CN108753815A - A kind of method that big flux screening plant virus replicates required host's gene - Google Patents

Abstract

A kind of method that big flux screening plant virus replicates required host's gene, belongs to gene engineering technology field.It is low that the present invention is directed to virus infection efficiency in current infectious clone construction method, gene expression problem rambunctious, the present invention provides a kind of methods that big flux screening plant virus replicates required host's gene, inducible promoter structure induction type viral infectivity is specifically utilized to clone, pass through agrobacterium mediation converted plant, mutant library is built with genetically modified plants, after mutant germination, virus infection is induced using derivant, carry out the screening of mutant, obtain the mutant plant that virus cannot be induced, in conjunction with the positioning of the method for map based cloning and genome sequencing and the required host's gene of cloning plant virus duplication, the present invention is suitable for disposable big flux screening plant virus and replicates required host's gene.

Description

A kind of method that big flux screening plant virus replicates required host's gene

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of big required host of flux screening plant virus duplication The method of gene.

Background technology

The RNA of plant positive sense RNA virus have infectivity, into plant cell after can be used as ribosomes of the mRNA by cell Translation generates virus replication required protein (Flasinski et al., 1996), and then starts the duplication of virus, movement Etc. life processes.Plant positive sense RNA virus can be transformed by building infectious clone to facilitate long-term preservation (Flasinski et al.,1996,Takahashi et al.,1997).Infecting there are many plant positive sense RNA virus at present Property clone be reported, summary method can be classified as two classes：1) cDNA of the full-length genome of virus is inserted into constitutive promoter, Zou cauliflowers mosaic virus (CaMV) 35S promoter downstream is such as spent, virus genome RNA is expressed in plant by Agrobacterium；2) will The cDNA of the full-length genome of virus is inserted into the downstream protokaryon transcripting promoter (such as SP6 and T7), and virus is obtained by in-vitro transcription Then full-length genome RNA passes through mechanical friction inoculated plant.There is following defects for above two method：1) composing type starts Son starts to express once entering cell, therefore controls the time of expression with being unable to conditionity；2) due to the physical feature of RNA, The viral RNA that protokaryon transcripting promoter transcription generates is very easy to be degraded by RNA enzyme in environment, causes virus infection efficiency Lowly.

Plant base can theoretically be covered by screening a kind of plant virus and replicating required host's gene and need to build one Because organizing the mutant library of all genes, then by virus inoculation one by one, the mutant of the viral not reproducible of screening obtains.It is this Method needs largely to plant mutant to certain plant height and carries out virus inoculation one by one, need there are an apparent defect Very big manpower, speed is slow, greenhouse and experiment material.

Invention content

Low, gene expression problem rambunctious, this hair for virus infection efficiency in current infectious clone construction method It is bright to provide a kind of method that big flux screening plant virus replicates required host's gene, include the following steps：

1) structure of induction type viral infectivity clone：The expression vector includes agrobatcerium T-DNA left margin sequence, agriculture Bacillus T-DNA right border sequences, inducible promoter, plant positive sense RNA virus full-length cDNA, eukaryotic transcription terminator, large intestine Bacillus and Agrobacterium replicate required element and resistance screening gene, wherein plant positive sense RNA virus full-length cDNA is located at induction type Between promoter and eukaryotic transcription terminator sequence, agrobatcerium T-DNA right border sequence is located at inducible promoter upstream, agriculture bar Bacterium T-DNA left margin sequences are located at eukaryotic transcription terminator downstream；The induction type viral infectivity Cloning Transformation large intestine bar of acquisition In bacterium host strain, positive transformant is obtained；

2) genetic transformation of induction type viral infectivity clone：The positive transformant that step 1) is obtained imports Agrobacterium, Then induction type viral infectivity clone is transferred in plant, obtains genetically modified plants；

3) plant virus replicates the big flux screening of indispensable gene：Mutant library, mutant hair are built with genetically modified plants After bud, virus infection is induced using derivant, carries out the screening of mutant, obtains the mutant plant that virus cannot be induced, Determine that the plant virus of screening replicates indispensable gene in conjunction with the method for map based cloning and genome sequencing.

Preferably, the host strain of the Escherichia coli is Escherichia coli Trans10, and the Agrobacterium is Agrobacterium GV3101。

Preferably, the agrobatcerium T-DNA left margin sequence such as SEQ ID NO:Shown in 1；On the right of the agrobatcerium T-DNA Boundary's sequence such as SEQ ID NO:Shown in 2；The Escherichia coli and Agrobacterium replicate the sequence such as SEQ ID NO of required element:3 It is shown.

Preferably, step 1) the plant positive sense RNA virus is potyvirus or potexvirus disease Poison；The potyvirus include Brassica 2 et 4, marmor upsilon, marmor solani, plumpox virus, Papaya ringspot virus, soybean mosaic virus and marmor erodens；The potexvirus virus includes potato X Virus, potato Ao Guba mosaic virus, garcinia mangostana mosaic virus, plantago mosaic virus and foxtail mosaic virus.

Preferably, step 1) the plant positive sense RNA virus full-length cDNA is the full-length cDNA of Brassica 2 et 4, sequence Row such as SEQ ID NO:Shown in 4.

Preferably, step 1) the plant positive sense RNA virus full-length cDNA is the full-length cDNA of potato virus X, sequence Row such as SEQ ID NO:Shown in 5.

Preferably, the inducible promoter described in step 1) is estrogen-inducible promoter XVE, sequence such as SEQ ID NO:Shown in 6.

Preferably, step 1) the resistance screening gene is hygromycin gene, sequence such as SEQ ID NO:It is described shown in 7 Hygromycin gene, upstream are estrogen-inducible promoter XVE-1, sequence such as SEQ ID NO:It is female downstream shown in 8 Hormone inducible promoter XVE-2, sequence such as SEQ ID NO:Shown in 9；Plant positive sense RNA virus overall length described in step 1) The promoter of cDNA, upstream are estrogen-inducible promoter XVE-2.

Preferably, step 1) the eukaryotic transcription terminator is T3A terminators, sequence such as SEQ ID NO:Shown in 10.

Preferably, the mutant screening described in step 3) uses estrogen revulsion.

It is to utilize agrobacterium-mediated transformation that induction type viral infectivity clone described in step 1) of the present invention, which is transferred in plant, Or particle bombardment realization, the conversion plant described in step 2) is the arabidopsis Col-0 ecotypes or Nicotiana tabacum；Step 3) is described Mutant library construction method use T-DNA methods or EMS mutagenesis；The plant virus replicates the screening of required host's gene It is to determine target gene in conjunction with map based cloning and genome sequencing method, when the plant of conversion is arabidopsis, passes through gene The gene obtained is positioned, is inserted into the T-DNA sequences with herbicide resistance gene in the gene, it is determined that it is multiple for plant virus The required host's gene of system；When the plant of conversion is tobacco, by the gene of assignment of genes gene mapping acquisition, the gene and reference gene (i.e. unmutated gene) nucleotide sequence is compared to that can cause, coded amino acid changes or coded amino acid shifts to an earlier date eventually Coding mutation only, it is determined that it replicates required host's gene for plant virus.

Advantageous effect

The present invention relates to a kind of big flux screening plant positive sense RNA virus to replicate, moves relevant required host's gene Method.This method can disposably carry out the screening of mass mutation body, therefore can save a large amount of time, experiment material and funds, Improve efficiency.

Description of the drawings

Fig. 1 induction type TuMV infectious clone carriers XVE::TuMV-GFP Vector maps.

Fig. 2 induction type PVX infectious clone carriers XVE::PVX-GFP Vector maps.

Specific implementation mode

LB culture mediums：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, PH7.5；

LB solid mediums：Every liter of LB liquid medium adds 15g~20g agar powders；

MS solid mediums：Purchased from PhytoTech companies of the U.S., article No.：M401；

Gibson Assembly Master Mix kits are bought from NEB Biolabs companies, article No.：E2611；

Escherichia coli Trans10 competent cells are bought from Quan Shijin Bioisystech Co., Ltd, article No.：CD101；

The extraction of EasyPure Plasmid MiniPrep Kit kits is bought from Quan Shijin Bioisystech Co., Ltd, Article No.：EM101；

The extraction of EasyPure PCR Purification Kit kits is bought from Quan Shijin Bioisystech Co., Ltd, Article No.：EP101；

Estrogen derivant is bought from Sigma-Aldrich, article No.：E8875；

Herbicide is bought from Beijing Ku Laibo Science and Technology Ltd.s, article No.：CB2471；

Growth chamber is bought from Wuhan Rui Hua instrument and equipments Co., Ltd, article No.：AR1200 is used for cultivating quasi- The condition of southern mustard is：Photoperiod is illumination in 18 hours, 6 hours dark；Temperature is 23 DEG C；

Black light lamp is bought from UVP companies of the U.S., article No. UVGL-55；

Other reagents or instrument unless otherwise specified, can be bought by existing commercialization approach and be obtained.

Embodiment 1：The method that big flux screening Brassica 2 et 4 replicates required host's gene.

Step 1. induction type Brassica 2 et 4 (Turnip mosaic virus, TuMV) infectivity expression vector establishment.

1) in order to which more intuitive simplicity has the genetically modified crops offspring of induction type viral vectors to screen conversion, this reality It is template to apply example and utilize the TuMV infectivity plasmids pCambia-TuMV-GFP with green fluorescent label gene GFP labels, should One is contained in pCambia-TuMV-GFP plasmids between encoding P1 and HcPro albumen inserted with encoding green fluorescent protein (GFP) the TuMV genomes (TuMV-GFP) of sequence, are shown in SEQ ID NO:Shown in 11, the infectivity plasmid pCambia- TuMV-GFP is documented in Cotton S, Grangeon R, Thivierge K, et al., 2009.Turnip mosaic virus RNA replication complex vesicles are mobile,align with microfilaments,and are each derived from a single viral genome.J Virol 83,10460-71。

Fluorescent marker gene is not added, can equally realize the performance of above-mentioned vector construction and function vector, mutant It identifies then after Genetic Transformation in Higher Plants, using corresponding screening pressure or inductive condition (such as the screening of herbicide or estrogen Under induction) it is carried out by the methods of character analysis and Molecular Detection.

With following primer sequence is denoted as to carrying out PCR reactions, the sequence between amplification TuMV-GFP genomes 1-6487nt A, including the segment total size of estrogen-inducible promoter sequence is 6507bp.

The primer pair is：

Primer 1：(lower-case portion is 5 '-gttcatttcatttggagaggAAAAAATATAAAAACTCAACATAAC-3 ' Estrogen-inducible promoter sequence)；

Primer 2：5′-TTGATGGTGAACTGCCTCAAGAGC-3′；

With following primer to carrying out PCR reactions, TuMV genome 6464-10578nt and polyA sequences are expanded, are denoted as Sequence B, including the segment total size of TuMV-GFP genome polyA sequences and pMDC sequences are 4213bp.

The primer pair is：

Primer 3：5′-GCTCTTGAGGCAGTTCACCATCAA-3′；

Primer 4：(upper-case portion comes from 5 '-gacgaaagctgggaggcctgGTCGACTCTAGGGGGATCCGA-3 ' PCambia-TuMV-GFP plasmids, lower-case portion come from pMDC7)；

Above-mentioned PCR reaction conditions are：95 DEG C of pre-degeneration 3min, into PCR cycle：95 DEG C of denaturation 15sec, 65 DEG C of annealing 10sec, 72 DEG C of extension 3.30min, 30 cycles；Last 72 DEG C of extensions 7min.

It is detected with 1% agarose gel electrophoresis after PCR reactions and recycles PCR product.The PCR product recycling is using complete The EasyPure PCR Purification Kit kits of Shi Jin Bioisystech Co., Ltd.

2) using the plant binary expression vector pMDC7 with estrogen-inducible promoter XVE as masterplate, which records In Curtis MD, Grossniklaus U, 2003.A gateway cloning vector set for high- throughput functional analysis of genes in planta.Plant Physiol 133,462-9。

With following primer to carrying out PCR reactions, expand in pMDC7 comprising XVE inducible promoters, plant hygromycin selection Gene, agrobatcerium T-DNA left margin, Escherichia coli and Agrobacterium replicate and resistance screening sequence, Agrobacterium T DNA right margins with And sequence between T3A terminators, it is denoted as sequence C, clip size 11447bp.

The primer pair is：

Primer 5：5′-cctctccaaatgaaatgaacttc-3′；

Primer 6：5′-caggcctcccagctttcgtccgtatc-3′.

PCR reaction conditions：95 DEG C of pre-degeneration 3min, into PCR cycle：95 DEG C of denaturation 15sec, 65 DEG C of 10sec that anneal, 72 DEG C extend 6min, 30 cycle；Last 72 DEG C of extensions 7min.

3) with reference to Gibson Assembly Master Mix kit specifications, using the method for homologous recombination by sequence A, sequence B and sequence C are spliced into an induction type TuMV infectious clone carrier, and sequence D is named as XVE::TuMV-GFP is shown in Shown in Fig. 1, wherein plant hygromycin selection gene order is located inside inducible promoter XVE, hygromycin gene upstream Promoter be estrogen-inducible promoter XVE-1, sequence such as SEQ ID NO:Shown in 8；On TuMV full length cDNA sequences The promoter of trip is estrogen-inducible promoter XVE-2, sequence such as SEQ ID NO:Shown in 9；The full-length cDNA position of TuMV Between XVE inducible promoter XVE-2 and T3A terminators, 1229-1972nt in the full-length cDNA of TuMV-GFP, altogether 744nt, for the GFP fluorescent marker genes of insertion, agrobatcerium T-DNA right margin RB is located at estrogen-inducible promoter XVE-1 Upstream, agrobatcerium T-DNA left margin LB are located at the downstreams eukaryotic transcription terminator T3A.

The 258-1709bp of wherein XVE-1 encodes a manual transcription factor, and the manual transcription factor is by large intestine bar The DNA binding structural domains of bacterium LexA albumen, 3 part group of the protein transcription activation structure domains VP16 and human estrogen receptor structural domain At.The DNA binding structural domains of LexA albumen have the function of identifying XVE-2 sequences, the protein transcription activation structure domains VP16 and people Oestrogen receptor domain component part has estrogen-dependent transcriptional activation function, i.e., can be with initial when combining estrogen The transcription of TuMV full-length cDNAs gene after XVE-2.

The agrobatcerium T-DNA left margin LB, sequence such as SEQ ID NO:Shown in 1；Agrobatcerium T-DNA right border sequence RB, sequence such as SEQ ID NO:Shown in 2；Escherichia coli and Agrobacterium replicate required element, sequence such as SEQ ID NO:3 institutes Show；The full-length cDNA of TuMV, sequence such as SEQ ID NO:Shown in 4；Estrogen-inducible promoter XVE, sequence such as SEQ ID NO:Shown in 6；Resistance screening gene hygromycin gene, sequence such as SEQ ID NO:Shown in 7；Estrogen-inducible promoter XVE-1, sequence such as SEQ ID NO:Shown in 8；Estrogen-inducible promoter XVE-2, sequence such as SEQ ID NO:9 institutes Show；Eukaryotic transcription terminator T3A, sequence such as SEQ ID NO:Shown in 10.

By XVE::TuMV-GFP carriers convert in Escherichia coli Trans10 competent cells, and operating method is said according to product Bright book carries out.After conversion, the LB solids training containing kanamycins (final concentration of 50 μ g/ml) is applied to after being cultivated 1 hour in 37 DEG C Support base, 37 DEG C of overnight incubation cultures.

With bacterium colony PCR screening positive clones.Bacterium colony PCR reaction primer pair used is：Primer 7：5′- ggctttcaagcgccagtgcgag-3′；Primer 8：5′-gaagtgctcttgcacaagggtg-3′；The target fragment of amplification is big Small is 824bp.

Bacterium colony PCR reaction systems are 50 μ L, wherein 2 × Taq PCR mix, 25 μ L, 8 each 1 μ L of primer 7 and primer, masterplate For the escherichia coli cloning of conversion, remaining is aqua sterilisa.Reaction condition：94 DEG C of pre-degeneration 3min, into PCR cycle：94 DEG C of changes Property 30sec, 60 DEG C annealing 20sec, 72 DEG C extension 1min, 30 cycle；Last 72 DEG C of extensions 7min.

The positive colony of acquisition, with the LB liquid medium containing kanamycins (final concentration of 50 μ g/ml), 37 DEG C overnight Culture, EasyPure Plasmid MiniPrep Kit kits extract plasmid, serve Hai Sheng works Bioisystech Co., Ltd into Row sequencing identifies that correct plasmid is named as XVE::TuMV-GFP, i.e. induction type Brassica 2 et 4 (Turnip Mosaic virus, TuMV) infectivity expression vector.

The genetic transformation of step 2. induction type Brassica 2 et 4 (TuMV) infectivity expression vector

1) reference《Molecular Cloning:A Laboratory guide》, with electric shocking method by XVE::TuMV-GFP imports Agrobacterium GV3101 strains (purchased from Beijing Hua Yue ocean biology), is coated on after conversion containing final concentration of 50 μ g/ml kanamycins and final concentration of 100 μ g/ On the LB solid mediums of ml rifampins, 28 DEG C culture to grow clone.6 clones of random picking carry out PCR detections, reactant System, reaction condition isogeneous induction type Brassica 2 et 4 (Turnip mosaic virus, TuMV) infectious clone construction step 1 In 3) described in bacterium colony PCR.

2) with containing XVE::The Agrobacterium of TuMV-GFP plasmids impregnates arabidopsis Colombia type environmental (Col-0) flower After sequence 10sec, growth chamber is put back to until solid, the seed for the inflorescence that collection is handled through Agrobacterium.The arabidopsis dips in flower Method for transformation is with reference to Clough SJ, Bent AF, 1998.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The Plant journal:for cell and molecular biology 16,735-43.。

3) reference Harrison SJ, Mott EK, Parsley K, Aspinall S, Gray JC, Cottage A, 2006.A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation.Plant Methods 2, 19.Takahashi Y,Takahashi T,Uyeda I,1997.A cDNA clone to clover yellow vein The arabidopsis seed that potyvirus genome is highly infectious.Virus genes 14,235-43. are provided The arabidopsis seed of collection is after 10% hydrogen peroxide carries out disinfection by the method for disinfection and screening with volume fraction, and tiling is to containing On the MS solid mediums for having the final concentration of 15mg/L of hygromycin, after putting to 4 DEG C of vernalization 2 days 22 DEG C of plant incubator cultures into Row illumination in 6 hours, dark, illumination in 1 day photoperiod processing in 2 days, filter out resistant plant, acquisition turns XVE::TuMV-GFP's Arabidopsis positive plant.

4) positive plant is transferred in cultivation soil, plantation is to solid, then carries out the screening of the second generation and the sieve of the third generation Choosing, until obtaining the homozygous third generation genetically modified plants no longer detached, the screening of the second generation, the third generation is the same as the first generation Screening technique.

TuMV replicates the big flux screening of required host's gene in step 3. arabidopsis

1) with from arabidopsis Bio-Centers (ABRC；https://www.arabidopsis.org) purchase pDAP101 matter Grain converts Agrobacterium GV3101 strains, and Herbicid resistant is carried in the pDAP101, between D-DNA right boundary sequences, The same reference of concrete operations《Molecular Cloning:A Laboratory guide》, electric shocking method conversion Agrobacterium.

2) using step 2 obtain transgenic arabidopsis as material, in step 2 2) described in method, conversion is had The Agrobacteriums of pDAP101 plasmids carries out the ecotypic conversions of arabidopsis Col-0, and to after solid, collection is handled through Agrobacterium for culture Inflorescence seed；

3) 2) after the arabidopsis seed of middle collection being soaked in water, after 4 DEG C of placement vernalization 2 days, directly sowing is native to cultivation In, it is sprayed with the herbicide basta aqueous solution of 10mg/L after all germinateing and (every other day sprays once, spray 3 altogether It is secondary), transfer-gen plant of the screening with antiweed Basta resistances obtains a large amount of anti-transgenic arabidopsis generation seeds；By institute There is generation seed to be planted, and collects second generation seed, structure Arabidopsis Mutants library.

4) according in step 2 3) described in the second generation seed collected of method processing, tiling is on MS solid mediums, 22 DEG C illumination cultivation sprays 1 times/day to germinateing with the estrogen aqueous solution derivant of 10mg/L, is sprayed in the daily same time, even It is 2 days continuous, Brassica 2 et 4 genomic expression is induced using estrogen derivant, is sieved under dark condition with black light lamp after 2 weeks The Arabidopsis Mutants for selecting no GFP expression, are transferred in cultivation soil, plant to solid.

5) 4) the middle Arabidopsis Mutants that obtain are planted to blooming, the ripe flower shears for exposing petal is gone, is left Calyx, petal, stamen are all removed with tip tweezers, only leave with pistillate ovary, carefully handling by also immature flower The ripe flower from wildtype Arabidopsis thaliana orchid moral Burger environmental (Ler) is buckled in complete ovary, allows Col-0 arabidopsis mutants The ovum of body and the pollen of Ler are hybridized, and hybrid seed is obtained；All first generation seeds are planted, and collect second For seed, structure filial generation group.

6) 5) the middle progeny population that obtains is planted, every plant of individually sampling, using full formula gold biotechnology EasyPure Plant Genomic DNA Kit extraction genomic DNAs, reference Li Q, Shen WY, Zheng Q, Tan YF, Gao J,Sheng JX,Wei YD,Kunst L,Zou JT.2017.Effects of eIFiso4G1mutation on seed oil biosynthesis.Plant J,90(5):966-978, by the method for map based cloning and genome sequencing, The position positioning of target gene is carried out, when detecting the gene for inserting the sequences of the T-DNA with herbicide resistance gene, then really Fixed its replicates required host's gene for plant virus.

The method that 2. big flux screening potato virus X of embodiment replicates required host's gene.

Embodiment 1 is repeated, is with the difference of embodiment 1, the induction type viral infectivity clone described in the present embodiment For potato virus X (Potato virus X, PVX) infectious clone with GFP labels：With the Ma Ling with GFP labels Potato X viral infectivities clone is masterplate (PVX.GFP-2A-CP), sees SEQ ID NO：Shown in 12, the infectivity plasmid is recorded In Cruz SS, Chapman S, Roberts AG, Roberts IM, Prior DAM, Oparka KJ.1996.Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.Proc.Natl.Acad.Sci.USA,93:6286-6290. primers 9 and primer 10 expand 1-3953nt sequences, It is denoted as sequence E (including estrogen-inducible promoter partial sequence, total length 3973nt), is expanded with primer 11 and primer 12 3933-3987nt sequences are denoted as sequence F (including T3A terminators partial sequence in polyA and pMDC7 carriers, total length 3109nt)。

Fluorescent marker gene is not added, can equally realize the performance of above-mentioned vector construction and function vector, mutant Identification then by character analysis and is divided after Genetic Transformation in Higher Plants using corresponding inductive condition (under the induction of such as estrogen) Son detection the methods of come carry out.

Primer 9：5 '-gttcatttcatttggagaggGAAAACTAAACCATACACCACCAACACAACC-3 ' (small letters Part is estrogen-inducible promoter sequence)；

Primer 10：GATAACGTGCCTAGGAAAATCTG

Primer 11：GATTTTCCTAGGCACGTTATC

Primer 12：(upper-case portion comes from 5 '-gacgaaagctgggaggcctgGGAAATTCGCGGTACCCACTAG-3 ' In PVX.GFP-2A-CP plasmids, lower-case portion comes from pMDC7)

With reference to Gibson Assembly Master Mix kit specifications, using homologous recombination method by sequence E, Sequence F and sequence C are spliced into an induction type PVX infectious clone carrier, are named as XVE::PVX-GFP, as shown in Figure 2, with 1 difference of embodiment is that in the present embodiment in constructed induction type PVX infectious clone carriers, plant positive sense RNA virus is complete Long cDNA is the full-length cDNA of potato virus X, sequence such as SEQ ID NO:Shown in 5.In the full-length cDNA of PVX-GFP 5671-6210nt, total 540nt, for the GFP fluorescent marker genes of insertion.

The plant of conversion is tobacco, the XVE described in step 2::PVX passes through agrobacterium mediation converted to tobacco In (Nicotiana tobacum) cell, specific method is with reference to Zhao Li, Zhong Ming, Ma Hui, Li Haoge, Chen Lijing, stroke .2011. The foundation of agriculture bacillus mediated tobacco efficient genetic trasformation system, Jiangsu's agriculture science .39 (3):67-68,74；In step 3) into Row, when PVX replicates the big flux screening of indispensable gene, mutant library will use EMS revulsions；The method according to Shi Yuewei, The brave .2009. gamma-rays of Ren Xueliang, Wang Yi, Sun Yanxia, Wu Xiao and the mutagenesis that EMS is single and combined processing is to tobacco seed vigor Effect Guizhou Agricultural Sciences, 12:The method of 62-65, description carry out.The mutant library of structure is without herbicide screening, directly Estrogen aqueous solution (15mg/L) induction expressing viral is sprayed, is sprayed once in the daily identical time, continuous 2 days, 2 Zhou Houyong Black light lamp screens the tobacco mutant body of no GFP expression under dark condition, is transferred in cultivation soil, plants to solid.Hybridization Tobacco used is three lives cigarette (Nicotiana tobacci cv.Samsun).Remaining method is with reference to embodiment 1.

By the method for map based cloning and genome sequencing, the position positioning of target gene is carried out, when detecting the base Because with reference gene (sequence do not change before gene) nucleotide sequence compared to carrying and coded amino acid can be caused to change Or the coding mutation that coded amino acid terminates in advance, it is determined that it replicates required host's gene for plant virus.

Using identical strategy and method, big flux screening plant virus of the present invention replicates required host's gene Method is suitable for other potyvirus in addition to Brassica 2 et 4 (TuMV) and potato virus X (PVX) With potexvirus virus.The potyvirus includes TuMV, marmor upsilon (Potato virus Y, PVY；Genbank accession number：NC_001616.1), marmor solani (Potato virus A, PVA；Genbank is logged in Number：NC_004039.1), plumpox virus (Plum pox virus, PPV；Genbank accession number：NC_001445.1), papaya Ring spot virus (Papaya ring spot virus, PRSV；Genbank accession number：NC_001785.1), soybean mosaic virus (Soybean mosaic virus, SMV；Genbank accession number：) and marmor erodens (Tobacco NC_002634.1 Etch virus, TEV；Genbank accession number：NC_001555.1)；The potexvirus virus includes PVX, Ma Ling Potato Ao Guba mosaic virus (Potato aucuba mosaic virus, PAMV；Genbank accession number：NC_003632.1), Garcinia mangostana mosaic virus (Pepino mosaic virus, PepMV；Genbank accession number：NC_004067.1), Asiatic plantain floral leaf Virus (Plantago asiatica mosaic virus, PlAMV；Genbank accession number：) and foxtail NC_003849.1 Mosaic virus (Foxtail mosaic virus, FoMV；Genbank accession number：NC_001483.1).

The genome cDNA overall length of above-mentioned virus is obtained by PCR amplification, with reference to step 1 institute in embodiment 1 and embodiment 2 The method stated is terminated using homologous recombination technique structure containing inducible promoter, plant positive sense RNA virus cDNA and T3A The induction type viral infectivity expression vector of son.

With reference to apply 2 step 2 of example 1 and embodiment, the method described in 3 screens the plant virus and replicates required host's gene.

Present approach provides a kind of methods that big flux screening plant virus replicates required host's gene, using we Method can be achieved at the same time is carried out at the same time screening to a large amount of (10,000 plants or more plants).Every plant of carry out machinery of mutant is connect originally relatively The method of kind, can greatly improve screening efficiency.

Sequence table

<110>Northeast Agricultural University

<120>A kind of method that big flux screening plant virus replicates required host's gene

<130>

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213>Agrobatcerium T-DNA left margin

<400> 1

gtttacacca caatatatcc tgcca 25

<210> 2

<211> 26

<212> DNA

<213>Agrobatcerium T-DNA right border sequence

<400> 2

gtttacccgc caatatatcc tgtcaa 26

<210> 3

<211> 6510

<212> DNA

<213>Escherichia coli and Agrobacterium replicate the sequence of required element

<400> 3

ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact cgatacaggc 60

agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca gactttgctc 120

atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa acacggatga 180

tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc tgtgatcaat 240

tcgggcacga acccagtgga cataagcctc gttcggttcg taagctgtaa tgcaagtagc 300

gtaactgccg tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc 360

agtggcggtt ttcatggctt cttgttatga catgtttttt tggggtacag tctatgcctc 420

gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca 480

acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg ggggaagcgg 540

tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg 600

aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac 660

acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag 720

ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg 780

ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc 840

gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag 900

ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct 960

tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg 1020

cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa 1080

atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga 1140

aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac 1200

ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc 1260

agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataat 1320

gtctagctag aaattcgttc aagccgacgc cgcttcgccg gcgttaactc aagcgattag 1380

atgcactaag cacataattg ctcacagcca aactatcagg tcaagtctgc ttttattatt 1440

tttaagcgtg cataataagc cctacacaaa ttgggagata tatcatgcat gaccaaaatc 1500

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 1560

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 1620

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 1680

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 1740

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 1800

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 1860

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 1920

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 1980

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 2040

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 2100

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 2160

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 2220

gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 2280

cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg 2340

atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc 2400

agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg 2460

actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 2520

gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 2580

agaggttttc accgtcatca ccgaaacgcg cgaggcaggg tgccttgatg tgggcgccgg 2640

cggtcgagtg gcgacggcgc ggcttgtccg cgccctggta gattgcctgg ccgtaggcca 2700

gccatttttg agcggccagc ggccgcgata ggccgacgcg aagcggcggg gcgtagggag 2760

cgcagcgacc gaagggtagg cgctttttgc agctcttcgg ctgtgcgctg gccagacagt 2820

tatgcacagg ccaggcgggt tttaagagtt ttaataagtt ttaaagagtt ttaggcggaa 2880

aaatcgcctt ttttctcttt tatatcagtc acttacatgt gtgaccggtt cccaatgtac 2940

ggctttgggt tcccaatgta cgggttccgg ttcccaatgt acggctttgg gttcccaatg 3000

tacgtgctat ccacaggaaa gagacctttt cgaccttttt cccctgctag ggcaatttgc 3060

cctagcatct gctccgtaca ttaggaaccg gcggatgctt cgccctcgat caggttgcgg 3120

tagcgcatga ctaggatcgg gccagcctgc cccgcctcct ccttcaaatc gtactccggc 3180

aggtcatttg acccgatcag cttgcgcacg gtgaaacaga acttcttgaa ctctccggcg 3240

ctgccactgc gttcgtagat cgtcttgaac aaccatctgg cttctgcctt gcctgcggcg 3300

cggcgtgcca ggcggtagag aaaacggccg atgccgggat cgatcaaaaa gtaatcgggg 3360

tgaaccgtca gcacgtccgg gttcttgcct tctgtgatct cgcggtacat ccaatcagct 3420

agctcgatct cgatgtactc cggccgcccg gtttcgctct ttacgatctt gtagcggcta 3480

atcaaggctt caccctcgga taccgtcacc aggcggccgt tcttggcctt cttcgtacgc 3540

tgcatggcaa cgtgcgtggt gtttaaccga atgcaggttt ctaccaggtc gtctttctgc 3600

tttccgccat cggctcgccg gcagaacttg agtacgtccg caacgtgtgg acggaacacg 3660

cggccgggct tgtctccctt cccttcccgg tatcggttca tggattcggt tagatgggaa 3720

accgccatca gtaccaggtc gtaatcccac acactggcca tgccggccgg ccctgcggaa 3780

acctctacgt gcccgtctgg aagctcgtag cggatcacct cgccagctcg tcggtcacgc 3840

ttcgacagac ggaaaacggc cacgtccatg atgctgcgac tatcgcgggt gcccacgtca 3900

tagagcatcg gaacgaaaaa atctggttgc tcgtcgccct tgggcggctt cctaatcgac 3960

ggcgcaccgg ctgccggcgg ttgccgggat tctttgcgga ttcgatcagc ggccgcttgc 4020

cacgattcac cggggcgtgc ttctgcctcg atgcgttgcc gctgggcggc ctgcgcggcc 4080

ttcaacttct ccaccaggtc atcacccagc gccgcgccga tttgtaccgg gccggatggt 4140

ttgcgaccgt cacgccgatt cctcgggctt gggggttcca gtgccattgc agggccggca 4200

gacaacccag ccgcttacgc ctggccaacc gcccgttcct ccacacatgg ggcattccac 4260

ggcgtcggtg cctggttgtt cttgattttc catgccgcct cctttagccg ctaaaattca 4320

tctactcatt tattcatttg ctcatttact ctggtagctg cgcgatgtat tcagatagca 4380

gctcggtaat ggtcttgcct tggcgtaccg cgtacatctt cagcttggtg tgatcctccg 4440

ccggcaactg aaagttgacc cgcttcatgg ctggcgtgtc tgccaggctg gccaacgttg 4500

cagccttgct gctgcgtgcg ctcggacggc cggcacttag cgtgtttgtg cttttgctca 4560

ttttctcttt acctcattaa ctcaaatgag ttttgattta atttcagcgg ccagcgcctg 4620

gacctcgcgg gcagcgtcgc cctcgggttc tgattcaaga acggttgtgc cggcggcggc 4680

agtgcctggg tagctcacgc gctgcgtgat acgggactca agaatgggca gctcgtaccc 4740

ggccagcgcc tcggcaacct caccgccgat gcgcgtgcct ttgatcgccc gcgacacgac 4800

aaaggccgct tgtagccttc catccgtgac ctcaatgcgc tgcttaacca gctccaccag 4860

gtcggcggtg gcccatatgt cgtaagggct tggctgcacc ggaatcagca cgaagtcggc 4920

tgccttgatc gcggacacag ccaagtccgc cgcctggggc gctccgtcga tcactacgaa 4980

gtcgcgccgg ccgatggcct tcacgtcgcg gtcaatcgtc gggcggtcga tgccgacaac 5040

ggttagcggt tgatcttccc gcacggccgc ccaatcgcgg gcactgccct ggggatcgga 5100

atcgactaac agaacatcgg ccccggcgag ttgcagggcg cgggctagat gggttgcgat 5160

ggtcgtcttg cctgacccgc ctttctggtt aagtacagcg ataaccttca tgcgttcccc 5220

ttgcgtattt gtttatttac tcatcgcatc atatacgcag cgaccgcatg acgcaagctg 5280

ttttactcaa atacacatca cctttttaga cggcggcgct cggtttcttc agcggccaag 5340

ctggccggcc aggccgccag cttggcatca gacaaaccgg ccaggatttc atgcagccgc 5400

acggttgaga cgtgcgcggg cggctcgaac acgtacccgg ccgcgatcat ctccgcctcg 5460

atctcttcgg taatgaaaaa cggttcgtcc tggccgtcct ggtgcggttt catgcttgtt 5520

cctcttggcg ttcattctcg gcggccgcca gggcgtcggc ctcggtcaat gcgtcctcac 5580

ggaaggcacc gcgccgcctg gcctcggtgg gcgtcacttc ctcgctgcgc tcaagtgcgc 5640

ggtacagggt cgagcgatgc acgccaagca gtgcagccgc ctctttcacg gtgcggcctt 5700

cctggtcgat cagctcgcgg gcgtgcgcga tctgtgccgg ggtgagggta gggcgggggc 5760

caaacttcac gcctcgggcc ttggcggcct cgcgcccgct ccgggtgcgg tcgatgatta 5820

gggaacgctc gaactcggca atgccggcga acacggtcaa caccatgcgg ccggccggcg 5880

tggtggtgtc ggcccacggc tctgccaggc tacgcaggcc cgcgccggcc tcctggatgc 5940

gctcggcaat gtccagtagg tcgcgggtgc tgcgggccag gcggtctagc ctggtcactg 6000

tcacaacgtc gccagggcgt aggtggtcaa gcatcctggc cagctccggg cggtcgcgcc 6060

tggtgccggt gatcttctcg gaaaacagct tggtgcagcc ggccgcgtgc agttcggccc 6120

gttggttggt caagtcctgg tcgtcggtgc tgacgcgggc atagcccagc aggccagcgg 6180

cggcgctctt gttcatggcg taatgtctcc ggttctagtc gcaagtattc tactttatgc 6240

gactaaaaca cgcgacaaga aaacgccagg aaaagggcag ggcggcagcc tgtcgcgtaa 6300

cttaggactt gtgcgacatg tcgttttcag aagacggctg cactgaacgt cagaagccga 6360

ctgcactata gcagcggagg ggttggatca aagtactttg atcccgaggg gaaccctgtg 6420

gttggcatgc acatacaaat ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc 6480

gttattctaa taaacgctct tttctcttag 6510

<210> 4

<211> 9834

<212> DNA

<213>The full-length cDNA of Brassica 2 et 4 TuMV（Without polyA tails）

<400> 4

aaaaaatata aaaactcaac ataacataca caaaacgatt aaagcaaaca caaatctttc 60

aaagcattca agcaatcaaa gattctcaaa tctttcatcg ttatcaaagc aatcaccaac 120

agcaaaccaa atggcagcag ttacattcgc atcagctatc accaacgcca tcaccagcaa 180

accagcactc accggaatgg tgcagtttgg gagtttccca ccaatgccat tgcgatccac 240

caccgtcacc acagtcgcca cttcagtggc gcaacctaaa ctgtacacag tgcagtttgg 300

aagccttgac ccagtagtcg tcaagagtgg agcagggtcc cttgctaagg caacacgcca 360

gcagcctaac gttgaaatag acgttagcct cagtgaagcc gcagctctgg aggttgcgaa 420

acctagatcg aatgccgtgt tgaggatgca cgaggaagca aacaaggaga gagcactctt 480

tttggactgg gaggctagtt tgaagagaag ctcgtatgga attgctgagg acgagaaggt 540

tgtaatgaca actcatggcg tcagcaagat agtgcccaga agttcaaggg caatgaagct 600

aaagcgcgca agggagaggc gtagagcgca gcaaccaatt atattaaagt gggagcccaa 660

attgagcggg atctcaatcg gaggagggct ctctgcgagc gtaatcgaag cagaagaggt 720

tcgcacaaag tggccgcttc ataagacacc gtcaatgaag aagaggacgg tgcacagaat 780

atgcaagatg aacgaccaag gagttgacat gttgacacga tccctggtta agattttcaa 840

gactaagagt gccaacattg aatacatcgg aaagaagtcg attaaggtcg atttcatcag 900

aaaagaacga acgaaattcg caagaatcca agtagcacac ttactcggga agagagcaca 960

gcgcgacttg ttaactggaa tggaagaaaa ccattttatt gacattctca gtaagtactc 1020

aggtaacaaa acaaccataa atcctggagt agtttgcgca ggttggagtg gcatagtcgt 1080

tggaaatgga attctaaccc agaaacgaag cagaagtcca tcagaggcct ttgtaattag 1140

aggtgagcac gaaggcaagt tgtacgatgc caggatcaaa gtcacgagga caatgagtca 1200

caagattgtg cactttagtg ccgcgggagc caacttctgg aaaggcttcg acagatgctt 1260

tctcgcatac cgtagtgaca atcgcgagca tacatgctat tcagggctag atgtcactga 1320

gtgcggcgag gtggcagcac tgatgtgttt ggctatgttc ccatgcggaa agataacctg 1380

ccctgactgt gtaacagata gtgagctatc ccaaggacaa gcaagcggac catctatgaa 1440

gcacaggttg acacagctac gcgatgtcat caagtcaagc tacccacgct tcaagcatgc 1500

agtgcagata ctagataggt atgagcaatc actgagcagt gcaaacgaga actaccaaga 1560

tttcgcagaa atccagagca taagcgatgg agttgaaaaa gctgcattcc cacacgtcaa 1620

caagctaaac gcaatattga tcaaaggggc cacagtaaca ggagaggaat tctcgcaggc 1680

tacgaagcac ttgctcgaga tagcacgata cctgaagaac agaaccgaga acattgagaa 1740

gggttcactg aagtcctttc gcaacaagat ttcccagaaa gcgcacatca acccaacact 1800

aatgtgtgac aaccagctcg atagaaatgg aaatttcata tggggtgaga gaggatacca 1860

tgcaaaacga ttcttcagca actactttga aataatcgat ccaaagaaag gctacaccca 1920

atacgagaca agagcggtac caaatgggtc acggaaactt gcaatcggca aactaatagt 1980

cccaacgaac ttcgaagttt taagggaaca gatgaaaggc gaaccggtag aaccataccc 2040

agtaacagtc gagtgtgtga gcaagttaca gggtgacttc gtccatgcat gttgttgtgt 2100

cacaacagaa tcaggcgacc cagtcttgtc tgagatcaaa atgccaacca aacaccatct 2160

agtgattggt aacagcggtg atccaaagta catagatctc cctgagatcg aggagaataa 2220

aatgtacata gcgaaagaag gttattgtta catcaatatc ttcctagcca tgttggtaaa 2280

tgtcaaggag tcgcaggcaa aggagttcac gaaagttgtt agggacaaac tagttggcga 2340

acttggcaag tggcccactc tgttagatgt agcaaccgct tgttatttcc tgaaagtatt 2400

ttacccagac gttgctaacg ccgaattgcc acgcatgcta gtggaccata agacaaagat 2460

aattcatgtc gttgattcat atgggtcact gtcaactgga tatcatgtcc ttaagacaaa 2520

cactgtggaa caactcatca aattcacgag atgtaatttg gagtcaagct tgaaacacta 2580

ccgcgttgga ggaacagaat gggaggacac tcatggatcc agcaacatag ataatccaca 2640

gtggtgcatc aagaggctca taaaaggagt ctacaaacca aagcaactga aagaagacat 2700

gttggcaaac cctttcttac cactatatgc tctactgtca ccaggtgtca tcctggcatt 2760

ttacaatagt ggctctctag agtacttgat gaaccattac atcagggtgg acagcaacgt 2820

cgccgttttg ttggtcgttt tgaaatctct agcgaagaag gtgtcaacta gtcagagtgt 2880

gttagcccag cttcaaatca ttgaacgaag tctaccagaa ctcatcgaag caaaggctaa 2940

tgttaatggg ccagatgacg cagccactcg cgcgtgtaac agattcatgg gcatgcttct 3000

gcatatggca gaaccaaact gggagcttgc ggatggtgga tacacaattc tgagggatca 3060

tagcatctcc attttggaaa aaagttatct acaaatcttg gacgaagcat ggaacgagtt 3120

aagttggtcg gagcgctgtg ctataagata ctactcgtca aagcaagcaa tctttacaca 3180

gaaagatttg ccaatgaaaa gcgaagccga tttaggcggc agatacagcg tgtcagtcat 3240

gtcatcttac gaacggagta agcaatgtat gaaaagcgtg cactctagta taggtaatag 3300

attacgtagt agtatgtctt ggactagtag caaggtgtcg aatagtgtgt gtaggactat 3360

taactattta gtaccagatg tgttcaagtt tatgaatgta ctcgtttgta tcagcttact 3420

aatcaagatg actgccgagg cgaatcacat cgtcaccacg caaagaaggc tcaaactaga 3480

tgtcgaggag acagagcgca ggaaaataga atgggagctt gcattccacc atgccattct 3540

gacgcagagt gcaggtcaac acccaacgat agacgagttc agagcgtaca tcgccgacaa 3600

ggcaccacat ctaagtgagc atatcgagcc tgaagaaaag gcggtggttc atcaagcgaa 3660

gagacaatcc gagcaagaac tcgagcgtat aatagcattt gttgcattgg tgctcatgat 3720

gttcgatgca gaacgaagcg actgtgtcac aaagattctc aacaagctta agggactagt 3780

cgccactgtg gaacctacag tctaccatca gactctcaat gatatagagg atgacttgag 3840

tgagaggaac ctcttcgtcg attttgagct tagcagcgat ggagatatgc tccaacagct 3900

tccagccgaa aagacatttg cctcatggtg gagtcatcaa ctaagcagag gattcacaat 3960

cccacactac aggacagaag ggaagttcat gactttcacc agagcaactg ccacggaagt 4020

cgcgggtaaa atagcacacg agagtgacaa agacatatta ctaatgggag cagtaggatc 4080

aggtaagtca actggcttgc catatcatct ctccagaaaa gggaacgtat tactccttga 4140

gccgactcgg ccacttgcag aaaacgtaca caagcagttg tcgcaggcac cgttccatca 4200

gaacacaact cttaggatgc gcggactaac agcattcggg tcggcaccaa tctcagtgat 4260

gaccagtggt tttgcactca attactttgc aaacaacaga atgcgaattg aagaatttga 4320

ctttgtcata tttgatgaat gtcacgttca tgacgccaat gcaatggcga tgagatgttt 4380

gctacatgag tgtgactatt ctggcaaaat tatcaaagtt tcagccacac caccaggtcg 4440

agaagttgag ttctccactc aataccccgt gtcgataagc acagaagaca cactatcgtt 4500

tcaggatttt gtgaacgcac agggtagtgg aagcaattgt gatgtgattt caaaaggaga 4560

caatatcctc gtgtatgtag caagctacaa tgaggtagac gcgctttcaa aacttctaat 4620

tgaaagagac ttcaaagtca cgaaggttga tggaagaacg atgaaagttg gaaacatcga 4680

gatcaccaca agtggaacac ctagtaagaa gcacttcata gttgcaacca acatcataga 4740

gaacggtgtt actctagaca tcgatgtggt tgctgatttt ggaacgaagg tactcccata 4800

tcttgataca gacagcagaa tgctgagcac aactaagaca agcatcaatt atggggaacg 4860

tatccaaagg ctaggaagag tcggaaggca caagccaggt cacgctctgc gaataggtca 4920

cacagagaag gggttgagcg aagttccaag ttgtattgca acagaagcag ctttaaagtg 4980

cttcacttat gggcttccag tgatcaccaa caacgtctcg acaagtattc ttggtaatgt 5040

aacggtaaag caggcacgaa caatgtctgt atttgagata acaccgttct acacaagcca 5100

agtggtgaga tatgatggct ccatgcatcc acaggtgcac gcactcttaa agagattcaa 5160

actcagagac tctgagattg ttttgaataa attagccata cctcaccgag gagtgaacgc 5220

ttggctcaca gctagtgagt atgcacgact tggcgcgaat gttgaagata ggcgtgacgt 5280

tcgaattcct tttatgtgtc gcgacatccc agaaaaactt catctagaca tgtgggatgt 5340

gattgttaaa ttcaaaggtg atgcaggttt tggtcggctt tcaagcgcca gtgcgagcaa 5400

ggtagcttat actctacaga cggacgtcaa ctccatacag cgaacagtca ctatcataga 5460

tacactaatc gctgaggaga gaaggaagca ggaatacttc aagacggtaa cctccaactg 5520

tgtctcttct tcgaacttct cactgcagag cataacaaat gcgataaaat ctcgtatgat 5580

gaaagatcac acgtgcgaga acatatcagt gcttgaagga gcgaagtcac agttactcga 5640

gtttagaaac ctgaatgctg atcactcatt tgctacaaaa accgatggaa tatctcggca 5700

tttcatgagt gagtatggag ctcttgaggc agttcaccat caaaacacca gcgacatgag 5760

caaattcctc aagcttaagg gcaaatggaa taaaacgcta atcacgcgag atgtgctggt 5820

actttgtgga gttcttggag gtggattgtg gatggttatt cagcacctgc ggtcaaagat 5880

gtccgaaccc gtaacccatg aagcgaaagg taagaggcaa aggcagaaac taaaatttcg 5940

caatgcccga gacaacaaaa tgggtagaga agtgtacgga gatgatgata ccatagagca 6000

tttcttcggt gatgcctaca caaagaaagg gaagagcaag ggtaggacac gtggtatcgg 6060

acacaaaaac aggaagttca tcaacatgta tgggtttgat cctgaagatt tctctgcagt 6120

tcgtttcgtg gatccactca caggagcgac gttggacgac aacccgctca cagacatcac 6180

ccttgtgcaa gagcacttcg gcaacataag aatggactta ctcggggagg atgagctgga 6240

ctcaaatgaa atacgtgtga ataagactat tcaagcctac tacatgaaca ataaaacagg 6300

caaggctttg aaggtggatc tgacaccaca catacctctc aaggtgtgtg atcttcacgc 6360

aaccattgct ggattcccag agcgagaaaa cgagctgagg cagactggaa aggctcagcc 6420

catcaacata gacgaagtgc caagagctaa caacgaactc gtcccagtgg accacgagag 6480

taactccatg ttcagagggt tgcgtgacta caacccaata tcaaacaaca tttgtcatct 6540

cacaaatgtt tcagatggag catcaaactc gttatatgga gtcggtttcg gaccactcat 6600

attaacgaac cgacacctct ttgagcggaa taacggtgaa ctcgtaataa aatcacgaca 6660

tggtgagttc gtgattaaaa acacaactca gctacacttg ctaccgattc cagacagaga 6720

tcttctgcta atccggttac caaaggacgt cccacccttt ccacagaaat tgggtttcag 6780

gcaacctgag aaaggtgaac gaatttgcat ggtggggtcc aatttccaaa ccaagagcat 6840

aacgagtata gtctctgaga ctagtacaat aatgccagtg gagaacagtc agttttggaa 6900

acactggatt agcactaaag acggccaatg cggaagtcca atggtgagca cgaaagacgg 6960

gaaaatactc ggattacaca gcctagcgaa cttccagaac tccatcaatt actttgctgc 7020

tttcccagat gattttgccg agaagtatct tcataccatt gaagcacacg agtgggtcaa 7080

gcactggaag tataacacta gcgccatcag ttggggctct ttgaatatac aagcatcgca 7140

accgtccggc ttgttcaaag taagcaagct aatctcagac ctcgacagca cggcagtcta 7200

cgcacaaacc cagcagaatc ggtggatgtt cgagcagctc aacgggaacc taaaagcgat 7260

agcacactgc cctagccagc ttgtgacaaa gcacacagtt aaaggaaaat gtcagatgtt 7320

tgacttgtat ctcaagttgc atgatgaagc acgagagtat ttccaaccga tgctgggcca 7380

gtatcaaaag agcaaactca atcgagaagc atatgcaaag gatcttctga aatatgcaac 7440

gccaatcgaa gcaggaaaca tcgactgtga tctgtttgaa aagacagttg aaatagtcgt 7500

atcagatctg cgaggttatg gtttcgaaac atgcaattat gtcactgatg agaatgacat 7560

attcgaagct cttaacatga aatccgcagt tggagcgttg tataaaggaa agaagaagga 7620

ttacttcgct gagttcacac ccgagatgaa agaagaaata ctgaaacaaa gttgtgaacg 7680

gctcttccta ggaaagatgg gagtgtggaa cggctcgctg aaggcagagt tgcgaccact 7740

agaaaaagtg gaagcaaaca aaacacggac gtttactgcc gcaccactag acacactgtt 7800

gggtggaaaa gtttgcgtgg atgatttcaa caaccagttc tatgatcaca accttagagc 7860

tccttggagc gttggcatga caaagtttta ttgtggttgg gatcgcttgt tggagtcgtt 7920

gccagatggt tgggtgtatt gcgatgctga tggctcacag ttcgacagct cgctatcgcc 7980

atacttgatc aacgcagtac tcaacatccg cttaggattc atggaagagt gggacatagg 8040

ggaggtaatg ctgagaaatt tgtacaccga aatcgtgtat acccctattt ctacaccaga 8100

tggtacactc gtcaagaagt tcaaaggaaa caatagcgga cagccatcga ctgttgtgga 8160

caacacgctc atggtcatat tggcagtcaa ctattcactc aagaaaagcg gaattccaag 8220

tgagttgcgc gacagcatca tcagattctt cgtcaacgga gatgatttac tgctaagcgt 8280

acacccagag tatgagtata ttcttgacac tatggcagac aactttcgtg aactgggcct 8340

gaagtatact ttcgactcaa gaaccaggga aaaaggagac ctctggttta tgtcgcacca 8400

ggggcacaaa agagagggaa tctggattcc caagctcgag ccagagcgaa tagtatcgat 8460

tctagaatgg gatcggtcga aagagccatg ccatcgacta gaggcaatct gcgcagcgat 8520

gattgagtcg tggggatacg acaagttaac tcacgagata cgcaagttct acgcgtggat 8580

gattgaacaa gctccattta gctccctagc acaagaaggg aaagctcctt acatagcgga 8640

aacagcgctg aggaagctct accttgataa ggaaccagct caagaggatc tcacccatta 8700

tttgcaagca atctttgagg attatgaaga tggtgctgag gcttgtgttt atcaccaggc 8760

aggtgaaacg cttgatgcag gtttgacaga cgagcaaaag caggcagaga aggagaagaa 8820

ggagagagag aaggcagaaa aggaacgaga gaggcaaaag cagttggcac tcaagaaagg 8880

caaggatgtt gcacaagaag agggaaaacg cgacaaggaa gtaaacgctg gaacctctgg 8940

aactttcagt gtacccagac tcaagagtct gacaagcaag atgcgcgtgc caagatacga 9000

gaaaagagtg gctctaaacc tcgatcatct aatcctatac acgccggagc agacggatct 9060

atccaacaca cgttcaacgc gaaagcagtt tgacacatgg tttgaaggtg taatggctga 9120

ttacgaactg acggaggaca aaatgcaaat cattctcaat ggtttaatgg tctggtgcat 9180

tgagaacgga accttcccga acataaacgg aatgtgggtg atgatggacg gcgacgatca 9240

ggtggaattc ccgatcaaac cgctcattga ccacgccaaa cccacattta ggcagataat 9300

ggcccatttc agtgacgtag ctgaagcgta cattgaaaag cgtaaccaag accgaccata 9360

catgccacga tatggtcttc agcgcaattt aaccgacatg agcttagctc gatacgcatt 9420

tgatttctat gaaatgactt ctaggactcc aatacgtgcg agagaggcac acatccagat 9480

gaaagcagca gcactgcgtg gcgcaaataa taatttgttc ggcttggatg gaaacgttgg 9540

tacaacggta gagaacacgg agaggcatac gaccgaggac gttaatcgga acatgcataa 9600

cttactgggc gttcaggggt tgtgaagttg tatgctggta gactataagt atttaagttt 9660

actcgttagt attctcgctt atgggaaata tgtaagtttg ttaaagcagc cagtgtgact 9720

ttgtcatgtg tgttgttgtt actttctgta ttttcgccga acattttatt ggtgttagcg 9780

catgtagtga ggatcgtcct cgattgcctt aacatttgat aggatgcaag ggac 9834

<210> 5

<211> 6443

<212> DNA

<213>The full-length cDNA of potato virus X（Without polyA tails）

<400> 5

gaaaactaaa ccatacacca ccaacacaac caaacccacc acgcccaatt gttacacacc 60

cgcttgaaaa agaaagttta acaaatggcc aaggtgcgcg aggtttacca atcttttaca 120

gactccacca caaaaactct catccaagat gaggcttata gaaacattcg ccccatcatg 180

gaaaaacaca aactagctaa cccttacgct caaacggttg aagcggctaa tgatctagag 240

gggttcggca tagccaccaa tccctatagc attgaattgc atacacatgc agccgctaag 300

accatagaga ataaacttct agaggtgctt ggttccatcc taccacaaga acctgttaca 360

tttatgtttc ttaaacccag aaagctaaac tacatgagaa gaaacccgcg gatcaaggac 420

attttccaaa atgttgccat tgaaccaaga gacgtagcca ggtaccccaa ggaaacaata 480

attgacaaac tcacagagat cacaacggaa acagcataca ttagtgacac tctgcacttc 540

ttggatccga gctacatagt ggagacattc caaaactgcc caaaattgca aacattgtat 600

gcgaccttag ttctccccgt tgaggcagcc tttaaaatgg aaagcactca cccgaacata 660

tacagcctca aatacttcgg agatggtttc cagtatatac caggcaacca tggtggcggg 720

gcataccatc atgaattcgc tcatctacaa tggctcaaag tgggaaagat caagtggagg 780

gaccccaagg atagctttct cggacatctc aattacacga ctgagcaggt tgagatgcac 840

acagtgacag tacagttgca ggaatcgttc gcggcaaacc acttgtactg catcaggaga 900

ggagacttgc tcacaccgga ggtgcgcact ttcggccaac ctgacaggta cgtgattcca 960

ccacagatct tcctcccaaa agttcacaac tgcaagaagc cgattctcaa gaaaactatg 1020

atgcagctct tcttgtatgt taggacagtc aaggtcgcaa aaaattgtga catttttgcc 1080

aaagtcagac aattaattaa atcatctgac ttggacaaat actctgctgt ggaactggtt 1140

tacttagtaa gctacatgga gttccttgcc gatttacaag ctaccacctg cttctcagac 1200

acactttctg gtggcttgct aacaaagacc cttgcaccgg tgagggcttg gatacaagag 1260

aaaaagatgc agctgtttgg tcttgaggac tacgcgaagt tagtcaaagc agttgatttc 1320

cacccggtgg atttttcttt caaagtggaa acttgggact tcagattcca ccccttgcaa 1380

gcgtggaaag ccttccgacc aagggaagtg tcggatgtag aggaaatgga aagtttgttc 1440

tcagatgggg acttgcttga ttgcttcaca agaatgccag cttatgcggt aaacgcagag 1500

gaagatttag ctgcaatcag gaaaacgccc gagatggatg tcggtcaaga agttaaagaa 1560

cctgcaggag acagaaatca atactcaaac cctgcagaaa ctttcctcaa caagctccac 1620

aggaaacaca gtagggaggt gaaacaccag gccgcaaaga aagctaaacg cctagctgaa 1680

atccaggagt caatgagagc tgaaggtgat gccgaaccaa atgaaataag cgggggcatg 1740

ggggcaatac ccagcaacgc cgaacttcct ggcacgaatg atgccagaca agaactcaca 1800

ctcccaacca ctaaacctgt ccctgcaagg tgggaagatg cttcattcac agattctagt 1860

gtggaagagg agcaggttaa accccttgga aaagaaaccg ttgaaacagc gacgcaacaa 1920

gtcatcgaag gacttccttg gaaacactgg attcctcaat taaatgctgt tggattcaag 1980

gcgctggaaa ttcagaggga taggagtgga acaatgatca tgcccatcac agaaatggtc 2040

tccgggctgg aaaaagagga cttccctgaa ggaactccaa aagagttggc acgagaattg 2100

ttcgctatga acagaagccc tgccaccatc cctttggacc tgcttagagc cagagactac 2160

ggcagtgatg taaagaacaa gagaattggt gccatcacaa agacacaggc aacgagttgg 2220

ggcgaatact tgacaggaaa gatagaaagc ttgactgaga ggaaagttgc gacttgtgtc 2280

attcatggag ctggaggttc tggaaaaagt catgccatcc agaaggcatt gagagaaatt 2340

ggcaagggct cggacatcac tgtagtcctg ccgaccaatg aactgcggct agattggagt 2400

aagaaagtgc ctaacactga gccctatatg ttcaagacct atgaaaaggc gttaattggg 2460

ggaacaggca gcatagtcat ctttgacgat tactcaaaac ttcctcccgg ttacatagaa 2520

gccttagtct gtttctactc taaaatcaag ctaatcattc taacaggaga tagcagacaa 2580

agcgtctacc atgaaactgc tgaggacgcc tccatcaggc atttgggacc agcaacagag 2640

tacttctcaa aatactgccg atactatctc aatgccacac accgcaacaa gaaagatctt 2700

gcgaacatgc ttggtgtcta cagtgagaga acgggagtca ccgaaatcag catgagcgcc 2760

gagttcttag aaggaatccc aactttggta ccctcggatg agaagagaaa gctgtacatg 2820

ggcaccggga ggaatgacac gttcacatac gctggatgcc aggggctaac taagccgaag 2880

gtacaaatag tgttggacca caacacccaa gtgtgtagcg cgaatgtgat gtacacggca 2940

ctttctagag ccaccgatag gattcacttc gtgaacacaa gtgcaaattc ctcggccttc 3000

tgggaaaagt tggacagcac cccttacctc aagactttcc tatcagtggt gagagaacaa 3060

gcactcaggg agtacgagcc ggcagaggca gagccaattc aagagcctga gccccagaca 3120

cacatgtgtg tcgagaatga ggagtccgtg ctagaagagt acaaagagga actcttggaa 3180

aagtttgaca gagagatcca ctctgaatcc catggtcatt caaactgtgt ccaaactgaa 3240

gacacaacca ttcagttgtt ttcgcatcaa caagcaaaag atgagaccct cctctgggcg 3300

actatagatg cgcggctcaa gaccagcaat caagagacaa acttccgaga attcctgagc 3360

aagaaggaca ttggggacgt tctgttttta aactaccaaa aagctatggg tttacccaaa 3420

gagcgtattc ctttttccca agaggtctgg gaagcttgtg cccacgaagt acaaagcaag 3480

tacctcagca agtcaaagtg caacttgatc aatgggactg tgagacagag cccagacttc 3540

gatgaaaata agattatggt attcctcaag tcgcagtggg tcacaaaggt ggaaaaacta 3600

ggtctaccca agattaagcc aggtcaaacc atagcagcct tttaccagca gactgtgatg 3660

ctttttggaa ctatggctag gtacatgcga tggttcagac aggctttcca gccaaaagaa 3720

gtcttcataa actgtgagac cacgccagat gacatgtctg catgggcctt gaacaactgg 3780

aatttcagca gacctagctt ggctaatgac tacacagctt tcgaccagtc tcaggatgga 3840

gccatgttgc aatttgaggt gctcaaagcc aaacaccact gcataccaga ggaaatcatt 3900

caggcataca tagatattaa gactaatgca cagattttcc taggcacgtt atcaattatg 3960

cgcctgactg gtgaaggtcc cacttttgat gcaaacactg agtgcaacat agcttacacc 4020

catacaaagt ttgacatccc agccggaact gctcaagttt atgcaggaga cgactccgca 4080

ctggactgtg ttccagaagt gaagcatagt ttccacaggc ttgaggacaa attactccta 4140

aagtcaaagc ctgtaatcac gcagcaaaag aagggcagtt ggcctgagtt ttgtggttgg 4200

ctgatcacac caaaaggggt gatgaaagac ccaattaagc tccatgttag cttaaaattg 4260

gctgaagcta agggtgaact caagaaatgt caagattcct atgaaattga tctgagttat 4320

gcctatgacc acaaggactc tctgcatgac ttgttcgatg agaaacagtg tcaggcacac 4380

acactcactt gcagaacact aatcaagtca gggagaggca ctgtctcact ttcccgcctc 4440

agaaactttc tttaaccgtt aagttacctt agagatttga ataagatgga tattctcatc 4500

agtagtttga aaagtttagg ttattctagg acttccaaat ctttagattc aggacctttg 4560

gtagtacatg cagtagccgg agccggtaag tccacagccc taaggaagtt gatcctcaga 4620

cacccaacat tcaccgtgca tacactcggt gtccctgaca aggtgagtat cagaactaga 4680

ggcatacaga agccaggacc tattcctgag ggcaacttcg caatcctcga tgagtatact 4740

ttggacaaca ccacaaggaa ctcataccag gcactttttg ctgaccctta tcaggcaccg 4800

gagtttagcc tagagcccca cttctacttg gaaacatcat ttcgagttcc gaggaaagtg 4860

gcagatttga tagctggctg tggcttcgat ttcgagacca actcaccgga agaagggcac 4920

ttagagatca ctggcatatt caaagggccc ctactcggaa aggtgatagc cattgatgag 4980

gagtctgaga caacactgtc caggcatggt gttgagtttg ttaagccctg ccaagtgacg 5040

ggacttgagt tcaaagtagt cactattgtg tctgccgcac caatagagga aattggccag 5100

tccacagctt tctacaacgc tatcaccagg tcaaagggat tgacatatgt ccgcgcaggg 5160

ccataggctg accgctccgg tcaattctga aaaagtgtac atagtattag gtctatcatt 5220

tgctttagtt tcaattacct ttctgctttc tagaaatagc ttaccccacg tcggtgacaa 5280

cattcacagc ttgccacacg gaggagctta cagagacggc accaaagcaa tcttgtacaa 5340

ctccccaaat ctagggtcac gagtgagtct acacaacgga aagaacgcag catttgctgc 5400

cgttttgcta ctgactttgc tgatctatgg aagtaaatac atatctcaac gcaatcatac 5460

ttgtgcttgt ggtaacaatc atagcagtca ttagcacttc cttagtgagg actgaacctt 5520

gtgtcatcaa gattactggg gaatcaatca cagtgttggc ttgcaaacta gatgcagaaa 5580

ccataagggc cattgccgat ctcaagccac tctccgttga acggttaagt ttccattgat 5640

actcgaaaga ggtcagcacc agctagcatg cccgcgagca caacacagcc catagggtca 5700

actacctcaa ctaccacaaa aactgcaggc gcaactcctg ccacagcttc aggcctgttc 5760

actatcccgg atggggattt ctttagtaca gcccgtgcca tagtagccag caatgctgtc 5820

gcaacaaatg aggacctcag caagattgag gctatttgga aggacatgaa ggtgcccaca 5880

gacactatgg cacaggctgc ttgggactta gtcagacact gtgctgatgt aggatcatcc 5940

gctcaaacag aaatgataga tacaggtccc tattccaacg gcatcagcag agctagactg 6000

gcagcagcaa ttaaagaggt gtgcacactt aggcaatttt gcatgaagta tgccccagtg 6060

gtatggaact ggatgttaac taacaacagt ccacctgcta actggcaagc acaaggtttc 6120

aagcctgagc acaaattcgc tgcattcgac ttcttcaatg gagtcaccaa cccagctgcc 6180

atcatgccca aagaggggct catccggcca ccgtctgaag ctgaaatgaa tgctgcccaa 6240

actgctgcct ttgtgaagat tacaaaggcc agggcacaat ccaacgactt tgccagccta 6300

gatgcagctg tcactcgagg tcgtatcact ggaacaacaa ccgctgaggc tgttgtcact 6360

ctaccaccac cataactacg tctacataac cgacgcctac cccagtttca tagtattttc 6420

tggtttgatt gtatgaataa tat 6443

<210> 6

<211> 2477

<212> DNA

<213>Estrogen-inducible promoter XVE

<400> 6

tagtttaaac tgaaggcggg aaacgacaat ctgatccaag ctcaagctaa gcttgcatgc 60

ctgcaggata tcgtggatcc aagcttgcca cgtgccgcca cgtgccgcca cgtgccgcca 120

cgtgcctcta gaggatccat ctccactgac gtaagggatg acgcacaatc ccactatcct 180

tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgggat 240

ccccaattcc gggcggaatg aaagcgttaa cggccaggca acaagaggtg tttgatctca 300

tccgtgatca catcagccag acaggtatgc cgccgacgcg tgcggaaatc gcgcagcgtt 360

tggggttccg ttccccaaac gcggctgaag aacatctgaa ggcgctggca cgcaaaggcg 420

ttattgaaat tgtttccggc gcatcacgcg ggattcgtct gttgcaggaa gaggaagaag 480

ggttgccgct ggtaggtcgt gtggctgccg gtgaaccgtc gagcgccccc ccgaccgatg 540

tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg 600

cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta 660

ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga 720

tgtttaccga tgcccttgga attgacgagt acggtgggga tccgtctgct ggagacatga 780

gagctgccaa cctttggcca agcccgctca tgatcaaacg ctctaagaag aacagcctgg 840

ccttgtccct gacggccgac cagatggtca gtgccttgtt ggatgctgag ccccccatac 900

tctattccga gtatgatcct accagaccct tcagtgaagc ttcgatgatg ggcttactga 960

ccaacctggc agacagggag ctggttcaca tgatcaactg ggcgaagagg gtgccaggct 1020

ttgtggattt gaccctccat gatcaggtcc accttctaga atgtgcctgg ctagagatcc 1080

tgatgattgg tctcgtctgg cgctccatgg agcacccagt gaagctactg tttgctccta 1140

acttgctctt ggacaggaac cagggaaaat gtgtagaggg catggtggag atcttcgaca 1200

tgctgctggc tacatcatct cggttccgca tgatgaatct gcagggagag gagtttgtgt 1260

gcctcaaatc tattattttg cttaattctg gagtgtacac atttctgtcc agcaccctga 1320

agtctctgga agagaaggac catatccacc gagtcctgga caagatcaca gacactttga 1380

tccacctgat ggccaaggca ggcctgaccc tgcagcagca gcaccagcgg ctggcccagc 1440

tcctcctcat cctctcccac atcaggcaca tgagtaacaa aggcatggag catctgtaca 1500

gcatgaagtg caagaacgtg gtgcccctct atgacctgct gctggagatg ctggacgccc 1560

accgcctaca tgcgcccact agccgtggag gggcatccgt ggaggagacg gaccaaagcc 1620

acttggccac tgcgggctct acttcatcgc attccttgca aaagtattac atcacggggg 1680

aggcagaggg tttccctgcc acagtctgag agctccctgg cgaattccca gagatgttag 1740

ctgaaatcat cactaatcag ataccaaaat attcaaatgg aaatatcaaa aagcttctgt 1800

ttcatcaaaa atgactcgac ctaactgagt aagctagctt gttcgagtat tatggcattg 1860

ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 1920

ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 1980

cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 2040

ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 2100

ctctaatgac cgaagttaat atgaggagta aaacatccca aacagcttgg gctgcaggtc 2160

gaggctaaaa aactaatcgc attatcatcc cctcgacgta ctgtacatat aaccactggt 2220

tttatataca gcagtactgt acatataacc actggtttta tatacagcag tcgacgtact 2280

gtacatataa ccactggttt tatatacagc agtactgtac atataaccac tggttttata 2340

tacagcagtc gaggtaagat tagatatgga tatgtatatg gatatgtata tggtggtaat 2400

gccatgtaat atgctcgact ctaggatctt cgcaagaccc ttcctctata taaggaagtt 2460

catttcattt ggagagg 2477

<210> 7

<211> 1637

<212> DNA

<213>Hygromycin gene

<400> 7

aggcgggaaa cgacaatctg atcatgagcg gagaattaag ggagtcacgt tatgaccccc 60

gccgatgacg cgggacaagc cgttttacgt ttggaactga cagaaccgca acgattgaag 120

gagccactca gccgcgggtt tctggagttt aatgagctaa gcacatacgt cagaaaccat 180

tattgcgcgt tcaaaagtcg cctaaggtca ctatcagcta gcaaatattt cttgtcaaaa 240

atgctccact gacgttccat aaattcccct cggtatccaa ttagagtctc atattcactc 300

tcaatccaaa taatctgcac cggatcccct agaatgaaaa agcctgaact caccgcgacg 360

tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 420

gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 480

gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 540

gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 600

tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 660

gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 720

acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 780

ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 840

gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 900

gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 960

cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 1020

gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 1080

gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 1140

ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 1200

cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 1260

gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 1320

aaccgacgcc ccagcactcg tccgagggca aaggaatagc gatcgttcaa acatttggca 1380

ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct 1440

gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 1500

ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 1560

gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcggggaat 1620

tgatcccccc tcgacag 1637

<210> 8

<211> 2143

<212> DNA

<213>Inducible promoter XVE-1

<400> 8

tagtttaaac tgaaggcggg aaacgacaat ctgatccaag ctcaagctaa gcttgcatgc 60

ctgcaggata tcgtggatcc aagcttgcca cgtgccgcca cgtgccgcca cgtgccgcca 120

cgtgcctcta gaggatccat ctccactgac gtaagggatg acgcacaatc ccactatcct 180

tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgggat 240

ccccaattcc gggcggaatg aaagcgttaa cggccaggca acaagaggtg tttgatctca 300

tccgtgatca catcagccag acaggtatgc cgccgacgcg tgcggaaatc gcgcagcgtt 360

tggggttccg ttccccaaac gcggctgaag aacatctgaa ggcgctggca cgcaaaggcg 420

ttattgaaat tgtttccggc gcatcacgcg ggattcgtct gttgcaggaa gaggaagaag 480

ggttgccgct ggtaggtcgt gtggctgccg gtgaaccgtc gagcgccccc ccgaccgatg 540

tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg 600

cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta 660

ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga 720

tgtttaccga tgcccttgga attgacgagt acggtgggga tccgtctgct ggagacatga 780

gagctgccaa cctttggcca agcccgctca tgatcaaacg ctctaagaag aacagcctgg 840

ccttgtccct gacggccgac cagatggtca gtgccttgtt ggatgctgag ccccccatac 900

tctattccga gtatgatcct accagaccct tcagtgaagc ttcgatgatg ggcttactga 960

ccaacctggc agacagggag ctggttcaca tgatcaactg ggcgaagagg gtgccaggct 1020

ttgtggattt gaccctccat gatcaggtcc accttctaga atgtgcctgg ctagagatcc 1080

tgatgattgg tctcgtctgg cgctccatgg agcacccagt gaagctactg tttgctccta 1140

acttgctctt ggacaggaac cagggaaaat gtgtagaggg catggtggag atcttcgaca 1200

tgctgctggc tacatcatct cggttccgca tgatgaatct gcagggagag gagtttgtgt 1260

gcctcaaatc tattattttg cttaattctg gagtgtacac atttctgtcc agcaccctga 1320

agtctctgga agagaaggac catatccacc gagtcctgga caagatcaca gacactttga 1380

tccacctgat ggccaaggca ggcctgaccc tgcagcagca gcaccagcgg ctggcccagc 1440

tcctcctcat cctctcccac atcaggcaca tgagtaacaa aggcatggag catctgtaca 1500

gcatgaagtg caagaacgtg gtgcccctct atgacctgct gctggagatg ctggacgccc 1560

accgcctaca tgcgcccact agccgtggag gggcatccgt ggaggagacg gaccaaagcc 1620

acttggccac tgcgggctct acttcatcgc attccttgca aaagtattac atcacggggg 1680

aggcagaggg tttccctgcc acagtctgag agctccctgg cgaattccca gagatgttag 1740

ctgaaatcat cactaatcag ataccaaaat attcaaatgg aaatatcaaa aagcttctgt 1800

ttcatcaaaa atgactcgac ctaactgagt aagctagctt gttcgagtat tatggcattg 1860

ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 1920

ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 1980

cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 2040

ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 2100

ctctaatgac cgaagttaat atgaggagta aaacatccca aac 2143

<210> 9

<211> 334

<212> DNA

<213>Estrogen-inducible promoter XVE-2

<400> 9

agcttgggct gcaggtcgag gctaaaaaac taatcgcatt atcatcccct cgacgtactg 60

tacatataac cactggtttt atatacagca gtactgtaca tataaccact ggttttatat 120

acagcagtcg acgtactgta catataacca ctggttttat atacagcagt actgtacata 180

taaccactgg ttttatatac agcagtcgag gtaagattag atatggatat gtatatggat 240

atgtatatgg tggtaatgcc atgtaatatg ctcgactcta ggatcttcgc aagacccttc 300

ctctatataa ggaagttcat ttcatttgga gagg 334

<210> 10

<211> 480

<212> DNA

<213>T3A terminators

<400> 10

caggcctccc agctttcgtc cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca 60

tcagtttcat tgcccacaca ccagaatcct actaagtttg agtattatgg cattggaaaa 120

gctgttttct tctatcattt gttctgcttg taatttactg tgttctttca gtttttgttt 180

tcggacatca aaatgcaaat ggatggataa gagttaataa atgatatggt ccttttgttc 240

attctcaaat tattattatc tgttgttttt actttaatgg gttgaattta agtaagaaag 300

gaactaacag tgtgatatta aggtgcaatg ttagacatat aaaacagtct ttcacctctc 360

tttggttatg tcttgaattg gtttgtttct tcacttatct gtgtaatcaa gtttactatg 420

agtctatgat caagtaatta tgcaatcaag ttaagtacag tataggcttt ttgtgtcgag 480

<210> 11

<211> 10578

<212> DNA

<213>TuMV-GFP sequences（1229-1972 nt are GFP genes）

<400> 11

aaaaaatata aaaactcaac ataacataca caaaacgatt aaagcaaaca caaatctttc 60

aaagcattca agcaatcaaa gattctcaaa tctttcatcg ttatcaaagc aatcaccaac 120

agcaaaccaa atggcagcag ttacattcgc atcagctatc accaacgcca tcaccagcaa 180

accagcactc accggaatgg tgcagtttgg gagtttccca ccaatgccat tgcgatccac 240

caccgtcacc acagtcgcca cttcagtggc gcaacctaaa ctgtacacag tgcagtttgg 300

aagccttgac ccagtagtcg tcaagagtgg agcagggtcc cttgctaagg caacacgcca 360

gcagcctaac gttgaaatag acgttagcct cagtgaagcc gcagctctgg aggttgcgaa 420

acctagatcg aatgccgtgt tgaggatgca cgaggaagca aacaaggaga gagcactctt 480

tttggactgg gaggctagtt tgaagagaag ctcgtatgga attgctgagg acgagaaggt 540

tgtaatgaca actcatggcg tcagcaagat agtgcccaga agttcaaggg caatgaagct 600

aaagcgcgca agggagaggc gtagagcgca gcaaccaatt atattaaagt gggagcccaa 660

attgagcggg atctcaatcg gaggagggct ctctgcgagc gtaatcgaag cagaagaggt 720

tcgcacaaag tggccgcttc ataagacacc gtcaatgaag aagaggacgg tgcacagaat 780

atgcaagatg aacgaccaag gagttgacat gttgacacga tccctggtta agattttcaa 840

gactaagagt gccaacattg aatacatcgg aaagaagtcg attaaggtcg atttcatcag 900

aaaagaacga acgaaattcg caagaatcca agtagcacac ttactcggga agagagcaca 960

gcgcgacttg ttaactggaa tggaagaaaa ccattttatt gacattctca gtaagtactc 1020

aggtaacaaa acaaccataa atcctggagt agtttgcgca ggttggagtg gcatagtcgt 1080

tggaaatgga attctaaccc agaaacgaag cagaagtcca tcagaggcct ttgtaattag 1140

aggtgagcac gaaggcaagt tgtacgatgc caggatcaaa gtcacgagga caatgagtca 1200

caagattgtg cactttagtg ccgcgggaat gagtaaagga gaagaacttt tcactggagt 1260

tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg 1320

agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg 1380

aaaactacct gttccatggc caacacttgt cactactttc tcttatggtg ttcaatgctt 1440

ttcaagatac ccagatcata tgaagcggca cgacttcttc aagagcgcca tgcctgaggg 1500

atacgtgcag gagaggacca tcttcttcaa ggacgacggg aactacaaga cacgtgctga 1560

agtcaagttt gagggagaca ccctcgtcaa caggatcgag cttaagggaa tcgatttcaa 1620

ggaggacgga aacatcctcg gccacaagtt ggaatacaac tacaactccc acaacgtata 1680

catcatggcc gacaagcaaa agaacggcat caaagccaac ttcaagaccc gccacaacat 1740

cgaagacggc ggcgtgcaac tcgctgatca ttatcaacaa aatactccaa ttggcgatgg 1800

ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt cgaaagatcc 1860

caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca 1920

tggcatggat gaactataca aagcttgtgt ctatcaccag gcagccgcgg gagccaactt 1980

ctggaaaggc ttcgacagat gctttctcgc ataccgtagt gacaatcgcg agcatacatg 2040

ctattcaggg ctagatgtca ctgagtgcgg cgaggtggca gcactgatgt gtttggctat 2100

gttcccatgc ggaaagataa cctgccctga ctgtgtaaca gatagtgagc tatcccaagg 2160

acaagcaagc ggaccatcta tgaagcacag gttgacacag ctacgcgatg tcatcaagtc 2220

aagctaccca cgcttcaagc atgcagtgca gatactagat aggtatgagc aatcactgag 2280

cagtgcaaac gagaactacc aagatttcgc agaaatccag agcataagcg atggagttga 2340

aaaagctgca ttcccacacg tcaacaagct aaacgcaata ttgatcaaag gggccacagt 2400

aacaggagag gaattctcgc aggctacgaa gcacttgctc gagatagcac gatacctgaa 2460

gaacagaacc gagaacattg agaagggttc actgaagtcc tttcgcaaca agatttccca 2520

gaaagcgcac atcaacccaa cactaatgtg tgacaaccag ctcgatagaa atggaaattt 2580

catatggggt gagagaggat accatgcaaa acgattcttc agcaactact ttgaaataat 2640

cgatccaaag aaaggctaca cccaatacga gacaagagcg gtaccaaatg ggtcacggaa 2700

acttgcaatc ggcaaactaa tagtcccaac gaacttcgaa gttttaaggg aacagatgaa 2760

aggcgaaccg gtagaaccat acccagtaac agtcgagtgt gtgagcaagt tacagggtga 2820

cttcgtccat gcatgttgtt gtgtcacaac agaatcaggc gacccagtct tgtctgagat 2880

caaaatgcca accaaacacc atctagtgat tggtaacagc ggtgatccaa agtacataga 2940

tctccctgag atcgaggaga ataaaatgta catagcgaaa gaaggttatt gttacatcaa 3000

tatcttccta gccatgttgg taaatgtcaa ggagtcgcag gcaaaggagt tcacgaaagt 3060

tgttagggac aaactagttg gcgaacttgg caagtggccc actctgttag atgtagcaac 3120

cgcttgttat ttcctgaaag tattttaccc agacgttgct aacgccgaat tgccacgcat 3180

gctagtggac cataagacaa agataattca tgtcgttgat tcatatgggt cactgtcaac 3240

tggatatcat gtccttaaga caaacactgt ggaacaactc atcaaattca cgagatgtaa 3300

tttggagtca agcttgaaac actaccgcgt tggaggaaca gaatgggagg acactcatgg 3360

atccagcaac atagataatc cacagtggtg catcaagagg ctcataaaag gagtctacaa 3420

accaaagcaa ctgaaagaag acatgttggc aaaccctttc ttaccactat atgctctact 3480

gtcaccaggt gtcatcctgg cattttacaa tagtggctct ctagagtact tgatgaacca 3540

ttacatcagg gtggacagca acgtcgccgt tttgttggtc gttttgaaat ctctagcgaa 3600

gaaggtgtca actagtcaga gtgtgttagc ccagcttcaa atcattgaac gaagtctacc 3660

agaactcatc gaagcaaagg ctaatgttaa tgggccagat gacgcagcca ctcgcgcgtg 3720

taacagattc atgggcatgc ttctgcatat ggcagaacca aactgggagc ttgcggatgg 3780

tggatacaca attctgaggg atcatagcat ctccattttg gaaaaaagtt atctacaaat 3840

cttggacgaa gcatggaacg agttaagttg gtcggagcgc tgtgctataa gatactactc 3900

gtcaaagcaa gcaatcttta cacagaaaga tttgccaatg aaaagcgaag ccgatttagg 3960

cggcagatac agcgtgtcag tcatgtcatc ttacgaacgg agtaagcaat gtatgaaaag 4020

cgtgcactct agtataggta atagattacg tagtagtatg tcttggacta gtagcaaggt 4080

gtcgaatagt gtgtgtagga ctattaacta tttagtacca gatgtgttca agtttatgaa 4140

tgtactcgtt tgtatcagct tactaatcaa gatgactgcc gaggcgaatc acatcgtcac 4200

cacgcaaaga aggctcaaac tagatgtcga ggagacagag cgcaggaaaa tagaatggga 4260

gcttgcattc caccatgcca ttctgacgca gagtgcaggt caacacccaa cgatagacga 4320

gttcagagcg tacatcgccg acaaggcacc acatctaagt gagcatatcg agcctgaaga 4380

aaaggcggtg gttcatcaag cgaagagaca atccgagcaa gaactcgagc gtataatagc 4440

atttgttgca ttggtgctca tgatgttcga tgcagaacga agcgactgtg tcacaaagat 4500

tctcaacaag cttaagggac tagtcgccac tgtggaacct acagtctacc atcagactct 4560

caatgatata gaggatgact tgagtgagag gaacctcttc gtcgattttg agcttagcag 4620

cgatggagat atgctccaac agcttccagc cgaaaagaca tttgcctcat ggtggagtca 4680

tcaactaagc agaggattca caatcccaca ctacaggaca gaagggaagt tcatgacttt 4740

caccagagca actgccacgg aagtcgcggg taaaatagca cacgagagtg acaaagacat 4800

attactaatg ggagcagtag gatcaggtaa gtcaactggc ttgccatatc atctctccag 4860

aaaagggaac gtattactcc ttgagccgac tcggccactt gcagaaaacg tacacaagca 4920

gttgtcgcag gcaccgttcc atcagaacac aactcttagg atgcgcggac taacagcatt 4980

cgggtcggca ccaatctcag tgatgaccag tggttttgca ctcaattact ttgcaaacaa 5040

cagaatgcga attgaagaat ttgactttgt catatttgat gaatgtcacg ttcatgacgc 5100

caatgcaatg gcgatgagat gtttgctaca tgagtgtgac tattctggca aaattatcaa 5160

agtttcagcc acaccaccag gtcgagaagt tgagttctcc actcaatacc ccgtgtcgat 5220

aagcacagaa gacacactat cgtttcagga ttttgtgaac gcacagggta gtggaagcaa 5280

ttgtgatgtg atttcaaaag gagacaatat cctcgtgtat gtagcaagct acaatgaggt 5340

agacgcgctt tcaaaacttc taattgaaag agacttcaaa gtcacgaagg ttgatggaag 5400

aacgatgaaa gttggaaaca tcgagatcac cacaagtgga acacctagta agaagcactt 5460

catagttgca accaacatca tagagaacgg tgttactcta gacatcgatg tggttgctga 5520

ttttggaacg aaggtactcc catatcttga tacagacagc agaatgctga gcacaactaa 5580

gacaagcatc aattatgggg aacgtatcca aaggctagga agagtcggaa ggcacaagcc 5640

aggtcacgct ctgcgaatag gtcacacaga gaaggggttg agcgaagttc caagttgtat 5700

tgcaacagaa gcagctttaa agtgcttcac ttatgggctt ccagtgatca ccaacaacgt 5760

ctcgacaagt attcttggta atgtaacggt aaagcaggca cgaacaatgt ctgtatttga 5820

gataacaccg ttctacacaa gccaagtggt gagatatgat ggctccatgc atccacaggt 5880

gcacgcactc ttaaagagat tcaaactcag agactctgag attgttttga ataaattagc 5940

catacctcac cgaggagtga acgcttggct cacagctagt gagtatgcac gacttggcgc 6000

gaatgttgaa gataggcgtg acgttcgaat tccttttatg tgtcgcgaca tcccagaaaa 6060

acttcatcta gacatgtggg atgtgattgt taaattcaaa ggtgatgcag gttttggtcg 6120

gctttcaagc gccagtgcga gcaaggtagc ttatactcta cagacggacg tcaactccat 6180

acagcgaaca gtcactatca tagatacact aatcgctgag gagagaagga agcaggaata 6240

cttcaagacg gtaacctcca actgtgtctc ttcttcgaac ttctcactgc agagcataac 6300

aaatgcgata aaatctcgta tgatgaaaga tcacacgtgc gagaacatat cagtgcttga 6360

aggagcgaag tcacagttac tcgagtttag aaacctgaat gctgatcact catttgctac 6420

aaaaaccgat ggaatatctc ggcatttcat gagtgagtat ggagctcttg aggcagttca 6480

ccatcaaaac accagcgaca tgagcaaatt cctcaagctt aagggcaaat ggaataaaac 6540

gctaatcacg cgagatgtgc tggtactttg tggagttctt ggaggtggat tgtggatggt 6600

tattcagcac ctgcggtcaa agatgtccga acccgtaacc catgaagcga aaggtaagag 6660

gcaaaggcag aaactaaaat ttcgcaatgc ccgagacaac aaaatgggta gagaagtgta 6720

cggagatgat gataccatag agcatttctt cggtgatgcc tacacaaaga aagggaagag 6780

caagggtagg acacgtggta tcggacacaa aaacaggaag ttcatcaaca tgtatgggtt 6840

tgatcctgaa gatttctctg cagttcgttt cgtggatcca ctcacaggag cgacgttgga 6900

cgacaacccg ctcacagaca tcacccttgt gcaagagcac ttcggcaaca taagaatgga 6960

cttactcggg gaggatgagc tggactcaaa tgaaatacgt gtgaataaga ctattcaagc 7020

ctactacatg aacaataaaa caggcaaggc tttgaaggtg gatctgacac cacacatacc 7080

tctcaaggtg tgtgatcttc acgcaaccat tgctggattc ccagagcgag aaaacgagct 7140

gaggcagact ggaaaggctc agcccatcaa catagacgaa gtgccaagag ctaacaacga 7200

actcgtccca gtggaccacg agagtaactc catgttcaga gggttgcgtg actacaaccc 7260

aatatcaaac aacatttgtc atctcacaaa tgtttcagat ggagcatcaa actcgttata 7320

tggagtcggt ttcggaccac tcatattaac gaaccgacac ctctttgagc ggaataacgg 7380

tgaactcgta ataaaatcac gacatggtga gttcgtgatt aaaaacacaa ctcagctaca 7440

cttgctaccg attccagaca gagatcttct gctaatccgg ttaccaaagg acgtcccacc 7500

ctttccacag aaattgggtt tcaggcaacc tgagaaaggt gaacgaattt gcatggtggg 7560

gtccaatttc caaaccaaga gcataacgag tatagtctct gagactagta caataatgcc 7620

agtggagaac agtcagtttt ggaaacactg gattagcact aaagacggcc aatgcggaag 7680

tccaatggtg agcacgaaag acgggaaaat actcggatta cacagcctag cgaacttcca 7740

gaactccatc aattactttg ctgctttccc agatgatttt gccgagaagt atcttcatac 7800

cattgaagca cacgagtggg tcaagcactg gaagtataac actagcgcca tcagttgggg 7860

ctctttgaat atacaagcat cgcaaccgtc cggcttgttc aaagtaagca agctaatctc 7920

agacctcgac agcacggcag tctacgcaca aacccagcag aatcggtgga tgttcgagca 7980

gctcaacggg aacctaaaag cgatagcaca ctgccctagc cagcttgtga caaagcacac 8040

agttaaagga aaatgtcaga tgtttgactt gtatctcaag ttgcatgatg aagcacgaga 8100

gtatttccaa ccgatgctgg gccagtatca aaagagcaaa ctcaatcgag aagcatatgc 8160

aaaggatctt ctgaaatatg caacgccaat cgaagcagga aacatcgact gtgatctgtt 8220

tgaaaagaca gttgaaatag tcgtatcaga tctgcgaggt tatggtttcg aaacatgcaa 8280

ttatgtcact gatgagaatg acatattcga agctcttaac atgaaatccg cagttggagc 8340

gttgtataaa ggaaagaaga aggattactt cgctgagttc acacccgaga tgaaagaaga 8400

aatactgaaa caaagttgtg aacggctctt cctaggaaag atgggagtgt ggaacggctc 8460

gctgaaggca gagttgcgac cactagaaaa agtggaagca aacaaaacac ggacgtttac 8520

tgccgcacca ctagacacac tgttgggtgg aaaagtttgc gtggatgatt tcaacaacca 8580

gttctatgat cacaacctta gagctccttg gagcgttggc atgacaaagt tttattgtgg 8640

ttgggatcgc ttgttggagt cgttgccaga tggttgggtg tattgcgatg ctgatggctc 8700

acagttcgac agctcgctat cgccatactt gatcaacgca gtactcaaca tccgcttagg 8760

attcatggaa gagtgggaca taggggaggt aatgctgaga aatttgtaca ccgaaatcgt 8820

gtatacccct atttctacac cagatggtac actcgtcaag aagttcaaag gaaacaatag 8880

cggacagcca tcgactgttg tggacaacac gctcatggtc atattggcag tcaactattc 8940

actcaagaaa agcggaattc caagtgagtt gcgcgacagc atcatcagat tcttcgtcaa 9000

cggagatgat ttactgctaa gcgtacaccc agagtatgag tatattcttg acactatggc 9060

agacaacttt cgtgaactgg gcctgaagta tactttcgac tcaagaacca gggaaaaagg 9120

agacctctgg tttatgtcgc accaggggca caaaagagag ggaatctgga ttcccaagct 9180

cgagccagag cgaatagtat cgattctaga atgggatcgg tcgaaagagc catgccatcg 9240

actagaggca atctgcgcag cgatgattga gtcgtgggga tacgacaagt taactcacga 9300

gatacgcaag ttctacgcgt ggatgattga acaagctcca tttagctccc tagcacaaga 9360

agggaaagct ccttacatag cggaaacagc gctgaggaag ctctaccttg ataaggaacc 9420

agctcaagag gatctcaccc attatttgca agcaatcttt gaggattatg aagatggtgc 9480

tgaggcttgt gtttatcacc aggcaggtga aacgcttgat gcaggtttga cagacgagca 9540

aaagcaggca gagaaggaga agaaggagag agagaaggca gaaaaggaac gagagaggca 9600

aaagcagttg gcactcaaga aaggcaagga tgttgcacaa gaagagggaa aacgcgacaa 9660

ggaagtaaac gctggaacct ctggaacttt cagtgtaccc agactcaaga gtctgacaag 9720

caagatgcgc gtgccaagat acgagaaaag agtggctcta aacctcgatc atctaatcct 9780

atacacgccg gagcagacgg atctatccaa cacacgttca acgcgaaagc agtttgacac 9840

atggtttgaa ggtgtaatgg ctgattacga actgacggag gacaaaatgc aaatcattct 9900

caatggttta atggtctggt gcattgagaa cggaaccttc ccgaacataa acggaatgtg 9960

ggtgatgatg gacggcgacg atcaggtgga attcccgatc aaaccgctca ttgaccacgc 10020

caaacccaca tttaggcaga taatggccca tttcagtgac gtagctgaag cgtacattga 10080

aaagcgtaac caagaccgac catacatgcc acgatatggt cttcagcgca atttaaccga 10140

catgagctta gctcgatacg catttgattt ctatgaaatg acttctagga ctccaatacg 10200

tgcgagagag gcacacatcc agatgaaagc agcagcactg cgtggcgcaa ataataattt 10260

gttcggcttg gatggaaacg ttggtacaac ggtagagaac acggagaggc atacgaccga 10320

ggacgttaat cggaacatgc ataacttact gggcgttcag gggttgtgaa gttgtatgct 10380

ggtagactat aagtatttaa gtttactcgt tagtattctc gcttatggga aatatgtaag 10440

tttgttaaag cagccagtgt gactttgtca tgtgtgttgt tgttactttc tgtattttcg 10500

ccgaacattt tattggtgtt agcgcatgta gtgaggatcg tcctcgattg ccttaacatt 10560

tgataggatg caagggac 10578

<210> 12

<211> 6987

<212> DNA

<213>PVX-GFP (5671-6210 nt are GFP genes)

<400> 12

gaaaactaaa ccatacacca ccaacacaac caaacccacc acgcccaatt gttacacacc 60

cgcttgaaaa agaaagttta acaaatggcc aaggtgcgcg aggtttacca atcttttaca 120

gactccacca caaaaactct catccaagat gaggcttata gaaacattcg ccccatcatg 180

gaaaaacaca aactagctaa cccttacgct caaacggttg aagcggctaa tgatctagag 240

gggttcggca tagccaccaa tccctatagc attgaattgc atacacatgc agccgctaag 300

accatagaga ataaacttct agaggtgctt ggttccatcc taccacaaga acctgttaca 360

tttatgtttc ttaaacccag aaagctaaac tacatgagaa gaaacccgcg gatcaaggac 420

attttccaaa atgttgccat tgaaccaaga gacgtagcca ggtaccccaa ggaaacaata 480

attgacaaac tcacagagat cacaacggaa acagcataca ttagtgacac tctgcacttc 540

ttggatccga gctacatagt ggagacattc caaaactgcc caaaattgca aacattgtat 600

gcgaccttag ttctccccgt tgaggcagcc tttaaaatgg aaagcactca cccgaacata 660

tacagcctca aatacttcgg agatggtttc cagtatatac caggcaacca tggtggcggg 720

gcataccatc atgaattcgc tcatctacaa tggctcaaag tgggaaagat caagtggagg 780

gaccccaagg atagctttct cggacatctc aattacacga ctgagcaggt tgagatgcac 840

acagtgacag tacagttgca ggaatcgttc gcggcaaacc acttgtactg catcaggaga 900

ggagacttgc tcacaccgga ggtgcgcact ttcggccaac ctgacaggta cgtgattcca 960

ccacagatct tcctcccaaa agttcacaac tgcaagaagc cgattctcaa gaaaactatg 1020

atgcagctct tcttgtatgt taggacagtc aaggtcgcaa aaaattgtga catttttgcc 1080

aaagtcagac aattaattaa atcatctgac ttggacaaat actctgctgt ggaactggtt 1140

tacttagtaa gctacatgga gttccttgcc gatttacaag ctaccacctg cttctcagac 1200

acactttctg gtggcttgct aacaaagacc cttgcaccgg tgagggcttg gatacaagag 1260

aaaaagatgc agctgtttgg tcttgaggac tacgcgaagt tagtcaaagc agttgatttc 1320

cacccggtgg atttttcttt caaagtggaa acttgggact tcagattcca ccccttgcaa 1380

gcgtggaaag ccttccgacc aagggaagtg tcggatgtag aggaaatgga aagtttgttc 1440

tcagatgggg acttgcttga ttgcttcaca agaatgccag cttatgcggt aaacgcagag 1500

gaagatttag ctgcaatcag gaaaacgccc gagatggatg tcggtcaaga agttaaagaa 1560

cctgcaggag acagaaatca atactcaaac cctgcagaaa ctttcctcaa caagctccac 1620

aggaaacaca gtagggaggt gaaacaccag gccgcaaaga aagctaaacg cctagctgaa 1680

atccaggagt caatgagagc tgaaggtgat gccgaaccaa atgaaataag cgggggcatg 1740

ggggcaatac ccagcaacgc cgaacttcct ggcacgaatg atgccagaca agaactcaca 1800

ctcccaacca ctaaacctgt ccctgcaagg tgggaagatg cttcattcac agattctagt 1860

gtggaagagg agcaggttaa accccttgga aaagaaaccg ttgaaacagc gacgcaacaa 1920

gtcatcgaag gacttccttg gaaacactgg attcctcaat taaatgctgt tggattcaag 1980

gcgctggaaa ttcagaggga taggagtgga acaatgatca tgcccatcac agaaatggtc 2040

tccgggctgg aaaaagagga cttccctgaa ggaactccaa aagagttggc acgagaattg 2100

ttcgctatga acagaagccc tgccaccatc cctttggacc tgcttagagc cagagactac 2160

ggcagtgatg taaagaacaa gagaattggt gccatcacaa agacacaggc aacgagttgg 2220

ggcgaatact tgacaggaaa gatagaaagc ttgactgaga ggaaagttgc gacttgtgtc 2280

attcatggag ctggaggttc tggaaaaagt catgccatcc agaaggcatt gagagaaatt 2340

ggcaagggct cggacatcac tgtagtcctg ccgaccaatg aactgcggct agattggagt 2400

aagaaagtgc ctaacactga gccctatatg ttcaagacct atgaaaaggc gttaattggg 2460

ggaacaggca gcatagtcat ctttgacgat tactcaaaac ttcctcccgg ttacatagaa 2520

gccttagtct gtttctactc taaaatcaag ctaatcattc taacaggaga tagcagacaa 2580

agcgtctacc atgaaactgc tgaggacgcc tccatcaggc atttgggacc agcaacagag 2640

tacttctcaa aatactgccg atactatctc aatgccacac accgcaacaa gaaagatctt 2700

gcgaacatgc ttggtgtcta cagtgagaga acgggagtca ccgaaatcag catgagcgcc 2760

gagttcttag aaggaatccc aactttggta ccctcggatg agaagagaaa gctgtacatg 2820

ggcaccggga ggaatgacac gttcacatac gctggatgcc aggggctaac taagccgaag 2880

gtacaaatag tgttggacca caacacccaa gtgtgtagcg cgaatgtgat gtacacggca 2940

ctttctagag ccaccgatag gattcacttc gtgaacacaa gtgcaaattc ctcggccttc 3000

tgggaaaagt tggacagcac cccttacctc aagactttcc tatcagtggt gagagaacaa 3060

gcactcaggg agtacgagcc ggcagaggca gagccaattc aagagcctga gccccagaca 3120

cacatgtgtg tcgagaatga ggagtccgtg ctagaagagt acaaagagga actcttggaa 3180

aagtttgaca gagagatcca ctctgaatcc catggtcatt caaactgtgt ccaaactgaa 3240

gacacaacca ttcagttgtt ttcgcatcaa caagcaaaag atgagaccct cctctgggcg 3300

actatagatg cgcggctcaa gaccagcaat caagagacaa acttccgaga attcctgagc 3360

aagaaggaca ttggggacgt tctgttttta aactaccaaa aagctatggg tttacccaaa 3420

gagcgtattc ctttttccca agaggtctgg gaagcttgtg cccacgaagt acaaagcaag 3480

tacctcagca agtcaaagtg caacttgatc aatgggactg tgagacagag cccagacttc 3540

gatgaaaata agattatggt attcctcaag tcgcagtggg tcacaaaggt ggaaaaacta 3600

ggtctaccca agattaagcc aggtcaaacc atagcagcct tttaccagca gactgtgatg 3660

ctttttggaa ctatggctag gtacatgcga tggttcagac aggctttcca gccaaaagaa 3720

gtcttcataa actgtgagac cacgccagat gacatgtctg catgggcctt gaacaactgg 3780

aatttcagca gacctagctt ggctaatgac tacacagctt tcgaccagtc tcaggatgga 3840

gccatgttgc aatttgaggt gctcaaagcc aaacaccact gcataccaga ggaaatcatt 3900

caggcataca tagatattaa gactaatgca cagattttcc taggcacgtt atcaattatg 3960

cgcctgactg gtgaaggtcc cacttttgat gcaaacactg agtgcaacat agcttacacc 4020

catacaaagt ttgacatccc agccggaact gctcaagttt atgcaggaga cgactccgca 4080

ctggactgtg ttccagaagt gaagcatagt ttccacaggc ttgaggacaa attactccta 4140

aagtcaaagc ctgtaatcac gcagcaaaag aagggcagtt ggcctgagtt ttgtggttgg 4200

ctgatcacac caaaaggggt gatgaaagac ccaattaagc tccatgttag cttaaaattg 4260

gctgaagcta agggtgaact caagaaatgt caagattcct atgaaattga tctgagttat 4320

gcctatgacc acaaggactc tctgcatgac ttgttcgatg agaaacagtg tcaggcacac 4380

acactcactt gcagaacact aatcaagtca gggagaggca ctgtctcact ttcccgcctc 4440

agaaactttc tttaaccgtt aagttacctt agagatttga ataagatgga tattctcatc 4500

agtagtttga aaagtttagg ttattctagg acttccaaat ctttagattc aggacctttg 4560

gtagtacatg cagtagccgg agccggtaag tccacagccc taaggaagtt gatcctcaga 4620

cacccaacat tcaccgtgca tacactcggt gtccctgaca aggtgagtat cagaactaga 4680

ggcatacaga agccaggacc tattcctgag ggcaacttcg caatcctcga tgagtatact 4740

ttggacaaca ccacaaggaa ctcataccag gcactttttg ctgaccctta tcaggcaccg 4800

gagtttagcc tagagcccca cttctacttg gaaacatcat ttcgagttcc gaggaaagtg 4860

gcagatttga tagctggctg tggcttcgat ttcgagacca actcaccgga agaagggcac 4920

ttagagatca ctggcatatt caaagggccc ctactcggaa aggtgatagc cattgatgag 4980

gagtctgaga caacactgtc caggcatggt gttgagtttg ttaagccctg ccaagtgacg 5040

ggacttgagt tcaaagtagt cactattgtg tctgccgcac caatagagga aattggccag 5100

tccacagctt tctacaacgc tatcaccagg tcaaagggat tgacatatgt ccgcgcaggg 5160

ccataggctg accgctccgg tcaattctga aaaagtgtac atagtattag gtctatcatt 5220

tgctttagtt tcaattacct ttctgctttc tagaaatagc ttaccccacg tcggtgacaa 5280

cattcacagc ttgccacacg gaggagctta cagagacggc accaaagcaa tcttgtacaa 5340

ctccccaaat ctagggtcac gagtgagtct acacaacgga aagaacgcag catttgctgc 5400

cgttttgcta ctgactttgc tgatctatgg aagtaaatac atatctcaac gcaatcatac 5460

ttgtgcttgt ggtaacaatc atagcagtca ttagcacttc cttagtgagg actgaacctt 5520

gtgtcatcaa gattactggg gaatcaatca cagtgttggc ttgcaaacta gatgcagaaa 5580

ccataagggc cattgccgat ctcaagccac tctccgttga acggttaagt ttccattgat 5640

actcgaaaga ggtcagcacc agctagcatg aaacggcatg actttttcaa gagtgccatg 5700

cccgaaggtt atgtacagga aagaactata tttttcaagg atgacgggaa ctacaagaca 5760

cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt 5820

gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac 5880

aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga 5940

cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt 6000

ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg 6060

aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg 6120

attacacatg gcatggatga actatacaaa tccggatcta gaaattttga ccttcttaag 6180

cttgcgggag acgtcgagtc caaccctggg cccgcgagca caacacagcc catagggtca 6240

actacctcaa ctaccacaaa aactgcaggc gcaactcctg ccacagcttc aggcctgttc 6300

actatcccgg atggggattt ctttagtaca gcccgtgcca tagtagccag caatgctgtc 6360

gcaacaaatg aggacctcag caagattgag gctatttgga aggacatgaa ggtgcccaca 6420

gacactatgg cacaggctgc ttgggactta gtcagacact gtgctgatgt aggatcatcc 6480

gctcaaacag aaatgataga tacaggtccc tattccaacg gcatcagcag agctagactg 6540

gcagcagcaa ttaaagaggt gtgcacactt aggcaatttt gcatgaagta tgccccagtg 6600

gtatggaact ggatgttaac taacaacagt ccacctgcta actggcaagc acaaggtttc 6660

aagcctgagc acaaattcgc tgcattcgac ttcttcaatg gagtcaccaa cccagctgcc 6720

atcatgccca aagaggggct catccggcca ccgtctgaag ctgaaatgaa tgctgcccaa 6780

actgctgcct ttgtgaagat tacaaaggcc agggcacaat ccaacgactt tgccagccta 6840

gatgcagctg tcactcgagg tcgtatcact ggaacaacaa ccgctgaggc tgttgtcact 6900

ctaccaccac cataactacg tctacataac cgacgcctac cccagtttca tagtattttc 6960

tggtttgatt gtatgaataa tataaat 6987

<210> 13

<211> 45

<212> DNA

<213>Primer 1

<400> 13

gttcatttca tttggagagg aaaaaatata aaaactcaac ataac 45

<210> 14

<211> 24

<212> DNA

<213>Primer 2

<400> 14

ttgatggtga actgcctcaa gagc 24

<210> 15

<211> 24

<212> DNA

<213>Primer 3

<400> 15

gctcttgagg cagttcacca tcaa 24

<210> 16

<211> 41

<212> DNA

<213>Primer 4

<400> 16

gacgaaagct gggaggcctg gtcgactcta gggggatccg a 41

<210> 17

<211> 23

<212> DNA

<213>Primer 5

<400> 17

cctctccaaa tgaaatgaac ttc 23

<210> 18

<211> 26

<212> DNA

<213>Primer 6

<400> 18

caggcctccc agctttcgtc cgtatc 26

<210> 19

<211> 22

<212> DNA

<213>Primer 7

<400> 19

ggctttcaag cgccagtgcg ag 22

<210> 20

<211> 22

<212> DNA

<213>Primer 8

<400> 20

gaagtgctct tgcacaaggg tg 22

<210> 21

<211> 51

<212> DNA

<213>Primer 9

<400> 21

gttcatttca tttggagagg gaaaactaaa ccatacacca ccaacacaac c 51

<210> 22

<211> 23

<212> DNA

<213>Primer 10

<400> 22

gataacgtgc ctaggaaaat ctg 23

<210> 23

<211> 21

<212> DNA

<213>Primer 11

<400> 23

gattttccta ggcacgttat c 21

<210> 24

<211> 42

<212> DNA

<213>Primer 12

<400> 24

gacgaaagct gggaggcctg ggaaattcgc ggtacccact ag 42

Claims (10)

1. a kind of method that big flux screening plant virus replicates required host's gene, which is characterized in that include the following steps：

1) structure of induction type viral infectivity clone：The expression vector includes agrobatcerium T-DNA left margin sequence, Agrobacterium T-DNA right border sequences, inducible promoter, plant positive sense RNA virus full-length cDNA, eukaryotic transcription terminator, Escherichia coli Required element and resistance screening gene are replicated with Agrobacterium, wherein plant positive sense RNA virus full-length cDNA is located at induction type startup Between son and eukaryotic transcription terminator sequence, agrobatcerium T-DNA right border sequence is located at inducible promoter upstream, Agrobacterium T- DNA left margin sequences are located at eukaryotic transcription terminator downstream；The induction type viral infectivity Cloning Transformation Escherichia coli place of acquisition In main bacterium, positive transformant is obtained；

2) genetic transformation of induction type viral infectivity clone：The positive transformant that step 1) is obtained imports Agrobacterium, then Induction type viral infectivity clone is transferred in plant, genetically modified plants are obtained；

3) plant virus replicates the big flux screening of indispensable gene：Mutant library is built with genetically modified plants, after mutant germination, Virus infection is induced using derivant, carries out the screening of mutant, the mutant plant that virus cannot be induced is obtained, in conjunction with figure The method of position clone and genome sequencing determines that the plant virus of screening replicates indispensable gene.

2. the method that a kind of big flux screening plant virus according to claim 1 replicates required host's gene, feature It is, the host strain of the Escherichia coli is Escherichia coli Trans10, and the Agrobacterium is Agrobacterium GV3101.

3. the method that a kind of big flux screening plant virus according to claim 1 replicates required host's gene, feature It is, the agrobatcerium T-DNA left margin sequence such as SEQ ID NO:Shown in 1；The agrobatcerium T-DNA right border sequence is such as SEQ ID NO:Shown in 2；The Escherichia coli and Agrobacterium replicate the sequence such as SEQ ID NO of required element:Shown in 3.

4. the method that a kind of big flux screening plant virus according to claim 1 replicates required host's gene, feature It is, step 1) the plant positive sense RNA virus is potyvirus or potexvirus virus；The horse It includes Brassica 2 et 4, marmor upsilon, marmor solani, plumpox virus, papaya ring spot that bell potato Y virus, which belongs to virus, Poison, soybean mosaic virus and marmor erodens；The potexvirus virus includes potato virus X, potato Austria Cuba's mosaic virus, garcinia mangostana mosaic virus, plantago mosaic virus and foxtail mosaic virus.

5. the method that a kind of big flux screening plant virus according to claim 4 replicates required host's gene, feature It is, step 1) the plant positive sense RNA virus full-length cDNA is the full-length cDNA of Brassica 2 et 4, sequence such as SEQ ID NO:Shown in 4.

6. the method that a kind of big flux screening plant virus according to claim 4 replicates required host's gene, feature It is, step 1) the plant positive sense RNA virus full-length cDNA is the full-length cDNA of potato virus X, sequence such as SEQ ID NO:Shown in 5.

7. the method that a kind of big flux screening plant virus according to claim 1 replicates required host's gene, feature It is, the inducible promoter described in step 1) is estrogen-inducible promoter XVE, sequence such as SEQ ID NO:Shown in 6.

8. the method that a kind of big flux screening plant virus according to claim 7 replicates required host's gene, feature It is, step 1) the resistance screening gene is hygromycin gene, sequence such as SEQ ID NO:Shown in 7, the hygromycin gene, Its upstream is estrogen-inducible promoter XVE-1, sequence such as SEQ ID NO:It is downstream estrogen-inducible shown in 8 Promoter XVE-2, sequence such as SEQ ID NO:Shown in 9.

9. the method that a kind of big flux screening plant virus according to claim 8 replicates required host's gene, feature It is, the plant positive sense RNA virus full-length cDNA described in step 1), the promoter of upstream is estrogen-inducible promoter XVE-2。

10. the method that a kind of big flux screening plant virus according to claim 1 replicates required host's gene, feature It is, step 1) the eukaryotic transcription terminator is T3A terminators, sequence such as SEQ ID NO:Shown in 10.