Cymbidium sinense CsCCD4 gene and effect of cymbidium sinense CsCCD4 gene in aroma regulation

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a cymbidium sinense CsCCD4 gene and an effect of the cymbidium sinense CsCCD4 gene in aroma regulation. The invention provides a related CsCCD4 gene for synthesizing a substance for enhancing orchid aroma. The nucleotide sequence of the related CsCCD4 gene is shown as SEQ ID NO. 1. According to the invention, the full length of a gene sequence is obtained through cloning, an over-expression vector is constructed on the basis, and instantaneous conversion of butterfly orchid petals and stable conversion of tobacco grandiflora are carried out. The result shows that the substance content of the aroma substance beta-ionone in the instantaneous phalaenopsis petals and the stably converted Bensi tobacco petals is remarkably increased compared with a control group CK, and it is indicated that CsCCD4 plays an important role in enhancing the aroma substance of orchid. As an important CsCCD4 gene in the synthesis process of substances for enhancing orchid aroma, the CsCCD4 gene can be used for improving ornamental characters and creating genetic varieties in orchid genetic engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant molecular biology, and particularly relates to a Cymbidium hybridum CsCCD4 gene and its role in regulating fragrance. BACKGROUND

[0002] Cymbidium is one of the ten traditional famous flowers in China, mainly including Cymbidium ensifolium, Cymbidium hybridum, Cymbidium goeringii, Cymbidium faberi, Cymbidium lancifolium, Cymbidium kanran, Cymbidium sinense and Cymbidium devonianum. Cymbidium carries a deep cultural heritage, and has been cultivated and inherited for thousands of years, and is deeply loved by the Chinese and people all over the world. The unique fragrance determines the core value of Chinese orchid. Different Cymbidium has different characteristic fragrance components, and currently the genes related to the synthesis of these characteristic fragrance substances need to be screened and functionally identified to provide gene resources for further development and utilization.

[0003] Carotenoid cleavage dioxygenase (CCD) family members can oxidatively cleave carotenoids at one or both ends of the molecule, thereby generating different apocarotenoids, which is one of the main pathways for carotenoid degradation in organisms. CCD family members all exhibit oxidative cleavage catalytic activity with carotenoids as substrates, but different CCDs have great differences in substrate specificity and double bond cleavage site. Among the CCD family members, the members that can catalyze carotenoids to form volatile substances with fragrance are CCD1 and CCD4. CCD4 usually has higher substrate selection specificity than CCD1, and mainly specifically cleaves β-carotene at 9, 10 and 9', 10' double bond sites to generate volatile apocarotenoids β-ionone with rich fragrance, which is one of the main components of plant flower fragrance. Currently, the reports on characteristic fragrance substances of Cymbidium hybridum find that β-ionone plays an important role in the formation of Cymbidium hybridum flower fragrance, but there is no report on the synthesis of β-ionone in Cymbidium hybridum. Therefore, it is of great significance to study and explore the genes in the genome of Cymbidium hybridum that regulate the synthesis of flower fragrance substances for later improvement and enhancement of the ornamental traits of Orchidaceae plants. SUMMARY

[0004] In view of the above problems existing in the prior art, the technical problems to be solved by the present application are: the first technical problem is to provide a Cymbidium hybridum CsCCD4 gene related to the synthesis of fragrance substances; the second technical problem is to provide an expression protein of the Cymbidium hybridum CsCCD4 gene related to the synthesis of fragrance substances; and the third technical problem is to provide a specific application of the Cymbidium hybridum CsCCD4 gene related to the synthesis of fragrance substances.

[0005] The present application is realized by the following technical solutions: A Cymbidium hybridum CsCCD4 gene, wherein the CsCCD4The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1 Furthermore, the aforementioned CsCCD4 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.2.

[0007] SEQ ID NO.2 MDTLFSTLLKSTSCTSFLPGKSGILISPMKPISPPPAPFISAVETKEKPQRERRTKSDSADNYGNTSSSAGSSSPQQLSQLKPQYQSPRSRYREPSAAAAFCNALDNLINNFIDPPHLRPSFDPRHLLSDNWAPADELPPTACPVIRGVIPTCL RGGAYIRNGPNPQYLPRGTHHLFDGDGMLNALLLPRSGDGTIPATLCSRYVRTYKYLLERDAGGPVLPNVFSGFNGTAGIARGAVAAARILTGQMNPAAGVGLANTSLAFFGDRLYALGESDLPYAVRLSPDDGNITTIGRCDFEGRLSIGMTAH PKKDPATGELFAFRYGPVPPFLTYFRFDAVGNYKTGEDVPIFSLTQPSMVHDFAITERFAIFPDIQIVMKPMDMVAPGRGSPVGPDRGKVPRLGVLPKYARSESGMRWFEVPGFNIMHTLNAWEEEGGDAIILVAPNVLSIEHALERIELVHSS VEMVRIDLQSGAVSRTPLSAENLDFGVIHPNYVGRKNRFAYLGVGNPMPKISGVRKLDFSLCGRGDCVVASRDFGPNCYGGEPFFVPSGKRGEEDDGYVVSYLHDEGIRESKFIVMDARSPQLDVVAEVLLPSRVPYGFHGLFLTQAEIMAQRQD A type containing the above-mentioned Cymbidium goeringii CsCCD4 The gene expression vector, wherein the starting vector of the expression vector is a binary vector that can be used for Agrobacterium-mediated transformation of plants or a vector that can be used for plant micro-bombardment.

[0008] Furthermore, the starting vector is a pOCA30 vector and a PHB vector.

[0009] A recombinant Agrobacterium containing the above expression vector, wherein the Agrobacterium is strain GV3101.

[0010] One of the described Cymbidium orchids CsCCD4 The application of genes in enhancing plant aroma synthesis includes the following steps: (1) Construct a structure containing the following as shown in SEQ ID NO.1 CsCCD4 Recombinant gene expression vectors; (2) Transform the recombinant expression vector obtained in step (1) into Agrobacterium to obtain recombinant Agrobacterium; (3) Introduce recombinant Agrobacterium into the target plant via transient or stable transformation methods; (4) After the transiently transformed plant has grown normally for 5 days, the petals of the transiently transformed plant were collected for GC-MS aroma substance determination; (5) On the second day after the stable transformed plant has grown normally and flowered, the petals of the stable transformed plant were collected for GC-MS aroma substance determination.

[0011] Furthermore, the starting vector of the expression vector described in step (1) is the pOCA30 plasmid.

[0012] Furthermore, the Agrobacterium mentioned in step (2) is strain GV3101.

[0013] Furthermore, the target plant for the instantaneous transformation is a Phalaenopsis orchid petal; The target plant for the stable transformation is Nicotiana macrocarpa.

[0014] The present invention has the following advantages over the prior art: This invention provides a method for enhancing the synthesis of orchid fragrance substances. CsCCD4 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, was cloned to obtain the full-length gene sequence. Based on this, an overexpression vector was constructed and transient transformation of Phalaenopsis orchid petals and stable transformation of Nicotiana benthamiana were performed. The results showed that the content of the aroma compound β-ionone in the transiently transformed Phalaenopsis orchid petals and the stably transformed Nicotiana benthamiana petals was significantly increased compared to the control group (CK), indicating that... CsCCD4 It plays an important role in enhancing the fragrance compounds of orchids. As an important component in the synthesis of fragrance compounds in orchids... CsCCD4 Genes can be used in orchid genetic engineering to improve ornamental traits and create genetic varieties. Attached Figure Description

[0015] Figure 1 For the target gene CsCCD4 Agarose gel electrophoresis image of the amplification products; Figure 2 Phylogenetic tree analysis of CCD proteins from six plant species; Figure 3 for CsCCD4 Analysis of gene expression patterns at different developmental stages; Figure 4 For target genes CsCCD4Image of a positive single colony after transformation of the amplified fragment ligated into the PHB vector; Figure 5 Agarose gel electrophoresis image of bacteria after transformation with Agrobacterium GV1301; Figure 6 A comparison diagram of the aroma components of plants undergoing transient transformation with those of a control group; Figure 7 Agarose gel electrophoresis image of tobacco plants that tested positive; Figure 8 A comparison chart of stable transformation plant aroma components and control aroma components. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments. In the following embodiments, operations not described in detail are routine biological experimental procedures, which can be performed with reference to molecular biology experimental manuals and existing publicly available journal articles, or according to kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc., of the embodiments can be selected according to local conditions without substantially affecting the results.

[0017] The materials used in this application are flowers of Phalaenopsis V3 during its peak bloom period, and the large-flowered tobacco used was provided by Nanjing Zhongding Biotechnology Co., Ltd.

[0018] Example 1 CsCCD4 Gene cloning and sequence analysis 1. RNA extraction and cDNA acquisition Approximately 2 g of floral organs from the Cymbidium goeringii cultivar 'Yingchunsu' during its peak flowering period (day 2 of flowering) were flash-frozen in liquid nitrogen, ground, and then total RNA was extracted from the floral organs using the Novizan RNA Extraction Kit (Fastpure Universal Plant Total RNA). The extracted total RNA was then stored at -80°C. The extracted floral organ total RNA was used to perform reverse transcription using the HiScript III 1 reverse transcription kit from Novizan Biotechnology Co., Ltd. (Nanjing Novizan Biotechnology Co., Ltd.). st The Strand cDNA Synthesis Kit (+gDNA wiper) is used to reverse transcribe cDNA and store it at -20°C for later use.

[0019] 2. Target gene CsCCD4 Cloning Using the cDNA obtained above as a template, PCR primers were used... CsCCD4 -F(GGGCTACTGAGTTTGCTTGT) and CsCCD4-R (ATCTTGTCGTTGCGCCAT) was amplified by PCR using Novizan's high-fidelity enzyme (2×Phanta Flash MasterMix (Dye Plus)). The PCR reaction mixture was as follows: cDNA 2 uL, high-fidelity enzyme 25 uL. CsCCD4 -F primer 2 μL, CsCCD4 -R primer 2 uL, add ddH2O to 50 uL. PCR reaction program is as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 56℃ annealing for 5 s, 72℃ extension for 5 s, 35 cycles; final extension at 72℃ for 1 min. The obtained PCR amplification products are subjected to 1% agarose gel electrophoresis (…). Figure 1 The DNA was extracted from the gel using the Novizan FastPure Gel DNA Extraction Mini Kit. The extracted product was ligated into a cloning vector (Novozymes 5 min TA / Blunt-Zero Cloning Kit) and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results revealed that the amplified fragment contained the complete CDS sequence of the target gene, consisting of 1857 bases, with the nucleotide sequence shown in SEQ ID NO.1. This gene was named the *Cymbidium goeringii* aroma regulatory gene. CsCCD4 Using SnapGene software to CsCCD4 The nucleotide sequence was translated into a protein sequence (as shown in SEQ ID NO.2), which encodes a protein amino acid sequence consisting of 618 amino acid residues, named the orchid aroma regulating protein CsCCD4. The obtained protein contains... CsCCD4 The Escherichia coli was preserved at the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences.

[0020] 3. CsCCD4 Gene sequence analysis Analysis of the conserved domains of the CsCCD4 protein using the Conserve Domain Search function in the NCBI database revealed... CsCCD4 has A conserved NCED domain (belonging to the RPE65 superfamily) is one of the main characteristics of oxidative lyases involved in the biosynthesis of apocarotenoids.

[0021] At the whole-genome level, all genes containing the PF03055 domain in Arabidopsis thaliana, rice, and four orchid species (Shenzhen Orchid, Dendrobium officinale, Cymbidium sinense, and Phalaenopsis orchid) were identified, and a phylogenetic tree was constructed based on these genes. Figure 2 Finally, candidate gene Mol015121 was identified as... CsCCD4 .

[0022] Example 2 CsCCD4Gene expression patterns during Cymbidium orchid bud development 1. RNA extraction and cDNA acquisition Approximately 2g of flower organs from the Cymbidium goeringii cultivar 'Yingchunsu' at five representative flower development stages from bud to senescence (bud stage (S1), early bloom stage (S2), half-bloom stage (S3), full bloom stage (S4), and withering stage (S5)) were flash-frozen in liquid nitrogen, then ground. Total RNA was extracted from the flower organs using the Novizan RNA extraction kit (Fastpure Universal Plant Total RNA) and stored at -80℃. The extracted total RNA was then used to perform reverse transcription using the HiScript III 1 reverse transcription kit from Novizan Biotechnology Co., Ltd. (Nanjing Novizan Biotechnology Co., Ltd.). st The Strand cDNA Synthesis Kit (+gDNA wiper) generates cDNA through reverse transcription and is stored at -20℃ for later use.

[0023] 2. qRT-PCR detection Using the forward primer qRT-CsCCD4-F (TCAACCTTGCTGAAGTCCAC) and the reverse primer qRT-CsCCD4-R (CGGGCTTTGATATTGAGGCT), the development of Cymbidium orchid organs at five representative flower development stages from bud to senescence was studied. CsCCD4 Gene expression levels were analyzed by qRT-PCR. Actin was amplified using qRT-CsActin-F (GGTGATGTGCTGGATCCTTT) and qRT-CsActin-R (TACTGGATTGGTTGGGCTTG) primers as an internal control. The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for 40 cycles. Real-time quantitative PCR was performed using a qTOWER 2.2 PCR instrument from Jena, Germany, following the instructions for the Taq Pro Universal SYBR qPCR Master Mix kit.

[0024] 3. Expression Analysis Analysis of the qRT-PCR results revealed that... CsCCD4 Gene expression analysis showed that, along with the flower bud opening process, CsCCD4 Gene expression levels peaked at time S3 and then declined. Figure 3 ).

[0025] Example 3 CsCCD4 Construction of gene overexpression vectors 1. Obtain the target gene fragment The amplification in Example 1 CCD4 Using a full-length TA vector as a template, the target gene fragment was amplified using the following primers.

[0026] F:ccagtctctctctcaagcttATGGATACTTTGTTCTCAACCTTGC R:ggatcaattcgagctcTCACAATGAATTTGGACTCTCTAAT 2. PCR amplification PCR amplification was performed using Q5® high-fidelity DNA polymerase.

[0027] Table 1 PCR reaction system ; Table 2 Amplification Procedure ; The PCR products after the run were detected by gel electrophoresis on a 1.0% low-melting-point agarose gel and observed using a UV gel imaging device. The correct bands were then cut and recovered.

[0028] 3. Plasmid double enzyme digestion and product purification Table 3 Enzyme digestion system ; Reaction conditions: 37°C, overnight. The enzyme digestion product was then purified and recovered.

[0029] Reconnection Table 4 Reorganization Linkage System ; Use a pipette to gently aspirate and mix (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube.

[0030] Single-fragment recombination reaction, 37°C, 30 min; cool to 4°C or immediately on ice.

[0031] 5. Transformation of recombinant plasmids A 1.5 mL centrifuge tube containing *E. coli* chemocompetent cells was removed from a -80°C freezer and placed on ice for 2 min. The ligation product was aspirated into the *E. coli* chemocompetent cells and mixed thoroughly. The mixed *E. coli* cells were then placed on ice and incubated for 30 min. Next, the tube was heat-shocked in a 42°C metal bath for 1 min, immediately removed, and placed on ice for 5 min. Then, 800 μL of sterile LB broth was added to the centrifuge tube, and the tube was incubated at 37°C in a shaker for 30 min. After centrifugation, the supernatant was discarded in a laminar flow hood. The mixture was then added to LB agar plates containing 50 mg / L Kan and spread evenly. The tubes were incubated upside down at 37°C for 14–18 h. Five single colonies were picked and transferred to centrifuge tubes containing 500 μL of LB broth containing 50 mg / L Kan, and incubated at 37°C in a shaker for 3 h. Then, bacterial PCR verification was performed. Figure 4 ).

[0032] 6. Detection of positive clones Table 5 PCR reaction system ; Table 6 PCR Reaction Conditions ; The PCR products were detected by gel electrophoresis with 1% low melting point agarose (WIX-EP300), and the presence of target bands was observed using an imaging system (Shanghai ChemiScope 6100).

[0033] Sequencing was performed on bacterial cultures containing the target band.

[0034] 7. Plasmid extraction Select the correct bacterial culture with the correct band and extract plasmid DNA according to the instructions of the plasmid extraction kit (Meiji Biotechnology P1001-02). The DNA can be used directly for Agrobacterium transformation or stored at -20℃.

[0035] 8. Agrobacterium-mediated transformation The recombinant plasmid was transformed into Agrobacterium GV3101 via freeze-thaw method. After overnight culture, single colonies were picked from the plate for colony PCR identification. Figure 5 Positive clones were incubated overnight in 500 μL LB solution containing 50 mg / L kan and 50 mg / L rif, and then stored in glycerol at a 1:1 ratio for subsequent experiments.

[0036] Example 4: Instantaneous transformation of Phalaenopsis orchid petals (1) Shaking: The bacterial culture of PHB empty vector and PHB-CCD4 target gene fusion expression vector transformed into Agrobacterium GV3101 was taken out at -80℃ and thawed at room temperature to a state of ice water mixture, and then placed in ice to thaw. 300 μL of bacterial culture was added to 30 mL of LB liquid medium (containing 50 μg·mL Kana) -1 Rif 25 μg·mL -1 Incubate at 28℃ and 200 rpm in the dark with shaking until the bacterial culture reaches 0D. 600 = Between 0.6 and 0.8.

[0037] (2) Preparation of mixed bacterial suspension: Weigh 0.196 g of acetylsuccinone (As) powder and dissolve and mix it in 10 mL of dimethyl sulfoxide (operation in a fume hood) to obtain a 100 mM As solution. Weigh 0.195 g of 2-morpholinoethanesulfonic acid (MES) and 0.095 g of magnesium chloride (MgCl2) powder and dissolve them in 1 mL of water to obtain 1 M MES solution and MgCl2 solution. Filter the above solutions through a 0.22 μm filter membrane, and add 100 μL of each solution to an Erlenmeyer flask. Make up to 100 mL with sterile water to obtain a suspension. Centrifuge the bacterial suspension (4℃, 5000 rpm, 10 min), discard the supernatant, collect the bacterial cells, and resuspend them using the suspension prepared on the same day until the resuspended solution has an OD of 0. 600 = Between 0.8 and 1.0. Place in the dark for 2 hours.

[0038] (3) Infection: Use a 1 mL disposable syringe to inject the mixed bacterial solution into the back of the petals of a healthy white Phalaenopsis orchid.

[0039] (4) Culture: Place the transiently transformed plants injected with the target gene mixed bacterial solution and the transiently transformed plants injected with the empty vector bacterial solution in the dark overnight, and take them out in the sunlight the next day for 3 days.

[0040] (5) Determination of β-ionone content in transiently transformed plants Table 7 Instruments ; (1.1) Standards and reagents Standard: β-ionone (source leaf, CAS: 14901-07-6) Reagent: Methanol (chromatographic grade, Shanghai Xingke).

[0041] (1.2) Draw the standard curve Accurately weigh an appropriate amount of the standard sample and prepare a standard stock solution with a concentration of 100 μg / mL using methanol. Then dilute the stock solution with methanol to a series of standard solutions with concentrations of 0.1, 0.20, 0.5, 1, 2, and 5 μg / mL.

[0042] (1.3) Sample extraction Accurately weigh approximately 0.1 g of sample (0.1 g of Phalaenopsis petals transferred into PHB empty vector, three biological replicates; 0.1 g of Phalaenopsis petals transferred into PHB-CCD4 vector, three biological replicates) into a centrifuge tube, add 1 mL of methanol, sonicate on ice for 30 min, then centrifuge at 12000 rpm and 4℃ for 10 min, take the supernatant, filter through a membrane, and then perform analysis.

[0043] (1.4) Chromatographic conditions Inlet temperature: 250℃; Carrier gas: Helium, flow rate: 1.2 mL / min; Injection volume: 1 μL; Purging flow rate: 5 mL / min; Table 8 Heating Program ; (1.5) Mass spectrometry parameters Mass spectrometry conditions: The ionization method is electron bombardment ion source: EI source; Transmission line temperature 250℃; Ion source temperature 250℃; Table 9 Qualitative and quantitative ionic parameters of ionone ; (1.6) Data Analysis The results showed that transient expression in Phalaenopsis orchid petals CsCCD4 Compared with the control containing only the empty vector, the accumulation of (E)-β-ionone increased by 1.44 ± 0.03 times (n = 3; P < 0.001), with a peak value of 30.69 μg·g. -1 ·DW ( Figure 6 ).

[0044] Example 5: Stable conversion of Tobacco Monogatna 1. Sterile tobacco seedling culture Place tobacco seeds in 1.5 mL centrifuge tubes, add 50% 84 disinfectant solution, invert the tubes to ensure full contact between the seeds and the disinfectant solution, sterilize for 10 min, then rinse 3-4 times with sterile water in a clean bench. Sow the sterile tobacco seeds onto bottled MS solid medium, one seed per bottle. Place the culture bottles in a light incubator at 25℃ with a photoperiod of 16 h / 8 h (light / dark) for cultivation.

[0045] 2. Preparation and pre-culture of explants Prepare robust, sterile tobacco seedlings that have grown for 60-70 days. Select large, thick, fully expanded green leaves near the top of the plant. Lay the leaves flat on sterile filter paper, fix them with tweezers, and cut off the leaf edges and veins with a blade. Then cut the leaves into 5 mm × 5 mm squares and place them on a pre-culture medium. Pre-culture for 3-4 days under light / dark conditions (16 / 8 h).

[0046] 3. Preparation of Agrobacterium Agrobacterium containing the expression vector of the target gene was inoculated onto the corresponding resistant solid medium and incubated upside down at 28°C for 3 days. Then, single colonies were picked and transferred to 1 mL of resistant liquid LB medium and incubated at 200 rpm in a shaker at 28°C for 24–48 h. After the bacterial culture was well shaken, the shaken bacterial culture was added to 50 mL of resistant liquid LB medium at a ratio of 1:100 and incubated at 200 rpm in a shaker at 28°C until the OD of the bacterial culture was reached. 600 The value is approximately 0.6, and it is reserved.

[0047] 4. Preparation of Infection Solution The Agrobacterium tumefaciens bacterial suspension that has reached the OD value is centrifuged at 5000 rpm for 5 min. The supernatant is discarded in an ultra-clean workbench, and tobacco infection solution is added to resuspend the bacteria. The suspension is then centrifuged again at 5000 rpm for 10 min, and finally resuspended in the infection solution to adjust the OD value of the bacterial suspension. 600 = Between 0.4 and 0.6, pending use.

[0048] 5. Infection and Co-cultivation Take 50 mL of the prepared bacterial inoculation solution into an Erlenmeyer flask, put the cut tobacco leaves into it, shake to ensure that the bacterial inoculation solution is in full contact with the leaves, inoculate for 8-10 min, shaking the Erlenmeyer flask several times during the process, then use tweezers to remove the leaves, place them on sterile filter paper, blot dry the bacterial inoculation solution on their surface, inoculate them onto a co-culture medium, and incubate at 25℃ in a light / dark 16 / 8 h environment for 2-3 days.

[0049] 6. Screening and Cultivation (1) Remove the leaves from the co-culture medium, wash them three times with sterile water, and then soak them in 200 mg / L Timetin water for 2 min. If the washing solution is turbid, repeat this step 2 to 3 times until the liquid is clear. Then use sterile filter paper to dry the water on the surface of the leaves and inoculate them on the primary screening medium for 1 to 2 weeks, with 10 to 14 leaves per plate. (2) After the initial screening, the explants are transferred to the secondary screening medium, with 4-5 explants per plate. They are cultured at 25°C under light, and subcultured every 2 weeks until new shoots are generated. (3) At this time, cut off the regenerated buds and place them on the seedling screening medium for about a week.

[0050] 7. Rooting Culture When the buds grow to more than 1 cm, cut off the callus tissue at the base of the buds and transfer them to a rooting medium containing antibiotics for further culture.

[0051] 8. Genomic DNA extraction from transgenic seedlings Extract DNA according to the instructions of the Magen RaPure Plant DNA Mini Kit: (1) Grind the plant sample into powder using liquid nitrogen, and transfer 50~150 mg of fresh / frozen sample or 15~40 mg of dried sample into a 2 mL centrifuge tube.

[0052] (2) Immediately add 700 μL of preheated Buffer PAL to the sample, vortex vigorously to fully disperse the sample, and bathe in a 65ºC water bath for 20 min, mixing 2 to 3 times during the process.

[0053] (3) Add 700 μL of Buffer BDP to the sample and vortex mix for 15 s.

[0054] (4) Centrifuge at 12,000 × g for 5 min at room temperature, and carefully transfer the supernatant to a new centrifuge tube.

[0055] (5) Add 700 μL Buffer GWP to the supernatant and mix by inverting 10 to 15 times.

[0056] (6) Load the DNA column into the collection tube and transfer half the volume of the mixture into the column. Centrifuge at 12,000 × g for 60 s.

[0057] (7) Discard the filtrate, put the column back into the collection tube, and transfer the remaining mixture into the column. Centrifuge at 12,000 × g for 60 s.

[0058] (8) Discard the filtrate, put the column back into the collection tube, and add 500 μL of Buffer PW1 to the column. Centrifuge at 12,000 × g for 60 s.

[0059] (9) Discard the filtrate, put the column back into the collection tube, and add 500 μL of Buffer PW2 into the column. Centrifuge at 12,000 × g for 60 s.

[0060] (10) (Optional) Discard the filtrate, reassemble the column into the collection tube, and add 500 μL of Buffer PW2 to the column. Centrifuge at 12,000 × g for 60 s.

[0061] (11) Discard the filtrate and put the column back into the collection tube. Centrifuge at 12,000 × g for 2 min to remove residual ethanol from the column.

[0062] (12) Transfer the column to a new 1.5 mL centrifuge tube and add 40-75 μL of preheated 65ºC Buffer AE to the center of the membrane in the column. Let stand at room temperature for 2 min, then centrifuge at 12,000 × g for 1 min.

[0063] (13) (Optional) Add 40-75 μL of preheated Buffer AE to the center of the membrane in the column. Let stand for 2 min. Centrifuge at 12,000 × g for 1 min.

[0064] (14) Discard the DNA binding column and store the DNA at -20°C.

[0065] 9. PCR identification The transgenic seedlings were identified by PCR using the following PCR amplification system. The results showed that all nine transgenic tobacco plants tested were positive. Figure 7 ).

[0066] Table 10 PCR System and Amplification System ; Table 11 Reaction Procedure ; 10. Determination of β-ionone content in stably converted Tobacco mono (1) Instruments 0.0001 g electronic balance (mL-204, METTLER-TOLEDO, USA) Gas chromatograph-tandem mass spectrometer (Thermo Trace 1300 / ISQ7000, ThermoFisher, USA) Rotary mixer (VORTEX-5, Kylin-Bell, China) Sample processing Weigh 5 g of sample (5 g of whole Nicotiana macrophylla flowers already transferred into PHB empty vector, three biological replicates; 5 g of whole Nicotiana macrophylla flowers already transferred into PHB-CCD4 vector, three biological replicates), add 2 g of sodium chloride, add 10 mL of pure water and place in a headspace vial. Equilibrate in a 60°C water bath for 60 min in an electrically heated constant temperature stirrer. Finally, insert the extraction head into the GC-MS injector for desorption.

[0067] GC-MS operation After the sample extraction is complete, quickly insert the extraction head into the gas chromatography-mass spectrometry (GC-MS) inlet.

[0068] GC conditions: Chromatographic column: TG-5MS (30 m × 0.25 mm × 0.25 μm); Heating program: Initial temperature 60℃, hold for 1 min, increase to 160℃ at a rate of 3℃ / min, and increase to 230℃ at a rate of 20℃ / min.

[0069] Inlet temperature: 250℃; Carrier gas: High-purity helium, 99.99% purity; Flow rate: 1.0 mL / min, splitless injection.

[0070] MS conditions: The ionization method was EI, the mass detector was used in 70 V electron collision mode, and the ion source temperature was 230 °C.

[0071] Data Analysis Compared with the control containing only the empty vector, the (E)-β-ionone content in transgenic tobacco flowers was significantly increased, by 5.81 ± 0.29 times (P < 0.01; n = 3), with the highest content reaching 2.69 μg·g⁻¹. -1 ·DW ( Figure 8 ).

[0072] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Cymbidium goeringii CsCCD4 Genes, characterized by, The CsCCD4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A type of Cymbidium goeringii according to claim 1 CsCCD4 Genes, characterized by, The CsCCD4 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.

2.

3. A type of orchid containing the orchid described in claim 1 CsCCD4 Gene expression vectors, characterized in that, The starting vector of the expression vector is a binary vector that can be used for Agrobacterium-mediated transformation of plants or a vector that can be used for plant micro-bombardment.

4. The Cymbidium goeringii according to claim 3 CsCCD4 Gene expression vectors, characterized in that, The starting carriers are pOCA30 and PHB carriers.

5. A recombinant Agrobacterium containing the expression vector of claim 3, characterized in that, The Agrobacterium was strain GV3101.

6. A type of Cymbidium as described in claim 1 CsCCD4 The application of genes in enhancing plant aroma synthesis is characterized by, Includes the following steps: (1) Construct a structure containing the following as shown in SEQ ID NO.1 CsCCD4 Recombinant gene expression vectors; (2) Transform the recombinant expression vector obtained in step (1) into Agrobacterium to obtain recombinant Agrobacterium; (3) Introduce recombinant Agrobacterium into the target plant via transient or stable transformation methods; (4) After the transiently transformed plant has grown normally for 5 days, the petals of the transiently transformed plant were collected for GC-MS determination of aroma substances; (5) On the second day after the stable transformed plant has grown normally and flowered, the petals of the stable transformed plant were collected for GC-MS aroma substance determination.

7. The Cymbidium goeringii according to claim 6 CsCCD4 The application of genes in enhancing plant aroma synthesis is characterized by, The starting vector for the expression vector mentioned in step (1) is the pOCA30 plasmid.

8. The Cymbidium goeringii according to claim 6 CsCCD4 The application of genes in enhancing plant aroma synthesis is characterized by, The Agrobacterium mentioned in step (2) is strain GV3101.

9. The Cymbidium goeringii according to claim 6 CsCCD4 The application of genes in enhancing plant aroma synthesis is characterized by, The target plant for the instantaneous transformation is the petal of a Phalaenopsis orchid; The target plant for the stable transformation is Nicotiana macrocarpa.

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