Cigar CYP82E10 gene mutant G796A and application thereof
By screening the CYP82E10 gene mutant G796A in cigars using EMS mutagenesis technology, the problem of high nicotine conversion rate in cigars was solved, and the nicotine conversion rate was significantly reduced, creating cigar materials with low nicotine conversion rate.
Patent Information
- Application Number
- CN202511126542.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-11-18
AI Technical Summary
In existing technologies, the nicotine conversion rate in cigars is high, resulting in a high content of TSNAs, which affects safety. There is a lack of effective methods to reduce the nicotine conversion rate in cigars.
The mutant G796A of the CYP82E10 gene in cigar tobacco was screened using EMS mutagenesis. The nucleotide sequence is SEQ ID NO:2 and the encoding amino acid sequence is SEQ ID NO:4. The screening methods included EMS treatment, DNA pool construction, PCR amplification and detection, and homozygous screening to obtain cigar tobacco material with low nicotine conversion rate.
Significantly reduces the nicotine conversion rate in cigars by no less than 38%, creating cigar materials with low nicotine conversion rates.
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Figure CN120966853A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of genetic engineering, in particular to a cigar CYP82E10 gene mutant G796A and application thereof. BACKGROUND
[0002] With the development of global economy, the share of cigar in tobacco market is gradually increasing, and the sales of domestic cigar in China are rapidly growing. High-quality cigar not only requires good smoking quality, but also needs to have low harm and safety. High content of tobacco-specific nitrosamines (TSNAs) is the primary safety problem that needs to be solved in the development of cigar. It is reported that the content of TSNAs in cigar is 1-2 orders of magnitude higher than that in burley and flue-cured tobacco.
[0003] N-nitrosonornicotine (NNN) in TSNAs is listed as a class I carcinogen. NNN is mainly formed through the nitrosation of nornicotine. As the main direct precursor of NNN, reducing the content of nornicotine is the most direct means to reduce NNN. The CYP82E4, CYP82E5 and CYP82E10 genes of the CYP82E2 subfamily of burley tobacco can encode active nornicotine demethylase, which is a key enzyme for nicotine transformation. Lewis et al. obtained burley tobacco materials with mutations in CYP82E4, CYP82E5 and CYP82E10 by EMS mutagenesis, and found that the mutations in CYP82E5 and CYP82E10 basically did not affect the nicotine conversion rate, and the nicotine conversion rate in the mutant strain with simultaneous mutations in the three genes was much lower than that in the control strain. The above studies show that CYP82E4 is the key gene that determines the nicotine conversion rate in burley tobacco, while CYP82E5 and CYP82E10 genes have no significant effect on nicotine conversion. The mechanisms of nicotine transformation in cigar and burley tobacco are different, and there is currently no relevant research on cigar. The method for reducing the nicotine conversion rate of cigar still needs to be further explored. SUMMARY
[0004] The present application aims at least to solve one of the problems in the prior art or related art.
[0005] To this end, the first aspect of the present application provides a cigar CYP82E10 gene mutant G796A, the nucleotide sequence of which is shown in SEQ ID NO: 2, wherein the cigar CYP82E10 gene mutant G796A is obtained by changing the 796th nucleotide of the cigar CYP82E10 gene from G to A.
[0006] Further, the amino acid sequence encoded by the cigar CYP82E10 gene mutant G796A is shown in SEQ ID NO: 4.
[0007] Further, the screening method of the cigar CYP82E10 gene mutant G796A comprises the following steps:
[0008] EMS mutagenesis: cigar seeds are treated with ethyl methanesulfonate to obtain M1 generation seeds;
[0009] DNA pool construction: the M1 generation seeds are selfed to obtain M2 generation seeds, and DNA of leaves of single plants of the M2 generation is extracted to construct a DNA pool;
[0010] PCR amplification and detection: specific primers of the cigar CYP82E10 gene are designed, and DNA samples of the DNA pool are subjected to PCR amplification, and M2 generation plants of the cigar CYP82E10 gene mutant G796A are screened by capillary electrophoresis;
[0011] Homozygote screening: the M2 generation plants of the cigar CYP82E10 gene mutant G796A are cultivated in a greenhouse to obtain M3 generation plants, and plants containing homozygous CYP82E10 gene mutant G796A are obtained by sequencing and selfed to obtain seeds.
[0012] Further, the EMS mutagenesis comprises:
[0013] The rinsed cigar seeds are soaked in a 0.5% ethyl methanesulfonate solution for 10-15 hours, and then washed with deionized water to obtain M1 generation seeds; wherein the cigar seeds are cigar seeds containing the CYP82E10 gene.
[0014] Further, the DNA pool construction comprises:
[0015] The M1 generation seeds are sown, and the M2 generation seeds are obtained by selfing, and DNA of leaves of single plants of the M2 generation is extracted, diluted to 40 ng / μl, and mixed to construct the DNA pool.
[0016] Further, the specific primers comprise E10-F and E10-R; wherein the nucleotide sequence of E10-F is shown as SEQ ID NO: 5, and the nucleotide sequence of E10-R is shown as SEQ ID NO: 6.
[0017] Further, the tobacco leaf containing the cigar CYP82E10 gene mutant G796A has a lower nicotine conversion rate than the tobacco leaf containing the cigar CYP82E10 gene.
[0018] Further, the nicotine conversion rate of the tobacco leaf comprising the mutant G796A of the cigar CYP82E10 gene is reduced by not less than 38% compared with the nicotine conversion rate of the tobacco leaf comprising the cigar CYP82E10 gene.
[0019] In a second aspect, the application provides a use of the mutant G796A of the cigar CYP82E10 gene in reducing the nicotine conversion rate of a cigar.
[0020] Further, the mutant G796A of the cigar CYP82E10 gene is used to create a cigar material with a low nicotine conversion rate.
[0021] Compared with the prior art, the application has at least the following beneficial effects:
[0022] The mutant G796A of the cigar CYP82E10 gene provided by the application can significantly reduce the nicotine conversion rate of a cigar and can be used to create a cigar material with a low nicotine conversion rate. Tests prove that the nicotine conversion rate of the tobacco leaf comprising the mutant G796A of the cigar CYP82E10 gene is reduced by 39% compared with the nicotine conversion rate of the tobacco leaf comprising the wild-type cigar CYP82E10 gene. BRIEF DESCRIPTION OF DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed in the embodiment description. Obviously, the drawings in the following description are only some embodiments described in the embodiments of the application, and other drawings can also be obtained by those skilled in the art according to these drawings.
[0024] Figure 1 The sequencing results of the mutant G796A of the cigar CYP82E10 gene are shown. Mu1020 represents the mutant, and Wild represents the wild type.
[0025] Figure 2 The nucleotide sequence alignment results of the coding region of the mutant G796A of the cigar CYP82E10 gene and the wild-type cigar CYP82E10 gene are shown. Mu1020 represents the mutant, and Wild represents the wild type.
[0026] Figure 3 The amino acid sequence alignment results of the protein encoded by the mutant G796A of the cigar CYP82E10 gene and the protein encoded by the wild-type cigar CYP82E10 gene are shown. Mu1020 represents the mutant, and Wild represents the wild type.
[0027] Figure 4The nicotine conversion rates of the cigar CYP82E10 gene mutant G796A and wild-type cigar are shown; wherein Mu1020 represents the mutant, Wild represents the wild-type tobacco, and ** represents a significant difference (P<0.01). DETAILED DESCRIPTION
[0028] In order to better understand the above technical solutions, the technical solutions of the embodiments of the present application will be described in detail below through the drawings and specific embodiments. It should be understood that the specific features in the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of the present application, and are not limitations of the technical solutions of the present application. In the case of no conflict, the technical features in the embodiments of the present application and the embodiments can be combined with each other.
[0029] In the following examples, the reagents not specifically specified are conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. The experimental methods and conditions not specifically specified are conventional experimental methods and conditions in the art, which can be referred to relevant experimental manuals, known literature or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.
[0030] In a first aspect of the embodiments of the present application, a cigar CYP82E10 gene mutant G796A is provided, the nucleotide sequence of which is shown in SEQ ID NO: 2. The cigar CYP82E10 gene mutant G796A is obtained by changing the 796th nucleotide of the cigar CYP82E10 gene shown in SEQ ID NO: 1 from G to A.
[0031] Further, the amino acid sequence encoded by the cigar CYP82E10 gene mutant G796A is shown in SEQ ID NO: 4, and compared with the amino acid sequence encoded by the cigar CYP82E10 gene shown in SEQ ID NO: 3, the 266th amino acid is changed from glutamic acid (E) to lysine (K).
[0032] In some embodiments, the screening method of the cigar CYP82E10 gene mutant G796A comprises:
[0033] EMS mutagenesis: ethyl methanesulfonate is used to treat cigar seeds to obtain M1 generation seeds;
[0034] DNA pool construction: M1 generation seeds are self-crossed to obtain M2 generation seeds, and DNA of leaves of single plants of M2 generation is extracted to construct a DNA pool;
[0035] PCR amplification and detection: specific primers of CYP82E10 gene of cigar tobacco are designed, and DNA samples of the DNA pool are subjected to PCR amplification, and M2 generation plants of CYP82E10 gene mutant G796A of cigar tobacco are screened through capillary electrophoresis;
[0036] Homozygote screening: M2 generation plants of CYP82E10 gene mutant G796A of cigar tobacco are cultivated in a greenhouse, and M3 generation plants are obtained, and plants containing homozygous CYP82E10 gene mutant G796A are obtained through sequencing, and are self-crossed to obtain seeds.
[0037] In the formula, the EMS mutagenesis comprises: soaking the rinsed cigar tobacco seeds in a 0.5% ethyl methanesulfonate solution for 10-15 hours, and then washing with deionized water to obtain M1 generation seeds; wherein the cigar tobacco seeds are cigar tobacco seeds containing CYP82E10 gene.
[0038] In the formula, the DNA pool construction comprises: sowing the M1 generation seeds, self-crossing to obtain M2 generation seeds, extracting DNA from leaves of single plants of the M2 generation, diluting to 40 ng / μl, and mixing 8 parts of DNA to construct the DNA pool.
[0039] In the formula, the specific primers comprise E10-F and E10-R, the nucleotide sequence of E10-F is shown as SEQ ID NO: 5, and the nucleotide sequence of E10-R is shown as SEQ ID NO: 6.
[0040] Specifically, the screening method has high throughput, saves time and labor.
[0041] In a second aspect of the present application, the above-mentioned CYP82E10 gene mutant G796A of cigar tobacco is provided for use in reducing the nicotine conversion rate of cigar tobacco.
[0042] Specifically, the tobacco leaf containing CYP82E10 gene mutant G796A of cigar tobacco has a lower nicotine conversion rate than the tobacco leaf containing CYP82E10 gene of cigar tobacco, and the reduction amount of nicotine conversion rate is not less than 40%.
[0043] Example 1
[0044] In this embodiment, the tobacco containing the above-mentioned CYP82E10 gene mutant G796A of cigar tobacco is obtained by treating cigar tobacco seeds with EMS and screening mutants using TILLING technology.
[0045] 1. EMS treatment of cigar tobacco seeds
[0046] The CYP82E10 gene-containing Virginia tobacco seeds are soaked in 50% commercially available bleaching water for 12 minutes, then spin centrifuged and filtered dry; the seeds are rinsed with deionized water to remove the bleaching water, so that the seeds are not affected by the chemical components of the bleaching agent, then spin centrifuged and filtered dry again; the rinsed seeds are soaked in deionized water at a water temperature of 10-30°C for 10-15 hours, the tobacco seeds are germinated to facilitate uniform mutagenesis treatment of the seeds, then spin centrifuged and filtered dry; the seeds are soaked in 0.5% EMS (ethyl methanesulfonate) solution for 10-15 hours, then spin centrifuged and filtered dry; then rinsed with deionized water for 5-8 times; finally, the seeds are filtered and dried using a Buchner funnel and filter paper.
[0047] 2. Mutant screening
[0048] Mutants are screened using the TILLING technology. The EMS-treated seeds (M1 generation) are sown in a field, individually bagged, and selfed to obtain M2 generation, and the M2 generation seeds harvested from each M1 generation plant are sown one seed per plant. The leaves of the M2 generation plants are taken, and genomic DNA is extracted using a DNA extraction kit (QIAGEN, 69106) according to the kit instructions. The obtained DNA samples are arranged in order. 2 μl of each DNA sample is taken, and concentration determination is performed on a 16-channel Tecan infinite M200 instrument. Then the concentration of all DNA samples is diluted to 40 ng / μl, and 8-fold DNA pools are prepared by mixing 8 DNA samples each, for TILLING analysis.
[0049] The TILLING analysis primers E10-F / E10-R for the CYP82E10 gene of Virginia tobacco are designed using Primer 3 software, and the nucleotide sequences are as follows:
[0050] E10-F: 5'-GTCAAATACCACCTCTTAATAGTAA-3' (SEQ ID NO: 5);
[0051] E10-R: 5'-AAAAGTCCCTATTGGTAGGAAGTGC-3' (SEQ ID NO: 6).
[0052] The size of the target fragment amplified by the above primers is 1136 bp. The above DNA samples are amplified according to the following reaction system and reaction procedure.
[0053] Table 1 Reaction system
[0054]
[0055] Reaction procedure: 95°C 3 min; (94°C 30 s, 63°C 30 s, -1°C / cycle, 72°C 1 min) 7 cycles; (94°C 30 s, 58°C 30 s, 72°C 1 min) 40 cycles; 72°C 5 min; 99°C 10 min; (70°C 20 s, -0.3°C / cycle) 70 cycles; 4°C storage.
[0056] After the reaction, the amplification products were analyzed by capillary electrophoresis to screen single strains of nucleotide mutations, and sequencing verification was performed, as shown in Figure 1
[0057] A mutant was obtained, in which the coding sequence (CDS) of the CYP82E10 gene was changed from G to A at position 796, as shown in Figure 2 Figure 3 The amino acid sequence encoded by the gene was changed from glutamic acid (E) to lysine (K) at position 266, as shown in
[0058] Example 2
[0059] In this example, the nicotine and nornicotine contents of the wild-type tobacco and the tobacco containing the homozygous CYP82E10 gene mutant G796A obtained in Example 1 were determined according to the Tobacco Industry Standard YC / T382-2010, and the nicotine conversion rate was calculated.
[0060] The instruments used for the determination were Shimadzu GCMS-QP2020 gas chromatograph mass spectrometer (Shimadzu Corporation, Japan), Millipore ultrapure water machine (Millipore Simplicity Corporation, USA), Eofo-945008 vortex mixing oscillator (Tallboys Corporation, USA), and Eppendorf 5804 high-speed centrifuge (Eppendorf Corporation, Germany).
[0061] The reagents used in the assay: the internal standard quinoline purity greater than 98%, purchased from Sigma-Aldrich Company; the internal standard nornicotine-D4 purity greater than 98%, purchased from the United States CATO Company; nornicotine purity greater than 98%, purchased from Sigma-Aldrich Company; nicotine purity greater than 98%, purchased from the Canadian TRC Company; sodium hydroxide is analytical pure, purchased from Shantou Xilong Chemical Factory in Guangdong; dichloromethane, methanol are chromatographic pure, purchased from the United States Fisher Company.
[0062] The determination method: wild type CYP82E10 gene containing Virginia tobacco and homozygous CYP82E10 gene mutant G796A containing Virginia tobacco were planted in the greenhouse, and the plants were topped when they grew to the flowering stage. 15 days after topping, the middle leaves were taken, 10 single plants with consistent growth were selected for each material, and the tobacco leaves were killed and dried. 0.3 g of crushed Virginia tobacco leaf sample was weighed in a 15 mL centrifuge tube, 0.05 mL of internal standard solution containing quinoline and nornicotine-d4 was added, and 2 mL of 5% sodium hydroxide aqueous solution was added, and then shaken and mixed, and then placed for 20 min. Then 10.0 mL of dichloromethane-methanol extraction solution (v:v=4:1) was added, sealed and placed in a vortex oscillator, vortexed at 2000 rpm for 40 min, and then the lower organic phase was transferred to a chromatographic analysis bottle for instrument analysis after standing for 1 h.
[0063] The chromatographic conditions are as follows:
[0064] Chromatographic column: BR-5MS (30 m x 0.25 mm x 0.25 μm); injection volume: 1 μL, split ratio 60:1; injection port: 250℃; temperature program: initial temperature 110℃, 10℃ / min to 185℃, 50℃ / min to 280℃, hold for 2 min; carrier gas: helium, 1.0 mL / min.
[0065] The mass spectrometry conditions are as follows:
[0066] Transfer line temperature: 250℃, ion source temperature: 230℃; ionization mode: electron impact ionization (EI); bombardment energy: 70 eV; solvent delay time: 4 min; scanning mode: selected ion monitoring mode (SIM) scan, the retention time, quantitative and qualitative selection ion parameters of nicotine, nornicotine and internal standard are as shown in Table 2.
[0067] Table 2 Retention time, quantitative and qualitative selection ion parameters of nicotine, nornicotine and internal standard
[0068]
[0069] The results of nicotine and nornicotine detection of cigar CYP82E10 gene mutant G796A and wild type material are shown in Table 3. Nicotine conversion rate was calculated according to the following formula: nicotine conversion rate = nornicotine content / (nicotine content + nornicotine content) x 100%.
[0070] Table 3 nicotine and nornicotine detection results
[0071]
[0072] Note: "**" in the table indicates significant difference (P < 0.01).
[0073] From Table 3 and Figure 4 It can be seen that the tobacco leaf containing the cigar CYP82E10 gene mutant G796A has a nicotine conversion rate reduced by nearly 39% compared with the tobacco leaf containing the wild type gene.
[0074] Those skilled in the art can easily understand that the above advantageous modes can be freely combined and superimposed without conflict. The above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application. The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and modifications without departing from the technical principles of the present application, and these improvements and modifications should be considered as the protection scope of the present application.
Claims
1. A cigar tobacco CYP82E10 gene mutant G796A, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:
2. The CYP82E10 gene mutant G796A of cigar cigarettes is obtained by changing the 796th nucleotide of the CYP82E10 gene of cigar cigarettes from G to A.
2. The CYP82E10 gene mutant G796A for cigars according to claim 1, characterized in that, The amino acid sequence encoded by the CYP82E10 gene mutant G796A of the cigar is shown in SEQ ID NO:
4.
3. The CYP82E10 gene mutant G796A for cigars according to claim 1, characterized in that, The screening method for the CYP82E10 gene mutant G796A in cigars includes: EMS mutagenesis: Cigar seeds were treated with ethyl methanesulfonate to obtain M1 generation seeds; DNA pool construction: M2 generation seeds were obtained by self-pollination of M1 generation seeds, and DNA was extracted from leaves of M2 generation single plants to construct a DNA pool. PCR amplification and detection: Specific primers for the CYP82E10 gene of cigar tobacco were designed, and DNA samples from the DNA pool were amplified by PCR. M2 generation plants of the CYP82E10 gene mutant G796A of cigar tobacco were obtained by capillary electrophoresis screening. Homozygous selection: M2 generation plants of the CYP82E10 gene mutant G796A of the cigar tobacco were cultivated in a greenhouse to obtain M3 generation plants. Plants containing the homozygous CYP82E10 gene mutant G796A were obtained by sequencing and self-pollination for seed collection.
4. The CYP82E10 gene mutant G796A for cigars according to claim 3, characterized in that, The EMS mutagenesis includes: The rinsed cigar seeds were soaked in a 0.5% ethyl methanesulfonate solution for 10-15 hours, and then washed with deionized water to obtain M1 generation seeds; wherein the cigar seeds are cigar seeds containing the CYP82E10 gene.
5. The CYP82E10 gene mutant G796A for cigars according to claim 3, characterized in that, The DNA pool construction includes: The M1 generation seeds were sown, and the M2 generation seeds were obtained by self-pollination. DNA was extracted from the leaves of the M2 generation single plants, diluted to 40 ng / μl, and 8 DNA samples were mixed to construct the DNA pool.
6. The CYP82E10 gene mutant G796A for cigars according to claim 3, characterized in that, The specific primers include E10-F and E10-R; wherein the nucleotide sequence of E10-F is shown in SEQ ID NO:5, and the nucleotide sequence of E10-R is shown in SEQ ID NO:
6.
7. The CYP82E10 gene mutant G796A for cigars according to any one of claims 1-6, characterized in that, Tobacco leaves containing the G796A mutant of the CYP82E10 gene in cigars have a lower nicotine conversion rate compared to tobacco leaves containing the CYP82E10 gene in cigars.
8. The CYP82E10 gene mutant G796A for cigars according to claim 7, characterized in that, Compared with tobacco leaves containing the CYP82E10 gene mutant G796A, tobacco leaves containing the CYP82E10 gene of cigars have a nicotine conversion rate that is reduced by no less than 38%.
9. The application of a CYP82E10 gene mutant G796A in cigars, characterized in that, The application of the CYP82E10 gene mutant G796A of cigar tobacco as described in any one of claims 1-8 in reducing the nicotine conversion rate of cigar tobacco.
10. The application according to claim 9, characterized in that, The CYP82E10 gene mutant G796A for cigars is used to create cigar materials with low nicotine conversion rates.