CN107475289A - Apple necrosis mosaic virus full length infectious cDNA and its construction method - Google Patents
Apple necrosis mosaic virus full length infectious cDNA and its construction method Download PDFInfo
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Abstract
The invention discloses a kind of apple necrosis mosaic virus full length infectious cDNA and its construction method.The method of structure apple necrosis mosaic virus full length infectious cDNA provided by the invention comprises the following steps：By cDNA sequence corresponding to RNA1, RNA2 and RNA3 in the triad genome of apple necrosis mosaic virus, (6) sequence 13, or sequence 4 are cloned into plant binary expression vector respectively, obtain three recombinant expression carriers；Three recombinant expression carriers form apple necrosis mosaic virus infectious cDNA clone.The infectious cDNA clone for the ApNMV that the present invention is built can successfully infect this life cigarette.Using this method, ApNMV infectious clone more can be fast and efficiently constructed, is advantageous to the follow-up biological characteristics and pathogenesis for carrying out ApNMV.
Description
Technical field
The invention belongs to plant genetic engineering field, is related to a kind of apple necrosis mosaic virus full length infectious cDNA
And its construction method.
Background technology
Infectious cDNA clone is cDNA or cDNA of the RNA virus with the infectivity in-vitro transcription with infectivity
Product, it is the basis to be conducted a research by reverse genetics system to RNA virus.Due to carrying out the something lost of RNA virus on rna level
It is more difficult to pass work study, greatly limit the progress to its pathogenesis etc., and infectious cDNA clone
Success structure can effectively realize the development RNA virus research on DNA level.In general, infectious cDNA clone structure be by
The full-length cDNA fragment of virus is connected into from cauliflower mosaic virus (Cauliflower mosaic virus, CaMV)
The downstream of 35S strength promoters, viral cDNA is started with this and transcribed in eukaryote.Profit in this way, bromegrass mosaic disease
Malicious (Brome mosaic virus, BMV) turns into the first and is built into infectious cDNA clone and the plant with bioactivity
Thing virus.At present, although the report that existing various plants virus or viroid infectious clone successfully construct both at home and abroad, entirely
The infectious cDNA clone constructing technology of long property is stronger, and construction work still has difficulty.
Apple necrosis mosaic virus (Apple necrotic mosaic virus, ApNMV) is reported recently in apple
A kind of new plant virus identified in floral leaf sample, it is single stranded positive-sense RNA virus, belongs to Bromoviridae
(Bromoviridae) subgroup (Subgroup 3) of Ilarvirus category (Ilarvirus) the 3rd, its genome structure
For triad, including RNA1, RNA2 and RNA3.The virus and apple mosaic virus (Apple mosaic virus, ApMV) and
Prunus necrotic bad spot virus (Prunus necrotic ring spot virus, PNRSV) homology is higher, and it is in China's apple
Recall rate in fruit floral leaf sample is up to 92.1%, and the raising to China's Apple yield and quality is a serious prestige
The side of body.Therefore, it is necessary to its it is pathogenic further investigate, for its popular pests occurrence rule research and Control Technology exploitation reason is provided
By basis.Because apple tree is perennial woody plant, viral Combined Infection situation in field apple sample under field conditions (factors)
Generally, single virus infection plant can quickly be obtained by artificial constructed viral infectious clone, is research pathogenicity
Effective means.
The content of the invention
It is an object of the invention to provide a kind of apple necrosis mosaic virus full length infectious cDNA and its construction method.
The method of structure apple necrosis mosaic virus full length infectious cDNA provided by the present invention, including following step
Suddenly：CDNA sequence corresponding to RNA1, RNA2 and RNA3 in the triad genome of apple necrosis mosaic virus is cloned respectively
Into plant binary expression vector, three recombinant expression carriers are obtained；Three recombinant expression carriers form apple necrosis
Mosaic virus infectious cDNA clone.
Wherein, cDNA sequence corresponding to the RNA1 in the triad genome of apple necrosis mosaic virus is sequence in sequence table
Row 1 or sequence 4；CDNA sequence corresponding to RNA2 in the triad genome of apple necrosis mosaic virus is sequence in sequence table
2 or sequence 5；CDNA sequence corresponding to RNA3 in the triad genome of apple necrosis mosaic virus is sequence 3 in sequence table
Or sequence 6.
In three recombinant expression carriers, in the triad genome for starting the apple necrosis mosaic virus
The promoter that cDNA sequence corresponding to RNA1, RNA2 and RNA3 is transcribed can be 35S promoter.
In one embodiment of the invention, the plant binary expression vector is specially pCASS4-Rz carriers.Accordingly
, the multiple cloning sites are specially Stu I and Xba I.
More specifically, RNA1, RNA2 and RNA3 in the triad genome of the apple necrosis mosaic virus are corresponding
CDNA sequence be cloned into respectively between restriction enzyme site Stu I and the Xba I of the pCASS4-Rz carriers.
The apple necrosis mosaic virus infectious cDNA clone being prepared using methods described falls within the guarantor of the present invention
Protect scope.
In the apple necrosis mosaic virus infectious cDNA clone, three recombinant expression carrier mol ratios can be
1:1:1。
Recombinant bacterium containing the apple necrosis mosaic virus infectious cDNA clone falls within protection scope of the present invention.
In one embodiment of the invention, the recombinant bacterium is specially to contain the apple necrosis mosaic virus infectivity
The Agrobacterium (such as EHA105) of cDNA clone.
The apple necrosis mosaic virus infectious cDNA clone or the recombinant bacterium are preparing single virus infection plant
Application in model falls within protection scope of the present invention；The single virus is apple necrosis mosaic virus.
The apple necrosis mosaic virus infectious cDNA clone or the recombinant bacterium are in research apple necrosis mosaic virus
Application in pathogenesis falls within protection scope of the present invention.
The apple necrosis mosaic virus infectious cDNA clone or the recombinant bacterium are in research apple necrosis mosaic virus
Protection scope of the present invention is fallen within the application in host plant interaction.
The plant can be apple or this life cigarette etc..
The present invention constructs ApNMV full length infectious cDNA, and can successfully infect this life cigarette.Utilize the party
Method, ApNMV infectious clone more can be fast and efficiently constructed, be advantageous to the follow-up biological characteristics for carrying out ApNMV
And pathogenesis.
Brief description of the drawings
Fig. 1 expands for ApNMV full length sequences.M：AL5000Marker；1,2,3 represents sample AM27ApNMV respectively
RNA1, RNA2 and RNA3, equal loading holes；4,5,6 represent sample AM75ApNMV RNA1, RNA2 and RNA3 respectively.In swimming lane
There is the purpose band for meeting expected size.Wherein, due to ApNMV RNA1 and RNA2 5 ' end and 3 ' terminal sequence uniformity compared with
Height, therefore have two bands in RNA1, RNA2 expand loading swimming lane.
Fig. 2 is digestion products electrophoresis detection figure.M：AL5000Marker；1,2,3 represents sample AM27ApNMV respectively
RNA1, RNA2 and RNA3 recovery product are through Xba I digestion results；4,5,6 represent respectively sample AM75ApNMV RNA1, RNA2 and
RNA3 recovery products are through Xba I digestion results；7 be the pCASS4-Rz plasmids through Stu I and Xba I double digestions, holes；8 is not
Digestion pCASS4-Rz plasmids.
Fig. 3 is RT-PCR methods detection inoculation this life cigarette ApNMV.A, B is respectively to be inoculated with AM27, AM75 infectious cDNA clone
Testing result after 21d；C is to be tied by being detected after resumption infection AM27, AM75ApNMV infectious cDNA clone this life cigarette 7d that rubs
Fruit；M：D2000Marker；H：Healthy this life Tobacco Leaves；E：It is inoculated with pCASS4-Rz empty carriers；P：Feel ApNMV Apple Leaves；N：
Healthy Apple Leaves.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Main agents：Polysaccharide polyphenol plant total RNA extraction reagent box (Polysaccharides＆Polyphenolics-
Rich RNAprep Pure) purchased from Tiangeng biotechnology (Beijing) company, restriction enzyme it is purchased from precious biotech firm
(TaKaRa), RT-PCR amplification needed for reagent essentially from Pu Luomaige (Beijing) Bioisystech Co., Ltd (PROMEGA),
AxyPrep DNA gels QIAquick Gel Extraction Kit and the small extraction reagent kit of AxyPrep DNAs (healthy and free from worry life science Co., Ltd), zero
The flat end Cloning Kits of background pTOPO-Blunt (Beijing Ai Delai bio tech ltd), pCASS4-Rz carriers are by sea
Southern university professor Cui Hongguang give, goat-anti rabbit ELIAS secondary antibody (article No. 458) is purchased from Beijing Bo Ermai Bioisystech Co., Ltd.
Key instrument：Bio-Rad PCR instruments, KCBIO-2800 gel imaging systems, DYY-6B type voltage stabilizings electrophoresis apparatus, constant temperature
Water-bath, constant incubator, AIRTECH SW-CJ-1FD superclean benches, ultraviolet specrophotometer, micro uv-spectrophotometric
MeterND-1000UV-Vis Spectrophotometer (NanoDrop, America), Synergy 4 are multi-functional
ELIASA (America).
Primer synthesis is sequenced by raw work bioengineering (Shanghai) stock by Beijing Liuhe Huada Genomics Technology Co., Ltd
Part Co., Ltd.
Embodiment 1, the structure of ApNMV full length infectious cDNAs and bioactivity detection
First, the structure of ApNMV full length infectious cDNAs
1st, plant Total RNAs extraction
Plant Total RNAs extraction uses polysaccharide polyphenol plant total RNA extraction reagent box (Tiangeng), and concrete operation method is illustratively
Book is carried out.Originally it is the Apple Leaves for having infected apple necrosis mosaic virus AM27 or AM75 for sample.
2nd, ApNMV genome sequences amplimer designs
Apple necrosis mosaic virus AM27 and AM75 the full-length genome sequence amplified based on inventor's early stage place team
Row, separately design RNA1, RNA2 and RNA3 full length sequence amplimer, and phosphorylation modification, anti-sense primer are done in the end of sense primer 5 '
5 ' ends introduce Xba I restriction enzyme sites and protection base (table 1).
ApNMV RNA1 of table 1, RNA2 and RNA3 full length sequence amplimers
* the end of sense primer 5 ' carries out phosphorylation modification, underlines, thickened portion represents restriction enzyme Xba I positions
Point.
3rd, RT-PCR cloning RNAs 1, RNA2 and RNA3 full length sequences and construction of recombinant plasmid
Reverse transcription system：
Mixed liquor is mixed, 37 DEG C of incubation 1h, then enters performing PCR reaction (reaction system is as follows) or -20 DEG C saves backup.
PCR reaction cycle parameters：98 DEG C of pre-degeneration 50sec, 98 DEG C of denaturation 10sec, Tm annealing 30sec, 72 DEG C extend
(RNA1, RNA2, RNA3 each extend over 2min, 1min 30sec and 1min), circulate 35 times；72 DEG C re-extend 8min, 4 DEG C of guarantors
Deposit.PCR primer is analyzed with 1% agarose gel electrophoresis, and electrophoresis 30min is left under DYY-6B type voltage stabilizing electrophoresis apparatuses, 120V
The right side, running gel are taken a picture in KCBIO-2800 gel image analysers, and cut purpose band (Fig. 1), use AxyPrep DNA
Gel reclaims kit carries out the purifying recovery of amplified production.
(" Annamalai P, Rao A L are recorded in using plant binary expression vector pCASS4-Rz
N.Replication-independent expression of genome components and capsid protein
of brome mosaic virus in planta:A functional role for viral replicase in RNA
packaging[J].Virology,2005,338:The texts of 96-111. " one, the public can obtain at applicant, can only be used to repeat
Present invention experiment uses) construction recombination plasmid, it is transferred in methylase deletion mycopremna JM110 competent cells to enter first
After row mass propgation, extraction target plasmid, Xba I and Stu I double digestions are carried out.Meanwhile to ApNMV geneome RNAs 1, RNA2
Xba I digestions are carried out with RNA3 recovery products.Reaction is as follows：
After flicking mixing, 37 DEG C of incubation 11h, 5 μ 10 × loading of l buffer are added to terminate endonuclease reaction, digestion products
Analyzed with 1% agarose gel electrophoresis, electrophoresis 30min or so under DYY-6B type voltage stabilizing electrophoresis apparatuses, 120V, running gel in
Taken a picture in KCBIO-2800 gel image analysers, and cut purpose band (Fig. 2), reclaimed and tried using AxyPrep DNA gels
Agent box carries out the purifying recovery of amplified production.
It is as follows to prepare coupled reaction system：
After mixing, 4 DEG C of connections overnight.Connection liquid is transformed into 100 μ l JM110 competent cells, coated plate, 37 DEG C of cultures
Afterwards, picking monoclonal is in 1ml (sulfur acid kanamycins, 100mg/L) LB fluid nutrient mediums, after bacterium is shaken in 37 DEG C of concussions, bacterium solution
PCR method identifies positive recombinant, and send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced positive colony, checking
The correctness of catenation sequence.Show the small fragment between restriction enzyme site Stu I and the Xba I by pCASS4-Rz carriers through sequencing
Replace with the recombinant plasmid in sequence table after DNA fragmentation shown in sequence 1 and be named as pCASS4-R1-1；By pCASS4-Rz carriers
Small fragment between restriction enzyme site Stu I and Xba I replaces with the recombinant plasmid life after DNA fragmentation shown in sequence 2 in sequence table
Entitled pCASS4-R2-1；Small fragment between restriction enzyme site Stu I and the Xba I of pCASS4-Rz carriers is replaced with into sequence table
Recombinant plasmid after DNA fragmentation shown in middle sequence 3 is named as pCASS4-R3-1；By the restriction enzyme site Stu of pCASS4-Rz carriers
The recombinant plasmid that small fragment between I and Xba I replaces with sequence table after DNA fragmentation shown in sequence 4 is named as pCASS4-
R1-2；Small fragment between restriction enzyme site Stu I and the Xba I of pCASS4-Rz carriers is replaced with sequence table shown in sequence 5
Recombinant plasmid after DNA fragmentation is named as pCASS4-R2-2；By restriction enzyme site Stu I and the Xba I of pCASS4-Rz carriers it
Between the recombinant plasmid that replaces with sequence table after DNA fragmentation shown in sequence 6 of small fragment be named as pCASS4-R3-2.Wherein, sequence
DNA fragmentation shown in row 1-3 is respectively RNA1, RNA2 and RNA3 couple in apple necrosis mosaic virus AM27 triad genome
The cDNA sequence answered；Sequence 4-6 be respectively RNA1, RNA2 in apple necrosis mosaic virus AM75 triad genome and
CDNA sequence corresponding to RNA3.
Then, select the bacterium solution after checking and expand culture, use the small extraction reagent kit of DNA (AxyPrepTM Plasmid
Miniprep Kit 250-prep AxyPrep) each recombinant plasmid is extracted respectively, -20 DEG C save backup.
Certainly, can be with DNA fragmentation shown in direct labor's composition sequence 1-6 when each recombinant plasmid is prepared, and pass through
Common molecular cloning approach is inserted it between restriction enzyme site Stu I and the Xba I of pCASS4-Rz carriers.
2nd, recombinant plasmid is transferred to Agrobacterium competent cell
1st, each recombinant expression carrier for taking 2 μ L step 1 to prepare is added separately to the impression of 100 μ L Agrobacterium EHA105 strains
In state, 30min is placed on ice；
2nd, 5min is placed in liquid nitrogen, 37 DEG C incubate 5min, cooled on ice 5min；
3rd, add 800 μ L LB liquid mediums, 28 DEG C, 180rpm shake bacterium 3-4h；
4th, 500 μ L bacterium solutions are taken to be coated with LB flat boards (sulfur acid kanamycins, 100mg/L), 28 DEG C of culture 48h；
5th, picking monoclonal shakes bacterium, PCR identifications in 2mL LB culture mediums (sulfur acid kanamycins, 100mg/L)；
6th, 500 μ L bacterium solutions are taken to shake 24h in 30mL LB culture mediums (sulfur acid kanamycins, 100mg/L) transfer；
7th, 4000rpm centrifuges 10min, abandons supernatant；
8th, with infiltration injection buffer solution (10mM MgCl2, 5.7,200 μM of Acetosyringone of 10mM MES pH) and weight
Outstanding thalline, adjusts OD600To 1.0 or so, the component of RNA1, RNA2, RNA3 tri- from AM27 (or AM57) mixes in equal volume, room
Temperature stands 2-3h；Inject this life cigarette seedling, every plant of 1mL or so, to inject pCASS4-Rz empty carriers and not inject tobacco as feminine gender
Control.
3rd, ApNMV infectious cDNA clones bioactivity detects
Utilize specific primer (ApNMV-CP (+)：5’-ATGGTGTGCAATCGCTGTCA-3’；ApNMV-CP(-)：5’-
CATCGACCATAAGGATATCA-3 ') by RT-PCR methods detection inoculation this life cigarette plant ApNMV, reaction system and condition are such as
Under：
PCR reaction cycle parameters：94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend
50sec, circulate 35 times；72 DEG C re-extend 10min, 4 DEG C of preservations.PCR primer is analyzed with 1.5% agarose gel electrophoresis,
Electrophoresis 30min or so under DYY-6B type voltage stabilizing electrophoresis apparatuses, 120V, running gel is clapped in KCBIO-2800 gel image analysers
According to.
Using ApNMV specific primers, to this life cigarette system leaf after inoculation AM27, AM75 infectious cDNA clone 21d
Carry out RT-PCR detections.As a result show, this life cigarette whole (16/16) for being inoculated with AM27 infectious clones detects ApNMV, is inoculated with
This life cigarette ApNMV positive rates of AM75 infectious clones are up to 93.3% (14/15) (A and B in Fig. 3).In addition, utilize disease plant
After blade rubbing is inoculated into healthy plant 7d, detection finds that AM27 and AM75 infection rate is respectively 20% (1/5) and 80% (4/
5) (C in Fig. 3).
It is multiple that result above synthesis illustrates that the ApNMV infectious clones constructed by the present invention can be completed in this life cigarette
System, protein expression, parcel assemble the processes such as complete virion.Using this method, more can fast and efficiently build
Go out ApNMV total length infectious cDNA clone, for the checking of further Koch's Postulates, viral pathogenesis and its and host plant
Interaction research lay the foundation.
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>Apple necrosis mosaic virus full length infectious cDNA and its construction method
<130> GNCLN171370
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 3380
<212> DNA
<213>Apple necrosis mosaic virus
<400> 1
gttttatttt cgtggttgaa aataaagaac agtgaccaag acattcacga tatggacccg 60
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gccataggaa aagtattcgc cgatagaggt gtggcgttag ttgaacgatc attaccgaag 180
gctgaaggtg agagactaaa tgttagtttc taccaaaccc ccgagcagaa ggctctgttg 240
gaacgaaact tcccgggtcg agttattcac tttacgaata aagactcgtc aagtcacagt 300
ttcgcagcag cgcataggtt gttggaaacc gattacatct accgatgttt cggtactacc 360
gaggaaccga taatcgatct aggggggaat tttgtttctc atatgaagca aaagaggtat 420
aacgtgcact cgtgttgccc acttctcgat gatagggatg gagcacgttt taccgagagg 480
ctgatcaatt tgaagactta ccttagatca cataaggaag aattgcattc agccgactat 540
tgtgagcaca gattcgaagt gtgtgacagg aaggccgagt acgtgatggc aattcacgct 600
acgagtgacc ttccgatcac agaactgtgc aaagcattgg ccaacaaggg aactaagaag 660
atgatctttt cgattatgat agatccgagt atgttgatca gggatagtgg tattatcccg 720
aatttcgatg tacgttggga aatcgatcat gcaaccgact tagtcacttt tgacttcatc 780
ggggctccat gtttgggtta ccaacacaag ttctctgtgt taaaacagta cctaactacg 840
aatgcagtag ttgtcggtga caaacaggct ttccgagtcg aaaggaaatc cgattttggt 900
ggagtcatga taattgacat cacggcagtc gccgggtaca aaccgggtat ggtggtcggt 960
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gagggtgtgg aagactggtg gtacgatgtt accaagcggt cgaaggtact ggttgacact 1080
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gagagcatga tacaaaacat tgctactatg ctgtcgtcat ctactaacta tacagtcatt 1200
aacggtgtga ctcttcaggc tggtgagagt ttacattttg acgactatgt tagcatcgct 1260
accaccatat acgtcaggac gaagaagatg ttcgaatcac tcccacagaa catcgataag 1320
ttgcaggaac aacggatggt gcgaatgaga gaccatacaa atggtcaagt ctacggtaaa 1380
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cctagaggaa ggggacgaga gatcgcttat gatttgcagt ccaagtcctg gtcggaagag 1500
acgagaaatt tcctctatag ttgtctaggg cggcagggag cgccccaaac actgattaat 1560
gatccagatc tattcttacc actatctgtg gtattgtcta cgaagtggga aggagcggca 1620
acattgtccg tgtccgatgt ctacgatcag gtagtgaaac ctcagtatga cagaatagag 1680
gatgagatca accggcgtcg agaggatgct gagaagaatg acggttttca gaggaatctg 1740
ctgaccatag cgaaatggat cgaaaatcac ccgacaggcg aaatgccgaa aggattgggt 1800
aagatcgcag ctttggtgcc tgacctggtg gatatccgag aacaggtggt cgagaaacca 1860
agtgatttgg ttgtgaataa atatgcttcc gagattcagg aggctattac ttattttgag 1920
accgaaatgg acattagcac cagaaagtta cagtccgtgg gtgagcattg tgagtggaag 1980
aaacagaagt tgatgaccat atggtcatct gatgagtcca ggagagttta cgatcctagt 2040
accaactctt ggtttggtcc accatcaccg catagagttc aaccgactgc gaggtatgaa 2100
cgagggctga cgaaggatgg ttttgttccg atcttgtgga aaaccgttgg tgaggttggg 2160
agtttccgaa aaacgactaa gtttgaggat gctaactact tcgaagtgga cgaaaattgt 2220
cgccgaaatt taatgcgata tccgtgtgtg ttttttgata gctcttgtga gttttcacct 2280
ggtacgaggt tagtacctgt gttgaaagaa gcattacaca tggaggcgaa tttcgagaga 2340
aagttggtcg atggtatcgc gggatgcggt aagacgacga agatactcgc cgaagcaaaa 2400
atggtcaccg aagatcctga tttggttttg accagtaatc gatcatctgc catggaattg 2460
cgtgagaaac tgcctggtag tcaattggtc aaggcgcaaa gagtccgaac ctgtgattcc 2520
tatttaatga acggtaagaa ggtacactcg aagagagtga tcttcgatga gtgttttttg 2580
caacacgccg gatgtgtgta cgccgcggcc actttggcga aggcggatga gctgattatg 2640
tttggcgaca catcgcaaat tccgttcgtg tcgaggatac cgaatatgag acttgttaat 2700
cacaaggtgg gttgtgatga aaagcgtgat tacaacctca cctataggtg tccgatagat 2760
accacgatgg ctttgtccaa gtggttttat aggaagaaca taaagacagc caatagagtg 2820
atgcgaagca tgtcgattaa gccgatttcg tcagtcaatc aaatagatgc atctcacgat 2880
ctatttttga ctcacactca ggcagacaag tgcacgctct tagcaacggg aaaatttccc 2940
cgagatagag tctttacttc agctgaggcg caagggaaga ccgaagggaa cgttgccttc 3000
gtgagattga acaggaccag tattaatttg tatactggga aagacccgtt gatgggtccc 3060
tgtcatagtt tggtagccat gtcgagacac acaaaaaggt tcacgtatta tactacggct 3120
gaaaccgatg gtgatgattt gatggtgaaa gctattaggg atgtgagtag tgcaaatgac 3180
gagcgagtca ttagctacct acataccgat tacaaattgt aggttcgatg cagaaccgaa 3240
ccccgattag ggtagaaccg gaccaccgcg tggatttttc cggttatctc cgagagtcgc 3300
tttccagact catgctaagg agattaccta cgaaagtaga tgcccaatcc gtgaaggatt 3360
ggatgccccg ttagggaagc 3380
<210> 2
<211> 2786
<212> DNA
<213>Apple necrosis mosaic virus
<400> 2
gtttttattt tcgtggttga aaataaagaa gaattcctta atacagattt caatttctat 60
ggacccactc taccatcttg tgaagcacgc accgtctgct gtgagatgtg ccttgtctaa 120
cagagctgcg cttgagcacg tcagagaaca gcagagtctc gagtttagag accattgggt 180
tgaaactgca cgtcagatgg gagatgtgaa gaccttcaac gatttagttg ttggttacgt 240
catggtcgaa ttttttagac cgaagtttga aagaattttc aaacaattcg tgggaaccga 300
agcaggacca tggatagtgg acagatgggc aatgttgttt gacatttcgt caccacccat 360
cgaagtcgaa tacactcgac agtcggttag catgtacgaa cagtacgctt atatcgacga 420
agagtatgag gagatattgc gattggaggc tctcgaacta gccgaagcac agggtcagga 480
atacaatacg aatgttccga cgtacccact tgagaatctg gatgaatcga ccctcgatcg 540
aaatgatgaa gacgaggagg ttctgacacc accaccagca acgagcagtt cagattgtgg 600
tgctgaatta gattccccga tcgatgtgaa cgaaccaatg atcgatgtag aagcagacat 660
gcacgcactc gacgatgagg ttccagctct accgaagtcg agtctgcaaa gggtgaccgt 720
atcgggttta ccgttggtac ccatgatcga aagcttcgaa accaatcaat tgatgttggt 780
taaggagatt gttgtcgata agttcgaaat gttgctggaa aatcacgata gggttttggc 840
aaggaatgac tggcgatctt tagccatgta tgttccgaat tgtcaagtgc ccgattgtag 900
tgatattttg gagatgcctg tgagacacac gcaacccgag ttcatccaga tggctataga 960
tgaactgtta ccaggtgttg ctgacgtcga tgacaggttc ttccaggaac tagttgagac 1020
ggacgatatt tgcttggagt tagatagggc tactatcgat cagtccgtgt tcaacgattg 1080
ggttccgaaa tcagacaaga aactgcaagg tgtgtttcga actggtaatg cctctaagcg 1140
agtcccgaca tttagggaag cagctctggc tatcaagaaa cgaaacctta atgttccaga 1200
tttgaagcaa gtcatgtttg atgacgatga ggctaggaag attgctaaca ggttcattaa 1260
cactgttatt gacccgaata agttggcgca gtttccggga tatatttctg aaggggaagt 1320
gggttattat aataaatatc tcactggaaa gaatatagac ccagatctct tcgtggatcc 1380
ttgtgctctt gtgtcaatgg ataaatacag gcacatgatc aagacgcagt tgaaacctgt 1440
tgaagacaca tcgcaggttt ttgaacgaag tcttccggcg actataacgt accacgataa 1500
gggtaaagtt atgtcgacgt ccccgatatt cctgatgatg tgtaatcgat tgttgttgtg 1560
tctgaacgac aagatttcca tcccatcagg aaagtaccat cagttgtttt cgctggaccc 1620
gtttgctttc gagatgacga aagaatttaa agagatagat ttctcgaaat tcgataagtc 1680
tcaacagagg ttgcaccatt tgatccaatt ccatatattc acagctcttg gagccgatca 1740
tgattttctg gatatgtggt ttggatctca tgagatatcg cacattaggg atggtccttg 1800
cggtataggg tttagcgtga actatcagcg tagaacaggg gatgcttgca cgtacttagg 1860
gaacacgata atcacactgt cagctttggc ttacatgtat gatttgttgg acccgaatat 1920
aactttcgtc atagcatctg gtgacgacag tttaattggt agtgtcaagc ccctgaatag 1980
ggacaccgag tttatgttta ccacgttgtt caattttgag gcgaagtttc ctcataatca 2040
agctttcgtg tgcagtaagt ttttgtgctt actccctaca aagtcaggtg gtaaaaaggt 2100
gttagcagtt ccgaatgcac ttaaactgaa tattaagtta ggtgtgaggg atttagcgcc 2160
cgaagcattc gatgcttggt atgagtcctg gttggactta acatggtact tcaaacacta 2220
tcttgtggtc tcgatgatga gagattacat aagtcacagg tatgcgagga agcaaactct 2280
ctttcaagag ggagctatgt tggcttatgg gacaattttc gcgaataagg aaaaatgtct 2340
caagagcgtg tttggcatta catcgaagga tttagagaga atgaaaccta aatcgagatg 2400
taatgacgtt aagactcaac gagggaagat agatgcgtca aaaaccccga ggaagtgggc 2460
cgatgttgta gagcacaccg agagtctgaa tcgcgatgtt agcgatgaga agatcacgag 2520
aagtgatctg actattatcg atatagataa gaaaaagatt tttccagtga atcgaagcag 2580
gagacttagt aaaaagaata ggtctgttgc ggagaaaact ggttaggttg atttgaacac 2640
aaccgaaccc cgataagggt agaaccggac caccatgtgg gttttccggt tatctccgag 2700
agtcgccttc cagacttgtg ttcaggagat tacctacgaa agtagatgcc caatccgtga 2760
aggattggat gccccgttag ggaggc 2786
<210> 3
<211> 1954
<212> DNA
<213>Apple necrosis mosaic virus
<400> 3
gttaaatttc tatttcttta attggcgtaa catccgtttc ggttgtcaat ttctttaatt 60
gacgttacca acgaaaggat gtcaattgaa tttaaccagt cagcgaaagt acagatcgaa 120
gaaattcgta ttagactttg tgtaacatcc gattttccgg ttgcatcgtc gaagtacttt 180
tctgcgaaat ggcagtagct gacttgaaac gctcttcaga ctttaccgtc aaagaggttt 240
cgatgtccga atatgagcgc atctcgggag acttacacca actaatgatc tccaaagaga 300
tgcgcgatct accgacaaag ggttgtcatg ttctgcattt gcagaatctc ccgaaatcca 360
atgtgttaga attggaatct cgtgaacaac gaggttttct aaccaaaaca gcggatagaa 420
tccgcgagaa ggtataccgc gacacgggaa gaattttctt cgtgtatgtc ccagttattc 480
agaggacgag tagcggtaca atcacgttaa agctacaaaa taccgatact ggtgaggtct 540
ccgatgtggt gaccgatgca ccagcagagc gtgctttcgt ggttatggac agatggggta 600
gatcactgac atcaaaagca aagttggtat tgctttattc gatcttttgc cccgaaataa 660
ggcccgaggc tcgagttgga gacatgtgtg tgttttggga tgaacacatg tcgaaacaaa 720
tgacttatgt caagaagccg aatcccataa tgtttccgat tgaggaaaca catccagcgc 780
aatatctaaa ggataagaag ctgttgatga gcatgatccg aggcaggata gcgttggggg 840
ccgaagggac ggatttgcgt cccgacgttt tgaaagtgga gagtgcgagt agtggtcgga 900
aattgatcac tattgaaccg aaggacagtc tgaaaccacc gaaaatagaa gaggttggag 960
agacgtccgg aacagacgtt catttggaag agacggatcg caccgtgagg gctgatccta 1020
taaatgcttg attgatattt gggcgaaggg tactgtgtac ccgaccgtgc gcgtttgcgc 1080
gggcgtcttt tatctttctg atttatattt tcttctatct cgcttgcaat cgatatggtg 1140
tgcaatcgct gtcatcacac tcacgctggt gggtgtcgtt cctgccgaca gtgccacccg 1200
agagatgctg ctccaccacc accgagagct cgagctagag ctcaaaatgt ggtagcgcga 1260
ggtttggcgc gaccagcgac ttcagctggg gagccgagaa ggcttcagtg gaccgtgata 1320
ggtccgaatg aggtaccacg agtaccgaga ggatatgtgg cacacagcaa cagggaagtt 1380
gttgcgacta gtgctgggaa gttcctacac gtgaacttca gcacgacatt cccacaccta 1440
ctgggattga atcttaggat tctctccgta gtagttcgag ccagctgcct agtgtctgct 1500
gggtgggtgg ggatggtaga ggaccatgtt gagactgatc tcaggggtcc gagtgccctg 1560
tccaggaagg ggtttcgcca agaccaaccg agaggttggc agtggttggc tccttccgat 1620
ttagaatacg ataggtttgc gaactcgcac cgattagtat tcgaagtcaa gaacgaattc 1680
gcggcaggtg tgaaagttct tgtgagggat atttacgtag tggtaagtga tttaccacga 1740
attgtgatcc cgaatgatat cctaatggtc gatgaagacc ttttggatgt ctaggttgag 1800
atgaacacaa ccgaaccccg ataagggtag aaccggacca ccatgtgggt tttccggtta 1860
tctccgagag tcgctttcca gactcgtgtt taggagatta cctacgaaag tagatgccca 1920
atccgtgaag gattggatgc cccgttaggg aagc 1954
<210> 4
<211> 3378
<212> DNA
<213>Apple necrosis mosaic virus
<400> 4
gttttatttt cgtggttgaa aataaagaat agtgactaga tattcacgat atggacccag 60
ttgctttatc cgagttgcta gtatccacgc taagcaaaca atgtgcagac gagacgtctg 120
ccataggaaa agtattcgcc gatagaggtg tggcgttagt tgaacgctca ttaccgaagg 180
cggaaggtga gagactaaat gttagtttct accaaacccc cgagcagaag gctctgttgg 240
aacgaaattt tccgggtcga gttattcact ttacgaataa ggactcgtca agtcacagtt 300
tcgcagcagc acataggttg ttggaaaccg attacatcta ccgatgtttc ggtaccaccg 360
aggaaccgat aatcgactta gggggaaatt ttgtttctca tatgaaacaa aagaggtata 420
acgtgcactc gtgttgtcca cttctcgatg atagagatgg agcacgtttt accgagaggc 480
tgatcagttt gaagacttac cttagatcac ataaggaaga actgcattca gccgactatt 540
gtgaacacag attcgaagag tgtgacagga aggccgagta cgtgatggca atacacgcta 600
cgagtgacct tccgatcaca gaactgtgca aagcattagc caacaaggga actaagaaga 660
tgatcttttc gattatgata gatccgagta tgttgattag ggatagtggc attatcccga 720
atttcgatgt acgttgggaa atcgatcatg cagccgattt agtcactttt gacttcattg 780
gggctccatg tttgggttac caacataagt tctctgtgtt aaaacagtac ctaactacga 840
atgcagtagt tgtcggtgac aaacaggcat tccgagttga aaggaaatcc gattttggtg 900
gagtcatgat aattgacatc acagcagtcg ccgggtacaa accgggtatg gtggtcggtg 960
gaagtagatc ctgtgcatgg agcagtcttg tgcagggtaa gacagtagta cacaccgtgg 1020
aaggtgtgga agactggtgg tacgatatta ccaggcggtc gaaggtgctg gttgatacaa 1080
aggttttaac gagagttctg gaagcttcat tcagacagta taaaccgaca actagtcccg 1140
agagcatgat acaaaatatt gctactatgc tgtcgtcatc tacgaactac acagtcatta 1200
atggtgtgac tcttcaggct ggtgagagtc tgcattttga tgactatgtt agcatcgcta 1260
ccaccatata cgttaggacg aagaagatgt tcgagtcact cccacagaac atcgataagt 1320
tgcaggaaca acgaatagtg cgaatgagag accacacaga tggtcaagtc tacggtaaat 1380
tgggttcgtt tatcggagat gtgctcttgc ctaatacagt tgaacaacga gtgtccgatc 1440
ctagaggaaa gggacgagag atcgtttatg atttgcagtc caaatcttgg tcggaagaaa 1500
cgagaaattt cctctatagt tgtttagggc ggcagggagc gccccaaaca ttgattaatg 1560
atccagatct attcttacca ctatctgtgg tactgtctac aaagtgggaa ggagcggcaa 1620
cgttgtccgt gtccgatgtc tacgatcaag tggtgaaacc tcagtatgac agaatagagg 1680
atgagatcag acagcgtcga gaggatgctg aaaagaacga cggtttccag aggaatctgc 1740
tgaccatagc gaaatggatc gaaaatcacc cgacaggcga aatgccgaaa ggattgggta 1800
agatcgcagc tttggtgccc gacctggtag atatccgaga acaggttgtc gaaaaaccaa 1860
gtgatttggt tgtgaataag tatgcttccg agattcagga ggccatcact tatttcgaga 1920
ctgaaatgga cattagcacc agaaagttac agtccgtggg tgaacattgt gagtggaaga 1980
aacagaagtt gatgaccata tggtcatctg acgagtccag gagagtttac gatcctagca 2040
ctaactcttg gtttggtcca ccatcaccgc atagagttca accgactgcg aggtatgaac 2100
gagggctgac gaaggatggt tttgtcccga ttttgtggaa aaccgttggt gaggttggga 2160
gtttccgaaa aacgactaag tttgaggatg ctaactactt cgaagtggac gaaacttgtc 2220
gccgaaattt aatgcgatat ccgtgtgtgt ttttcgatag ctcttgtgag ttttcacctg 2280
gtacgagatt agtacctgta ttgaaagaag cattacacat ggaggcgaat ttcgagagaa 2340
agttggtcga tggtatcgct ggttgcggta agacgacgaa gatactcgcc gaagcaaaaa 2400
tggtcaccga agatcctgat ttggttttga ccagtaatcg atcatctgcc atggaattgc 2460
gagagaagct acctggtagt caattgatca aggcgcaaag agtccgaact tgcgattcct 2520
atttaatgaa cggtaagaag gtacactcga agagagtgat cttcgatgaa tgttttttgc 2580
aacacgctgg gtgtgtgtac gccgcggcca ctttggcgaa ggcggacgaa ctaattatgt 2640
ttggtgacac atcgcaaatt ccgttcgtgt cgaggatacc gaatatgaga cttgttaatc 2700
ataaggtggg ttgtgatgag aaacgtgatc ataaccttac ctataggtgt ccgatagaca 2760
ccacaatggc tttgtccaag tggttttata ggaagaacat aaagacagcc aatagagtga 2820
tgcgaagtat gtcgattaaa ccgatttcgt cagtcaatca aatagatgca tctcatgatc 2880
tatttttgac tcacactcag gcagacaagt gcacactctt agcaacaggg aaatttcccc 2940
gagatagagt ctttacttct gccgaggcgc aagggaagac tgaggggaac gttgcttttg 3000
ttagattgaa caggaccagt attaatttgt atactgggaa agacccgctg atgggtccct 3060
gtcatagctt ggtagccatg tcgagacaca cgaagaggtt tacgtattat actacggctg 3120
aaaccgatag tgacgatttg atggtgaaag ctattagaga tgtgagtagt gcgaatgacg 3180
agcgagtcat tagttactta cataccgatt acaaattgta ggttcaatgc agaaccgaac 3240
cccgattagg gtagaaccgg accaccgtgt gggttttccg gttatctccg agagtcgctt 3300
tctggactca cgctaaggag attacctacg aaagtagatg cccaatccgt gaaggattgg 3360
atgccccgtt agggaagc 3378
<210> 5
<211> 2778
<212> DNA
<213>Apple necrosis mosaic virus
<400> 5
gttttatttt cgtggttgaa aataaagaag gaaactgaaa tattcaattt ctatggaccc 60
actctaccat cttgtgaagc acgcgccttc agctgtgaga tgtgccttgt caagaagagc 120
tgagcttgag cacatcagag aacagcagag tctcgaattc agagaccatt gggttgaaac 180
tgctcgccga atgggagatg tgaaaacctt caacgattta gttgttggtt atgttcttgt 240
cgaattcttc agaccgaagt tcgaaagaat ttttaaacaa ttcgtgggta ccgaagcagg 300
accatggata gtggatagat gggcaatgtt gtttgacatt ccgacaccgc ctatcgagat 360
cgaatacact cgaccgtcag tcagtatgta cgaacagtac gcttatattg acgaagagta 420
tgaagagata ctgcgattgg aggctcttga actggccgaa gcacaaggtc aggaacataa 480
cgatgttccg atgtacccta ctgagagtcc ggatgaatcg atcctcgatc gaaattatga 540
agatgaggag gttctgacac cactaccaac aacgagcagt tcagattgtg gtgctgaatt 600
aggttccccg accgatgtga acgatctaac aatcgaagta gaaacagaca tgcatgcact 660
cgacgatgag gttccagctc taccgaagtc gagtctgcaa agggtgaccg tatcgacctt 720
accgttggta cccatgatcg aaggtttcga aaccaatcaa ttgatgttgg tgaaggagat 780
tgttgttgat aagttcgaaa tgttgttgga aagtcacgat agagtcctgg cgaggaatga 840
ttggcggtct ttagctatgt acattccgaa ctgtcaagta ccggactgta gtgacatttt 900
ggagatgcct gtgagacata cacaacccga attcatccag atggctatag atgaattgtt 960
accaggtgtt gctgatgtcg acgacaggtt cttccaggaa ctagttgaga cggacgatat 1020
ttgtttggaa ttagataggg caactatcga tcagtctgtg ttcaacgatt gggttccgag 1080
atcagacaag aaactgcaag gtgtgtttcg aactggtaat acctctaagc gagtcccgac 1140
gtttagggaa gcagctttag ctattaagaa acgaaacctc aatgttccag atttgaagca 1200
agtcatgttt gatgacgatg aagctaggaa aattgccaat aggttcatta acactgttat 1260
tgacccgaat aagttggcgc agtttccggg ttatatttct gaaggggagg tgggatatta 1320
taacaaatat cttactggaa agaatataga cccagatctc tttgtagatc cttgtgcact 1380
tgtgtcgatg gataagtata gacacatgat caagacgcag ttaaaacctg ttgaagatac 1440
atcgcaggtt tttgaacgaa gtctcccggc gactataacc tatcacgata agggtaaagt 1500
tatgtcgacg tccccgatat tcctgatgat gtgtaatcga ttgttattgt gtctgaatga 1560
caagatttcc attccatcag gaaagtacca tcagctgttc tcactggacc cgtttgcatt 1620
cgagatgacg aaagaattta aagagataga tttctcgaaa ttcgataagt ctcaacagag 1680
attgcatcat ttgatccaat tccacatatt tacagccctt ggagccgatc atgagttttt 1740
agatatgtgg ttcggatctc acgagatatc gcacattaga gacggtcctt gtggtatagg 1800
gttcagtgtg aactaccaac gcagaactgg agacgcttgc acgtacctag ggaacacaat 1860
aattacattg tcagccttgg cctacatgta tgatctgttg gacccgaata taactttcgt 1920
catagcatct ggtgacgata gtttaattgg tagtgtcaat cccttgaata gggacaccga 1980
gtttatgttt accacgcttt ttaattttga ggcgaaattt cctcataatc aggcttttgt 2040
gtgtagtaag tttttgtgct tactccccac aaagtcgggt ggtaagaagg tgttagccgt 2100
tccgaatgca cttaagttga atataaaatt aggtgtgaag gatttggcac ccgaagcttt 2160
cgatgcttgg tatgaatcct ggttggattt aacatggtac ttcaaacact atcttgtggt 2220
ctcgatgatg agagattaca taagtcacag gtatgcgagg aagcaaactc tctttcagga 2280
gggagctatg ttggcttatg ggacaatttt cgcgaataag gaaaaatgtc tcaagagcgt 2340
gtttggtatc acatcaaaag atttagagaa gatgaaacct aaatcgagat gtggtgaagt 2400
tagggctcaa cgagggaaga cagatttgtc gaaaaccccg agaaagtgga ccgatgttgt 2460
agagcacact gagagtctga atcgcgatga tagcgatgaa aggatcacga gaagtgatct 2520
gattcctatc tatgaagaag gaaaaatttt gtcaccagtc aatcgaagta gaagacctag 2580
taaaaagaat aggtcgattg cgaagaaaac tggtaaggtt gagatgaaca caaccgaacc 2640
ccgataaggg tagaaccgga ccaccgtgtg ggttttccgg ttgtctccga gagtcgcttt 2700
ccagactcgt gttttaggag attacctacg aaagtagatg cccaatccgt gaaggattgg 2760
atgccccgtt agggaagc 2778
<210> 6
<211> 1916
<212> DNA
<213>Apple necrosis mosaic virus
<400> 6
gtttaaattt gtatcactgg cgttacatcc gtttcggttg tcagtcgatc ttttattccg 60
gtcagcgaaa gtacatttca aagattaatt tgtatcagac tctacgtaac atccgtaacc 120
ggttgtattg tcgaagtact tttctgcgaa atggcagtag ctgacctgaa atcttcttca 180
gactttaccg tcgagaaggt ttcgatgtcc gaatatgagc gcatctcggg agacttgcac 240
caactattga tctctaatga gatgcgcgat ctaccgacaa agggttgtca tgttctgcat 300
ttgcagaatc tcccgaaatc caaagtgtta gaactagaat ctcgcgaaca gcggggtttt 360
ctatccaagg tagcggacag gatccgcgag aaggtatacc gcgatacggg aagaatcttc 420
ttcgtgtacg tcccagtcat ccaagggacg agtagcggta cgatcacgtt aaagctgcaa 480
aatactgata ccggtgaggt ttccgatgtg gttaccgacg caccggctga acgggctttt 540
gtgatcatgg acagatgggg taggtcttta acgtcgaaag caaagttggt gttgctttat 600
tcgatttttt gccccgaaat aaggcctgag gctcgagttg gagatatgtg cgtgttttgg 660
gatgaacaca tgtcgaaaca gatgacttat atcaagaagc cgaatcccat catgtttccg 720
atcgaggaaa cgtatccagc gcaatatctg aaggataaga agttgctgat gagtatgatc 780
cgaggtagga tcgcgttggg ggccgaaggg gcggacatac gtcccgatgt tttgaaagtg 840
gagagtgcga gtagtggtcg gaaattgatc actattgaac cgaaggatag tccgaagcca 900
ccgaaaatag aagaggtagg tgggacgtcc gggatagacg ttcatgtgga agaaacggat 960
cgtgccgtga gggctgatcc tataaatgct tgatttgata tttggttaga gggtactttg 1020
tacccgaccg tgcgcatttg cgcgggcgtc cttttatctt tctacattta tcattccatc 1080
ttgcgtgtaa tcgatatggt gtgcaatcgc tgtcatcaca ctcacgctgg tgggtgtcgg 1140
tcctgccgac agtgccaccc gagagatgct gctccaccac caccgagagc tcgagctaga 1200
gctcaaaacg tggtagcgcg aggtttagcg cgaccagaga cttcagctag ggagccgagg 1260
aggcttcaat ggaccgtgat aggtccgaat gaggtaccac gagtaccgag gggatacgtg 1320
gcacatagca acagagaagt tgttgcgact agcgctggga agtttctaca tgtgaacttc 1380
agcacgactt tcccgcaact attaggattg aatcttagga ttctctccgt ggtagttcga 1440
gccagctgcc tggtatccgc tgggtgggtg ggaatgttgg aggactttga tgagaatcat 1500
ctcagaggtc cgagtgcctt gtccaggaag ggttttcgcc aagaccaacc gagaggttgg 1560
caatggttgg ctccttccga tttagaatac gatacgtttg cgaactcgca ccgtttagta 1620
ttcgaagtca agaacgaatt cgcggcaggc gcgaaagttc ttgtgaggga catctatata 1680
gtggtaaatg atttaccacg aattgtgatc ccgaatgata tccttatggt cgatgaagac 1740
cttttggatg tctaggttga gatgaacgca accgaacccc gataagggta gaaccggacc 1800
accgcgtggg tttttccggt tatctccgag agtcgctttc cagactcatg tttaggagat 1860
tacctacgaa agtagatgcc caatccgtga aggattggat gccccgttag ggaagc 1916
Claims (10)
1. a kind of method for building apple necrosis mosaic virus full length infectious cDNA, comprises the following steps：Apple is bad
CDNA sequence corresponding to RNA1, RNA2 and RNA3 in the triad genome of dead mosaic virus is cloned into plant binary table respectively
Up in carrier, three recombinant expression carriers are obtained；
Three recombinant expression carriers form apple necrosis mosaic virus infectious cDNA clone；
Wherein, cDNA sequence corresponding to the RNA1 in the triad genome of apple necrosis mosaic virus is sequence 1 in sequence table
Or sequence 4；CDNA sequence corresponding to RNA2 in the triad genome of apple necrosis mosaic virus be sequence table in sequence 2 or
Sequence 5；CDNA sequence corresponding to RNA3 in the triad genome of apple necrosis mosaic virus is sequence 3 or sequence in sequence table
Row 6.
2. according to the method for claim 1, it is characterised in that：In three recombinant expression carriers, start the apple
The promoter of cDNA sequence transcription is corresponding to RNA1, RNA2 and RNA3 in the triad genome of fruit necrosis mosaic virus
35S promoter.
3. method according to claim 1 or 2, it is characterised in that：The plant binary expression vector carries for pCASS4-Rz
Body.
4. the apple necrosis mosaic virus infectious cDNA clone being prepared using any methods described in claim 1-3.
5. apple necrosis mosaic virus infectious cDNA clone according to claim 4, it is characterised in that：In the apple
In downright bad mosaic virus infectious cDNA clone, the mol ratio of three recombinant expression carriers is 1:1:1.
6. the recombinant bacterium containing the apple necrosis mosaic virus infectious cDNA clone of claim 4 or 5.
7. recombinant bacterium according to claim 6, it is characterised in that：The recombinant bacterium is to contain the apple necrosis mosaic disease
The Agrobacterium of malicious infectious cDNA clone.
8. the restructuring described in the apple necrosis mosaic virus infectious cDNA clone of claim 4 or 5 or claim 6 or 7
Application of the bacterium in single virus infection plant model is prepared；The single virus is apple necrosis mosaic virus.
9. the restructuring described in the apple necrosis mosaic virus infectious cDNA clone of claim 4 or 5 or claim 6 or 7
Application of the bacterium in apple necrosis mosaic virus pathogenesis is studied.
10. the restructuring described in the apple necrosis mosaic virus infectious cDNA clone of claim 4 or 5 or claim 6 or 7
Bacterium is in research apple necrosis mosaic virus and the application in host plant interaction.
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