CN106636146A - CYP82E5 gene mutant for reducing nicotine conversion rate and application thereof - Google Patents

CYP82E5 gene mutant for reducing nicotine conversion rate and application thereof Download PDF

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CN106636146A
CN106636146A CN201710082843.4A CN201710082843A CN106636146A CN 106636146 A CN106636146 A CN 106636146A CN 201710082843 A CN201710082843 A CN 201710082843A CN 106636146 A CN106636146 A CN 106636146A
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cyp82e5
leu
gene
nicotine conversion
conversion ratio
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CN106636146B (en
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宋中邦
王丙武
李文正
高玉龙
李永平
孔光辉
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Yunnan Academy of Tobacco Agricultural Sciences
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Yunnan Academy of Tobacco Agricultural Sciences
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    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc

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Abstract

The invention discloses a CYP82E5 gene mutant for reducing nicotine conversion rate and an application thereof. Compared with a CYP82E5 gene whose nucleotide sequence is SEQ ID No.1, the CYP82E5 gene mutant for reducing nicotine conversion rate contains a point mutation of G which is at the 392th position in the CYP82E5 gene sequence and is substituted by A, and the CYP82E5 gene mutant for reducing nicotine conversion rate has a nucleotide sequence as shown in SEQ ID No.3. The CYP82E5 gene mutant for reducing nicotine conversion rate can be applied to tobacco plants with reduced nicotine conversion rate. A Yunyan 87 material obtained by the CYP82E5 gene mutant for reducing nicotine conversion rate, leaf nicotine conversion rate is reduced by about 20%, and the conversion rate of nicotine is substantially reduced.

Description

A kind of CYP82E5 gene mutation bodies for reducing nicotine conversion ratio and its application
Technical field
The invention belongs to genetic engineering technology field, and in particular to a kind of CYP82E5 genes of reduction nicotine conversion ratio Mutant and its application.
Background technology
Nicotine（nicotine）, nornicotine（nornicotine）, anabasine（anabasine）And new cigarette Alkali（anatabine）It is tobacco（Nicotiana tobacum）In main alkaloid, wherein nicotine is primary biology Alkali, accounts for the 90-95% of biological total alkali content, and nornicotine content is usually less than the 3.5% of TA, is that nicotine passes through Demethylation is converted into.Nornicotine can produce harm to health, and it is cigarette cigarette to be mainly manifested in it Potential carcinogen matter nitroso nornicotine in gas（nitrosonornicotine, NNN）Synthesis precursor, may lead Cause the cancer of the esophagus, the generation of carcinoma of mouth.Nornicotine also can directly induce the Aberrant glycosylation of albumen in smoker's blood plasma, There are some researches show that it can occur covalent reaction with conventional steroid drugs, affect drug effect and toxicity.Therefore, reduce nicotine to turn Metaplasia reduces nicotine conversion ratio and is significant into nornicotine.
There is two kinds of tobacco extensively to apply aborning in the world, burley tobaccos and flue-cured tobacco, wherein burley tobaccos production master Will be in western developed country and South America, flue-cured tobacco is mainly planted by China.The CYP82E4 of burley tobaccos CYP82E2 subfamilies, CYP82E5, CYP82E10 gene can encode active nicotine demethyl enzyme, be the key enzyme of nicotine conversion. Gavilano etc. suppresses strong " transformant " CYP82E4 of burley tobaccos and its homogenic expression, demethyl Buddhist nun by RNAi technology Gu Ding synthesis is significantly inhibited, the nicotine conversion ratio minimum only 0.8% of transfer-gen plant, even below " non-turn of burley tobaccos The conversion ratio of the universal about 3-5% of change strain ".Julio et al. screen in 1132 EMS mutagenic mutants obtain 10 plants Nicotine conversion ratio is down to extremely in the tobacco of CYP82E4 locus generation point mutation, wherein nonsense mutation and missense mutation strain Minor levels.Lewis et al. obtains respectively what CYP82E4, CYP82E5, CYP82E10 underwent mutation with the method for EMS mutagenesis Burley tobaccos material, it is found that CYP82E5, CYP82E10 gene mutation does not affect nicotine conversion ratio, three gene simultaneous mutations Nicotine conversion ratio is well below control strain in mutant strain.The studies above shows that CYP82E4 is to determine that nicotine turns in burley tobaccos The key gene of rate, CYP82E5, CYP82E10 gene has no significant effect.Nicotine is shifted to new management mechanisms not in burley tobaccos and flue-cured tobacco Together, the functional study that flue-cured tobacco nicotine demethyl enzyme gene but is there is no at present is delivered, and reduces the method for its nicotine conversion ratio still Need to explore.
The content of the invention
The first object of the present invention is to provide a kind of CYP82E5 gene mutation bodies for reducing nicotine conversion ratio；Second Purpose is the application of the CYP82E5 gene mutation bodies of the reduction nicotine conversion ratio for providing described.
The first object of the present invention is achieved in that and be classified as shown in SEQ ID NO.1 relative to nucleotides sequence CYP82E5 genes, in the CYP82E5 gene orders that the described CYP82E5 gene mutation bodies for reducing nicotine conversion ratio contain The G of 392 is replaced by A and there occurs point mutation, the nucleotides of the CYP82E5 gene mutation bodies of described reduction nicotine conversion ratio Sequence is as shown in SEQ ID NO.3.
The second object of the present invention is achieved in that the CYP82E5 gene mutations of described reduction nicotine conversion ratio Application of the body in the tobacco plant for obtaining nicotine low-conversion.
In the material of cloud and mist 87 that the CYP82E5 gene mutation bodies for reducing nicotine conversion ratio of the present invention are obtained, leaf Piece nicotine conversion ratio declines about 20% than control, significantly reduces the conversion ratio of nicotine.
Description of the drawings
Fig. 1 is CYP82E5 gene mutation capillary electrophoresis detections；
Fig. 2 is the comparison of mutant material sequencing result；
Fig. 3 is the sequencing peak figure of mutant strain 572；
Fig. 4 is the analysis of mutant material nicotine conversion ratio.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but never in any form to the present invention in addition Limit, based on present invention teach that any conversion for being made or replacement, belong to protection scope of the present invention.
The CYP82E5 gene mutation bodies for reducing nicotine conversion ratio of the present invention, relative to nucleotides sequence SEQ is classified as CYP82E5 genes shown in ID NO.1, what the described CYP82E5 gene mutation bodies for reducing nicotine conversion ratio contained The G of 392 there occurs point mutation, the described CYP82E5 for reducing nicotine conversion ratio by A replacements in CYP82E5 gene orders The nucleotide sequence of gene mutation body is as shown in SEQ ID NO.3.
The amino acid sequence such as SEQ ID of the CYP82E5 gene mutation bodies coding of described reduction nicotine conversion ratio Shown in NO.4.
The CYP82E5 gene mutation body applications for reducing nicotine conversion ratio of the present invention are described reduction nicotine Application of the CYP82E5 gene mutation bodies of conversion ratio in the tobacco plant for obtaining nicotine low-conversion.
Below to be embodied as case, the present invention will be further described：
Embodiment 1
Flue-cured tobacco mutant library is formulated
First, tobacco seed EMS process
Flue-cured tobacco（Kind：Cloud and mist 87）After seed is soaked 12 minutes with 50% commercial goods bleaching liquid, deionized water is quickly clear Wash, and soak 12 hours in deionized water.Ionized water is discarded, the EMS of equal-volume 0.5% is added（Ethylmethane sulfonate）Process 12 Hour.Treatment fluid is abandoned, adds deionized water to clean 6-8 time, every time about 1 minute.Seed collection is drained stand-by in Buchner funnel.
2nd, M1 plant field planting
The EMS seeds that are disposed are seeded in floating disc, per one, cave seed, transplant after emerging to field, normal agronomic measures pipe Reason.Individual plant bagging numbering sowing obtains M2 seeds after buddingging.
3rd, mutant gene group DNA is extracted and sample mixing
Genomic DNA is extracted using kit, step is as follows：
Weigh the fresh samples of 0.1g, liquid nitrogen grinding, it is in small, broken bits after, in proceeding to 2.0 ml sample cells, add immediately 600 μ L AP1 buffer solutions and 4 μ L RNaseA store liquid（100 mg/ml）.Water bath processing 10 minutes in 65 DEG C, period reverse EP pipes 2-3 time.Add 190 μ L AP2 buffer solutions, mixing is placed in 5 minutes on ice, and 14000 rpm room temperatures are centrifuged 5 minutes.Aspirate supernatant is to QIAshredder Mini posts, 14000 rpm room temperatures are centrifuged 2 minutes.450-650 μ L filtered fluids are taken into 2.0ml centrifuge tubes, 675-900 is added Buffer A P3/E of μ L.Take in the DNeasy posts of 650 μ L mixtures to 2 ml, room temperature is with >=8000rpm centrifugation 1-2 minutes.Will Above-mentioned DNeasy posts are put into 2 new ml collecting pipes, add 500 μ L AW buffer solutions, are centrifuged 2 minutes with >=8000rpm, Discarding efflux, then with AW buffer solutions repeated washing once.DNeasy posts are placed in 1.5 ml centrifuge tubes, 100 μ L AE are added Buffer solution, room temperature is placed 5 minutes, and centrifugation gained filtrate is genomic DNA.
About 2200 parts of M2 of field planting gather blade and extract genome using said method for EMS mutant plants DNA, by all samples DNA concentration 40 ng/ μ l are diluted to, and DNA libraries of the M2 for the mutant of cloud and mist 87 are finally set up, per 8 parts of DNA Sample mixing, constitutes 8 times of mixing pits, in being stored in 96 orifice plates.
Embodiment 2
CYP82E5 gene end screening mutants
First, Tilling primers
CYP82E5 genes have two extrons, using the mutant of first exon region of Tilling technology screenings.According to The genome sequence of mesh ground gene,
Forward primer is CYP82E5_F:5’-GGTAATTTTGTATTTATTATATTATGCG-3’；
Reverse primer is CYP82E5_R：5’-TCATCCTTAGTATTTAGATAATCTAATT-3’.
2nd, PCR amplification conditions
PCR reaction systems are as follows：Cumulative volume is 10 μ L, wherein the μ L of 20 ng/ μ L DNA samples 1.0,10 × PCR buffer The μ L of 1.0 μ L, dNTPs 0.8, each 0.16 μ L of primer, the μ L of Taq DNA enzymatics 0.1, the μ L of ddH2O 6.78.
PCR response procedures are as follows：95 DEG C of denaturations 3 minutes；94 DEG C of denaturation 30 seconds, 62 DEG C are annealed 30 seconds（Each is followed Ring declines 1 DEG C）, 72 DEG C extend 90 seconds, run 7 circulations；94 DEG C of denaturation 30 seconds, 58 DEG C are annealed 30 seconds, and 72 DEG C extend 90 Second, run 40 circulations；Last 72 DEG C extend 5 minutes.PCR amplified productions can be in 4 DEG C of preservations.
3rd, PCR primer digestion and electrophoresis
The characteristic of heteroduplex is cut using CEL I enzyme spcificitys, digestion is carried out to PCR primer, digestion system is as follows：PCR is produced The μ L of thing 4,10 × buffer 1 μ L, the μ L of CEL I enzymes 0.5, supplement H2O to cumulative volume is 10 μ L.Digestion system utilizes automatic hair Cons electrophoresis system is separated, and separation condition is as follows：Sample loading voltage is 9KV, and the loading of the sec of loading 30, Marker is electric Press as 7.5KV, loading 5sec, prerunning voltage 9KV, the kv of separation electrophoresis voltage 9, the min of run time 80, electrophoresis result use The software analysis of Prosize 2.0.Tilling screenings obtain altogether 9 CYP82E5 gene mutation bodies, wherein the individual plant of numbering 572, has One codon sports TAG from TGG, is the mutation of CYP82E5 gene ends, the results are shown in Table 1 and Fig. 1.
CYP82E5 gene mutation bodies analysis in the mutant library of 1 cloud and mist of table 87
Embodiment 3
The mutation checking of CYP82E5 gene ends
First, M3 is extracted and PCR amplifications for mutant gene group DNA
According to M2 for plant Tilling the selection results, CYP82E5 gene targets region is selected to undergo mutation the seed of individual plant（M3 Generation, numbering 572）, sow in seedlings nursing plate.Seedling leaves genomic DNA is extracted using RNA isolation kit.With CYP82E5_F and CYP82E5_R primers, with genomic DNA as first exon region of template amplification CYP82E5 genes.PCR reaction systems are such as Under：Cumulative volume is 25 μ L, wherein the μ L of 20 ng/ μ L DNA samples 1.0, the μ L of 10 × PCR buffer 2.5, the μ of dNTPs 2 L, each 0.5 μ L of primer, Taq DNA enzymatics 0.3 μ L, ddH2O 18.2 μL.PCR response procedures are as follows：95 DEG C of denaturations 3 Minute；94 DEG C of denaturation 30 seconds, 55 DEG C are annealed 30 seconds（Each circulation declines 1 DEG C）, 72 DEG C extend 90 seconds, and operation 30 is followed Ring；72 DEG C extend 5 minutes.PCR amplified productions can be in 4 DEG C of preservations.
2nd, PCR primer TOPO clone and sequencing
PCR primer connects pTOPO carriers after reclaiming（Invitrogen）, system is as follows：PCR primer 4 μ L, pCR-BluntII- The μ L of TOPO plasmids 1, salt Solution 1 μ L.25 DEG C of incubation 30min of reactive component, turn to be mixed in E.coli. competent cells Even, ice bath 30min is immediately placed in 2min in ice bath after 42 DEG C of heat shocks 90sec, adds the LB fluid nutrient mediums of 0.35mL, and 37 DEG C 210rpm shaken cultivations 1h.Centrifugation 1min（7500rpm）, abandon supernatant and mix to about 100 μ l, it is spread evenly across the training of Km resistances On foster base, 37 DEG C of incubated overnights.Selecting positive colony to extract after plasmid carries out Sanger sequencings, as a result sees Fig. 2 and Fig. 3.
Embodiment 4
CYP82E5 gene end mutant nornicotine content analysis
First, prepared by mutant material sample
Tobacco growing rounds the 60 DEG C of drying of plant tobacco sample to the maturity period, crushes 60 mesh sieves to be measured.Accurately weigh cigarette sample 0.5 G adds the NaOH solution of 5 mL 10% in 50 mL centrifuge tubes, shakes up, and soaks 15 min, adds the extraction of 20 mL containing the internal standards Liquid, ultrasonic 60 min, on centrifuge 5000 r/min be centrifuged 5 min, take 2 mL lower floors dichloromethane clear liquids cross equipped with 2 g without After the millipore filter of aqueous sodium persulfate, analyzed with gas chromatography tandem mass spectrometry instrument.
2nd, blade nornicotine content analysis
Using nornicotine content in GC-MS-MS methods analysis blade.Transmission line temperature：230 DEG C, ion source temperature：210 ℃；Ionization pattern：Electron impact ionization（EI）；Bombarding energy：70 eV；Heater current：50 μ A, electron multiplier voltage 1200 V；Collision gas：Argon gas（Purity >=99.999%）；Collision cell pressure：0.3Pa）；The solvent delay time：4 min；Data acquisition module Formula：MRM.Retention time（min）：10.48；Quota ion pair：119 ﹥ 92；Impact energy (eV)：15；Qualitative ion pair：119 ﹥ 65；Impact energy (eV)： 25；Residence time (s)： 0.15.CYP82E5 gene pures stop mutation body blade nicotine conversion Rate is 1.34, and control cloud and mist 87 is 1.69, and mutant declines about 20%.Nicotine conversion ratio=[nornicotine/demethyl Nicotine+nicotine] * 100%, concrete outcome is shown in Fig. 4.
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>A kind of CYP82E5 gene mutation bodies for reducing nicotine conversion ratio and its application
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1554
<212> DNA
<213>CYP82E5 gene nucleotides
<400> 1
atggtttctc ccgtagaagc cattgtagga ctagtaaccc ttacacttct cttctacttc 60
ctatggccca aaaaatttca aataccttca aaaccattac caccgaaaat tcccggaggg 120
tggccggtaa tcggccatct tttctacttc gatgatgacg gcgacgaccg tccattagct 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcactttccg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctgtaaaag actgcttctc tacaaatgac 300
gccattttct ccaatcgtcc agcttttctt tacggtgaat accttggcta caataatgcc 360
atgctatttt tgacaaaata cggaccttat tggcgaaaaa atagaaaatt agtcattcag 420
gaagttctct ctgctagtcg tctcgaaaaa ttgaagcacg tgagatttgg taaaattcaa 480
acgagcatta agagtttata cactcgaatt gatggaaatt cgagtacgat aaatctaact 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 agagaaaaaa tggaggttaa tgcacaaggg 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggttactctc gtgatactgt cataaaagca acagtgttta gtttggtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattac tgataaacaa tcaacatgcc 1020
ttgaagaaag cacaagaaga gatcgataaa aaagttggta aggaaagatg ggtagaagag 1080
agtgatatta aggatttggt ctacctccaa gctattgtta aagaagtgtt acgattatat 1140
ccaccaggac ctttattagt acctcatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattgcagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gtttgatcca gagagattct tcgctgatga tattgactac 1320
cgtggtcagc actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcat tacaagtgga acacctaaca atagcacatt tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaaa 1500
gtaaatcctg tagaagtgac aattacggct cgcctggcac ctgagcttta ttaa 1554
<210> 2
<211> 517
<212> PRT
<213>The acid of CYP82E5 aminopeptidase genes
<400> 2
Met Val Ser Pro Val Glu Ala Ile Val Gly Leu Val Thr Leu Thr Leu
1 5 10 15
Leu Phe Tyr Phe Leu Trp Pro Lys Lys Phe Gln Ile 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 Gly 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 Val 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
Pro Tyr Trp Arg Lys Asn Arg Lys Leu Val Ile Gln Glu Val Leu Ser
130 135 140
Ala Ser Arg Leu Glu Lys Leu Lys His Val Arg Phe Gly Lys Ile Gln
145 150 155 160
Thr Ser Ile Lys Ser 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 Arg Glu
260 265 270
Lys Met Glu Val Asn Ala Gln 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 Glu Arg Trp Val Glu Glu Ser Asp Ile Lys Asp Leu Val Tyr
355 360 365
Leu Gln Ala 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 Asp Asp Ile Asp Tyr 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 Leu
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 Val Glu Val Thr Ile Thr Ala Arg Leu
500 505 510
Ala Pro Glu Leu Tyr
515
<210> 3
<211> 1554
<212> DNA
<213>CYP82E5 gene mutation body nucleotides
<400> 3
atggtttctc ccgtagaagc cattgtagga ctagtaaccc ttacacttct cttctacttc 60
ctatggccca aaaaatttca aataccttca aaaccattac caccgaaaat tcccggaggg 120
tggccggtaa tcggccatct tttctacttc gatgatgacg gcgacgaccg tccattagct 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcactttccg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctgtaaaag actgcttctc tacaaatgac 300
gccattttct ccaatcgtcc agcttttctt tacggtgaat accttggcta caataatgcc 360
atgctatttt tgacaaaata cggaccttat tagcgaaaaa atagaaaatt agtcattcag 420
gaagttctct ctgctagtcg tctcgaaaaa ttgaagcacg tgagatttgg taaaattcaa 480
acgagcatta agagtttata cactcgaatt gatggaaatt cgagtacgat aaatctaact 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 agagaaaaaa tggaggttaa tgcacaaggg 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggttactctc gtgatactgt cataaaagca acagtgttta gtttggtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattac tgataaacaa tcaacatgcc 1020
ttgaagaaag cacaagaaga gatcgataaa aaagttggta aggaaagatg ggtagaagag 1080
agtgatatta aggatttggt ctacctccaa gctattgtta aagaagtgtt acgattatat 1140
ccaccaggac ctttattagt acctcatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattgcagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gtttgatcca gagagattct tcgctgatga tattgactac 1320
cgtggtcagc actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcat tacaagtgga acacctaaca atagcacatt tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaaa 1500
gtaaatcctg tagaagtgac aattacggct cgcctggcac ctgagcttta ttaa 1554
<210> 4
<211> 130
<212> PRT
<213>CYP82E5 gene mutation body amino acid
<400> 4
Met Val Ser Pro Val Glu Ala Ile Val Gly Leu Val Thr Leu Thr Leu
1 5 10 15
Leu Phe Tyr Phe Leu Trp Pro Lys Lys Phe Gln Ile 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 Gly 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 Val 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
Pro Tyr
130
<210> 5
<211> 28
<212> DNA
<213> CYP82E5_F
<400> 5
ggtaattttg tatttattat attatgcg 28
<210> 6
<211> 28
<212> DNA
<213> CYP82E5_R
<400> 6
tcatccttag tatttagata atctaatt 28

Claims (3)

1. it is a kind of reduce nicotine conversion ratio CYP82E5 gene mutation bodies, it is characterised in that be relative to nucleotide sequence CYP82E5 genes shown in SEQ ID NO.1, the CYP82E5 gene mutation bodies of described reduction nicotine conversion ratio contain CYP82E5 gene orders in the G of 392 replaced by A and there occurs point mutation, described reduction nicotine conversion ratio The nucleotide sequence of CYP82E5 gene mutation bodies is as shown in SEQ ID NO.3.
2. it is according to claim 1 reduce nicotine conversion ratio CYP82E5 gene mutation bodies, it is characterised in that it is described The amino acid sequence of the CYP82E5 gene mutation bodies coding of reduction nicotine conversion ratio is as shown in SEQ ID NO.4.
3. a kind of CYP82E5 gene mutation body applications of the reduction nicotine conversion ratio described in claim 1 or 2, its feature exists In the described CYP82E5 gene mutation bodies for reducing nicotine conversion ratio in the tobacco plant for obtaining nicotine low-conversion Using.
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