McMYC2 gene.

[0061] 1. McMYC2 protein structure analysis

[0062] In order to preliminarily understand the basic structure of McMYC2 protein, the present invention analyzes the secondary structure of McMYC2 protein by SOPMA online tool. The results show that the protein contains classical secondary structures such as α-helix, β-fold, extended chain and random coil, accounting for 36.47%, 1.94%, 15.72% and 45.87% of the total protein, respectively. The tertiary structure of McMYC2 protein was predicted by SWISS-MODEL online tool. The prediction model shows that the global model quality estimation (GMQE) of the protein is 0.61, indicating that the prediction result has a certain degree of credibility. The whole protein model is mainly composed of extended chain and random coil, α-helix and β-fold are distributed on both sides of the model, and β-fold is wrapped by α-helix.

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

[0064] In order to clarify the similarities and differences between the functions of the mint McMYC2 protein and the MYC2 protein of other species, the present invention systematically analyzed its amino acid sequence and evolution. The amino acid sequence comparison showed that the McMYC2 protein had certain similarities with the MYC2 proteins of other species (Artemisia annua, Aquilaria sinensis, Arabidopsis thaliana, Catharanthus roseus, and Salvia miltiorrhiza), and all had highly conserved JID (JAZ Interaction Domain) domains, transcription activation domains (AD), HLH domains, and nuclear localization signals (Nuclear Localization Signaling, NLS). Studies have shown that the JID domain is responsible for the interaction with the JA signal inhibitor JAZ protein, while the transcription activation domain is responsible for the regulation of its downstream target genes, and the NLS domain is responsible for its localization in the nucleus. It is suggested that the McMYC2 protein is very likely to regulate the mint essential oil synthase gene in the nucleus by responding to the JA signal.

[0065] 3. McMYC2 protein evolution tree analysis

[0066] Download the NCBI online website of Arabidopsis thaliana, Hevea brasiliensis, V. vinifera, P. trifoliata, M. domestica, Aquilaria sinensis, T. wilfordii, M. truncatula, A. annua, T. cinerariifolium, G. rigescens, and A. acuminate The amino acid sequences of MYC2 proteins from tobacco (N. attenuata), tobacco (N. tabacum), withania somnifera (W. somnifera), Mexican pepper (C. annuum), potato (S. tuberosum), tomato (S. lycopersicum; L. esculentum), salvia (S. miltiorrhiza), mint (M. longifolia), rice (O. sativa), freesia (F. hybrida), Dendrobium officinale (D. officinale), lily (Lilium hybrid'Sorbonne'), cabbage (B. rapa), northeastern yew (T. cuspidata), southern yew (T. chinensis), etc. At the same time, considering that mint is an allopolyploid, the amino acid sequence of McMYC2 protein was used again to perform local amino acid alignment on the third-generation transcriptome data of mint, and a candidate gene with high homology to McMYC2 was obtained, named McMYC2-like. Subsequently, the MEGA5.0 software was used to construct the evolutionary tree of the MYC2 gene among the above-mentioned different species. The results showed that McMYC2 and diploid European mint MlMYC2 were clustered in one branch, with the highest homology and the closest relationship, while Salvia miltiorrhiza, which is also in the Lamiaceae family, was second ( Figure 2 ). In addition, through consulting the data, it was found that in many plants such as Arabidopsis, MYC2 protein is involved in regulating the synthesis of secondary metabolites, which further indicates that McMYC2 may serve as the core transcription factor of mint JA signaling and has a potential role in regulating the synthesis of mint essential oil.

[0067] Example 4

[0068] This example provides analysis of the expression characteristics of the mint McMYC2 gene.

[0069] 1. Analysis of the organization and leaf order of McMYC2 gene expression in mint

[0070] Two-month-old mint plants with consistent growth were selected, and different tissues were sampled, including roots, stems, leaves, flowers, buds, and stem tips. At the same time, five-week-old mint plants with consistent growth were selected, and different leaf sequences (morphologically the 1st to 8th leaves from top to bottom (marked as L1 to L8) were sampled. Each group of samples was repeated 3 times, and then quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator.

[0071] qRT-PCR primers were designed according to the McMYC2 gene sequence (see qMcMYC2-F and qMcMYC2-R in Table 5) to analyze the McMYC2 gene expression in different tissues and leaf sequences, with β-actin as the internal reference gene. Fluorescence quantitative experiments were 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 replicated 3 times. 2 -ΔΔCT The relative mRNA expression level of McMYC2 gene was calculated by the method, and the 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×SYBR Green Pro Taq HS Premix 5μL F Primer 0.5μL R Primer 0.5μL cDNA 2μL <![CDATA[ddH 2 The]]> up to 10 μL

[0077] Table 7. qRT-PCR program

[0078]

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

[0080] The expression of McMYC2 gene in different leaf sequences of mint is shown in Figure 4 , Figure 4The results showed that the expression of McMYC2 gene in different leaves fluctuated with the aging of leaves, and generally showed a trend of increasing expression with the aging of leaves. The above results indicate that McMYC2 may be involved in the development or aging process of mint leaves in addition to the synthesis of peppermint essential oil.

[0081] 2. Analysis of the response of mint McMYC2 gene and essential oil synthesis genes to MeJA

[0082] Select mint seedlings with consistent growth, pick the aboveground parts for hydroponics, and when the root system grows luxuriantly (about 3 weeks), select mint plants with consistent growth again, refer to the MeJA treatment method of Ma et al., spray the leaves with 100 μmol / L MeJA solution for induction treatment, sample the leaves and roots at 0, 1, 3, 6, 12, and 24 h after treatment, quickly freeze them in liquid nitrogen at -80°C for later use, and set up 3 biological replicates for each treatment.

[0083] The qRT-PCR primers of McMYC2 gene were used, and specific qRT-PCR primers of essential oil synthase genes (McGPPS-LSU, McGPPS-SSU, McLS, McL3OH, MciPD, MciPR, McPR, McMMR, McMFS) were designed (see Table 5). The experimental conditions such as internal reference genes, qRT-PCR reaction system, and procedures were the same as those of “Expression analysis of McMYC2 gene tissue and leaf sequence in mint”.

[0084] Figure 5 The expression patterns of McMYC2 gene in mint leaves (A) and roots (B) under MeJA treatment. The results showed that MeJA treatment significantly induced the expression of McMYC2 gene in leaves and roots. Figure 5 In A), the expression of McMYC2 gene reached a peak after 1 hour of MeJA treatment, followed by a slight decrease, a second expression peak at 12 hours of treatment, and then dropped to a level with no significant difference from the control; in the root ( Figure 5 In B), the expression of McMYC2 gene showed a gradual increasing trend, indicating that MeJA activated the expression of mint McMYC2 at the transcriptional level.

[0085] Figure 6This is the co-expression analysis of McMYC2 gene and essential oil synthase genes under MeJA treatment. CK1 and CK2 were the control group; Mock was the expression of McMYC2 at different time points under 0.1% alcohol aqueous solution treatment. The results showed that after 3h of MeJA treatment, the expression of mint essential oil synthase genes McGPPS-LSU, McL3OH, MciPD, MciPR, McPR and McMFS showed a gradual upward trend and reached the highest level at 24h; the expression of McGPPS-SSU and McLS reached the highest level at 6h of MeJA treatment, and maintained a high expression level as the treatment time prolonged; the expression of McMMR increased slightly at 6h and 24h; and the expression of McMYC2 was significantly upregulated and reached a peak at 1h of MeJA treatment. The above results show that MeJA treatment rapidly induces McMYC2 expression, and the change of McMYC2 transcription level is earlier than that of essential oil synthase genes. In summary, McMYC2 is an early response gene to the MeJA signal, and is induced by MeJA treatment together with the peppermint essential oil synthase gene, showing a co-induced expression pattern. It is speculated that McMYC2 may be involved in the regulation of essential oil synthesis.

[0086] Example 5

[0087] This example provides McMYC2 protein feature analysis.

[0088] 1. Subcellular localization of McMYC2 protein

[0089] In order to further clarify the subcellular localization of McMYC2, the present invention constructed McMYC2 with the stop codon removed and Arabidopsis thaliana AtMYC2 genes into the pCAMBIA2300-GFP vector and performed transient expression by Agrobacterium-mediated transient transformation. The specific operation is as follows:

[0090] Homologous recombination PCR amplification primers of McMYC2 and Arabidopsis AtMYC2 gene sequences were designed (see SEQ ID NOs: 27-30 in Table 8), and PCR amplification was performed using pClone007-McMYC2 plasmid or Arabidopsis leaf cDNA as template. The reaction system included 2×ApexHF FS PCR Master Mix 12.5 μL, F and R primers 0.5 μL each, template plasmid / cDNA 1 μL, ddH 2 0.5 μL. PCR reaction program: 94°C for 5 min; 95°C for 15 s, 50°C for 20 s, 72°C for 45 s (1 kb / 5 s), 34 cycles; 72°C for 10 min, 4°C for 1 min.

[0091] Table 8. Homologous recombination PCR amplification primers

[0092]

[0093]

[0094] Table 9. Double enzyme digestion system

[0095] Components Dosage <![CDATA[10×Cutone TM buffer]]> 5μL Fast Dicer 1 5μL Fast Dicer 2 5μL Plasmids 5μg <![CDATA[ddH 2 The]]> upto50μL

[0096] Table 10. Homologous recombination reaction system

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

[0098] Table 11. Subcellular localization infection fluid system

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

[0100] The pCAMBIA2300-GFP (VC) vector was double-digested with BamHI and Sal I. The digestion system is shown in Table 9. The PCR product and the VC vector digestion product were separated by agarose gel electrophoresis and the gel was recovered for later use. IIOneStep Cloning Kit (C112, vazyme) was used to perform homologous recombination reaction. The reaction system is shown in Table 10. The PCR product was connected to the VC vector by in vitro homologous recombination. The connection, transformation and identification methods were the same as in Example 2 to confirm the correctness of the sequence.

[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 and centrifuged and then resuspended in infection solution (see Table 11 for infection solution system). The OD value of the resuspended solution was 0. 600 Adjusted to 0.6. Then, the Agrobacterium GV3101 resuspension carrying the fusion expression vector and VC vector was mixed evenly with the Agrobacterium GV3101 resuspension containing the nuclear localization vector at a volume ratio of 1:1. 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 the dark for 16 hours, followed by normal culture for 2 days. The green fluorescent protein (GFP) or red fluorescent protein (mCherry) signal was detected using a laser confocal microscope. Each experiment was repeated 3 times.

[0103] 2) Subcellular localization in mint leaves: Agrobacterium EHA105 carrying the fusion expression vector pCAMBIA2300-McMYC2-GFP was propagated and centrifuged and then resuspended in infection solution (see Table 11 for infection solution system). The OD value of the resuspended solution was 0. 600 The concentration of Agrobacterium tumefaciens EHA105 was adjusted to 0.7. Similarly, the Agrobacterium tumefaciens EHA105 resuspension carrying the fusion expression vector and the VC vector was mixed evenly with the Agrobacterium tumefaciens EHA105 resuspension containing the nuclear localization vector in a volume ratio of 1:1. After standing at room temperature for 1 hour, the mixture was injected into the middle leaves of mint seedlings (plant height of about 6 cm). After culturing in the dark at 23°C for 16 hours, it was 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 A in the figure represents the expression of McMYC2 and AtMYC2 in tobacco leaves. The results show that the green fluorescence signal of the empty vector VC group is widely distributed in the cytoplasm, cell membrane and cell nucleus. In tobacco leaves containing the fusion expression vector pCAMBIA2300-McMYC2-GFP, the green fluorescence signal can only be observed in the cell nucleus, which coincides with the red fluorescence signal, indicating that McMYC2, like Arabidopsis AtMYC2, is localized in the cell nucleus of tobacco cells. Figure 7 B in the figure indicates that McMYC2 is expressed in mint leaves. The results show that, consistent with the results in tobacco, McMYC2 is also localized in the nucleus in mint leaf cells. In summary, McMYC2 is localized in the nucleus and may have the function of regulating the transcription level of downstream genes.

[0105] 2. McMYC2 protein transcriptional autoactivation analysis

[0106] In order to analyze the transcriptional activation activity of McMYC2 protein and its self-activation functional region, the present invention constructed the full-length McMYC2 gene and the truncated nucleic acid sequences encoding each structural region into the pGBKT7 (BD) vector, transformed the yeast AH109, ​​and observed its growth after 3 days on the screening medium to analyze its transcriptional self-activation ability. The details are as follows:

[0107] Design of McMYC2 complete coding sequence ( Figure 8 A in (see annotation 1) and the primers encoding the N-terminus of McMYC2 protein (containing PRD+JID+AD domains, Figure 8 A in (see annotation 2), C-terminus (HLH domain, Figure 8 A in (see annotation 6), PRD+JID domain ( Figure 8 A in (see annotation 3), JID+AD domain ( Figure 8A in (see annotation 4), AD domain ( Figure 8 A in (see annotation 5) was used as the homologous recombination PCR amplification primers (see SEQ ID NOs: 31 to 39 in Table 8), and PCR amplification was performed using the pClone007-McMYC2 plasmid as the template. The pGBKT7 (BD) vector was double-digested with Nde I and EcoR I, and the enzyme digestion system was the same as in Table 9. The PCR amplification product and the vector double-digestion product were separated by gel electrophoresis, recovered and purified by the steps described in the Biomed DNA gel recovery kit (DH103-01), and the DNA was purified according to the method described in Table 9. IIOneStep Cloning (C112, vazyme) kit instructions, PCR amplification products and vector double enzyme digestion products were homologously recombined in vitro to obtain recombinant vectors, and the connection, transformation and identification methods were the same as in Example 2 to confirm the correctness of the sequence. pGBKT7 (BD) vector was used as a control ( Figure 8 A in (see note 7).

[0108] Transform yeast using the PEG / LiAC method: 1 μg of pGBKT7 (BD) vector and the above recombinant vector were mixed with 50 μL of AH109 yeast competent cells, 346 μL of PEG / LiAC mixture was added, and the mixture was shaken at 30°C for 30 min and 42°C for 20 min. 500 μL of YPDA liquid medium was added to recover the culture for 90 min and then centrifuged. ddH 2 O, resuspended with 200 μL 0.9% NaCl, evenly spread on SD / -Trp nutrient deficiency medium, and cultured at 30°C for 3 days. After the culture, pick an appropriate amount of bacteria and resuspend them in 100 μL 0.9% NaCl solution. Take 5 μL of yeast cell resuspension and spot on nutrient deficiency medium SD / -Trp, SD / -Trp / -His and SD / -Trp / -His / -Ade, culture at 30°C for 2 days, observe the growth of yeast, determine the transcriptional activation activity of McMYC2 and its transcriptional activation functional region, and set up 5 replicates for each experimental group.

[0109] The results showed that all combinations could grow normally on SD / -Trp medium, indicating that all combinations were successfully transformed. Further analysis showed that, like Arabidopsis AtMYC2, yeast transfected with the full-length McMYC2 gene could grow normally on the selective medium SD / -Trp / -His and SD / -Trp / -His / -Ade, indicating that mint McMYC2 can activate the expression of reporter genes His and Ade in yeast and has transcriptional activation activity ( Figure 8B in the figure). The results of the sequence truncation experiment showed that after transforming yeast AH109 with PRD and JID alone, and HLH alone, the yeast could not grow on the selection medium. However, after transforming yeast with the fragment vector containing McMYC2AD structure and AD structure alone, the yeast could grow normally on SD / -Trp / -His medium, indicating that the AD structure is the key region for McMYC2 transcriptional activation. In addition, when McMYC2 retains PRD, JID and AD structures at the same time, the transformed yeast can grow normally on the more rigorous SD / -Trp / -His / -Ade culture, indicating that the integrity of the N-terminus of McMYC2 protein has a certain effect on its transcriptional activation ability. The above results show that McMYC2 protein has transcriptional self-activation activity, and it can be used as a transcription factor to regulate the expression of downstream genes in the cell nucleus.

[0110] Example 6

[0111] This example 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 (test) and the empty vector pCAMBIA 2300-GFP (control) were respectively transferred into EHA105 Agrobacterium by freeze-thaw method, and the EHA105 Agrobacterium containing these two vectors were respectively injected into the lower epidermis of opposite leaves in the middle of mint seedlings (plant height of about 6 cm) according to the method of Example 5. After culturing at 23° C. for 3 days, the leaves were collected, RNA was extracted and reverse transcribed into cDNA, the method is shown in Example 2, and specific primers were designed: sMcMYC2-F: 5'-AGTAGTGAGTTGGAGTTGC T-3' (SEQ ID NO: 40); sGFP-R: 5'-GTCGTCCTTGAAGAAGATGG-3' (SEQ ID NO: 41).

[0113] RT-PCR was used to detect whether McMYC2 was normally expressed, and 6 biological replicates were set. Referring to the method described in Example 4, qRT-PCR (primers 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 group and the test group.

[0114] RT-PCR results showed that the forward primer sMcMYC2-F on the McMYC2 gene and the reverse primer sGFP-R on the GFP gene (the action site is shown in Fig. 9 ), a PCR fragment with the predicted size was successfully detected, while the target fragment was not amplified in the control group (VC) ( Fig.10). qRT-PCR analysis results showed that the expression of McMYC2 in mint leaves with transient overexpression of McMYC2 was significantly upregulated compared with the control group ( Fig.11 ), indicating that McMYC2 was successfully overexpressed in mint leaves.

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

[0116] Example 7

[0117] This example provides the effect of stable overexpression of McMYC2 gene on the growth and number of glandular hairs in mint. Homologous recombination primers for connecting McMYC2 gene to overexpression vector pCAMBIA1300-GFP were designed:

[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] Referring to the method for constructing a transgenic vector by Ma et al., a McMYC2 overexpression vector was constructed, and the McMYC2 overexpression vector was transformed into EHA105 by the freeze-thaw method. Stably transformed peppermint was obtained by Agrobacterium infection and resistance bud differentiation screening. The total DNA of wild-type and transgenic peppermint was obtained by the method described in the Bomade CTAB Plant Genomic DNA Rapid Extraction Kit (DL114-01), and specific primers were designed: McM YC2-T1F-E06: 5'-AAAGCCTAGGAAACGGGGGA-3' (SEQ ID NO: 44); p1300-GFP-R: 5'-GCTGAACTTGTGGCCGTTTAC-3' (SEQ ID NO: 45).

[0120] DNA level identification was performed by PCR and gel electrophoresis separation. The PCR amplification procedure is shown in Table 3. RNA level identification was performed according to the method of Ma et al. (primers are shown in Table 5) to obtain positive plants. The following analysis was performed:

[0121] 1. Gene expression: Wild-type and transgenic peppermint plants with similar growth were selected and repeated three times. The expression of the McMYC2 gene in wild-type and transgenic peppermint plants was analyzed by qRT-PCR.

[0122] 2. Agronomic traits: In addition, 8 cm aboveground parts of wild-type and transgenic peppermint plants with the same growth were cut for cuttings. After culturing at 25°C, 16 h light / 8 h dark conditions for three weeks, the plant height and morphology of the third leaf from top to bottom, leaf fresh weight, leaf length, leaf area and leaf width were observed and counted. This was repeated six times.

[0123] 3. Number of glandular hairs: Take three replicate samples and divide the peppermint leaves into three areas: the upper part of the leaf, the middle part of the leaf, and the lower part of the leaf. Use a stereo microscope to observe the growth of glandular hairs.

[0124] Through Agrobacterium infection, differentiation and resistance screening of regenerated seedlings, 14 strains of peppermint were obtained, which were named McMYC2OE-1, McMYC2OE-2...McMYC2OE-14. The present invention extracted leaf DNA from transgenic peppermint seedlings and wild-type peppermint (wild-type, WT) seedlings, and detected the integration of the McMYC2 gene by genomic-PCR technology using the forward primer McMYC2-T1F-E06 on the McMYC2 gene and the reverse primer p1300-GFP-R on the GFP gene. Fig.13 The results show that compared with the wild type, except for McMYC2OE-5 and McMYC2OE-6 lines, the target fragment McMYC2-GFP can be detected at the DNA level in the remaining transgenic offspring plants, indicating that the present invention has obtained transgenic positive peppermint plants that stably overexpress the McMYC2 gene.

[0125] In addition, two transgenic peppermint strains, McMYC2OE-7 and McMYC2OE-14, were selected using qRT-PCR technology to identify the expression level of McMYC2 ( Fig.14 ), the results showed that the transcription levels of McMYC2 in McMYC2OE-7 and McMYC2OE-14 were higher than that in WT peppermint, indicating that McMYC2 was successfully transferred into peppermint and could be stably overexpressed.

[0126] Fig.15The plant height, leaf fresh weight, leaf length, leaf area, and leaf width of WT peppermint and two transgenic peppermints were shown. It was found that McMYC2OE-7 and McMYC2OE-14 had no obvious phenotypic differences in plant height, leaf fresh weight, leaf length, leaf area, and leaf width compared with WT peppermint, indicating that overexpression of the McMYC2 gene did not affect the agronomic traits of mint.

[0127] Fig.16 The results of microscopic observation of secretory glandular hairs of WT peppermint and two transgenic peppermints were shown in Figure 2. The results showed that there was no significant difference in the density of secretory glandular hairs among WT, McMYC2OE-7 and McMYC2OE-14 in the upper, middle and lower leaf regions. Further statistical analysis ( Fig.17 ) found that there was no significant difference in the number of secretory glandular hairs in 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 embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

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

1.

2. The gene encoding the mint 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 use of the mint transcription factor McMYC2 according to claim 1, characterized in that: The mint transcription factor McMYC2 is used to regulate the transcription level of related genes in the mint essential oil synthesis pathway, thereby increasing the content of mint essential oil.

4. The use according to claim 3, characterized in that: The related genes include: McGPPS-LSU, McGPPS-SSU, McLS, and McPR.

5. A recombinant vector, characterized in that: The recombinant vector comprises the coding gene according to claim 2.

6. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria comprises the recombinant vector according to claim 5.

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

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