CN103820465A - Tobacco recessive PVY (Potato Virus Y) resistance gene eIF4E-1 and application thereof - Google Patents
Tobacco recessive PVY (Potato Virus Y) resistance gene eIF4E-1 and application thereof Download PDFInfo
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Abstract
The invention discloses a tobacco recessive PVY (Potato Virus Y) resistance gene eIF4E-1 and application thereof. The tobacco recessive PVY resistance gene eIF4E-1 comprises the DNA (Deoxyribonucleic Acid) fragment of an eIF4E-1 gene coding region, wherein the DNA fragment is obtained by PCR (Polymerase Chain Reaction) amplification of a specific primer to a primer 4E-1F (SEQIDNO.3) and a primer 4E-1R (SEQIDNO.4); a nucleotide sequence SEQIDNO.1 of a tobacco PVY resistance gene eIF4E-1 coding region and the amino acid sequence SEQIDNO.2 of protein coded by the tobacco PVY resistance gene are obtained first; the eIF4E-1 gene is a recessive disease resistance gene; the eIF4E-1 gene of infected tobacco is lost by a conventional breeding means, so that the non-GMO (Genetically Modified Organism) disease resistance tobacco is obtained; an interference vector plasmid is built by using the eIF4E-1 gene and converted to the infected tobacco; expression of the eIF4E-1 gene of the infected tobacco is inhibited, so that genetically-modified tobacco with obviously improved disease resistance is obtained; the gene has an important application prospect in cultivating the PVY viral disease resistance tobacco.
Description
Technical field
The present invention relates to a kind of Resistance In Tobacco PVY gene eIF4E-1 and application thereof.
Background technology
Marmor upsilon (Potato virus Y, PVY) can infect 34 and belong to 170 various plants, endangers heavier to solanaceous crops (as: tobacco, potato, capsicum, tomato etc.).The disease that PVY infects tobacco is called again tobacco arteries and veins pinta, is a kind of global popular disease, is difficult to implement owing to lacking disease-resistant main breed and comprehensive preventive health measures, and in cigarette district of China, harm is serious.Infecting of PVY can make tobacco production reduce 7%-18%, and the output value reduces 11%-80%.1989, tobacco arteries and veins pinta caused Canadian tobacco direct economic loss to reach 1,000,000 dollars.Within 1993, arteries and veins pinta comes into vogue at China Shandong Yan Qu, becomes first tobacco diseases by 1998.PVY in Henan, the harm in the local cigarette district in Shaanxi, Heilungkiang, Liaoning, Anhui, Guizhou and Yunnan is serious, brings greater loss to leaf tobacco production.
The tobacco bred of the anti-PVY of seed selection is the most economical effective means of this disease of control.The research of the mutation Virgin A Mutant (VAM) of U.S. G.Koellent to cultivar Virgin A finds that the resistance of the PVY of VAM mainly controlled by recessive single-gene (va) afterwards, and multiple strains to PVY, particularly Necrosis Strain cording has resistance.Noguchis S. etc. to the research of VAM genetic locus find afterwards VAM to the resistance of PVY because the chromosome deletion of large section causes.This finds that deriving from X-ray mutant with VAM matches.VAM is also unclear at present to the molecule mechanism of PVY resistance.
The U.S., European Union and day other anti-sources of similar high resistance of this application VAM, select burley tobaccos kind TN86, TN90 and flue-cured tobacco cultivars NC55, the NC102 of anti-PVY.Due to reasons such as the Linkage drag of disease-resistant gene or the ecological suitabilities of kind, above-mentioned flue-cured tobacco cultivars is little at the popularizing planting area of China.Obtain the report that rarely has of anti-PVY tobacco by conventional breeding in China.In capsicum and tomato, find recessive anti-PVY gene.The albumen (eIF4E) of capsicum pvr Recessive alleles (pvr1, pvr2, pvr5) coding can interact with the VPg of PVY, suppresses virus replication and movement.In tomato, the eIF4E albumen (lacking the 2 3rd exon part) after sudden change can provide the resistance of tomato PVY.Tobacco, capsicum and tomato belong to solanaceous crops together, infect identical viral PVY.Have and in bibliographical information tobacco, have equally eIF4E gene.Therefore, infer eIF4E gene in tobacco and to feel PVY relevant, but the anti-PVY gene of recessiveness in tobacco has no bibliographical information.
Summary of the invention
The object of the invention is to lack in separating clone tobacco bred TN86 one has sense PVY gene (recessive anti-PVY gene) the eIF4E-1 coding region (SEQ ID No.1) of biological function.
Another object of the present invention is to provide the protein amino acid sequence (SEQ ID No.2) of above-mentioned anti-PVY coded by said gene.
Another object of the present invention is to provide Auele Specific Primer for the above-mentioned anti-PVY gene that increases to SEQ ID NO.3 and SEQ ID NO.4, it is characterized in that: the nucleotides sequence of SEQ ID NO.3 is classified CGCGGATCCATGGCAGAGGAAGCTGAGAAATT as, the nucleotides sequence of SEQ ID NO.4 is classified GCTAGAGCTCCTATACTGTATAACGATTCTTGG as.
4E-1F：CGCGGATCCATGGCAGAGGAAGCTGAGAAATT（SEQ?ID?No.3）；
4E-1R：GCTAGAGCTCCTATACTGTATAACGATTCTTGG（SEQ?ID?No.4）。
Object of the present invention is achieved through the following technical solutions:
The recessive anti-PVY gene eIF4E-1 of tobacco, is characterized in that, the DNA fragmentation that comprises eIF4E-1 gene coding region, this DNA fragmentation by Auele Specific Primer to primer 4E-1F(SEQ ID No.3) and primer 4E-1R(SEQ ID No.4) obtain by pcr amplification; The disappearance of this DNA fragmentation disappearance or this fragment coding albumen can be given tobacco the susceptibility of the caused disease of marmor upsilon (Potato virus Y) is lost; The nucleotide sequence of this DNA fragmentation is as shown in SEQ ID NO.1, and the aminoacid sequence of its coded protein is as shown in SEQ ID NO.2.
The sequence table of the DNA fragmentation of described eIF4E-1 gene coding region as shown in SEQ ID NO.1, or be substantially equivalent to the DNA sequence dna shown in SEQ ID NO.1, or its function is equivalent to the subfragment shown in SEQ ID NO.1.
Described SEQ ID NO.1 nucleotides sequence is classified as:
ATGGCAGAGGAAGCTGAGAAATTGCGGGTAGATGAAGTAGAAGTAGTCGACGATGGACCTGAAGAAGGAGAAATTGTGGATGAATCTGATGATACGGCGTCGTATTTGGGCAAAGAAATCAAACCTAAGCATCCATTAGAGAATTCTTGGACTTTTTGGTTTGATAATCCTATGGCTAAATCTAGACAAGCTGCTTGGGGCAGTTCCCTTCGCGAACTTTACACTTTTTCCACTGTCGAAGATTTTTGGGGTGTTTACAATAATATCAACCACCCAAGCAAGTTAGTTGTGGGAGCAGACTTTCATTGTTTTAAGCATAAAATTGAGCCAAAGTGGGAAGATCCTGTATGTGCGAATGGAGGGAATTGGACAATGAGCTTTAGTAAGGGTAAATCTGATACCAGCTGGCTATACACGCTGCTGGCAATGATTGGACATCAATTCGATCATGGAGAGGAAATTTGTGGAGCAGTAGTTAGCGTCCGAAATAAGGGGGATAAAATAGCTTTATGGACCAAGAATGCTGCAAATGAAACAGCTCAGGTTAGCATTGGTAAGCAATGGAAGGAGTTTCTGGATTACAGCAACTCGATTGGCTTCATATTTCATGACGACTCAATGAGGCTCGGCAGAGGTGCCAAGAATCGTTATACAGTATAG。
The aminoacid sequence SEQ ID NO.2 of its described coded protein is:
MAEEAEKLRVDEVEVVDDGPEEGEIVDESDDTASYLGKEIKPKHPLENSWTFWFDNPMAKSRQAAWGSSLRELYTFSTVEDFWGVYNNINHPSKLVVGADFHCFKHKIEPKWEDPVCANGGNWTMSFSKGKSDTSWLYTLLAMIGHQFDHGEEICGAVVSVRNKGDKIALWTKNAANETAQVSIGKQWKEFLDYSNSIGFIFHDDSMRLGRGAKNRYTV。
The Auele Specific Primer of described anti-PVY gene is classified CGCGGATCCATGGCAGAGGAAGCTGAGAAATT as to the nucleotides sequence of SEQ ID NO.3, and the nucleotides sequence of SEQ ID NO.4 is classified GCTAGAGCTCCTATACTGTATAACGATTCTTGG as.
In separating clone tobacco bred TN86, lack one has the anti-PVY gene of recessiveness of biological function, crossing the DNA fragmentation of expressing eIF4E-1 gene coding region makes the tobacco bred TN86 of anti-PVY show as susceptible, be indicated as tobacco sense PVY gene, namely the recessive anti-PVY gene of tobacco.
The application of the recessive anti-PVY gene eIF4E-1 of tobacco, first obtain the nucleotides sequence SEQ ID NO.1 of Resistance In Tobacco PVY gene eIF4E-1 coding region, and the aminoacid sequence SEQ ID NO.2 of the protein of coding, described eIF4E-1 gene is recessive disease-resistant gene, by conventional breeding means, susceptible tobacco eIF4E-1 gene is lost eIF4E-1 gene, obtain the disease-resistant tobacco of non-transgenic, utilize the gene constructed interference vector plasmid of eIF4E-1, and be transformed into susceptible tobacco, suppress the expression of susceptible tobacco eIF4E-1 gene, obtain the transgene tobacco that disease resistance obviously strengthens.
The present invention relates to separate and apply the DNA fragmentation of a kind of eIF4E-1 of comprising gene coding region, this fragment is by primer 4E-1F(SEQ ID No.3) and primer 4E-1R(SEQ ID No.4) obtain by pcr amplification.The afunction of this fragment deletion or this fragment coding albumen can be given tobacco the susceptibility of the caused disease of potyvirus (Potyviruses) is lost.Wherein, described fragment, as shown in sequence table SEQ ID NO.1, or is equivalent to the DNA sequence dna shown in SEQ ID NO.1 substantially, or its function is equivalent to the subfragment shown in SEQ ID NO.1.A kind of eukaryotic translation initiation factor (eIF4E) albumen of this DNA sequence encoding, its aminoacid sequence is as shown in SEQ ID NO.2.
According to eIF4E-1 coding sequence information provided by the invention (SEQ ID NO.1), those skilled in the art can easily obtain the gene being equal to eIF4E-1 by the following method: (1) obtains by genomic data library searching; (2) take eIF4E-1 gene coding region fragment as probe, screening tobacco gene group library or cDNA library obtain; (3), according to eIF4E-1 coding sequence information design Oligonucleolide primers, from genome, mRNA and the cDNA of tobacco, obtain by pcr amplification method; (4) on the basis of eIF4E-1 coding sequence, obtain with gene engineering method transformation; (5) obtain this gene by the method for chemosynthesis.
The present invention has following beneficial effect:
Tobacco PVY recessive resistance genes eIF4E-1 provided by the invention has important using value.By conventional breeding means, susceptible tobacco eIF4E-1 gene is lost, can be obtained the disease-resistant tobacco of non-transgenic.Utilize the gene constructed interference vector plasmid of eif4e-1, and be transformed into susceptible tobacco, can obtain the transgene tobacco that disease resistance obviously strengthens.Described eIF4E-1 coding sequence, by sudden change, can be obtained to the disease-resistant tobacco material of gene described in inactivation, thereby is applied to production.The present invention has major application prospect to cultivating the tobacco bred of anti-PVY.
Accompanying drawing explanation
Fig. 1 is the coding region agarose gel electrophoresis figure that adopts primer 4E-1F and the 4E-1R eIF4E-1 that pcr amplification goes out from Zhongyan-100, wherein: M:DNA Marker (2000bp, precious biotech firm).
Fig. 2 is eIF4E-1 gene fragment (194bp) the PCR product agarose gel electrophoresis figure for building RNAi expression vector, wherein: M:100bp DNA Marker (precious biotech firm).
Fig. 3 is the XbaI enzyme cutting agarose gel electrophoresis figure of pK7GWIWG2-eIF4E1 plasmid, wherein: swimming lane 1:DNA marker (2000bp, precious biotech firm); Swimming lane 2:pK7GWIWG2 plasmid; Swimming lane 3:pK7GWIWG2 plasmid single endonuclease digestion; Swimming lane 4:pK7GWIWG2-eIF4E1 plasmid; Swimming lane 5:pK7GWIWG2-eIF4E1 plasmid single endonuclease digestion, swimming lane 6:DNA marker (λ DNA/Hind III)
Fig. 4 is that TN86-crosses the symptom of expressing 21d after eIF4E-1 plant inoculation PVY, wherein: A: a left side: TN86-crosses expression plant (TN86-35S ∷ eIF4E-1), and morbidity, performance floral leaf and vein necrosis; Right: TN86 adjoining tree, not morbidity.B a: left side: TN86-crosses expression (TN86-35S ∷ eIF4E-1) plant leaf, morbidity, performance floral leaf and vein necrosis; ; Right: TN86 contrasts blade, not morbidity.
Fig. 5 is that TN86-crosses the symptom of expressing 35d after eIF4E-1 plant inoculation PVY, wherein: a left side: TN86-crosses expression plant (TN86-35S ∷ eIF4E-1), and morbidity, performance floral leaf and vein necrosis; Right: TN86 adjoining tree, not morbidity.
Fig. 6 is the symptom of 21d after cloud and mist 87RNAi transfer-gen plant inoculation PVY, wherein: A: a left side: cloud and mist 87-RNAi plant, not morbidity; Right: cloud and mist 87 adjoining trees, morbidity, shows as floral leaf and vein necrosis.B a: left side: cloud and mist 87-RNAi plant leaf, not morbidity; Right: cloud and mist 87 adjoining tree blades, morbidity, shows as floral leaf and vein necrosis.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent company.The anti-PVY tobacco of tobacco bred TN86(), the anti-PVY tobacco of NC55(), tobacco bred Zhongyan-100 (sense PVY tobacco) and tobacco bred cloud and mist 87(sense PVY tobacco): be that business is planted tobacco bred, the public can obtain from Yunnan Academy of Tobacco Agricultural Science.
Embodiment 1
PCR-BluntII-TOPO carrier, Gateway LR clonase Enzyme Mix kit, pENTR 2B carrier are purchased from Invitrogen company, and pK7GWIWG2 expression vector is purchased from VIB (www.gateway.psb.ugent.be).Agrobacterium C58C1 (pMP90) (seeing document McKersie, etc., 1999, Plant Physiol 119:839-847), Agrobacterium GV3101 is purchased from Invitrogen company.Extraction of plasmid DNA test kit, sepharose DNA reclaim test kit, DNA fragmentation purification kit purchased from QIAGEN company.Intestinal bacteria (Escherichia coli) DH5 α; Restriction enzyme, Klenow Fragment; Reverse transcription test kit, DNA Marker, LA Taq archaeal dna polymerase, T4 archaeal dna polymerase and T4 DNA ligase, spectinomycin are all purchased from the precious biotech firm in Dalian and Roche company.RNA extracts test kit Trizol purchased from Invitrogen company, and the ELISA test kit of detection PVY and test strip are purchased from Agdia company (www.agdia.com).
One, the extraction of the total RNA of tobacco
Get respectively the 100mg young leaflet tablet of Zhongyan-100 and NC55 and put into the mortar with DEPC water treatment; The special extraction reagent of RNA that adds 1mlTrizol(Invitrogen company to produce), grind to form homogenate, be transferred in the EP centrifuge tube of DEPC water treatment, room temperature leaves standstill 5min; Add 0.2ml trichloromethane, vibration 15sec, room temperature leaves standstill 3min; The centrifugal 15min of 12000g at 4 ℃; Shift supernatant liquor to new EP pipe, shift the maximum 400 μ L of supernatant liquor; In supernatant liquor, add 0.5ml Virahol, leave standstill 10min; The centrifugal 15min of 12000g at 4 ℃; Abandon Virahol, remaining Virahol sucking-off is abandoned with liquid-transfering gun, add the ethanol of 1ml75%; The centrifugal 2min of 7500g at 4 ℃; Remaining ethanol sucking-off is abandoned with liquid-transfering gun to drying at room temperature 2-3min; Add 30ul DEPC to process water dissolution precipitation piece, put into 55 ℃ of water-bath water-bath 10min if not soluble;-80 ℃ of preservations are stand-by.
Two, the clone of eIF4E-1 gene
1. the first chain cDNA's is synthetic
In EP pipe, add the total RNA of above-mentioned tobacco approximately 0.1 μ g-5 μ g, oligo (dT) 50ng, 10mM dNTP mixture 1 μ L, process water with DEPC and complement to 10 μ L, mix rear of short duration centrifugal.Be placed in 65 ℃ of water-bath 5min, take out ice bath 10min, add reaction mixture 9 μ L(5 × reaction buffer 4 μ L, 25mM MgCl
22 μ L, 0.1M DTT 2 μ L, RNA enzyme inhibitors 1 μ L), mix rear of short duration centrifugal.25 ℃ of insulation 2min, add the precious biotech firm of 1 μ L M-MLV Reverse Transcriptase(to produce), mix rear of short duration centrifugal.25 ℃ of insulation 20min, then 42 ℃ of insulation 70min, the synthetic of cDNA completes, and-20 ℃ save backup.
The clone of 2.eIF4E-1 gene coding region
According to the sequences Design special primer of eIF4E-1 gene, on primer, introduce BamHI and SacI site with reference to the restriction enzyme site on pBI121 carrier simultaneously.Primer sequence is:
4E-1F：CGCGGATCCATGGCAGAGGAAGCTGAGAAATT（SEQ?No.3）
4E-1R：GCTAGAGCTCCTATACTGTATAACGATTCTTGG（SEQ?No.4）
Take the cDNA of Zhongyan-100 as template, with Taq enzyme (precious biotech firm produces) and primer 4E-1F and 4E-1R(SEQ No.3 and SEQ No.4), by the eIF4E-1 gene fragment of pcr amplification susceptible variety Zhongyan-100, size is 660bp, and PCR reaction system is as follows: cumulative volume is 20 μ L, wherein 50ng/ μ L DNA sample 2.5 μ L, 10 × PCR buffer, 2.0 μ L, dNTPs 1.2 μ L, the each 1.5 μ L of primer 4E-1F and 4E-1R, Ex-Taq DNA enzyme 0.3 μ L, ddH2O 12.6 μ L.Agents useful for same is purchased from precious biotech firm.
PCR temperature cycle parameter is: 94 ℃ of 3min, 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 1min, 35 circulations, 72 ℃ of 7min.PCR reaction is carried out on BIO-RAD gene-amplificative instrament (specifications and models are C1000 Thermal Cycler).
PCR product retrieve and purification: by PCR product electrophoresis on 1.5% sepharose, electrophoretic buffer is 1 х TBE, in the time that electrophoresis indicator tetrabromophenol sulfonphthalein migrates to enough DNA isolation fragments under 120V and 60min condition, take off gel, by gel image analysis system log (SYSLOG) result, as shown in Figure 1.Under ultraviolet lamp, cut off containing target DNA fragment gel.Reclaim test kit (QIAGEN company product) with glue and reclaim DNA.
Three, eIF4E-1 over-express vector builds
DNA fragmentation good purifying is connected on pCR-BluntII-TOPO carrier by T4 ligase enzyme, transform bacillus coli DH 5 alpha competent cell, select positive colony, extract plasmid and adopt BamHI and SacI enzyme to cut detection, select enzyme and cut correct plasmid, send the order-checking of the precious biotech firm in Dalian.The correct pCR-BluntII-TOPO plasmid that contains eIF4E-1 gene and the pBI121 plasmid of order-checking all spent the night with BamHI and SacI double digestion, enzyme is cut rear gel electrophoresis, cut respectively glue and reclaim target gene fragment and carrier segments, by T4DNA ligase enzyme, eIF4E-1 gene fragment is connected on pBI121 carrier, obtain the plant expression vector that contains eIF4E-1 gene fragment.Transform Agrobacterium GV3101, select positive colony, for transformation of tobacco.
Four, eIF4E-1 interferes (RNAi) expression vector establishment
(1) pcr amplification of eIF4e-1 gene fragment and purifying
According to eIF4e-1 gene order, with reference to the position of restriction enzyme site on intermediate carrier, on forward primer and reverse primer, introduce respectively BamH I and Xho I restriction enzyme site.Forward primer eIF4E1_194_F_Bam:5 '-CG
gGATCCaTGGCAGAGGAAGCTGAGAAATTG-3 ', reverse primer eIF4E1_194_R_Xho:5 '-CCG
cTCGAGgCAGCTTGTCTAGATTTAGCC-3 '.
Take the cDNA of cloud and mist 87 as template, carry out pcr amplification with above-mentioned primer.PCR reaction system is: cumulative volume is 20 μ L, wherein 50ng/ μ L DNA sample 2.5 μ L, 10 × PCR buffer, 2.0 μ L, dNTPs 1.2 μ L, the each 1.5 μ L of primer 4E-1F and 4E-1R, Ex-Taq DNA enzyme 0.3 μ L, ddH2O 12.6 μ L.Agents useful for same is purchased from precious biotech firm.
PCR temperature cycle parameter is: 94 ℃ of 3min, 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations, 72 ℃ of 5min.PCR reaction is carried out on BIO-RAD gene-amplificative instrament (specifications and models C1000 Thermal Cycler).
PCR product retrieve and purification: by PCR product electrophoresis on 1.5% sepharose, electrophoretic buffer is 1 х TBE, (120V in the time that electrophoresis indicator tetrabromophenol sulfonphthalein migrates to enough DNA isolation fragments, 60min), take off gel, answer gel image analysis system log (SYSLOG) result, as shown in Figure 2.Under ultraviolet lamp, cut off containing target DNA fragment gel.Reclaim test kit (QIAGEN company product) with glue and reclaim DNA.
(2) target fragment connects pCR-BluntII-TOPO carrier
The object fragment of purifying is connected on pCR-BluntII-TOPO carrier, transforms bacillus coli DH 5 alpha competent cell.Select positive colony, extraction plasmid carries out enzyme and cuts evaluation, selects enzyme and cuts the plasmid that evaluation is correct, the order-checking of Song Bao biotech firm.Obtain pTOPO-eIF4E1-194 plasmid.
(3) target fragment connects pENTR 2B carrier
PTOPO-eIF4E1-194 plasmid and pENTR 2B plasmid are all used to BamH I and Xho I double digestion, and target gene fragment and carrier segments are reclaimed in rubber tapping respectively, are connected and are obtained p2B-eIF4E1-194 entry clones by T4DNA ligase enzyme.
(4) react and obtain plant RNA i carrier by LR
Adopt Gateway LR clonase Enzyme Mix kit(Invitrogen company to produce), structure is obtained to entry clones and pK7GWIWG2 expression vector (VIB) and carry out LR reaction and obtain RNAi carrier.Reaction system is: p2B-eIF4E1-194 entry clones (300ng/ μ L) 2 μ L, pK7GWIWG2 expression vector (200ng/ μ L) 1 μ L, 5 × LR Clonase reaction buffer, 1 μ L, LR Clonase enzyme mix 1 μ L.25 ℃ spend the night after transformed competence colibacillus cell Escherichia coli DH5 α.Select positive colony, extract plasmid.The band producing after plasmid XbaI enzyme cutting is consistent with expection, sees Fig. 3, the order-checking of plasmid Song Bao biotech firm.Obtain the plant RNA i carrier pK7GWIWG2-eIF4E1 that order-checking is correct.Transform Agrobacterium C58C1 (pMP90), select positive colony, for transformation of tobacco.
Five, Transformation of tobacco
Picking is cloned (containing spectinomycin Spe 100 μ g/mL) in the LB substratum that is inoculated in 50ml containing the Agrobacterium of target expression vector, and 180rpm cultivates 24h, bacterium liquid OD for 28 ℃
600to 1.0 left and right, the centrifugal 10min of 3000rpm, precipitation thalline.Suspend with the MS liquid nutrient medium of 10ml left and right again, the centrifugal 10min of 3000rpm, precipitation thalline.Repeat above operation 2～3 times.Finally, with the MS liquid nutrient medium resuspension that adds certain volume, make the OD of thalline
600value is 0.5.Adopt leaf dish method to transform, choose the blade of sterile culture Resistance In Tobacco PVY kind TN86 and sense PVY kind cloud and mist 87, in Agrobacterium bacterium liquid, contaminate 15min to 20min, after blotting with aseptic thieving paper, be laid in dark on MS1 substratum and cultivate altogether 2d, explant is transferred to the bud inducing culture row filter as enterprising in MS4 containing carboxylic Bian penicillin 500 μ g/mL and kantlex 200 μ g/mL, and about 15d subculture once.After having blastogenesis to become, proceed on the root media MS containing carboxylic Bian penicillin 250 μ g/mL and kantlex 100 μ g/mL, carry out root induction growth.After well developed root system, be transplanted in greenhouse, carry out Routine Management, obtain transgene tobacco T
0dai Miao.The T obtaining
0for transfer-gen plant, adopt kalamycin resistance gene (NPT II) primer to carry out Genomic PCR detection, determine the transfer-gen plant of the NPT II positive, detect for PVY resistance.
Six, cross and express the resistance detection of eIF4E-1 transgenosis TN86 tobacco to PVY
To 60 strain T
0transfer-gen plant and transgenosis blank TN86 carry out PVY Resistance Identification.Adopt 40 times of the sick leaf saps of the downright bad strain ZT-5 of high-pressure spray gun inoculation marmor upsilon (PVY) seedling stage.14d, 21d investigation incidence after inoculation, and gather leaf sample, adopt ELISA test kit and the test strip of Agdia company, detect PVY.
After inoculation, the symptoms of the 21st day and sampling ELISA detected result show: cross the TN86 transgenic seedling performance PVY symptom of expressing eIF4E-1, as shown in Figure 4 and Figure 5, ELISA and ELISA test strip are the PVY positive, as table 1.Contrast TN86 plant is without PVY symptom, and ELISA and ELISA test strip are PVY feminine gender.Crossing and express eIF4E-1 that the tobacco bred TN86 of anti-PVY is shown as is susceptible, show that eIF4E-1 possesses biological function, is tobacco sense PVY gene, the namely recessive anti-PVY gene of tobacco.
Table 1 is crossed after the TN86 seedling inoculation PVY that expresses eIF4E-1 the symptom and the detection of PVY virus of the 21st day
M: represent floral leaf, VN: represent vein necrosis
Seven, interfere and express the resistance detection of eIF4E-1 transgenosis cloud and mist 87 tobaccos to PVY
To 47 strain T
0transfer-gen plant and transgenosis blank cloud and mist 87 carry out PVY Resistance Identification.Adopt 40 times of the sick leaf saps of the downright bad strain ZT-5 of high-pressure spray gun inoculation marmor upsilon (PVY) seedling stage.14d, 21d investigation incidence after inoculation, and gather leaf sample, adopt ELISA test kit and the test strip of Agdia company, detect PVY.
After inoculation, the symptoms of the 21st day and sampling ELISA detected result show: interfere cloud and mist 87 transgenic seedlings of eIF4E-1 without PVY symptom, the results are shown in Figure 6, ELISA and ELISA test strip is PVY feminine gender, the results are shown in Table 2.Contrast cloud and mist 87 plant performance PVY symptoms, are floral leaf and vein necrosis, and ELISA and ELISA test strip are the PVY positive.The tobacco bred cloud and mist 87 of interfering eIF4E-1 to make to feel PVY shows as disease-resistant, shows that eIF4E-1 possesses biological function, is tobacco sense PVY gene, namely the recessive anti-PVY gene of tobacco.
Table 2 is interfered after the cloud and mist 87 seedlings inoculations PVY of eIF4E-1 the symptom and PVY detection of the 21st day
M: represent floral leaf, VN: represent vein necrosis
EIF4E ?1 genes encoding region nucleotide sequence (SEQ No.1):
ATGGCAGAGGAAGCTGAGAAATTGCGGGTAGATGAAGTAGAAGTAGTCGACGATGGACCTGAAGAAGGAGAAATTGTGGA
TGAATCTGATGATACGGCGTCGTATTTGGGCAAAGAAATCAAACCTAAGCATCCATTAGAGAATTCTTGGACTTTTTGGTTTG
ATAATCCTATGGCTAAATCTAGACAAGCTGCTTGGGGCAGTTCCCTTCGCGAACTTTACACTTTTTCCACTGTCGAAGATTTTT
GGGGTGTTTACAATAATATCAACCACCCAAGCAAGTTAGTTGTGGGAGCAGACTTTCATTGTTTTAAGCATAAAATTGAGCCA
AAGTGGGAAGATCCTGTATGTGCGAATGGAGGGAATTGGACAATGAGCTTTAGTAAGGGTAAATCTGATACCAGCTGGCTA
TACACGCTGCTGGCAATGATTGGACATCAATTCGATCATGGAGAGGAAATTTGTGGAGCAGTAGTTAGCGTCCGAAATAAGG
GGGATAAAATAGCTTTATGGACCAAGAATGCTGCAAATGAAACAGCTCAGGTTAGCATTGGTAAGCAATGGAAGGAGTTTCT
GGATTACAGCAACTCGATTGGCTTCATATTTCATGACGACTCAATGAGGCTCGGCAGAGGTGCCAAGAATCGTTATACAGTAT
AG。
EIF4E ?1 aminoacid sequence (SEQ No.2):
maeeaeklrvdevevvddgpeegeivdesddtasylgkeikpkhplenswtfwfdnpmaksrqaawgsslrelytfstvedfwgvynninhpsklvvgadf
hcfkhkiepkwedpvcanggnwtmsfskgksdtswlytllamighqfdhgeeicgavvsvrnkgdkialwtknaanetaqvsigkqwkefldysnsigfifhd
dsmrlgrgaknrytv*。
Primer sequence is:
4E-1F：CGCGGATCCATGGCAGAGGAAGCTGAGAAATT（SEQ?No.3）；
4E-1R：GCTAGAGCTCCTATACTGTATAACGATTCTTGG（SEQ?No.4）。
Claims (7)
1. the recessive anti-PVY gene eIF4E-1 of tobacco, is characterized in that, the DNA fragmentation that comprises eIF4E-1 gene coding region, this DNA fragmentation by Auele Specific Primer to primer 4E-1F(SEQ ID No.3) and primer 4E-1R(SEQ ID No.4) obtain by pcr amplification; The disappearance of this DNA fragmentation disappearance or this fragment coding albumen can be given tobacco the susceptibility of the caused disease of marmor upsilon (Potato virus Y) is lost; The nucleotide sequence of this DNA fragmentation is as shown in SEQ ID NO.1, and the aminoacid sequence of its coded protein is as shown in SEQ ID NO.2.
2. the recessive anti-PVY gene eIF4E-1 of the tobacco as described in claim 1, it is characterized in that, the sequence table of the DNA fragmentation of described eIF4E-1 gene coding region as shown in SEQ ID NO.1, or be substantially equivalent to the DNA sequence dna shown in SEQ ID NO.1, or its function is equivalent to the subfragment shown in SEQ ID NO.1.
3. the recessive anti-PVY gene eIF4E-1 of the tobacco as described in claim 1, is characterized in that, described SEQ ID NO.1 nucleotides sequence is classified as:
ATGGCAGAGGAAGCTGAGAAATTGCGGGTAGATGAAGTAGAAGTAGTCGACGATGGACCTGAAGAAGGAGAAATTGTGGATGAATCTGATGATACGGCGTCGTATTTGGGCAAAGAAATCAAACCTAAGCATCCATTAGAGAATTCTTGGACTTTTTGGTTTGATAATCCTATGGCTAAATCTAGACAAGCTGCTTGGGGCAGTTCCCTTCGCGAACTTTACACTTTTTCCACTGTCGAAGATTTTTGGGGTGTTTACAATAATATCAACCACCCAAGCAAGTTAGTTGTGGGAGCAGACTTTCATTGTTTTAAGCATAAAATTGAGCCAAAGTGGGAAGATCCTGTATGTGCGAATGGAGGGAATTGGACAATGAGCTTTAGTAAGGGTAAATCTGATACCAGCTGGCTATACACGCTGCTGGCAATGATTGGACATCAATTCGATCATGGAGAGGAAATTTGTGGAGCAGTAGTTAGCGTCCGAAATAAGGGGGATAAAATAGCTTTATGGACCAAGAATGCTGCAAATGAAACAGCTCAGGTTAGCATTGGTAAGCAATGGAAGGAGTTTCTGGATTACAGCAACTCGATTGGCTTCATATTTCATGACGACTCAATGAGGCTCGGCAGAGGTGCCAAGAATCGTTATACAGTATAG。
4. the recessive anti-PVY gene eIF4E-1 of the tobacco as described in claim 1, is characterized in that, the aminoacid sequence SEQ ID NO.2 of its described coded protein is:
maeeaeklrvdevevvddgpeegeivdesddtasylgkeikpkhplenswtfwfdnpmaksrqaawgsslrelytfstvedfwgvynninhpsklvvgadfhcfkhkiepkwedpvcanggnwtmsfskgksdtswlytllamighqfdhgeeicgavvsvrnkgdkialwtknaanetaqvsigkqwkefldysnsigfifhddsmrlgrgaknrytv。
5. the recessive anti-PVY gene eIF4E-1 of the tobacco as described in claim 1, it is characterized in that, the Auele Specific Primer of described anti-PVY gene is classified CGCGGATCCATGGCAGAGGAAGCTGAGAAATT as to the nucleotides sequence of SEQ ID NO.3, and the nucleotides sequence of SEQ ID NO.4 is classified GCTAGAGCTCCTATACTGTATAACGATTCTTGG as.
6. the recessive anti-PVY gene eIF4E-1 of tobacco, it is characterized in that, in separating clone tobacco bred TN86, lack one has the anti-PVY gene of recessiveness of biological function, crossing the DNA fragmentation of expressing eIF4E-1 gene coding region makes the tobacco bred TN86 of anti-PVY show as susceptible, be indicated as tobacco sense PVY gene, namely the recessive anti-PVY gene of tobacco.
7. the application of the recessive anti-PVY gene eIF4E-1 of tobacco, it is characterized in that, first obtain the nucleotides sequence SEQ ID NO.1 of Resistance In Tobacco PVY gene eIF4E-1 coding region, and the aminoacid sequence SEQ ID NO.2 of the protein of coding, described eIF4E-1 gene is recessive disease-resistant gene, by conventional breeding means, susceptible tobacco eIF4E-1 gene is lost eIF4E-1 gene, obtain the disease-resistant tobacco of non-transgenic, utilize the gene constructed interference vector plasmid of eIF4E-1, and be transformed into susceptible tobacco, suppress the expression of susceptible tobacco eIF4E-1 gene, obtain the transgene tobacco that disease resistance obviously strengthens.
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