Mutant CYP82e4-1 of cigar CYP82E4 gene and application of mutant CYP82e4-1

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a mutant CYP82e4-1 of a cigar CYP82E4 gene and application of the mutant CYP82e4-1. The nucleotide sequence of the mutant CYP82e4-1 is as shown in SEQ ID NO: 2; the mutant CYP82e4-1 is obtained by changing the 139th nucleotide of a cigar CYP82E4 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 CYP82e4-1 is as shown in SEQ ID NO: 4. The cigar CYP82E4 gene mutant (mutant CYP82e4-1) 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.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a mutant of a cigar tobacco CYP82E4 gene CYP82e4-1 and application thereof. BACKGROUND

[0002] High-quality cigar tobacco not only requires good smoking quality, but also needs to have low harm and safety characteristics. High content of tobacco-specific nitrosamines (TSNAs) is the primary safety problem that needs to be solved in the development of cigar tobacco.

[0003] NNN (N-nitrosonornicotine) in TSNAs is listed as a class I carcinogen. NNN is mainly formed through nitrosation of nornicotine. As the main direct precursor of NNN, reducing the content of nornicotine is the most direct means to reduce NNN. Nornicotine in tobacco is formed by catalysis of cytochrome P450 enzyme CYP82E4 from nicotine, which is usually referred to as "conversion". The conversion rate (nornicotine content / (nicotine content + nornicotine content)) is generally used to describe the degree of conversion of nicotine to nornicotine. Therefore, reducing or inhibiting the expression of CYP82E4 gene can effectively reduce the conversion of nicotine to nornicotine. However, the low-conversion tobacco created by transgenic means cannot be applied to tobacco production. Amino acid changes in the target gene caused by EMS mutagenesis can effectively reduce the function of the gene, and the mutant material created can be directly used in production. SUMMARY

[0004] In view of the above technical problems, the present application provides a mutant of a cigar tobacco CYP82E4 gene CYP82e4-1 and application thereof.

[0005] The present application is achieved by the following technical solutions: A mutant of a cigar tobacco CYP82E4 gene CYP82e4-1 , the nucleotide sequence of the mutant CYP82e4-1 is shown as SEQ ID NO: 2; the mutant CYP82e4-1 is a mutant of a cigar tobacco CYP82E4 gene with a nucleotide sequence shown as SEQ ID NO: 1, in which the 139th nucleotide is changed from C to T.

[0006] Further, the amino acid sequence encoded by the mutant CYP82e4-1 is shown as SEQ ID NO: 4. Compared with the amino acid sequence encoded by the cigar tobacco CYP82E4 gene shown as SEQ ID NO: 3, the 47th amino acid is changed from leucine (L) to phenylalanine (F).

[0007] Furthermore, the mutant CYP82e4-1 It was obtained by amplification using the following primer pair, the nucleotide sequence of which is: Upstream primer: TTTTCTATTTCGGTACGAC, as shown in SEQ ID NO:5; Downstream primer: TAACTTTACAGTTGCTCCA, as shown in SEQ ID NO:6.

[0008] Furthermore, including the mutant CYP82e4-1 Tobacco leaves and cigar tobacco CYP82E4 Compared to tobacco leaves with different genes, the nicotine conversion rate is lower.

[0009] The present invention also provides a mutant CYP82e4-1 Application in obtaining cigars with low nicotine conversion rate.

[0010] Furthermore, including the mutant CYP82e4-1 Tobacco leaves and cigar tobacco CYP82E4 Compared to tobacco leaves with different genes, the nicotine conversion rate is lower.

[0011] Beneficial technical effects of the present invention: The cigar provided by this invention CYP82E4 Gene mutants (mutants) CYP82e4-1 It can significantly reduce the nicotine conversion rate in cigars and can be used to create cigar materials with low nicotine conversion rates.

[0012] Tests have proven that it contains cigar smoke. CYP82E4 Gene mutants (mutants) CYP82e4-1 Compared to tobacco leaves containing wild-type genes, tobacco leaves from this group showed a 33% reduction in nicotine conversion. Attached Figure Description

[0013] Figure 1 Showing cigars CYP82E4 Sequencing results of gene mutants; Figure 2 Showing cigars CYP82E4 Nicotine conversion rate between gene mutants and wild-type cigars; where ** indicates extremely significant difference (…). P <0.01). Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0015] On the contrary, the present application covers any alternatives, modifications, equivalent methods and solutions made within the spirit and scope of the present application defined by the claims. Further, in order to make the public better understand the present application, some specific details are described in the following detailed description of the present application. The present application can also be fully understood without the description of these details by those skilled in the art.

[0016] In the following examples, the reagents not specifically specified are all 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 all conventional experimental methods and conditions in the art, which can be referred to relevant experimental manuals, known literatures or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0017] Example 1: The present example provides a gene mutant for reducing the conversion rate of nicotine in cigar tobacco leaves CYP82E4 The gene mutant is named mutant CYP82E4 -1, the nucleotide sequence of mutant CYP82e4 -1 is shown as SEQ ID NO: 2. CYP82e4 As shown in

[0018] compared with the wild-type cigar tobacco Figure 1 gene with the nucleotide sequence shown as SEQ ID NO: 1, the mutant CYP82E4 -1 is changed from C to T at the 139th position. CYP82e4 Compared with the amino acid sequence (SEQ ID NO: 3) encoded by the wild-type cigar tobacco

[0019] gene, the amino acid sequence (SEQ ID NO: 4) encoded by the mutant cigar tobacco CYP82E4 gene is changed from leucine (L) to phenylalanine (F) at the 47th position. CYP82E4 The coding sequence (1554 bp) of the wild-type cigar tobacco

[0020] gene is as follows: CYP82E4 The coding sequence (1554 bp) of the wild-type cigar tobacco

[0021] Cigars CYP82E4 The coding sequence of the gene mutant (1554 bp) is as follows:

[0022] Wild-type cigar CYP82E4 The amino acid sequence encoded by the gene (517 aa) is as follows: MLSPIEAIVGLVTFTFLFFFLWTKKSQKPSKPLPPKIPGGWPVIGHLFHFNDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGDYLGYNNAMLFLANYGPYWRKNRKLVIQEVLSASRLEKFKHVRFARIQASIKNLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKKAFKDFMILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHINKREKMEVNAEGNEQDFIDVVLSKMSNEYLGEGYSRDTVIKATVFSLVLDAADTVALHINWGMALLINNQKALTKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSDPDTFDPERFIATDIDFRGQYYKYIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNYRTPNDEPLDMKEGAGITIRKVNPVELIIAPRLAPELY (SEQ ID NO: 3) Cigar CYP82E4 The amino acid sequence encoded by the mutant gene (517 aa) is as follows: MLSPIEAIVGLVTFTFLFFFLWTKKSQKPSKPLPPKIPGGWPVIGHFFHFNDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGDYLGYNNAMLFLANYGPYWRKNRKLVIQEVLSASRLEKFKHVRFARIQASIKNLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKKAFKDFMILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHINKREKMEVNAEGNEQDFIDVVLSKMSNEYLGEGYSRDTVIKATVFSLVLDAADTVALHINWGMALLINNQKALTKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSDPDTFDPERFIATDIDFRGQYYKYIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNYRTPNDEPLDMKEGAGITIRKVNPVELIIAPRLAPELY (SEQ ID NO: 4) The present example obtains a tobacco plant comprising the above-mentioned Nicotiana sylvestris gene mutant by treating Nicotiana sylvestris seeds with EMS and screening mutants using TILLING technology. CYP82E4 The present example obtains a tobacco plant comprising the above-mentioned Nicotiana sylvestris gene mutant by treating Nicotiana sylvestris seeds with EMS and screening mutants using TILLING technology.

[0023] EMS treatment of Nicotiana sylvestris seeds: Wild-type Nicotiana sylvestris seeds are soaked in 50% commercially available bleaching water for 12 minutes, then spun and filtered dry; the seeds are rinsed with deionized water to remove the bleaching agent chemical composition, so that the seeds are not affected by the bleaching agent chemical composition, then spun and filtered dry again; the rinsed seeds are soaked in deionized water at a water temperature of 10-30°C for 10-15 hours, and the tobacco seeds are germinated to facilitate uniform mutagenesis treatment of the seeds, then spun and filtered dry; the seeds are soaked in a 0.5% EMS (ethyl methanesulfonate) solution for 10-15 hours, then spun and filtered dry; rinsed with deionized water for 5-8 times; finally, the seeds are filtered and dried using a Buchner funnel and filter paper.

[0024] 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.

[0025] Cigar smoke was designed using Primer 3 software. CYP82E4 The nucleotide sequence of the TILLING analysis primers E4-F / E4-R is as follows: E4-F: 5'-TTTTCTATTTCGGTACGAC-3' (SEQ ID NO: 5); E4-R: 5'-TAACTTTACAGTTGCTCCA-3' (SEQ ID NO: 6).

[0026] The target fragment amplified by the above primers was 1148 bp in size. The above DNA samples were amplified according to the reaction system (as shown in Table 1) and reaction procedure.

[0027] Table 1 Reaction System 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; and stored at 4℃.

[0028] After the reaction, the amplification products were analyzed by capillary electrophoresis to screen for single strains with nucleotide mutations, and then sequenced for verification (e.g., Figure 1 (As shown).

[0029] A mutant was obtained and named mutant. CYP82e4 -1, its CYP82E4 A change from C to T at position 139 of the coding sequence (CDS) of the gene alters the amino acid sequence encoded by the gene, changing leucine (L) to phenylalanine (F) at position 47. This mutant was grown in a greenhouse, resulting in the M3 generation of plants. Nucleotide sequencing was used to screen for plants containing homozygous mutants.CYP82E4 Tobacco plants of the mutant of the gene, self-crossing to collect seeds.

[0030] Example 2: The mutant according to Example 1 CYP82e4 -1 in obtaining low nicotine conversion rate in cigar tobacco.

[0031] Tobacco leaves containing the mutant CYP82e4 -1 have a lower nicotine conversion rate compared to tobacco leaves containing cigar tobacco CYP82E4 containing the gene.

[0032] This example determines the nicotine and nornicotine content of wild type tobacco and tobacco containing homozygous CYP82E4 mutants of the gene according to tobacco industry standard YC / T382-2010, and calculates the nicotine conversion rate.

[0033] 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 (Germany Eppendorf Company).

[0034] Reagents used for determination: internal standard quinoline purity greater than 98%, purchased from Sigma-Aldrich Company; internal standard nornicotine-D4 purity greater than 98%, purchased from American CATO Company; nornicotine purity greater than 98%, purchased from Sigma-Aldrich Company; nicotine purity greater than 98%, purchased from Canadian TRC Company; sodium hydroxide is analytical pure, purchased from Shantou Xilong Chemical Factory in Guangdong; dichloromethane, methanol are chromatographically pure, purchased from American Fisher Company.

[0035] Determination method: wild type cigar tobacco and mutants containing homozygous CYP82E4 genes are planted in the greenhouse, and the plants are topped when they reach the flowering stage. 15 days after topping, the middle leaves are taken, 10 single plants with consistent growth are selected for each material, and the tobacco leaves are killed and dried. 0.3 g of crushed cigar tobacco leaf sample is weighed into a 15 mL centrifuge tube, 0.05 mL of internal standard solution containing quinoline and nornicotine-d4 is added, and 2 mL of 5% sodium hydroxide aqueous solution is added, shaken and mixed, then placed for 20 min. Then add 10.0 mL dichloromethane-methanol extraction solution (v;v=4:1), seal after covering, place in vortex oscillator, vortex at 2000 rpm for 40 min, stand for 1 h, then take the lower organic phase to a chromatographic analysis bottle for instrument analysis.

[0036] 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.

[0037] Mass spectrometric 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; scan mode: selected ion monitoring mode (SIM) scan, retention time, quantitative and qualitative selected ion parameters of nicotine, nornicotine and internal standard were as shown in Table 2.

[0038] Table 2. Retention time, quantitative and qualitative selected ion parameters of nicotine, nornicotine and internal standard Cigar CYP82E4 The results of nicotine and nornicotine detection of the genetic mutant and wild type material are shown in Table 3 and Figure 2 The nicotine conversion rate was calculated according to the following formula: nicotine conversion rate = nornicotine content / (nicotine content + nornicotine content) x 100%.

[0039] Table 3. Cigar CYP82E4 The results of nicotine and nornicotine detection of the genetic mutant and wild type material Note: "**" in the table indicates that the difference is extremely significant P <0.01) It has been proved by experiments that the tobacco leaf comprising the genetic mutant of the cigar CYP82E4 The nicotine conversion rate of the tobacco leaf comprising the genetic mutant is reduced by 33% as compared with the tobacco leaf comprising the wild type gene.

[0040] The above description is merely preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A cigar CYP82E4 mutant of a gene CYP82e4-1 characterized in that, The mutant CYP82e4-1 The nucleotide sequence of the mutant is shown as SEQ ID NO: 2; the mutant CYP82e4-1 The mutant is a tobacco plant with a nucleotide sequence of the CYP82E5 CYP82E4 Gene at position 139 changed from C to T.

2. A cigar according to claim 1 CYP82E4 Mutants of genes CYP82e4-1 characterized in that, The mutant CYP82e4-1 The encoded amino acid sequence is shown as SEQ ID NO:

4.

3. The cigar of claim 1 CYP82E4 mutants of genes CYP82e4-1 characterized in that, The mutant CYP82e4-1 was amplified by the following primer pair, the nucleotide sequence of which is: Upstream primer: TTTTCTATTTCGGTACGAC, as set forth in SEQ ID NO: 5; Downstream primer: TAACTTTACAGTTGCTCCA, as set forth in SEQ ID NO:

6.

4. The cigar of claim 1 CYP82E4 mutants of genes CYP82e4-1 characterized in that, Tobacco leaves comprising said mutant CYP82e4-1 have a lower nicotine conversion rate than tobacco leaves comprising the gene for a CYP82E4 Cavendish tobacco.

5. The mutant according to any one of claims 1 to 4 CYP82e4-1 Use in obtaining low nicotine conversion in a cigar.

6. Use according to claim 5, characterized in that, Tobacco leaves comprising said mutant CYP82e4-1 have a lower nicotine conversion rate than tobacco leaves comprising the gene for a CYP82E4 Cavendish tobacco.

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