CcCYP81bp12 protein of camphor tree, encoding gene thereof and application in sesamin synthesis

Abstract

The application discloses a Cinnamomum camphora CcCYP81BP12 protein, a coding gene thereof and application of the CcCYP81BP12 protein in sesamin synthesis, and belongs to the field of genetic engineering. The application provides the CcCYP81BP12 protein with an amino acid sequence as shown in SEQ ID NO. 2 and the gene with a nucleotide sequence as shown in SEQ ID NO. 1 CcCYP81BP12 The in-vitro experiment shows that the protein can catalyze various substrates to generate sesamin. CcCYP81BP12 The experiment result shows that, compared with a non-transgenic strain, The sesamin content of the transgenic strain is significantly increased from 2.12 μg / g to 797.96 μg / g, and the gene is a key gene for positively regulating sesamin synthesis. The application provides a gene resource and an operation target point with important application value for plant sesamin synthesis regulation, and can be applied to metabolic engineering and synthetic biology research.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a camphor tree CcCYP81BP12 protein, its encoding gene, and its application in sesamin synthesis. Background Technology

[0002] Camphor tree ( Cinnamomum camphora ) belongs to the genus Cinnamomum of the family Lauraceae. Cinnamomum Pininol is an evergreen tree, an important timber and special economic tree species in subtropical evergreen broad-leaved forests. Not only is its wood of excellent quality, but its roots, bark, branches, leaves, and fruits can all be used to extract terpenes, polyphenols, and other secondary metabolites, making them important raw materials for medicine, chemicals, food, and spices. Currently, pininol... β Various lignan polyphenolic compounds, such as D-glucoside, sesamin, and pinoresinol, have been isolated, purified, and identified from camphor tree extracts, and have been verified to have antibacterial, anti-hepatocellular carcinoma, anti-inflammatory, anthelmintic, anti-allergic, and antioxidant functions.

[0003] Sesame( Sesamum indicum Camphor tree leaves are a major source of important antioxidant lignans such as sesamin, sesaminin, sesaminol, and sesamol. The sesamin content in camphor tree leaves can reach (2.596±0.27) mg / g, which is comparable to the average sesamin content in sesame (2.54±1.57) mg / g, making it an ideal plant material for extracting sesamin.

[0004] Sesamin belongs to the ditetrahydrofuran lignan class of polyphenols. The common lignan precursor, coniferyl alcohol, is synthesized from phenylalanine or tyrosine via the phenylpropane pathway. Coniferyl alcohol dimerizes under the combined action of laccase and dirigent protein, stereoselectively producing pinoresinol. Pinoresinol is then catalyzed by enzymes such as cytochrome P450 to form sesamin and other compounds. Cytochrome P450 is a superfamily of heme-thiolate proteins, one of the largest enzyme groups in plants, capable of catalyzing a wide variety of redox reactions, such as CH hydroxylation, C=C double bond epoxidation, N- and S- oxidation, O-, N- and S- dealkylation, and C / C bond cleavage. However, there are few reports on the application of cytochrome P450 in sesamin synthesis in camphor trees. Therefore, studying its sequence characteristics and catalytic products is of great significance for regulating the biosynthesis of sesamin and related active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a camphor tree CcCYP81BP12 protein and its encoding gene and its application in sesamin synthesis, in order to solve the problems existing in the prior art. This invention provides a gene resource and operational target with important application value for the regulation of plant sesamin synthesis, which can be used in metabolic engineering and synthetic biology research.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a camphor tree CcCYP81BP12 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides a gene encoding the above-mentioned camphor tree CcCYP81BP12 protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides an expression vector comprising the above-described coding gene.

[0009] The present invention also provides a recombinant bacterium comprising the above-described expression vector.

[0010] The present invention also provides an application of the above-mentioned camphor tree CcCYP81BP12 protein in the synthesis of sesamin.

[0011] The present invention also provides a method for synthesizing sesamin, comprising the steps of using (+)-pinene, (-)-pinene, (+)-epistene, or (-)-epistene as substrates and catalytically synthesizing it using the above-mentioned camphor tree CcCYP81BP12 protein.

[0012] The present invention also provides the application of the above-mentioned camphor tree CcCYP81BP12 protein, the above-mentioned encoding gene, the above-mentioned expression vector, or the above-mentioned recombinant bacteria in any of the following: (1) Application in regulating the sesamin content in plants; (2) Application in the cultivation of transgenic plants with increased sesamin content.

[0013] Furthermore, the expression level of the encoding gene is upregulated in the plant, thereby increasing the sesamin content of the plant; The plant in question is a camphor tree.

[0014] The present invention also provides a method for increasing the sesamin content in plants, comprising the step of upregulating the expression level of the gene encoding the camphor tree CcCYP81BP12 protein in the plant to promote the synthesis of sesamin in the plant; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0015] Furthermore, the plant in question is a camphor tree.

[0016] This invention also provides a breeding method for transgenic plants with increased sesamin content, comprising the following steps: The gene encoding the camphor tree CcCYP81BP12 protein was overexpressed in plant cells, the plant cells were then cultured, and the plant cells were used to regenerate plants, thus obtaining the transgenic plant with increased sesamin content. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0017] Furthermore, the plant in question is a camphor tree.

[0018] The present invention discloses the following technical effects: This invention provides the camphor tree CcCYP81BP12 protein with the amino acid sequence shown in SEQ ID NO.2 and the nucleotide sequence shown in SEQ ID NO.1. CcCYP81BP12 Gene. In vitro experiments revealed that this protein can catalyze the production of sesamin from various substrates. Experimental results showed that, compared to non-transgenic lines, CcCYP81BP12 The sesamin content in the transgenic lines significantly increased from 2.12 μg / g to 797.96 μg / g, indicating that this gene is a key gene positively regulating sesamin synthesis. This invention provides a gene resource and manipulative target with significant application value for the regulation of plant sesamin synthesis, and can be used in metabolic engineering and synthetic biology research. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for CcCYP81BP12 Image of PCR amplification products of the gene; Figure 2 for CcCYP81BP12 The results of gene expression pattern analysis are shown in the figure; among them, Bor is the dextrorotatory borneol type of camphor tree; Eur is the eucalyptol type of camphor tree; Cit is the citral type of camphor tree; Ner is the trans-nerolidol type of camphor tree; Lin is the linalool type of camphor tree; Figure 3 Figure 1 shows the LC-MS identification results of the CcCYP81BP12 recombinant protease reaction solution; (A) shows the LC-MS identification results using (+)-pineneol as the substrate; (B) shows the LC-MS identification results using (-)-pineneol as the substrate; (C) shows the LC-MS identification results using (+)-episteneol as the substrate; (D) shows the LC-MS identification results using (-)-episteneol as the substrate. Figure 4 for CcCYP81BP12 The results of gene expression level analysis in transiently transformed camphor trees are shown in the figure; among them, compared with the control group, P <0.01; Figure 5 for CcCYP81BP12 LC-MS results of sesamin content in camphor trees after transient gene transformation; among them, compared with the control group, P <0.05. Detailed Implementation Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 1 . CcCYP81BP12 Gene cloning 1.1 Experimental Materials Eucalyptus oil type camphor tree leaves.

[0026] 1.2 Experimental Methods 1.2.1 RNA Extraction Camphor tree leaves are rich in polysaccharides, polyphenols, and lipids, making RNA extraction challenging. This invention is based on and optimized from the instructions of an RNA extraction kit (Reagent Kit for Difficult-to-Extract Plant RNA, Guangzhou Meiji Biotechnology Co., Ltd.).

[0027] The specific steps are as follows: All consumables (centrifuge tubes, mortar and pestle, spatula, grinding hammer, etc.) are sterilized at 121℃ for 30 min and then dried for later use; the leaf samples are ground into powder using liquid nitrogen. Weigh 0.1-0.2 g of leaf sample and grind it quickly into powder in liquid nitrogen. Transfer the powder to a pre-cooled 2.0 mL centrifuge tube (the centrifuge tube is numbered in advance) and quickly freeze it in liquid nitrogen; add 750 μL of PAL lysis buffer (which already contains 2% mercaptoethanol preheated at 65℃) to the ground sample, vortex to disperse the sample, and lyse at 65℃ for 10 min; add 750 μL of nucleic acid extraction buffer, centrifuge at 13000 rpm, 4℃ for 6 min; transfer 600 μL of supernatant to a new centrifuge tube, add 900 μL of binding buffer (GXP2), gently pipette 10 times to mix, and transfer to an RNA purification column; centrifuge at 12000 rpm for 60 s, remove the filtrate, and repeat the operation until all the solution is filtered; add 700 μL of PAL lysis buffer (GXP2) to the sample. Centrifuge at 12000 rpm for 60 s with 750 μL RW1, discard the filtrate, then add 750 μL RW2, centrifuge at 12000 rpm for 60 s, and repeat the washing once; centrifuge at 14000 rpm for 2 min, and air dry in a 1.5 mL centrifuge tube for 2 min; add 28 μL sterile water, cover and let stand for 1 min, then centrifuge at 12000 rpm for 60 s.

[0028] 1.2.2 Gene Amplification Using cDNA obtained from reverse transcription of camphor leaf RNA as a template, forward and reverse primers were designed to amplify the amplified cDNA. CcCYP81BP12 The primer sequences are as follows: Forward primer: 5'-ATGGACGAACTCTTCTTCTTC-3', SEQ ID NO.3; Reverse primer: 5'-AAGACGAGAAAGGACCTCAAC-3', SEQ ID NO.4.

[0029] PCR amplification was performed using MCLAB high-fidelity DNA polymerase. The reaction system was as follows: cDNA 2 μL, 2×H Buffer 25 μL, forward primer 2 μL, reverse primer 2 μL, ddH2O 19 μL.

[0030] The PCR amplification conditions were: 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 52℃ annealing for 30 seconds, 72℃ extension for 2 minutes, 35 cycles; 72℃ final extension for 5 minutes. PCR products were detected by 1.5% agarose gel electrophoresis. CcCYP81BP12 The gene length is 1536 bp, and the results are as follows: Figure 1 As shown, Figure 1 The marker is DL2000, and the two lanes outside the marker are... CcCYP81BP12 Amplification products.

[0031] Agarose gel recovery was performed using the gel DNA recovery kit (brand: SIMGEN, catalog number: 2001050) from Hangzhou Xinjing Biological Reagent Development Co., Ltd., following the instructions in its manual.

[0032] The recovered fragments were then ligated to the BGV008 vector. The specific steps are as follows: use Kpn I and Nco The BGV008-GFP vector was double-digested with a fast digester, and the reaction system is shown in Table 1.

[0033] Table 1. BGV008-GFP vector double enzyme digestion reaction system The reaction system was incubated at 37°C for 60 min. DNA purification was then performed immediately according to the DNA purification kit instructions.

[0034] Will CcCYP81BP12 The amplified products were ligated with the double-digested BGV008-GFP linearized vector according to the seamless cloning instructions. The reaction system is shown in Table 2.

[0035] Table 2. Reaction system for ligation of amplification products with BGV008-GFP linearized vector The reaction system was incubated at 50 °C for 30-45 min.

[0036] CcCYP81BP12 After ligation of the amplified product with the BGV008-GFP vector, *E. coli* transformation was immediately performed. Positive bacteria verified by colony PCR were sent to Youkang Biotechnology Co., Ltd. for plasmid extraction and sequencing, and alignment was performed using Snapgene software. CcCYP81BP12 The CDS sequence was obtained through sequencing and cloning. CcCYP81BP12The nucleotide sequence of the gene is shown in SEQ ID NO.1. It contains 1536 bases and encodes a CcCYP81BP12 protein with a total length of 511 amino acids. The amino acid sequence of the CcCYP81BP12 protein is shown in SEQ ID NO.2.

[0037] SEQ ID NO.1:

[0038] SEQ ID NO.2: MDELFFFSALLFFISFFLISKLLFTDRSRHPKTPVPPSLPILGHLHLLKKPLHRSLTALSSRYGPLLSLRFGSRRVVVVSSPALVEECFTKNDIIFANRPRLLAGKYIGFNYTTLGWSSYGPHWRNLR RIAVTQILSSNRVHMFSFVRSDEVRALVKELFRDSYLGKVNMKQKFSELALNNMMQMIAGKRYYGERVVDLEEAKRFRENMRGSLAVSGMPNLLDFLPVVRWIGLKGLEKRMVGLAKRRDVLLQGLID EHTKKKMDGTFEIGEEKKKTTLIDVLLSLRETEEPEYYTDDIIKGLIMVMLGAGTETSSLTMEWAMSLLLNNPEVLKKAKAELDIQVGKDRLLDESDFDKLRYLHCIINETLRLYPAAPLLLPHDSSEE CTLGGLHVPRGTMLLVNVWAIHRDPDLWPDPTSFKPERFEGVEKDKEGLDFKFLPFGYGRRSCPGMGMAKKVVGLALGALIQCFEWERVGEEKVDMKDGGGLTLPKAQPLEAIYRPYADMVEVLSRL.

[0039] 2. Different chemical types of camphor trees CcCYP81BP12 qRT-PCR analysis of genes 2.1 Experimental Materials Camphor tree leaves of different chemical types (dextral borneol type, eucalyptol type, citral type, trans-nerolidol type and linalool type).

[0040] 2.2 CcCYP81BP12 qRT-PCR analysis of genes The PrimeScript™ RT reagent Kit (Takara, model RR047Q) was used for reverse transcription of the samples. Specific primers were designed using Primer 3.0 online software (https: / / bioinfo.ut.ee / primer3-0.4.0 / ), and the specificity of the CcCYP81BP12 gene primers was retested using Tbtools and NCBI. qRT-PCR was performed using the TB Green PremixEx Taq II FAST qPCR kit (Takara, model CN830A). The real-time quantitative PCR system contained 10 μL of premix, 1 μL each of upstream and downstream primers, 1 μL of cDNA template, and 7 μL of sterile water, for a total of 20 μL. The amplification program was performed for 40 cycles according to the Takara reagent instructions. Actin was used as an endogenous reference gene, and 2... -ΔΔCT Method Calculation CcCYP81BP12 The relative expression level of genes.

[0041] like Figure 2 As shown, the results indicate that CcCYP81BP12 The gene was expressed in the leaves of camphor trees of different chemotypes. CcCYP81BP12 The gene was specifically highly expressed in the eucalyptol type in July and August, and specifically highly expressed in the eucalyptol and trans-nerolidol types in September. Furthermore, it showed a high correlation with sesamin content, with correlations of 0.97, 0.75, and 0.79 with sesamin content in July, August, and September, respectively. CcCYP81BP12 Genes may play an important role in the biosynthesis of sesamin.

[0042] 3. CcCYP81BP12 Construction of yeast expression vectors for gene expression, transformation of yeast and expression in eukaryotes 3.1 Carrier Construction pESC-Ura- CcCYP81BP12 The specific steps for constructing the carrier are as follows: use BamH I and Nhe The expression vector pESC-URA was digested with restriction endonuclease I and purified to obtain a linearized vector. Design specific primers to amplify primers containing homologous arms. CcCYP81BP12 The complete coding sequence (CDS) of the gene is used as the insertion fragment; Prepare the following recombination reaction system on ice: After brief centrifugation, react at 37°C for 30 min and then immediately place on ice to cool; All 10 μL of the recombinant product was transformed into competent E. coli cells; After a single colony grows, pick a single colony and mix it in 20 μL of sterile water by pipetting. Take 1 μL as a template for colony PCR verification. After shaking a single colony with the correct band size and extracting the plasmid, it was sent for sequencing. The plasmid with successful ligation and correct reading frame is the expression vector pESC-Ura- CcCYP81BP12 .

[0043] 3.2 Transformation of competent cells in Saccharomyces cerevisiae pESC-Ura empty vector and pESC-Ura- CcCYP81BP12 The recombinant expression vector plasmid was transformed into Saccharomyces cerevisiae WAT11 competent cells (which had been overexpressed with Arabidopsis thaliana-derived CYP450 reductase CPR) by chemical transformation.

[0044] 3.3 Yeast Conversion Operation Pretreatment of Carrier DNA: Insert the Carrier DNA into a 95°C metal bath for 5 min, then quickly insert it into ice after heating.

[0045] Take 100 µL of WAT11 competent cells thawed on ice, add 2-5 µg of pre-cooled target plasmid, 10 µL of pretreated carrier DNA, and 500 µL of PEG / LiAc, and mix by pipetting several times. Incubate at 30°C for 30 min (invert 6-8 times at 15 min to mix).

[0046] Place the centrifuge tubes in a 42°C water bath for 15 minutes (tumble 6-8 times at 7.5 minutes to mix).

[0047] Centrifuge at 5000 rpm for 40 s and discard the supernatant. Resuspend in 400 µL of ddH2O and centrifuge for 30 s, then discard the supernatant.

[0048] Resuspend in 50 µL of ddH2O, spread on defective SD-Ura solid medium, and incubate at 29°C for 48-96 h.

[0049] 4. Biochemical functions of CcCYP81BP12 yeast expressed protein 4.1 CcCYP81BP12 yeast expressed protein Select empty pESC-Ura vectors or pESC-Ura- CcCYP81BP12 A single positive colony of Saccharomyces cerevisiae was cultured in 1 mL of SD-Ura deficient liquid medium and shaken at 30°C and 220 rpm for 24 h. Transfer 400 μL of bacterial culture to 50 mL of SD Deficit Liquid Medium and incubate at 30°C and 220 rpm for 24 h. Centrifuge at 5000 rpm at room temperature for 5 min, then discard the supernatant; Resuspend in 10 mL of sterile water, centrifuge at 5000 rpm for 5 min at room temperature, and discard the supernatant; Repeat the previous step twice; All cells were transferred to 50 mL of SGR / -Ura induction expression medium containing galactose and raffinose, and cultured at 28°C and 220 rpm for 12 h. Add a final concentration of 50 μM of substrate (substrate is (+)-pinene, (-)-pinene, (+)-epistene, or (-)-epistene), and react at 25 °C and 220 rpm for 96 h. Add an equal volume of ethyl acetate for extraction, vortex for 5 min, sonicate for 30 min, repeat three times, and combine the extracts. The extract was concentrated to dryness by vacuum centrifugation, 200 μL of pure methanol was added, the mixture was vortexed for 5 min, centrifuged at 13000 rpm for 10 min, and the supernatant was collected for targeted quantitative detection.

[0050] 4.2 Targeted quantitative detection of sesamin High-resolution liquid chromatography-mass spectrometry (UPLC-Q-Orbitrap MS / MS) was used to detect compounds such as sesamin. Plant sample preparation: Weigh an appropriate amount of sample, add 1 mL of pure methanol and grinding beads, grind for 5 min, vortex for 10 min, centrifuge at 13000 rpm for 10 min at 4℃, filter the supernatant through a 0.22 μm filter membrane, and inject the filtrate for analysis. Accurately weigh appropriate amounts of each standard and prepare stock solutions of 2.00 mg / mL for later use. Dilute sequentially with pure methanol to prepare a series of working solutions for the standard curve at varying concentrations.

[0051] The specific testing parameters are as follows: The chromatographic conditions were as follows: column: Agela Venusil C18 Plus 50×2.1 mm, 5 μm; flow rate: 0.4 mL / min; aqueous phase: 0.1% formic acid water; organic phase: pure methanol; injection solution: methanol; column oven temperature: 35℃; autosampler temperature: 8℃; injection volume: 5.00 μL; and the chromatographic gradient is shown in Table 3.

[0052] Table 3 Chromatographic gradient Mass spectrometry conditions were as follows: Ion source: electrospray ionization (ESI); Scan mode: positive ion scan; Detection mode: PRM; Spray voltage: 4000 V (Positive); Capillary temperature: 320℃; Collision gas: high-purity argon (purity ≥99.999%); Sheath gas pressure: nitrogen (purity ≥99.999%), 50 Arb; Aux gas pressure: nitrogen (purity ≥99.999%), 15 Arb; Data acquisition time: 5.0 min.

[0053] The results are as follows Figure 3 As shown, the CcCYP81BP12 protein can catalyze the conversion of substrates (+)-pinene, (-)-pinene, (+)-epistene, and (-)-epistene into sesamin.

[0054] 5. CcCYP81BP12 Construction of gene overexpression vectors and Agrobacterium-mediated transformation CcCYP81BP12 For details on constructing the gene overexpression vector, please refer to the "1.2.2 Gene Amplification" section above. The steps for transforming Agrobacterium competent cells GV3101 are as follows: Thaw the competent cells on ice, add 2 μL of plasmid (approximately 100-500 ng) to 50 μL of competent cells, and gently tap the centrifuge tube.

[0055] Sequentially, 5 min on ice, 5 min in liquid nitrogen, 5 min at 37℃, and 5 min in ice.

[0056] Add 500 μL of antibiotic-free LB to a shaker at 28°C and 180 rpm for 2-3 h.

[0057] Centrifuge at 10,000 rpm for 10 s, discard the supernatant, and retain approximately 100 μL to spread on LB agar plates containing the appropriate antibiotic.

[0058] Incubate in the dark at 28℃ for about 48 hours, then pick single colonies for colony PCR identification.

[0059] 6. Determination of instantaneous transformation and sesamin content in camphor tree leaves Single colonies containing the target gene from the BGV008-GFP vector and those without the vector were picked and cultured in 2 mL LB liquid medium (containing 50 μg / L kanamycin and 25 μg / L rifampin) at 28°C and 200 rpm for 14–18 h until OD500. 600 It is 0.8.

[0060] Centrifuge at 5000 rpm for 2 min, discard the supernatant, resuspend the Agrobacterium cells in the infection solution, and centrifuge again. Resuspend the infection solution to OD. 600 =0.8, incubate at 28℃ for 2-4 hours.

[0061] The camphor seedlings to be transformed were placed in a vacuum desiccator, with their leaves completely immersed in the inoculum, and vacuum permeated for 20 minutes under a pressure of approximately 0.8 kPa. The permeated leaves were then completely wrapped in aluminum foil and kept in the dark. The treated seedlings were then returned to their original culture environment for further cultivation, and the aluminum foil was removed after 24 hours.

[0062] Samples were collected 60 h after treatment and used for real-time quantitative PCR to analyze gene expression levels and perform targeted quantitative detection of sesamin. The detection method was the same as in "4.2 Targeted Quantitative Detection of Sesamin".

[0063] The results are as follows Figure 4 As shown, RT-qPCR analysis indicated that, compared with the control (leaf transiently transformed with BGV008 empty vector), the transiently transformed camphor tree showed higher levels of [something unclear - possibly related to cell count or morphology]. CcCYP81BP12 Gene expression levels were significantly increased by approximately 6-fold. LC-MS analysis of camphor tree leaves revealed overexpression of gene genes in camphor trees. CcCYP81BP12 Afterwards, the sesamin content increased significantly, with a sesamin content 2.12 μg·g higher than that of the control group. -1 Increased to 797.96 μg·g -1 ( Figure 5 ).

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A camphor tree CcCYP81BP12 protein, characterized in that, The amino acid sequence of the camphor tree CcCYP81BP12 protein is shown in SEQ ID NO.

2.

2. The gene encoding the camphor tree CcCYP81BP12 protein as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. An expression carrier, characterized in that, It includes the coding gene as described in claim 2.

4. A recombinant bacterium, characterized in that, It includes the expression vector as described in claim 3.

5. The application of the camphor tree CcCYP81BP12 protein as described in claim 1 in the synthesis of sesamin.

6. A method for synthesizing sesamin, characterized in that, The method includes a step of catalytic synthesis using the camphor tree CcCYP81BP12 protein of claim 1 as a substrate, with (+)-pinene, (-)-pinene, (+)-epistene, or (-)-epistene as a substrate.

7. The use of the encoding gene of claim 2, the expression vector of claim 3, or the recombinant bacteria of claim 4 in any of the following: (1) Application in regulating the sesamin content in plants; (2) Application in the cultivation of transgenic plants with increased sesamin content.

8. The application as described in claim 7, characterized in that, Upregulate the expression level of the encoding gene in plants to increase the sesamin content in the plants; The plant in question is a camphor tree.

9. A method for increasing the sesamin content in plants, characterized in that, The method includes the step of upregulating the expression of the gene encoding the CcCYP81BP12 protein in camphor trees to promote the synthesis of sesamin in the plant. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

10. A breeding method for a transgenic plant with increased sesamin content, characterized in that, Includes the following steps: The gene encoding the camphor tree CcCYP81BP12 protein was overexpressed in plant cells, the plant cells were then cultured, and the plant cells were used to regenerate plants, thus obtaining the transgenic plant with increased sesamin content. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

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