Csdbr4 gene and application thereof

Abstract

The application discloses a dihydro-beta-ionone synthetase gene CsDBR4 of Iris domestica and application thereof. The dihydro-beta-ionone synthetase gene CsDBR4 is cloned and identified from Iris domestica, the full-length cDNA sequence of the gene is shown as SEQ ID NO:1, the coding sequence is shown as SEQ ID NO:2, and the amino acid sequence is shown as SEQ ID NO:3. The CsDBR4 gene is a key gene for biosynthesis and release of dihydro-beta-ionone which is a main component of Iris domestica flower fragrance, can catalyze beta-ionone to generate dihydro-beta-ionone in vitro and in vivo, and is connected to a plant transformation vector to be transferred into plants, so that transgenic plants expressing the gene and having dihydro-beta-ionone flower fragrance can be obtained, and the transgenic plants can be used for identifying plants containing dihydro-beta-ionone flower fragrance and for genetic improvement of plant flower fragrance, which has important significance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology. More specifically, it relates to a moss dihydro-β-ionone synthase gene, CsDBR4, and its applications. Background Technology

[0002] Cymbidium sinense is a perennial herbaceous plant belonging to the genus Cymbidium in the family Orchidaceae. Its elegant flowers, graceful leaves, and rich fragrance make it a highly prized ornamental plant. Research indicates that dihydro-β-ionone is an important aroma compound in Cymbidium sinense, possessing a mellow woody aroma. Due to its unique scent, dihydro-β-ionone is widely used as an additive in food, cosmetics, toiletries, and detergents. It is also used as a substrate in the chemical synthesis of theaspirane, a major aroma compound in cigarettes (Liu et al., 2011). Furthermore, dihydro-β-ionone has been shown to possess herbicidal properties and can be used as a weed suppressant (Lun et al., 2023). Therefore, dihydro-β-ionone has broad application prospects and significant commercial value.

[0003] Due to the increasing consumer demand for organic products, chemical extraction from plant sources is the main method for the commercial production of dihydro-β-ionone. Biotechnological methods are also used to synthesize dihydro-β-ionone in vitro. For example, the pET-28a-DBR1 recombinant strain can catalyze the reduction of β-ionone to dihydro-β-ionone in a whole-cell system (Zhang et al., 2018); a single-pot synthesis system using β-apo-8'-carotene aldehyde as a substrate, with the simultaneous addition of CCD1 (carotenoid cleavage dioxygenase 1) and AaDBR1 (double bond reductase 1), has been used, and HPLC analysis revealed the formation of both β-ionone and dihydro-β-ionone (Qi et al., 2022).

[0004] However, research on the synthesis of dihydro-β-ionone in plants is limited, with only the OfWRKY36 gene in Osmanthus fragrans being used for its synthesis. As an important aroma compound in Cymbidium goeringii, the synthesis of dihydro-β-ionone in Cymbidium goeringii has not yet been reported in this plant, the relevant genes involved remain unclear, and its application in Cymbidium goeringii is lacking. Therefore, identifying key genes related to the biosynthesis of dihydro-β-ionone is crucial for the genetic improvement of plant fragrance and is of great significance for promoting molecular breeding and industrial development in orchids. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the insufficiency of dihydro-β-ionone synthesis in plants and to provide a dihydro-β-ionone synthase gene CsDBR4 from Cymbidium goeringii and its application.

[0006] The first objective of this invention is to provide a moss dihydro-β-ionone synthase gene, CsDBR4.

[0007] A second objective of this invention is to provide a moss dihydro-β-ionone synthase CsDBR4.

[0008] A third objective of this invention is to provide the moss dihydro-β-ionone synthase gene CsDBR4 or its expression promoter, or the application of moss dihydro-β-ionone synthase CsDBR4.

[0009] A fourth objective of this invention is to provide a recombinant vector and a recombinant bacterium containing the recombinant vector.

[0010] A fifth object of the present invention is to provide a formulation that promotes the synthesis of dihydro-β-ionone.

[0011] The sixth object of the present invention is to provide a method for cultivating fragrant plants containing dihydro-β-ionone.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention marks the first time that a dihydro-β-ionone synthase gene, CsDBR4, has been cloned and identified in Cymbidium goeringii. This gene belongs to the medium-chain dehydrogenase / reductase (MDR) protein superfamily and is a key gene for the biosynthesis and release of dihydro-β-ionone, the main component of Cymbidium goeringii fragrance. The CsDBR4 gene can catalyze the formation of dihydro-β-ionone fragrance in Cymbidium goeringii. It is highly expressed during the full bloom stage of Cymbidium goeringii and has the lowest expression level during the bud stage. Moreover, the expression level of CsDBR4 at different flowering stages of Cymbidium goeringii is consistent with the trend of dihydro-β-ionone release. The full-length cDNA of the CsDBR4 gene is 1064 bp, and its nucleotide sequence is shown in SEQ ID NO:1. The coding region (CDS) is 1029 bp, and its nucleotide sequence is shown in SEQ ID NO:2. It is presumed to encode 343 amino acids, and its amino acid sequence is shown in SEQ ID NO:3. The presumed protein molecular weight is 38.07 kDa, the isoelectric point (pI) is 5.13, and the amino acid sequence contains typical conserved domains of the MDR superfamily.

[0014] Based on the CsDBR4 gene sequence information provided by the present invention, those skilled in the art can easily obtain a gene equivalent to CsDBR4 by the following methods: (1) obtaining it through database retrieval; (2) obtaining it by screening genomic libraries or cDNA libraries of Cymbidium or other plants using CsDBR4 gene fragments as probes; (3) obtaining it from the genome, mRNA and cDNA of Cymbidium or other plants by designing oligonucleotide primers based on CsDBR4 gene sequence information and using PCR amplification; (4) obtaining it by modifying it using genetic engineering methods based on the CsDBR4 gene sequence; (5) obtaining the gene by chemical synthesis.

[0015] This invention provides a primer pair for amplifying the moss dihydro-β-ionone synthase gene CsDBR4, the primer sequences of which are shown in SEQ ID NO:4-5.

[0016] Studies have shown that the CsDBR4 gene can catalyze the formation of dihydro-β-ionone aroma in Cymbidium goeringii, and can catalyze the production of dihydro-β-ionone from β-ionone in vitro and in vivo. The construction of a recombinant expression vector for CsDBR4 in Cymbidium goeringii demonstrates that the exogenous recombinant protein of CsDBR4 can catalyze the production of dihydro-β-ionone from β-ionone, and can be used to prepare dihydro-β-ionone. Connecting CsDBR4 to a plant transformation vector and introducing it into Cymbidium goeringii or other plant cells yields transgenic plants expressing the CsDBR4 gene and possessing a dihydro-β-ionone aroma.

[0017] Therefore, the present invention provides the following applications of the moss dihydro-β-ionone synthase gene CsDBR4 or its expression promoter, or the moss dihydro-β-ionone synthase CsDBR4:

[0018] Application in promoting the synthesis of dihydro-β-ionone in plants.

[0019] Application in the preparation of formulations that promote the synthesis of dihydro-β-ionone.

[0020] Application in the identification of floral plants containing dihydro-β-ionone.

[0021] Application in the preparation of dihydro-β-ionone.

[0022] Application in cultivating fragrant plants containing dihydro-β-ionone.

[0023] This invention provides a recombinant vector containing the moss dihydro-β-ionone synthase gene CsDBR4.

[0024] The present invention provides a recombinant bacterium containing the above-mentioned recombinant expression vector; and a cell line containing the recombinant bacterium.

[0025] This invention provides the application of the CsDBR4 gene in identifying fragrant plants containing dihydro-β-ionone. Specific molecular markers can be generated based on the CsDBR4 gene sequence information, including but not limited to SNPs (single nucleotide polymorphisms), SSRs (simple sequence repeat polymorphisms), RFLPs (restriction endonuclease length polymorphisms), and CAPs (cutting amplification fragment polymorphisms). These markers can be used to identify the dihydro-β-ionone synthase gene in Cymbidium or other plants, and can be used for marker-assisted selection breeding, thereby improving the selection efficiency of breeding.

[0026] In addition, the present invention provides a formulation for promoting the synthesis of dihydro-β-ionone, the formulation containing a reagent that promotes the expression of the moss dihydro-β-ionone synthase gene CsDBR4, or containing an exogenous recombinant protein of moss dihydro-β-ionone synthase CsDBR4.

[0027] The present invention also provides a method for cultivating plants with dihydro-β-ionone fragrance, wherein the dihydro-β-ionone synthase gene CsDBR4 of Cymbidium goeringii is linked to a plant transformation vector and transformed into plants, thereby obtaining plants with dihydro-β-ionone fragrance; or the expression of the dihydro-β-ionone synthase gene CsDBR4 of Cymbidium goeringii is promoted in plants.

[0028] Furthermore, the CsDBR4 gene sequence can be ligated into any plant transformation vector, and the CsDBR4 gene can be introduced into Cymbidium or other plant cells using conventional or any transformation method in the art, thereby obtaining transgenic plants capable of expressing the gene. In particular, when constructing the CsDBR4 gene provided by this invention into a plant transformation vector, the CsDBR4 gene or its regulatory sequence can be appropriately modified. Simultaneously, other promoters can be used to replace the original promoter to broaden and enhance the plant's ability to produce dihydro-β-ionone.

[0029] Preferably, the primer sequences for constructing the CsDBR4 gene prokaryotic expression vector are shown in SEQ ID NO:10-11.

[0030] Preferably, the primer sequences for constructing the CsDBR4 gene recombinant expression vector are shown in SEQ ID NO:12-13.

[0031] The present invention has the following beneficial effects:

[0032] This invention marks the first cloning of the dihydro-β-ionone synthase gene CsDBR4 in Cymbidium goeringii. This gene is a key gene for the biosynthesis and release of dihydro-β-ionone, the main component of Cymbidium goeringii's fragrance, and can catalyze the conversion of β-ionone to dihydro-β-ionone both in vitro and in vivo. The construction of a recombinant expression vector for CsDBR4 in Cymbidium goeringii demonstrates that the exogenous recombinant protein of CsDBR4 can catalyze the conversion of β-ionone to dihydro-β-ionone, which can be used for the preparation of dihydro-β-ionone and its further application as an additive in food, cosmetics, and other production processes. This is of great significance for further utilizing microbial metabolic engineering to prepare dihydro-β-ionone for industrial production. Ligating CsDBR4 into a plant transformation vector and then introducing it into Cymbidium goeringii or other plant cells yields transgenic plants expressing the CsDBR4 gene and possessing a dihydro-β-ionone fragrance, contributing to the improvement of plant fragrance and promoting molecular breeding of plant fragrances. It can also be used to identify the fragrance genotype of Cymbidium goeringii varieties and their hybrid offspring, and to improve breeding efficiency through molecular marker-assisted selection breeding. It has great application prospects and is of great significance in overcoming the problem of not being able to transfer genes between plant species in traditional breeding. Attached Figure Description

[0033] Figure 1 Cloning of the CsDBR4 gene in *Arabidopsis thaliana* and analysis of its amino acid sequence homology (A: Agarose gel electrophoresis of the *Arabidopsis thaliana* gene clone; B: Amino acid sequence homology analysis of *Arabidopsis thaliana*, where the homologous genes are: AtDBR (*Arabidopsis thaliana*, AAY25470.1); DoDBR (*Dendrobium catenatum*, XP_020685484.1); VvDBR (*Vitis vinifera*, XP_002275595.1); PtDBR (*Pinus taeda*, ABG91753.1); CaDBR (*Colchicum autumnale*, UZM07734.1)).

[0034] Figure 2 The relative expression level of the CsDBR4 gene and the release of dihydro-β-ionone at different flowering stages of Cymbidium sinense (S1: bud stage; S2: semi-open stage; S3: full bloom stage).

[0035] Figure 3SDS-PAGE gel electrophoresis of CsDBR4 recombinant protein and in vitro catalysis of β-ionone to dihydro-β-ionone (A: SDS-PAGE gel electrophoresis of CsDBR4 recombinant protein (where M is the marker; K is the pET-32a empty vector; S is the supernatant after bacterial lysis of recombinant protein; C is the precipitate after bacterial lysis of recombinant protein; E is the elution buffer of recombinant protein); B: Chromatogram and mass spectrum of CsDBR4 catalyzing the product of β-ionone in vitro; C: Empty vector control).

[0036] Figure 4 Functional identification of CsDBR4 in transient transformation of tobacco leaves (A: Chromatograms and mass spectra of CsDBR4 and the product generated from the injected substrate β-ionone in transient transformation of tobacco; B: Empty control). Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] The Cymbidium goeringii used in the following examples was purchased from Yutian Horticulture Farm in Wengyuan County, Guangdong Province, and planted in the germplasm resource nursery of the Flower Research Center of South China Agricultural University. It is growing well.

[0040] Example 1: Obtaining the full-length cDNA of the CsDBR4 gene

[0041] 1. Extraction of total RNA from Cymbidium goeringii flowers

[0042] Total RNA was extracted from Cymbidium goeringii flowers using the OMEGA (Plant RNA Kit R6827) kit. The specific steps are as follows: After grinding the sample in liquid nitrogen, ≤100 mg of plant sample was collected into a centrifuge tube. 500 μL of RB Buffer / 10 μL of β-mercaptoethanol was added, and the mixture was immediately vortexed. A gDNA Filter column was inserted into a 2 mL collection tube, and the mixture was transferred to the gDNA Filter column and centrifuged at 14000 × g at room temperature for 5 min. The filtrate was transferred to a new 1.5 mL centrifuge tube, and 0.5 times the volume of anhydrous ethanol was added. The mixture was vortexed at high speed for 20 s. If precipitation occurred, it was pipetted 10–15 times. Insert the RNA Mini binding column into a 2 mL collection tube, and transfer 700 μL of the mixture from step 3 to... Centrifuge at 12000×g at room temperature for 1 min in an RNA Mini binding column, discard the filtrate; repeat the previous step until all the mixture has been transferred through the column; The RNA Mini binding column was placed into the same 2mL collection tube, 400μL of RWF Wash Buffer was added, and the tube was centrifuged at 10000×g at room temperature for 30s. The filtrate was discarded. Insert the RNA Mini binding column into the same 2mL collection tube, add 500μL RNA Wash Buffer II (pre-diluted with anhydrous ethanol), centrifuge at 10000×g at room temperature for 30s, discard the filtrate; repeat the previous step.

[0043] Will The RNA Mini binding column was inserted into the same 2mL collection tube, centrifuged at room temperature at maximum speed for 2 minutes, and then dried. RNA Mini binds to column matrix; Place the RNA Mini binding column into a new 1.5 mL centrifuge tube, and accurately add 50–100 μL of DEPC Water. RNA Minis were bound to the center of the column membrane, incubated at room temperature for 2 minutes, centrifuged at 10000×g for 1 minute at room temperature, and eluted. After RNA extraction, the integrity of the RNA was detected by 1% agarose gel electrophoresis, and its concentration and purity were determined by micro-spectrophotometry. The RNA was stored at -80℃ for later use.

[0044] 2. PCR amplification and agarose gel electrophoresis detection

[0045] Total RNA from Cymbidium goeringii flowers was used as a template to synthesize single-stranded cDNA using Evo M-MLV reverse transcriptase from Aikerui. Primers were designed using Primer Premier 6.0 software based on gene sequences annotated in the Cymbidium goeringii transcriptome database and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are as follows: upstream primer P1: 5'-GATGGCAGGGAAGAAGGTTG-3' (as shown in SEQ ID NO:4); downstream primer P2: 5'-CTATTCCCTATCCACCGCCA-3' (as shown in SEQ ID NO:5).

[0046] Using the synthesized cDNA as a template, PCR amplification was performed using Phanta high-fidelity enzyme. The PCR amplification system consisted of 35 cycles: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 15 s, 60℃ annealing for 15 s, and 72℃ extension for 60 s, followed by a final extension at 72℃ for 10 min. After the PCR reaction, the presence of the target fragment band in the PCR product was preliminarily detected by 1.0% agarose gel electrophoresis.

[0047] After PCR amplification products were detected by 1% agarose gel electrophoresis, gel blocks containing the target fragment were cut off under UV light using a scalpel and recovered using a DNA gel recovery kit (SanPrep column-based DNA gel recovery kit, Sangon Biotech). The recovery method was basically in accordance with the kit instructions. The recovered products were subjected to an A-tailing reaction using rTaq DNA polymerase (TaKaRa) at 72℃ for 30 min.

[0048] 3. Cloning and Identification

[0049] The target fragment was ligated to the cloning vector TaKaRa pMD19-T, with the molar ratio of target DNA to cloning vector controlled at approximately 3:1. Ligation was carried out at 16℃ for 3–6 hours. Competent DH5α (TaKaRa) cells were removed from a -80℃ freezer and placed on ice to thaw naturally. 10 μL of ligation buffer was added to the centrifuge tube containing the competent cells. After incubating on ice for 30 minutes, a heat shock was performed at 42℃ for 90 seconds, followed by immediate placement on ice for 2–5 minutes. Then, 1 mL of LB liquid medium was added, mixed well, and cultured at 37℃ with shaking at 180 rpm for 1 hour. An appropriate amount of transformation buffer was spread on the surface of an LB solid medium plate containing 100 μg / mL ampicillin. After complete absorption of the transformation buffer, the plate was inverted and cultured overnight at 37℃. Results were observed after approximately 16 hours.

[0050] Using a sterile pipette tip, pick a single white colony from an LB agar plate and inoculate it into LB liquid medium containing 100 μg / mL ampicillin. Incubate the culture in a temperature-controlled shaking incubator at 37°C and 180 rpm for 3–6 h. Then, perform PCR amplification on the bacterial culture using universal primers M13-47 and M13-48 for the pMD19-T vector. Finally, detect the PCR products by 1% agarose gel electrophoresis.

[0051] The bacterial culture identified as a positive clone was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to determine whether the target gene fragment was incorporated into the pMD19-T vector. The sequencing results were compared with the original transcriptome sequence using the Multalin online database, and further alignment and homology analysis were performed using NCBI to confirm that the obtained gene sequence was a complete full-length sequence. The protein sequence was also deduced from the cDNA sequence.

[0052] 4. Results

[0053] Amplification results as follows Figure 1 As shown in Figure A, the target gene fragment size is 1029 bp. Homology comparison analysis shows that the cloned gene is homologous to genes from Arabidopsis thaliana (AtDBR), Dendrobium officinale (DoDBR), grape (VvDBR), pine pine (PtDBR), and autumn crocus (CaDBR), belonging to the MDR superfamily. The comparison results are as follows. Figure 1As shown in B, it was named the Cymbidium CsDBR4 gene.

[0054] Sequencing results showed that the full-length cDNA sequence of the CsDBR4 gene is shown in SEQ ID NO:1, with a full-length cDNA of 1064 bp; the coding region (CDS) is shown in SEQ ID NO:2, totaling 1029 bp; and its amino acid sequence is shown in SEQ ID NO:3. Further research revealed that CsDBR4 amino acids contain conserved domains of the MDR superfamily: the Rossmann fold domain (…). Figure 1 The Rossmann fold domain (within the red dashed line in section B) and the AXXGXXG conserved domain. The Rossmann fold domain contains 7 α-helices and 6 β-sheets, which together form a typical six-stranded NADP(H) sheet with 3 helices on each side; the AXXGXXG conserved domain is a typical domain that binds NADP or NADP(H). It also includes three key amino acid residues: Lys-192, Try-208, and Cys-254. Figure 1 (B, marked with a red dot) can interact with NADP, forming hydrogen bonds and substrate-binding domains between the phosphate group and the ribose ring. Figure 1 In B, the red rectangles mark the substrate binding domains (Gly-281, Phe-282, Val-284).

[0055] Example 2: Expression analysis of the CsDBR4 gene

[0056] Total RNA was extracted from Cymbidium goeringii flowers at the bud, semi-open, and full-open stages using the OMEGA (Plant RNA Kit R6827) kit, following the same method as in Example 1. Quantitative real-time PCR was performed using the Hieff qPCR SYBR Green Master Mix (Low Rox) kit; the principle and method are described in the instruction manual.

[0057] Following the principles of quantitative real-time PCR (qPCR) primer design, TBtools software was used to design qPCR primers and perform specificity detection. qPCR was used to detect mismatches, primer dimers, and amplification efficiency. A pair of optimal primers was selected: P1: 5'-CAGAGGGTTCGCGGTGTT-3' (as shown in SEQ ID NO:6); P2: 5'-CGTTCTCAAGGCCCTCCG-3' (as shown in SEQ ID NO:7). For the internal reference gene ACTIN, qPCR primers were designed using TBtools software according to the same principles, and specificity was detected: ACTIN-P1: 5'-CTGGGATGGTGAAGGCCG-3' (as shown in SEQ ID NO:8); ACTIN-P2: 5'-TCTGTCCCATCCCGACCA-3' (as shown in SEQ ID NO:9).

[0058] The reaction mixture consisted of 10.0 μL Hieff qPCR SYBR Green Master Mix (Low Rox), 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 2.0 μL cDNA, and 7.2 μL ddH2O. The assay program was 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 55℃ annealing for 20 s, and 72℃ extension for 20 s, for 40 cycles.

[0059] Detection was performed using quantitative real-time PCR, and a standard curve was constructed to screen samples based on whether the amplification efficiency (E) was within the range of 90-110%. Using cDNA from each sample as a template, quantitative real-time PCR reactions were performed on an ABI 7500 instrument. Each sample had three biological replicates and three technical replicates, with ddH2O as a negative control. After the reaction, 2... －△△Ct The data were analyzed using the method (Livak et al., 2001) to calculate the expression of CsDBR4 in different samples.

[0060] Simultaneously, the release of dihydro-β-ionone from Cymbidium goeringii flowers at different flowering stages was determined. Whole flowers at the bud, half-open, and full-bloom stages were cut and placed in 300 mL glass containers. 0.865 μg of ethyl decanoate was added as an internal standard, and the containers were immediately sealed with aluminum foil. The floral aroma was collected for 1 hour, followed by extraction with SPME (PDMS DVB / CAR, 50 / 30 μm) for 1 hour. The GC model was Agilent 7890A, the MSD model was Agilent 5975C, the column model was Agilent DB-5MS (30 m × 0.25 mm), and the carrier gas was helium. The operating procedure was as follows: split ratio 20:1, injection port temperature 250℃, held at 100℃ for 2 min, increased to 170℃ at a rate of 10℃ / min, held for 2 min, then increased to 250℃ at a rate of 5℃ / min, and finally held at 280℃ for 5 min. The mass spectrometry detector conditions were 1 sec / scan, m / z 35-500, and 1 kV. The ion chromatograms were compared with those from the NIST mass spectrum library (NIST 2008) and literature to determine the types of aromatic compounds in Cymbidium goeringii. The content of aroma substances was calculated using a relative quantification method, with the following formula (Fan Yanping et al., 2020):

[0061] Content of a certain component (μg·gFW) -1 ·h -1 = Internal standard mass * peak area of ​​a certain component / (internal standard peak area * sample mass).

[0062] Gene expression analysis and dihydro-β-ionone release, as shown in the following figures: Figure 2 As shown, the CsDBR4 gene is highly expressed during the full bloom stage of Cymbidium goeringii, and its expression level is lower during the bud stage. Dihydro-β-ionone is released at the highest level during the full bloom stage of Cymbidium goeringii, and at the lowest level during the bud stage. The relative expression of the CsDBR4 gene at different flowering stages of Cymbidium goeringii is consistent with the trend of dihydro-β-ionone release at different flowering stages. The results indicate that CsDBR4 is a gene involved in the synthesis of dihydro-β-ionone in Cymbidium goeringii.

[0063] Example 3: Prokaryotic expression of the CsDBR4 gene

[0064] 1. Carrier Construction

[0065] Based on the coding region of the CsDBR4 gene obtained in Example 1, PCR amplification was performed using homologous recombination primers containing BamHI and HindIII restriction sites: P1: 5'-gccatggctgatatcggatccATGGCAGGGAAGAAGGTTG TT-3' (as shown in SEQ ID NO: 10), P2: 5'-ctcgagtgcggccgcaagcttTTCCCTATCCAC CGCCACC-3' (as shown in SEQ ID NO: 11). The pET-32a prokaryotic expression vector was double-digested with BamHI and HindIII restriction enzymes in a 37°C digestion program for 2 h followed by 65°C inactivation for 15 min. The digestion products were purified using the SanPrep Column PCR Product Purification Kit to remove small DNA fragments and salts from the digestion system, yielding the linear vector DNA fragment. II. Homologous recombination of the gene fragment and the vector was performed, followed by ligation at 37°C for 30 min and then on ice for 5 min. All ligation products were transformed into DH5α competent cells. After positive clone screening, bacterial PCR identification, and gene sequencing to confirm their accuracy, the recombinant prokaryotic expression vector was obtained.

[0066] 2. Recombinant protein expression

[0067] Rosetta (DE3) competent cells were transformed with identified recombinant plasmid DNA. Single colonies were picked and inoculated into 1 mL of LB broth (containing 100 mg / L Kan) and cultured at 37°C and 180 rpm for 6–8 hours. 50 μL of the seed culture was then transferred to 5 mL of fresh LB broth (containing 100 mg / L Kan) and cultured at 37°C for 12 hours. Another 1 mL of the seed culture was then transferred to 100 mL of fresh LB broth (containing 100 mg / L Kan) and cultured at 37°C and 180 rpm until OD (Organic Dose) was reached. 600 The pH value was 0.4–0.6. 10 μL of IPTG was added, and the cells were induced at 16°C for 20 h. A control group without IPTG induction was also included. Cells were collected by centrifugation, resuspended in 5 mL of lysis buffer (50 mM phosphate buffer, pH 8.0), cooled, and sonicated on ice. The cells were then centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was transferred to a new centrifuge tube, the precipitate was washed once with double-distilled water, and then resuspended in 5 mL of lysis buffer. 50 μL of supernatant (S) and precipitate (C) were collected and stored at -20°C.

[0068] 3. Purification of recombinant proteins

[0069] 0.5 mL of Ni-NTA resin was packed into the chromatography column. After the resin precipitated, the internal liquid was drained, 5 mL of ddH2O was added, and the column was washed three times. Then, 5 mL of Wash buffer was added, and the column was washed three times. The column was pre-cooled at 4°C. 5 mL of cell lysis supernatant was added to the pre-cooled chromatography column, mixed thoroughly, and incubated on a low-speed shaker at 4°C for 1 h. The liquid was collected in a centrifuge tube containing flow-through buffer F. 2 mL of Wash buffer was added, and the column was eluted three times, collecting the W1 and W2 eluents respectively. 1 mL of Elution buffer was added, and the column was washed four times, collecting the E1, E2, and E3 eluents respectively. A 12.5% ​​SDS-PAGE gel was prepared, and 16 μL of each of the collected liquids were loaded sequentially for SDS-PAGE gel electrophoresis analysis. After electrophoresis, the column was stained with Coomassie Brilliant Blue for 30 min, then destained with destaining solution for 24 h. The experimental results were observed and recorded.

[0070] The SDS-PAGE gel electrophoresis results of CsDBR4 recombinant protein are as follows: Figure 3 As shown in Figure A, clear bands of the CsDBR4 recombinant protein can be seen in the supernatant S, precipitate C, and elution buffers E2 and E3. The band size is consistent with the predicted size of the recombinant protein, indicating that the CsDBR4 recombinant protein has been successfully purified.

[0071] 4. Identification of enzyme catalytic function

[0072] Add 10 μL of 1M Tirs-HCl (pH 7.5), 1.66 μL of 12 mM NADPH, 20 μL of protein extraction buffer, and 1 μL of β-ionone to 167.34 μL of [presumably a specific solution]. Add ddH₂O to a final volume of 200 μL into a sample vial and seal. React at 37°C for 1 h. Insert a 75 μm polydimethyloxane (PMDS) extraction fiber into the glass vial and perform headspace solid-phase microextraction for 1 h. After the reaction, place the extraction fiber into a gas chromatograph-mass spectrometer for analysis. The gas chromatographic conditions were: HP-1NNOWAX column (30 m × 0.25 mm); high-purity helium as carrier gas; split ratio of 20:1; injection port temperature of 250°C; hold at 100°C for 2 min; increase to 170°C at 10°C / min and hold for 2 min; increase to 250°C at 5°C / min and hold at 280°C for 5 min; mass spectrometry detector conditions were 1 sec / scan, m / z 35-500, 1 kV. The acquired mass spectra were analyzed using the WILLEY / MAINLIB library.

[0073] Chromatographic and mass spectra of CsDBR4 catalyzing the production of β-ionone from the substrate in vitro are shown below. Figure 3As shown in B, when β-ionone was used as a substrate, the product of the in vitro enzyme-catalyzed reaction of pET-32a-CsDBR4 was identified by mass spectrometry as dihydro-β-ionone (the results of the empty control are shown in Figure B). Figure 3 (As shown in C). This indicates that the CsDBR4 gene encodes an enzyme that can catalyze the production of dihydro-β-ionone from β-ionone.

[0074] Example 4: CsDBR4 instantaneous conversion of tobacco leaves

[0075] 1. Vector construction and Agrobacterium-mediated transformation

[0076] Based on the cDNA sequence of the CsDBR4 gene, specific primers containing BamHI and HindIII restriction sites were designed. The upstream primer P1: cgctctagaactagtggatccATGGCAGGGAAGAAGGTTGTT (as shown in SEQ ID NO:12); the downstream primer P2: gtcgacggtatcgataagcttTTCCCTATCCACCG CCACC (as shown in SEQ ID NO:13). PCR amplification was performed using Phanta high-fidelity enzyme. After recovery, the amplified product was double-digested and ligated into the pGreenII 62-SK vector, transformed into DH5α, and after sequencing confirmation, the recombinant plasmid was transformed into Agrobacterium GV3101psoup strain.

[0077] 2. Agrobacterium culture and transient transformation of tobacco

[0078] Single colonies of Agrobacterium were picked and inoculated into LB liquid medium containing Rif (50 μg / mL) + Kan (50 μg / mL), and cultured at 28°C with shaking at 200 rpm for 24 h. The culture was centrifuged at 5000 × g for 10 min, the supernatant was discarded, and the bacterial suspension was resuspended in pure water. The suspension was then centrifuged again at 5000 × g for 10 min, and the supernatant was discarded. Agrobacterium was resuspended in osmosis buffer (10 mM MES, pH 5.2, 10 mM MgCl2, 0.1 mM acetylsyleugenone) to OD0.05. 600 The concentration was approximately 0.4, and the solution was left at room temperature for 3 hours. A 1 mL needleless syringe was used to draw up the Agrobacterium suspension and infiltrate the tobacco leaves. After infection, the leaves were placed in a culture room for further incubation.

[0079] 3. Detection of volatiles in tobacco leaves

[0080] After 3 days of cultivation, 3 mM β-ionone was injected into tobacco leaves infected with Agrobacterium using a 1 mL needleless syringe. The entire tobacco plant was then sealed in a 1000 mL glass bottle, and the tobacco volatiles were extracted using solid-phase microextraction for 12 h. GC-MS analysis was then performed under the same gas chromatography and mass spectrometry conditions as in Example 3. The types of products were determined by comparing the retention times and mass spectra of the reaction products.

[0081] 4. Results

[0082] Chromatographic and mass spectrometric results of volatile components in tobacco leaves are as follows: Figure 4 As shown in Figure A, after transient transformation of the CsDBR4 gene and simultaneous injection of the substrate β-ionone, a large amount of dihydro-β-ionone was detected in the leaf volatiles (results of the empty control are shown in Figure A). Figure 4 (As shown in B), this indicates that CsDBR4 also has the function of catalyzing the production of dihydro-β-ionone from β-ionone in plants.

[0083] In summary, this invention marks the first time that the dihydro-β-ionone synthase gene CsDBR4 has been cloned from Cymbidium goeringii. CsDBR4 is a key gene for the biosynthesis and release of dihydro-β-ionone, the main component of Cymbidium goeringii's fragrance. The CsDBR4 gene catalyzes the formation of dihydro-β-ionone aroma in Cymbidium goeringii. The results show that the CsDBR4 gene is highly expressed during the full bloom stage of Cymbidium goeringii, with the lowest expression level during the bud stage. Furthermore, the expression level at different flowering stages of Cymbidium goeringii is consistent with the trend of dihydro-β-ionone release. The construction of a recombinant expression vector for CsDBR4 in Cymbidium goeringii demonstrates that the exogenous recombinant protein of CsDBR4 can catalyze the production of dihydro-β-ionone from β-ionone. This protein can be used for the preparation of dihydro-β-ionone and its further application as an additive in the production of food and cosmetics. Linking CsDBR4 to a plant transformation vector and introducing it into Cymbidium goeringii or other plant cells yields transgenic plants expressing the CsDBR4 gene and exhibiting a dihydro-β-ionone fragrance. Furthermore, based on the gene sequence information, specific molecular markers can be studied and designed to identify the fragrance genotypes of Cymbidium goeringii varieties and their hybrids, enabling marker-assisted selection breeding and improving breeding efficiency, thus showing great application potential.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A gene for synthase of moss dihydro-β-ionone CsDBR4 Its characteristics are, The full-length cDNA sequence of the gene is shown in SEQ ID NO:1, and the gene coding sequence is shown in SEQ ID NO:

2.

2. A moss dihydro-β-ionone synthase CsDBR4, characterized in that, The amino acid sequence of the synthase is shown in SEQ ID NO:

3.

3. The moss dihydro-β-ionone synthase gene according to claim 1 CsDBR4 Or the use of the Cymbidium goeringii dihydro-β-ionone synthase CsDBR4 as described in claim 2 in promoting the synthesis of dihydro-β-ionone in Cymbidium goeringii or in the preparation of formulations that promote the synthesis of dihydro-β-ionone.

4. The moss dihydro-β-ionone synthase gene according to claim 1 CsDBR4 Or the application of the molan dihydro-β-ionone synthase CsDBR4 as described in claim 2 in the preparation of dihydro-β-ionone.

5. The moss dihydro-β-ionone synthase gene according to claim 1 CsDBR4 Or the application of the Cymbidium dihydro-β-ionone synthase CsDBR4 as described in claim 2 in the cultivation of fragrant plants containing dihydro-β-ionone, characterized in that, The recombinant expression vector containing the dihydro-β-ionone synthase CsDBR4 from *Cymbidium goeringii* was transferred into a plant. If the plant did not contain the substrate β-ionone, the substrate β-ionone needed to be injected into the plant. The recombinant expression vector catalyzed the production of dihydro-β-ionone from β-ionone. The plant was tobacco.

6. A recombinant vector, characterized in that, Contains the molan dihydro-β-ionone synthase gene as described in claim 1 CsDBR4 .

7. A recombinant bacterium, characterized in that, It contains the recombinant vector as described in claim 6.

8. A formulation, characterized in that, The formulation contains the gene of claim 1, or the vector of claim 7, or the exogenous recombinant protein of the enzyme molluscum dihydro-β-ionone synthase CsDBR4 of claim 2.

9. A method for cultivating fragrant plants containing dihydro-β-ionone, characterized in that, The moss dihydro-β-ionone synthase gene as described in claim 1 CsDBR4 The gene was ligated into a plant expression vector and transformed into plants to obtain plants with dihydro-β-ionone fragrance; or the dihydro-β-ionone synthase gene of Cymbidium goeringii was promoted in plants. CsDBR4 The expression of β-ionone; if the plant does not contain β-ionone, then the substrate β-ionone needs to be injected into the plant; the plant is tobacco.

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