Mutant CYP82e5-2 of cigar CYP82E5 gene and application of mutant CYP82e5-2

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a mutant CYP82e5-2 of a cigar CYP82E5 gene and application of the mutant CYP82e5-2. The nucleotide sequence of the mutant CYP82e5-2 is as shown in SEQ ID NO: 2; the mutant CYP82e5-2 is obtained by changing the 505th nucleotide of a cigar CYP82E5 gene of which the nucleotide sequence is shown as SEQ ID NO: 1 into T from C. The amino acid sequence coded by the mutant CYP82e5-2 is as shown in SEQ ID NO: 4. The cigar CYP82E5 gene mutant (mutant CYP82e5-2) provided by the invention can obviously reduce the conversion rate of nicotine in cigars, and can be used for creating cigar materials with low nicotine conversion rate. Tests prove that compared with tobacco leaves containing wild type genes, tobacco leaves containing the cigar CYP82E5 gene mutant have the advantage that the nicotine conversion rate is reduced by 48%.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a cigar cigarette. CYP82E5 Mutants of genes CYP82e5-2 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 and flue-cured cigarettes.

[0003] NNN (N-nitrosonornicotinic acid) in TSNA is classified as a Group 1 carcinogen. NNN is primarily formed through the nitrosation of nornicotinic acid. Nornicotinic acid is a major direct precursor to NNN, and reducing its content is the most direct way to reduce NNN levels. Burley tobacco. CYP82E2 The sub-family CYP82E4 , CYP82E5 , CYP82E10 The gene encodes an active nicotine demethylase, a key enzyme in nicotine conversion. Lewis et al. obtained these enzymes using EMS mutagenesis. CYP82E4 , CYP82E5 , CYP82E10 The mutated burley tobacco material was found to have... CYP82E5 , CYP82E10 Gene mutations have virtually no impact on nicotine conversion rate; however, mutant strains with simultaneous mutations in all three genes exhibit significantly lower nicotine conversion rates than control strains. The above research indicates that in Burley tobacco... CYP82E4 It is a key gene that determines nicotine conversion rate, and CYP82E5 , CYP82E10 Genes have no significant effect on nicotine conversion.

[0004] 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

[0005] To address the aforementioned technical problems, the present invention provides a cigar cigarette. CYP82E5 Mutants of genes CYP82e5-2 And its applications.

[0006] This invention is achieved through the following technical solution: A type of cigar CYP82E5 Mutants of genesCYP82e5 -2, the mutant CYP82e5 The nucleotide sequence of -2 is shown in SEQ ID NO:2; the mutant CYP82e5 -2 is the nucleotide sequence of a cigar as shown in SEQ ID NO:1. CYP82E5 The 505th nucleotide of the gene is obtained by changing C to T.

[0007] Furthermore, the mutant CYP82e5 The amino acid sequence encoded by -2 is shown in SEQ ID NO:4. Compared to the cigar shown in SEQ ID NO:3... CYP82E5 The amino acid sequence encoded by the gene has its arginine (R) replaced by a stop codon at position 169.

[0008] Furthermore, the mutant CYP82e5 -2 was obtained by amplification using the following primer pair, the nucleotide sequences of which are: Upstream primer: GGTAATTTTGTATTTATTATATTATGCG, as shown in SEQ ID NO:5; Downstream primer: TCATCCTTAGTATTTAGATAATCTAATT, as shown in SEQ ID NO:6.

[0009] Furthermore, including the mutant CYP82e5 -2 tobacco leaves with cigar tobacco CYP82E5 Compared to tobacco leaves with different genes, the nicotine conversion rate is lower.

[0010] The present invention also provides a mutant. CYP82e5 -2 Application in obtaining cigars with low nicotine conversion.

[0011] Furthermore, including the mutant CYP82e5 -2 tobacco leaves with cigar tobacco CYP82E5 Compared to tobacco leaves with different genes, the nicotine conversion rate is lower.

[0012] Beneficial technical effects of the present invention: The cigar provided by this invention CYP82E5 Gene mutants (mutants) CYP82e5 -2) It can significantly reduce the nicotine conversion rate in cigars and can be used to create cigar materials with low nicotine conversion rate.

[0013] Tests have proven that the cigar tobacco contained therein... CYP82E5 Compared with tobacco leaves containing the wild-type gene, the nicotine conversion rate of the mutant tobacco leaves was reduced by 48%. Attached Figure Description

[0014] Figure 1 For the cigar tobacco leaf of the embodiments of the present application CYP82E5 The sequencing results of the gene mutants.

[0015] Figure 2 The cigar tobacco leaves of the embodiments of the present application CYP82E5 The nicotine conversion rates of the gene mutants and the wild-type cigar tobacco; wherein ** indicates extremely significant difference P <0.01 DETAILED DESCRIPTION

[0016] In order to make the objects, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0017] On the contrary, the present application covers any substitutions, modifications, equivalent methods and solutions made within the essence and scope of the present application defined by the claims. Further, in order to make the public have a better understanding of the present application, some specific details are described in detail in the following detailed description of the present application. The present application can also be completely understood without the description of these details by those skilled in the art.

[0018] The technical solutions of the present application are described in detail below in combination with examples. It should be understood that the following examples are only used to explain and illustrate the present application, and are not used to limit the scope of the present application.

[0019] In the following examples, the reagents not specifically described 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 described are conventional experimental methods and conditions in the art, which can be referred to relevant experimental manuals, known documents or manufacturer's instructions. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those skilled in the art to which the present application belongs.

[0020] Example 1: The present example provides a gene mutant for reducing the nicotine conversion rate in the cigar tobacco leaf CYP82E5 The gene mutant of the cigar tobacco CYP82E5 The gene mutant is named mutant CYP82e5 -2, the nucleotide sequence of mutant CYP82e5 -2 is shown in SEQ ID NO: 2.

[0021] As Figure 1 shown, compared with the wild-type cigar tobacco CYP82E5 gene with the nucleotide sequence shown in SEQ ID NO: 1, the cigar tobacco CYP82E5 gene mutant (i.e. mutant CYP82e5 -2) has a C to T change at position 505.

[0022] The wild-type Nicotiana attenuata CYP82E5 The amino acid sequence encoded by the wild-type Nicotiana attenuata CYP82E5 The amino acid sequence encoded by the mutant of the gene (SEQ ID NO: 4) is changed from arginine (R) at position 169 to a stop codon.

[0023] The wild-type Nicotiana attenuata CYP82E5 The coding sequence of the gene (1554 bp) is as follows: Cigars CYP82E5 The coding sequence of the gene mutant (1554 bp) is as follows: Wild-type cigar tobacco CYP82E5 The amino acid sequence encoded by the gene (517 aa) is as follows: MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFRKAFKDFIILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHVKKREKMEVNAQGNEQDFIDVVLSKMSNEYLDEGYSRDTVIKATVFSLVLDAADTVALHMNWGMALLINNQHALKKAQEEIDKKVGKERWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSNPDKFDPERFFADDIDYRGQHYEFIPFGSGRRSCPGMTYALQVEHLTIAHLIQGFNYKTPNDEPLDMKEGAGLTIRKVNPVEVTITARLAPELY (SEQ ID NO: 3) Cigar tobacco CYP82E5 The amino acid sequence encoded by the mutant gene (168 aa) is as follows: MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYT (SEQ ID NO: 4) This example obtained a tobacco comprising the above-mentioned cigar tobacco CYP82E5 gene mutant by treating cigar tobacco seeds with EMS and screening mutants using TILLING technology.

[0024] EMS treatment of cigar seeds: Wild-type cigar seeds are soaked in 50% commercially available bleach for 12 minutes, then centrifuged and drained. The seeds are then rinsed with deionized water to remove the bleach and prevent the seeds from being affected by the bleaching chemicals. The seeds are then centrifuged again and drained. The rinsed seeds are then soaked in deionized water at 10℃-30℃ for 10-15 hours to promote germination and ensure uniform mutagenesis. The seeds are then centrifuged and drained. The seeds are then soaked in 0.5% EMS (ethyl methanesulfonate) solution for 10-15 hours, then centrifuged and drained. The seeds are then rinsed 5-8 times with deionized water. Finally, the seeds are filtered and dried using a Buchner funnel and filter paper.

[0025] Mutant screening: TILLING technology was used to screen for mutants. EMS-treated seeds (M1 generation) were sown in the field, individually bagged, and self-pollinated to obtain M2 generation. One seed from each M2 generation seed harvested from each M1 generation plant was sown. Leaves from individual M2 generation plants were collected, and genomic DNA was extracted 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 was 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.

[0026] Cigar smoke was designed using Primer 3 software. CYP82E5 The nucleotide sequences of the TILLING analysis primers E5-F / E5-R are as follows: E5-F: 5'-GGTAATTTTGTATTTATTATATTATGCG-3' (SEQ ID NO: 5); E5-R: 5'-TCATCCTTAGTATTTAGATAATCTAATT-3' (SEQ ID NO: 6).

[0027] The target fragment amplified by the above primers was 1136 bp in size. The DNA samples were amplified according to the reaction system (as shown in Table 1) and reaction procedure.

[0028] Table 1 Reaction System Reaction procedure: 95℃ 3 min; (94℃ 30 s, 63℃ 30 s, -1℃ / cycle, 72℃ 1 min) 7 cycles; (94℃ 30 s, 58℃ 30 s, 72℃ 1 min) 40 cycles; 72℃ 5 min; 99℃ 10 min; (70℃ 20 s, -0.3℃ / cycle) 70 cycles; 4℃ storage.

[0029] 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

[0030] A mutant was obtained, named mutant CYP82e5 -2, which CYP82E5 The 505th site of the coding sequence (CDS) of the gene was changed from C to T, so that the 169th site of the amino acid sequence coded by the gene was changed from arginine (R) to a stop codon. The mutant was planted in a greenhouse, and M3 generation plants were obtained. Through nucleotide sequencing screening, a tobacco plant containing a homozygous CYP82E5 gene mutant was obtained, and was self-crossed to produce seeds.

[0031] Example 2: Application of mutant CYP82e5 -2 according to example 1 in obtaining low nicotine conversion rate cigar.

[0032] Tobacco leaves containing the mutant CYP82e5 -2 have a lower nicotine conversion rate than tobacco leaves containing cigar CYP82E5 genes.

[0033] In this example, according to the tobacco industry standard YC / T382-2010, the nicotine and nornicotine contents of wild type tobacco and tobacco containing homozygous CYP82E5 gene mutant obtained in example 1 were determined, and the nicotine conversion rate was calculated.

[0034] Instrument equipment used for determination: Shimadzu GCMS-QP2020 gas chromatograph mass spectrometer (Japan Shimadzu Corporation), Millipore ultrapure water machine (American Millipore Simplicity Company), Eofo-945008 vortex mixing oscillator (American Tallboys Company), Eppendorf 5804 high-speed centrifuge (German Eppendorf Company).

[0035] ​Reagents used in the assay: The internal standard quinoline purity is greater than 98%, purchased from Sigma-Aldrich Company; the internal standard nornicotine-D4 purity is greater than 98%, purchased from CATO Company in the United States; nornicotine purity is greater than 98%, purchased from Sigma-Aldrich Company; nicotine purity is greater than 98%, purchased from TRC Company in Canada; sodium hydroxide is analytical pure, purchased from Shantou Xilong Chemical Factory in Guangdong; dichloromethane and methanol are chromatographic pure, purchased from Fisher Company in the United States.

[0036] Method of determination: Wild-type cigar tobacco and mutants containing homozygous CYP82E5 genes were planted in the greenhouse, and the plants were topped when they reached the flowering stage. Fifteen days after topping, the middle leaves were taken, and 10 single plants with consistent growth were selected for sampling from each material. The tobacco leaves were killed and dried. 0.3 g of crushed cigar tobacco leaf sample was weighed into 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. After shaking and mixing, it was allowed to stand for 20 min. Then 10.0 mL of dichloromethane-methanol extraction solution (v;v=4:1) was added, and the sealed tube was placed in a vortex shaker at a speed of 2000 rpm for 40 min. After standing for 1 h, the lower organic phase was transferred to a chromatographic analysis bottle for instrument analysis.

[0037] Chromatographic conditions 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 °C; temperature program: initial temperature 110 °C, 10 °C / min to 185 °C, 50 °C / min to 280 °C, hold for 2 min; carrier gas: helium, 1.0 mL / min.

[0038] Mass spectrometry conditions Transfer line temperature: 250 °C, ion source temperature: 230 °C; ionization mode: electron impact ionization (EI); bombardment energy: 70 eV; solvent delay time: 4 min; scanning mode: selected ion monitoring mode (SIM) scanning, nicotine, nornicotine and internal standard retention time, quantitative and qualitative selection ion parameters are as shown in Table 2.

[0039] Table 2 Nicotine, nornicotine and internal standard retention time, quantitative and qualitative selection ion parameters Cigar tobacco CYP82E5 The detection results of nicotine and nornicotine in gene mutant and wild-type material are shown in Table 3 and Figure 2The nicotine conversion rate is calculated according to the following formula: nicotine conversion rate = reduced nicotine content / (nicotine content + reduced nicotine content) x 100%.

[0040] Table 3 Cigar Tobacco CYP82E5 Nicotine and nornicotine detection results of gene mutant and wild type material Note: "**" in the table indicates extremely significant difference P <0.01) It can be seen that the tobacco leaf containing the CYP82E5 gene mutant of the cigar tobacco has a nicotine conversion rate reduced by 48% compared with the tobacco leaf containing the wild type gene.

[0041] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cigar CYP82E5 Mutants of the gene CYP82e5 -2, characterized in that, The mutant CYP82e5 -2 is shown in SEQ ID NO: 2; the mutant CYP82e5 -2 is a tobacco variety having a nucleotide sequence shown in SEQ ID NO: 1 CYP82E5 the 505th nucleotide of the gene is changed from C to T.

2. A cigar according to claim 1 CYP82E5 Mutants of genes CYP82e5 -2, characterized in that, The mutant CYP82e5 The amino acid sequence encoded by the -2 gene is shown in SEQ ID NO:

4.

3. The cigar of claim 1 CYP82E5 Mutants of genes CYP82e5 -2, characterized in that, The mutant CYP82e5 -2 was amplified from the following primer pair, the nucleotide sequences of which are: Upstream primer: GGTAATTTTGTATTTATTATATTATGCG, as set forth in SEQ ID NO: 5; Downstream primer: TCATCCTTAGTATTTAGATAATCTAATT, as set forth in SEQ ID NO:

6.

4. The cigar of claim 1 CYP82E5 mutants of genes CYP82e5 -2, characterized in that, comprising said mutant CYP82e5 Tobacco leaves comprising the mutant of the application have a lower nicotine conversion rate compared to tobacco leaves comprising a wild type NtNIA2 gene. CYP82E5 Tobacco leaves comprising the mutant of the application have a lower nicotine conversion rate compared to tobacco leaves comprising a wild type NtNIA2 gene.

5. The mutant according to any one of claims 1 to 4 CYP82e5 -2 in obtaining low nicotine conversion rate in cigar.

6. Use according to claim 5, characterized in that, comprising said mutant CYP82e5 Tobacco leaves comprising the mutant of the gene of the application have a lower nicotine conversion rate compared to tobacco leaves comprising the wild type of the gene of the application. CYP82E5 Tobacco leaves comprising the mutant of the gene of the application have a lower nicotine conversion rate compared to tobacco leaves comprising the wild type of the gene of the application.

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