Method for improving aroma quality of tobacco

Abstract

The invention discloses a method for improving tobacco aroma quality, and belongs to the technical field of gene engineering. The limitation that instantaneous transformation verification is mostly adopted in the prior art is broken through, a technical system for stably transforming the tobacco through the PrGES gene is established, the obtained transgenic tobacco can continuously and stably accumulate geraniol, fragrance is derived from self metabolism, exogenous addition is not needed, and the defect that tobacco body components cannot be improved through traditional exogenous perfuming is thoroughly overcome.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to a method for improving the aroma quality of tobacco. Background Technology

[0002] Flavored cigarettes have become an important growth point and a competitive area for differentiation in the global tobacco market. Currently, floral-flavored cigarettes are relatively scarce, and most are dominated by rose and jasmine. Peony, as a representative of "national beauty and heavenly fragrance," possesses a dignified and rich aroma (a sweet floral fragrance with a slight powdery note), exhibiting distinct Chinese characteristics and cultural recognizability. Developing peony-flavored cigarettes can create a unique memory point, standing out among numerous fruit and mint flavors and filling the market gap for distinctive floral aromas. Geraniol is a high-value monoterpene compound with a sweet floral aroma, but tobacco lacks this key component. Global annual demand for geraniol exceeds 10,000 tons, widely used in fragrances, cosmetics, and food additives. However, industrial production mainly relies on chemical synthesis, which involves complex processes and the potential introduction of non-natural ingredients. Direct extraction from plants is limited by raw material resources, cost, and supply stability. Adding flavorings to tobacco products cannot fundamentally improve the aroma quality of the tobacco raw materials themselves.

[0003] Flare tree peony (FTP) is an ornamental and economic crop with extremely high geraniol content. Geraniol accounts for the highest proportion of total volatile components in its flowers, making it a natural resource for geraniol synthesis genes. However, current research on geraniol synthesis in peonies is limited to aroma component identification; there are no reports of cloning geraniol synthase genes from peonies and achieving stable genetic transformation and functional verification in tobacco. Therefore, there is an urgent need to explore efficient geraniol synthase genes from peonies. GES Genes provide new genetic resources and technological pathways for improving the aroma of tobacco.

[0004] Targeted enhancement of endogenous geraniol synthesis in plants through metabolic engineering is considered a more promising fundamental solution. Geraniol is mainly synthesized via the MEP pathway in plastids, where geraniol synthase (GES) directly catalyzes the conversion of the precursor geraniyl diphosphate (GPP) into geraniol. Previous studies have shown that heterologous transformation of tobacco with GES or terpene synthase (TPS) genes from different plant sources can promote the accumulation of monoterpenes. However, most studies have significant limitations: either transient transformation is used for preliminary functional verification, failing to obtain stably inherited plants; or the gene source is not a peony variety with high geraniol content, resulting in limited regulatory efficiency. Furthermore, transgenic peony-flavored tobacco has several advantages over traditional flavoring additives: firstly, traditional flavorings need to be sprayed or injected during processing, which can easily lead to aroma distortion or attenuation due to volatilization and pyrolysis; while transgenic tobacco synthesizes its own aroma substances, allowing the aroma to naturally blend with the tobacco matrix, resulting in a more even and richer release during smoking. Secondly, while synthetic flavorings (such as vanillin) are compliant within limited dosage limits, their antibacterial and cytotoxic properties remain controversial (e.g., damaging cell membranes and interfering with metabolic enzyme activity). Genetically modified tobacco generates aroma endogenously, eliminating the need for exogenous compounds, thus avoiding concerns about flavoring residues and potential toxicity, and aligning better with the trend of "harm reduction" products. Traditional flavorings are limited by extraction processes (e.g., low reduction rates of natural fragrances) and reaction efficiency (e.g., Maillard reaction conversion rates are only 25%–40%). Gene editing can target and enhance specific metabolic pathways; for example, by inserting geraniol synthesis genes, we can directly impart rare floral aromas like peony to tobacco, modify terpene synthesis pathways, and enhance the sweet or woody base notes of tobacco itself. Finally, flavoring preparation relies on complex chemical synthesis or natural extraction (e.g., the cost of natural vanillin extraction is hundreds of times that of synthetic products), while genetically modified tobacco can continuously express flavor with a single breeding, resulting in significant long-term cost optimization. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, the present invention provides a method for improving the aroma quality of tobacco.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a method for improving the aroma quality of tobacco, which includes the following steps: (1) Extract from peony as shown in SEQ ID NO.1 GES Gene; (2) To GES The gene and vector were double-digested, and the digested products were recovered separately. GES Gene fragments and vector fragments are then ligated together using homologous recombinase and transformed into competent E. coli cells; (3) Screen positive clones to obtain those containing GESThe recombinant plasmid of the gene was then transformed into competent Agrobacterium tumefaciens cells, and positive Agrobacterium tumefaciens were obtained by screening. (4) Immerse tobacco explants in a culture of Agrobacterium tumefaciens, remove excess culture after soaking, and then culture to obtain GES Tobacco with improved aroma quality and stable gene expression.

[0007] Furthermore, the peony in step (1) is a purple-spotted peony, and the variety is 'Jingyunxiang'.

[0008] Furthermore, the vector in step (2) is the plant binary expression vector pCAMBIA1304.

[0009] Furthermore, the competent cells in step (2) are *Escherichia coli*. DH5α Competent cells.

[0010] Furthermore, in step (3), the recombinant plasmid is transformed into competent Agrobacterium tumefaciens cells by freeze-thaw method.

[0011] Furthermore, the Agrobacterium tumefaciens in step (3) is Agrobacterium tumefaciens GV3101.

[0012] Furthermore, in step (4), the tobacco variety is tobacco 'NC89'.

[0013] Furthermore, in step (4), the explant is a tender leaf of a tobacco 'NC89' seedling, cut into a leaf disc of 0.5 cm × 0.5 cm.

[0014] Furthermore, the cultivation process in step (4) is as follows: S1. Place the soaked tobacco explants in a co-culture medium and incubate in the dark at 25°C for 2 days; the co-culture medium is MS basal medium + 3% sucrose + 100 μM acetylsalicylic acid. S2. The co-cultured explants were transferred to the selection medium and cultured at 25℃ with a photoperiod of 16h / 8h. The selection medium was replaced with fresh selection medium every 2-3 weeks to induce the differentiation of resistant callus and shoots. The selection medium consisted of MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 300 mg / L cephalosporin + 50 mg / L hygromycin. S3. When the resistant buds grow to 2-3 cm, cut them off and transfer them to the rooting medium to induce rooting. After obtaining complete plants, harden them off in the greenhouse for 1 week and then transplant them into the soil for normal cultivation. The rooting medium is 1 / 2 MS basal medium + 0.1 mg / L NAA + 50 mg / L hygromycin.

[0015] The beneficial effects of this invention are: 1. First cloning and application of the purple-spotted peony source PrGES This gene discovery fills a gap in the research and application of genes key to the synthesis of geraniol in peony. PrGES The gene is derived from Paeonia suffruticosa, a plant with high geraniol content. Its catalytic efficiency and specificity are significantly better than those of synthase genes from non-Paeonia suffruticosa sources, providing a new high-quality gene resource for geraniol synthesis in plants.

[0016] 2. This invention overcomes the limitations of existing technologies that mostly employ instantaneous conversion verification, and establishes... PrGES The technology system for stable gene transformation of tobacco produces transgenic tobacco that sustainably and stably accumulates geraniol. The aroma originates from its own metabolism, requiring no exogenous addition, thus completely overcoming the shortcomings of traditional exogenous flavoring methods that cannot improve the composition of tobacco itself. By directly introducing the key enzyme gene that catalyzes geraniol synthesis into tobacco, without relying on upstream transcription factor regulation, the technical path is more direct, the operation is simpler, and it is easy to apply on a large scale to tobacco breeding practices.

[0017] 3. Cloned by this invention PrGES Genes can not only be used to improve the aroma of tobacco, but also to enhance the geraniol synthesis capacity of other ornamental plants and spice crops, providing new technological support for the natural fragrance industry. Attached Figure Description

[0018] Figure 1 for PrGES Gene cloning and sequencing comparison and amino acid domain detection diagram; Figure 2 Wild type (WT) and PrGES In genetically modified tobacco plants PrGES RT-PCR detection results of the gene; Figure 3 Wild type (WT) and PrGES In genetically modified tobacco plants PrGES Gene qRT-PCR detection results; Figure 4 Wild type (WT) and PrGES GC-MS detection image of linalool in transgenic tobacco plants; Figure 5 Wild type (WT) and PrGES Figure showing the results of linalool release from genetically modified tobacco plants. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] RT-PCR: Reverse Transcription-Polymerase Chain Reaction, used to detect gene transcription levels.

[0021] qRT-PCR: Quantitative Real-time RT-PCR is used to accurately quantify the number of gene transcripts.

[0022] GC-MS: Gas Chromatography-Mass Spectrometry, used for the separation and qualitative and quantitative analysis of volatile components in complex mixtures.

[0023] HS-SPME: Headspace Solid-Phase Microextraction, a pretreatment method for enriching volatile components in the headspace of a sample.

[0024] Agrobacterium: Here it specifically refers to Agrobacterium tumefaciens (Gastrointestinal rust) Agrobacterium tumefaciens ), a type of bacteria commonly used as a vector for plant genetic transformation.

[0025] CaMV 35S: Cauliflower Mosaic Virus 35S Promoter, a strongly constitutive promoter widely used in dicotyledonous plants.

[0026] CDS: Coding Sequence, refers to the portion of DNA or RNA that encodes a protein.

[0027] Examples of Improving Tobacco Aroma Quality 1. Peony PrGES Cloning of genes (1) Plant materials and RNA extraction Fresh petals of the purple-spotted peony cultivar 'Jingyunxiang' during its peak bloom (S4) were collected, immediately flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA was extracted from the petals using a commercially available plant total RNA extraction kit, following the manufacturer's instructions. RNA purity and integrity were assessed by agarose gel electrophoresis and Nanodrop.

[0028] (2) cDNA synthesis and gene amplification Total RNA was reverse transcribed into first-strand cDNA using reverse transcriptase. This was based on data obtained from transcriptome sequencing. PrGESDesign specific primers for gene coding sequence (CDS) (forward primer contains...) NcoI Enzyme cleavage site, reverse primer contains SpeI (Enzyme restriction sites). Using cDNA as a template, high-fidelity polymerase chain reaction (PCR) amplification is performed to obtain... PrGES Full-length CDS fragment of the gene.

[0029]

[0030] (3) PCR product purification and sequencing verification The PCR products were subjected to agarose gel electrophoresis, the target band was recovered, and ligated into the pEASY-Blunt cloning vector for sequencing. The sequence alignment results are shown in [Figure number missing]. Figure 1 .

[0031] like Figure 1 As shown, sequence alignment confirmed that the cloned gene sequence matched the target sequence. PrGES The gene sequences are completely identical and without mutations. The protein sequences, after comparison with other species, contain the conserved catalytic domains (DDxxD and NSE / DTE) unique to geraniol synthase.

[0032] 2. Construction of plant expression vectors (1) Enzyme digestion of vector and gene fragment use NcoI and SpeI Restriction endonucleases were used to treat purified samples containing the correct enzymes. PrGES The sequence cloning vector plasmid and the plant binary expression vector pCAMBIA1304 were double-digested with enzymes, and the digested enzymes were recovered. PrGES Gene fragments and large vector fragments.

[0033] (2) Connection and transformation Enzyme digestion and recovery PrGES The gene fragment was ligated with the pCAMBIA1304 vector fragment, which had also undergone enzyme digestion and recovery, using homologous recombinase. The ligation product was then transformed into *E. coli*. DH5α competent cells.

[0034] (3) Screening and validation of recombinant plasmids In the presence of kanamycin ( Kanamycin Positive clones were screened on LB agar medium, and plasmids were extracted. Confirmation was achieved through double enzyme digestion and sequencing. PrGES The gene has been correctly inserted downstream of the CaMV 35S promoter and upstream of the Nos terminator in the pCAMBIA1304 vector. This recombinant plasmid is named pCAMBIA1304- 35S : PrGES .

[0035] 3. Agrobacterium-mediated genetic transformation and plant regeneration in tobacco (1) Agrobacterium transformation The recombinant plasmid pCAMBIA1304- was prepared using a freeze-thaw method. 35S:PrGES Transformation into Agrobacterium tumefaciens ( Agrobacteriumtumefaciens In competent cells of strain GV3101, positive Agrobacterium strains were verified by PCR.

[0036] (2) Preparation and co-culture of tobacco explants Young leaves from sterile cultured tobacco 'NC89' seedlings were cut into leaf discs of approximately 0.5 cm × 0.5 cm. The leaf discs were immersed in Agrobacterium tumefaciens bacterial suspension containing the target plasmid and activated and resuspended in the infection solution (containing 100 μM acetylsyleugenol) for 10 minutes. After removing the discs, excess bacterial suspension was aspirated, and the discs were placed on co-culture medium (MS basal medium + 3% sucrose + 100 μM acetylsyleugenol) and incubated in the dark at 25°C for 2 days.

[0037] (3) Induction of resistant callus and bud After co-culture, the leaf discs were transferred to selection medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 300 mg / L cephalosporin + 50 mg / L hygromycin) and cultured at 25℃ with a photoperiod of 16h / 8h. Fresh selection medium was replaced every 2-3 weeks to induce the differentiation of resistant callus and buds.

[0038] (4) Rooting and hardening off seedlings When the resistant shoots reach 2-3 cm in length, cut them off and transfer them to rooting medium (1 / 2 MS + 0.1 mg / L NAA + 50 mg / L hygromycin) to induce rooting. After obtaining complete plants, harden them off in a greenhouse for one week before transplanting them into soil for normal cultivation.

[0039] 4. Molecular identification of transgenic tobacco plants (1) DNA level identification (PCR detection) Genomic DNA was extracted from the leaves of transplanted tobacco plants to... PrGES Genes and hygromycin resistance genes ( Hyg PCR amplification was performed using specific primers for the target band, with wild-type tobacco as a negative control. Plants that could amplify the target band were considered positive.

[0040] (2) RNA level identification (RT-PCR detection) Total RNA was extracted from the leaves of positive plants and reverse transcribed into cDNA for further processing. PrGES Gene-specific primers were used for RT-PCR detection to verify the transcriptional expression of the gene (see...). Figure 2 ).

[0041] like Figure 2 As shown, it can only be detected in transgenic plants. PrGES The transcript of the gene proves that the gene is successfully expressed in tobacco.

[0042] (3) Quantitative analysis of gene expression (qRT-PCR detection) Using qRT-PCR technology, NtActinThis is an internal reference gene, used to detect [genes] in transgenic plants. PrGES The relative expression levels of genes were analyzed to differentiate gene expression among different transgenic lines (see...). Figure 3 ).

[0043] like Figure 3 As shown, in transgenic plants PrGES Gene expression was significantly upregulated compared to the wild-type control.

[0044] 5. Phenotypic analysis of transgenic tobacco plants (1) Extraction of aroma substances Take 1g each of mature leaves or full-blown flowers of genetically modified and wild-type tobacco, place them in a headspace vial, add 3-octanol as an internal standard, equilibrate at 40℃ for 40 minutes, and then adsorb headspace volatiles using a 100μm PDMS solid-phase microextraction fiber head.

[0045] (2) GC-MS analysis After thermal desorption of the fiber head, separation and detection were performed using an Agilent 5975C GC-MS system with an HP-1MS capillary column and helium as the carrier gas. The programmed temperature conditions were: 40℃ for 3 minutes, 5℃ / min to 120℃ (hold for 1 minute), 10℃ / min to 180℃ (hold for 3 minutes), and 10℃ / min to 280℃ (hold for 1 minute).

[0046] (3) Qualitative and quantitative analysis Geraniol was identified by NIST14 mass spectrometry library comparison, and its content was calculated using the internal standard method. The differences between transgenic and wild-type plants were statistically analyzed, and a significance analysis (one-way ANOVA) was performed. Results are shown below. Figure 4 and Figure 5 .

[0047] like Figure 4 and Figure 5 As shown, compared with the wild-type control, overexpression PrGES In the leaves of the genetically modified tobacco, the content of geraniol was significantly increased to approximately 50 ng / g / h (fresh weight). The content of some terpenoid-related substances also increased synergistically. Sensory evaluation revealed that the leaves of the genetically modified plants had a unique sweet floral aroma, namely the "peony fragrance".

[0048] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for improving the aroma quality of tobacco, characterized in that, Includes the following steps: (1) Extract from peony as shown in SEQ ID NO.1 GES Gene; (2) To GES The gene and vector were double-digested, and the digested products were recovered separately. GES Gene fragments and vector fragments are then ligated together using homologous recombinase and transformed into competent E. coli cells; (3) Screen positive clones to obtain those containing GES The recombinant plasmid of the gene was then transformed into competent Agrobacterium tumefaciens cells, and positive Agrobacterium tumefaciens were obtained by screening. (4) Immerse tobacco explants in a culture of Agrobacterium tumefaciens, remove excess culture after soaking, and then culture to obtain GES Tobacco with improved aroma quality and stable gene expression.

2. The method according to claim 1, characterized in that, The peony in step (1) is a purple-spotted peony, and the variety is 'Jingyunxiang'.

3. The method according to claim 1, characterized in that, The vector in step (2) is the plant binary expression vector pCAMBIA1304.

4. The method according to claim 1, characterized in that, The competent cells in step (2) are Escherichia coli. DH5α Competent cells.

5. The method according to claim 1, characterized in that, In step (3), the recombinant plasmid is transformed into competent Agrobacterium tumefaciens cells by freeze-thaw method.

6. The method according to claim 1 or 5, characterized in that, The Agrobacterium tumefaciens in step (3) is Agrobacterium tumefaciens GV3101.

7. The method according to claim 1, characterized in that, In step (4), the tobacco variety is tobacco 'NC89'.

8. The method according to claim 7, characterized in that, In step (4), the explant is the tender leaf of tobacco 'NC89' seedling, which is cut into leaf discs of 0.5 cm × 0.5 cm.

9. The method according to claim 1, characterized in that, The cultivation process in step (4) is as follows: S1. Place the soaked tobacco explants in a co-culture medium and incubate in the dark at 25°C for 2 days; the co-culture medium is MS basal medium + 3% sucrose + 100 μM acetylsalicylic acid. S2. The co-cultured explants were transferred to the selection medium and cultured at 25℃ with a photoperiod of 16h / 8h. The selection medium was replaced with fresh selection medium every 2-3 weeks to induce the differentiation of resistant callus and shoots. The selection medium consisted of MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 300 mg / L cephalosporin + 50 mg / L hygromycin. S3. When the resistant buds grow to 2-3 cm, cut them off and transfer them to the rooting medium to induce rooting. After obtaining complete plants, harden them off in the greenhouse for 1 week and then transplant them into the soil for normal cultivation. The rooting medium is 1 / 2 MS basal medium + 0.1 mg / L NAA + 50 mg / L hygromycin.

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