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CN108424922B - CYP82E10 gene missense mutant M271 capable of reducing nicotine conversion rate and application thereof - Google Patents

CYP82E10 gene missense mutant M271 capable of reducing nicotine conversion rate and application thereof Download PDF

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CN108424922B
CN108424922B CN201810360474.5A CN201810360474A CN108424922B CN 108424922 B CN108424922 B CN 108424922B CN 201810360474 A CN201810360474 A CN 201810360474A CN 108424922 B CN108424922 B CN 108424922B
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cyp82e10
nicotine conversion
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CN108424922A (en
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宋中邦
李梅云
王丙武
高玉龙
李文正
吴兴富
赵璐
隋学艺
李永平
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Yunnan Academy of Tobacco Agricultural Sciences
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

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Abstract

The invention discloses a method for nicotine conversion rateCYP82E10A missense mutant M271 and application thereof. Said method for reducing nicotine conversionCYP82E10The missense mutant M271 has the nucleotide sequence shown in SEQ ID NO.1CYP82E10Genes for reducing nicotine conversionCYP82E10Missense mutant M271 containsCYP82E10The C at position 385 in the gene sequence is replaced by T, and point mutation is generated, so that the nicotine conversion rate is reducedCYP82E10The nucleotide sequence of the missense mutant M271 is shown as SEQ ID NO. 2. The use ofCYP82E10Application of the missense mutant M271 in obtaining tobacco plants with low nicotine conversion rate. For reducing nicotine conversion as described in the present inventionCYP82E10In the cloud smoke 87 material obtained by the missense mutant M271, the nicotine conversion rate of the leaf blades is respectively reduced by about 62 percent compared with that of a control, and the nicotine conversion rate is obviously reduced.

Description

CYP82E10 gene missense mutant M271 capable of reducing nicotine conversion rate and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for reducing nicotine conversion rateCYP82E10A missense mutant M271 and application thereof.
Background
Nicotine (nicotine), nornicotine (nornicotine), anabasine (anabasene), and neonicotine (anabasene) are tobacco (Nicotine), nornicotine (nornicotine), and Nicotine (anabasene)Nicotiana tobacum) The main alkaloid in the total alkaloid contains nicotine 90-95%, and demethylated nicotine content is usually lower than 3.5% of total alkaloid, and is converted from nicotine by demethylation reaction. The demethylated nicotine can cause harm to human health, and is mainly shown in that the demethylated nicotine (NNN) is a synthetic precursor of nitroso-demethylated nicotine (NNN) which is a potential carcinogen in cigarette smoke and can cause esophageal cancer and oral cancer. Demethylated nicotine also directly induces abnormal glycosylation of proteins in the plasma of smokers, and studies show that it can generate covalent reaction with common steroid drugs, thereby affecting drug efficacy and toxicity. Therefore, reducing nicotine conversion to demethylated nicotine, i.e. reducing nicotine conversion, is an important goal for tobacco harm reduction.
There are two types of tobacco widely used internationally in production, burley tobacco and flue-cured tobacco, with burley tobacco production being the major concern in western developed countries and south america, and flue-cured tobacco being the major concern in our country. Burley tobaccoCYP82E2Of subfamiliesCYP82E4、CYP82E5、CYP82E10The gene can code active nicotine demethylase, and is a key enzyme for nicotine conversion. Gavilano et al "transformants" for inhibiting Burley tobacco by RNAi technique "CYP82E4And the expression of homologous genes thereof, the synthesis of demethylated nicotine is obviously inhibited, and the gene is transgenicThe nicotine conversion rate of the plants is only 0.8 percent at the lowest, and is even lower than the common conversion rate of about 3 to 5 percent of burley tobacco non-transformant. Julio et al screened 10 of 1132 EMS-mutagenized mutantsCYP82E4Tobacco with point mutations at the locus, wherein the nicotine conversion in nonsense and missense mutants is reduced to a very trace level. Respectively obtained by the method of EMS mutagenesis for Lewis and the likeCYP82E4、CYP82E5、CYP82E10Mutated burley tobacco material, foundCYP82E5、CYP82E10The gene mutation does not affect the nicotine conversion rate, and the nicotine conversion rate in the mutant strain with the simultaneous mutation of the three genes is far lower than that of a control strain. The above studies indicate that in burley tobaccoCYP82E4Is a key gene for determining the conversion rate of nicotine,CYP82E5、CYP82E10the gene has no obvious influence.
The flue-cured tobacco is the main raw material of the Chinese style cigarette, and the reduction of the nicotine conversion rate and the NNN content of the flue-cured tobacco is of great significance to the harm reduction of the Chinese style cigarette. However, the research on the synthesis of the demethylated nicotine of the flue-cured tobacco is less so far, and whether the synthesis mechanism of the flue-cured tobacco and the burley tobacco is consistent or not is not reported in documents at present, and how to reduce the conversion rate of the nicotine of the flue-cured tobacco still needs to be explored.
Disclosure of Invention
It is a first object of the present invention to provide a method for reducing nicotine conversionCYP82E10Missense mutant M271; the second purpose is to provide the nicotine conversion rate reductionCYP82E10Application of missense mutant M271.
The first object of the invention is achieved in that said nicotine conversion is reducedCYP82E10The missense mutant M271 has the nucleotide sequence shown in SEQ ID NO.1CYP82E10Genes for reducing nicotine conversionCYP82E10Missense mutant M271 containsCYP82E10The C at position 385 in the gene sequence is replaced by T, and point mutation is generated, so that the nicotine conversion rate is reducedCYP82E10The nucleotide sequence of the missense mutant M271 is shown as SEQ ID NO. 2.
The second object of the invention is achieved in that said nicotine conversion is reducedCYP82E10Missense of geneApplication of the mutant M271 in obtaining tobacco plants with low nicotine conversion rate.
For reducing nicotine conversion as described in the present inventionCYP82E10In the cloud smoke 87 material obtained by the missense mutant M271, the nicotine conversion rate of the leaf blades is respectively reduced by about 62 percent compared with that of a control, and the nicotine conversion rate is obviously reduced.
Drawings
FIG. 1 is a drawing ofCYP82E10Detecting gene mutation by capillary electrophoresis;
figure 2 is an analysis of mutant material nicotine conversion.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
For reducing nicotine conversion as described in the present inventionCYP82E10The missense mutant M271 has the nucleotide sequence shown in SEQ ID NO.1CYP82E10Genes for reducing nicotine conversionCYP82E10Missense mutant M271 containsCYP82E10The C at position 385 in the gene sequence is replaced by T, and point mutation is generated, so that the nicotine conversion rate is reducedCYP82E10The nucleotide sequence of the missense mutant M271 is shown as SEQ ID NO. 2.
Said method for reducing nicotine conversionCYP82E10The amino acid sequence coded by the missense mutant M271 is shown in SEQ ID NO. 3.
The invention for reducing nicotine conversionCYP82E10Application of missense mutant M271 to reduce nicotine conversion rateCYP82E10Application of the missense mutant M271 in obtaining tobacco plants with low nicotine conversion rate.
Example 1
Flue-cured tobacco mutant library creation
EMS treatment of flue-cured tobacco seeds
Flue-cured tobacco (variety: Yunyan 87) seeds are soaked in 50% commercial bleaching solution for 12 minutes, then quickly cleaned by deionized water, and soaked in the deionized water for 12 hours. Deionized water was discarded and an equal volume of 0.5% EMS (ethyl methanesulfonate) was added for 12 hours of treatment. Discarding the treatment solution, adding deionized water to wash for 6-8 times, each time for about 1 minute. The seeds were collected in a buchner funnel and drained for use.
Second, M1 plant field planting
After EMS treatment, the seeds are sowed on a floating disc, one seed is planted in each hole, after emergence of seedlings, the seeds are transplanted to a field, and normal agricultural measures are taken for management. After budding, the single plants are bagged, numbered and harvested to obtain M2 seeds.
Third, mutant genome DNA extraction and sample mixing
The kit is used for extracting genome DNA and comprises the following steps:
0.1g of fresh sample was weighed, ground with liquid nitrogen, finely ground and transferred to a 2.0ml sample tube, and 600. mu.L of AP1 buffer and 4. mu.L of RNaseA stock solution (100 mg/ml) were immediately added. The water bath treatment was carried out at 65 ℃ for 10 minutes, during which the EP tube was inverted 2-3 times. 190. mu.L of AP2 buffer was added, mixed well and placed on ice for 5 minutes, and centrifuged at 14000rpm for 5 minutes at room temperature. The supernatant was pipetted onto a QIAshredder Mini column and centrifuged at 14000rpm for 2 minutes at room temperature. 450 to 650 μ L of the filtrate was taken into a 2.0ml centrifuge tube, and 675-900 μ L of buffer AP3/E was added. A650. mu.L mixture was taken into a 2ml DNeasy column and centrifuged at > 8000rpm for 1-2 minutes at room temperature. The DNeasy column was placed in a new 2ml collection tube, 500. mu.L of AW buffer was added, centrifuged at > 8000rpm for 2 minutes, the effluent discarded and washed again with AW buffer. The DNeasy column was placed in a 1.5ml centrifuge tube, 100. mu.L of AE buffer was added, the mixture was left at room temperature for 5 minutes, and the filtrate obtained by centrifugation was genomic DNA.
About 2200 parts of EMS mutant plants of M2 generation are planted in a field, leaves are collected, genome DNA is extracted by the method, the DNA concentration of all samples is diluted to 40 ng/mu l, finally, a DNA library of the M2 generation Yunyan 87 mutant is established, 8 parts of DNA are mixed to form an 8-time mixing pool, and the mixing pool is stored in a 96-well plate.
Example 2
CYP82E10Gene mutant screening
First, Tilling primer
CYP82E10The gene has two outer genesAnd (3) displaying the exons, and screening mutants of the first exon region by using the Tilling technology. Depending on the genomic sequence of the target gene,
the forward primer isCYP82E10_Tilling_F：GTCAAATACCACCTCTTAATAGTAA，
The reverse primer isCYP82E10_Tilling_R：AAAAGTCCCTATTGGTAGGAAGTGC。
Second, PCR amplification conditions
The PCR reaction system is as follows: the total volume was 10. mu.L, wherein 20 ng/. mu.L of DNA sample was 1.0. mu.L, 10 XPCR buffer was 1.0. mu.L, dNTPs were 0.8. mu.L, each of primers was 0.16. mu.L, Taq DNase was 0.1. mu.L, ddH2O 6.78μL。
The PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 62 ℃ for 30 seconds (1 ℃ drop per cycle), extension at 72 ℃ for 90 seconds, run for 7 cycles; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 90 seconds, and running for 40 cycles; final extension at 72 ℃ for 5 min. The PCR amplification product can be stored at 4 ℃.
Third, PCR product enzyme digestion and electrophoresis
And (2) carrying out enzyme digestion on the PCR product by utilizing the characteristic of specific heteroduplex cleavage of the CEL I enzyme, wherein the enzyme digestion system is as follows: PCR product 4. mu.L, 10 XBuffer 1. mu.L, CEL I enzyme 0.5. mu.L, supplemented with H2O to a total volume of 10. mu.L. The enzyme digestion system is separated by an automatic capillary electrophoresis system, and the separation conditions are as follows: the sample loading voltage is 9KV, the sample loading voltage is 30sec, the sample loading voltage of Marker is 7.5KV, the sample loading voltage is 5sec, the pre-electrophoresis voltage is 9KV, the separation electrophoresis voltage is 9KV, the running time is 80min, and the electrophoresis result is analyzed by using Prosize 2.0 software. A total of 11 mutants were obtained by Tilling screening, and the M271 individual strain was mutated at P129L, the results of which are shown in Table 1 and FIG. 1.
TABLE 1 Yunyan 87 mutant libraryCYP82E10Analysis of Gene mutants
Figure DEST_PATH_IMAGE001
Example 3
CYP82E10Verification of missense mutation of gene
First, M3 generation mutant genome DNA extraction and PCR amplification
Selecting according to the Tiling screening result of M2 generation plantsCYP82E10Seeds of the mutant individuals (M3 generation) were sown in the seedling trays. Extracting the seedling leaf genome DNA by a kit method. To be provided withCYP82E10A Tiling _ F andCYP82E10tiling R primer, using genomic DNA as template to amplifyCYP82E10The first exon region of the gene. The PCR reaction system is as follows: the total volume was 25. mu.L, wherein 20 ng/. mu.L of DNA sample was 1.0. mu.L, 10 XPCR buffer was 2.5. mu.L, dNTPs were 2. mu.L, each of primers was 0.5. mu.L, Taq DNase was 0.3. mu.L, ddH2O18.2. mu.L. The PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds (1 ℃ per cycle), extension at 72 ℃ for 90 seconds, and running for 30 cycles; extension at 72 ℃ for 5 minutes. The PCR amplification product can be stored at 4 ℃.
Secondly, cloning and sequencing TOPO (top of the polymerase chain reaction) product
The pTOPO vector (Invitrogen) was ligated after recovery of the PCR product as follows: PCR product 4. mu.L, pCR-BluntII-TOPO plasmid 1. mu.L, salt Solution 1. mu.L. The reaction components are incubated at 25 ℃ for 30min, transferred into E.coli competent cells and mixed evenly, ice-bathed for 30min, immediately placed in the ice-bath for 2min after heat shock at 42 ℃ for 90sec, added with 0.35mL of LB liquid medium, and shake-cultured at 37 ℃ and 210rpm for 1 h. Centrifuging for 1min (7500 rpm), discarding the supernatant to about 100. mu.l, mixing, spreading on Km resistant medium, and culturing overnight at 37 deg.C. Positive clones were selected for plasmid extraction and Sanger sequencing was performed.
Example 4
CYP82E10Analysis of Gene missense mutant Nicotine conversion
First, preparation of mutant Material sample
And (3) when the tobacco grows to the mature period, taking a whole tobacco leaf sample, drying at 60 ℃, crushing, and sieving with a 60-mesh sieve to be detected. Accurately weighing 0.5g of a tobacco sample into a 50mL centrifuge tube, adding 5mL of 10% NaOH solution, shaking uniformly, soaking for 15min, adding 20mL of extract containing an internal standard, carrying out ultrasonic treatment for 60min, centrifuging for 5min at 5000r/min on a centrifuge, taking 2mL of lower-layer dichloromethane clear liquid, passing through a microporous filter filled with 2g of anhydrous sodium sulfate, and analyzing by using a gas chromatography-tandem mass spectrometer.
Second, leaf demethylation nicotine content analysis
The content of demethylated nicotine in the leaves was analyzed by GC-MS-MS method. Transmission line temperature: 230 ℃, ion source temperature: at 210 ℃; ionization mode: electron impact ionization (EI); bombardment energy: 70 eV; filament current: 50 muA, electron multiplier voltage 1200V; collision gas: argon (the purity is more than or equal to 99.999%); collision cell pressure: 0.3 Pa); solvent delay time: 4 min; and (3) data acquisition mode: and (4) MRM. Retention time (min): 10.48; and (3) quantitative ion pair: 119 > 92; collision energy (eV): 15; and (3) qualitative ion pair: 119 > 65; collision energy (eV): 25; residence time(s): 0.15. in M4 generation plants, the nicotine conversion rate of M271 material is 38% of that of Yunyan 87. The nicotine conversion rate is 100% (demethyl nicotine/demethyl nicotine + nicotine), and the specific results are shown in fig. 2.
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> CYP82E10 gene missense mutant M271 with nicotine conversion rate and application thereof
<130> 2018
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1554
<212> DNA
<213> CYP82E10 Gene
<400> 1
atggtttctc ccgtagaagc catcgtagga ctagtaactc ttacacttct cttctacttc 60
atacggacca aaaaatctca aaaaccttca aaaccattac caccgaaaat ccccggaggg 120
tggccggtaa tcggccatct tttctatttc gatgacgaca gcgacgaccg tccattagca 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcacttttcg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctataaaag actgcttctc tacaaatgat 300
gccattttct ccaatcgtcc agcttttctt tatggcgaat accttggcta caataatgcc 360
atgctatttt tgacaaaata cggaccttac tggcgaaaaa atagaaaatt agtcattcag 420
gaagttctct gtgctagtcg tctcgaaaaa ttgaagcacg tgagatttgg tgaaattcag 480
acgagcatta agaatttata cactcgaatt gatggaaatt cgagtacgat aaatctaacc 540
gattggttag aagaattgaa ttttggtctg atcgtgaaaa tgatcgctgg gaaaaattat 600
gaatccggta aaggagatga acaagtggag agatttagga aagcgtttaa ggattttata 660
attttatcaa tggagtttgt gttatgggat gcttttccaa ttccattgtt caaatgggtg 720
gattttcaag gccatgttaa ggccatgaaa aggacattta aggatataga ttctgttttt 780
cagaattggt tagaggaaca tgtcaagaaa aaagaaaaaa tggaggttaa tgcagaagga 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggctactctc gtgatactgt cataaaagca acagtgttta gtttagtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattat tgataaacaa tcaacatgcc 1020
ttgaagaaag cgcaagaaga gatagataaa aaagttggta aggatagatg ggtagaagag 1080
agtgatatta aggatttggt atacctccaa actattgtta aagaagtgtt acgattatat 1140
ccaccgggac ctttattagt accccatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattacagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gttcgatcca gagagatttt tcgctgctga tattgacttt 1320
cgtggtcaac actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcaa tgcaagtgga acacctaaca atcgcacact tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaag 1500
gtaaatccta tagaagtggt aattacgcct cgcctgacac ctgagcttta ttaa 1554
<210> 2
<211> 1554
<212> DNA
<213> nucleotide sequence of mutant M271
<400> 2
atggtttctc ccgtagaagc catcgtagga ctagtaactc ttacacttct cttctacttc 60
atacggacca aaaaatctca aaaaccttca aaaccattac caccgaaaat ccccggaggg 120
tggccggtaa tcggccatct tttctatttc gatgacgaca gcgacgaccg tccattagca 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcacttttcg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctataaaag actgcttctc tacaaatgat 300
gccattttct ccaatcgtcc agcttttctt tatggcgaat accttggcta caataatgcc 360
atgctatttt tgacaaaata cggactttac tggcgaaaaa atagaaaatt agtcattcag 420
gaagttctct gtgctagtcg tctcgaaaaa ttgaagcacg tgagatttgg tgaaattcag 480
acgagcatta agaatttata cactcgaatt gatggaaatt cgagtacgat aaatctaacc 540
gattggttag aagaattgaa ttttggtctg atcgtgaaaa tgatcgctgg gaaaaattat 600
gaatccggta aaggagatga acaagtggag agatttagga aagcgtttaa ggattttata 660
attttatcaa tggagtttgt gttatgggat gcttttccaa ttccattgtt caaatgggtg 720
gattttcaag gccatgttaa ggccatgaaa aggacattta aggatataga ttctgttttt 780
cagaattggt tagaggaaca tgtcaagaaa aaagaaaaaa tggaggttaa tgcagaagga 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggctactctc gtgatactgt cataaaagca acagtgttta gtttagtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattat tgataaacaa tcaacatgcc 1020
ttgaagaaag cgcaagaaga gatagataaa aaagttggta aggatagatg ggtagaagag 1080
agtgatatta aggatttggt atacctccaa actattgtta aagaagtgtt acgattatat 1140
ccaccgggac ctttattagt accccatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattacagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gttcgatcca gagagatttt tcgctgctga tattgacttt 1320
cgtggtcaac actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcaa tgcaagtgga acacctaaca atcgcacact tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaag 1500
gtaaatccta tagaagtggt aattacgcct cgcctgacac ctgagcttta ttaa 1554
<210> 3
<211> 517
<212> PRT
<213> amino acid sequence encoded by mutant M271
<400> 3
Met Val Ser Pro Val Glu Ala Ile Val Gly Leu Val Thr Leu Thr Leu
1 5 10 15
Leu Phe Tyr Phe Ile Arg Thr Lys Lys Ser Gln Lys Pro Ser Lys Pro
20 25 30
Leu Pro Pro Lys Ile Pro Gly Gly Trp Pro Val Ile Gly His Leu Phe
35 40 45
Tyr Phe Asp Asp Asp Ser Asp Asp Arg Pro Leu Ala Arg Lys Leu Gly
50 55 60
Asp Leu Ala Asp Lys Tyr Gly Pro Val Phe Thr Phe Arg Leu Gly Leu
65 70 75 80
Pro Leu Val Leu Val Val Ser Ser Tyr Glu Ala Ile Lys Asp Cys Phe
85 90 95
Ser Thr Asn Asp Ala Ile Phe Ser Asn Arg Pro Ala Phe Leu Tyr Gly
100 105 110
Glu Tyr Leu Gly Tyr Asn Asn Ala Met Leu Phe Leu Thr Lys Tyr Gly
115 120 125
Leu Tyr Trp Arg Lys Asn Arg Lys Leu Val Ile Gln Glu Val Leu Cys
130 135 140
Ala Ser Arg Leu Glu Lys Leu Lys His Val Arg Phe Gly Glu Ile Gln
145 150 155 160
Thr Ser Ile Lys Asn Leu Tyr Thr Arg Ile Asp Gly Asn Ser Ser Thr
165 170 175
Ile Asn Leu Thr Asp Trp Leu Glu Glu Leu Asn Phe Gly Leu Ile Val
180 185 190
Lys Met Ile Ala Gly Lys Asn Tyr Glu Ser Gly Lys Gly Asp Glu Gln
195 200 205
Val Glu Arg Phe Arg Lys Ala Phe Lys Asp Phe Ile Ile Leu Ser Met
210 215 220
Glu Phe Val Leu Trp Asp Ala Phe Pro Ile Pro Leu Phe Lys Trp Val
225 230 235 240
Asp Phe Gln Gly His Val Lys Ala Met Lys Arg Thr Phe Lys Asp Ile
245 250 255
Asp Ser Val Phe Gln Asn Trp Leu Glu Glu His Val Lys Lys Lys Glu
260 265 270
Lys Met Glu Val Asn Ala Glu Gly Asn Glu Gln Asp Phe Ile Asp Val
275 280 285
Val Leu Ser Lys Met Ser Asn Glu Tyr Leu Asp Glu Gly Tyr Ser Arg
290 295 300
Asp Thr Val Ile Lys Ala Thr Val Phe Ser Leu Val Leu Asp Ala Ala
305 310 315 320
Asp Thr Val Ala Leu His Met Asn Trp Gly Met Ala Leu Leu Ile Asn
325 330 335
Asn Gln His Ala Leu Lys Lys Ala Gln Glu Glu Ile Asp Lys Lys Val
340 345 350
Gly Lys Asp Arg Trp Val Glu Glu Ser Asp Ile Lys Asp Leu Val Tyr
355 360 365
Leu Gln Thr Ile Val Lys Glu Val Leu Arg Leu Tyr Pro Pro Gly Pro
370 375 380
Leu Leu Val Pro His Glu Asn Val Glu Asp Cys Val Val Ser Gly Tyr
385 390 395 400
His Ile Pro Lys Gly Thr Arg Leu Phe Ala Asn Val Met Lys Leu Gln
405 410 415
Arg Asp Pro Lys Leu Trp Ser Asn Pro Asp Lys Phe Asp Pro Glu Arg
420 425 430
Phe Phe Ala Ala Asp Ile Asp Phe Arg Gly Gln His Tyr Glu Phe Ile
435 440 445
Pro Phe Gly Ser Gly Arg Arg Ser Cys Pro Gly Met Thr Tyr Ala Met
450 455 460
Gln Val Glu His Leu Thr Ile Ala His Leu Ile Gln Gly Phe Asn Tyr
465 470 475 480
Lys Thr Pro Asn Asp Glu Pro Leu Asp Met Lys Glu Gly Ala Gly Leu
485 490 495
Thr Ile Arg Lys Val Asn Pro Ile Glu Val Val Ile Thr Pro Arg Leu
500 505 510
Thr Pro Glu Leu Tyr
515
<210> 4
<211> 25
<212> DNA
<213> CYP82E10_Tilling_F
<400> 4
gtcaaatacc acctcttaat agtaa 25
<210> 5
<211> 25
<212> DNA
<213> CYP82E10_Tilling_R
<400> 5
aaaagtccct attggtagga agtgc 25

Claims (2)

1. A method for reducing nicotine conversionCYP82E10The missense mutant M271 is characterized in that relative to the nucleotide sequence shown as SEQ ID NO.1CYP82E10Genes of reducing nicotine conversionCYP82E10Missense mutant M271 containsCYP82E10The C at position 385 in the gene sequence is replaced by T, so that point mutation is generated, and the nicotine conversion rate is reducedCYP82E10The nucleotide sequence of the missense mutant M271 is shown as SEQ ID NO.2, and the amino acid sequence thereof is shown as SEQ ID NO. 3.
2. A method of reducing nicotine conversion according to claim 1CYP82E10The gene missense mutant M271 is applied to obtaining tobacco plants with low nicotine conversion rate, and the variety of the tobacco plants is Yunyan 87.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
CN102858983A (en) * 2010-01-15 2013-01-02 北卡罗来纳州立大学 Compositions And Methods For Minimizing Nornicotine Synthesis In Tobacco
CN106636146A (en) * 2017-02-16 2017-05-10 云南省烟草农业科学研究院 CYP82E5 gene mutant for reducing nicotine conversion rate and application thereof
CN106998787A (en) * 2014-09-26 2017-08-01 菲利普莫里斯生产公司 Tobacco-specific nitrosamines are reduced by changing nitrate assimilation path
WO2018067985A1 (en) * 2016-10-07 2018-04-12 Altria Client Services Llc Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (tsnas)

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CN102858983A (en) * 2010-01-15 2013-01-02 北卡罗来纳州立大学 Compositions And Methods For Minimizing Nornicotine Synthesis In Tobacco
CN106998787A (en) * 2014-09-26 2017-08-01 菲利普莫里斯生产公司 Tobacco-specific nitrosamines are reduced by changing nitrate assimilation path
WO2018067985A1 (en) * 2016-10-07 2018-04-12 Altria Client Services Llc Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (tsnas)
CN106636146A (en) * 2017-02-16 2017-05-10 云南省烟草农业科学研究院 CYP82E5 gene mutant for reducing nicotine conversion rate and application thereof

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Title
Development of CAPS and dCAPS markers for CYP82E4,CYP82E5v2 and CYP82E10 gene mutants reducing nicotine to nornicotine conversion in tobacco;Li等;《Mol Breeding》;20110411;第29卷;第589-599页 *
烟草尼古丁去甲基化酶基因及其在育种中的应用;宋中邦等;《广西植物》;20121130;第32卷(第6期);第849-853页 *

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