Mutant CYP82e4-2 of cigar CYP82E4 gene and application of mutant CYP82e4-2

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a mutant CYP82e4-2 of a cigar CYP82E4 gene and application of the mutant CYP82e4-2. The nucleotide sequence of the mutant CYP82e4-2 is as shown in SEQ ID NO: 2; the mutant CYP82e4-2 is obtained by changing the 134 nucleotide of a cigar CYP82E4 gene of which the nucleotide sequence is as shown in SEQ ID NO: 1 into A from G. The amino acid sequence coded by the mutant CYP82e4-2 is as shown in SEQ ID NO: 4. The cigar CYP82E4 gene mutant (mutant CYP82e4-2) 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.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a mutant of a cigar tobacco CYP82E4 gene CYP82e4-2 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. 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. 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 transgenic low-conversion tobacco created by the current transgenic means cannot be applied to tobacco production. The change of amino acid in the target gene 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 application provides a mutant of a cigar tobacco CYP82E4 gene CYP82e4-2 and application thereof.

[0005] The application is realized by the following technical scheme: A mutant of a cigar tobacco CYP82E4 gene CYP82e4-2 , the nucleotide sequence of the mutant CYP82e4-2 is shown in SEQ ID NO: 2; the mutant CYP82e4-2 is a mutant of a cigar tobacco CYP82E4 gene with the nucleotide sequence shown in SEQ ID NO: 1, in which the 134th nucleotide is changed from G to A.

[0006] Further, the encoded amino acid sequence of the mutant CYP82e4-2 is shown in SEQ ID NO: 4. Compared with the cigar tobacco CYP82E4The amino acid sequence encoded by the gene is changed from glycine (G) to aspartic acid (D) at the 45th position.

[0007] Further, the mutant CYP82e4-2 is amplified by the following primer pair, the nucleotide sequence of which is: The upstream primer is TTTTCTATTTCGGTACGAC, as shown in SEQ ID NO: 5. The downstream primer is TAACTTTACAGTTGCTCCA, as shown in SEQ ID NO: 6.

[0008] Further, the tobacco leaf containing the mutant CYP82e4-2 has a lower nicotine conversion rate than the tobacco leaf containing the wild-type gene. CYP82E4

[0009] The application also provides a mutant CYP82e4-2 for use in obtaining a low-nicotine conversion rate cigar.

[0010] Further, the tobacco leaf containing the mutant CYP82e4-2 has a lower nicotine conversion rate than the tobacco leaf containing the wild-type gene. CYP82E4 The application has the beneficial technical effects that:

[0011] The mutant gene of the cigar can significantly reduce the nicotine conversion rate in the cigar, and can be used to create a low-nicotine conversion rate cigar material. CYP82E4 CYP82e4-2

[0012] Experiments have proved that the tobacco leaf containing the mutant gene of the cigar CYP82E4 has a nicotine conversion rate that is 48% lower than that of the tobacco leaf containing the wild-type gene. CYP82e4-2 BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The sequencing results of the mutant gene of the cigar CYP82E4 are shown. Figure 2 The nicotine conversion rates of the mutant gene of the cigar CYP82E4 and the wild-type cigar are shown; wherein ** indicates a very significant difference (P<0.01). P DETAILED DESCRIPTION

[0014] ​​​​​In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are only intended to explain the present application and not to limit the present application.

[0015] On the contrary, the present application covers any substitution, modification, equivalent method and solution made within the gist 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 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.

[0016] In the following examples, the reagents not specifically described 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 described 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, 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.

[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 of the cigar tobacco CYP82E4 The gene mutant of the cigar tobacco CYP82e4 is named mutant CYP82e4 -2, the nucleotide sequence of mutant -2 is shown as SEQ ID NO: 2.

[0018] As shown in Figure 1 compared with the wild-type cigar tobacco CYP82E4 gene with the nucleotide sequence shown as SEQ ID NO: 1, the 134th of the cigar tobacco CYP82E4 gene mutant (mutant CYP82e4 -2) is changed from G to A.

[0019] Compared with the amino acid sequence (SEQ ID NO: 3) encoded by the wild-type cigar tobacco CYP82E4 gene, the 45th of the amino acid sequence (SEQ ID NO: 4) encoded by the cigar tobacco CYP82E4 gene mutant is changed from glycine (G) to aspartic acid (D).

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

[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).

[0023] Cigar CYP82E4 The amino acid sequence encoded by the mutant gene (517 aa) is as follows: MLSPIEAIVGLVTFTFLFFFLWTKKSQKPSKPLPPKIPGGWPVIDHLFHFNDDGDDRPLARKLGDLADKYGPVFTFRLGL 1568 PLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGDYLGYNNAMLFLANYGPYWRKNRKLVIQEVLSASRLEKFKHVRFARIQ 1568 ASIKNLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKKAFKDFMILSMEFVLWDAFPIPLFKWV 1568 DFQGHVKAMKRTFKDIDSVFQNWLEEHINKREKMEVNAEGNEQDFIDVVLSKMSNEYLGEGYSRDTVIKATVFSLVLDAA 1568 DTVALHINWGMALLINNQKALTKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGY 1568 HIPKGTRLFANVMKLQRDPKLWSDPDTFDPERFIATDIDFRGQYYKYIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNY 1568 RTPNDEPLDMKEGAGITIRKVNPVELIIAPRLAPELY (SEQ ID NO: 4) The present example obtains a tobacco plant comprising the above-mentioned Nicotiana attenuata CYP82E4 gene mutant by treating Nicotiana attenuata seeds with EMS and screening mutants using TILLING technology.

[0024] 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 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.

[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. 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).

[0027] 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.

[0028] 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℃.

[0029] 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).

[0030] A mutant was obtained and named mutant. CYP82e4 -2, its CYP82E4 The coding sequence (CDS) of the gene was changed from G to A at position 134, causing the amino acid sequence encoded by the gene to change from glycine (G) to aspartic acid (D) at position 45. This mutant was grown in a greenhouse, resulting in the M3 generation plants. Nucleotide sequencing was used to screen for plants containing homozygous mutants.CYP82E4 Tobacco plants of the genetic mutant, selfed to produce seed.

[0031] Example 2: The mutant according to Example 1 CYP82e4 -2 in obtaining low nicotine conversion rate in cigar tobacco.

[0032] Tobacco leaves comprising the mutant CYP82e4 -2 have a lower nicotine conversion rate compared to tobacco leaves comprising cigar tobacco CYP82E4 genes.

[0033] This example determines the nicotine and nornicotine content of wild type tobacco and tobacco comprising homozygous CYP82E4 gene mutants obtained in Example 1 according to the Tobacco Industry Standard YC / T382-2010, and calculates the nicotine conversion rate.

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

[0035] 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 CATO Company, USA; nornicotine purity greater than 98%, purchased from Sigma-Aldrich Company; nicotine purity greater than 98%, purchased from TRC Company, Canada; sodium hydroxide is analytical pure, purchased from Shantou Xilong Chemical Factory, Guangdong; dichloromethane, methanol are chromatographically pure, purchased from Fisher Company, USA.

[0036] Determination method: wild type cigar tobacco and mutants comprising homozygous CYP82E4 genes were planted in the greenhouse, and the plants were topped when they reached 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 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, and then the mixture was shaken and mixed, and then stood 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 a speed of 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.

[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 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 shown in Table 2.

[0039] 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 were 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%.

[0040] Table 3. Cigar CYP82E4 The results of nicotine and nornicotine detection of the genetic mutant and wild type material Note: "**" in the table means that the difference is extremely significant P <0.01) It has been proved by experiments that the tobacco leaf containing the genetic mutant of the cigar CYP82E4 The nicotine conversion rate of the tobacco leaf containing the genetic mutant of the cigar is reduced by 48% compared with the tobacco leaf containing the wild type gene.

[0041] The above description is only the preferred embodiment of the present application, and is not used to limit the present application, 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 CYP82e4-2 characterized in that, The mutant CYP82e4-2 The nucleotide sequence of the mutant is shown as SEQ ID NO: 2; the mutant CYP82e4-2 The mutant is a tobacco plant with a nucleotide sequence of the Cigar gene shown as SEQ ID NO: 1 CYP82E4 The 134th nucleotide of the Cigar gene is changed from G to A.

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

4.

3. The cigar of claim 1 CYP82E4 mutants of genes CYP82e4-2 characterized in that, The mutant CYP82e4-2 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 CYP82e4-2 characterized in that, Tobacco leaves comprising said mutant CYP82e4-2 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 CYP82e4-2 Use in obtaining low nicotine conversion in a cigar.

6. Use according to claim 5, characterized in that, Tobacco leaves comprising said mutant CYP82e4-2 have a lower nicotine conversion rate than tobacco leaves comprising the gene for a CYP82E4 Cavendish tobacco.

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