Plmyc2 gene, protein encoded by the plmyc2 gene and application thereof

Abstract

This application belongs to the field of plant genetic engineering and molecular biology, specifically providing a PlMYC2 gene. The protein encoded by the PlMYC2 gene can specifically bind to and activate the G-box cis-acting element in the promoter of the terpene synthase gene, and activate the promoter activity of the terpene synthase gene. Overexpression of the PlMYC2 gene can increase the content of linalool and / or caryophyllene in plants. Specifically, the PlMYC2 gene is selected from (a) or (b): (a) having the CDS sequence shown in SEQ ID NO:1; (b) a polynucleotide that hybridizes with the CDS sequence shown in SEQ ID NO:1 under strict hybridization conditions, and the protein encoded by it has the above-mentioned functions of binding the G-box cis-acting element, activating promoter activity, and increasing the content of linalool and / or caryophyllene in plants.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering and molecular biology technology, and in particular relates to a PlMYC2 gene and its encoded protein and its applications. Background Technology

[0002] Floral fragrance is a crucial quality trait of ornamental plants, directly influencing their ornamental and economic value. Terpenoids, especially monoterpenes (such as linalool) and sesquiterpenes (such as caryophyllene), are the main components of the fragrance in many ornamental flowers (such as roses, lilies, and peonies). Linalool, with its fresh and pleasant lily-of-the-valley aroma, is one of the most widely used fragrance ingredients globally.

[0003] Methyl jasmonate (MeJA) is a potent activator of the jasmonic acid (JA) pathway. As a key plant signaling molecule, it has been widely demonstrated to significantly induce the synthesis of volatile terpenoids in various plants, such as strawberries, grapes, and tea. In peonies, the decline in fragrance during postharvest vase life is a prominent issue, and exogenous MeJA treatment can effectively promote the release of terpenoids such as linalool from flowers, offering potential for postharvest preservation and quality control. However, the specific molecular regulatory mechanisms by which MeJA induces linalool synthesis in peonies, especially the key transcriptional regulators, remain unclear.

[0004] The biosynthesis of terpenoids is ultimately catalyzed by specific terpene synthases (TPS). Transcriptional regulation of TPS genes is crucial in determining the diversity and release of terpenoid products. Studies have shown that the MYC2 transcription factor is a core regulatory element of the JA signaling pathway, capable of responding to MeJA-induced activation of downstream target gene expression. For example, in Arabidopsis thaliana, AtMYC2 activates the sesquiterpene synthase genes TPS21 and TPS11; in lavender, LaMYC7 is a positive regulator of linalool and caryophyllene biosynthesis; and in grape, VvMYC2 has been shown to directly activate the monoterpene synthase gene VvTPS453, thereby promoting linalool accumulation. These findings suggest that MYC2 may have an evolutionarily conserved function in the regulation of terpene synthesis. However, whether a MYC2 homologous gene responds to MeJA and regulates linalool synthesis exists in peony, an important traditional flower, and its specific target and molecular mechanism, remains unreported.

[0005] Therefore, the study identified two key linalool synthase genes, PlTPS4 and PlTPS8, in the peony variety 'WHLY', thereby elucidating the transcriptional regulatory mechanism of MeJA-induced linalool synthesis in peonies and isolating and identifying key transcription factors. This can provide genetic resources and technical foundation for molecular breeding and postharvest regulation of the fragrance quality of peonies and other ornamental plants, which is of great significance. Summary of the Invention

[0006] To address the above issues, this application provides a PlMYC2 gene, its encoded protein, and its applications.

[0007] In a first aspect, this application provides a PlMYC2 gene, wherein the protein encoded by the PlMYC2 gene can specifically bind to and activate the G-box cis-acting element in the promoter of the terpene synthase gene, and activate the promoter activity of the terpene synthase gene, and overexpression of the PlMYC2 gene can increase the content of linalool and / or caryophyllene in plants. Specifically, the PlMYC2 gene is selected from (a) or (b): (a) Has the CDS sequence shown in SEQ ID NO: 1; (b) A polynucleotide with the CDS sequence shown in SEQ ID NO: 1 under strict hybridization conditions, and the protein encoded therein has the functions of binding G-box cis-acting elements, activating promoter activity and increasing the content of linalool and / or caryophyllene in plants.

[0008] Secondly, this application provides an isolated protein encoded by the aforementioned PlMYC2 gene.

[0009] Thirdly, this application provides a recombinant vector containing the aforementioned PlMYC2 gene, wherein the PlMYC2 gene is operatively linked to a regulatory sequence that functions in plant cells.

[0010] Fourthly, this application provides a recombinant host cell containing the above-mentioned PlMYC2 gene or the above-mentioned recombinant vector; preferably, the host cell is a plant cell, such as the cells of plants like peony, tobacco, Arabidopsis, rose, and lily.

[0011] Fifthly, this application provides a method for increasing the content of linalool and / or caryophyllene in plants, including the step of increasing the expression level and / or activity of the above-mentioned proteins in the plants.

[0012] Furthermore, the step of enhancing expression includes: introducing and expressing the above-mentioned PlMYC2 gene or the above-mentioned recombinant vector into the plant cells.

[0013] Furthermore, the method also includes the step of applying methyl jasmonate to the plant or plant cells.

[0014] Sixthly, this application provides the application of the above-mentioned PlMYC2 gene or the above-mentioned protein in regulating the synthesis of plant terpenoids.

[0015] Furthermore, the regulation is a positive regulation of the synthesis of linalool and / or caryophyllene.

[0016] In a seventh aspect, this application provides a plant material obtained by the method described in the fifth aspect, which, compared to its unmodified wild-type control, exhibits a significantly enhanced ability to synthesize linalool and / or caryophyllene. The plant material may be peony, or other ornamental plants or economic crops capable of synthesizing linalool or caryophyllene.

[0017] Compared with the prior art, this application has the following beneficial effects: 1. This application systematically demonstrated the function of PlMYC2 as a key transcription factor in the jasmonic acid signaling pathway using molecular biology, biochemistry, and genetic methods. It clarified that PlMYC2 activates the transcription of the two linalool synthase genes by directly binding to the G-box elements in the promoter regions of PlTPS4 and PlTPS8, thereby revealing the core molecular mechanism of MeJA-induced linalool synthesis in paeony.

[0018] 2. This application, through various experiments including yeast one-hybrid assay, dual-luciferase reporter system, and electrophoretic mobility shift analysis (EMSA), confirmed that the molecular basis of the interaction between PlMYC2 and target DNA is the highly conserved cis-regulatory element G-box (CACGTG). This provides new evidence for understanding the commonalities in the regulation of secondary metabolism by MYC2 family transcription factors in different plants.

[0019] 3. The experiments in this application show that overexpression of the PlMYC2 gene derived from peony not only effectively increases the content of linalool and caryophyllene in this species (peony petals), but also specifically upregulates the expression of endogenous linalool synthase gene NtTPS67 and caryophyllene synthase gene NtTPS7 in the heterologous plant tobacco, and significantly increases the accumulation of the corresponding products. This fully demonstrates the evolutionary conservation of the regulatory function of PlMYC2 and its broad application potential.

[0020] 4. This study elucidates the role of the PlMYC2 gene and its encoded protein as a highly efficient "molecular switch," providing key targets and powerful tools for the targeted improvement of the fragrance quality of peonies and other ornamental plants through genetic engineering. Whether through transgenic technology to cultivate new "fragrant" peony varieties or by developing inducers to activate endogenous PlMYC2 and combining them with postharvest MeJA treatment, these approaches open up entirely new technological pathways to solve the industry problem of fragrance decay in cut peony flowers, possessing significant theoretical and practical value. Attached Figure Description

[0021] Figure 1 This diagram shows the results of MeJA treatment on linalool release and related gene expression at different developmental stages of peony 'Wuhua Longyu' flowers in Example 1 of this application. Figure 1A: Phenotypic comparison of flowers in the NG (natural growth), CK (water propagation control), and MeJA treatment groups at five developmental stages (S1-S5); Figure 1 B: Dynamic curves of linalool release in the NG, CK and MeJA treatment groups, with asterisks indicating significant differences between the NG and MeJA groups; Figure 1 C: Bar chart of relative expression patterns of PlTPS4 gene in different treatment groups (with NG-S1 as 1), different lowercase letters indicate significant differences between groups (P<0.05). Figure 1 D: Bar chart showing the relative expression patterns of the PlTPS8 gene in different treatment groups; Figure 1 E: Schematic diagram of the analysis of cis-acting elements in the promoter regions of the PlTPS4 and PlTPS8 genes, indicating the location of key elements such as the G-box.

[0022] Figure 2 This figure shows the expression pattern analysis results of candidate transcription factors selected by transcriptome screening and RT-qPCR verification in Example 2 of this application under CK and MeJA treatments. The expression patterns of PlMYC2 and PlMYB306 are highlighted in red.

[0023] Figure 3 This is a graph showing the results of verifying the binding and regulation functions of PlMYC2 and PlMYB306 on the PlTPS4 / PlTPS8 promoters in Embodiment 3 of this application. Figure 3 A: The growth results of the yeast one-hybrid (Y1H) plate experiment show that PlMYC2 can bind to the PlTPS4 and PlTPS8 promoters, while no obvious binding to the above promoters was detected in PlMYB306; Figure 3 B: Schematic diagram of the construction of effector vectors (p62SK series) and reporter vectors (pLUC series) for dual-luciferase (Dual-LUC) experiments; Figure 3 C: Bar chart of quantitative results from dual-luciferase assay, showing that PlMYC2 can strongly activate the promoter activities of PlTPS4 and PlTPS8; Figure 3 D: Electrophoretic mobility shift analysis (EMSA) results verifying the specific binding of His-PlMYC2 protein to the PlTPS4 promoter G-box probe; Figure 3 E: EMSA verification results of the specific binding of His-PlMYC2 protein to the PlTPS8 promoter G-box probe.

[0024] Figure 4 This is a diagram illustrating the spatiotemporal expression pattern and molecular characteristics of PlMYC2 in Example 4 of this application. Figure 4A: Under natural growth conditions (NG), the superimposed graph of the expression pattern of the PlMYC2 gene (bar graph) and the linalool release (line graph) in the five developmental stages shows that the two change trends are consistent. Figure 4 B: A schematic diagram of the seven different parts of a peony flower in the S3 stage (leaf, stem, petals, pistil, receptacle, calyx, stamen); Figure 4 C: The superimposed graph of the expression level of the PlMYC2 gene (bar chart) and the linalool release (line chart) in the seven parts of the S3 stage shows that both are highest in the petals; Figure 4 D: Phylogenetic tree of PlMYC2 protein and Arabidopsis bHLH family members, showing that PlMYC2 belongs to subgroup IIIe (MYC2 class). Figure 4 E: Confocal microscopy image of the subcellular localization of PlMYC2 protein in the epidermal cells of Nicotiana benthamiana leaf, showing that the green fluorescence signal of the PlMYC2-GFP fusion protein is mainly colocalized with the blue fluorescence signal of the nuclear dye DAPI.

[0025] Figure 5 This is a graph showing the results of the analysis of loss-of-function (silencing) and gain-of-function (overexpression) of PlMYC2 in peony petals using transient conversion technology in Example 5 of this application. Figure 5 A: Bar chart of RT-qPCR analysis of expression levels of PlMYC2 and its downstream related genes in petals after silencing PlMYC2 using virus-induced gene silencing (VIGS) technology; Figure 5 B: Schematic diagram of representative gas chromatography-mass spectrometry (GC-MS) total ion chromatograms (TIC) of terpenoids released from petals in the silent group and the control group; Figure 5 C: A bar chart showing the changes in the release of major terpenoid compounds (linalool, caryophyllene, citronellol, etc.) in petals after silencing PlMYC2, based on quantitative analysis by GC-MS. Figure 5 D: Bar chart of RT-qPCR analysis of expression levels of related genes in petals after transient overexpression of PlMYC2; Figure 5 E: Schematic diagram of representative GC-MS total ion chromatograms (TIC) of terpenoids released from petals in the overexpression group and the control group; Figure 5 F: Bar chart showing the changes in the release of major terpenoid compounds in petals after overexpression of PlMYC2.

[0026] Figure 6 This is a functional analysis diagram of the stably overexpressed PlMYC2 gene in tobacco in Example 6 of this application. Figure 6 A: Electrophoresis diagram of genomic DNA PCR detection in wild-type (WT) and three transgenic overexpression lines (OE-1, OE-3, OE-5); Figure 6B: Comparison of flowering phenotypes between WT and three OE lines; Figure 6 C: Bar chart of RT-qPCR analysis of relative expression levels of the PlMYC2 gene in flowers of WT and OE lines; Figure 6 D: Schematic diagram of representative GC-MS total ion current (TIC) chromatograms of volatile components in flowers of WT and OE strains; Figure 6 E: Bar chart for quantitative analysis of linalool content in flowers of WT and OE strains; Figure 6 F: Bar chart of quantitative analysis of caryophyllene content in flowers of WT and OE lines; Figure 6 G- Figure 6 M: Bar chart showing RT-qPCR analysis of expression levels of key genes (NtDXS, NtDXR, NtGPPS, NtTPS67, NtHMGRL, NtFPPS, NtTPS7) in the tobacco endogenous terpene synthesis pathway in flowers of WT and OE lines. Detailed Implementation

[0027] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] When using “including,” “having,” and “contains” as described herein, the intention is to cover non-exclusive inclusion, unless explicit qualifying terms such as “only,” “consisting of,” etc. are used, other components may be added.

[0030] The terms "preferred," "more preferably," "better," and "even better" used in this application refer to embodiments of this application that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this application. That is, in this application, "preferred," "more preferably," "better," and "even better" are merely descriptions of implementations or examples with better effects, but do not constitute a limitation on the scope of protection of this application.

[0031] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0032] In this application, "at least one" means one or more, such as one, two, or more. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layered" means at least two layers, such as two layers, three layers, etc., unless otherwise explicitly specified. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0033] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0034] Unless otherwise specified, all steps in this application may be performed sequentially or in any order. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if it is mentioned that the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c) in that order, or it may include steps (a), (c), and (b) in that order, or it may include steps (c), (a), and (b), etc. Unless otherwise stated, singular terms may include plural forms and should not be construed as having a quantity of one.

[0035] In this application, "above" or "below" includes the number itself. For example, "below 1" includes 1.

[0036] In this application, room temperature refers to 0~40℃, including but not limited to 10~40℃, or further to 20~30℃.

[0037] The applicant has conducted extensive experimental research and provided information on the PlMYC2 transcription factor, its encoding gene, and its applications.

[0038] In a first aspect, this application provides a PlMYC2 gene, wherein the protein encoded by the PlMYC2 gene can specifically bind to the G-box cis-acting element in the promoter of the terpene synthase gene and activate the promoter activity of the terpene synthase gene, and overexpression of the PlMYC2 gene can increase the content of linalool and / or caryophyllene in plants. Specifically, the PlMYC2 gene is selected from any of the following: (a) Has the CDS sequence shown in SEQ ID NO: 1; (b) A polynucleotide that hybridizes with the CDS sequence shown in SEQ ID NO: 1 under strict hybridization conditions, and whose encoded protein has the functions of binding G-box cis-acting elements, activating promoter activity and increasing the content of linalool and / or caryophyllene in plants.

[0039] Among them, SEQ ID NO: 1 is the complete coding sequence (CDS) of the PlMYC2 gene cloned from the peony variety 'Wuhua Longyu', with a length of 2040bp.

[0040] Specifically: SEQ ID NO.1: Secondly, this application provides an isolated protein encoded by the aforementioned PlMYC2 gene.

[0041] This protein is a transcription factor in the jasmonic acid signaling pathway. It can be located in the cell nucleus and specifically recognizes and binds to G-box elements in the promoter regions of target genes through its bHLH domain, thereby activating the transcription of downstream terpene synthase genes.

[0042] Thirdly, this application provides a recombinant vector containing the aforementioned PlMYC2 gene, wherein the PlMYC2 gene is operatively linked to a regulatory sequence that functions in plant cells.

[0043] The regulatory sequences include, but are not limited to: promoters (such as constitutive promoter CaMV 35S, tissue-specific promoters, and inducible promoters), enhancers, terminators, and poly(A) tailing signals. The vector is preferably a plant expression vector, such as pCAMBIA1300, pBI121, or the pGreenII series. The construction of the vector is achievable by those skilled in the art using conventional molecular cloning techniques.

[0044] Fourthly, this application provides a recombinant host cell containing the above-mentioned PlMYC2 gene or the above-mentioned recombinant vector; preferably, the host cell is a plant cell, such as the cells of plants like peony, tobacco, Arabidopsis, rose, and lily.

[0045] Fifthly, this application provides a method for increasing the content of linalool and / or caryophyllene in plants, including the step of increasing the expression level and / or activity of the above-mentioned proteins in the plants.

[0046] Furthermore, the step of enhancing expression includes: introducing and expressing the above-mentioned PlMYC2 gene or the above-mentioned recombinant vector into the plant cells.

[0047] Furthermore, the method also includes the step of applying methyl jasmonate to the plant or plant cells.

[0048] In a preferred embodiment, the step of enhancing expression includes: introducing and expressing the PlMYC2 gene described in the first aspect or the recombinant vector described in the third aspect into the plant cells or tissues. The introduction method can be conventional plant genetic transformation methods in the art, such as Agrobacterium-mediated transformation, gene gun transformation, PEG-mediated transformation, or electroporation.

[0049] In another preferred embodiment, the method further includes the step of applying methyl jasmonate (MeJA) to the plant or plant cells. The application of MeJA can be performed simultaneously with the step of increasing PlMYC2 expression, or before or after it, to achieve a synergistic effect. Application methods may include spraying, soaking, or stem absorption.

[0050] Sixthly, this application provides the application of the above-mentioned PlMYC2 gene or the above-mentioned protein in regulating the synthesis of plant terpenoids.

[0051] Furthermore, the regulation is a positive regulation of the synthesis of linalool and / or caryophyllene.

[0052] Preferably, the regulation is positive regulation, i.e., promoting the synthesis of terpenoid compounds. More preferably, the terpenoid compounds are linalool and / or caryophyllene. The applications include, but are not limited to: breeding transgenic plants with enhanced floral fragrance, developing biological agents to improve the postharvest aroma quality of flowers, or using them as molecular markers to screen plant germplasm resources with excellent floral fragrance traits.

[0053] In a seventh aspect, this application provides a plant material obtained by the method described in the fifth aspect, which, compared to its unmodified wild-type control, exhibits a significantly enhanced ability to synthesize linalool and / or caryophyllene. The plant material may be peony, or other ornamental plants or economic crops capable of synthesizing linalool or caryophyllene.

[0054] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0055] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, or made by the methods, equipment, and materials in the embodiments of this application may be used to implement this application.

[0056] Example 1 This embodiment tests the MeJA treatment-induced linalool release from peony and the expression of linalool synthase genes PlTPS4 / PlTPS8, as detailed below: 1.1 Plant materials and hormone treatment This embodiment uses the peony variety 'WHLY' (also known as 'Five-Flower Dragon Jade') with a strong fragrance and a significant response to MeJA as the experimental material. The flower development stages are divided into 5 periods: S1 (bud stage), S2 (half-open stage), S3 (full bloom stage), S4 (initial decline stage), and S5 (decline stage).

[0057] During S1, the sepals split open, revealing the outer petals. The sepals move to the middle of the bud, and the bud softens. During S2, two days after S1, the flower is half-open. During S3, three days after S1, the flower is fully open. During S4, two days after S3, the flower begins to age. During S5, six days after S3, the petals begin to fall off.

[0058] Configure the following three groups of processes: Naturally grown group (NG): Healthy flowers at each developmental stage from S1 to S5 are directly picked from the field.

[0059] Water-propagated control group (CK): Flowers were picked at stage S1 and the base of the flower stems were immediately immersed in conical flasks containing deionized water.

[0060] MeJA treatment group: S1 stage flowers were treated by immersing the stems in bottles containing 100 μM MeJA.

[0061] In addition, plants in the S3 stage were divided into seven parts: pistil, stamen, receptacle, sepals, petals, stem, and leaves. Pistils, stamens, receptacle, sepals, petals, stems, and leaves were collected from the S3 stage. The collected petals were also analyzed by HS-SPME-GC-MS to determine the effect of MeJA on the release of volatile substances.

[0062] Three 5-year-old plants with consistent growth were selected for each period, and one central flower was collected from each plant. Samples were collected on sunny mornings between 10:00 and 11:00 AM. The collected samples were divided into two categories: fresh samples and frozen samples. Fresh samples were immediately analyzed for volatile compounds using HS-SPME-GC-MS, while frozen samples were immediately placed in liquid nitrogen and then stored at -80 °C for subsequent experiments.

[0063] 1.2 HS-SPME-GC-MS Conditions The SPME extraction head (50 / 30 µm DVB / CAR / PDMS, Supelco) was aged at 250°C for 2 hours. The concentration was 0.41 mg / mL. −1Using 3-octanol as an internal standard, 0.5 g of sample was weighed and a certain amount of saturated saline was added to a glass bottle. The sample and 10 μL of internal standard were placed together in a 40 mL transparent glass bottle, sealed with aluminum foil, and equilibrated at 40 °C for 10 min. The aged extraction head was inserted into the sealed glass bottle, and extraction was performed at 40 °C for 30 min. Then, the head was inserted into the GC-MS (ISQ&TRACE GC Ultra, Thermo Fisher) inlet, and desorption was performed at 250 °C for 3 min. The experiment was performed in three biological replicates.

[0064] GC conditions are as follows: Carrier gas: Helium; Column: DB-5MS (30m×0.25mm×0.25µm, Agilent); Carrier gas flow rate: 1 mL·min -1 ; Inlet temperature: 250℃; Injection method: Splitless; Column temperature: Initial temperature 40℃, hold for 2.5 min, then increase at 5℃·min. -1 Raise the temperature to 230°C and maintain for 5 minutes.

[0065] MS conditions are as follows: Electron ionization: EI source; Electron bombardment energy: 70 eV; Ion source temperature: 240℃; Interface temperature: 240℃; Data collection started 2.5 minutes ago; Mass range: 35-450 m / z.

[0066] 1.3 Qualitative and quantitative analysis of floral fragrance components Thermo Scientific Xcalibur software is used for the acquisition and processing of volatile compounds, identifying the volatile components in each sample by combining the National Institute of Standards and Technology (NIST) mass spectrometry library. The content of each component is determined by the internal standard method, and the calculation formula is as follows: Component content (ng·g) -1 = [(peak area of ​​each component × internal standard content (ng·μL)] -1 [×Internal standard volume (μL) / internal standard peak area] / sample weight (g).

[0067] Test results are as follows Figure 1 As shown, there were significant differences in the linalool release pattern. In the NG group, no release was detected in stages S1 and S2. Release peaked in stage S3 when the flower was fully open (421.10 ng·g). -1 ), and then gradually declines as the aging process progresses ( Figure 1B). Conversely, no linalool release was detected in the CK group at any stage ( Figure 1 B) indicates that the in vitro environment may affect the normal physiological metabolic balance and signal regulation network of plants, resulting in linalool release levels that are undetectable. The MeJA group showed a significantly different pattern: the release peak occurred in the S2 phase (6842.26 ng·g). -1 The release levels were 16.25 times higher than the peak levels in the NG group, subsequently decreasing with increasing treatment time. Release levels at all stages were significantly higher than those in the NG group at the corresponding time points. Figure 1 B). Furthermore, MeJA treatment resulted in a darker color in the petals ( Figure 1 A). In addition, short-term MeJA treatment is more effective than long-term treatment in increasing linalool release.

[0068] These results confirm that MeJA promotes linalool release from peony 'WHLY'. In 'WHLY', genes involved in linalool synthesis include PlDXS1-3, PlDXR1, PlMCT1, PlCMK1, PlMDS1, PlHDS1, PlHDR1, PlGPPS1-4, PlTPS4, and PlTPS8. Among these, PlTPS4 and PlTPS8 convert GPP to linalool, and MeJA did not activate these genes in 'WHLY'. However, it is unclear whether MeJA can activate PlTPS4 and PlTPS8, suggesting that MeJA likely promotes linalool release by inducing the expression of PlTPS4 and PlTPS8.

[0069] Subsequently, the expression levels of PlTPS4 and PlTPS8 in the NG, CK, and MeJA treatment groups were measured (with NG-S1 as the reference, set as 1) to further explore the association between their expression patterns and linalool release dynamics. The results showed that in S1, there were no significant differences in the expression of PlTPS4 or PlTPS8 among the groups. However, from S2 onwards, significant differentiation in expression patterns emerged: the expression levels of both genes in the CK group were significantly lower than those in the NG and MeJA groups, while the expression levels in the MeJA group were significantly higher than those in the NG and CK groups. Figure 1 CD). This trend is highly consistent with the linalool release dynamics. Further analysis revealed that in the MeJA group, the expression peaks of both genes occurred in the S2 phase, and their expression patterns highly matched the linalool release profile. Figure 1 These results indicate that MeJA can induce upregulation of PlTPS4 and PlTPS8 expression.

[0070] After cutting, the variety and quantity of floral compounds in 'WHLY' flowers decreased. However, MeJA treatment increased terpenoids and decreased benzene ring / phenylpropane compounds, while having no significant effect on fatty acid derivatives (Zhao et al., 2025a). Since this study focuses on linalool, changes in other compounds are not discussed here.

[0071] To determine whether PlTPS4 and PlTPS8 directly respond to MeJA, their promoters were cloned using chromosome walking. Besides the core CAAT and TATA boxes, the PlTPS4 promoter contains one G-box, five stress-responsive elements, one gibberellin-responsive element, and one light-responsive element; while the PlTPS8 promoter contains one MYB binding site, one G-box, and one light-responsive element. Figure 1 E). Analysis showed that none of their promoters contained MeJA response elements. Since the MYB binding site and G-box are known to be cis-elements that interact with specific transcription factors, and the MeJA signaling pathway usually mediates downstream gene expression through transcription factors, it is speculated that MeJA may regulate linalool release by inducing the expression of PlTPS4 and PlTPS8 through specific transcription factors.

[0072] Example 2 This embodiment identifies transcription factors that respond to MeJA and screens the transcriptome database of peony 'WHLY' (NCBI SRA accession number SRP287892).

[0073] Specific experimental methods: 2.1 Extraction of total RNA Total RNA was extracted using Trizol reagent (Invitrogen, Thermo Fisher), and the specific method is as follows: (1) Take petals from S1, S2 and S3 respectively and grind them thoroughly in liquid nitrogen. Transfer the ground powder to a 2 mL centrifuge tube, add 800 μL Trizol, vortex and let stand at room temperature for 10 min. (2) Add 200 μL of chloroform, mix well, and centrifuge at 12000 rpm for 10 min at 4℃. (3) Remove the upper aqueous phase and add phenol:chloroform (25:24) at a volume ratio of 1:1. Centrifuge at 12000 rpm for 10 min at 4℃. (4) Take out the upper aqueous phase, add chloroform at a volume ratio of 1:1, and centrifuge at 4℃ and 12000rpm for 10min; (5) Take out the upper aqueous phase, add isopropanol at a volume ratio of 1:1, place it in a -20℃ environment for 1 hour, and centrifuge at 4℃ and 12000rpm for 10 minutes. (6) Carefully remove the supernatant, add 800 μL of 75% ethanol, and centrifuge at 8000 rpm for 5 min at 4°C; (7) Repeat (6); (8) Discard the supernatant, add 30 μL of RNase-free ddH2O, and incubate at room temperature for 10 min. Store at -80℃ for long-term storage.

[0074] The quality, integrity, and purity of total RNA were assessed using agarose gel electrophoresis, a micro spectrophotometer (N50Touch, Implen), and a bioanalyzer (Agilent 2100).

[0075] Given that the promoter regions of PlTPS4 and PlTPS8 contain G-box and MYB binding elements, bZIP, bHLH, and MYB transcription factors whose expression patterns matched the linalool release profile were preferentially screened. Ultimately, nine transcription factors whose expression patterns were closely related to the linalool release trend under NG conditions were selected, including seven MYB family members and two bHLH family members.

[0076] Subsequently, the expression levels of these genes in the CK group and the MeJA treatment group at five stages were analyzed by RT-qPCR, and the results are as follows: Figure 2 As shown, one MYC transcription factor (Isoform0006058, a member of the bHLH family) and one MYB transcription factor (Isoform0030218) showed significantly induced expression under MeJA treatment. Figure 2 Based on the transcriptome annotation results, the MYC transcription factor (Isoform0006058) was named PlMYC2, and the MYB transcription factor (Isoform0030218) was named PlMYB306.

[0077] Example 3 This embodiment verifies whether PlMYC2 and PlMYB306 bind to the promoters of PlTPS4 and PlTPS8 by performing a yeast one-hybrid (Y1H) experiment. The promoter regions of PlTPS4 and PlTPS8 were cloned from genomic DNA and inserted into the pHIS2 vector as bait, respectively. The CDS of PlMYC2 and PlMYB306 were cloned and ligated into the pGADT7 vector. The bait and prey vectors were co-transformed into yeast strain Y187, and selection was performed on selective medium (SD / -Leu / -Trp / -His) containing 50 mM 3-AT. Yeast cells containing p53-pHIS2 + pGADT7-p53 served as a positive control. The experimental results are as follows: Figure 3 As shown.

[0078] The results showed that PlMYC2 could bind to the promoters of PlTPS4 and PlTPS8, while no obvious binding to either promoter was detected in PlMYB306. Figure 3 A). Next, effector vectors containing PlMYC2 or PlMYB306 and reporter vectors containing PlTPS4 or PlTPS8 promoters were constructed for use in dual-luciferase (Dual-LUC) experiments. Figure 3 B). The above-mentioned effector and reporter vectors were co-transferred into tobacco leaves to determine the effects of PlMYC2 and PlMYB306 on the activity of the two promoters. Dual-luciferase assays further verified the results: PlMYC2 activated both promoters (increasing PlTPS4 promoter activity by 9.12-fold and PlTPS8 promoter activity by 12.53-fold), while PlMYB306 did not activate either promoter. Figure 3 C).

[0079] Since both PlTPS4 and PlTPS8 promoters contain a G-box element (CACGTG), electrophoretic mobility shift analysis (EMSA) was performed to determine whether PlMYC2 binds to this element. A 36 bp probe containing the G-box region of each promoter was used. When the labeled probe containing the CACGTG motif was incubated with the control protein, the probe migrated freely without band shift. Conversely, when His-PlMYC2 protein was incubated with the labeled CACGTG probe, a protein-probe complex with delayed migration was formed. Unlabeled cold probes competed with labeled probes for binding to His-PlMYC2 protein, and the competition was stronger at higher concentrations. Furthermore, His-PlMYC2 protein did not bind to the mutated CACGTG probe. Figure 3 These results indicate that PlMYC2 specifically binds to the G-box elements in the PlTPS4 and PlTPS8 promoters.

[0080] These results indicate that PlMYC2 activates the promoters of PlTPS4 and PlTPS8 by binding to the G-box (CACGTG) element. Therefore, PlMYC2 may be a key regulator of MeJA-induced linalool release in peony flowers.

[0081] Example 4 The expression level of the PlMYC2 gene was measured in five developmental stages under NG conditions, and its expression trend was found to be highly consistent with the linalool release pattern, reaching its peak in stage S3. Figure 4 A). Considering that the release is highest in stage S3, seven parts from this stage (leaf, stem, petals, pistil, receptacle, calyx, and stamen) were further analyzed for volatile substances. Figure 4 B).

[0082] The results are as follows Figure 4 As shown, linalool is mainly released in the petals during the S3 stage (626.94 ng·g). -1 Stamen release was low (17.09 ng·g). -1 ), while linalool was not detected in other parts ( Figure 4 C). Simultaneously, analysis of PlMYC2 expression levels in these sites revealed that it was most highly expressed in petals, correlated with organ-specific release patterns of linalool, suggesting that PlMYC2 may be a candidate regulator of linalool synthesis.

[0083] The coding sequence (CDS) of PlMYC2 is 2040 bp long, encoding a 679-amino acid protein. ExPASy analysis predicts its theoretical isoelectric point to be 5.70 and its molecular weight to be 74.65 kDa. Sequence alignment shows that PlMYC2 is similar to other MYC2 proteins, possessing a conserved bHLH-MYC_N domain at the N-terminus and an HLH DNA-binding domain at the C-terminus. These features support its classification as a typical member of the MYC family. Phylogenetic analysis indicates that PlMYC2 belongs to subgroup IIIe (represented by MYC2) and shows a close phylogenetic relationship with Arabidopsis thaliana AtbHLH006 (AtMYC2). Figure 4 D). Subcellular localization experiments showed that the protein was located in the nucleus of epidermal cells of *Nicotiana benthamiana* leaves. Figure 4 E).

[0084] Example 5 This embodiment uses 'WHLY' flower petals as experimental material to verify the function of PlMYC2 in the biosynthesis of linalool in peony. Virus-induced gene silencing (VIGS) based on tobacco brittle virus (TRV) and Agrobacterium-mediated transient overexpression were employed to silence or overexpress PlMYC2. The corresponding empty vector served as a negative control. After three days of infiltration, the petals significantly enlarged. Samples were taken at this time, and positive petals carrying the corresponding vector fragment were identified by PCR. The PCR-positive samples were then analyzed by RT-qPCR and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0085] The results are as follows Figure 5As shown, the structural genes involved in linalool biosynthesis, besides PlTPS4 and PlTPS8, include PlDXS1-3, PlDXR1, PlMCT1, PlCMK1, PlMDS1, PlHDS1, PlHDR1, and PlGPPS1-4. Expression analysis of these genes showed that only the expression levels of PlDXS2, PlDXS3, PlGPPS1, and PlGPPS3 were positively correlated with the floral fragrance release pattern, and therefore were considered key genes involved in 'WHLY' floral fragrance synthesis. Compared with the control group, gene silencing significantly reduced the expression of PlMYC2. However, this treatment did not significantly affect the expression of PlDXS2, PlDXS3, PlGPPS1, or PlGPPS3. Figure 5 A).

[0086] Terpenoids in peony are mainly synthesized by five TPS genes (PlTPS1, PlTPS4, PlTPS6, PlTPS8, and PlTPS9), among which PlTPS4 and PlTPS8 catalyze the biosynthesis of linalool. In this application, silencing PlMYC2 significantly reduced the expression levels of PlTPS4 and PlTPS8, while the expression of PlTPS1 / 6 / 9 remained significantly unchanged. Figure 5 A).

[0087] Analysis of terpenoid compounds released from petals before and after PlMYC2 silencing revealed five compounds: four monoterpenes (linalool, citronellol, geraniol, and citral) and one sesquiterpene (caryophyllene). Citronellol, geraniol, and citral are collectively referred to as geraniol and its derivatives. The results indicate that PlMYC2 silencing significantly inhibited the production of linalool and caryophyllene, reducing their release, while geraniol and its derivatives remained unaffected. Figure 5 BC).

[0088] Overexpression of PlMYC2 in 'WHLY' significantly upregulated its own expression, while enhancing the transcriptional abundance of PlTPS4 and PlTPS8, resulting in a significant increase in the release of linalool and caryophyllene from the petals. Figure 5 The above results indicate that PlMYC2 positively regulates the expression of PlTPS4 and PlTPS8, as well as the biosynthesis of linalool and caryophyllene.

[0089] PlTPS4 specifically catalyzes the biosynthesis of linalool in peony. Simultaneously, in vitro enzyme activity assays showed that PlTPS8 can synthesize six monoterpenes, including caryophyllene, and nine sesquiterpenes. Notably, no other terpenoids besides linalool, caryophyllene, and geraniol and their derivatives were detected in this application. This may be due to reduced release of volatile substances from isolated tissues and interannual variations in flower fragrance composition regulated by multiple factors. Furthermore, other terpenoids derived from PlTPS8 accumulate at low levels in peony flowers, and may be below the detection limit after in vitro analysis.

[0090] The biosynthesis of caryophyllene in peony is mainly catalyzed by PlTPS6, while PlTPS8 has low catalytic efficiency for this compound. This application found that PlMYC2 increased the release of caryophyllene without affecting the expression of PlTPS6. The applicant believes that although PlTPS8 has a weak ability to produce caryophyllene, PlMYC2 increases the expression of PlTPS8, which is sufficient to promote the release of caryophyllene. The aforementioned MeJA treatment experiments showed that, under MeJA induction, in addition to linalool and caryophyllene, other terpenoids derived from PlTPS8, such as α-pinene, α-terpineol, geraniol-D, and nerolidol, also showed increased content. Therefore, PlMYC2 likely increases the content of various PlTPS8-derived terpenoids by activating PlTPS8 expression.

[0091] Example 6 To further elucidate the function of PlMYC2, this embodiment conducted a stable expression analysis in tobacco (Nicotiana tabacum). After DNA testing confirmed positive transgenic plants, the applicant ultimately selected three overexpression lines (OE lines, ...). Figure 6 A). Compared with the wild type (WT), the flower color or flower shape of the three OE lines (OE-1, OE-2, and OE-5) remained unchanged. Subsequent analysis showed that the content of volatile terpenoids in the flowers of the three OE lines differed significantly from that of WT. The expression level of PlMYC2 in the WT and OE lines was determined using RT-qPCR.

[0092] The results are as follows Figure 6 As shown, compared with WT, the expression of PlMYC2 was significantly increased in all three OE lines ( Figure 6 C).

[0093] Volatile terpenoids in the flowers of WT and OE lines were analyzed using HS-SPME-GC-MS. Linalool and caryophyllene were the main compounds detected. Figure 6D). Overexpression of PlMYC2 significantly increased the content of these two terpenoids in tobacco. The linalool content in the OE-1, OE-2, and OE-5 lines was 7.77, 9.79, and 6.75 times that of the total wt, respectively. The caryophyllene content in the OE-1, OE-2, and OE-5 lines was 1.36, 1.74, and 1.70 times that of the total wt, respectively. Figure 6 EF).

[0094] The applicant also measured the expression levels of terpene synthesis structural genes in the WT and OE lines. For genes involved in monoterpene synthesis, including NtDXS, NtDXR, NtGPPS, and the linalool synthase gene NtTPS67, only NtTPS67 expression was significantly increased in the three OE lines compared to the WT lines. Figure 6 GJ). Among the genes involved in sesquiterpene synthesis, including NtHMGRL, NtFPPS, and the caryophyllene synthase gene NtTPS7, only NtTPS7 expression was significantly increased in all three OE lines compared to WT. Figure 6 (KM). Therefore, overexpression of PlMYC2 in tobacco upregulated the expression of NtTPS67 and NtTPS7, and increased the content of linalool and caryophyllene. These results indicate that PlMYC2 regulates the biosynthesis of terpenoids in both peony and tobacco by upregulating the corresponding TPS genes.

[0095] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A PlMYC2 gene, characterized in that, The protein encoded by the PlMYC2 gene can specifically bind to and activate the G-box cis-acting element in the promoter of the terpene synthase gene, and activate the promoter activity of the terpene synthase gene. Overexpression of the PlMYC2 gene can increase the content of linalool and / or caryophyllene in plants. The PlMYC2 gene is selected from (a) or (b): (a) Has the CDS sequence shown in SEQ ID NO: 1; (b) A polynucleotide that hybridizes with the CDS sequence shown in SEQ ID NO: 1 under strict hybridization conditions, and whose encoded protein has the functions of binding G-box cis-acting elements, activating promoter activity and increasing the content of linalool and / or caryophyllene in plants.

2. An isolated protein encoded by the PlMYC2 gene as described in claim 1.

3. A recombinant vector, characterized in that, It includes the PlMYC2 gene of claim 1, wherein the PlMYC2 gene is operatively linked to a regulatory sequence that functions in plant cells.

4. A recombinant host cell, characterized in that, It contains the PlMYC2 gene of claim 1 or the recombinant vector of claim 3; preferably, the host cell is a plant cell.

5. A method for increasing the content of linalool and / or caryophyllene in plants, characterized in that, The step includes increasing the expression level and / or activity of the protein of claim 2 in the plant.

6. The method according to claim 5, characterized in that, The step of enhancing expression includes: introducing and expressing the PlMYC2 gene of claim 1 or the recombinant vector of claim 3 into the plant cells.

7. The method according to claim 5 or 6, characterized in that, The method also includes the step of applying methyl jasmonate to the plant or plant cells.

8. The use of the PlMYC2 gene of claim 1 or the protein of claim 2 in regulating the synthesis of plant terpenoids.

9. The application according to claim 8, characterized in that, The regulation is a positive regulation of the synthesis of linalool and / or caryophyllene.

10. A plant material, characterized in that, It is obtained by the method of any one of claims 5-7 and has enhanced ability to synthesize linalool and / or caryophyllene compared to its wild type.

Images