Mutant CYP82e5-1 of cigar CYP82E5 gene and application of mutant CYP82e5-1

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a mutant CYP82e5-1 of a cigar CYP82E5 gene and application of the mutant CYP82e5-1. The nucleotide sequence of the mutant CYP82e5-1 is as shown in SEQ ID NO: 2; the mutant CYP82e5-1 is obtained by mutating the 590th nucleotide of a cigar CYP82E5 gene of which the nucleotide sequence is shown as SEQ ID NO: 1 from G to A. The amino acid sequence coded by the mutant CYP82e5-1 is as shown in SEQ ID NO: 4. The cigar CYP82E5 gene mutant disclosed 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 44%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a mutant of a gene of a cigar tobacco CYP82E5 CYP82e5-1 and application thereof. BACKGROUND

[0002] With the development of global economy, the share of cigar tobacco in the tobacco market is gradually increasing. High-quality cigar tobacco 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 tobacco. It is reported that the content of TSNAs in cigar products is 1-2 orders of magnitude higher than that in burley tobacco and flue-cured tobacco.

[0003] NNN (N-nitrosonornicotine) 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. Burley tobacco CYP82E2 subfamily of CYP82E4 , CYP82E5 , CYP82E10 genes can encode active nornicotine demethylase, which is a key enzyme for nornicotine conversion. Lewis et al. obtained burley tobacco materials with mutations in CYP82E4 , CYP82E5 , CYP82E10 genes by EMS mutagenesis, and found that the mutation in CYP82E5 , CYP82E10 gene basically does not affect the nornicotine conversion rate, and the nornicotine conversion rate in the mutant strain with simultaneous mutation in the three genes is much lower than that in the control strain. The above studies show that CYP82E4 is the key gene that determines the nornicotine conversion rate in burley tobacco, and CYP82E5 , CYP82E10 genes have no obvious effect on nornicotine conversion.

[0004] The nornicotine conversion mechanisms in cigar tobacco and burley tobacco are different, and there is currently no relevant research on cigar tobacco. The method for reducing the nornicotine conversion rate of cigar tobacco still needs to be further explored. SUMMARY

[0005] In view of the above technical problems, the present application provides a mutant of a gene of a cigar tobacco CYP82E5 CYP82e5-1 and application thereof.

[0006] The present application is realized by the following technical solutions: A mutant of a gene of a cigar tobacco CYP82E5 CYP82e5 -1, the mutant​​​CYP82e5 the nucleotide sequence of the mutant is shown as SEQ ID NO: 2; the mutant CYP82e5 -1 is a mutant of the tobacco gene shown as SEQ ID NO: 1. CYP82E5 the 590th nucleotide of the gene is mutated from G to A.

[0007] further, the mutant CYP82e5 -1 encodes the amino acid sequence shown as SEQ ID NO: 4; compared with the tobacco gene shown as SEQ ID NO: 3 CYP82E5 the amino acid sequence encoded by the gene is mutated from glycine (G) at the 197th position to glutamic acid (E).

[0008] further, the mutant CYP82e5 -1 is amplified by the following primer pair, the nucleotide sequence of which is as follows: upstream primer: GGTAATTTTGTATTTATTATATTATGCG, shown as SEQ ID NO: 5; downstream primer: TCATCCTTAGTATTTAGATAATCTAATT, shown as SEQ ID NO: 6.

[0009] further, the tobacco leaf containing the mutant CYP82e5 -1 has a lower nicotine conversion rate than the tobacco leaf containing the tobacco gene CYP82E5 -1 has a lower nicotine conversion rate than the tobacco leaf containing the tobacco gene

[0010] The application also provides a mutant CYP82e5 -1 in obtaining a tobacco gene mutant with a low nicotine conversion rate.

[0011] further, the tobacco leaf containing the mutant CYP82e5 -1 has a lower nicotine conversion rate than the tobacco leaf containing the tobacco gene CYP82E5 -1 has a lower nicotine conversion rate than the tobacco leaf containing the tobacco gene

[0012] The beneficial technical effects of the application are as follows: The tobacco gene mutant of the application CYP82E5 can significantly reduce the nicotine conversion rate of the tobacco gene mutant, and can be used to create a tobacco material with a low nicotine conversion rate. It has been proved by tests that the tobacco leaf containing the tobacco gene mutant CYP82E5 has a nicotine conversion rate reduced by 44% compared with the tobacco leaf containing the wild-type gene. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The sequencing results of the tobacco gene mutant in the embodiments of the application are shown in the following table. CYP82E5 The sequencing results of the tobacco gene mutant in the embodiments of the application are shown in the following table.

[0014] Figure 2 Cigar tobacco CYP82E5 Nicotine conversion rate of gene mutant and wild type cigar tobacco; wherein ** indicates extremely significant difference P <0.01. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present application more clear, 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.

[0016] On the contrary, the present application covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present application as 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.

[0017] The technical scheme of the present application is 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.

[0018] 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 literature or manufacturer's instructions. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those skilled in the art to which the present application belongs.

[0019] Example 1: The present example provides a method for reducing nicotine conversion rate in cigar tobacco leaves CYP82E5 Gene mutant, the cigar tobacco CYP82E5 The gene mutant is named mutant CYP82e5 -1, mutant CYP82e5 The nucleotide sequence of mutant -1 is shown in SEQ ID NO: 2.

[0020] 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 -1) has G changed to A at position 590.

[0021] Compared with the wild type cigar tobacco CYP82E5The amino acid sequence encoded by the gene (SEQ ID NO: 3) is compared to CYP82E5 The 197th amino acid of the amino acid sequence encoded by the gene mutant (SEQ ID NO: 4) is changed from glycine (G) to glutamic acid (E).

[0022] Wild-type Virginia tobacco CYP82E5 The coding sequence (1554 bp) of the gene is as follows: Cigars CYP82E5 The coding sequence of the gene mutant (1554 bp) is as follows: Wild-type cigar CYP82E5 The amino acid sequence encoded by the gene (517 aa) is as follows: MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFRKAFKDFIILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHVKKREKMEVNAQGNEQDFIDVVLSKMSNEYLDEGYSRDTVIKATVFSLVLDAADTVALHMNWGMALLINNQHALKKAQEEIDKKVGKERWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSNPDKFDPERFFADDIDYRGQHYEFIPFGSGRRSCPGMTYALQVEHLTIAHLIQGFNYKTPNDEPLDMKEGAGLTIRKVNPVEVTITARLAPELY (SEQ ID NO: 3) Cigar CYP82E5 The amino acid sequence encoded by the mutant gene (517 aa) is as follows: MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYTRIDGNSSTINLTDWLEELNFGLIVKMIAEKNYESGKGDEQVERFRKAFKDFIILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHVKKREKMEVNAQGNEQDFIDVVLSKMSNEYLDEGYSRDTVIKATVFSLVLDAADTVALHMNWGMALLINNQHALKKAQEEIDKKVGKERWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSNPDKFDPERFFADDIDYRGQHYEFIPFGSGRRSCPGMTYALQVEHLTIAHLIQGFNYKTPNDEPLDMKEGAGLTIRKVNPVEVTITARLAPELY (SEQ ID NO: 4) In this embodiment, tobacco seeds are treated by EMS and mutants are screened by TILLING technology to obtain tobacco containing the mutant of the above-mentioned Nicotiana attenuata CYP82E5 tobacco.

[0023] EMS treatment of Nicotiana attenuata seeds: wild-type Nicotiana attenuata 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 and prevent the seeds from being affected by the chemical components of the bleaching agent, 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 to facilitate seed germination and uniform mutagenesis treatment, then spun and filtered dry; the seeds are soaked in 0.5% EMS (ethyl methanesulfonate) solution for 10-15 hours, then spun and filtered dry; the seeds are 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. 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).

[0026] 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.

[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. CYP82e5 -1, its CYP82E5The 590th position of the coding sequence (CDS) of the gene is changed from G to A, so that the 197th position of the amino acid sequence coded by the gene is changed from glycine (G) to glutamic acid (E). The mutant is planted in a greenhouse, M3 generation plants are obtained, and nucleotide sequencing screening is performed to obtain tobacco plants containing homozygous CYP82E5 The tobacco plants of the gene mutant are selfed to obtain seeds.

[0030] Example 2: The mutant according to Example 1 CYP82e5 -1 is applied in obtaining low nicotine conversion rate in cigar tobacco.

[0031] Tobacco leaves containing the mutant CYP82e5 -1 have lower nicotine conversion rate compared with tobacco leaves containing cigar tobacco CYP82E5 containing the gene.

[0032] 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 (i.e. mutant CYP82e5 -1) obtained in Example 1 are determined, and the nicotine conversion rate is calculated.

[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 (German Eppendorf Company).

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

[0035] Determination method: wild type cigar tobacco and tobacco containing homozygous CYP82E5Mutants of the gene, the tobacco plants were grown to the flowering stage and topped, 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 cigar tobacco 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, shaken and mixed, then placed for 20 min. Then add 10.0 mL dichloromethane-methanol extraction solution (v;v=4:1), seal with cover, place in vortex oscillator, vortex at 2000 rpm for 40 min, stand for 1 h, then take the lower organic phase and transfer it to a chromatographic analysis bottle for instrument analysis.

[0036] Chromatographic conditions: 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 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; scan mode: selected ion monitoring mode (SIM) scan, nicotine, nicotine and internal standard retention time, quantitative and qualitative selection ion parameters as shown in Table 2.

[0038] Table 2 Nicotine, nicotine and internal standard retention time, quantitative and qualitative selection ion parameters Cigar tobacco CYP82E5 The results of nicotine and nornicotine detection of gene 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 tobacco CYP82E5 The results of nicotine and nornicotine detection of gene mutant and wild type material are shown in Table 3 and Note: “**” in the table indicates extremely significant difference P <0.01).

[0040] It can be seen that the nicotine conversion rate of tobacco leaves containing the CYP82E5 gene mutant of the cigar tobacco is reduced by 44% compared with the tobacco leaves containing the wild type gene.

[0041] The above description is only the preferred embodiment 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 -1, characterized in that, The mutant CYP82e5 -1 has a nucleotide sequence as shown in SEQ ID NO: 2; the mutant CYP82e5 -1 is a cigar tobacco having a nucleotide sequence as shown in SEQ ID NO: 1 CYP82E5 The 590th nucleotide of the gene is mutated from G to A.

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

4.

3. The cigar of claim 1 CYP82E5 Mutants of genes CYP82e5 -1 characterized in that, The mutant CYP82e5 -1 is 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 -1, 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 NOS gene. CYP82E5 Tobacco leaves comprising the mutant of the application have a lower nicotine conversion rate compared to tobacco leaves comprising a wild type NOS gene.

5. The mutant according to any one of claims 1 to 4 CYP82e5 -1 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 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.

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