KR101570765B1 - Mixture comprising Agrobacterium tumefaciens species for causing infection activity of Broad bean wilt virus 2 - Google Patents

Abstract

The present invention relates to a Agrobacterium tumefaciens mixture capable of reproducing the infectious activity of Broad bean wilt virus 2 (BBWV2) in a plant. More specifically, the present invention relates to an Agrobacterium tumefaciens mixture, The present invention relates to an Agrobacterium tumefaciens mixture comprising two Agrobacterium tumefaciens transformed with SEQ ID NO: 1 or SEQ ID NO: 2, respectively, which can reproduce the infectious activity of the fake virus 2 in plants. The present invention also provides Agrobacterium tumefaciens of Accession No. KACC91892P or KACC91893P.

Description

The present invention relates to an agrobacterium tumefaciens mixture for reproducing the infectious activity of the bean curd virus 2,

The present invention is broad bean forged virus 2 (Broad bean wilt virus 2; BBWV2) can be reproduced in a plant. The present invention also relates to an Agrobacterium tumefaciens mixture.

Numerous technologies are needed for biotechnology research, among which transgenic technology is an essential technique that is indispensable. Transformation is the introduction of a trait (gene) that does not literally have it, or the removal of the original trait. This allows us to analyze the function of the trait.

In particular, plant transformation technology is an important technology that can be very useful for analyzing the function or expression of a plant gene. It can identify various phenomena involved in the development and differentiation of plants, or develop new functional varieties through introduction of useful genes Has been used as a technology to In general, transformation of plants has been developed based on tissue culture technology. However, when tissue culture is used, there is a disadvantage that time and effort are required because regeneration period is long and somatic cell mutation or natural teratogenesis Such as the appearance of a serious problem has been raised. Thus, a plant transformation technique that does not involve tissue culture has been developed, which involves transforming cells located at the growth point of a plant in the state of growing plants without tissue culture, and then transforming the transformed cells Is a technology that can acquire genetically stable transgenic plants. One technique derived from this technique is the Agro-infiltration method using the Agrobacterium strain.

Agrobacterium ( Agrobacterium < RTI ID = 0.0 > tumefaciens ) has been known for many years as soil bacteria causing Crown gall disease in many dicotyledonous plants. In the 1970s, the Ti plasmid of Agrobacterium was associated with pathogenicity, and T-DNA, a part of the Ti plasmid, was found to integrate into the plant genome. Later, it was found that T-DNA contains hormone synthesis genes (cytokinins and auxins) necessary for the formation of colon carcinomas and these genes are derived from bacteria but expressed in plants. A group of genes located within the toxic region (Vir region) of the Ti plasmid are required for the ablation of T-DNA and their transfer to the plant, and also the right border sequence located at the opposite ends of the T-DNA, It has also been found that border sequences called border sequences are required for abstinence.

Forged virus 2 ( Broad bean wilt virus 2; BBWV2) has become a problem in many crops, including Como virus (Comoviridae) and paba virus (since it was first reported in 1947 Broad separated from a spherical virus of about 25mm in diameter belonging to the genus Fabavirus), Solanaceae the world, including Europe. It is transmitted easily by nonphysiology by aphids, and the transmission rate by peach aphid and cotton aphid is 60 ~ 90%. In Korea, it is a virus that continues to grow mainly in the main cultivation areas of red pepper and pigmented sweet pepper. Since the first outbreak in Jeonbuk province in 1999, it has spread to the main cultivation areas of pigmented sweet peppers such as Chonnam and Gyeongnam in 2002 and has become a problem after PepMoV since 2005. The symptoms are similar to those of PepMoV disease in the early stages of development. In general, however, typical features are mosaic symptoms with little or no symptoms in the leaves, whereas gingivitis and gangrene stripe symptoms are seen in the fruit.

BBWV2 virion is an icosahedral particle composed of two proteins (large coat protein and small coat protein; LCP and SCP) and two single-stranded positive-sense RNAs of about 6 and 4 kb RNA) molecules. The two RNAs are translated from the cleavage of the functional protein by a hydrolytic cleavage into a polyprotein. RNA 1 encodes proteins involved in genetic replication and expression, and RNA 2 encodes migrating proteins and two CPs (Lisa and Boccardo, 1996).

International Patent Application No. PCT / JP1999 / 005386 (international filing date September 30, 1999) relates to a plant transformation vector, which comprises transforming a transformed Agrobacterium strain to raise the recognition of the border sequences of the vector by the vir proteins of the Agrobacterium And thus reduce the likelihood of transduction of DNAs other than T-DNA into plant chromosomes, in particular, these vectors contain right and left border sequences that can be recognized by the vir proteins of Agrobacterium, A T-DNA sequence located between the border sequences, wherein the gene to be introduced into the plant can be inserted therein, and a cloning start point enabling cloning of the vectors in the bacteria, wherein a plurality of left border sequences ≪ / RTI > are used for plant transformation.

However, the technology for reproducing the infectious activity into plants by introducing the infectious clone of the fake virus into the binary vector as an agro-infiltration into the plant has not been studied so far.

An object of the present invention is broad bean forged virus 2 (Broad bean wilt virus 2; It is an object of the present invention to provide an Agrobacterium tumefaciens mixture which can reproduce the infectious activity of BBWV2 in plants.

In order to achieve the above object, the present invention is broad bean forged virus 2 (Broad bean wilt virus 2; 0.0 > 1 < / RTI > or 2 < RTI ID = 0.0 > ID < / RTI > transformed with Agrobacterium tumefaciens ). < / RTI >

The present invention also provides Agrobacterium tumefaciens transformed with SEQ ID NO: 1 of Accession No. KACC91892P and Agrobacterium tumefaciens transformed with SEQ ID NO: 2 of Accession No. KACC91893P.

The present invention also provides an agro-infiltration kit comprising Agrobacterium tomefaciens strain transformed with SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

Preferably, the plant is pepper ( Capsicum annuum ) or paprika ( Capsicum annuum there is . angulosum , which is an agrobacterium tomeepathis mixture.

The present invention forgery broad bean virus 2 (Broad pepper and paprika bean wilt virus 2; BBWV2) can be continuously reproduced to study the function of the protein expressed by the virus, to identify pathogenic factors, and to utilize it as a basic data for cultivating resistant varieties.

FIG. 1 is pCassRz vector and Broad fake virus 2 (Broad bean wilt virus 2; BBWV2) ((A) pBBWV2-PAP1-R1 in which BBWV2 RNA1 is cloned, and (B) pBBWV2-PAP1-R2 in which BBWV2 RNA2 is cloned.
FIG. 2 is a graph showing the results of a comparison between BBWV2 infected Hot pepper, Quarri green pepper, Paprika and Nicotiana benthamiana ) plant.
FIG. 3 is a photograph showing the BBWV2 infectivity using RT-PCR (lane 1-2: Hot pepper, lane 3-4: Quarri green pepper, lane 5-6: Paprika, lane 7: Nicotiana benthamiana).
Figs. 4 to 8 are the nucleotide sequences of SEQ ID NO: 1, and Figs. 9 to 13 are the nucleotide sequences of SEQ ID NO: 2 (upper case: virus sequence, lower case: vector sequence).

Embodiment of the first aspect of the present invention is broad bean forged virus 2 (Broad bean wilt virus 2; ( Agrobacterium tumefaciens ) transformed with SEQ ID NO: 1 or SEQ ID NO: 2, respectively, which can reproduce the infectious activity of BBWV2 in plants, and Agrobacterium tumefaciens do.

Preferably, the plant is characterized in that pepper (Capsicum annuum) or paprika in Agrobacterium-to mepa sieon's mixture, characterized in that (Capsicum annuum var. Angulosum).

A second aspect of the invention provides Agrobacterium tumefaciens of Accession No. KACC91892P and Agrobacterium tumefaciens of Accession No. KACC91893P.

A strain containing pBBWV2-PAP1-R1 (SEQ ID NO: 1) or pBBWV2-PAP1-R2 (SEQ ID NO: 2) gene was deposited at KACC (Agricultural Microbiology Resource Center) on Nov. 1, 2013 and deposited at accession numbers KACC91892P and KACC91893P Respectively.

The term "Agro-infiltration" used in the present invention means a method for introducing a gene into a plant and expressing it or producing a desired protein in a plant. A suspension of Agrobacterium having the gene to be transferred is injected into the leaves of a plant by using a needle-free syringe to transfer the desired gene to the plant cell. This has the advantage of being quick and convenient compared to traditional plant transformation.

As used herein, the term " vector " refers to a nucleic acid molecule capable of binding another nucleic acid to it for delivery. A preferred vector is a vector capable of self-replication and expression of the nucleic acid to which it is coupled.

As used herein, the term " transformation " means that the foreign DNA or RNA is absorbed into the cell and the genotype of the cell is changed. Host cells include, but are not limited to, plant cells, prokaryotic cells, yeast cells, insect cells.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Design and production of recombinant vectors for plant transformation using infectious clones of Drosophila virus 2

A pCassRz vector was used as a binary vector for producing an infectious clone of BBWV2 (Fig. 1). The pCassRz vector consists of the left border of T-DNA, double 35S promoter of cauliflower mosaic virus (CaMV), multiple cloning site (MCS; Stu I, Kpn I, Xba I, Bam HI), a cis- cleaving ribozyme sequence (Rz), a 35S terminator (T), and a right border of T-DNA. Thus, Agrobacterium can be transformed and the desired gene can be transferred to plants through agro-filtration. In addition, the pCassRz vector contains a kanamycin resistance gene and can be selectively cultured when transformed into E. coli and Agrobacterium.

A BBWV2 isolate isolated from domestic paprika was used to produce a BBWV2 infectious clone capable of agro-filtration using a binary vector. Total RNA was extracted from paprika infected by the BBWV2 isolate. CDNAs for BBWV2 RNA1 and RNA2 were synthesized by reverse transcription using primers (5'-CATCGGAACTCGGTACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3 ') from this total RNA. PCR was performed using primers 5'-GTTTTAATAAAATATTAAAACAAACAGCTTTCGTT-3 'and 5'-CACATCGGAACTCGGTACCT-3' to amplify the entire genome of BBWV2 RNA1 and RNA2 from the synthesized cDNA. Each primer was denatured at 94 ° C for 3 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 8 minutes in one cycle, followed by reaction at 72 ° C for 10 minutes, The DNA fragment containing the base sequence of the RNA was amplified.

to cloned into the vector pCassRz were each cloned by treating the PCR product of BBWV2 RNA1 and RNA2 with Kpn I by using the vector of the StuI and KpnI restriction enzyme site. The resulting BBWV2 RNA1 clone was named pBBWV2-PAP1-R1 (SEQ ID NO: 1) and the RNA2 clone was named pBBWV2-PAP1-R2 (SEQ ID NO: 2). SEQ ID NO: 1 is shown in FIGS. 4 to 8, and SEQ ID NO: 2 is shown in FIG. 9 to FIG. The uppercase nucleotide sequence shown in Figs. 4 to 13 is the sequence of the virus, and the lowercase nucleotide sequence is the vector sequence.

The strains containing the pBBWV2-PAP1-R1 (SEQ ID NO: 1) and pBBWV2-PAP1-R2 (SEQ ID NO: 2) genes were deposited at KACC (Agricultural Microbiology Resource Center) on Nov. 1, 2013 with accession numbers KACC91892P and KACC91893P .

BBWV2  Infectious DNA  Agrobacterium containing clones Two Mephasians Recombinant  making

Agrobacterium strain GV2260 was transformed by electroporation using the plasmid DNA of the BBWV2 infectious DNA clone. Agrobacterium transformed with pBBWV2-PAP1-R1 or pBBWV2-PAP1-R2, respectively, was shake cultured in 5 ml of YEP liquid medium (containing 100 mg / ml kanamycin and 50 mg / ml rifampicin) at 30 ° C for 16 hours 1 ml of the primary culture was inoculated into 50 ml of fresh YEP medium (containing 50 mg / ml of kanamycin, 50 mg / ml of rifampicin and 20 uM of acetosyrinone) and cultured at 30 ° C for 6 hours with shaking. The culture broth was centrifuged at 4800G for 10 minutes to precipitate Agrobacterium and then resuspended in MMA infiltration buffer (MS salts, 10 mM MES, pH 5.6, 200 uM Acetosilin Zone) at a wavelength of 600 nm at a concentration of OD 0.7 Lt; / RTI > The suspension was then incubated with shaking at 30 DEG C for 2 hours.

Agro - Infiltration (Agro-infiltration)  Inoculation of infectious clone and confirmation of infection

Agrobacterium broth transformed with pBBWV2-PAP1-R1 or pBBWV2-PAP1-R2, respectively, were mixed in the same ratio. Then, the pelleted surface of red pepper, chilli pepper, paprika and Nicotiana botanicum leaves was pressurized with a 1 ml syringe Infiltration.

In the plants infected with BBWV2, symptoms and infectivity were confirmed. The BBWV2 infectious clone was inoculated into various plants through agro-infiltration, and the symptoms caused by BBWV2 after 7 to 12 days were observed (FIG. 2). In addition, the total RNA was extracted from the upper lobes showing symptoms and RT-PCR was performed using primers (primer_1: 5-TGAATCCAGATGGCACATGG-3 and primer_2: 5-CCCTCACTACTGAAATTTACTTG-3) capable of specifically detecting RNA1 of BBWV2 And examined for infection. As a result, a PCR product of a size expected for a plant inoculated with the BBWV2 infectious clone was confirmed, and thus it was confirmed that the plant was systemically infected by BBWV2 (FIG. 3).

National Institute of Agricultural Science KACC91892P 20131101 National Institute of Agricultural Science KACC91893P 20131101

<110> Rural development administration <120> Mixture comprising Agrobacterium tumefaciens species causing causing          infection activity of Broad bean wilt virus 2 <130> 2013-0410-10-A (P13-264) <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 16236 <212> RNA <213> pBBWV2-PAP1-R1 <400> 1 gttttaataa aatattaaaa caaacagctt tcgttccgaa aacagctttc agttactaaa 60 cagctttcgt tccgattaaa cagctttcaa acaaacagct ttcatttcat tcagccattc 120 ttggtttgtg tggcttcgga aagaacccgg aaaaagggag tgtgattaaa agcgcaccat 180 atatttcgaa gatcattttg aatttcattt tgttacctta attttaagat ggatttcagt 240 atgatgcaaa tgactgtggg ttttctcaaa acttcaatgg gtttacaatc cattaaggac 300 attgtggaaa aagcctgcgt ggcggagaag gacaaaacct tgctccacat tcatttatgc 360 tttttccacg ccaatgagat ggctcgggac tataatgaag gtatggacat ggggcagata 420 caaagttctg ctgcaatcaa ataccgagct gcggtgttaa agcgccatgt tttaatgaat 480 gtggaaacgg gaagatatga tcgtgcacgc atgatgcagt acaataatga aacatgtgtt 540 aattggttct cgtgcaggat cgtcgaagaa aaggttggaa aagtgcttga cgaagaggag 600 gagcaagagg atgacattgt tgataatttt ctcaagaatt gtgcgatttc tgagaaggtt 660 ggccagacat caggagaagg cgctgagcat tgtgcctttg gccagggttt gtatgagtat 720 gt; gatgattttc tggacaaagt gtatgcagtt atggcacagg ttttcgcggc ttggatgcct 840 agaattagag ctgcgtttca gtggttcgag aacattaaag aagtcattaa gagatgggcc 900 aatacgatgc atgaaaaaat caatttcatt ctagttggca tggaagattg tctgtacatg 960 ggcgctggtc tcgttgctgc tacttgcatc gtcacattgc ttgaaaaatt catgatagca 1020 attgggattt taaccaagcc ttgtggggcg gctactctct tcctaacaac tgcaatggcg 1080 gctatttcag ccacatacgt gtgtggaaaa gcattggaaa agtcagctat gctctccaat 1140 ttgttactct ttgtaaccac caactgccga gttgtgttgg atacgctatt caatcacgaa 1200 ttaggaaggc aggagcagga gatgagccac aatggtgaga aagccccgac aagctctggt 1260 cagtttggca tttcaacaat gctacaagat gtagccaatt tagtttcaac gtggtcaaca 1320 agttcggtca cagaaattgg gaaaacattt ggtgccattt cacagattaa aaatggaatt 1380 ctagctttaa gggacatggt gtactttgtg tttgaaaagt tgagtgaatt ggctcacaga 1440 gtgcttgggt ttgaatctca agtattggct gatttgtcaa ttttgcttgg agagaatgtg 1500 gcagactggt tatctgaatg cgattgcatg gtggcttata tgcttgagtt taactccaga 1560 aatagagaga tttttgatag actgtctcag ctaatagaga aaggcagaat gataagaaca 1620 ggagttttgc gctcaagtca tcgtgggtcg tcacaggtca tgtcgcttgt tactaaggct 1680 ttggagaagc taatagagtt gcataactca gttgtgatgg caggttcaaa cacaactagg 1740 aagtctccct ttatggtttt cttcacaggt gcctctggta cagggaaaac ttcagttgtt 1800 caaagagttg ctataaattg gctgcaggaa gaacagcttg ggacgagtga aatatacgct 1860 cgcaatgggc aggatccttt ttggtctggt tacaaaaggc atgcggttgt cacgtatgat 1920 gatttcggag ccgttccagg cacaacttca aatgaagctg aaattatcaa cgtcatatca 1980 agaaacccat atgcaacagt gatggcaggc ttggcggaaa aaggaatgta ttttgactca 2040 agattggttt tggctagcag caatttcctt gctgccaacc cagagtcagg agttcatgat 2100 tcagaggcgt acgagagacg aaggcacgca gtggtaagag tttcattgaa accaggagtt 2160 ccttacaatg ccaatgatcc gtgcgccaat caaaattaca ccttgttaga gtccaaaaca 2220 ccctttaatg aaattcaaac atttgaaaca tatgcggaac tctggtcttt tttgtacaca 2280 aggtttaagg agcacgagga acaagaggaa ttgtacctaa agtccttgcc aatattggat 2340 tcagacaaaa aggaagctct ggagggtttg ataggtctca cggttatagc cacttcattt 2400 gctccgaagg cagtcatgca gtttggagca gaaaagtttc caggatacca ttttctcatt 2460 tcggatggtg agtattgcta tttttggcat ggagatgggt cggtggaatt tgtcagtgta 2520 gagcgaatga aattgagcaa gcaagatata attcagttga agcagcaagg actatccacg 2580 gccatgatgt ataaggatct ggctaaggcc tttcccactt tgaattcatt ggctgtgctg 2640 tatgccaaaa atattgtggt aaagcgctgg attggtgcag atttggagcc cacaaagagc 2700 tgtgaagatg tttacatgcg tgagcaaata ggcaatttgc ccaaatggca gagggcgtat 2760 ttgcacgtgc tcagcaagta tttaacagtt caaagccctc gaggatggtt tatggaatgt 2820 ttagaagaag caaagaggaa tttgcgggca acatacttat gggaatacaa acaatggcca 2880 ttgcctttga aattggcatt gggttcattg attgccatca tggcgggtgg agccatttgg 2940 tattcactac agtctttatg gtgtgtgtct ggggacgctt cttttatagc gggagcagca 3000 acggtttttt ctgtttcttc ttttgctgga caaagtgatg tgccaaaccg ggataacaca 3060 gaaagatcgt tcagaaacag gaagatccgt gctagaacat ggcaaggtca gagttcgtgt 3120 tttggagact cggcactttg gatcgcggaa acttgtatgg caacattgac cttctcaaat 3180 gtaagaacac aagtgtgctt agcacctggg agaggtttct ttggagtgaa ccattgcctt 3240 gctgtcattc ccgcaggtgt gatggtcaag ctggattcga gtataggagt aacttatttt 3300 gtgtgggaga aggaaaaact gatacagttt gaaggcaatg aaatagcact gtatatgaca 3360 agcacaatgc ccaagaccgt ggattctctt ttgagcagaa ttcactttga tgctgagact 3420 ctgcccaaga cctttagtgc agttttcttt tcctacaagt atgatccagt aattcagcaa 3480 atggtgcctg agttgggaag cgtcacgtgc aagattcaca acaaagcttt cacattggca 3540 catggtgaat ataggcggga aattccccag agccttacgt atgaggcaag cactgtggct 3600 ggtgattgtg gctctttaat attggcggaa attgaaggga aattcaagct agtgggtatg 3660 catgtggcat ttaatggcaa ggaaggaagt gcaagtttta tgccgtacca tgctctttta 3720 gatcaaaaag tgggccaagg agattttgtg ttgaagtacc aagaatgggc tgagccaaag 3780 attttgggtc caggttgcag ggcgatggga ctcatagatc cagaacatgc tttggcagct 3840 agtgggaaga ctacatttgt tgaaacccca gaggagtggc atttgaatta cccatgtgac 3900 aaacttccta gtgttttggc aaggggtgat cccagattgg cgggcacagt tcatgccgac 3960 tacgacccat ttgccgcagg catgagcaaa tatgcaaaag aggcagggcc ttttgaagct 4020 gccagcttga agcaggtgtg tttgggaata actgaagtat gggaagatgc ttcagcagat 4080 tttccaatgg atgaagttga tttggacact gctatcaatg gcttggaaaa tgtcgaattc 4140 tttgatgcct tggttttagg aacttctgag gggtttcctt accggctaga tcgtggtcca 4200 ggcgagaaag gaaagagcag gtatgtgtct ggtgagagtg ggaattttaa aatcactgat 4260 gaaggtgtgc tctcggacat taactggttc gaggagacaa gcaagacaca ggtgcctgac 4320 ctgtattgta ttgaatgcgt caaggatgag cgcttgccaa ttaggaaagt gctgcatgaa 4380 cccaaaagtc ggttgttcac agtacttcca atgtcctaca acatagccat taggaagaag 4440 tttttgaact ttgtcagatt tttcatgaaa aggagggatg tgttacctgc ccaggttgga 4500 gttaatccct attctcggga gtggacccga atagccaata agttgttgag caaaggtaac 4560 aacattctgt gttgtgatta cagcaggttt gatggttttt tgcccaagtg tgttatgaat 4620 gagattgggg acatgatagc cagggttatg cgagcaaatg atgagtccaa gactcagatc 4680 aagaatctta tgcttgcgtg taccagtcga tatgccatgt gcaaccgagt cttgtacaga 4740 gttgaaaatg gtatcccttc tggctttccg ctcactgtcg ttgtgaattc catcctgaat 4800 gagatcttgg ttaaatatgc gtattggcat tgctttgtgg ataatccaac tgttcaaagt 4860 aactttgatg cgcatgtttc catggtggta tatggtgatg acaatttgat atcagtatcg 4920 gatgcaataa gctcaaagtt tgatggaaat ttccttgtga agttcatgga aggacttgga 4980 attaaggtta ctgatggaat tgacaaaaca aaaattggaa tagagttccg tcgcctggag 5040 aattgtgatt tccttaagcg ctctttcaag ttgaatccag atggcacatg gcgcagcccc 5100 atgtctaagg agagcttatg gccacagctg cactatgtga aagcaaaaaa attggaaatg 5160 gctgaggctt acataaacaa ttgcaataat atcttgcgtg agctgtggtt gcatgatgtt 5220 aaagaggcca aggaattccg caataaagtg ctgagaagtt taaaatggat tggccatgag 5280 caattgctca atatgcaaca attggcagtg tttcatagtg agcaaatgaa cggggtgagt 5340 gattttctat ccacttgtgt gacagttgat agcatttcat taatggatcc cctggttcct 5400 ggtttgctac cggtcaagac ttgtgaagtg attcctcgag tttttgtggc ggcagaaaag 5460 cattttgaag gaaattttga ggatttcttc acagtatcca taaccacaag tcgtaaattc 5520 aaggaggaca agggttttgt tcttttgttc ccatatggag caggcagagg tggattgcca 5580 acaacacaat tcatgaaaga gaatgtggta aggaaagggt gttccataca aaagaaattc 5640 aggcaggcat atgagagagg agagaaaatt ttgttcatct cgcagagctc agtggttcca 5700 gcttacgtgt ttgctgtcat gctactacat tcaattggag caatttccag attgacgagc 5760 aataaagctc tcacacaagc aatgcaaact tgcaagaaat tagagtattt gcctaaagag 5820 tatgaagatt tcttttgaat ttaattttat tttgttgtgt gtgcatttta attattaatc 5880 actacttata ttgtataata tgtattatgc agggttgaaa tgcctttccc aagtaaattt 5940 cagtagtgag ggaaaaaaaa aaaaaaaaag gtacctctag aggatccgat accctgtcac 6000 cggatgtgtt ttccggtctg atgagtccgt gaggacgaaa caggactgtc ctgcaggagc 6060 tcgaattcgg tacgctgaaa tcaccagtct ctctctacaa atctatctct ctctattttc 6120 tccataaata atgtgtgagt agtttccgat aaggaaatta gggttcttat agggtttcgt 6180 catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc 6240 aataaaattt ctaattccta aaaccaaaat cagtactaaa tcagatcccc aattcactgg 6300 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 6360 cagcacatcc ccctttcgcc agtggcactg gccgtcgttt tacaacgtcg tgactgggaa 6420 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 6480 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 6540 tgctagagca gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca 6600 gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac 6660 ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac 6720 cacagggttc ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag 6780 tcggcttctg acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag 6840 ttacgcgaca ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc 6900 gcataaagta gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg 6960 ccgctggcct gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac 7020 gggccgaact gcacgcggcc ggctgcacca agctgttttc cgagaagatc accggcacca 7080 ggcgcgaccg cccggagctg gccaggatgc ttgaccacct acgccctggc gacgttgtga 7140 cagtgaccag gctagaccgc ctggcccgca gcacccgcga cctactggac attgccgagc 7200 gcatccagga ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc gacaccacca 7260 cgccggccgg ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc 7320 taatcatcga ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg 7380 gcccccgccc taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg 7440 aaggccgcac cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg accctgtacc 7500 gcgcacttga gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc 7560 gtgaggacgc attgaccgag gccgacgccc tggcggccgc cgagaatgaa cgccaagagg 7620 aacaagcatg aaaccgcacc aggacggcca ggacgaaccg tttttcatta ccgaagagat 7680 cgaggcggag atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt 7740 gcggctgcat gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct ggccggccag 7800 cttggccgct gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa 7860 cagcttgcgt catgcggtcg ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa 7920 ggggaacgca tgaaggttat cgctgtactt aaccagaaag gcgggtcagg caagacgacc 7980 atcgcaaccc atctagcccg cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat 8040 tccgatcccc agggcagtgc ccgcgattgg gcggccgtgc gggaagatca accgctaacc 8100 gttgtcggca tcgaccgccc gacgattgac cgcgacgtga aggccatcgg ccggcgcgac 8160 ttcgtagtga tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca 8220 gccgacttcg tgctgattcc ggtgcagcca agcccttacg acatatgggc caccgccgac 8280 ctggtggagc tggttaagca gcgcattgag gtcacggatg gaaggctaca agcggccttt 8340 gtcgtgtcgc gggcgatcaa aggcacgcgc atcggcggtg aggttgccga ggcgctggcc 8400 gggtacgagc tgcccattct tgagtcccgt atcacgcagc gcgtgagcta cccaggcact 8460 gccgccgccg gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc 8520 caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa 8580 tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg 8640 caacgttggc cagcctggca gacacgccag ccatgaagcg ggtcaacttt cagttgccgg 8700 cggaggatca caccaagctg aagatgtacg cggtacgcca aggcaagacc attaccgagc 8760 tgctatctga atacatcgcg cagctaccag agtaaatgag caaatgaata aatgagtaga 8820 tgaattttag cggctaaagg aggcggcatg gaaaatcaag aacaaccagg caccgacgcc 8880 gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc 8940 tgccggccct gcaatggcac tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa 9000 accatccggc ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa 9060 ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg 9120 gcaagcggcc gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc 9180 gtcgattagg aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta 9240 tgacgtgggc acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa 9300 gcgtgaccga cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt 9360 ttccgcaggg ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt 9420 ttcccatcta accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg 9480 cgtgttccgt ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa 9540 gcagaaagac gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca 9600 gcgtacgaag aaggccaaga acggccgcct ggtgacggta tccgagggtg aagccttgat 9660 tagccgctac aagatcgtaa agagcgaaac cgggcggccg gagtacatcg agatcgagct 9720 agctgattgg atgtaccgcg agatcacaga aggcaagaac ccggacgtgc tgacggttca 9780 ccccgattac tttttgatcg atcccggcat cggccgtttt ctctaccgcc tggcacgccg 9840 cgccgcaggc aaggcagaag ccagatggtt gttcaagacg atctacgaac gcagtggcag 9900 cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct 9960 gccggagtac gatttgaagg aggaggcggg gcaggctggc ccgatcctag tcatgcgcta 10020 ccgcaacctg atcgagggcg aagcatccgc cggttcctaa tgtacggagc agatgctagg 10080 gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta 10140 cattgggaac ccaaagccgt acattgggaa ccggaacccg tacattggga acccaaagcc 10200 gtacattggg aaccggtcac acatgtaagt gactgatata aaagagaaaa aaggcgattt 10260 ttccgcctaa aactctttaa aacttattaa aactcttaaa acccgcctgg cctgtgcata 10320 actgtctggc cagcgcacag ccgaagagct gcaaaaagcg cctacccttc ggtcgctgcg 10380 ctccctacgc cccgccgctt cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc 10440 tggcctacgg ccaggcaatc taccagggcg cggacaagcc gcgccgtcgc cactcgaccg 10500 ccggcgccca catcaaggca ccctgcctcg cgcgtttcgg tgatgacggt gaaaacctct 10560 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 10620 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt 10680 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact 10740 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 10800 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 10860 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 10920 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 10980 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 11040 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 11100 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 11160 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 11220 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 11280 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 11340 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 11400 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 11460 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 11520 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 11580 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 11640 gattttggtc atgcattcta ggtactaaaa caattcatcc agtaaaatat aatattttat 11700 tttctcccaa tcaggcttga tccccagtaa gtcaaaaaat agctcgacat actgttcttc 11760 cccgatatcc tccctgatcg accggacgca gaaggcaatg tcataccact tgtccgccct 11820 gccgcttctc ccaagatcaa taaagccact tactttgcca tctttcacaa agatgttgct 11880 gtctcccagg tcgccgtggg aaaagacaag ttcctcttcg ggcttttccg tctttaaaaa 11940 atcatacagc tcgcgcggat ctttaaatgg agtgtcttct tcccagtttt cgcaatccac 12000 atcggccaga tcgttattca gtaagtaatc caattcggct aagcggctgt ctaagctatt 12060 cgtataggga caatccgata tgtcgatgga gtgaaagagc ctgatgcact ccgcatacag 12120 ctcgataatc ttttcagggc tttgttcatc ttcatactct tccgagcaaa ggacgccatc 12180 ggcctcactc atgagcagat tgctccagcc atcatgccgt tcaaagtgca ggacctttgg 12240 aacaggcagc tttccttcca gccatagcat catgtccttt tcccgttcca catcataggt 12300 ggtcccttta taccggctgt ccgtcatttt taaatatagg ttttcatttt ctcccaccag 12360 cttatatacc ttagcaggag acattccttc cgtatctttt acgcagcggt atttttcgat 12420 cattttttc aattccggtg atattctcat tttagccatt tattatttcc ttcctctttt 12480 ctacagtatt taaagatacc ccaagaagct aattataaca agacgaactc caattcactg 12540 ttccttgcat tctaaaacct taaataccag aaaacagctt tttcaaagtt gttttcaaag 12600 ttggcgtata acatagtatc gacggagccg attttgaaac cgcggtgatc acaggcagca 12660 acgctctgtc atcgttacaa tcaacatgct accctccgcg agatcatccg tgtttcaaac 12720 ccggcagctt agttgccgtt cttccgaata gcatcggtaa catgagcaaa gtctgccgcc 12780 ttacaacggc tctcccgctg acgccgtccc ggactgatgg gctgcctgta tcgagtggtg 12840 attttgtgcc gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg 12900 gtgtaaacaa attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact 12960 gaattaacgc cgaattaatt cgggggatct ggattttagt actggatttt ggttttagga 13020 attagaaatt ttattgatag aagtatttta caaatacaaa tacatactaa gggtttctta 13080 tatgctcaac acatgagcga aaccctatag gaaccctaat tcccttatct gggaactact 13140 cacacattat tatggagaaa ctcgagcttg tcgatcgaca gatccggtcg gcatctactc 13200 tatttctttg ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct 13260 acaccccat cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc 13320 ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa 13380 ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg 13440 agtcgtggcg atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc 13500 atacaagcca accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg 13560 aacatcgcct cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg 13620 ttggagccga aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc 13680 atcagctcat cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc 13740 cagtgataca catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga 13800 ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg 13860 atcgcatcca tagcctccgc gaccggttgt agaacagcgg gcagttcggt ttcaggcagg 13920 tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctaaac 13980 tccccaatgt caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca 14040 taacgatctt tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct 14100 cctacatcga agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag 14160 acgctgtcga acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt 14220 ttcatatctc attgcccccc gggatctgcg aaagctcgag agagatagat ttgtagagag 14280 agactggtga tttcagcgtg tcctctccaa atgaaatgaa cttccttata tagaggaagg 14340 tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt cagtggagat atcacatcaa 14400 tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt 14460 gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta 14520 tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga 14580 cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc 14640 tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg 14700 tcgtgctcca ccatgttatc acatcaatcc acttgctttg aagacgtggt tggaacgtct 14760 tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag 14820 gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt gccaccttcc 14880 ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc gaggaggttt 14940 cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga gactgtatct 15000 ttgatattct tggagtagac gagagtgtcg tgctccacca tgttggcaag ctgctctagc 15060 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 15120 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 15180 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 15240 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgaattc tggcacgaca 15300 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 15360 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 15420 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc 15480 tgcaggtcaa catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag 15540 tctcagaaga ccagagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc 15600 tcggattcca ttgcccagct atctgtcact tcatcgaaag gacagtagaa aaggaagatg 15660 gcttctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat gcctctaccg 15720 acagtggtcc caaagatgga cccccaccca cgaggaacat cgtggaaaaa gaagacgttc 15780 caaccacgtc ttcaaagcaa gtggattgat gtgatggtca acatggtgga gcacgacact 15840 ctcgtctact ccaagaatat caaagataca gtctcagaag accagagggc tattgagact 15900 tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac 15960 ttcatcgaaa ggacagtaga aaaggaagat ggcttctaca aatgccatca ttgcgataaa 16020 ggaaaggcta tcgttcaaga tgcctctacc gacagtggtc ccaaagatgg acccccaccc 16080 acgaggaaca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga 16140 tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc gcaagaccct 16200 tcctctatat aaggaagttc atttcatttg gagagg 16236 <210> 2 <211> 13901 <212> RNA <213> pBBWV2-PAP1-R2 <400> 2 gttttaataa aatattaaaa caaacagctt tcgttccgaa aacagctttc agttactaaa 60 cagctttctt ttggaaaaca gctttcagat actttgaagt tttcaatttg aacccggaaa 120 aagggagtgt gatttaaagc gcaccatata ttttgaaact tcttttctct cattttctat 180 cttcttttct tgtaagtgca tactacattt aagatttgat cttgaacatg ctgtaatgcg 240 tcccgaactt gttgcagtgc tagatagata tttctcagaa atcataagtt gcttctttct 300 aggttggtta ttaaattttc tgcttgtttg gttctgttcc acaaagagca cttttctttt 360 gtggtcagtg tttttgtacg tttgttacta catattgagg atcgaatttg tgtatatcgt 420 tgcacccttc ttaaaaacga tatacacaaa tagttctcaa tatcatactg tagattgggc 480 aaacgcgtat acggcattgc ctaaaggatt gtgggagcaa atcactgatt acaattactg 540 ttataatttc cctcctcctt gtgtcgaagg ttttgtgtct gatttctcac cgagattcac 600 acttaaagaa cttgaaatca tgaatgaggc aaatatcact ccagtgcata caattcccaa 660 agacacattg cttaagagag cgagtgacta caaattagct gtggagagga agaaatccat 720 cttgccaaga gtgcaagatt tgtatgaaat ggacaagtgg cacaacttga gaagtaagtt 780 gagcaagaac gttcctagct acgtcacgac atcagagatt gctgttggtg cgatgtcagg 840 tgcgggaaac actaaattgg cgataccagt tgtggagaag tacactgagg aagtggctga 900 cgacagattg ccagacaagg ttcgtgctaa agctgatcag ataatggttg cagctattga 960 gttggtggcg gatggttttg cttcagtcaa ttcggatgtt accatggctg gtgcactcta 1020 cgataagcgc cacaagacca ttgcaagttc ttttaaggga gctttcgcat ctcgggcaag 1080 tggtgtccct tcacatgtaa tttattttcc aatgcataga gtccctgcat gtgatgaccc 1140 gaacacaact ttggagcttt caatggtgag ccgagattca gattttgatg agggctacac 1200 attggccaac atatcagctc gcactctcta cgttcgtgca aaaggacctg aaaaggtgac 1260 tgaaacaaga catcttttga aagccaagac tgaggatgtg gtcaaagcgc gccagtttgc 1320 tagcgaagca caagttgttt ttgcaacgcc acgattgttt cctgaagtaa atttggacaa 1380 ttacaatttg cctgggccta gcaatgcaca acatacagag gctattacca ctgatagagg 1440 aatattgttt ccaaagccaa aattcaaagg caatgaagtg gtacttaatt acacaggctc 1500 agggagaatc aggaacattg gccctcagag atttgagaag aaaaccgcca ctggtgaaca 1560 gtttgtcaga tgtgtcgatg atcttggatg cctgtctgat gaggatggca aagattatag 1620 atatggtcaa ggtctgatgg aggaggatgt tttgaatgtg caaacaaata attttgccat 1680 agagtctgct acagagacca tgcgcttgct gtttagtggg tatgcaagca ttccactgaa 1740 cgtgatacct ggaacaaaaa tcactgtggc ttacttaaat gaactatcca aacatagcgc 1800 tgtgcatact ggtttgctga acatgttgag taagatccct ggctctttaa aagttaagat 1860 taattgccag gtggctccga catgcggtat tgggctggca gtcagctatg ttgaagggaa 1920 tgaaagtgcg aacttgggtt ctagtctggg acgcttgttg ggcattcagc actacaaatg 1980 gaatccagcc atagagccat atgtggagtt tgttttcaaa cctttttctt gtgcagattg 2040 gtggaatatg cattacttgg ggtctttcaa atttgcccct gtggtggtga tacaaacgct 2100 ttccaaatgg ctgaatgctc cgaaggtaga tgctagaata agctttgcaa tctactatga 2160 acccaccgtt atgctgccta agcaaatagc aactcttgaa aatgctccag cgttcatgtt 2220 tcgcaaagag ttggggacat tggctttcaa acaaggagag cgtgtggctt attcctttga 2280 agttaatttt ggcaaaccac agacagatgg gaaggaagtg acctcaactt ttgcctcttc 2340 atattgtggt cttagtcagt atatgcaatc ggatgtcatt ttagatttta ctcttatgag 2400 tagccctatg attggaggta ctttctcaat tgcatatgtt gctggagcct acattgaaaa 2460 ggttgggaac atgcagattc ttgattcgct gccccacatc gattttactt tttcctcagg 2520 atctaagagc acgcgttccg tgcgctttcc taaagaagtc tttggagtgt accaagcgct 2580 tgatagatgg gatttggatt cggcaagagg ggatgatgtc tcaggaaact tcgtacttta 2640 ccaaagagac gcggtgtcga gtgctttgga aggagagctt acattcagaa tagcagctcg 2700 tttatctgga gatatcagtt ttacaggagt gagcgcaggt tacccaacga cgatcacacg 2760 catagggaag ggcaagacta taggaagatc gcttaatcct gaaatcagaa aacctttgag 2820 gtatatgctt gggcaagcgc atgtgacacc caaagatttt agttcagtgc gcttcgtgat 2880 gggccattgg aaatacaagg caggtttgta tcctgggagc aaatcggatg aagacattca 2940 tcctttttct ttgaaaatgc gcctagatgg atctaagagc agcgagaact ttgaaattat 3000 acattcccct tttgtgcgtt tgttgcagaa ttgtgcatgg atgagagga ctttgaagtt 3060 ttacgtggtg gcccgtgcca gttctgacta tatgagttac cgtagaacct cccaattgat 3120 agtttcagct catgaaaata gtcttagttc caatcagttc tacagtggag ttttgacgag 3180 tccaagtggt gagttgagtt tctccagaga agtggttggt cccgtggatg gttttgcttc 3240 tatgggttgg aacgttcgtg ggagcaagaa gttctataag atacacgtgg agatggggaa 3300 tgttcatgag tatgatacagi tagtgttgta cggacaattt ggctccaatg tcgaatttgc 3360 tggccaacag aaaggtgggc attacctgtt ggagaaggaa actcccatat ttaagacaat 3420 taagtattaa taattcaaat agttatttgg catttgagtt ttaataaaaa ggcgtaggtc 3480 actagcctcc cattaaaatg ggttccatga cctttatttg attgtaaaat ttactatttt 3540 taaaacacat gtgtgctgtg ttaggttaaa gcttcctata tgtataggct ttaatagtat 3600 tttaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaggtacc tctagaggat ccgataccct 3660 gtcaccggat gtgttttccg gtctgatgag tccgtgagga cgaaacagga ctgtcctgca 3720 ggagctcgaa ttcggtacgc tgaaatcacc agtctctctc tacaaatcta tctctctcta 3780 ttttctccat aaataatgtg tgagtagttt ccgataagga aattagggtt cttatagggt 3840 ttcgtcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 3900 ctatcaataa aatttctaat tcctaaaacc aaaatcagta ctaaatcaga tccccaattc 3960 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 4020 ccttgcagca catccccctt tcgccagtgg cactggccgt cgttttacaa cgtcgtgact 4080 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 4140 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 4200 gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac 4260 tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga 4320 ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg 4380 ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc gctgctatag 4440 tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc 4500 ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt 4560 tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca tgaacaagag 4620 cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg acttgaccaa 4680 ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga agatcaccgg 4740 caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc ctggcgacgt 4800 tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac tggacattgc 4860 cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt gggccgacac 4920 caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg 4980 ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa 5040 gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg agctgatcga 5100 ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct 5160 gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc 5220 cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga atgaacgcca 5280 agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt cattaccgaa 5340 gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc gcacgtctca 5400 accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg 5460 gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag 5520 taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata 5580 cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga 5640 cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag 5700 tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc 5760 taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc 5820 gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca 5880 aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg 5940 ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg 6000 cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc 6060 tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag 6120 gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg 6180 aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga 6240 gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa 6300 ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt 6360 gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac 6420 cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga 6480 gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg 6540 acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg 6600 ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgacggt 6660 cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag 6720 ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa 6780 tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt 6840 gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg 6900 ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg 6960 tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta 7020 gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg 7080 gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc 7140 ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc 7200 ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc 7260 atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc 7320 ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc 7380 gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg 7440 gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca 7500 cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt 7560 ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat 7620 gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg 7680 cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg 7740 ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc tgtggatagc 7800 acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca 7860 aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc 7920 gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt 7980 gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg 8040 ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa 8100 atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc 8160 gccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa 8220 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 8280 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 8340 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 8400 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 8460 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 8520 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 8580 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 8640 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 8700 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 8760 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 8820 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 8880 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 8940 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 9000 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 9060 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 9120 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 9180 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 9240 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 9300 taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa aatataatat 9360 tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc gacatactgt 9420 tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata ccacttgtcc 9480 gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt cacaaagatg 9540 ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt ttccgtcttt 9600 aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca gttttcgcaa 9660 tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg gctgtctaag 9720 ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat gcactccgca 9780 tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga gcaaaggacg 9840 ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa gtgcaggacc 9900 tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg ttccacatca 9960 taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc attttctccc 10020 accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca gcggtatttt 10080 tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta tttccttcct 10140 cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt 10200 cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt 10260 caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg 10320 cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt 10380 caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg 10440 ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag 10500 tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata 10560 ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat 10620 gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg attttggttt 10680 taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt 10740 tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct tatctgggaa 10800 ctactcacac attattatgg agaaactcga gcttgtcgat cgacagatcc ggtcggcatc 10860 tactctattt ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta 10920 cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga 10980 cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat 11040 cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg 11100 cccggagtcg tggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct 11160 gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat 11220 ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga 11280 cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc 11340 aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag 11400 tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt 11460 attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg 11520 cagcgatcgc atccatagcc tccgcgaccg gttgtagaac agcgggcagt tcggtttcag 11580 gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc 11640 taaactcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat 11700 aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac 11760 gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt 11820 cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag 11880 gctttttcat atctcattgc cccccgggat ctgcgaaagc tcgagagaga tagatttgta 11940 gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc ttatatagag 12000 gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg gagatatcac 12060 atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg 12120 tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg atagcctttc 12180 ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc cttttgatga 12240 agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc ctttgttgaa 12300 aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg gagtagacga 12360 gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac gtggttggaa 12420 cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg 12480 cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac 12540 cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga 12600 ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg 12660 tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg gcaagctgct 12720 ctagccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 12780 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 12840 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 12900 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattctggca 12960 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 13020 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 13080 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 13140 atgcctgcag gtcaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga 13200 tacagtctca gaagaccaga gggctattga gacttttcaa caaagggtaa tatcgggaaa 13260 cctcctcgga ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga 13320 agatggcttc tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc 13380 taccgacagt ggtcccaaag atggaccccc acccacgagg aacatcgtgg aaaaagaaga 13440 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat ggtcaacatg gtggagcacg 13500 acactctcgt ctactccaag aatatcaaag atacagtctc agaagaccag agggctattg 13560 agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct 13620 gtcacttcat cgaaaggaca gtagaaaagg aagatggctt ctacaaatgc catcattgcg 13680 ataaaggaaa ggctatcgtt caagatgcct ctaccgacag tggtcccaaa gatggacccc 13740 cacccacgag gaacatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 13800 attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 13860 acccttcctc tatataagga agttcatttc atttggagag g 13901

Claims (6)

The infection activity of broad bean wilt virus 2 (BBWV2) was detected in Capsicum spp. An Agrobacterium tumefaciens mixture comprising two Agrobacterium tumefaciens transformed with SEQ ID NO: 1 or SEQ ID NO: 2, respectively, which can be reproduced in plants. The Agrobacterium tumefaciens mixture according to claim 1, wherein the Agrobacterium tumefaciens transformed with SEQ ID NO: 1 is the accession number KACC91892P. 2. The Agrobacterium tumefaciens mixture according to claim 1, wherein the Agrobacterium tumefaciens transformed with SEQ ID NO: 2 is the accession number KACC91893P. An agro-infiltration kit comprising Agrobacterium tomefaciens strain transformed with SEQ ID NO: 1 of claim 1. An agro-infiltration kit comprising Agrobacterium tomefaciens strain transformed with SEQ ID NO: 2 of claim 1. 2. The Agrobacterium tumefaciens mixture according to claim 1, wherein the plant of the genus Capsicum is pepper or paprika.

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