Cinnamomum camphora MYB transcription factor gene and application thereof

Abstract

The application discloses a Cinnamomum camphora MYB transcription factor gene and belongs to the technical field of forest metabolic regulation. The nucleotide sequence of the gene is shown in SEQ ID NO. 5. The amino acid sequence of the transcription factor is shown in SEQ ID No. 7. The application is one of key genes in the metabolic process of Cinnamomum camphora and can regulate flavonoid metabolism, terpenoid metabolism, organic acid metabolism and the like. The application provides an important theoretical basis for future industrial production in the fields of Cinnamomum camphora breeding, medicine and spices and has wide application prospect and great economic value.

Description

Technical Field

[0001] This invention belongs to the field of forest tree metabolism regulation technology, specifically relating to a camphor tree MYB transcription factor gene and its application. Background Technology

[0002] Camphor trees are large trees belonging to the genus Cinnamomum in the family Lauraceae. They have beautiful shapes, emerald green leaves, and emit a unique fragrance, making them a popular choice for landscaping. Their wood is hard, durable, and has natural anti-corrosion and insect-repellent properties. The roots, branches, and leaves contain various medicinal and aromatic components, which can be used to make medicines, spices, and fragrances.

[0003] The MYB transcription factor family refers to a class of transcription factors containing the MYB domain. It is one of the largest transcription factor families in plants, participating in the biosynthesis of primary and secondary metabolisms in numerous plants such as Arabidopsis thaliana, maize, and rice, influencing cell division and differentiation, tissue and organ development, hormone responses, and adaptation to biotic and abiotic stresses. Although researchers have discovered a large number of MYB transcription factors in different species, many functions remain to be determined, and the functions of MYB transcription factors with different structures vary considerably among different species.

[0004] Currently, the molecular regulatory mechanisms of biosynthesis of medicinal components and aromatic substances in camphor trees have not been thoroughly elucidated. Therefore, it is necessary to investigate whether there are MYB transcription factors regulating the synthesis pathways of related substances in camphor trees, further reveal the biosynthetic pathways of medicinal components and aromatic substances in camphor trees, and find key genes in their synthesis pathways, thereby providing targets for molecular breeding and genetic improvement of camphor trees. Summary of the Invention

[0005] This invention discloses a camphor MYB transcription factor gene, which participates in the metabolic regulation of flavonoids, terpenes, organic acids, etc. in camphor.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A camphor MYB transcription factor gene, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0008] The terpene synthase (TPS) family of enzymes are key enzymes in the biosynthesis of monoterpenoid and sesquiterpene essential oils. According to a literature report (Wang X, Xu C, Zheng Y, et al. Chromosome-level genome assembly and resequencing of camphor tree (Cinnamomum camphora) provides insight into phylogeny and diversification of terpenoid and triglyceride biosynthesis of Cinnamomum[J]. Hortic Res, 2022, 9: 216), CcTPS16 in camphor tree catalyzes the production of 1,8-eucalyptus oil. The camphor MYB transcription factor gene of this invention was cloned from the camphor variety 'Gantong 2'. By constructing an overexpression vector and transforming camphor leaves with it, the expression of the eucalyptus oil synthase gene TPS16 was verified. Furthermore, the overexpression vector of the camphor MYB transcription factor gene of this invention was used to transform camphor callus and leaves, and metabolomics differential metabolite analysis was performed to verify its regulatory effect on the synthesis of various flavonoids, terpenes, organic acids and other metabolites.

[0009] A camphor MYB transcription factor, the amino acid sequence of which is shown in SEQ ID NO.7.

[0010] The camphor MYB transcription factor of this invention is a hydrophilic protein located in the cell nucleus. It is expressed in camphor leaves, stems and roots, with the expression level in roots being significantly higher than that in stems and leaves.

[0011] Primers for cloning the above-mentioned camphor MYB transcription factor gene are shown as follows: upstream primer as shown in SEQ ID No. 1 and downstream primer as shown in SEQ ID No. 2.

[0012] A kit comprising primers for cloning the above-mentioned camphor MYB transcription factor gene. The primers in the kit of the present invention can be designed according to the basic principles of primer design for the above-mentioned camphor MYB transcription factor gene, or the primers shown in SEQ ID No. 1 and SEQ ID No. 2 can be used.

[0013] Recombinant expression vectors, expression cassettes, or host bacteria containing the above-mentioned camphor MYB transcription factor gene.

[0014] This invention utilizes primers designed for the camphor MYB transcription factor gene and kits prepared to facilitate genetic transformation operations and verification during the cultivation and screening of camphor varieties. Genetic transformation can be achieved by constructing recombinant expression vectors, expression cassettes, or host bacteria containing the camphor MYB transcription factor gene.

[0015] The aforementioned genes, transcription factors, primers, kits, or recombinant expression vectors, expression cassettes, or host bacteria can be used for the metabolic regulation or variety selection of camphor trees.

[0016] Furthermore, the aforementioned metabolic regulation includes one or more of the following: camphor flavonoid metabolic regulation, terpene metabolic regulation, or organic acid metabolic regulation.

[0017] Furthermore, the above-mentioned regulation of flavonoid metabolism includes regulating the metabolism of flavonoids or their derivatives;

[0018] Terpenoid metabolism regulation includes regulating the metabolism of terpenes or their derivatives, such as geraniol D, trans-bergamot glycerene, caryophyllene, (-)-Alpha-pinene, canocorside C, alanji glycoside, 3-O-[β-D-glucose(1→2)-β-D-glucose(1→4)-β-D-galactose]-(25R)-12-oxo-5α-spirost-3β-ol;

[0019] Organic acid metabolism regulation includes regulating the metabolism of organic acids such as gambogeylic acid, malic acid, methylmalonic acid, cis-5, and cis-12-octadecadienoic acid.

[0020] Dioonflavone is a natural flavonoid compound with antioxidant, anti-inflammatory, and antibacterial effects. Isoscutellarein 4'-methylether 8-(2,4”-disulfatoglucuronide) is a sulfated glycoside compound of flavonoids with a unique sulfonated glycoside and flavonoid skeleton structure. The flavonoid core can usually scavenge free radicals and inhibit oxidative stress; the sulfonated glycoside may affect the release of inflammatory factors.

[0021] Germacrene D possesses antibacterial and antifungal activities and can be used as an adjuvant for aminoglycosides and azoles, as well as as a fragrance. Trans-Bergamotene is a monoterpene compound widely found in plant volatile oils and exhibits various pharmacological effects. Caryophyllene is a bicyclic sesquiterpene compound that can be used as a fragrance. α-Santalum (Tricyclo[2.2.1.0(2,6)]heptane,1,7-dimethyl-7-(4-methyl-3-pentenyl)-,(-)-) is one of the representative sesquiterpenes, possessing good antibacterial, antioxidant, and antitumor pharmacological activities; it is also a major component of sandalwood essential oil and has important applications in medicine, food, and fragrances. (-)-Alpha-pinene (Copaene) is a sesquiterpene hydrocarbon that can be used as a volatile fragrance. 3-O-[β-D-glucose(1→2)-β-D-glucose(1→4)-β-D-galactose]-(25R)-12-oxo-5α-spirostan-3β-ol (3-O-(Glcb1-2Glcb1-4Galb)-(25R)-12-oxo-5alpha-spirostan-3beta-ol) is a steroidal saponin with anti-inflammatory, immunomodulatory, and antitumor activities. Kanokoside C is an iridoid glycoside with anti-inflammatory, antioxidant, hepatoprotective, and neuroprotective effects. Alangiside is an iridoid glycoside with anti-inflammatory, analgesic, and antitumor effects. Iridoid glycosides are a subclass of terpenes, specifically referring to monoterpenoid glycosides with a cyclic ether structure. They consist of an iridoid skeleton and a glycoside, and are monoterpene derivatives. They are an important branch of the terpene biosynthesis network, and their synthesis relies on the MEP / MVA pathway common to terpenes, with GPP (geraniyl pyrophosphate, C10) as the direct precursor. GPP can also be used to synthesize other monoterpenes (such as limonene and pinene), or further condensed into sesquiterpenes (FPP), diterpenes (GGPP), etc.

[0022] Garcinolic acid has strong anti-cancer activity and is effective against breast cancer, lymphoma, skin cancer, and gastric cancer, while having no effect on the normal hematopoietic system and white blood cells.

[0023] The above metabolites have been shown to be significantly upregulated or downregulated in camphor callus or leaves overexpressing the above-mentioned camphor MYB transcription factor gene. Therefore, the camphor MYB transcription factor gene of this invention can be used as a target to regulate the above-mentioned metabolites such as flavonoids, terpenes, and organic acids, thereby expanding the application potential of camphor medicinal components and aromatic substances.

[0024] Furthermore, the above-mentioned metabolic regulation also includes regulation by alkaloids or amide metabolites.

[0025] Somniferine is an isoquinoline alkaloid with anti-inflammatory, analgesic, and sedative activities. It was significantly upregulated in camphor callus tissue overexpressing the MYB transcription factor gene. Haplophytine is a rare, complex indole alkaloid, mainly found in Apocynaceae plants. It possesses both insecticidal and antitumor activities, has a complex structure, and is extremely difficult to synthesize. It was significantly upregulated in camphor leaves overexpressing the MYB transcription factor gene. Grossamide is a lignan amide compound with antioxidant, neuroprotective, and anti-inflammatory activities. It was significantly upregulated in camphor callus tissue overexpressing the MYB transcription factor gene.

[0026] Thalipindione (CAS No. 74690-97-4) is a diketone alkaloid with heat-clearing, fire-purging, and detoxifying effects. It was significantly upregulated in camphor leaves overexpressing the MYB transcription factor gene. Caseadine (CAS No. 34413-12-2) is an isoquinoline alkaloid with multi-target and multi-pathway pharmacological activities, including anti-inflammatory, antibacterial, antitumor, hypoglycemic, neuroprotective, and cardiovascular regulatory effects. It was also significantly upregulated in camphor leaves overexpressing the MYB transcription factor gene.

[0027] Furthermore, malic acid, as the starting material of the tricarboxylic acid cycle, directly participates in cellular energy production; changes in malic acid concentration in vacuoles can regulate stomatal opening and affect photosynthetic efficiency; H+ produced by malic acid metabolism... +Malic acid plays a crucial role in maintaining cell membrane potential and balancing the ion gradient caused by active transport. Furthermore, malic acid metabolism can be activated by environmental stress, enhancing plant resistance by regulating related transport proteins. Methylmalonic acid (MMA) is an organic acid produced during plant metabolism, primarily derived from the metabolism of propionic acid and thymine. Within plants, it is converted to succinic acid through the action of methylmalonyl-CoA mutase and vitamin B12, participating in the tricarboxylic acid cycle to maintain energy metabolism and growth. Cis-5 and cis-12-octadecadienoic acid (CEA) are unsaturated fatty acids produced during plant metabolism. Unsaturated fatty acid metabolism typically regulates cell membrane properties, activates defense mechanisms, and enhances plant resistance. Therefore, this invention utilizes the *Cinnamomum camphora* MYB transcription factor gene as a target to regulate organic acid metabolites such as malic acid, methylmalonic acid, cis-5, and cis-12-octadecadienoic acid. Optimizing organic acid metabolic pathways through genetic engineering or metabolic engineering techniques holds promise for regulating *Cinnamomum camphora* growth and development and improving its resistance.

[0028] In addition, L-serine-L-alanyl-L-serine-L-phenylpropionyl-L-leucine-L-phenylpropionyl-L-serine (L-Seryl-L-alanyl-L-seryl-L-leucyl-L-phenylalanyl-L-serine) is a short peptide chain, which is significantly upregulated in the camphor callus tissue overexpressing the above-mentioned camphor MYB transcription factor gene. It can be used as a target to study its effects on the growth, development and stress resistance of camphor.

[0029] Pentafluoroethyl radical (PFC) is a highly reactive free radical that was significantly upregulated in camphor callus tissue overexpressing the aforementioned camphor MYB transcription factor gene. It may induce oxidative stress, activate disease resistance and stress tolerance pathways, and regulate growth and development. Therefore, this provides a basis for using the camphor MYB transcription factor gene of this invention as a target to regulate the growth and development of camphor trees and improve their stress resistance through genetic engineering or metabolic engineering.

[0030] Furthermore, the aforementioned regulation includes both positive and negative regulation.

[0031] Furthermore, through metabolic pathway enrichment analysis, this invention shows that overexpression of the above-mentioned camphor MYB transcription factor gene affects glyoxylate and dicarboxylic acid metabolism, nitrogen metabolism, citric acid cycle (TCA cycle), Calvin cycle carbon fixation, pyruvate metabolism, and degradation of valine, leucine, and isoleucine.

[0032] In summary, the camphor MYB transcription factor of this invention participates in the regulation of camphor flavonoid metabolism, terpene metabolism, and organic acid metabolism. The gene provided by this invention can be used as a target for molecular breeding and genetic improvement of camphor. Simultaneously, it provides a research direction for further elucidating the biosynthetic pathways of various metabolites such as camphor flavonoids, terpenes, and organic acids. Attached Figure Description

[0033] Figure 1 The image shown is a map of the PBI121-GUS plasmid.

[0034] Figure 2 The image shows an agarose gel electrophoresis diagram of total RNA from camphor trees; 1-4 in the figure represent four samples of total RNA from camphor trees.

[0035] Figure 3 The image shown is a PCR electrophoresis diagram of the CcMYBs gene; M is the marker, 7 is CcMYB7, and 8 is CcMYB8.

[0036] Figure 4 The image shows the subcellular localization map of CcMYBs in camphor trees.

[0037] Figure 5 The figure shows the expression levels of CcMYBs in tissues; different letters in the column represent differences of p<0.05.

[0038] Figure 6 The image shown is an agarose gel electrophoresis image of total RNA from transgenic leaves; M represents Marker 2000, and OE represents overexpression.

[0039] Figure 7 The figure shows the gene expression in the transgenic leaves; OE represents overexpression, and asterisks indicate significant differences from the control group (nsp>0.05, *p<0.05, **p<0.01).

[0040] Figure 8 The image shown is an agarose gel electrophoresis diagram of total RNA from transgenic callus; M represents Marker 2000, and OE represents overexpression.

[0041] Figure 9 The figure shows the expression of transgenic callus genes; OE in the figure represents overexpression, and asterisks indicate significant differences from the control group (**p<0.01).

[0042] Figure 10 The following are metabolic pathway enrichment analyses: a. Glyoxylate and dicarboxylic acid metabolism; b. Nitrogen metabolism; c. Citric acid cycle (TCA cycle); d. Calvin cycle carbon fixation; e. Pyruvate metabolism; f. Degradation of valine, leucine, and isoleucine.

[0043] Figure 11The figure shows the TIC (total ion current) intensity of some differential metabolites;

[0044] Among them, A is a differential metabolite TIC in callus tissue, and a, b, c, and d are Kanokoside C, Somniferine, Alangiside, and Grossamide, respectively.

[0045] B represents some differential metabolites TIC found in the leaves; e, f, g, and h represent Thalipindione, Haplophytine, Caseadine, and 3-O-(Glcb1-2Glcb1-4Galb)-(25R)-12-oxo-5alpha-spirostan-3beta-ol, respectively.

[0046] An asterisk indicates a significant difference from the control group (*p<0.05, **p<0.01).

[0047] Figure 12 The image shows a matchstick plot analysis of differentially expressed metabolites by GC-MS; the vertical axis represents the differentially expressed metabolite number, and the horizontal axis shows the fold change after logarithmic transformation. The intensity of the dot color represents the VIP value, and * indicates significance (* 0.01). <p<0.05,**0.001<p<0.01,***p<0.001)。 Detailed Implementation

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] The camphor tree used in this embodiment of the invention is the camphor tree plant 'Gantong No. 2' planted in the greenhouse of the Chinese Academy of Forestry (Zhong Yongda, Wu Zhaoxiang, Hu Miao, et al. New ornamental camphor tree variety 'Gantong No. 2' [J]. Journal of Horticulture, 2020, 47(S2):3130-3131).

[0050] The terpenoid components and content in the leaves of 'Gantong No. 2' were detected by gas chromatography-mass spectrometry (GC-MS). 1.0 g of crushed camphor leaf powder was placed in a 10 mL headspace vial, sealed, and injected into the headspace vial injector under the following conditions: sample vial heating temperature 120℃; sample loop temperature 100℃; transfer line temperature 100℃; sample vial heating time 20 min.

[0051] Chromatographic conditions:

[0052] Column: Agilent 122-7032:DB-WAX (30m×250μm, 0.25μm); Carrier gas: Helium; Temperature program: 40℃ for 5 min, then ramp to 180℃ at 5℃ / min and hold for 1 min; Injector temperature: 200℃; Split mode (split ratio 10:1) was used, with a split flow rate of 10 mL / min and a constant gas flow rate of 1.0 mL / min.

[0053] Mass spectrometry conditions:

[0054] EI source; electron energy 70 eV; transfer line temperature 240℃; ion source temperature 230℃. Scan type: MS2, mass scan range m / z 45~650.

[0055] The results showed that the main volatile components in the leaves of 'Gantong No. 2' were eucalyptol (34.27%), sabinene (22.58%), trans-β-ocimene (11.32%), β-pinene (7.46%), α-terpineol (4.23%), and myrcene (3.25%), and contained no camphor, borneol, linalool, or nerolidol, or contained very little of it.

[0056] The tobacco used in the examples was cultured in an artificial climate chamber for about 30 days.

[0057] Strains used in the examples:

[0058] Escherichia coli competent cells DH5α were purchased from Tiangen Biotech (Beijing) Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd.; Agrobacterium competent cells GV3101 were purchased from Beijing Huayueyang Biotechnology Co., Ltd.

[0059] Carrier used in the embodiments:

[0060] M5 HiPerpTOPO-Blunt CloningKit blunt-end cloning vector (MF021) was purchased from Beijing Polymer Biotechnology Co., Ltd.; pCAMBIA1300-35S-EGFP was purchased from Beijing Coolplay Technology Co., Ltd.; PBI121-GUS was a laboratory-preserved vector, and the plasmid map is shown below. Figure 1 As shown.

[0061] Main reagents used in the examples:

[0062] 2×CTAB extraction buffer and β-mercaptoethanol were purchased from Beijing Coollab Technology Co., Ltd.; FastKing cDNA First Strand Synthesis Kit (Genomic De-generated) (KR116-02), SuperReal Quantitative Real-Time Premixed Real-Time Enhanced Version (SYBR Green) (FP205), EasyGeno Single Fragment Recombinant Cloning Kit (VI202-02), and Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) were purchased from Tiangen Biotech (Beijing) Co., Ltd.; M5 Hiper Gel Extraction Kit (MF029-01), Multicolor Endotoxin-Free Plasmid Miniature Extraction Kit (MF077-01), and M5 Hiper PCR & DNA Fragment Purification Kit (MF030-01) were purchased from Beijing Polymer Biotechnology Co., Ltd. BamH I(JB101-01), Quick Gel Extraction Kit(EG101-01)、 The PCR Purification Kit (EP101) was purchased from Beijing TransGen Biotech Co., Ltd.; KOD One TM PCRMaster Mix-Blue (KMM-201) was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; BamH I-HF (R3136S) and Sac I-HF (R3156S) were purchased from Beijing Bailinke Biotechnology Co., Ltd.

[0063] All other materials and reagents not otherwise specified are standard materials and reagents used in this field.

[0064] Example 1: Cloning and Sequence Characterization of Cinnamomum camphora CcMYBs Gene

[0065] 1. Extraction of total RNA from camphor trees

[0066] Total RNA was extracted from different tissues (roots, stems, and leaves) of *Cinnamomum camphora* using the CTAB method. The extracted RNA was detected using a NanoDrop 8000 spectrophotometer, with concentrations ranging from 382.6 to 393.6 ng / μL. RNA integrity was assessed by agarose gel electrophoresis. Figure 2 As shown, the electrophoresis showed obvious 28S and 18S bands, indicating that the extracted total RNA was intact and of high quality and concentration.

[0067] 2. Synthesis of the first strand of camphor cDNA

[0068] RNA was reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (Genomic De-transfer). See the instruction manual for the method and steps. The resulting cDNA can be used for subsequent experiments or stored at -20°C.

[0069] 3. Cloning of the CcMYBs gene of camphor tree

[0070] (1) PCR amplification

[0071] Using camphor cDNA as a template, the target gene was amplified using the primers in Table 1. The primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0072] Table 1 Primers used for PCR

[0073]

[0074] Amplification system: 5 μL template cDNA, KOD One TM PCR Master Mix-Blue 25 μL, primer F 1.5 μL, primer R 1.5 μL, ddH2O 17 μL (to a final volume of 50 μL); Amplification program: pre-denaturation, 98℃ for 3 min; denaturation, 98℃ for 10 s; annealing, 58℃ for 5 s; extension, 68℃ for 10 s, 30 cycles; final extension, 72℃ for 5 min; 8℃, infinity.

[0075] (2) Gel extraction and recovery of PCR products

[0076] After the PCR reaction, electrophoresis was performed on a 1.2% agarose gel to confirm successful cloning of the target fragment. The gel containing the target fragment was excised and recovered using a gel extraction kit. The procedure is described in the instructions for the M5 HiperGel Extraction Kit (MF029-01).

[0077] (3) Linking the purified gel product to a T-vector

[0078] A 10 μL cloning system was prepared using the M5 HiPerpTOPO-Blunt Cloning Kit blunt-ended cloning vector (MF021) at room temperature (20℃-30℃): 0.5-8 μL of gel-recovered product, 1 μL of M5 HiPerpTOPO-Blunt Vector, 1 μL of 10×Enhancer, and ddH2O to a final volume of 10 μL. Ligation was carried out at room temperature (20℃-30℃) for 5 min to obtain the ligation reaction solution.

[0079] (4) The ligation reaction product was transformed into competent E. coli DH5α cells.

[0080] Take 100 μL of competent cells, thaw at room temperature, and gently shake several times to suspend the cells evenly after complete thawing; add 10 μL of ligation reaction solution to the competent cells, mix gently, incubate on ice for 5 min, heat shock at 42℃ for 90 s, and then incubate on ice for 3 min; add 400 μL of LB liquid medium (without antibiotics), and incubate at 37℃ with shaking for about 30 min; take 60 μL of bacterial culture, spread it evenly on LB solid medium plates containing 50 μg / mL of Amp using a spreader, and incubate upside down at 37℃ for about 12 h.

[0081] (5) Identify clones and extract plasmids

[0082] Single colonies grown on the plate were picked and added to LB liquid medium (containing 50 μg / mL of Amp), and cultured at 37°C with shaking for 8-12 h. The bacterial culture was then verified by PCR using universal primers (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3').

[0083] The PCR products were subjected to agarose gel electrophoresis, and the results are as follows: Figure 3 As shown, two MYB genes were cloned, denoted as CcMYB7 and CcMYB8, respectively. The bacterial culture containing the target band was sent to Sino-American Taihe Biotechnology (Beijing) Co., Ltd. for sequencing. The sequencing results confirmed that the ORF of CcMYB7 was 876 bp, and the ORF of CcMYB8 was 909 bp.

[0084] The CcMYB7 nucleotide sequence is shown in SEQ ID NO.5:

[0085] ATGTCCACTGTCACCAAGAAAGGGTGCAACCCAGAAGAGGACATTGAGCTTAGGAGAGGACCATGGACTCTTGAAGAAGACACTCTCCTCATCCACTACATTGCTTGTCATGGTGAAGGACGTTGGAATCTTGTAGCCAGATGTTCAGGACTGAAGAGAACCGGCAAGAGTTGCAGATTGAGATGGCTGAATTACTTGAAACCCGATGTGAAGCGTGGGAATCTTACGCCAGAAGAACAGCTCGTGATTCTTGAACTCCATTCCAAGTGGGGCAATAGGTGGTCTAAGATTGCACAACATCTTCCAGGAAGAACAGACAATGAAATAAAGAACTATTGGCGAACGAGGGTGCAAAGACAAGCACGCCAGCTCAAGATTGAAACAAACAGCGCTAGGTTTCGAGATGTGGTCCGGTGGTTTTTGATGCCTAAATTGCTTCAAAAGATGCCATATCAGTCATCAATCCAATCTCTAAATTCAACCCCTATTCAACTCAACACACCCCCAGAGTCACTCAGCTTTATTGAACAAAATACAGTAAACAATCCCTACTATTCTGACAAATCCATCTCAGAAGGTTGGAGCCCCAGCATCTGTTCAAACTCCCTTAACATCACCCAATTCCCTCAAATCTCAGAATTCCCAGCAACTCCTCAGGATTTTGGCTATATTGACCACAGTCCACTTTTGAAAGAAAGCTACAATAGCTATGAGTCAGAGGCTATCCAACTAGCTTCCACCGCAACACCAGGCTTCAATTATCCTTACTATGATTCCTCAATGACTGAGAACCCCTGTATTGATGACATGGCAGACAGTCTATGGAACATGGATGAGTTTTGGCAGTTACAAAAGCTACAAGGAAGGAGCATCTAA;

[0086] The nucleotide sequence of CcMYB8 is shown in SEQ ID NO.6:

[0087] ATGTCTTCGGCAACCAAGAGGGGGTGCAAACAAGATGAGGAAGACACTGAGCTGAGG

[0088] AGAGGGCCATGGACTCTTGACGAAGACACCCTTCTCATTCATTACATTGCTTGTCATGGG

[0089] GAAGGGCGTTGGAACCTTGCTGCAAGAAGTTCAGGATTGAAGAGAACTGGCAAGAGCT

[0090] GCAGATTGAGATGGCTAAATTACTTGAAACCTGATGTGAAGCGAGGGAACCTTACACCA

[0091] GAGGAGCAGCTTGTCATTCTTGAGCTCCATTCCAAGTGGGGGAACAGGTGGTCTAAGAT

[0092] TGCAAAACAACTCCCAGGGAGAACAGACAATGAAATAAAGAATTATTGGAGAACTAGG

[0093] GTGCAAAAACAAGCACGGCAGCTCAAGATCGATGCGAACAGCTCTAGGTTTAAAGATAT

[0094] GATCCGTTTGTTGTGGATGCCTAGATTGCTTGAGAAGATGACATCTCCTCCATCTATCCAA

[0095] TCTCTAAATCCAACCTCCACTCTACTCAACTTTGTCCCTCAATCATATCCATCACCTCCAG

[0096] AGCTTCTTAGTGCTAGTGAGCAAACTACGGTTACCACATCCTGTTATTCTCACAAATCAT

[0097] TCTCAGAGAACTGCAGCCCCAGCATCTCTTCAAATTCCATGAACACCACCCAACTCCCT

[0098] CAAATCTCAGAATTCCCAACAAGTCCTAAAGACTTTGGCAATATTGACCACACCCAACT

[0099] TTTGAAAGAATATTGTTATGTGGACGAAAGCTTTGACTTAGAGGACTTACACCTTTCTTC

[0100] CACATCTACTGCAGCACTCAATTATCCGACCTCTGATTTCTCAATGACACAGAACATTTG

[0101] TGTTGACGACATGGTAGATAGTCTATGGAACATGGATGAGCTATGGCAGTTTAGAAAGCTGCAAGGAAGGTGCATCTAG.

[0102] Add the correct single-clone bacterial culture to 10 mL of LB liquid medium (containing 50 μg / mL of Amp), and incubate at 37°C with shaking for 8-12 h. Extract the plasmid using the Multicolor Endotoxin-Free Plasmid Mini-Prep Kit (MF077-01), following the instructions in the manual, to obtain the T-vector plasmid containing the target gene.

[0103] 4. Bioinformatics Analysis

[0104] The physicochemical properties of proteins encoded by the CcMYBs gene were predicted and analyzed using the online tool ExpasyProtParam; as shown in Table 2, they were all predicted to be hydrophilic proteins.

[0105] Table 2 Predicted Physicochemical Properties of CcMYBs

[0106]

[0107] The amino acid sequence of CcMYB7 is shown in SEQ ID NO.7:

[0108] MSTVTKKGCNPEEDIELRRGPWTLEEDTLLIHYIACHGEGRWNLVARCSGLKRTGKSCRLRWLNYLKPDVKRGNLTPEEQLVILELHSKWGNRWSKIAQHLPGRTDNEIKNYWRTRVQRQARQLKIETNSARFRDVVRWFLMPKLL QKMPYQSSIQSLNSTPIQLNTPPESLSFIEQNTVNNPYYSDKSISEGWSPSICSNSLNITQFPQISEFPATPQDFGYIDHSPLLKESYNSYESEAIQLASTATPGFNYPYYDSSMTENPCIDDMADSLWNMDEFWQLQKLQGRSI.

[0109] The amino acid sequence of CcMYB8 is shown in SEQ ID NO.8:

[0110] MSSATKRGCKQDEEDTELRRGPWTLDEDTLLIHYIACHGEGRWNLAARSSGLKRTGKSCRLRWLNYLKPDVKRGNLTPEEQLVILELHSKWGNRWSKIAKQLPGRTDNEIKNYWRTRVQKQARQLKIDANSSRFKDMIRLLWMPRLLEKMT SPPSIQSLNPTSTLLNFVPQSYPSPPELLSASEQTTVTTSCYSHKSFSENCSPSISSNSMNTTQLPQISEFPTSPKDFGNIDHTQLLKEYCYVDESFDLEDLHLSSTSTAALNYPTSDFSMTQNICVDDMVDSLWNMDELWQFRKLQGRCI.

[0111] 5. Subcellular localization

[0112] (1) Constructing a recombinant vehicle

[0113] The homologous arm primers for the CcMYBs gene and the pCAMBIA1300-35S-EGFP vector were designed and are shown in Table 3. They were then sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0114] Table 3 Primers for the homologous arm of the target gene and pCAMBIA1300-35S-EGFP

[0115]

[0116] Using the T-vector plasmid containing the target gene as a template, PCR amplification was performed using the primers listed in Table 3. The amplification system consisted of 2 μL of template and KOD One. TM PCRMaster Mix-Blue 25 μL, primer F 1.5 μL, primer R 1.5 μL, ddH2O 20 μL (to a final volume of 50 μL); amplification program: 98℃ for 30 s, 98℃ for 10 s, 60℃ for 10 s, 68℃ for 5 s, 68℃ for 15 s, 32 cycles. After the reaction, the PCR products were verified by agarose gel electrophoresis. The target band was then excised and recovered to obtain the linearized insert.

[0117] The empty pCAMBIA1300-35S-EGFP plasmid was digested with enzymes. The digestion system consisted of 10 μL of pCAMBIA1300-35S-EGFP plasmid, 1.3 μL of BamHI-HF, 1.3 μL of SacHI-HF, 5 μL of rCutsmart buffer, and 32.4 μL of ddH2O (totaled to 50 μL). The reaction was carried out at 37°C for 1 h. After the reaction, electrophoresis was performed to check the enzyme digestion status, and the linearized vector was recovered by gel extraction.

[0118] Ligation system: 2 μL linearized vector, 1 μL linearized insert, 5 μL 2×EasyGenoSingleAssemblyMix, 2 μL ddH2O (total to 10 μL); after mixing the reaction solution thoroughly, centrifuge briefly to the bottom of the tube. Incubate at 50℃ for 15 min. After the reaction, briefly centrifuge the reaction solution in the centrifuge tube and cool it on ice before proceeding with subsequent transformation experiments.

[0119] (2) Transformation of E. coli competent cells DH5α with recombinant vector

[0120] Take 100 μL of competent cells, thaw at room temperature, and gently shake several times to suspend the cells evenly after complete thawing; add 10 μL of ligation product to the competent cells, mix gently, incubate on ice for 5 min, heat shock at 42℃ for 90 s, and then incubate on ice for 3 min; add 400 μL of LB liquid medium (without antibiotics), and incubate at 37℃ with shaking for about 30 min; take 100 μL of bacterial culture and spread it on LB solid medium plates (containing 50 μg / mL of Kan), and incubate upside down at 37℃ for 12-14 h.

[0121] Single colonies grown on the plate were picked and added to LB liquid medium (containing 50 μg / mL Kan), and cultured at 37°C with shaking for 8-12 h. Colony PCR was then performed for verification using universal primers (35S: GACGCCACAATCCCACTATCC, PEGFP-N-3: CGTCGCCGTCCAGCTCGACCAG). PCR products were run on agarose gel electrophoresis, and colonies showing the target band were sent to Sino-American Taihe Biotechnology (Beijing) Co., Ltd. for sequencing. Plasmids were extracted from the correct colony culture by shaking.

[0122] (3) Transformation of Agrobacterium GV3101 competent cells with recombinant vector

[0123] Thaw 100 μL of Agrobacterium tumefaciens 3101 on ice; add about 1 μg of the plasmid to be transformed, mix gently, and let stand on ice for 5 min; freeze rapidly in liquid nitrogen for 5 min; quickly place the centrifuge tube in a 28°C water bath for 5 min; keep in an ice bath for 5 min; add 700 μL of LB liquid medium (without antibiotics) under aseptic conditions, and shake at 28°C for 2-3 h; collect the bacteria by centrifugation at 6000 rpm for 1 min, and collect 100 μL of supernatant. After resuspending and mixing the bacteria, add it to an LB solid medium plate containing 50 μg / mL Kans and 20 μg / mL Rif. Spread evenly and incubate upside down at 28°C for 48-60 h.

[0124] Single colonies grown on the plate were picked and added to LB liquid medium (containing 50 μg / mL Kan + 20 μg / mL Rif), and cultured at 28°C with shaking for 8-12 h. PCR verification was then performed using universal primers (35S: GACGCCACAATCCCACTATCC, pEGFP-N-3: CGTCGCCGTCCAGCTCGACCAG). PCR products were run on agarose gel electrophoresis; positive clones with the target band could be used for subsequent experiments.

[0125] (4) Subcellular localization determination by Agrobacterium-mediated transient transformation of tobacco

[0126] Take 200 μL of positive clone bacterial culture and place it in a 15 mL centrifuge tube. Add 3 mL of LB liquid medium (containing 50 μg / mL Kan + 20 μg / mL Rif) and incubate overnight. Take a 50 mL centrifuge tube, add 10 mL of LB liquid medium (containing 50 μg / mL Kan + 20 μg / mL Rif), then add 3 mL of the overnight culture. Incubate at 28 °C with shaking for 6-8 h until OD (Organic Dose) is reached. 600 The concentration was approximately 0.6; the cells were centrifuged at 5000 rpm for 8 minutes, and the supernatant was removed; the cells were resuspended in Agrobacterium infection medium (tobacco-specific) (containing 100 μM acetylsuccinone AS), and the bacterial concentration was adjusted to OD. 600 The concentration is around 0.6. After placing the plant in the dark for 2 hours, gently shake the bacterial solution. Use a 1mL sterile syringe to draw up the bacterial solution, remove the needle, place the syringe tip against the back of the leaf, and gently inject the bacterial solution until the bacterial solution is observed to completely saturate the entire tobacco leaf. Remove the bacterial solution adhering to the leaf surface, place the plant in the dark for 12 hours, and then culture normally for 3-4 days.

[0127] Subcellular localization observation: Tobacco leaves infected with Agrobacterium were cut into small pieces approximately 0.5cm × 0.5cm, placed on a glass slide with the back side facing up, covered with a coverslip, and then observed under a laser confocal microscope to determine whether EGFP was expressed. When observing EGFP, Ex = 488nm and Em = 514nm. Results are as follows... Figure 4As shown, the control group showed green fluorescence throughout the entire cell, while the experimental group showed green fluorescence only in the cell nucleus, indicating that both CcMYB7 and CcMYB8 proteins are located in the cell nucleus.

[0128] 6. Tissue expression specificity analysis

[0129] CcMYBs gene expression analysis was performed on different tissues (roots, stems, and leaves) of camphor tree, with three biological replicates for each gene. Total RNA extraction and first-strand cDNA synthesis were performed using the same methods as before.

[0130] The qRT-PCR primers are shown in Table 4 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0131] Table 4 Primers used for qPCR

[0132]

[0133] The cDNA obtained from reverse transcription was diluted 5 times and used as a template. The camphor actin gene CcActin was used as an internal reference gene, and qRT-PCR was performed using SuperReal premixed real-enhanced version (SYBR Green).

[0134] qRT-PCR system: cDNA template 1 μL, 2×SuperReal PreMix Plus 5 μL, primer F 0.3 μL, primer R 0.3 μL, ddH2O 3.4 μL; qRT-PCR reaction program: pre-denaturation: 95℃ for 15 min; quantification analysis: 95℃ for 10 s, 55℃ for 30 s, 72℃ for 32 s, 40 cycles; melting curve analysis: 95℃ for 5 s, 55℃ for 1 min, then increase the temperature to 95℃ and collect the fluorescence signal; None: 50℃ for 30 s.

[0135] Each reaction was performed in triplicate (biological replicates) and triplicate (technical replicates), with water and DNA-free, untranscribed RNA used as templates as NTC and NRC negative controls, respectively. Amplification and melting curves of qRT-PCR were analyzed after the reactions. The raw data were processed using Microsoft Excel 2019 and analyzed using 2... －ΔΔCT The method performs calculations on the data and uses GraphPad Prism 9.5.0 software for subsequent data analysis and chart creation.

[0136] The results are as follows Figure 5 As shown, CcMYBs are expressed in different tissues. The expression level of CcMYB8 is not significantly different in the three tissues. CcMYB7 has the highest expression level in the root and the lowest expression level in the leaf.

[0137] Example 2: Functional analysis of CcMYBs genes in camphor trees

[0138] 1. Construction of overexpression vectors

[0139] Based on the BamHI restriction site of the PBI121-GUS plasmid, homologous arm primers for the CcMYBs gene and PBI121-GUS were designed, as shown in Table 5, and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0140] Table 5 Primers for the target gene and the homologous arm of PBI121-GUS

[0141]

[0142] Using the T-vector plasmid containing the target gene prepared in Example 1 as a template, PCR amplification was performed using the primers in Table 5. The amplification system consisted of 2 μL of template and KOD One. TM PCR Master Mix-Blue 25 μL, primer F 1.5 μL, primer R 1.5 μL, ddH2O 20 μL (to a final volume of 50 μL); amplification program: 98℃ for 30 s, 98℃ for 10 s, 60℃ for 10 s, 68℃ for 5 s, 68℃ for 15 s, 32 cycles. Electrophoresis was performed after the reaction to verify the result. The target band was then excised and recovered from the gel to obtain the linearized insert.

[0143] The empty PBI121-GUS plasmid was digested with a single enzyme. The digestion system consisted of 10 μL of PBI121-GUS plasmid. BamHⅠ 1μL, 5 μL buffer, 34 μL ddH2O (to a final volume of 50 μL); react at 37 °C for 15 min, then inactivate at 80 °C for 20 min. After the reaction, perform electrophoresis to check the enzyme digestion and then perform gel extraction to obtain the linearized vector.

[0144] Ligation system: 4 μL linearized vector, 1 μL linearized insert, 5 μL 2×EasyGeno SingleAssemblyMix; after mixing the reaction solution thoroughly, centrifuge briefly to the bottom of the tube. Incubate at 50℃ for 15 min. After the reaction, briefly detach the centrifuge tube and cool it on ice for use in transformation experiments.

[0145] 2. Transform E. coli competent cells DH5α with the overexpression vector, following the same steps as in the subcellular localization section of Example 1.

[0146] 3. Transform Agrobacterium GV3101 competent cells with the overexpression vector, following the same steps as in the subcellular localization section of Example 1.

[0147] 4. Agrobacterium-mediated transformation of camphor leaves

[0148] (1) Preparation of bacterial culture

[0149] Take 500 μL of the accurately identified Agrobacterium tumefaciens bacterial suspension and the Agrobacterium tumefaciens bacterial suspension containing the empty vector into 50 mL centrifuge tubes, add 15 mL of LB liquid medium (containing 50 μg / mL Kan + 20 μg / mL Rif), and incubate overnight at 28 °C. Centrifuge at 5000 rpm for 8 min to collect the bacteria, discard the supernatant, resuspend in resuspending buffer (1 / 2 MS + 100 μMAS), and adjust to OD200. 600 The value is around 0.8. Let it stand in the dark at room temperature for 2 hours.

[0150] (2) Infection of camphor leaves

[0151] The disinfected young camphor leaves were placed into the prepared bacterial solution, and a vacuum pump (with the pressure set to -0.09 MPa) was used to evacuate the air for 30 minutes to promote the penetration of Agrobacterium into the leaf cells. The bacterial solution was then filtered out using a cell sieve, and the infected leaves were washed with sterile water. The leaves were then dried with sterile filter paper and finally inoculated onto a co-culture medium: MS + sucrose 30 g / L + agar 6.0 g / L + AS 100 μM, and cultured in the dark for 4 days.

[0152] (3) RNA extraction from camphor leaves

[0153] After 4 days of co-culturing, camphor leaves were washed with sterile water to remove the bacterial suspension, then dried with sterile filter paper. The samples were collected in aluminum foil and placed in liquid nitrogen at -80℃. RNA was extracted from the leaves using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441), following the instructions. The extracted leaf RNA was analyzed for concentration and detected by agarose gel electrophoresis. The results showed a concentration between 91.6 and 424.6 ng / μL, with distinct 28S and 18S bands on the electrophoresis. Figure 6 The results indicate that the extracted total RNA was intact, of good quality, and of high concentration, and could be used for subsequent experiments. Five samples were taken from each treatment to extract RNA, and three of the RNA samples with high quality and concentration were selected for reverse transcription.

[0154] (4) qRT-PCR detection

[0155] The extracted RNA was reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (Genomic De-transfer). qRT-PCR was performed using the cDNA as a template to analyze the gene expression levels of *Cinnamomum camphora* CcMYBs and *Eucalyptus globulus* oil synthase gene CcTPS16. The method and procedures were the same as those in the tissue expression specificity analysis in Example 1. Leaves transformed with the empty vector served as the control group (CK). Specific primers for the CcTPS16 gene were: TPS16-F (quantitative): ACAATCTCCCAGTCCGCA, TPS16-R (quantitative): CATCACCCATTAACTTCTCG.

[0156] The results are as follows Figure 7 As shown, the expression level of CcMYB7 in the OE-CcMYB7 treatment group was 9.12 times that of the control group, indicating a significant increase and successful overexpression in leaves. The expression level of TPS16 in the OE-CcMYB7 group was significantly increased, reaching 19.40 times that of the control group. In the OE-CcMYB8 treatment group, the expression level of CcMYB8 showed no significant change, remaining 1.18 times that of the control group, but the expression level of TPS16 was significantly increased, reaching 26.77 times that of the control group. These results indicate that both CcMYB7 and CcMYB8 have certain regulatory effects on the eucalyptus oil synthase gene TPS16.

[0157] 5. Agrobacterium-mediated callus transformation in camphor trees

[0158] (1) Induction and culture of camphor callus

[0159] After disinfection, young camphor leaves were inoculated onto camphor callus induction medium: MS + 6-BA 5.0 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + agar 5.5 g / L, pH around 5.8, autoclaved at 121℃ for 20 min, and cultured for about 30 days; the induced callus was then subcultured onto subculture medium (the same as the induction medium), with a subculture cycle of about 4 weeks.

[0160] (2) Preparation of bacterial culture

[0161] Take 500 μL of the accurately identified Agrobacterium tumefaciens bacterial culture (successfully transformed into the overexpression vector) and the Agrobacterium tumefaciens bacterial culture containing the empty vector (CK) into 50 mL centrifuge tubes, add 15 mL of LB liquid medium (containing 50 μg / mL Kan + 20 μg / mL Rif), and incubate overnight at 28 °C; collect the bacteria by centrifugation at 5000 rpm for 8 min, discard the supernatant; resuspend in resuspending buffer (1 / 2 MS + 100 μMAS), and adjust to OD200. 600 The value is around 0.6. Let it stand in the dark at room temperature for 2 hours.

[0162] (3) Infection of callus tissue

[0163] Callus tissues subcultured for approximately 20 days were placed into prepared bacterial suspensions and inoculated for 30 minutes. The bacterial suspension was then filtered out using a cell strainer, and the surface of the callus tissues was blotted dry with sterile filter paper. Finally, the callus tissues were inoculated onto co-culture medium: MS + 6-BA 5.0 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + agar 6.0 g / L + AS 100 μM, and incubated in the dark for 3 days. Each sample was repeated 3 times.

[0164] (4) RNA extraction from callus tissue

[0165] After callus culture in the dark, the surface bacterial solution was washed 3-5 times with sterile water, and the surface moisture was blotted dry with sterile filter paper. The sample was then wrapped in aluminum foil and placed in liquid nitrogen at -80℃, following the same steps as in Agrobacterium-mediated transformation of camphor leaves. The RNA concentration was detected by spectrophotometry to be between 47.1-445.2 ng / μL, and RNA integrity was detected by agarose gel electrophoresis. The results are as follows. Figure 8 As shown, the electrophoresis revealed distinct 28S and 18S bands, indicating that the extracted total RNA was intact and of good quality. Samples that passed the test can be used for subsequent experiments or stored at -80°C.

[0166] (5) qRT-PCR detection

[0167] Extracted RNA was reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (Genomic De-transformation). qRT-PCR was performed using the cDNA as a template to analyze the expression level of the *C. camphor* CcMYBs gene, following the same steps as those involved in *Agrobacterium*-mediated transformation of *C. camphor* leaves. Each reaction was performed in triplicate (biological and technical replicates). Results are shown below. Figure 9 As shown, the expression levels of CcMYBs increased significantly by several times, and the expression level of CcMYB7 increased by more than 100 times.

[0168] 6. Metabolomics research

[0169] (1) The differences in metabolites in camphor callus / leaf tissues before and after CcMYB7 and CcMYB8 overexpression were detected by LC-MS.

[0170] 0.1 g to 0.12 g of each sample was added to a pre-cooled and thawed fixative solution (water-methanol-acetonitrile 1:4:4) at a ratio of 1 mg:10 μL, and steel balls were added. The sample was then placed in a grinder and ground at -20°C and 70 Hz for 180 s. After grinding, the sample was incubated at -20°C for 1 h. After incubation, the sample was centrifuged at 4°C and 12000 rpm for 15 min. The supernatant was then collected in a liquid chromatography vial. Simultaneously, aliquots of the supernatant from all samples were mixed to prepare the quality control (QC) sample.

[0171] Chromatographic conditions:

[0172] ACQUITY UPLC BEH AMIDE column (2.1 mm × 100 mm, 1.7 μm); mobile phase A: 25 mmol·L⁻¹ -1 Ammonium hydroxide (NH4OH) + 25 mmol·L -1 Ammonium acetate (NH4OAc) aqueous solution, mobile phase B is acetonitrile, gradient elution (0 min–1 min, 95% B; 1 min–14 min, 95%–65% B; 14 min–16 min, 65%–40% B; 16 min–18 min, 40% B; 18 min–18.1 min, 40%–95% B; 18.1 min–23 min, 95% B); flow rate 0.3 mL / min -1 The injection volume was 2 μL. All samples were injected randomly during data acquisition.

[0173] Mass spectrometry conditions:

[0174] LC-MS / MS analysis was performed using a UHPLC system (ExionL CAD, ABSCIEX, USA) equipped with an ACQUITYUPLC BEHAmide column (2.1 mm × 100 mm, 1.7 μm, Waters) coupled with a four-polarity time-of-flight mass spectrometer (TripleTOF 5600+, AB SCIEX, USA). The column temperature was set to 25 °C. Mobile phase A consisted of 25 mM ammonium hydroxide (NH4OH) + 25 mM ammonium acetate (NH4OAc) dissolved in water, and mobile phase B consisted of acetonitrile. Both positive (ESI+) and negative (ESI-) modes were suitable, with a flow rate of 0.3 mL / min. All samples were randomly injected during data acquisition using information-dependent acquisition (IDA) mode, with an injection volume of 2 μL. The source parameters were set as follows: Ion source gas 1 (GAS1) 50 psi (1 psi ≈ 6.895 kPa); Ion source gas 2 (GAS2) 50 psi; Curtain gas (CUR) 35 psi; Temperature (TEM) 500℃; Declustering potential (DP) 80 V in positive mode and -80 V in negative mode; Ion spray voltage float (ISVF) 5500 V in positive mode and -4500 V in negative mode. The TOF MS scan parameters were set as follows: Scan range 60 Da~1200 Da; Accumulation time 200 ms; Dynamic background subtraction enabled. The product ion scanning parameters were set as follows: scanning range 25 Da to 1200 Da; accumulation time 50 ms; collision energy 35 V in positive mode and -35 V in negative mode; collision energy spread 15; resolution UNIT; charge state 1:1; intensity 100 cps; exclusion of isotopes within 4 Da; mass tolerance 10 ppm; maximum number of candidate ions monitored per cycle 10; exclusion of the previous target ion within 4 s after it appears twice.

[0175] Raw data were processed using XCMSPlus v3.6.3 for peak detection, extraction, alignment, integration, and annotation. The centWave algorithm was used for peak picking at a resolution of 15 ppm, with peak widths ranging from 5 to 20 s and a signal-to-noise ratio of 6. The Obiwarp algorithm was used for retention time correction. Isotope ions were annotated. Metabolite annotation and data analysis were then performed using MSDIAL 5.1.2 and MetaboAnalyst 5.0, including principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Differential metabolites were screened based on preliminary data analysis results, using the following criteria: variable importance inprojection (VIP) > 1 in the OPLS-DA model, p < 0.05 in the T-test, and fold change (FC) ≥ 2 or FC ≤ 0.5. Finally, the screened metabolites were imported into MetaboAnalyst 5.0 for metabolic pathway enrichment analysis.

[0176] The results showed that 89 differentially expressed metabolites were screened from the camphor callus CK group (callus not overexpressing CcMYB7) compared to the MYB7 group (callus overexpressing CcMYB7), of which 38 were upregulated and 51 were downregulated. The key metabolic pathways screened included: glyoxylate and dicarboxylate metabolism, nitrogen metabolism, the citric acid cycle (TCA cycle), carbon fixation by the Calvin cycle, pyruvate metabolism, and valine, leucine, and isoleucine degradation. Figure 10 A total of 37 differentially expressed metabolites were screened from camphor tree leaves in the CK1 group (leaves not overexpressing CcMYB7) compared to the MYB7 group (leaves overexpressing CcMYB7), of which 21 were upregulated and 16 were downregulated. Some of the differentially expressed metabolites are shown in Table 6.

[0177] Table 6. Some differentially expressed metabolites

[0178]

[0179]

[0180] None of the differentially expressed metabolites in the table were present in the CcMYB8 overexpression group. Some differentially expressed metabolites, such as TIC (total ion current), were also present. Figure 11 As shown.

[0181] (2) GC-MS was used to detect the differences in metabolites in camphor callus tissue before and after CcMYB7 overexpression (the analysis was commissioned to Shanghai Baiqu Biomedical Technology Co., Ltd., with 5 samples per group). The results showed that 137 differentially expressed metabolites were detected in the CK group (non-overexpressing CcMYB7) compared to the OE-CcMYB7 group (overexpressing CcMYB7). Differentially expressed metabolites with VIP>1, p<0.05, and fold change FC>1 or FC<0.5 were selected. Among them, 3 were upregulated and 20 were downregulated. The results of the upregulated metabolites and the top 10 downregulated metabolites are presented below. Figure 12 Among them, differentially expressed metabolites with significant variations may correspond to enzyme gene expression that is strongly activated or strongly inhibited. The fold changes of differentially expressed metabolites are shown in Table 7.

[0182] Table 7. Fold change in differentially metabolites

[0183]

[0184] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0185] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A camphor MYB transcription factor gene, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

5.

2. A camphor MYB transcription factor, characterized in that, The amino acid sequence is shown in SEQ ID NO.

7.

3. The primer pair for cloning the gene of claim 1, characterized in that, The upstream primer is shown in SEQ ID No.

1. The downstream primer is shown in SEQ ID No.

2.

4. A reagent kit, characterized in that, It includes the primer pair described in claim 3.

5. A recombinant expression vector, expression cassette, or host bacterium containing the gene of claim 1.

6. The application of the gene of claim 1, the transcription factor of claim 2, the primer pair of claim 3, the kit of claim 4, or the recombinant expression vector, expression cassette, or host bacterium of claim 5 in the regulation of camphor tree metabolism; The metabolic regulation is as follows: (1) Regulation of flavonoid metabolism: The overexpression of the camphor MYB transcription factor gene negatively regulates the production of baicalein 4'-methyl ether 8-(2'',4''-disulfonamide glucuronide); (2) Regulation of terpenoid metabolism: The overexpression of the camphor MYB transcription factor gene negatively regulates geraniol D, trans-bergamot glycerene, caryophyllene, α-santalene, and (-)-Alpha-pinene, and positively regulates canocoridine C, arganin, and 3-O-[β-D-glucose(1→2)-β-D-glucose(1→4)-β-D-galactose]-(25R)-12-oxo-5α-spirost-3β-ol. (3) Regulation of organic acid metabolism: The overexpression of the camphor MYB transcription factor gene negatively regulates gambogeylic acid, malic acid, cis-5, and cis-12-octadecadienoic acid, and positively regulates methylmalonic acid. (4) Regulation of alkaloid metabolism: The overexpression of the camphor MYB transcription factor gene positively regulates dormantin, oleandrin, alpine thalidomide, and protoberberine alkaloids. (5) Regulation by amide metabolites: The overexpression of the camphor MYB transcription factor gene positively regulates clofamide; (6) The overexpression of the camphor MYB transcription factor gene positively regulates the short peptide chain L-serine-L-alanyl-L-serine-L-phenylpropionyl-L-leucine-L-phenylpropionyl-L-serine; (7) The overexpression of the camphor MYB transcription factor gene positively regulates pentafluoroethyl radicals.

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