KR101090458B1 - DNA Recombinant virus vector and Recombinant HYVV gene using Sweet Potato Leaf Curl Virus Korean Isolate - Google Patents

Abstract

The present invention relates to a domestic sweet potato leaf blight infection DNA virus gene and recombinant plasmid, more specifically, sweet potato leaf blight infection which is a geminivirus that causes leaf blight symptoms in sweet potatoes that are cultivated mainly in Korea. Confirmed infection of DNA virus (SPLCV), and obtained the entire nucleotide sequence of the virus from the sweet potato DNA to infect the sweet potato in the form of a recombinant virus to confirm the pathogenesis of artificially induced the induction of sweet potato leaf rotosis The present invention relates to a recombinant plasmid which can be used.

Sweet potato leaf disease infection DNA virus gene

Description

DNA Recombinant virus vector and Recombinant HYVV gene using Sweet Potato Leaf Curl Virus Korean Isolate

The present invention relates to domestic sweet potato leaf blight infection DNA virus gene and recombinant plasmid.

Sweet potato leaf curl is a disease that causes sweet potato leaves to curl up due to the infection of sweet potato with the Gemini virus. Is one of the few new diseases reported in the world. Long hosts have a dormant period of up to several months. It has been reported only recently in Sicily and China, along with the United States, and the diagnosis of the virus relies solely on PCR assays. Therefore, the research results are limited to the detection of viruses, and there are no cases of application using viruses.

Geminiviruses, which cause leaf swelling in sweet potatoes, belong to the Geminiviridae, which is divided into four genus, of which begomovirus, subgroup III. It has a characteristic mediated by whitefly [ Bemisia tabaci (Gennadius)]. Thus, reports on Geminiviruses are often made in tropical or subtropical climates where Garui is suitable. In Asia, geminiviruses were reported in tomato growing regions in China in the 1960s, but they were limited in some regions and the damage was minimal. However, there has been a surge of reports in recent decades that floury is observed in most tomato growing regions of major Asian countries, followed by the testing of geminiviruses.

Begomoviruses reported in Asia, including China, Taiwan, Japan, and India, include Tomato leaf curl virus (ToLCV), Tomato yellow leaf curl virus (TYLCV), Tobacco leaf curl virus (TbLCV), and Honeysuckle yellow vein mosaic virus (HYVMV). The back is dominant. They are generally classified as very specific viruses because they have only one genome, unlike other begomoviruses with two dogs. In addition, it has a common feature of requiring satellite DNA to complete replication. According to the literature and reports to date, one genome has been distributed in the old continent including Africa and Asia, and two genomes have been distributed in the new continent. This phenomenon appears to be interpreted as evolving from a virus with one genome to a virus with two genomes.

Gemini virus found in Korea is also likely to be a begomovirus with one genome. However, since the possibility of other viruses cannot be excluded, PCR was carried out by preparing primers with affinity for various kinds of begomovirus. As a result, Gemini virus isolated from Korea has a relatively high core region of CP. Although it was maintained as, the sequence was found to be many variations occur. Because of this variation, there are many difficulties in obtaining and analyzing the entire nucleotide sequence.

Gemini virus similar to TLCV has been identified in domestic crop cultivation environment, which has not been reported by Gemini virus or reported cases so far, and it is possible to introduce Gemini virus that can seriously damage domestic cultivation environment through various mutations. Or implies a change to an already introduced environment. In addition, the current distribution of the Gemini virus is still in the early stage of the introduction of the Gemini Virus due to the low level of separation of the Gemini Virus, and it may not be established in Korea anytime soon due to the change in recombination rate and infectivity. Since the damage caused by the Gemini virus is considered to continue, it is urgent to prepare countermeasures against these viruses through rapid identification and research.

Therefore, the present inventors have detected the SPLCV, using the unique characteristics of the region-specific strains of the virus, as a field that can be applied from the entire sequence of them as a field of research efforts to develop a vector system for sweet potato transformation, leaf curling, PCR was used to isolate a partial sequence of a virus having a single-stranded DNA genome in a plant from domestic sweet potato plants (Ipomoea batatas) showing the symptoms of (leaf curl) disease. A dimeric SPLCV genome is formed by recombining the entire sequence of viruses of the same length with each other to form a repeat sequence. The dimeric SPLCV genome is recombined with a cloning plasmid, transformed into E. coli, and the cultured transformed Escherichia coli is cultured to infect a non-toxic sweet potato. The result of the drying and infection symptoms inherent in the Gemini virus Confirmed by thereby completing the present invention. In addition, in Korea, RNA virus was usually identified, but DNA virus is the first to find out. In addition, although a high homology with the previously reported sweet potato infection SPLCV gene sequence, a dimer SPLCV genome which has never been attempted by using a source material that can be applied as a transporter that enables transformation into sweet potato It is clear that it is completed.

Accordingly, an object of the present invention is to provide a domestic sweet potato leaf rolling infection DNA virus (SPLCV) gene represented by SEQ ID NO: 1.

In addition, the present invention has a fundamental object to provide a recombinant plasmid for cloning comprising the dimer, the E. coli transformed with the recombinant plasmid.

The present invention is characterized by the domestic sweet potato leaf rolling infection DNA virus (SPLCV) gene represented by SEQ ID NO: 1.

In another aspect, the present invention is to provide a recombinant plasmid for cloning comprising the dimer, the E. coli transformed with the recombinant plasmid.

The present invention relates to a sweet potato plant infection single-chain DNA virus isolated for the first time in Korea, by first separating a virus that can cause sweet potato leaf curling disease in Korea, sweet potato leaf curling disease that can occur in the ecological environment and climatic conditions of Korea It is expected to be the foundation for preparing countermeasures in a hurry, and can be applied and developed as a transporter that can transfer specific genes to the sweet potato genome by using and replacing replication mechanisms and transcription control mechanisms in the host nucleus of general DNA viruses. It can contribute to breeding, and also a complex development such as the development of resistant sweet potato plants using the useful gene sequence of SPLCV virus and the isolation of viral factors that regulate the expression of foreign genes is possible.

Hereinafter, the present invention will be described in more detail.

The present invention confirms the infection of sweet potato foliar infection DNA virus (SPLCV), which is a geminivirus that causes leaf foliation symptoms in sweet potatoes cultivated mainly for food in Korea. The present invention relates to a recombinant plasmid capable of artificially inducing the induction of sweet potato leaf curling disease in Korea, by acquiring the entire nucleotide sequence and artificially infecting the sweet potato in the form of a recombinant virus to confirm the metastasis.

The present inventors have used oligonucleotide primer sets devised directly from domestic sweet potato plants ( Ipomoea batatas ) showing the symptoms of leaf curl disease, using a polymerase chain reaction (PCR) method. Partial sequences were identified and confirmed that the virus was replicating in plants using DNA hybridization techniques. In order to isolate the entire nucleotide sequence of the virus, the whole genomic DNA is extracted from the plant, and for separating the entire nucleotide sequence of the virus, amplification of two partial strands by a polymerase chain reaction method (SP1; 1632 bp, SP2; 2138 bp), which was recombined to obtain a sequence corresponding to approximately 3.4 kbp [see FIG. 3] nucleic acid length. The recombinant plasmid DNA sequence used for cloning was transformed into bacteria, followed by pure separation of a large amount of the recombinant sequence generated through cultivation of the bacteria, and a nucleotide sequence of 2828 bp corresponding to the entire nucleotide sequence through sequencing method. [SEQ ID NO: 1] was determined and analyzed through a homology test to confirm that the sequence has a homology of about 96% with the previously reported sweet potato leaf curl virus (SPLCV). Thus, value was recognized as a newly discovered sequence and [SEQ ID NO: 1] was registered in NCBI GENBANK [FJ560719.1].

Dimeric SPLCV genome (5656 bp) that recombines the entire sequence of viruses of the same length (2828 bp) that were differently truncated using restriction enzyme sites (Sac I and Kas I sites) that exist in the determined sequence to form a repeat sequence. E. coli DH5 alpha was transformed into the plasmid.

The transformed E. coli DH5 alpha was deposited with KACC on April 23, 2009 and received KACC91467P.

After mass cultivation of the transformed bacteria, the recombinant plasmid was isolated to ultra high purity, and then the plasmid was fired on the sweet potato leaf by using a gene gun in a particle bombardment method to the potato plant (Ipomoea batatas) corresponding to the host plant. As a result, symptoms of Geminivirus infection were shown.

The DNA hybridization method was used to confirm that the finally recombined virus carriers actually replicated in artificially infected sweet potato plants, thereby developing a plasmid carrier of recombinant SPLCV.

The present invention provides a system for enabling the breeding of genetically modified crops using the production of sweet potato infected DNA virus-resistant crops against the gemini virus and the development of stable transgenic carriers in sweet potato plants. In this case, the market is expected to form a market of 100 billion won annually through the commercialization of crops and systems, and in Korea, it is expected to reduce agricultural wastes of more than 10 billion won annually by using prevention and recovery measures for these viruses. .

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to the following Examples.

Example 1 Domestic SPLCV Gene Isolation and Sequencing

Genomic DNA was extracted from the sweet potato, which had been severely dried by the RDA Crop Science Division, using the Dellaporta method, and then, using the designed primer sequence, 95 ° C 3 minutes for the initial DNA denaturation process. 30 seconds at 30 ° C., 55 ° C. 30 seconds for primer binding, 72 ° C. 2 minutes for DNA chain extension], 30 times, and finally at 72 ° C. for 10 minutes for PCR chain extension. Sweet potato genomic DNA 2 microliter, 5'- primer and 3'- primer respectively 0.25 pmol, 1X PCR buffer, 0.125 mM dNTPs to form a final 20 microliter reaction solution and the sequence of SP1 and SP2 of SPLCV through PCR Amplified. The bands obtained by electrophoresis of the amplified sequences (FIG. 1) were recovered from the gel using an RBC gel extraction kit, and the T-vectors were prepared using Promega's pGEM-T easy vector ligation kit. 2992 bp). The ligation reaction solution was added to the bacterial E. coli DH5alpha strain to induce transformation by heat shock at 42 ° C. for 45 seconds and incubated for 12 hours in LB / amp solid medium at 37 ° C. , The resulting bacterial colonies were inoculated in LB / amp liquid medium and shaken for 12 hours at 37 ° C. to separate plasmids from them, and the sequencing reaction was performed using the sequencing primers described. After confirming the sequencing results repeated three times, the final sequence was determined and confirmed as a new type of SPLCV sequence existing in the final Korea through the NCBI BLAST search.

As shown in FIG. 1, SP1 (1632 bp) and SP2 (2138 bp) fragments of expected sizes were amplified through DNA polymerase chain reaction.

[Table 1]

In the case of SPLCV, the gene sequence amplified using the primers of FIG. 2 and Table 1, respectively, was cloned into the pGEM-T easy vector, and the clone including the entire sequence of the virus corresponding to 1.2 mer by EcoRV digestion from the SP1 and SP2 clones. Was produced. In the case of SPLCV, only two fragments having a size of 1 kb or more were cloned to determine the overall sequence. Therefore, primers for identifying the entire SPLCV sequence were devised as shown in Table 2 and FIG. 3 to perform a sequencing reaction to determine the total sequence.

TABLE 2

Sequencing primers for SPLCV full sequencing

primer Sequence (5 '→ 3') 5SpLCV2ndSeq GAT GTG TGG GTC CCT GTA AG (SEQ ID NO: 8) 5SpLCV2Seq_Rc CTT ACA GGG ACC CAC ACA TC (SEQ ID NO: 9) 5SpLCV3rdSeq AGT AAT CCT GTG TAT CAG AC (SEQ ID NO: 10) 5SpLCV4thSeq CTG TGC GTG AAT CCA TGC TG (SEQ ID NO: 11) 5SpLCV5thSeq CCT ATT CTG CTT GGG CCT TC (SEQ ID NO: 12) 5SpLCVlastSeq AAA GGC GGG CAC CGT ATT AA (SEQ ID NO: 13)

10 μg of isolated genomic DNA was electrophoresed on a 0.8% agarose gel, then transferred to a nylon membrane (Amersham Hybond-N +) for 16 hours, after which a probe DNA fragment made from an infectious clone was P ³² -labelled dCTP. After radiolabeling, hybridization reaction was performed. The ORF of the virus was identified based on the genomic sequence of the identified geminivirus, and the identified ORF was sequenced through homology search [BLAST] of sequences previously reported in GenBank.

Figure 5 shows a genomic map of SPLCV purely isolated from domestic sweet potatoes, the arrow indicates each gene (complementary gene strand; C1, C2, C3, C4, viral gene strand; V1, V2).

In addition, Figure 4 is the total nucleotide sequence of the SPLCV, the total sequence corresponding to the length of 2828 bp was determined, and the moiety is indicated by the respective restriction enzyme recognition sequence.

Example 2: Coding and Structure Prediction of Genes in SPLCV

Comparing the entire nucleotide sequence identified with the reference sequence (AF_104036), each ORF present in the identified total nucleotide sequence (SEQ ID NO: 1, FJ_560719) of SPLCV purely isolated from domestic sweet potatoes. As a result of the alignment of the identified amino acid sequence [SEQ ID NO: 14], it was found that some specific amino acid sequences differed from the reference sequence (see FIG. 6). The structural analysis through computer simulation shows that the three-dimensional structure of the protein is different from that of the reference sequence, so that the charge value at the isoelectric point (AF_104036; 6.68, FJ_560719; 6.64) and pH7 is different from those of the previously reported protein. (AF_104036; -1.22, FJ_560719; -1.33), etc., which may have new characteristics (see FIG. 7). Since these structural changes are also linked to the protein's functional features, isolation of new functional proteins can also be expected.

Example 3: Recombinant Plasmid Construction of SPLCV Gene

From the clones of the SP1 and SP2 fragments amplified from sweet potato genomic DNA, fragments of pSP1 were cloned into the fragments of pSP2 using EcoRV and PstI treatment to clone pSPLCV 1.2 clones and 2828 bp through SacI digestion. After acquiring the monomeric fragment, after treatment with Calp intestinal alkaline phosphatase (CIAP) of the SacI digested fragment (2992 bp) of the pGEM-Teasy self-ligated vector, the pSPLCV1.0 monomer clone was obtained by ligation with the monomer fragment. clone). The monomer clone pSPLCV1.0 was cut again into KasI, and the KasI digest monomer fragment of pSPLCV1.2 was ligated again to complete the dimeric clone pSPLCV2.0 [ 8, 9].

Example 4 Preparation of Transgenic E. Coli

0.1 microgram of dimer pSPLCV 2.0 DNA was heat shock transformed to DH5alpha competent cell at 42 ° C for 45 seconds and incubated in 37 ° C shaker for 1 hour by adding 900 µl of LB medium. After that, it was plated on ampicillin-added LB solid medium to form colonies in a 37 ° C. incubator for 12 hours. The colonies formed were inoculated into LB liquid medium containing 3 ml of ampicillin, incubated for 12 hours using a 37 ° C shaking incubator, and the mixture was added at a final concentration of 14% glycerol and then stored at -80 ° C. It was. In addition, the same culture solution was centrifuged to purify the plasmid using the plasmid separation method, and then mixed with 10 X buffer solution and the plasmid, followed by addition of Sac I enzyme to form a total 20 μl reaction solution, followed by reaction at 37 ° C. for 1 hour. After electrophoresis, the size of the sequence cut out from the vector was confirmed, the sequencing reaction was performed using the T7 and SP6 primer sequences in the pGEM-Teasy vectror, and the result of the sequencing was confirmed by NCBI BLAST search. Confirmed.

Example 5: Confirmation of Sweet Potato Leaf Dough Expression by Recombinant Gene

Infection is performed by firing 500 microliter pSPLCV2.0 recombinant plasmid purified at high purity (1 mg / ml) onto a shoot tip portion of sweet potato using a gene gun. As a result of observing the manifestation of the symptoms for 3 to 4 weeks, it was confirmed that the leaf symptom appeared [see FIG. 10].

10 [mu] g of isolated genomic DNA from the sweet potato leaf [Fig. 10] showing the curling symptoms was electrophoresed on a 0.8% agarose gel and then transferred to a nylon membrane (Amersham Hybond-N +) for 16 hours, followed by infection. Probe DNA fragments made from possible recombinant clones were radiolabeled using P32-labelled dCTP and then hybridized to expose X-ray films to confirm that viruses were replicating in some leaves. .

Overcoming the limitations of modern technology, which is difficult to apply the same method for the activation of academic / industrial applications and development of transformants for various plants due to the securing of unreported viral genetic resources in Korea, which is causing a great industrial damage worldwide. It is anticipated that the development and application as a transgenic carrier can be done.

Figure 1 shows the electrophoresis picture of the result of amplification of the partial gene sequence for SPLCV polymerase chain reaction assay and overall sequence determination. SP1 (1632 bp) and SP2 (2138 bp) fragments of expected sizes were amplified by DNA polymerase chain reaction.

Figure 2 shows a primer design for determining the overall sequence, a primer design schematic showing where the primer sequence is located on SPLCV using the overlapping portion of the amplified region to secure the entire virus sequence.

Figure 3 shows the design of the sequencing primer for identifying the SPLCV sequence purely isolated from domestic sweet potatoes.

Figure 4 is a total sequence corresponding to the length of 2828 bp of pure purified SPLCV from domestic sweet potatoes.

Figure 5 shows a genomic map of SPLCV purely isolated from domestic sweet potatoes (arrow indicates each gene (complementary gene strand; C1, C2, C3, C4, viral gene strand; V1, V2)).

Figure 6 shows the sequence alignment between the amino acid sequence (top) and reference sequence (AF_104036) of each ORF inferred from the SPLCV base sequence (FJ_560719) purely isolated from domestic sweet potatoes.

FIG. 7 compares the predicted protein structure (left) of AC1 in the ORF inferred from SPLCV sequences purely isolated from domestic sweet potatoes and the predicted protein structure (right) from the AC1 protein sequence of reference sequence (AF_104036).

8 shows a schematic diagram of the gene recombination process for the completion of the carrier. Using a plasmid from the entire sequence obtained (top left), a recombinant plasmid containing repeated viral sequences of different sizes (1.2 mer; pSPLCV1.2, monomer; pSPLCV1.0, dimer; pSPLCV2.0) was made. Was produced.

9 shows a cleavage map of the recombinant plasmid pSPLCV2.0.

FIG. 10 shows the plasmid pSPLCV2.0 containing a recombinant dimeric SPLCV gene with a gene gun using a particle bombardment method in a sweet potato. Sweet potato leaves showing symptoms of infection are shown (arrows indicate infection location (lower left) and infection symptoms (right)).

FIG. 11 illustrates a DNA hybridization reaction using genomic DNA extracted from sweet potato showing infection symptoms by infection with plasmid pSPLCV2.0 containing a recombinant dimer SPLCV gene using a gene gun. It is confirmed that the virus is replicated from the recombinant plasmid in the [-; no DNA control, +; Recombinant plasmid, I; Particle injection sites, 1-5; Genomic DNA extracted from each sweet potato leaf of FIG. 10].

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration          RURAL DEVELOPMENT ADMINISTRATION <120> DNA Recombinant virus vector and Recombinant HYVV gene using          Sweet Potato Leaf Curl Virus Korean Isolate <150> KR10-2008-0038413 <151> 2008-04-24 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 2828 <212> DNA <213> SPLCKV <400> 1 taatattacc ggtgcccgcc gcgcccttta atagtgggcc ccacaaggga ccacgcgtct 60 tttccgtact gtctttaatg attactttgc tttataagga ccaatcaggt ttccgtgctt 120 gggcgccaag aatggagcag ttgtgggacc ctttgcagaa cccactcccg gatactttat 180 acggttttag gtgtatgcta tccgtaaaat acttgcagag tattttgaag aaatacgagc 240 caggaacctt agggttcgag ctctgttcgg agttaatccg tatattcagg gtcaggcagt 300 atgacagggc gaattcccgt ttcgcggaga tttcatccat atggggggag accggtaaga 360 cggaggctga acttcgagac agctatcgtg ccctacactg ggaatgctgt cccaattgct 420 gcccgaagct atgtcccggt ttcaagaggc gtccggatga agagaaagag gggtgaccgc 480 atcccgaagg gatgtgttgg tccctgtaag gtccaggact atgagttcaa gatggacgtt 540 ccccacacgg gaacgtttgt ctgtgtctcg gattttacta ggggaactgg gcttactcat 600 cgcctgggta agcgtgtttg tgtgaagtcc atgggtatag atgggaaggt ctggatggat 660 gacaatgtgg ccaagagaga tcacaccaat atcatcacgt attggttgat tcgtgacaga 720 aggcccaata aggatccgct gaactttggc caagtcttca ctatgtatga caatgagccc 780 actactgcta agatccgaat ggatctgagg gatagaatgc aggtattgaa gaagttctct 840 gttacagttt caggaggtcc atacagccac aaggagcagg ccttaattag gaagtttttt 900 aagggtttgt ataatcatgt tacttacaat cataaggaag aagctaagta tgagaatcag 960 ttagaaaatg ctttgatgtt gtatagtgct agcagtcatg cgagtaatcc tgtgtatcag 1020 accctgcgtt gcaggtctta tttctatgat tcgcacaata attaataaaa ttatatttta 1080 ttaatacagt aatatcttta catcatcatt acaatctaca gtatctactt catctatcca 1140 agaacatact cttggaagat atctaataac aaaaactaaa ttaactaaac taaaaaaccc 1200 taaatttgct aattcattac atatacgcca tttaaggcgt tccaaaatac cagtccaact 1260 gtgaatagca ccagttagac gggtcgtcaa gatccggaat tggaggaaga tcttgtggaa 1320 tcccagttgc ctcctttccc tgtagttgac ccacatctgg aatttgagga tggttctccc 1380 ctgtgtgctc tcgtgggcat acataattct gagatggaac ggtgcagtcc gacccacaga 1440 catggggttg gtgtcgaatt cgagtgctct cgtagtctga gcatgactta gtgattcccc 1500 tgtgcgtgaa tccatgctga tacttgcagt ttggggtgat gaaggctgaa cagccgcaac 1560 ccttccacac tatccttgtc ctctgttcag gaactttcct cttggccttc ttcgcctctg 1620 cgtgcagtgg ctcctgaatg ggacacttcc tcttgattcc agaagggaga ttggacatcg 1680 cagaatatag cgtttgctgt tgcccaattc ttgagtgctc cttgctctgg tttgtccaac 1740 cagagtttaa atgaggagcc ctctcctgga ttgcagagga agatagtggg aattccacct 1800 ttaatttgaa ctggctttcc gtatttacag ttggattgcc agtccttctg ggcccccatg 1860 aattccttaa agtgctttag gtattggggg ttgacgtcat caatgacgtt ataccaagca 1920 ctgttgctgt atacttttgg gcttagatct aaatgcccac acaaataatt gtgagggccc 1980 aaagacctgg cccatactgt tttgcctatt ctgcttgggc cttcaataac aatggatatg 2040 ggtctatccg gccgcgcagc ggcatccatg acattttcag cggcccagtc gctgataatg 2100 tcaggaactg cattgaaaga agaagatgaa aaaggagaag aatatacaga aggaggagga 2160 gaaaaaatcc tatctaaatt actaactaaa ttgtgaaatt gaaacaaata tttttcaggg 2220 agtttctccc taattatttg caacgcagct tctttagaac ctgcgtttag agcctctgcg 2280 gctgcgtcat tagcagtctg ctggcctcct ctagcagatc tgccgtcgac ctggaattca 2340 ccccaggtga tggtatcccc gtccttgtca acgtaggact tgacatcgga cgaggattta 2400 gctccctgaa tgttggggtg aaagtgattc gatctgttcg gtgagaccag atcgaagaat 2460 ctgctatttg tgcagacgaa tttgccttcg aactgcacca gcacatggag gtgaggttcc 2520 ccatcctcgt gcagttctct tgctacgtgt atgtattttt tgttggtggg tgtttggata 2580 tttaggagtt gggctaggca gtcctctttt gagagagaac agcgtggata agtaataaaa 2640 taatttttgg cctgaatttt aaaacgtttt ggaggagcca tttggagaca cgcataagtt 2700 caaatgaatt ggagactgga gacaatatat agtatgtctc caaatggcat tctggtaatt 2760 tagaagatcc ttttagcttt aattcaaatt ccgacaactc tgggtccacc aaaaggcggg 2820 caccgtat 2828 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPF1 (forward) <400> 2 ctcgtgcagt tctcttgcta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPR1 (reverse) <400> 3 gcaactggga ttccacaaga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPF2 (forward) <400> 4 gtgtatcaga ccctgcgttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPR2 (reverse) <400> 5 atacggtgcc cgccttttgg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SPR3 (reverse) <400> 6 ccttcaaaag tgggccccac a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SPR4 (reverse) <400> 7 atgacagggc gaatgcgcgt t 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCV2ndSeq <400> 8 gatgtgtggg tccctgtaag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCV2Seq_Rc <400> 9 cttacaggga cccacacatc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCV3rdSeq <400> 10 agtaatcctg tgtatcagac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCV4thSeq <400> 11 ctgtgcgtga atccatgctg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCV5thSeq <400> 12 cctattctgc ttgggccttc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5SpLCVlastSeq <400> 13 aaaggcgggc accgtattaa 20 <210> 14 <211> 114 <212> PRT <213> SPLCKV-AV2 <400> 14 Met Glu Gln Leu Trp Asp Pro Leu Gln Asn Pro Leu Pro Asp Thr Leu   1 5 10 15 Tyr Gly Phe Arg Cys Met Leu Ser Val Lys Tyr Leu Gln Ser Ile Leu              20 25 30 Lys Lys Tyr Glu Pro Gly Thr Leu Gly Phe Glu Leu Cys Ser Glu Leu          35 40 45 Ile Arg Ile Phe Arg Val Arg Gln Tyr Asp Arg Ala Asn Ser Arg Phe      50 55 60 Ala Glu Ile Ser Ser Ile Trp Gly Glu Thr Gly Lys Thr Glu Ala Glu  65 70 75 80 Leu Arg Asp Ser Tyr Arg Ala Leu His Trp Glu Cys Cys Pro Asn Cys                  85 90 95 Cys Pro Lys Leu Cys Pro Gly Phe Lys Arg Arg Pro Asp Glu Glu Lys             100 105 110 Glu gly         <210> 15 <211> 254 <212> PRT <213> SPLCKV-AV1 <400> 15 Met Thr Gly Arg Ile Pro Val Ser Arg Arg Phe His Pro Tyr Gly Gly   1 5 10 15 Arg Pro Val Arg Arg Arg Leu Asn Phe Glu Thr Ala Ile Val Pro Tyr              20 25 30 Thr Gly Asn Ala Val Pro Ile Ala Ala Arg Ser Tyr Val Pro Val Ser          35 40 45 Arg Gly Val Arg Met Lys Arg Lys Arg Gly Asp Arg Ile Pro Lys Gly      50 55 60 Cys Val Gly Pro Cys Lys Val Gln Asp Tyr Glu Phe Lys Met Asp Val  65 70 75 80 Pro His Thr Gly Thr Phe Val Cys Val Ser Asp Phe Thr Arg Gly Thr                  85 90 95 Gly Leu Thr His Arg Leu Gly Lys Arg Val Cys Val Lys Ser Met Gly             100 105 110 Ile Asp Gly Lys Val Trp Met Asp Asp Asn Val Ala Lys Arg Asp His         115 120 125 Thr Asn Ile Thr Tyr Trp Leu Ile Arg Asp Arg Arg Pro Asn Lys     130 135 140 Asp Pro Leu Asn Phe Gly Gln Val Phe Thr Met Tyr Asp Asn Glu Pro 145 150 155 160 Thr Thr Ala Lys Ile Arg Met Asp Leu Arg Asp Arg Met Gln Val Leu                 165 170 175 Lys Lys Phe Ser Val Thr Val Ser Gly Gly Pro Tyr Ser His Lys Glu             180 185 190 Gln Ala Leu Ile Arg Lys Phe Phe Lys Gly Leu Tyr Asn His Val Thr         195 200 205 Tyr Asn His Lys Glu Glu Ala Lys Tyr Glu Asn Gln Leu Glu Asn Ala     210 215 220 Leu Met Leu Tyr Ser Ala Ser Ser His Ala Ser Asn Pro Val Tyr Gln 225 230 235 240 Thr Leu Arg Cys Arg Ser Tyr Phe Tyr Asp Ser His Asn Asn                 245 250 <210> 16 <211> 85 <212> PRT <213> SPLCKV-AC4 <400> 16 Met Gly Asn Leu Thr Ser Met Cys Trp Cys Ser Ser Lys Ala Asn Ser   1 5 10 15 Ser Ala Gln Ile Ala Asp Ser Ser Ile Trp Ser His Arg Thr Asp Arg              20 25 30 Ile Thr Phe Thr Pro Thr Phe Arg Glu Leu Asn Pro Arg Pro Met Ser          35 40 45 Ser Pro Thr Leu Thr Arg Thr Gly Ile Pro Ser Pro Gly Val Asn Ser      50 55 60 Arg Ser Thr Ala Asp Leu Leu Glu Glu Ala Ser Arg Leu Leu Met Thr  65 70 75 80 Gln Pro Gln Arg Leu                  85 <210> 17 <211> 144 <212> PRT <213> SPLCKV-AC3 <400> 17 Met Asp Ser Arg Thr Gly Glu Ser Leu Ser His Ala Gln Thr Thr Arg   1 5 10 15 Ala Leu Glu Phe Asp Thr Asn Pro Met Ser Val Gly Arg Thr Ala Pro              20 25 30 Phe His Leu Arg Ile Met Tyr Ala His Glu Ser Thr Gln Gly Arg Thr          35 40 45 Ile Leu Lys Phe Gln Met Trp Val Asn Tyr Arg Glu Arg Arg Gln Leu      50 55 60 Gly Phe His Lys Ile Phe Leu Gln Phe Arg Ile Leu Thr Thr Arg Leu  65 70 75 80 Thr Gly Ala Ile His Ser Trp Thr Gly Ile Leu Glu Arg Leu Lys Trp                  85 90 95 Arg Ile Cys Asn Glu Leu Ala Asn Leu Gly Phe Phe Ser Leu Val Asn             100 105 110 Leu Val Phe Val Ile Arg Tyr Leu Pro Arg Val Cys Ser Trp Ile Asp         115 120 125 Glu Val Asp Thr Val Asp Cys Asn Asp Asp Val Lys Ile Leu Leu Tyr     130 135 140 <210> 18 <211> 148 <212> PRT <213> SPLCKV-AC2 <400> 18 Met Ser Asn Leu Pro Ser Gly Ile Lys Arg Lys Cys Pro Ile Gln Glu   1 5 10 15 Pro Leu His Ala Glu Ala Lys Lys Ala Lys Arg Lys Val Pro Glu Gln              20 25 30 Arg Thr Arg Ile Val Trp Lys Gly Cys Gly Cys Ser Ala Phe Ile Thr          35 40 45 Pro Asn Cys Lys Tyr Gln His Gly Phe Thr His Arg Gly Ile Thr Lys      50 55 60 Ser Cys Ser Asp Tyr Glu Ser Thr Arg Ile Arg His Gln Pro His Val  65 70 75 80 Cys Gly Ser Asp Cys Thr Val Pro Ser Gln Asn Tyr Val Cys Pro Arg                  85 90 95 Glu His Thr Gly Glu Asn His Pro Gln Ile Pro Asp Val Gly Gln Leu             100 105 110 Gln Gly Lys Glu Ala Thr Gly Ile Pro Gln Asp Leu Pro Pro Ile Pro         115 120 125 Asp Leu Asp Asp Pro Ser Asn Trp Cys Tyr Ser Gln Leu Asp Trp Tyr     130 135 140 Phe Gly Thr Pro 145 <210> 19 <211> 364 <212> PRT <213> SPLCKV-AC1 <400> 19 Met Ala Pro Pro Lys Arg Phe Lys Ile Gln Ala Lys Asn Tyr Phe Ile   1 5 10 15 Thr Tyr Pro Arg Cys Ser Leu Ser Lys Glu Asp Cys Leu Ala Gln Leu              20 25 30 Leu Asn Ile Gln Thr Pro Thr Asn Lys Lys Tyr Ile His Val Ala Arg          35 40 45 Glu Leu His Glu Asp Gly Glu Pro His Leu His Val Leu Val Gln Phe      50 55 60 Glu Gly Lys Phe Val Cys Thr Asn Ser Arg Phe Phe Asp Leu Val Ser  65 70 75 80 Pro Asn Arg Ser Asn His Phe His Pro Asn Ile Gln Gly Ala Lys Ser                  85 90 95 Ser Ser Asp Val Lys Ser Tyr Val Asp Lys Asp Gly Asp Thr Ile Thr             100 105 110 Trp Gly Glu Phe Gln Val Asp Gly Arg Ser Ala Arg Gly Gly Gln Gln         115 120 125 Thr Ala Asn Asp Ala Ala Ala Glu Ala Leu Asn Ala Gly Ser Lys Glu     130 135 140 Ala Ala Leu Gln Ile Ile Arg Glu Lys Leu Pro Glu Lys Tyr Leu Phe 145 150 155 160 Gln Phe His Asn Leu Val Ser Asn Leu Asp Arg Ile Phe Ser Pro Pro                 165 170 175 Pro Ser Val Tyr Ser Ser Pro Phe Ser Ser Ser Ser Phe Asn Ala Val             180 185 190 Pro Asp Ile Ile Ser Asp Trp Ala Ala Glu Asn Val Met Asp Ala Ala         195 200 205 Ala Arg Pro Asp Arg Pro Ile Ser Ile Val Ile Glu Gly Pro Ser Arg     210 215 220 Ile Gly Lys Thr Val Trp Ala Arg Ser Leu Gly Pro His Asn Tyr Leu 225 230 235 240 Cys Gly His Leu Asp Leu Ser Pro Lys Val Tyr Ser Asn Ser Ala Trp                 245 250 255 Tyr Asn Val Ile Asp Asp Val Asn Pro Gln Tyr Leu Lys His Phe Lys             260 265 270 Glu Phe Met Gly Ala Gln Lys Asp Trp Gln Ser Asn Cys Lys Tyr Gly         275 280 285 Lys Pro Val Gln Ile Lys Gly Gly Ile Pro Thr Ile Phe Leu Cys Asn     290 295 300 Pro Gly Glu Gly Ser Ser Phe Lys Leu Trp Leu Asp Lys Pro Glu Gln 305 310 315 320 Gly Ala Leu Lys Asn Trp Ala Thr Ala Asn Ala Ile Phe Cys Asp Val                 325 330 335 Gln Ser Pro Phe Trp Asn Gln Glu Glu Val Ser His Ser Gly Ala Thr             340 345 350 Ala Arg Arg Gly Glu Glu Gly Gln Glu Glu Ser Ser         355 360  

Claims (4)

Sweet Potato Leaf Curl Virus gene of domestic sweet potato leaf curling disease represented by SEQ ID NO: 1. Recombinant plasmid containing a dimer of the gene of claim 1, represented by the cleavage map of FIG. 9. Escherichia coli DH5alpha transformed with the recombinant plasmid of claim 2 and deposited with KACC 91467P. Sweet Potato Leaf Curl Virus protein of domestic sweet potato leaf disease shown in SEQ ID NO: 14 to 19.

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