CN106636146B - It is a kind of reduce nicotine conversion ratio CYP82E5 gene mutation body and its application - Google Patents
It is a kind of reduce nicotine conversion ratio CYP82E5 gene mutation body and its application Download PDFInfo
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- CN106636146B CN106636146B CN201710082843.4A CN201710082843A CN106636146B CN 106636146 B CN106636146 B CN 106636146B CN 201710082843 A CN201710082843 A CN 201710082843A CN 106636146 B CN106636146 B CN 106636146B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- 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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- C—CHEMISTRY; METALLURGY
- 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
Abstract
The invention discloses a kind of CYP82E5 gene mutation body for reducing nicotine conversion ratio and its applications.The CYP82E5 gene mutation body of the reduction nicotine conversion ratio, CYP82E5 gene shown in SEQ ID NO.1 is classified as relative to nucleotides sequence, 392 G are replaced by A in the CYP82E5 gene order that the CYP82E5 gene mutation body of the reduction nicotine conversion ratio contains has occurred point mutation, and the nucleotide sequence of the CYP82E5 gene mutation body of the reduction nicotine conversion ratio is as shown in SEQ ID NO.3.Using application of the CYP82E5 gene mutation body for the reduction nicotine conversion ratio in the tobacco plant for obtaining nicotine low-conversion.In 87 material of cloud and mist that the CYP82E5 gene mutation body of the present invention for reducing nicotine conversion ratio obtains, blade nicotine conversion ratio significantly reduces the conversion ratio of nicotine than control decline about 20%.
Description
Technical field
The invention belongs to genetic engineering technology fields, and in particular to a kind of CYP82E5 gene for reducing nicotine conversion ratio
Mutant and its application.
Background technique
Nicotine (nicotine), nornicotine (nornicotine), anabasine (anabasine), He Xinyan
Alkali (anatabine) be 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 total alkaloid, is that nicotine passes through
Demethylation reaction is converted into.Nornicotine can generate harm to human health, and being mainly manifested in it is cigarette cigarette
The synthesis precursor of potential carcinogen matter nitroso nornicotine (nitrosonornicotine, NNN), may lead in gas
Cause the generation of the cancer of the esophagus, carcinoma of mouth.Nornicotine also can directly induce the Aberrant glycosylation of albumen in smoker's blood plasma,
Some researches show that it covalent reaction occurs with common steroid drugs, influences drug effect and toxicity.Therefore, nicotine is reduced to turn
Metaplasia is of great significance at nornicotine, i.e. reduction nicotine conversion ratio.
There are two types of the tobaccos of type to be widely applied in production in the world, burley tobaccos and flue-cured tobacco, wherein burley tobaccos production master
Flue-cured tobacco is mainly planted in western developed country and South America, China.Burley tobaccosCYP82E2SubfamilyCYP82E4、CYP82E5、CYP82E10Gene can encode active nicotine demethyl enzyme, be the key enzyme of nicotine conversion.
Gavilano etc. inhibits burley tobaccos strong " transformant " by RNAi technologyCYP82E4And its expression of homologous gene, demethyl Buddhist nun
Gu Ding synthesis is significantly inhibited, the nicotine conversion ratio of transgenic plant minimum only 0.8%, even lower than " non-turn of burley tobaccos
The conversion ratio of the universal about 3-5% of change strain ".Julio et al. screened in 1132 EMS mutagenic mutants obtain 10 plantsCYP82E4The tobacco of point mutation occurs for locus, and wherein nicotine conversion ratio is down to extremely in nonsense mutation and missense mutation strain
Minor levels.Lewis et al. is obtained respectively with the method for EMS mutagenesisCYP82E4、CYP82E5、CYP82E10It mutates
Burley tobaccos material, discoveryCYP82E5、CYP82E10Gene mutation does not influence nicotine conversion ratio, three gene simultaneous mutations
Nicotine conversion ratio is well below control strain in mutant strain.The studies above shows in burley tobaccosCYP82E4It is to determine that nicotine turns
The key gene of rate,CYP82E5、CYP82E10Gene has no significant effect.Nicotine is shifted to new management mechanisms not in burley tobaccos and flue-cured tobacco
Together, but at present it there is no the functional study of flue-cured tobacco nicotine demethyl enzyme gene to deliver, reduce the method for its nicotine conversion ratio still
It needs to explore.
Summary of the invention
The first object of the present invention is to provide a kind of CYP82E5 gene mutation body for reducing nicotine conversion ratio；Second
It is designed to provide the application of the CYP82E5 gene mutation body of the reduction nicotine conversion ratio.
The first object of the present invention is achieved in that be classified as shown in SEQ ID NO.1 relative to nucleotides sequence
CYP82E5 gene, in the CYP82E5 gene order that the CYP82E5 gene mutation body of the reduction nicotine conversion ratio contains
392 G are replaced by A has occurred point mutation, the nucleotide of the CYP82E5 gene mutation body of the 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 mutation of the reduction nicotine conversion ratio
Application of the body in the tobacco plant for obtaining nicotine low-conversion.
In 87 material of cloud and mist that the CYP82E5 gene mutation body of the present invention for reducing nicotine conversion ratio obtains, leaf
Piece nicotine conversion ratio significantly reduces the conversion ratio of nicotine than control decline about 20%.
Detailed description of the invention
Fig. 1 isCYP82E5Gene mutation capillary electrophoresis detection；
Fig. 2 is the comparison of mutant material sequencing result；
The sequencing peak figure that Fig. 3 is mutant strain 572；
Fig. 4 is the analysis of mutant material nicotine conversion ratio.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
The CYP82E5 gene mutation body of the present invention for reducing nicotine conversion ratio, is classified as SEQ relative to nucleotides sequence
CYP82E5 gene shown in ID NO.1, what the CYP82E5 gene mutation body of the reduction nicotine conversion ratio contained
392 G are replaced by A in CYP82E5 gene order has occurred point mutation, the CYP82E5 of the reduction nicotine conversion ratio
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 body coding of the reduction nicotine conversion ratio
Shown in NO.4.
It is the reduction nicotine that the CYP82E5 gene mutation body of the present invention for reducing nicotine conversion ratio, which is applied,
Application of the CYP82E5 gene mutation body of conversion ratio in the tobacco plant for obtaining nicotine low-conversion.
Case is embodied, the present invention will be further described below:
Embodiment 1
The initiative of flue-cured tobacco mutant library
One, tobacco seed EMS is handled
It is quick with deionized water after flue-cured tobacco (kind: cloud and mist 87) seed is impregnated 12 minutes with 50% commercial goods bleaching liquid
Cleaning, and impregnate 12 hours in deionized water.Ionized water is discarded, isometric 0.5% EMS(ethylmethane sulfonate is added) processing
12 hours.Treatment fluid is abandoned, deionized water is added and cleans 6-8 times, every time about 1 minute.Seed collection in Buchner funnel, drain to
With.
Two, M1 plant field planting
The EMS seed that is disposed is seeded in floating disc, and one, every cave seed, to field, normal agronomy is arranged for transplanting after emergence
Apply management.Single plant bagging number sowing obtains M2 seed after buddingging.
Three, mutant gene group DNA extraction and sample mixing
Genomic DNA is extracted using kit, steps are as follows:
Weigh the fresh sample of 0.1g, liquid nitrogen grinding, it is fine crushing after, be transferred in 2.0 ml sample cells, 600 μ L AP1 buffering be added immediately
Liquid and 4 μ L RNaseA storage liquid (100 mg/ml).During which water bath processing 10 minutes in 65 DEG C overturn EP pipe 2-3 times.It is added
190 μ L AP2 buffers, mixing are placed in 5 minutes on ice, and 14000 rpm room temperatures are centrifuged 5 minutes.Aspirate supernatant is extremely
QIAshredder Mini column, 14000 rpm room temperatures are centrifuged 2 minutes.Take 450-650 μ L filtered fluids into 2.0ml centrifuge tube,
Add the buffer solution A P3/E of 675-900 μ L.In the DNeasy column for taking 650 μ L mixtures to 2 ml, room temperature is with >=8000rpm
Centrifugation 1-2 minutes.Above-mentioned DNeasy column is put into 2 new ml collecting pipes, 500 μ L AW buffers are added, with >=
8000rpm is centrifuged 2 minutes, abandons efflux, then primary with AW buffer repeated washing.DNeasy column is placed on 1.5 ml centrifugation
Guan Zhong is added 100 μ L AE buffers, is placed at room temperature for 5 minutes, and centrifugation gained filtrate is genomic DNA.
About 2200 parts of M2 of field planting acquire blade and extract genome using the above method for EMS mutant plants
All samples DNA concentration is diluted to 40 ng/ μ l by DNA, finally establishes M2 for the DNA library of 87 mutant of cloud and mist, every 8 parts of DNA
Sample mixing constitutes 8 times of mixing pits, is stored in 96 orifice plates.
Embodiment 2
The screening of CYP82E5 gene end mutant
One, Tilling primer
There are two exons for CYP82E5 gene, utilize the mutant of first exon region of Tilling technology screening.
According to mesh the genome sequence of gene,
Forward primer is CYP82E5_F:5 '-GGTAATTTTGTATTTATTATATTATGCG-3 '；
Reverse primer is CYP82E5_R:5 '-TCATCCTTAGTATTTAGATAATCTAATT-3 '.
Two, PCR amplification condition
PCR reaction system is as follows: total volume is 10 μ L, wherein 20 ng/ μ L DNA sample, 1.0 μ L, 10 × PCR
1.0 0.8 μ L of μ L, dNTPs of buffer, 0.1 6.78 μ L of μ L, ddH2O of each 0.16 μ L, Taq DNA enzymatic of primer.
PCR response procedures are as follows: 95 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, and 62 DEG C of annealing (each follow for 30 seconds
Ring declines 1 DEG C), 72 DEG C extend 90 seconds, run 7 circulations；94 DEG C are denaturalized 30 seconds, and 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 production can be saved at 4 DEG C.
Three, PCR product digestion and electrophoresis
Using the characteristic of CEL I enzyme spcificity cutting heteroduplex, digestion is carried out to PCR product, digestion system is as follows:
41 μ L, CEL I enzyme of μ L, 10 × buffer of PCR product, 0.5 μ L supplements H2O to total volume be 10 μ L.Digestion system utilizes
Automatic capillary electrophoresis system is separated, and separation condition is as follows: sample loading voltage is 9KV, 30 sec of loading, Marker's
Loading voltage is 7.5KV, loading 5sec, prerunning voltage 9KV, 9 kv of separation electrophoresis voltage, 80 min of runing time, electrophoresis knot
Fruit is analyzed with 2.0 software of Prosize.Tilling screening obtains 9 CYP82E5 gene mutation bodies altogether, wherein number 572 is single
Strain, has a codon to sport TAG from TGG, is mutated for CYP82E5 gene end, the results are shown in Table 1 and Fig. 1.
In 1 cloud and mist of table, 87 mutant libraryCYP82E5Gene mutation body analysis
Embodiment 3
The mutation verifying of CYP82E5 gene end
One, M3 is for mutant gene group DNA extraction and PCR amplification
According to M2 for plant Tilling the selection result, the seed of CYP82E5 gene target region mutation single plant is selected
(M3 generation, number 572), is sowed in seedlings nursing plate.Seedling leaves genomic DNA is extracted using RNA isolation kit.With CYP82E5_F and
CYP82E5_R primer, using genomic DNA as first exon region of template amplification CYP82E5 gene.PCR reaction system is such as
Under: total volume is 25 μ L, wherein 20 ng/ μ L DNA sample, 1.0 μ L, 10 × PCR buffer, 2.5 2 μ of μ L, dNTPs
L, primer each 0.5 μ L, Taq DNA enzymatic 0.3 μ L, ddH2O 18.2 μL.PCR response procedures are as follows: 95 DEG C of initial denaturations 3
Minute；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds (each circulation declines 1 DEG C), and 72 DEG C extend 90 seconds, and operation 30 follows
Ring；72 DEG C extend 5 minutes.PCR amplified production can be saved at 4 DEG C.
Two, PCR product TOPO clone and sequencing
PTOPO carrier (Invitrogen) is connected after PCR product recycling, system is as follows: PCR product 4 μ L, pCR-
1 μ L, salt Solution of BluntII-TOPO plasmid, 1 μ L.25 DEG C of incubation 30min of reactive component, it is thin to turn E.coli. competence
It is mixed in born of the same parents, is immediately placed in 2min in ice bath after ice bath 30min, 42 DEG C of heat shock 90sec, the LB Liquid Culture of 0.35mL is added
Base, 37 DEG C of 210rpm shaken cultivation 1h.It is centrifuged 1min(7500rpm), it abandons supernatant and is mixed to about 100 μ l, it is anti-to be spread evenly across Km
On property culture medium, 37 DEG C are incubated overnight.Sanger sequencing is carried out after selecting positive colony to extract plasmid, as a result sees Fig. 2 and Fig. 3.
Embodiment 4
CYP82E5 gene end mutant nornicotine content analysis
One, prepared by mutant material sample
Tobacco growing is rounded 60 DEG C of strain tobacco sample drying, it is to be measured to crushed 60 meshes to the maturity period.Accurately weigh cigarette
0.5 g of sample is added the NaOH solution of 5 mL 10%, shakes up in 50 mL centrifuge tubes, impregnates 15 min, and 20 mL containing the internal standards are added
Extract liquor, 60 min of ultrasound, on centrifuge 5000 r/min be centrifuged 5 min, take 2 mL lower layer methylene chloride clear liquids to cross dress
After the millipore filter for there are 2 g anhydrous sodium sulfates, analyzed with gas chromatography tandem mass spectrometry instrument.
Two, blade nornicotine content analysis
Using nornicotine content in GC-MS-MS method analysis blade.Transmission line temperature: 230 DEG C, ion source temperature:
210 ℃；Ionization mode: 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 mode: 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 pure stops mutation body blade nicotine
Conversion ratio is 1.34, and control cloud and mist 87 is 1.69, mutant decline about 20%.Nicotine conversion ratio=[nornicotine/it goes
Methyl 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 body 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 nucleotide
<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
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<213>CYP82E5 aminopeptidase gene acid
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Met Val Ser Pro Val Glu Ala Ile Val Gly Leu Val Thr Leu Thr Leu
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Leu Phe Tyr Phe Leu Trp Pro Lys Lys Phe Gln Ile Pro Ser Lys Pro
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Leu Pro Pro Lys Ile Pro Gly Gly Trp Pro Val Ile Gly His Leu Phe
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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 nucleotide
<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. a kind of CYP82E5 gene mutation body for reducing nicotine conversion ratio, it is characterised in that the reduction nicotine conversion
The nucleotide sequence of the CYP82E5 gene mutation body of rate is classified as SEQ ID as shown in SEQ ID NO.3, relative to nucleotides sequence
CYP82E5 gene shown in NO.1, the CYP82E5 base that the CYP82E5 gene mutation body for reducing nicotine conversion ratio contains
Point mutation has occurred because 392 in sequence G are replaced by A.
2. the CYP82E5 gene mutation body according to claim 1 for reducing nicotine conversion ratio, it is characterised in that described
The amino acid sequence of CYP82E5 gene mutation body coding of nicotine conversion ratio is reduced as shown in SEQ ID NO.4.
3. a kind of CYP82E5 gene mutation body application of any of claims 1 or 2 for reducing nicotine conversion ratio, feature exist
In the CYP82E5 gene mutation body for reducing nicotine conversion ratio in the tobacco plant for obtaining nicotine low-conversion
Using.
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| CN108424922B (en) * | 2018-04-20 | 2021-04-16 | 云南省烟草农业科学研究院 | CYP82E10 gene missense mutant M271 capable of reducing nicotine conversion rate and application thereof |
| CN115404250A (en) * | 2021-05-29 | 2022-11-29 | 重庆博腾制药科技股份有限公司 | Method for preparing (S) -nicotine by reduction mode |
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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 |
| CN105517430A (en) * | 2013-01-11 | 2016-04-20 | 北卡罗莱纳州立大学 | Tobacco inbred plants K326 SRC, CMS K326 SRC, K346 SRC, CMS K346 SRC, NC1562-1 SRC, NCTG-61 SRC, CMS NCTG-61 SRC AND HYBRID NC196 SRC |
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| EP1851317B1 (en) * | 2005-02-23 | 2011-10-26 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes |
| US8319011B2 (en) * | 2006-12-15 | 2012-11-27 | U.S. Smokeless Tobacco Company Llc | Tobacco plants having reduced nicotine demethylase activity |
| CN106459923B (en) * | 2014-05-08 | 2020-08-11 | 菲利普莫里斯产品有限公司 | Reduction of nicotine conversion to nornicotine in plants |
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| CN101939431A (en) * | 2007-11-12 | 2011-01-05 | 北卡罗来纳州立大学 | Alteration of tobacco alkaloid content through modification of specific cytochrome P450 genes |
| 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 |
| CN105517430A (en) * | 2013-01-11 | 2016-04-20 | 北卡罗莱纳州立大学 | Tobacco inbred plants K326 SRC, CMS K326 SRC, K346 SRC, CMS K346 SRC, NC1562-1 SRC, NCTG-61 SRC, CMS NCTG-61 SRC AND HYBRID NC196 SRC |
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