CN107058335A - A kind of gene mutation bodies of NteIF4E 1 of generation PVY resistances and its application - Google Patents
A kind of gene mutation bodies of NteIF4E 1 of generation PVY resistances and its application Download PDFInfo
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- CN107058335A CN107058335A CN201710082839.8A CN201710082839A CN107058335A CN 107058335 A CN107058335 A CN 107058335A CN 201710082839 A CN201710082839 A CN 201710082839A CN 107058335 A CN107058335 A CN 107058335A
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- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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Abstract
The invention discloses a kind of generation PVY resistancesNteIF4E‑ 1Gene mutation body and its application.Described generation PVY resistancesNteIF4E‑1Gene mutation body, is classified as shown in SEQ ID NO.1 relative to nucleotides sequenceNteIF4E‑1Gene, described generation PVY resistancesNteIF4E‑1What gene mutation body containedNteIF4E‑1The G of 159 is replaced by A in gene order there occurs point mutation, described generation PVY resistancesNteIF4E‑1The nucleotide sequence of gene mutation body is as shown in SEQ ID NO.3.Using for described generation PVY resistancesNteIF4E‑1Gene mutation body obtain with PVY resistances tobacco plant in application.EMS mutant initiative of the present invention through flue-cured tobacco cultivars cloud and mist 87, utilizes Tilling technology screeningsNteIF4E‑1Gene mutation strain, in mutant material the sequence verification of targeted mutagenesis andNteIF4E‑1Gene pure stops mutation the PVY Analysis of Resistance of body material.Obtained using this methodNteIF4E‑1The flue-cured tobacco material that gene stops mutation can resist PVY viruses.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of generation PVY resistancesNteIF4E-1Gene mutation body and
It is applied.
Background technology
Marmor upsilon (Potato virus Y, PVY) be ten big most economic and scientific value plant viruses it
One.PVY viruses can infect the solanaceous crops such as potato, tomato, tobacco in world's distribution in extensive range.PVY is potato Y
Tobamovirus member, is maximum plant virus category.PVY is one of most important disease of tobacco in world wide, mainly passes through aphid
Worm is propagated, and causes Tobacco mosaic or blade necrosis.The susceptible gene of PVY host plants is mainly translation initiation factor, including
Eukaryotic initiation factor 4E（eIF4E）And 4G（eIF4G）.Vpg（VPg）It can be imitated with eIF4E interactions
The 5 ' of mRNAs-end cap sequence so that viral genome mRNA can be translated in host cell, complete the duplication of virus.
The anti-sources of main PVY are va recessive genes in tobacco, and the mutant resource VAM obtained is induced from UV.Research
Show that the chromosome segment that 1 Mb or so is there are about in VAM is lacked.Pass through conventional breeding means or dihaploid method, va genes
Many different types of tobaccos are transferred to, PVY resistant variety seed selections are widely used in.It has recently been demonstrated that the va in VAM resists
Performance is enough broken down into the recessive gene of two separation, and wherein va1 is responsible for limiting the iuntercellular transfer of PVY viruses, and va2 can press down
System virus accumulation.Although va genes are widely used, it is also adversely affected to tobacco, the blade of disease-resistant variety often compared with
Short, narrower, yield can also decline.This shows there be other important bases in the chromosome segment lacked in VAM in addition to va genes
Cause, causes tobacco phenotypes abnormal.In addition, in the disease-resistant variety of part, blade surface can not produce secretion, influence tobacco is fragrant
Gas quality.In eIF4E genes are found in tobacco to the RIL transcriptome analysis of PVY Resistant segregations being VAM deletion fragments
It is the susceptible gene of tobacco to be responsible for PVY recessive resistance, the i.e. gene.In susceptible burley tobaccos kind BB16NN EMS mutant library
Screening is obtainedeIF4EGene end mutant（W50*）, elisa assay shows that it has PVY resistances.
There are the extensive use in production of two kinds of tobacco, burley tobaccos and flue-cured tobacco, wherein burley tobaccos production master in the world
Flue-cured tobacco is mainly planted in western developed country and South America, China.The susceptible gene studies result hairs of flue-cured tobacco PVY are there is no at present
Table, flue-cured tobaccoeIF4ECan gene mutation cause its generation PVY resistance still to require study.
The content of the invention
The first object of the present invention is to provide a kind of generation PVY resistancesNteIF4E-1Gene mutation body；Second mesh
Be provide described in generation PVY resistancesNteIF4E-1Gene mutation body application.
The first object of the present invention is achieved in that and is classified as relative to nucleotides sequence shown in SEQ ID NO.1NteIF4E-1Gene, described generation PVY resistancesNteIF4E-1What gene mutation body containedNteIF4E-1In gene order
The G of 159 is replaced by A there occurs point mutation, described generation PVY resistancesNteIF4E-1The nucleotides sequence of gene mutation body
Row are as shown in SEQ ID NO.3.
The second purpose of the present invention is achieved in that described generation PVY resistancesNteIF4E-1Gene mutation body
The amino acid sequence of coding is as shown in SEQ ID NO.4.
EMS mutant initiative of the present invention through flue-cured tobacco cultivars cloud and mist 87, utilizes Tilling technology screeningsNteIF4E-1Base
Because of mutant strain, in mutant material the sequence verification of targeted mutagenesis andNteIF4E-1Gene pure stop mutation body material PVY resist
Property analysis.Obtained using this methodNteIF4E-1The flue-cured tobacco material that gene stops mutation can resist PVY viruses.
Brief description of the drawings
Fig. 1 isNteIF4E-1Gene mutation capillary electrophoresis detection；
Fig. 2 isNteIF4E-1Gene mutation position；
Fig. 3 is that peak figure is sequenced in mutant strain；
Fig. 4 is PVY Resistance Identification phenotypes（Whole strain）；
Fig. 5 is PVY Resistance Identification phenotypes（Blade）.
Embodiment
With reference to embodiment accompanying drawing, the present invention is further illustrated, but the present invention is not limited in any way
System, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Generation PVY resistances of the present inventionNteIF4E-1Gene mutation body, SEQ ID are classified as relative to nucleotides sequence
Shown in NO.1NteIF4E-1Gene, described generation PVY resistancesNteIF4E-1What gene mutation body containedNteIF4E-1
The G of 159 is replaced by A in gene order there occurs point mutation, described generation PVY resistancesNteIF4E-1Gene mutation body
Nucleotide sequence as shown in SEQ ID NO.3.
Described generation PVY resistancesNteIF4E-1Gene mutation body coding amino acid sequence such as SEQ ID NO.4
It is shown.
Generation PVY resistances of the present inventionNteIF4E-1Gene mutation body apply be described generation PVY resistances
'sNteIF4E-1Gene mutation body obtain with PV resistances tobacco plant in application.
So that case is embodied, the present invention will be further described below：
Embodiment 1
Flue-cured tobacco mutant library is formulated
First, tobacco seed EMS processing
Flue-cured tobacco（Kind：Cloud and mist 87）It is quickly clear with deionized water after seed is soaked 12 minutes with 50% commercial goods bleaching liquid
Wash, and soak 12 hours in deionized water.Ionized water is discarded, isometric 0.5% EMS is added（Ethylmethane sulfonate）Processing 12
Hour.Treatment fluid is abandoned, deionized water is added and cleans 6-8 times, 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, are transplanted 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, be transferred in 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, during which overturn EP pipes 2-3 times.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.Take 450-650 μ L filtered fluids into 2.0ml centrifuge tubes, add 675-900
μ L buffer A P3/E.In the DNeasy posts for taking 650 μ L mixtures to 2 ml, room temperature is centrifuged 1-2 minutes with >=8000rpm.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,
Efflux is abandoned, 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 the above method for EMS mutant plants
DNA, 40 ng/ μ l are diluted to by all samples DNA concentration, finally set up DNA libraries of the M2 for the mutant of cloud and mist 87, every 8 parts of DNA
Sample mixing, constitutes 8 times of mixing pits, is stored in 96 orifice plates.
Embodiment 2
NteIF4E-1Gene end screening mutant
First, Tilling primers
NteIF4E-1Gene C DS total length 660bp, comprising 5 extrons, first extron is most long, about 251bp.This research
For first exon region screening mutant,
Primer sequence is：
isoform_c_mF_1：tagccactaatattccaccgtc；
isoform_c_mR_1：tacattagtagtgtttgatttggt,
Amplification length is about 590bp or so.
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
1.0 0.8 μ L of μ L, dNTPs, the 6.78 μ L of μ L, ddH2O of each 0.16 μ L, Taq DNA enzymatic of primer 0.1.
PCR response procedures are as follows：95 DEG C of pre-degenerations 3 minutes；94 DEG C are denatured 30 seconds, and 62 DEG C are annealed 30 seconds（Each follow
Ring declines 1 DEG C）, 72 DEG C extend 90 seconds, run 7 circulations；94 DEG C are denatured 30 seconds, and 58 DEG C are annealed 30 seconds, 72 DEG C of extensions 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 41 μ L, CEL I enzymes of μ L, 10 × buffer of thing 0.5, supplement H2O to cumulative volume be 10 μ L.Digestion system utilizes automatic hair
Cons electrophoresis system is separated, and separation condition is as follows：Sample loading voltage is 9KV, loading 30 sec, Marker loading electricity
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 13 altogetherNteIF4E-1Gene mutation body, has 2 mutation to occur
Subregion is included, remaining 11 mutation cause amino acid to change.Wherein numbering 918# individual plants, have a codon prominent from TGG
It is changed into TGA（W53*）, it isNteIF4E-1Gene end is mutated, and the results are shown in Table 1 and Fig. 1, Fig. 2.
In the mutant library of 1 cloud and mist of table 87NteIF4E-1Gene mutation body is analyzed
Embodiment 3
NteIF4E-1Gene end mutation checking
First, M3 is extracted for mutant gene group DNA and PCR is expanded
According to M2 for plant Tilling the selection results, selectionNteIF4E-1Gene target region is undergone mutation the seed of individual plant
（M3 generations）, sowed in seedlings nursing plate.Seedling leaves genomic DNA is extracted using RNA isolation kit.With isoform_c_mF_1 and
Isoform_c_mR_1 primers, using genomic DNA as template amplificationNteIF4E-1First exon region of gene.PCR is anti-
Answer system as follows：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 L of dNTPs 2, each 0.5 μ L, Taq DNA enzymatic 0.3 μ L, ddH of primer2O 18.2 μL.PCR response procedures are as follows： 95℃
Pre-degeneration 3 minutes；94 DEG C are denatured 30 seconds, and 55 DEG C are annealed 30 seconds（Each circulation declines 1 DEG C）, 72 DEG C extend 90 seconds, operation
30 circulations；72 DEG C extend 5 minutes.PCR amplified productions can be in 4 DEG C of preservations.、
2nd, PCR primer TOPO clones 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 1 μ L, salt Solution of TOPO plasmids 1.25 DEG C of incubation 30min of reactive component, turn to mix in E.coli. competent cells
It is even, 2min in ice bath is immediately placed in after ice bath 30min, 42 DEG C of heat shock 90sec, 0.35mL LB fluid nutrient mediums, 37 is added
DEG C 210rpm shaken cultivations 1h.Centrifuge 1min（7500rpm）, abandon supernatant and mixed to about 100 μ l, be spread evenly across the training of Km resistances
Support on base, 37 DEG C of incubated overnights.Select positive colony to extract and Sanger sequencings are carried out after plasmid, as a result（See Fig. 3）Show M3 generations
In mutantNteIF4E-1Gene TGG sports TGA still by stable heredity, and screens homozygosis and stop mutation body（W53*）.
Embodiment 4
NteIF4E-1Gene end mutant PVY Analysis of Resistance
M3 is transplanted to small flower for homozygous mutation strain and is inoculated with PVY virus isolates, and inoculation uses elisa assay plant after 23 days
Resistance.Washing uses automatic board-washing instrument（Bio-Rad Products）Board-washing three times.50 min, 2hr are respectively in enzyme after addition substrate
Mark instrument（Benchmark types, Bio-Rad Products）Upper measure 405nm absorbance（OD values）, sample OD values and feminine gender are right
It is judged as the positive according to ratio >=2.0 of OD values（+）, ratio ＜ 1.5 is judged as feminine gender（-）, 1.5≤ratio ＜ 2.0 is judged as
+-.As a result show, homozygosis stops mutation the noninductive disease symptoms of strain after inoculation 23 days, and ELISA detections are PVY negative, control
The incidence of disease of cloud and mist 87 reaches that 100%, ELISA detections are PVY positive.This shows that the mutation of eif4E_1 gene ends can suppress
PVY viruses are replicated or transported in tobacco, susceptible flue-cured tobacco is obtained anti-PVY phenotypes, be the results are shown in Table 2.
The ELISA of table 2 detects M3 for homozygous mutation material PVY resistances
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>A kind of NteIF4E-1 gene mutation bodies of generation PVY resistances and its application
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 660
<212> DNA
<213>NteIF4E-1 gene nucleotides
<400> 1
atggcagagg aagctgagaa attgcgggta gatgaagtag aagtagtcga cgatggacct 60
gaagaaggag aaattgtgga tgaatctgat gatacggcgt cgtatttggg caaagaaatc 120
aaacctaagc atccattaga gaattcttgg actttttggt ttgataatcc tatggctaaa 180
tctagacaag ctgcttgggg cagttccctt cgcgaacttt acactttttc cactgtcgaa 240
gatttttggg gtgtttacaa taatatcaac cacccaagca agttagttgt gggagcagac 300
tttcattgtt ttaagcataa aattgagcca aagtgggaag atcctgtatg tgcgaatgga 360
gggaattgga caatgagctt tagtaagggt aaatctgata ccagctggct atacacgctg 420
ctggcaatga ttggacatca attcgatcat ggagaggaaa tttgtggagc agtagttagc 480
gtccgaaata agggggataa aatagcttta tggaccaaga atgctgcaaa tgaaacagct 540
caggttagca ttggtaagca atggaaggag tttctggatt acagcaactc gattggcttc 600
atatttcatg acgactcaat gaggctcggc agaggtgcca agaatcgtta tacagtatag 660
<210> 2
<211> 219
<212> PRT
<213>NteIF4E-1 aminopeptidase genes acid
<400> 2
Met Ala Glu Glu Ala Glu Lys Leu Arg Val Asp Glu Val Glu Val Val
1 5 10 15
Asp Asp Gly Pro Glu Glu Gly Glu Ile Val Asp Glu Ser Asp Asp Thr
20 25 30
Ala Ser Tyr Leu Gly Lys Glu Ile Lys Pro Lys His Pro Leu Glu Asn
35 40 45
Ser Trp Thr Phe Trp Phe Asp Asn Pro Met Ala Lys Ser Arg Gln Ala
50 55 60
Ala Trp Gly Ser Ser Leu Arg Glu Leu Tyr Thr Phe Ser Thr Val Glu
65 70 75 80
Asp Phe Trp Gly Val Tyr Asn Asn Ile Asn His Pro Ser Lys Leu Val
85 90 95
Val Gly Ala Asp Phe His Cys Phe Lys His Lys Ile Glu Pro Lys Trp
100 105 110
Glu Asp Pro Val Cys Ala Asn Gly Gly Asn Trp Thr Met Ser Phe Ser
115 120 125
Lys Gly Lys Ser Asp Thr Ser Trp Leu Tyr Thr Leu Leu Ala Met Ile
130 135 140
Gly His Gln Phe Asp His Gly Glu Glu Ile Cys Gly Ala Val Val Ser
145 150 155 160
Val Arg Asn Lys Gly Asp Lys Ile Ala Leu Trp Thr Lys Asn Ala Ala
165 170 175
Asn Glu Thr Ala Gln Val Ser Ile Gly Lys Gln Trp Lys Glu Phe Leu
180 185 190
Asp Tyr Ser Asn Ser Ile Gly Phe Ile Phe His Asp Asp Ser Met Arg
195 200 205
Leu Gly Arg Gly Ala Lys Asn Arg Tyr Thr Val
210 215
<210> 3
<211> 660
<212> DNA
<213>NteIF4E-1 gene mutation body nucleotides
<400> 3
atggcagagg aagctgagaa attgcgggta gatgaagtag aagtagtcga cgatggacct 60
gaagaaggag aaattgtgga tgaatctgat gatacggcgt cgtatttggg caaagaaatc 120
aaacctaagc atccattaga gaattcttgg actttttgat ttgataatcc tatggctaaa 180
tctagacaag ctgcttgggg cagttccctt cgcgaacttt acactttttc cactgtcgaa 240
gatttttggg gtgtttacaa taatatcaac cacccaagca agttagttgt gggagcagac 300
tttcattgtt ttaagcataa aattgagcca aagtgggaag atcctgtatg tgcgaatgga 360
gggaattgga caatgagctt tagtaagggt aaatctgata ccagctggct atacacgctg 420
ctggcaatga ttggacatca attcgatcat ggagaggaaa tttgtggagc agtagttagc 480
gtccgaaata agggggataa aatagcttta tggaccaaga atgctgcaaa tgaaacagct 540
caggttagca ttggtaagca atggaaggag tttctggatt acagcaactc gattggcttc 600
atatttcatg acgactcaat gaggctcggc agaggtgcca agaatcgtta tacagtatag 660
<210> 4
<211> 52
<212> PRT
<213>NteIF4E-1 gene mutation body amino acid
<400> 4
Met Ala Glu Glu Ala Glu Lys Leu Arg Val Asp Glu Val Glu Val Val
1 5 10 15
Asp Asp Gly Pro Glu Glu Gly Glu Ile Val Asp Glu Ser Asp Asp Thr
20 25 30
Ala Ser Tyr Leu Gly Lys Glu Ile Lys Pro Lys His Pro Leu Glu Asn
35 40 45
Ser Trp Thr Phe
50
<210> 5
<211> 22
<212> DNA
<213> isoform_c_mF_1
<400> 5
tagccactaa tattccaccg tc 22
<210> 6
<211> 24
<212> DNA
<213> isoform_c_mR_1
<400> 6
tacattagta gtgtttgatt tggt 24
Claims (3)
1. a kind of generation PVY resistancesNteIF4E-1Gene mutation body, it is characterised in that be classified as SEQ ID relative to nucleotides sequence
Shown in NO.1NteIF4E-1Gene, described generation PVY resistancesNteIF4E-1What gene mutation body containedNteIF4E-1
The G of 159 is replaced by A in gene order there occurs point mutation, described generation PVY resistancesNteIF4E-1Gene mutation body
Nucleotide sequence as shown in SEQ ID NO.3.
2. generation PVY resistances according to claim 1NteIF4E-1Gene mutation body, it is characterised in that described production
Raw PVY resistancesNteIF4E-1Gene mutation body coding amino acid sequence as shown in SEQ ID NO.4.
3. a kind of generation PVY resistances described in claim 1 or 2NteIF4E-1Gene mutation body application, it is characterised in that institute
The generation PVY resistances statedNteIF4E-1Gene mutation body obtain with PVY resistances tobacco plant in application.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371056A (en) * | 2018-09-16 | 2019-02-22 | 云南省烟草农业科学研究院 | A kind of method of breeding to the tobacco plant of capsicum arteries and veins mottle virus resistance |
| CN109371054A (en) * | 2018-09-16 | 2019-02-22 | 云南省烟草农业科学研究院 | A kind of method of breeding to the tobacco plant of marmor upsilon durable resistance |
| CN109371055A (en) * | 2018-09-16 | 2019-02-22 | 云南省烟草农业科学研究院 | A kind of breeding wide spectrum resistant to PVY belongs to the method for viral tobacco plant |
| CN109371056B (en) * | 2018-09-16 | 2022-04-08 | 云南省烟草农业科学研究院 | Method for breeding tobacco plant resistant to pepper vein mottle virus |
| CN109371054B (en) * | 2018-09-16 | 2022-04-08 | 云南省烟草农业科学研究院 | Method for breeding tobacco plant with lasting resistance to potato virus Y |
| CN109371055B (en) * | 2018-09-16 | 2022-06-10 | 云南省烟草农业科学研究院 | Method for breeding broad-spectrum potato virus Y resistant tobacco plant |
| CN111662912A (en) * | 2020-06-01 | 2020-09-15 | 云南省烟草农业科学研究院 | Tobacco NtARF6 gene mutant and molecular identification method and application |
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