Cigar CYP82E10 gene mutant G358A and application thereof
By introducing the CYP82E10 gene mutant G358A into cigar seeds, cigar materials with low nicotine conversion rate were screened out, solving the problem of high nicotine conversion rate in cigars and achieving a significant reduction in nicotine conversion rate and improved safety.
Patent Information
- Application Number
- CN202511122890.8
- 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 CYP82E10 gene mutant G358A was introduced into cigar seeds using EMS mutagenesis. Cigar materials with low nicotine conversion rate were screened out. The specific steps included EMS treatment, DNA pool construction, PCR amplification, and homozygous screening to obtain the CYP82E10 gene mutant G358A for cigars.
It significantly reduces the nicotine conversion rate in cigars by 41%, creating a cigar material with a low nicotine conversion rate and improving the safety of cigars.
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Figure CN120966852A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, specifically to a cigar tobacco CYP82E10 gene mutant G358A and its applications. Background Technology
[0002] With global economic development, cigars have gained a significant share of the tobacco market, and sales of domestically produced cigars in my country have grown rapidly. High-quality cigars not only require excellent smoking quality but also low harm and safety. The high content of tobacco-specific nitrosamines (TSNAs) is a primary safety issue that needs to be addressed in the development of cigars. Reports indicate that the TSNA content in cigar products is 1-2 orders of magnitude higher than that in Burley tobacco and flue-cured cigarettes.
[0003] N-nitrosonornicotinic acid (NNN) in TSNAs is classified as a Group 1 carcinogen. NNN is mainly formed through the nitrosation of nornicotinic acid. Nornicotinic acid is the main direct precursor of NNN, and reducing its content is the most direct way to reduce NNN levels. The CYP82E4, CYP82E5, and CYP82E10 genes of the CYP82E2 subfamily in Burley tobacco encode active nicotine demethylases, which are key enzymes in nicotine conversion. Lewis et al. obtained Burley tobacco materials with mutations in CYP82E4, CYP82E5, and CYP82E10 using EMS mutagenesis. They found that mutations in the CYP82E5 and CYP82E10 genes had little effect on nicotine conversion, while mutant strains with simultaneous mutations in all three genes had significantly lower nicotine conversion rates than control strains. These studies indicate that CYP82E4 is the key gene determining nicotine conversion in Burley tobacco, while the CYP82E5 and CYP82E10 genes have no significant impact on nicotine conversion. The nicotine conversion mechanisms in cigars and burley tobaccos are different. There is currently no relevant research on cigars, and methods to reduce the nicotine conversion rate in cigars still need to be further explored. Summary of the Invention
[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.
[0005] Therefore, in a first aspect, the present invention provides a cigar tobacco CYP82E10 gene mutant G358A, the nucleotide sequence of which is shown in SEQ ID NO:2. The cigar tobacco CYP82E10 gene mutant G358A is obtained by changing the 358th nucleotide of the cigar tobacco CYP82E10 gene from G to A.
[0006] Furthermore, the amino acid sequence encoded by the CYP82E10 gene mutant G358A of the cigar is shown in SEQ ID NO:4.
[0007] Furthermore, the screening method for the CYP82E10 gene mutant G358A in cigars includes:
[0008] EMS mutagenesis: Cigar seeds were treated with ethyl methanesulfonate to obtain M1 generation seeds;
[0009] 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.
[0010] 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 G358A of cigar tobacco were obtained by capillary electrophoresis screening.
[0011] Homozygous selection: M2 generation plants of the CYP82E10 gene mutant G358A of the cigar tobacco were cultivated in a greenhouse to obtain M3 generation plants. Plants containing the homozygous CYP82E10 gene mutant G358A were obtained by sequencing and self-pollination for seed collection.
[0012] Furthermore, the EMS mutagenesis includes:
[0013] 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.
[0014] Furthermore, the construction of the DNA pool includes:
[0015] 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.
[0016] Furthermore, 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.
[0017] Furthermore, tobacco leaves containing the G358A mutant of the cigar tobacco CYP82E10 gene have a lower nicotine conversion rate compared to tobacco leaves containing the CYP82E10 gene of cigar tobacco.
[0018] Furthermore, tobacco leaves containing the CYP82E10 gene mutant G358A of cigar tobacco have a nicotine conversion rate that is reduced by no less than 40% compared to tobacco leaves containing the CYP82E10 gene of cigar tobacco.
[0019] In a second aspect, the present invention provides the application of the above-mentioned cigar tobacco CYP82E10 gene mutant G358A in reducing the nicotine conversion rate of cigar tobacco.
[0020] Furthermore, the CYP82E10 gene mutant G358A of the cigar tobacco is used to create cigar tobacco materials with low nicotine conversion rate.
[0021] Compared with the prior art, the present invention has at least the following beneficial effects:
[0022] The CYP82E10 gene mutant G358A for cigars provided by this invention can significantly reduce the nicotine conversion rate in cigars and can be used to create cigar materials with low nicotine conversion rates. Experiments have shown that tobacco leaves containing the CYP82E10 gene mutant G358A have a 41% lower nicotine conversion rate compared to tobacco leaves containing the wild-type CYP82E10 gene. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0024] Figure 1 The sequencing results of the CYP82E10 gene mutant G358A for cigars are shown; Mu1135 represents the mutant and Wild represents the wild type.
[0025] Figure 2 The results show the nucleotide sequence alignment of the coding region of the CYP82E10 gene mutant G358A in cigars with that of the wild-type CYP82E10 gene; where Mu1135 represents the mutant and Wild represents the wild type.
[0026] Figure 3 The amino acid sequence alignment results of the protein encoded by the CYP82E10 gene mutant G358A of cigar tobacco and the protein encoded by the CYP82E10 gene of wild-type cigar tobacco are shown; where Mu1135 represents the mutant and Wild represents the wild type.
[0027] Figure 4The nicotine conversion rate of the CYP82E10 gene mutant G358A in cigar tobacco and wild-type cigar tobacco is shown; where Mu1135 represents the mutant, Wild represents wild-type tobacco, and * indicates significant difference (P<0.05). Detailed Implementation
[0028] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.
[0029] In the following examples, reagents not specifically mentioned are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and of laboratory purity. Experimental methods and conditions not specifically mentioned are all conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, publicly available literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0030] In a first aspect, the present invention provides a cigar tobacco CYP82E10 gene mutant G358A, the nucleotide sequence of which is shown in SEQ ID NO:2. The cigar tobacco CYP82E10 gene mutant G358A is obtained by changing the 358th nucleotide of the cigar tobacco CYP82E10 gene, as shown in SEQ ID NO:1, from G to A.
[0031] Furthermore, the amino acid sequence encoded by the CYP82E10 gene mutant G358A of cigar tobacco is shown in SEQ ID NO:4. Compared with the amino acid sequence encoded by the CYP82E10 gene of cigar tobacco shown in SEQ ID NO:3, position 120 is changed from alanine (A) to threonine (T).
[0032] In some embodiments, the screening method for the CYP82E10 gene mutant G358A in cigars includes:
[0033] EMS mutagenesis: Cigar seeds were treated with ethyl methanesulfonate to obtain M1 generation seeds;
[0034] DNA pool construction: M2 generation seeds were obtained by self-pollination of M1 generation seeds. DNA was extracted from leaves of M2 generation plants to construct a DNA pool.
[0035] 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 G358A of cigar tobacco were obtained by capillary electrophoresis screening.
[0036] Homozygous selection: M2 generation plants of the CYP82E10 gene mutant G358A of cigar tobacco were cultivated in a greenhouse to obtain M3 generation plants. Plants containing the homozygous CYP82E10 gene mutant G358A were obtained by sequencing and self-pollination for seed collection.
[0037] The EMS mutagenesis includes: soaking the rinsed cigar seeds in a 0.5% ethyl methanesulfonate solution for 10-15 hours, then washing them with deionized water to obtain M1 generation seeds; wherein the cigar seeds are cigar seeds containing the CYP82E10 gene.
[0038] The DNA pool construction includes: sowing the M1 generation seeds, obtaining the M2 generation seeds through self-pollination, extracting DNA from the leaves of the M2 generation single plants, diluting it to 40 ng / μl, and mixing 8 DNA samples to construct the DNA pool.
[0039] The specific primers include E10-F and E10-R. 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.
[0040] Specifically, the above screening method has high throughput, saves time and effort.
[0041] In a second aspect, the present invention provides the application of the aforementioned cigar tobacco CYP82E10 gene mutant G358A in reducing the nicotine conversion rate of cigar tobacco. Further, the cigar tobacco CYP82E10 gene mutant G358A is used to create cigar tobacco materials with low nicotine conversion rates.
[0042] Specifically, tobacco leaves containing the CYP82E10 gene mutant G358A of cigar tobacco have a lower nicotine conversion rate than tobacco leaves containing the CYP82E10 gene of cigar tobacco, with a reduction in nicotine conversion rate of not less than 40%.
[0043] Example 1
[0044] In this embodiment, tobacco containing the aforementioned CYP82E10 gene mutant G358A was obtained by treating cigar seeds with EMS and screening for mutants using the TILLING technique.
[0045] 1. EMS treatment of cigar seeds
[0046] Cigar seeds containing the CYP82E10 gene were soaked in 50% commercially available bleach for 12 minutes, then centrifuged and drained. The seeds were then rinsed with deionized water to remove the bleach and prevent further chemical effects, followed by another centrifugation and draining. The rinsed seeds were then soaked in deionized water at 10℃-30℃ for 10-15 hours to promote germination and ensure uniform mutagenesis. The seeds were then centrifuged and drained. The seeds were then soaked in 0.5% EMS (ethyl methanesulfonate) solution for 10-15 hours, followed by centrifugation and draining. They were then rinsed 5-8 times with deionized water. Finally, the seeds were filtered and dried using a Buchner funnel and filter paper.
[0047] 2. Mutant screening
[0048] Mutants were screened using the TILLING technique. EMS-treated seeds (M1 generation) were sown in the field, individually bagged, and self-pollinated to obtain M2 generation seeds. One seed was sown from each M2 generation seed harvested from each M1 generation plant. Genomic DNA was extracted from leaves of individual M2 generation plants using a DNA extraction kit (QIAGEN, 69106) according to the kit's instructions. The obtained DNA samples were arranged sequentially. 2 μl of each DNA sample was taken and its concentration determined on a 16-channel Tecan Infinite M200 instrument. All DNA samples were then diluted to 40 ng / μl, and eight samples were pooled to create an 8-fold DNA pool for TILLING analysis.
[0049] Primers E10-F / E10-R for tilling analysis of the CYP82E10 gene in cigar tobacco were designed using Primer 3 software. Their nucleotide sequences are as follows:
[0050] E10-F: 5'-GTCAAATAACCACCTCTTAATAGTAA-3' (SEQ ID NO: 5);
[0051] E10-R: 5'-AAAAGTCCCTATTGGTAGGAAGTGC-3' (SEQ ID NO: 6).
[0052] The target fragment amplified by the above primers is 1136 bp in size. The above DNA sample was amplified according to the following reaction system and procedure.
[0053] Table 1 Reaction System
[0054]
[0055] The reaction program was as follows: 95℃ for 3 min; (94℃ for 30 s, 63℃ for 30 s, -1℃ / cycle, 72℃ for 1 min) for 7 cycles; (94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min) for 40 cycles; 72℃ for 5 min; 99℃ for 10 min; (70℃ for 20 s, -0.3℃ / cycle) for 70 cycles; store at 4℃.
[0056] After the reaction, the amplification products were analyzed by capillary electrophoresis to screen for single strains with nucleotide mutations, and then sequenced for verification. Figure 1 ).
[0057] A mutant was obtained in which position 358 of the coding sequence (CDS) of the CYP82E10 gene was changed from G to A, as shown below. Figure 2 As shown, compared to the wild-type cigar tobacco CYP82E10 gene with the nucleotide sequence shown in SEQ ID NO:1, the mutant cigar tobacco CYP82E10 gene has a G-to-A change at position 358. This changes alanine (A) to threonine (T) at position 120 of the amino acid sequence encoded by the gene, as shown in SEQ ID NO:1. Figure 3 As shown, compared with the amino acid sequence encoded by the CYP82E10 gene in wild-type cigar tobacco (SEQ ID NO:3), the amino acid sequence encoded by the CYP82E10 gene mutant in cigar tobacco (SEQ ID NO:4) has a change at position 120 from alanine (A) to threonine (T). This mutant was grown in a greenhouse to obtain M3 generation plants. Tobacco plants containing homozygous CYP82E10 gene mutants were obtained through nucleotide sequencing and self-pollinated for seed production.
[0058] Example 2
[0059] In this embodiment, the nicotine and nonicotine contents of wild-type tobacco and tobacco containing the homozygous CYP82E10 gene mutant G358A 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 and equipment used for the determination were: Shimadzu GCMS-QP2020 gas chromatograph-mass spectrometer (Shimadzu Corporation, Japan), Millipore ultrapure water system (Millipore Simplicity Corporation, USA), Eofo-945008 vortex mixer (Tallboys Corporation, USA), and Eppendorf 5804 high-speed centrifuge (Eppendorf GmbH, Germany).
[0061] Reagents used in the assay: internal standard quinoline with a purity greater than 98% was purchased from Sigma-Aldrich; internal standard nornicotinic acid-D4 with a purity greater than 98% was purchased from CATO, USA; nornicotinic acid with a purity greater than 98% was purchased from Sigma-Aldrich; nicotine with a purity greater than 98% was purchased from TRC, Canada; sodium hydroxide was of analytical grade and purchased from Xilong Chemical Plant in Shantou City, Guangdong Province; dichloromethane and methanol were of chromatographic grade and purchased from Fisher Scientific, USA.
[0062] Methods: Wild-type cigar tobacco containing the CYP82E10 gene and cigar tobacco containing the homozygous CYP82E10 gene mutant G358A were grown in a greenhouse. The plants were topped at the flowering stage. Fifteen days after topping, the middle leaves were collected. Ten individual plants with consistent growth were sampled from each material. The tobacco leaves were then blanched and dried. 0.3 g of the crushed cigar tobacco leaf sample was weighed into a 15 mL centrifuge tube. 0.05 mL of an internal standard solution containing quinoline and nornicotine-d4, and 2 mL of 5% sodium hydroxide aqueous solution were added. The mixture was shaken and allowed to stand for 20 min. Then, 10.0 mL of dichloromethane-methanol extraction solution (v:v = 4:1) was added. The tube was sealed and placed in a vortex mixer. Extraction was performed by vortexing at 2000 rpm for 40 min. After standing for 1 h, the lower organic phase was transferred to a chromatographic analysis bottle for instrumental analysis.
[0063] The specific chromatographic conditions are as follows:
[0064] Column: BR-5MS (30m×0.25mm×0.25μm); Injection volume: 1μL, split ratio 60:1; Injector: 250℃; Temperature program: initial temperature 110℃, increase to 185℃ at 10℃ / min, increase to 280℃ at 50℃ / min, hold for 2min; Carrier gas: helium, 1.0mL / min.
[0065] The specific mass spectrometry conditions are as follows:
[0066] Transfer line temperature: 250℃, ion source temperature: 230℃; ionization mode: electron impact ionization (EI); impact energy: 70eV; solvent delay time: 4min; scanning mode: selected ion monitoring mode (SIM) scanning. The retention time, quantitative and qualitative selected ion parameters of nicotine, nornicotine and internal standard are shown in Table 2 below.
[0067] Table 2 Retention time, quantitative and qualitative selected ion parameters of nicotine, nornicotine and internal standard
[0068]
[0069] The results of nicotine and nornicotine content detection in the CYP82E10 gene mutant G358A and wild-type materials of cigars are shown in Table 3. Nicotine conversion rate was calculated using the following formula: Nicotine conversion rate = (Nonicotine content / (Nonicotine content + Nornicotine content)) × 100%.
[0070] Table 3. Results of nicotine and nornicotine tests
[0071]
[0072] Note: "*" in the table indicates a significant difference (P < 0.05).
[0073] From Table 3 and Figure 4 It can be seen that the nicotine conversion rate of tobacco leaves containing the CYP82E10 gene mutant G358A of the cigar tobacco was reduced by 41% compared with that of tobacco leaves containing the wild-type gene.
[0074] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.
Claims
1. A CYP82E10 gene mutant G358A of cigar, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:
2. The CYP82E10 gene mutant G358A of cigar cigarettes is obtained by changing the 358th nucleotide of the CYP82E10 gene of cigar cigarettes from G to A.
2. The cigar CYP82E10 gene mutant G358A of claim 1, wherein, The amino acid sequence encoded by the CYP82E10 gene mutant G358A of the cigar is shown in SEQ ID NO:
4.
3. The cigar CYP82E10 gene mutant G358A of claim 1, wherein, The screening method for the CYP82E10 gene mutant G358A 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 G358A of cigar tobacco were obtained by capillary electrophoresis screening. Homozygous selection: M2 generation plants of the CYP82E10 gene mutant G358A of the cigar tobacco were cultivated in a greenhouse to obtain M3 generation plants. Plants containing the homozygous CYP82E10 gene mutant G358A were obtained by sequencing and self-pollination for seed collection.
4. The CYP82E10 gene mutant G358A 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 G358A for cigars according to claim 3, characterized in that, The construction of the DNA pool 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 G358A 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 G358A for cigars according to any one of claims 1-6, characterized in that, Tobacco leaves containing the G358A 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 G358A for cigars according to claim 7, characterized in that, Compared with tobacco leaves containing the CYP82E10 gene mutant G358A, tobacco leaves containing the CYP82E10 gene of cigars have a nicotine conversion rate that is reduced by no less than 40%.
9. The application of a CYP82E10 gene mutant G358A in cigars, characterized in that, The application of the CYP82E10 gene mutant G358A 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 G358A of the cigar is used to create cigar materials with low nicotine conversion rate.