CN120905261A - Mutant CYP82E5-2 of cigar CYP82E5 gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a cigar CYP82E5 gene mutant CYP82E5-2 and application thereof. The nucleotide sequence of the mutant CYP82E5-2 is shown as SEQ ID NO. 2, and the mutant CYP82E5-2 is obtained by changing the 505 th nucleotide of a cigar CYP82E5 gene with the nucleotide sequence shown as SEQ ID NO. 1 from C to T. The amino acid sequence of the mutant CYP82e5-2 is shown in SEQ ID NO. 4. The cigar CYP82E5 gene mutant (mutant CYP82E 5-2) provided by the invention can obviously reduce the nicotine conversion rate in cigars, and can be used for creating cigar materials with low nicotine conversion rate. Experiments prove that compared with tobacco leaves containing wild type genes, the tobacco leaves containing the cigar CYP82E5 gene mutant have 48% of nicotine conversion rate reduced.

Description

Mutant CYP82E5-2 of cigar CYP82E5 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a cigar CYP82E5 gene mutant CYP82E5-2 and application thereof.

Background

With the development of global economy, the occupancy of cigars in tobacco markets is gradually increased, and the sales of cigars produced in China are rapidly increased. High quality cigars are required to have not only good smoking quality but also low hazard, safety and other characteristics. The high content of tobacco specific nitrosamines (Tobacco-specific nitrosamines, TSNAs) is the primary safety issue to be addressed in cigar development. TSNAs levels in cigar products have been reported to be 1-2 orders of magnitude higher than burley and flue-cured cigarettes.

NNN (N-nitrosonornicotine) in TSNA is listed as a class I carcinogen. NNN is formed mainly by nitrosation reactions of nornicotine. Reduced nicotine is the main direct precursor of NNN, reducing the reduced nicotine content being the most direct means of reducing NNN. The CYP82E4, CYP82E5 and CYP82E10 genes of the burley tobacco CYP82E2 subfamily can code active nicotine demethylase, and are key enzymes for nicotine conversion. The Lewis et al obtain the burley tobacco material mutated by CYP82E4, CYP82E5 and CYP82E10 by using an EMS mutagenesis method, and find that the mutation of CYP82E5 and CYP82E10 genes does not influence the nicotine conversion rate basically, and the nicotine conversion rate in mutant strains with three simultaneous genes is far lower than that of a control strain. The above studies indicate that CYP82E4 in rib smoke is a key gene determining nicotine conversion rate, while CYP82E5 and CYP82E10 genes have no obvious effect on nicotine conversion.

The conversion mechanism of nicotine in cigars is different from that of burley tobacco, so that no related research in cigars exists at present, and a method for reducing the conversion rate of nicotine in cigars still needs to be further explored.

Disclosure of Invention

Aiming at the technical problems, the invention provides a cigar CYP82E5 gene mutant CYP82E5-2 and application thereof.

The invention is realized by the following technical scheme:

A mutant CYP82E5-2 of a cigar CYP82E5 gene, wherein the nucleotide sequence of the mutant CYP82E5-2 is shown as SEQ ID NO. 2, and the mutant CYP82E5-2 is obtained by changing the 505 th nucleotide of the cigar CYP82E5 gene with the nucleotide sequence shown as SEQ ID NO. 1 from C to T.

Further, the amino acid sequence of the mutant CYP82e5-2 is shown in SEQ ID NO. 4. Compared with the amino acid sequence encoded by cigar CYP82E5 gene shown in SEQ ID NO. 3, the 169 th position of the amino acid sequence is changed from arginine (R) to a stop codon.

Further, the mutant CYP82e5-2 is amplified by the following primer pair, wherein the nucleotide sequences of the primer pair are as follows:

the upstream primer GGTAATTTTGTATTTATTATATTATGCG is shown in SEQ ID NO. 5;

the downstream primer TCATCCTTAGTATTTAGATAATCTAATT is shown in SEQ ID NO. 6.

Further, tobacco leaves comprising the mutant CYP82E5-2 have lower nicotine conversion than tobacco leaves comprising the cigar CYP82E5 gene.

The invention also provides an application of the mutant CYP82e5-2 in obtaining cigars with low nicotine conversion rate.

Further, tobacco leaves comprising the mutant CYP82E5-2 have lower nicotine conversion than tobacco leaves comprising the cigar CYP82E5 gene.

The beneficial technical effects of the invention are as follows:

the cigar CYP82E5 gene mutant (mutant CYP82E 5-2) provided by the invention can obviously reduce the nicotine conversion rate in cigars, and can be used for creating cigar materials with low nicotine conversion rate.

Experiments prove that compared with tobacco leaves containing wild type genes, the tobacco leaves containing the cigar CYP82E5 gene mutant have 48% of nicotine conversion rate reduced.

Drawings

FIG. 1 shows the sequencing results of the cigar CYP82E5 gene mutant in the embodiment of the invention.

FIG. 2 shows nicotine conversion rates of cigar CYP82E5 gene mutants versus wild type cigars, wherein the differences are shown to be very pronounced (P < 0.01)

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

On the contrary, the invention is intended to cover any alternatives, modifications, equivalents, and variations as may be included within the spirit and scope of the invention as defined by the appended claims. Further, in the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention. The present invention will be fully understood by those skilled in the art without the details described herein.

The technical scheme of the present invention will be described in detail with reference to examples. It is to be understood that the following examples are illustrative and explanatory only and are not intended to limit the scope of the invention.

In the following examples, reagents not specifically described are all conventional in the art, are commercially available or are formulated according to conventional methods in the art, and are of laboratory grade, and experimental methods and conditions not specifically described are all conventional in the art, and reference is made to the relevant experimental handbook, well-known literature or manufacturer's instructions. Unless defined otherwise, 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 belongs.

Example 1 this example provides a mutant CYP82E5 gene that reduces nicotine conversion in cigar leaves, designated mutant CYP82E5-2, the nucleotide sequence of which mutant CYP82E5-2 is shown in SEQ ID NO: 2.

As shown in FIG. 1, the 505 th position of the cigar CYP82E5 gene mutant (i.e., mutant CYP82E 5-2) is changed from C to T, compared with the wild type cigar CYP82E5 gene with the nucleotide sequence shown as SEQ ID NO: 1.

The 169 th position of the amino acid sequence (SEQ ID NO: 4) encoded by the cigar CYP82E5 gene mutant was changed from arginine (R) to a stop codon, as compared with the amino acid sequence (SEQ ID NO: 3) encoded by the wild-type cigar CYP82E5 gene.

The coding sequence of the wild cigar CYP82E5 gene (1554 bp) is as follows:

ATGGTTTCTCCCGTAGAAGCCATTGTAGGACTAGTAACCCTTACACTTCTCTTCTACTTCCTATGGCCCAAAAAATTTCAAATACCTTCAAAACCATTACCACCGAAAATTCCCGGAGGGTGGCCGGTAATCGGCCATCTTTTCTACTTCGATGATGACGGCGACGACCGTCCATTAGCTCGAAAACTCGGAGACTTAGCTGACAAATACGGCCCGGTTTTCACTTTCCGGCTAGGCCTTCCGCTTGTGTTAGTTGTAAGCAGTTACGAAGCTGTAAAAGACTGCTTCTCTACAAATGACGCCATTTTCTCCAATCGTCCAGCTTTTCTTTACGGTGAATACCTTGGCTACAATAATGCCATGCTATTTTTGACAAAATACGGACCTTATTGGCGAAAAAATAGAAAATTAGTCATTCAGGAAGTTCTCTCTGCTAGTCGTCTCGAAAAATTGAAGCACGTGAGATTTGGTAAAATTCAAACGAGCATTAAGAGTTTATACACTCGAATTGATGGAAATTCGAGTACGATAAATCTAACTGATTGGTTAGAAGAATTGAATTTTGGTCTGATCGTGAAAATGATCGCTGGGAAAAATTATGAATCCGGTAAAGGAGATGAACAAGTGGAGAGATTTAGGAAAGCGTTTAAGGATTTTATAATTTTATCAATGGAGTTTGTGTTATGGGATGCTTTTCCAATTCCATTGTTCAAATGGGTGGATTTTCAAGGCCATGTTAAGGCCATGAAAAGGACATTTAAGGATATAGATTCTGTTTTTCAGAATTGGTTAGAGGAACATGTCAAGAAAAGAGAAAAAATGGAGGTTAATGCACAAGGGAATGAACAAGATTTCATTGATGTGGTGCTTTCAAAAATGAGTAATGAATATCTTGATGAAGGTTACTCTCGTGATACTGTCATAAAAGCAACAGTGTTTAGTTTGGTCTTGGATGCTGCGGACACAGTTGCTCTTCACATGAATTGGGGAATGGCATTACTGATAAACAATCAACATGCCTTGAAGAAAGCACAAGAAGAGATCGATAAAAAAGTTGGTAAGGAAAGATGGGTAGAAGAGAGTGATATTAAGGATTTGGTCTACCTCCAAGCTATTGTTAAAGAAGTGTTACGATTATATCCACCAGGACCTTTATTAGTACCTCATGAAAATGTAGAGGATTGTGTTGTTAGTGGATATCACATTCCTAAAGGGACTAGACTATTCGCGAACGTTATGAAATTGCAGCGCGATCCTAAACTCTGGTCAAATCCTGATAAGTTTGATCCAGAGAGATTCTTCGCTGATGATATTGACTACCGTGGTCAGCACTATGAGTTTATCCCATTTGGTTCTGGAAGACGATCTTGTCCGGGGATGACTTATGCATTACAAGTGGAACACCTAACAATAGCACATTTGATCCAGGGTTTCAATTACAAAACTCCAAATGACGAGCCCTTGGATATGAAGGAAGGTGCAGGATTAACTATACGTAAAGTAAATCCTGTAGAAGTGACAATTACGGCTCGCCTGGCACCTGAGCTTTATTAA（SEQ ID NO:1）

The coding sequence of the cigar CYP82E5 gene mutant (1554 bp) is as follows:

ATGGTTTCTCCCGTAGAAGCCATTGTAGGACTAGTAACCCTTACACTTCTCTTCTACTTCCTATGGCCCAAAAAATTTCAAATACCTTCAAAACCATTACCACCGAAAATTCCCGGAGGGTGGCCGGTAATCGGCCATCTTTTCTACTTCGATGATGACGGCGACGACCGTCCATTAGCTCGAAAACTCGGAGACTTAGCTGACAAATACGGCCCGGTTTTCACTTTCCGGCTAGGCCTTCCGCTTGTGTTAGTTGTAAGCAGTTACGAAGCTGTAAAAGACTGCTTCTCTACAAATGACGCCATTTTCTCCAATCGTCCAGCTTTTCTTTACGGTGAATACCTTGGCTACAATAATGCCATGCTATTTTTGACAAAATACGGACCTTATTGGCGAAAAAATAGAAAATTAGTCATTCAGGAAGTTCTCTCTGCTAGTCGTCTCGAAAAATTGAAGCACGTGAGATTTGGTAAAATTCAAACGAGCATTAAGAGTTTATACACTTGAATTGATGGAAATTCGAGTACGATAAATCTAACTGATTGGTTAGAAGAATTGAATTTTGGTCTGATCGTGAAAATGATCGCTGGGAAAAATTATGAATCCGGTAAAGGAGATGAACAAGTGGAGAGATTTAGGAAAGCGTTTAAGGATTTTATAATTTTATCAATGGAGTTTGTGTTATGGGATGCTTTTCCAATTCCATTGTTCAAATGGGTGGATTTTCAAGGCCATGTTAAGGCCATGAAAAGGACATTTAAGGATATAGATTCTGTTTTTCAGAATTGGTTAGAGGAACATGTCAAGAAAAGAGAAAAAATGGAGGTTAATGCACAAGGGAATGAACAAGATTTCATTGATGTGGTGCTTTCAAAAATGAGTAATGAATATCTTGATGAAGGTTACTCTCGTGATACTGTCATAAAAGCAACAGTGTTTAGTTTGGTCTTGGATGCTGCGGACACAGTTGCTCTTCACATGAATTGGGGAATGGCATTACTGATAAACAATCAACATGCCTTGAAGAAAGCACAAGAAGAGATCGATAAAAAAGTTGGTAAGGAAAGATGGGTAGAAGAGAGTGATATTAAGGATTTGGTCTACCTCCAAGCTATTGTTAAAGAAGTGTTACGATTATATCCACCAGGACCTTTATTAGTACCTCATGAAAATGTAGAGGATTGTGTTGTTAGTGGATATCACATTCCTAAAGGGACTAGACTATTCGCGAACGTTATGAAATTGCAGCGCGATCCTAAACTCTGGTCAAATCCTGATAAGTTTGATCCAGAGAGATTCTTCGCTGATGATATTGACTACCGTGGTCAGCACTATGAGTTTATCCCATTTGGTTCTGGAAGACGATCTTGTCCGGGGATGACTTATGCATTACAAGTGGAACACCTAACAATAGCACATTTGATCCAGGGTTTCAATTACAAAACTCCAAATGACGAGCCCTTGGATATGAAGGAAGGTGCAGGATTAACTATACGTAAAGTAAATCCTGTAGAAGTGACAATTACGGCTCGCCTGGCACCTGAGCTTTATTAA（SEQ ID NO:2）

The amino acid sequence encoded by the wild cigar CYP82E5 gene (517 aa) is as follows:

MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFRKAFKDFIILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHVKKREKMEVNAQGNEQDFIDVVLSKMSNEYLDEGYSRDTVIKATVFSLVLDAADTVALHMNWGMALLINNQHALKKAQEEIDKKVGKERWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSNPDKFDPERFFADDIDYRGQHYEFIPFGSGRRSCPGMTYALQVEHLTIAHLIQGFNYKTPNDEPLDMKEGAGLTIRKVNPVEVTITARLAPELY（SEQ ID NO:3）

the amino acid sequence encoded by the cigar CYP82E5 gene mutant (168 aa) is as follows:

MVSPVEAIVGLVTLTLLFYFLWPKKFQIPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAVKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLTKYGPYWRKNRKLVIQEVLSASRLEKLKHVRFGKIQTSIKSLYT（SEQ ID NO:4）

this example obtained tobacco comprising cigar CYP82E5 gene mutants as described above by EMS treatment of cigar seeds and screening of the mutants using TILLING technology.

EMS treatment of cigar seeds, namely soaking the wild cigar seeds in 50% of commercial bleach water for 12 minutes, spin-centrifuging and filtering, rinsing the seeds with deionized water, removing the bleach water, enabling the seeds not to be affected by chemical components of the bleach, spin-centrifuging and filtering again, soaking the rinsed seeds in deionized water with the water temperature of 10-30 ℃ for 10-15 hours, sprouting and germinating tobacco seeds to facilitate uniform mutagenesis treatment of the seeds, spin-centrifuging and filtering, soaking the seeds in 0.5% EMS (ethyl methylsulfonate) solution for 10-15 hours, spin-centrifuging and filtering, washing the seeds with deionized water for 5-8 times, and finally filtering and drying the seeds by using Buchner funnels and filter papers.

Mutant screening mutants were screened using TILLING technology. Sowing seeds (M1 generation) treated by EMS into a field, bagging the seeds by a single plant, obtaining M2 generation by selfing and harvesting, and sowing 1 seed from the M2 generation seeds harvested from each M1 generation plant. The M2 generation individual leaves were taken and genomic DNA was extracted using a DNA extraction kit (QIAGEN, 69106) according to the kit instructions. The obtained DNA samples were arranged in order. Mu.l of each DNA sample was taken and concentration was determined on a 16 channel TECAN INFINITE M200 instrument. All DNA samples were then diluted to a concentration of 40 ng/. Mu.l and mixed to 8-fold DNA pools for TILLING analysis per 8 DNA samples.

The Primer 3 software is used for designing and obtaining a TILLING analysis Primer E5-F/E5-R of the cigar CYP82E5 gene, and the nucleotide sequence is as follows:

E5-F:5’-GGTAATTTTGTATTTATTATATTATGCG-3’(SEQ ID NO:5);

E5-R:5’-TCATCCTTAGTATTTAGATAATCTAATT-3’(SEQ ID NO:6)。

The size of the target fragment amplified by the primer is 1136bp. The above DNA samples were amplified according to the reaction system (shown in Table 1) and the reaction procedure.

TABLE 1 reaction system

The reaction procedure was 95℃3 min, (94℃30s, 63℃30s, -1℃per cycle, 72℃1 min) 7 cycles, (94℃30s, 58℃30s, 72℃1 min) 40 cycles, 72℃5min, 99℃10 min, (70℃20 s, -0.3℃per cycle) 70 cycles, and 4℃storage.

After the reaction, the amplified products were analyzed by capillary electrophoresis, and nucleotide mutant individuals were selected and verified for sequencing (as shown in FIG. 1).

A mutant, designated mutant CYP82E5-2, was obtained in which the 505 th position of the coding sequence (CDS) of the CYP82E5 gene was changed from C to T, and the 169 th position of the amino acid sequence encoded by the gene was changed from arginine (R) to a stop codon. The mutant is planted in a greenhouse to obtain M3 generation plants, and tobacco plants containing the homozygous CYP82E5 gene mutant are obtained through nucleotide sequencing screening, and self-bred seeds are obtained.

Example 2 use of the mutant CYP82e5-2 according to example 1 to obtain a low nicotine conversion cigar.

Tobacco lamina comprising the mutant CYP82E5-2 has a lower nicotine conversion rate than tobacco lamina comprising the cigar CYP82E5 gene.

This example performs nicotine and nornicotine content assays on wild-type tobacco and tobacco obtained in example 1 comprising homozygous CYP82E5 gene mutants, respectively, according to tobacco industry standard YC/T382-2010, and calculates the nicotine conversion.

The apparatus used for the measurement was a Shimadzu GCMS-QP2020 gas chromatograph mass spectrometer (Shimadzu corporation, japan), a Millipore ultra-pure water machine (Millipore Simplicity Co., ltd.), a Eofo-945008 vortex mixing shaker (Tallboys Co., ltd.), and an Eppendorf 5804 high-speed centrifuge (Eppendorf, germany).

The reagents used for the determination were more than 98% of internal standard quinoline purity, purchased from Sigma-Aldrich, more than 98% of internal standard nornicotine-D4 purity, purchased from CATO, more than 98% of nornicotine purity, purchased from Sigma-Aldrich, more than 98% of nicotine purity, purchased from TRC, canada, analytically pure sodium hydroxide, purchased from Guangdong's chemical plant of Chelongling, and chromatographically pure dichloromethane and methanol, all purchased from Fisher, U.S.A..

The method comprises the steps of planting wild cigars and mutants containing homozygous CYP82E5 genes in a greenhouse, topping after the tobacco plants grow to a flowering period, taking middle leaves 15 days after topping, sampling 10 individual plants with consistent growth vigor, and deactivating enzymes and drying tobacco leaves. 0.3 g of crushed cigar tobacco leaf samples are weighed into a 15 mL centrifuge tube, respectively added with 0.05 mL of internal standard solution containing quinoline and nornicotine-d 4 and 2mL of 5% sodium hydroxide aqueous solution, and after shaking and mixing uniformly, the cigar tobacco leaf samples are kept stand for 20 min. Then 10.0 mL methylene chloride-methanol extraction solution (v; v=4:1) was added, the mixture was capped and sealed, and then placed in a vortex shaker, and vortex shaking was performed at 2000: 2000 rpm to extract 40: 40 min, and after standing for 1:1 h, the lower organic phase was transferred to a chromatographic flask for instrumental analysis.

Chromatographic conditions

The chromatographic column comprises BR-5MS (30 m multiplied by 0.25 multiplied by mm multiplied by 0.25 mu m), sample injection amount of 1 mu L, split ratio of 60:1, sample injection port of 250 ℃, heating program of initial temperature of 110 ℃ to 185 ℃ in 10 ℃ per min, temperature of 50 ℃ per min to 280 ℃ in 2 min, and carrier gas of helium of 1.0 mL per min.

Mass spectrometry conditions

The transmission line temperature was 250 ℃, the ion source temperature was 230 ℃, the ionization mode was electron bombardment ionization (EI), the bombardment energy was 70 eV, the solvent delay time was 4 min, the scanning mode was selected for ion monitoring mode (SIM) scanning, the retention time of nicotine and internal standard, and the quantitative and qualitative selection of ion parameters were as shown in Table 2 below.

TABLE 2 Nicotine, retention time of Nicotine and internal Standard, quantitative and qualitative selection of ion parameters

Cigar CYP82E5 gene mutant and wild type material nicotine and nornicotine detection results are shown in Table 3 and FIG. 2. The nicotine conversion was calculated according to the following formula, nicotine conversion = nornicotine content/(nicotine content + nornicotine content) ×100%.

TABLE 3 cigar CYP82E5 Gene mutant and wild-type Nicotine and nornicotine detection results

Note that the table "×" indicates that the difference is very significant (P < 0.01)

It can be seen that tobacco lamina comprising the cigar CYP82E5 gene mutant has a 48% reduction in nicotine conversion compared to tobacco lamina comprising the wild-type gene.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (6)

1. A mutant of the CYP82E5 gene in cigars, CYP82e5-2 , characterized in that 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 the CYP82E5 gene in cigars, as shown in SEQ ID NO:1, from C to T. 2. The mutant CYP82e5-2 of the CYP82E5 gene of cigars according to claim 1, characterized in that the amino acid sequence encoded by the mutant CYP82e5-2 is as shown in SEQ ID NO:4. 3. The mutant CYP82e5-2 of the CYP82E5 gene for cigars according to claim 1, characterized in that the mutant CYP82e5-2 is obtained by amplification using the following primer pair, the nucleotide sequence of which is: Upstream primer: GGTAATTTTGTATTTATTATATTATGCG, as shown in SEQ ID NO:5; Downstream primer: TCATCCTTAGTATTTAGATAATCTAATT, as shown in SEQ ID NO:6. 4. The mutant CYP82e5-2 of the CYP82E5 gene for cigars according to claim 1, characterized in that tobacco leaves containing the mutant CYP82e5-2 have a lower nicotine conversion rate compared with tobacco leaves containing the CYP82E5 gene for cigars. 5. The use of the mutant CYP82e5-2 according to any one of claims 1 to 4 in obtaining cigars with low nicotine conversion. 6. The application according to claim 5, characterized in that the tobacco leaves containing the mutant CYP82e5-2 have a lower nicotine conversion rate compared with the tobacco leaves containing the cigar CYP82E5 gene.