KR101090459B1 - Korean sweet potato leaf curl virus isolated gene capable of suppressing posttranscriptional gene silencing - Google Patents

Abstract

The present invention relates to genes derived from domestic sweet potato leaf rot infection DNA virus having inhibitory activity of gene silencing after transcription, and more particularly, to develop resistant plants or to neutralize plant defense mechanisms for expression of foreign genes. The present invention relates to an AC2 gene derived from a domestic sweet potato leaf blight infection DNA virus that is applicable as a factor and can be used to increase the efficiency of a carrier upon introduction of a specific gene in a plant.

Sweet Potato Blight Infection DNA Virus, Gene Silencing After Transcription, AC2 Gene

Description

Korean sweet potato leaf curl virus isolated gene capable of suppressing posttranscriptional gene silencing

The present invention relates to a gene derived from a sweet potato leaf disease infected DNA virus having a inhibitory activity of gene silencing after transcription.

Geminiviruses, which cause leafiness 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 oneysuckle yellow vein mosaic virus (HYVMVH). 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.

In general, the AC2 gene present in the geminivirus is known as a protein having a function of regulating transcriptional activity. Recently, in some geminiviruses, the AC2 gene has a function of an inhibitory protein for posttranscriptional gene silencing (PTGS). It is known.

Therefore, the present inventors confirmed that the AC2 gene of the sweet potato leaf rot infection DNA virus (SPLCV) isolated from Korea has PTGS inhibitory activity, and thus shows a PTGS inhibitory activity as well as a systematic whole that can affect the whole plant. The present invention was completed by confirming that it exhibits an activity (systemic silencing activity) and exhibits appropriate activity that does not adversely affect plant growth.

Accordingly, an object of the present invention is to provide an AC2 gene derived from sweet potato leaf blight infected DNA virus having the nucleotide sequence of SEQ ID NO: 16.

Another object of the present invention is to provide an AC2 protein derived from sweet potato leaf blight infected DNA virus having the amino acid sequence of SEQ ID NO: 17 and a recombinant expression vector including the AC2 gene.

The present invention is characterized by the AC2 gene derived from sweet potato leaf blight infected DNA virus having the nucleotide sequence of SEQ ID NO: 16.

In another aspect, the present invention is to provide an AC2 protein derived from sweet potato leaf blight infected DNA virus having the amino acid sequence of SEQ ID NO: 17 and a recombinant expression vector comprising the AC2 gene.

The present invention relates to a gene having a gene silencing inhibitory activity after transcription isolated from sweet potato plant infection single-chain DNA virus isolated for the first time in Korea. Application and development as a carrier for the introduction of specific genes in plants applied to isolation and characterization studies are possible.

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

To identify genes with posttranscriptional gene silencing (PTGS) inhibitory activity after transcription, AC2 genes with the nucleotide sequence of SEQ ID NO: 16 were isolated from the isolated sweet potato leaf blight infection DNA virus (SPLCV). Then, sequence alignment was performed with the AC2 protein encoded by the gene and another AC2 protein. Through sequencing, it was found that the protein having the amino acid sequence of SEQ ID NO: 17 shows various differences in the overall sequence but also shares the characteristic sequences preserved by domains with the compared AC2 proteins.

It is judged that there will be a difference in PTGS inhibitory activity according to gene and amino acid sequence differences. These genes are amplified from the virus through a polymerase chain reaction, and the amplified sequences are recombined into the plant expression vector pBI121, thereby agrobacterium tume Passengens GV3101 was transformed and mass cultured through the agroinfiltration method to model tobacco ( Nicotiana) benthamiana ) was injected into the leaves to confirm the activity according to protein expression. At this time, the transformed Agrobacterium tumefaciens KYRT1 was deposited with KACC on April 24, 2009, and was given accession number KACC91470P.

As a control group, P19 protein of TBSV (Tomato bush stunt virus), a single-stranded RNA virus having a very high PTGS inhibitory activity, was compared. Although the activity was low, the AC2 protein of SPLCV also showed PTGS inhibitory activity.

The important point for genetic engineering in terms of PTGS inhibitory activity is that it shows systematic activity that can affect the whole plant and that the intensity of the activity is not too strong to adversely affect plant growth. There are two aspects to refrain from giving. The AC2 gene isolated from SPLCV exhibited the systematic activity of the former unlike P19, and is not suitable for genetic engineering because its strength is not high.

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 Sequence Analysis

Genomic DNA was extracted from the sweet potato, which had been severely dried by the RDA Crop Science Division, Department of Virus, using Dellaporta method, using the designed primer sequence at 95 ° C. for 3 minutes; 30 repetitions of 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 2 minutes; Under the PCR reaction conditions of 72 ° C. for 10 minutes, 2 μl of sweet potato genomic DNA, 0.25 pmol of 5′-primer and 3'-primer, 0.25 × mol, 1X PCR buffer, and 0.125 mM dNTPs were prepared. Amplify partial sequences of SP1 and SP2 of SPLCV, and recover the DNA from the gel using RBC gel extraction kit, and amplify the bands by electrophoresis of the amplified sequences, and then use Promega pGEM-T easy vector ligation kit. -Inserted into the vector. The conjugated reaction solution was added to E. coli DH5alpha strain to induce transformation by thermal shock at 42 ° C. for 45 seconds, and then cultured in LB / amp solid medium at 37 ° C. for 12 hours, and the resulting bacterial colonies were transferred to LB / amp liquid medium. Inoculation was carried out for 12 hours shaking culture at 37 ℃ to isolate the plasmid from them, and the sequencing reaction using a sequencing primer. After confirming the sequencing results repeated three times, the final sequence was determined and confirmed as the final SPLCV sequence by 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.

In the case of SPLCV, the gene sequences amplified using the primers of FIGS. 2 and 1 were cloned into pGEM-T easy vectors, respectively, and clones containing the entire sequence of the virus corresponding to 1.2 mer by EcoRV digestion from SP1 and SP2 clones. 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.

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.

Example 2 Acquisition of AC2 Gene from SPLCV

In order to isolate the AC2 gene reported as a transcriptional activator of geminivirus by PCR, a primer set as shown in Table 3 was designed and used.

Gene Primer Sequence (5 '→ 3') SPLCV AC2 5SpAC2 CCC GGA TCC CAA GCT TGC ATG TCC AAT CTC CCT TCT GGA ATC (SEQ ID NO: 14) 3SpAC2 CCC AAG CTT CGG ATC CGC TTA AGG CGT TCC AAA ATA CCA GTC (SEQ ID NO: 15)

Using the primer set of Table 3, it was possible to secure an AC2 gene of approximately 450 bp as shown in Figure 6, PCR product was confirmed by electrophoresis, cut out the band portion to recover the DNA by gel extraction kit, recovery The resulting DNA was ligation with pGEM-Teasy vetor using Promega's T-vector ligation kit. After reacting the ligation mixture overnight at 16 ℃, heat shock transformation method was applied to DH5alpha competent cell for 45 seconds at 42 ℃ and 900 ㎕ LB medium was added and incubated for 1 hour at 37 ℃ shaking incubator It was then plated in LB solid medium to which ampicillin was added to form colonies in a 37 ° C. incubator for 12 hours. The colonies formed were inoculated into LB medium containing 3 ml of ampicillin, incubated for 12 hours using a 37 ° C shaking incubator, and the culture solution was added at a final 14% of glycerol and mixed at -80 ° C. It was. In addition, the same culture solution was centrifuged to purify the plasmid using the plasmid separation method, and then, using EcoRI enzyme, mixed 10X buffer and plasmid, and then added EcoRI enzyme to form a total 20 μl reaction solution and then in a 37 ° C. incubator. After the reaction for about 1 hour, the size of the sequence cut out from the vector was confirmed by electrophoresis, and the sequencing reaction was performed using the T7 and SP6 primer sequences in the pGEM-Teasy vector. The SPLCV AC2 gene [SEQ ID NO: 16] was cloned from the SPLCV Korean isolate.

In addition, a protein having an amino acid sequence of SEQ ID NO: 17 was obtained by encoding each protein based on the entire nucleotide sequence determined from the obtained gene, and the domains of the Nuclear localization signal and the zinc-finger binding motif in the amino acid sequence were AC2 of SPLCV. It was confirmed that the gene is well preserved in all (FIG. 7).

Example 3: Preparation of Recombinant Vector Using AC2 Gene

In order to confirm the PTGS inhibitory activity of the AC2 gene of SPLCV of SEQ ID NO: 17 isolated in Example 2, the gene of SEQ ID NO: 16 was separated by PCR using the primer of Figure 8, plant transformation as shown in Figure 9 The vector was cloned into pBI121.

In order to recover the SPLCV AC2 gene contained in the pGEM-Teasy vector of Example 2, a gel extraction kit was obtained by electrophoresis of the SPLCV AC2 gene cut out from the T-vector using Sac I and Xba I restriction enzymes. The DNA was recovered, and the recovered DNA was ligation with the pBI121 vector using fragments digested with Sac I and Xba I restriction enzymes of pBI121 prepared in the same manner. After reacting the ligation mixture overnight at 16 ℃, heat shock transformation method was applied to DH5alpha competent cell for 45 seconds at 42 ℃ and 900 ㎕ LB medium was added and incubated for 1 hour at 37 ℃ shaking incubator It was then plated in LB solid medium to which ampicillin was added to form colonies in a 37 ° C. incubator for 12 hours. The colonies formed were inoculated into 3 ml of kanamycin-added LB medium, incubated for 12 hours using a 37 ° C. shaking incubator, and the mixture was mixed with glycerol to be 14% final, and then stored at -80 ° C. . In addition, after centrifugation of the same culture to purify the plasmid by using the plasmid separation method, using SacI and XbaI restriction enzyme, the mixture of 10 X buffer and the plasmid and then the two restriction enzymes were added to the total 20 ㎕ reaction solution After the composition was reacted in an incubator at 37 ° C. for about 1 hour, the size of the sequence cut out from the vector was confirmed by electrophoresis, and the sequencing reaction was performed using the 35s promoter and NOS terminator sequences in the pBI121 vector. The NCBI BLAST search confirmed that the SPLCV AC2 gene, which is the gene of SEQ ID NO: 16, was cloned into pBI121, a plant transformation vector.

Example 4: Preparation of transformants and recombinant PTGS inhibitory activity using recombinant vector

Agrobacterium tumefaciens KYRT1 was transformed into the pBI121 vector, which was recombined with the AC2 gene of SPLCV of SEQ ID NO: 16 of Example 3, and then expressed inverted repeat GFP and GFP by mass culture. Agrobacterium transformed with the vector was mixed with Agrobacterium transformed with the SPLCV AC2 gene (presumed as a suppressor_) at a concentration ratio of 0.1: 1: 1, and the TBSV-P19 gene as a positive control and a negative control. Agrobacteria transforming the vector and pBI121, respectively, were also mixed with IR-GFP and GFP at the same ratio as above, adding acetosyringone to each reaction solution to finally reach 150 μM. for 4 hours and injected into the leaves of tobacco (Nicotiana benthamiana) plants model after reaction at room temperature for up to 15 days (11 analyzing the Nikkei City), and observed under UV light, it was confirmed the activity of the protein.

As a control group, P19 protein of TBSV (Tomato bush stunt virus), a single-stranded RNA virus having a very high PTGS inhibitory activity, was compared. 10, the final 1 to P19, GUS, SPLCV-AC2 is GFP OD600 = 3, IR-GFP OD600 = 0.3 , and mixing to cell density ratio after the cultured to a final OD ₆₀₀ = 3, except for GFP by OD ₆₀₀ by: Coinfiltration with acrobacterium (agrobacterium) expressing both GFP and IR-GFP to 1: 0.1. In the case of GFP, OD ₆₀₀ = 1 was cultured to infiltration as a control. Inhibition of silencing of GFP, a fluorescent protein, confirmed that the inhibitory gene activity was almost absent in the GUS control group. Although the activity was lower than that of P19 derived from RNA virus, the AC2 protein of SPLCV also inhibited PTGS inhibitory activity. It can be seen that (Fig. 10). In addition, as a result of expression for more than 10 days, it was confirmed that the AC2 gene of SPLCV virus was able to suppress the systematic silencing activity as in other reports.

In order to molecularly assay the luminescence of GFP, the infiltrated region was finely ground in a mortar with liquid nitrogen, followed by 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 5% SDS, 20% sucrose, 5% 2- 500 microliters of mercaptoethanol was added thereto, stirred vigorously, and centrifuged at 15,000 rpm for 1 hour at 15,000 rpm to recover the supernatant and stored in a -80 ° C deep freezer. After protein quantification by Bradford method, each sample was diluted in sterile water in 96 well plate so that the final 0.1 μg / μl was dispensed by the final 100 microliter, and then fluorometer, emission 509nm, excitation 395 nm wavelength. The measurement was carried out. The average value of the measurement results was obtained from three repeated experiments, and the error value was calculated. 42.131 ± 1.812 RU for P19, 26.325 ± 3.245 RU for AC2, 7.312 ± 2.263 RU for GFP, 4.121 for GUS It was confirmed that the value represented ± 0.348 RU. Thus, although weaker than P19, AC2 also inhibited GFP from silencing, which was able to maintain expression for longer periods of time when the inhibitory gene activity was applied than the GFP expression time in the sample infiltrated with GFP alone. It was confirmed that it was possible to make (FIG. 11).

In addition, after extracting the total RNA from the same sample tissue using TRIzol reagent, and synthesized the cDNA with oligo-dT primers using the Promega reverse transcription kit, using the real-time RT-PCR method to express the amount of GFP protein expression Quantified by sample. Using a primer sequence designed for this [5'-aag cgt tca act agc aga cca-3 ', 3'-ccc agc agc tgt tac aaa ctc-5'], 95 ° C. for 1 minute; 2 μl of template cDNA prepared from each tissue sample at 0.25 ° C 10 sec, 55 ° C 10 sec, and 68 ° C 15 sec, 0.25 pmol of 5'-primer and 3'-primer, 1X PCR buffer, 0.125 mM dNTPs, 1X SYBR solution to prepare a final 10 μl reaction solution and quantified the gene expression by PCR (Fig. 12).

Figure 1 shows the results of electrophoresis of the polymerase chain reaction assay of the SPLCV gene purely isolated from domestic sweet potatoes and partial gene sequence amplification for the overall sequence determination. SP1 (1632 bp) and SP2 (2138 bp) fragments of expected sizes were amplified by DNA polymerase chain reaction.

2 shows the primer design for overall sequencing. As a primer design schematic showing where the primer sequences are located on SPLCV, overlapping regions of the amplified regions were used to obtain the entire virus sequence.

3 shows the design of sequencing primers for SPLCV sequence identification.

Figure 4 shows the total base sequence corresponding to the length 2828 bp of SPLCV.

Figure 5 shows a genomic map of the SPLCV of the present invention (arrow indicates each gene (complementary gene strand; C1, AC2, C3, C4, viral gene strand; V1, V2)).

Figure 6 shows the electrophoresis picture of the amplification result of the SPLCV AC2 gene sequence by the polymerase chain reaction. It can be seen that the entire gene sequence of 486 bp was amplified.

Figure 7 compares the AC2 amino acid sequence of the SPLCV of the present invention and other AC2 amino acid sequences. Consists of 148 amino acids, NLS (nuclear localization signal) at the amino terminus, zinc finger binding motif corresponding to the DNA binding region at the center, and the active domain at the carboxyl terminus It can be seen. NCBI_AC2 is the AC2 gene of sweet potato leaf blight virus (USA) (Genbank NP_808843.1) and CelTech_AC2 is the AC2 gene identified from domestic sweet potato leaf blight virus (Genbank ACL97985.1).

8 is a primer sequence used for sequence amplification of the SPLCV AC2 gene of the present invention, in which an Xba I (underlined italic) restriction enzyme recognition sequence is inserted before a start codon of a gene, and a stop codon is modified near a stop codon. Subsequently, his tag (underlined) was inserted for protein purification or various purposes, followed by insertion of a Sac I (underlined italic) restriction enzyme recognition sequence to easily recombine the plant expression vector pBI121.

Figure 9 is one of the examples illustrating the process of cloning the SPLCV AC2 gene of the present invention to the plant transformation vector pBI121.

Figure 10 shows the results of the inhibitory activity assay by agroinfiltration (agroinfiltration) method. When the GFP transformant Nicotiana benthamiana 16c was injected with GFP transgenic agrobacteria to induce posttranscriptional gene silencing (PTGS), and when the control group was injected with the control group P19 transgenic Agro bacteria and the experimental group SPLCV AC2 transgenic Agro, respectively. Gene transcription was suppressed after transcription to confirm GFP expression.

11 shows the results of inhibition activity assay after agroinfiltration, and extracting proteins from infiltrated regions of plants to measure relative fluorescence values using a fluorophotometer.

12 is a result of confirming the inhibitory activity assay results after agroinfiltration and extracting total RNA from infiltrated regions of plants to measure relative expression levels using quantitative real-time RT-PCR. The results of three replicates were plotted using three measurements.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration          RURAL DEVELOPMENT ADMINISTRATION <120> Korean sweet potato leaf curl virus isolated gene capable of          suppressing posttranscriptional gene silencing <150> KR10-2008-0038414 <151> 2008-04-24 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 2820 <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                                                                         2820 <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> SPR2 (reverse) <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> 42 <212> DNA <213> Artificial Sequence <220> <223> primer_5SpAC2 <400> 14 cccggatccc aagcttgcat gtccaatctc ccttctggaa tc 42 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer_3SpAC2 <400> 15 cccaagcttc ggatccgctt aaggcgttcc aaaataccag tc 42 <210> 16 <211> 444 <212> DNA <213> SPLCKV <400> 16 atgtccaatc tcccttctgg aatcaagagg aagtgtccca ttcaggagcc actgcacgca 60 gaggcgaaga aggccaagag gaaagttcct gaacagagga caaggatagt gtggaagggt 120 tgcggctgtt cagccttcat caccccaaac tgcaagtatc agcatggatt cacgcacagg 180 ggaatcacta agtcatgctc agactacgag agcactcgaa ttcgacacca accccatgtc 240 tgtgggtcgg actgcaccgt tccatctcag aattatgtat gcccacgaga gcacacaggg 300 gagaaccatc ctcaaattcc agatgtgggt caactacagg gaaaggaggc aactgggatt 360 ccacaagatc ttcctccaat tccggatctt gacgacccgt ctaactggtg ctattcacag 420 ttggactggt attttggaac gcct 444 <210> 17 <211> 148 <212> PRT <213> SPLCKV <400> 17 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> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5'-primer <400> 18 aagcgttcaa ctagcagacc a 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3'-primer <400> 19 cccagcagct gttacaaact c 21  

Claims (4)

AC2 protein from domestic sweet potato leaf blight infected DNA virus having the amino acid sequence of SEQ ID NO: 17. AC2 gene derived from the domestic sweet potato leaf blight infection DNA virus having the nucleotide sequence of SEQ ID NO: 16.  The AC2 gene derived from the domestic sweet potato leaf blight infection DNA virus according to claim 2, which has a gene silencing inhibitory activity after transcription. A recombinant vector deposited with KACC 91470P, comprising the gene of claim 2.

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