KR101934951B1 - Novel White clover mottle virus gene from white clover plant - Google Patents

Abstract

The present invention relates to a novel white clover mottle virus gene comprising nucleotide sequences of SEQ ID NO: 1, a recombinant vector comprising the white clover mottle virus gene, a host cell transformed by the recombinant vector, a composition for diagnosis of the white clover mottle virus and a diagnostic method. Since the use of the white clover mottle virus gene of the present invention enables the diagnosis of infection with white clover mottle virus, the novel white clover mottle virus gene can be used for the prediction of disease outbreak by applying the gene to the monitoring or the investigation of the occurrence of viral diseases in domestic crops or weeds. Especially, the reality of the disease occurring in host plants including the white clover can be clarified, and the gene can contribute to the dissemination and quality improvement of the disease-free host plants including the white clover.

Description

A novel white clover mottle virus gene derived from a white shamrock plant {Novel White clover mottle virus gene from a white clover plant}

The present invention relates to a white clover mottle virus gene comprising the nucleotide sequence of SEQ ID NO: 1, a recombinant vector comprising the white clover mottle virus gene and a host cell transformed with the recombinant vector, Compositions and methods of diagnosis.

The emergence and risk of emergent viruses and new viral diseases are increasing gradually due to ecosystem changes due to climate warming, evolution and variation of plants and viruses, and increased trade in agricultural products. About 1,000 plant viruses have been reported so far worldwide, and new viruses are still reported in various plants. At present, about 100 viruses have been reported in various plants in Korea, but detailed investigation of the viruses has been done only in some crops, and most of the crops and natural host weed plants have only a fragmentary investigation. Thus, it is not yet understood how many viral diseases are occurring in many major crops and weeds.

Plant diseases are abnormalities of the host cells and tissues that result from the continued influences of pathogens or environmental factors on the plant host, including abnormalities in plant morphology, physiology, etc., resulting in a reduction in the resulting economic value It is also said. Causes of these plant diseases include fungi, bacteria, Nematode, Mycoplasma, protozoa and viruses. However, the virus is a pathogen having a size of less than 1 占 퐉 and its substance can not be observed even with an optical microscope, and since it requires an electron microscope, it is difficult to diagnose a virus that has developed in a plant with the naked eye. Therefore, there is a need for genetic studies to diagnose these viruses. In particular, genetic studies for diagnosing viruses against native host plants are needed. Among these, white clover is a native host plant native to the whole country, and it is necessary to study the virus causing the disease.

Therefore, the present inventors prepared a cDNA library for plant samples infected with white shamrock plants native to the whole country, and obtained a PCR sequence of a primer set based on their contiguous nucleotide sequences. Finally, the present inventors confirmed the novel white clover mottle virus gene of about 6.2 kb in size, which causes disease in white clover .

Accordingly, an object of the present invention is to provide a method for screening a white shamrock mite virus gene comprising the nucleotide sequence of SEQ ID NO: 1, a recombinant vector containing the gene, a host cell transformed with the vector, .

In order to achieve the above object, the present invention provides a white shamrock mite virus gene comprising the nucleotide sequence shown in SEQ ID NO: 1.

In addition, the present invention provides a recombinant vector comprising the white clover mottle virus gene.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention also provides a composition for detecting a white shamroot mottle virus infection comprising an agent for detecting the white shamrock mite virus gene.

The present invention also provides a method for detecting white shrapnel mottle virus infection in a plant, comprising detecting a white shamrock mite virus gene comprising the nucleotide sequence of SEQ ID NO: 1.

Since the white shamrock mite virus gene of the present invention can be used to diagnose the infection of white shamrock mite virus, it can be used in the monitoring or investigation of the occurrence of viral disease occurring in domestic crops or weeds to be used for the prediction of disease occurrence. Especially, it is possible to clarify the reality of the disease occurring in the host plants including white shamrock, and to contribute to the improvement of the quality distribution and quality improvement of host plants including white shamrock.

FIG. 1 is a diagram showing alignment of the contiguous positions of white shrimp mottle virus using Chickpea chlorotic stunt virus CHN as a reference sequence.
Fig. 2 is a schematic diagram of the white shrapnel mottle virus gene.
FIG. 3 is a diagram showing the result of diagnosing a virus as a primer set by amplifying a white shamrock mite virus gene and the virus envelope protein region.

The present invention provides a white shamrock mite virus gene comprising the nucleotide sequence shown in SEQ ID NO: 1.

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

The white shamrock ( Trifolium repens L.) is a perennial plant belonging to the soybean family. It is a herb with three lobules with a stalk and a white pattern at the tip of the nipple.

The white shamrock mite virus gene derived from the white shrimp plant may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto, and the homologue of the nucleotide sequence is within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also provides a recombinant vector comprising a white shamroot mottle virus gene.

In the present invention, the white clover mottle virus gene nucleotide sequence can be inserted into a recombinant vector. The term " vector " is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term " carrier " is often used interchangeably with " vector ". The term " recombinant vector " means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. The recombinant vector of the present invention is preferably a plant expression vector. Any plasmid and vector may be used as long as the recombinant vector of the present invention can be cloned and stabilized in the host.

An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element. Enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters, which are available in eukaryotic cells, are well known in the art. The expression vector can be constructed by a method known in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis.

Expression vectors comprising the white shamoto mottle virus gene sequence and suitable transcription / translation control signals from the shamrock plant can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant gene, aadA gene, and the like, but are not limited thereto.

In the plant expression vector according to the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term " promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A " plant promoter " is a promoter capable of initiating transcription in plant cells. A " constitutive promoter " is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages.

In the plant expression vector according to the present invention, the terminator can be a conventional terminator such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaci Agrobacterium and the terminator of the Octopine gene of Tumefaciens, but the present invention is not limited thereto.

The present invention also provides a host cell transformed with the recombinant vector.

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells. The host cell is preferably a plant cell.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl ₂ method, one method (Hanahan, D., J. MoI. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell, And an electric drilling method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also provides a composition for detecting a white shamroot mottle virus infection comprising a preparation for detecting a white shamrock mottle virus gene.

The agent for detecting the white clover mottle virus gene of the present invention may be a primer or a probe that specifically binds to the white clover mottle virus gene. The composition may further comprise a reaction amplification mixture, wherein the reaction amplification mixture contains a reagent necessary for performing an amplification reaction, a thermostable DNA polymerase, a deoxynucleotide, a sterile water without nucleases, and a divalent metal cation Or the like, and preferably includes a reaction buffer, a deoxynucleotide, and a DNA polymerase.

When the probe of the present invention is used, a reporter such as a fluorescent substance can be labeled at the end of the probe.

The term " primer " in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a template of a complementary nucleic acid, Lt; RTI ID = 0.0 > nucleic acid < / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

In the design of the primer, there are various restrictions such as a ratio of A, G, C and T content of the primer, prevention of formation of a primer complex (dimer) and repetition of the same base sequence three times or more. the conditions such as the amount of template DNA, the concentration of the primer, the concentration of dNTP, the concentration of Mg ^{2 +} , the reaction temperature, and the reaction time should be appropriate.

Such primers may incorporate additional features that do not alter the basic properties. I.e. the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more nucleotides into nucleotides, and uncharged linkages such as phosphonates, phosphotriesters, phosphoramidates or carbamates, phosphorothioates or phosphorodithioates Or the like can be transformed into a charged nucleus. The nucleic acid may be at least one of a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalating agent such as acridine or psoralen, a chelating agent such as a metal, a radioactive metal, And may have additional covalently bonded moieties.

The primer sequences of the present invention can also be modified using a label that can directly or indirectly provide a detectable signal. The primers can include labels that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include 32P, fluorescent dyes, electron dense reagents, enzymes (commonly used in ELISA), biotin or haptens, and proteins that are available for antisera or monoclonal antibodies.

The primers of the present invention can be prepared by cloning and restriction cleavage of appropriate sequences and digestion with restriction enzymes such as the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68: 90-99), diethylphosphoramidate Including, for example, direct chemical synthesis methods, such as the solid support method of Tetrahedron Lett. 22: 1859-1862 (1981), and the solid support method of US 4458066.

The agent for detecting a viral gene of the present invention may comprise a primer set for the detection of a white shamrock mite virus infection comprising the nucleotide sequence shown in SEQ ID NO: 18 and SEQ ID NO: 19, And a primer set for the detection of a white shamroot virus infection including a base sequence. The primer set set forth in SEQ ID NO: 20 and SEQ ID NO: 21 is a primer set capable of detecting the coat protein (CP) of the white shamroot virus. Using the primer set, only the white shrapnel mottle virus can be specifically detected from Polerovirus -infected samples belonging to the genus such as white shrimp mottle virus.

The primer sets manufactured and used in the present invention are shown in Tables 1 and 2 below.

[Table 1]

[Table 2]

The present invention also provides a method for detecting white shrapnel mottle virus infection in a plant, comprising detecting a white shamrock mite virus gene comprising the nucleotide sequence of SEQ ID NO: 1.

The method of the present invention for diagnosing a white shamrock mite virus infection refers to a method for diagnosing whether a white shamrock mite virus is infected by confirming the presence of a viral gene consisting of the base sequence shown in SEQ ID NO: 1 in a plant. Preferably, the method for diagnosing infectious white poultry virus infection according to the present invention comprises the step of detecting the infection with a primate set of a primate set for the detection of P. shorelin virus infection comprising the nucleotide sequence shown in SEQ ID NO: 18 and SEQ ID NO: 19 or the nucleotide sequence shown in SEQ ID NO: And the like.

In carrying out the method of diagnosing white bladder mottle virus according to the present invention, the concentration of the primer set may be preferably 7 to 13 pmol / μl, more preferably 10 pmol / μl.

Also, in performing RT-PCR, reverse transcription was performed at 50 캜 for 30 minutes; Denaturation step at 95 DEG C for 10 minutes; Reacting at 95 ° C for 30 seconds, then reacting at 55 ° C for 30 seconds, and then amplifying at 72 ° C for 1 minute. This step is repeated 35 cycles and finally elongated at 72 ° C for 5 minutes Step can be performed.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  1. white shamrock Motley  Virus identification

1.1 The yield of the sample and the white shamrock Motley  Total RNA extraction of virus

Two white shamrock and five other plants were obtained in order to isolate and identify the plant virus infected with the plant native to the genus of the peach test site of Cheongdo-gun, Gyeongbuk province. The plant samples were each ground using liquid nitrogen. All samples were stored in a cryogenic freezer. Total RNA was extracted with Easy-Spin Total RNA Extraction Kit (iNtRON Biotechnology) after mixing the same amount of each sample in the same amount.

1.2 RNA Sequence of  Analysis, cDNA library production

Ribosomal RNA was removed from the extracted total RNA using Ribo-Zero ™ Magnetic Kit (plant leaf) (Epicenter). The prepared RNA was disrupted with short fragments and synthesized with double-stranded cDNA. The synthesized double-stranded cDNA was end-repaired and an adapter was attached for the cloning required for library production. This series of steps was performed using a TrueqRNA sample prep kit (Illumina), and an appropriate size purified insert DNA (purified by BluePippin ™ 2% agarose gel cassette (Sage Science) Respectively. Finally, the size and quantity / quality of the inserted DNA suitable for library production were measured using an Agilent 2100 BioAnalyzer 2100 (Agilent Technologies). The fragment size of the cDNA library was found to be approximately 350-450 bp. The library was sequenced using Illumina HiSeq2500 and raw data was generated at approximately 5 Gbp using a paired-end sequencing method.

1.3 Plant Virus Related Contiguous  making

After selecting good data from the raw data, plant virus-related readings were obtained through a de novo transcriptome analysis method to construct contigs. When the length of the conti is too short (<200bp), data with low coverage is removed when compared with a virus database (DataBase), and finally about 400 contigs are obtained.

1.4 primer  Genome and virus genome Sequence  Identification

In order to find the sequence of the entire gene of the virus, the step of confirming the most homologous virus was performed through the nucleotide sequence similarity test of the virus-related database (DataBase) obtained in Example 4. The four contigs showing high homology to Chickpea chlorotic stunt virus CHN isolate (CpCSV, NCBI GenBank accession no. JF507725) were identified and the contig position was aligned using the CpCSV virus as a reference sequence. Respectively.

In addition, a primer was prepared based on the nucleotide sequence of the contig, and PCR was performed on 7 plant samples using the primer. The band was confirmed by agarose gel electrophoresis and the sequence of the PCR product confirmed by band was sequenced to confirm the base sequence. Based on the nucleotide sequence obtained from the PCR result of the primer set based on the contig, another primer was prepared based on the obtained nucleotide sequence to obtain an additional base sequence, and the primers used for the whole genome crystal of the virus are shown in Table 3.

[Table 3]

1.5 Identification of the Nucleotide Sequence of a New Plant Virus

The viral sequences obtained through PCR of the primer set prepared in Example 5 were identified and are shown in SEQ ID NO: 1 and FIG.

The virus is approximately 6.2 kb (+) single-stranded RNA genome, ((+) single-stranded RNA genome) , and the base sequences of all the encoded protein (Open Reading Frame, ORF) other Polerovirus The virus was identified as a new virus according to the classification standard of the International Committee on Taxonomy of Viruses (ICTV). This virus was named White clover mottle virus (WClMoV).

Example  2. White shamrock Motley  Diagnosis of virus Primer  Performing production and diagnosis

Based on the determined base sequence, a primer for diagnosing white shrimp mottle virus was constructed. Specifically, a diagnostic primer capable of amplifying the coat protein (CP) region was prepared, and at the same time, the finally determined CP gene sequence was registered in NCBI GenBank. The sequences of the white clover mottle virus sequence primers are shown in Table 1, and the envelope protein (CP) primers of the viruses are shown in Table 2.

[Table 1]

[Table 2]

RT-PCR was performed to confirm whether only the white shrapnel mottle virus could be specifically detected from the same Polerovirus- infected sample through the above-mentioned final determined primer. The results are shown in FIG.

Multiplex RT-PCR was performed by Genetbio suprimescript RT-PCR premix SR-8000 (GeNeTBio Inc., Korea). To each 10 μl of RT-PCR premix SR-8000 (GeNeTBio Inc., Korea), add 1 μl of the forward / reverse primer to the concentration of 10 pmol / μl and add 7 μl of DNase / RNase-free water and 1 μl of template total RNA And RT-PCR was performed using 20 μl of the final reaction solution as a composition.

RT-PCR was performed under the following conditions. After a reverse transcription step at 50 DEG C for 30 minutes, a denaturation step at 95 DEG C for 10 minutes was performed. Thereafter, the reaction was carried out at 95 ° C for 30 seconds, followed by 30 seconds at 55 ° C, followed by 1 minute at 72 ° C, followed by 35 cycles and finally extension at 72 ° C for 5 minutes.

After performing the RT-RCR experiment, electrophoresis was performed on 1% agarose gel at 100 V for 40 minutes to confirm the amplification.

M is a marker, lane 1 is BWYV (Beet western yellows virus), lane 2 is PDMV (Panicum distortion mosaic virus), lane 3 is SeiyDV (Setaria yellow dwarf virus), lane 4 is CABYV (Cucurbit aphid-borne yellows virus) , Lane 5 for potato leafroll virus, lane 6 for BVG (barley virus G), and lane 7 for WClMoV (white clover mottle virus).

As shown in FIG. 3, the viruses belonging to the same genus Polerovirus or the diagnostic methods of the primers of the present invention can be used to detect only the white shrimp mottle virus from the Polerovirus -infected samples belonging to the genus such as white shrimp mottle virus And the diagnosis was made.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel White clover mottle virus gene from white clover plant <130> 1-297 <160> 21 <170> KoPatentin 3.0 <210> 1 <211> 6205 <212> DNA <213> Unknown <220> <223> white clover mottle virus <400> 1 caaaagaaag atagagggaa gctttgctgc tttagcgcaa gtttatgaat ttcttgatca 60 acaatcaaaa ttcacgaatc gaattaactt tttcgaactc tctttctcta gatacaagaa 120 gatctttaac tgctcttttc ctaatctcag cagagcagta tttacaaatt tctcacgagc 180 aacatggcac ccaaaatagc gttgcttatt gctctcttct tgtctgctcc ttttctactc 240 gggggattca atttccgaga tggcaaactt gtcatcccct cgagttcgag aaatcgccac 300 cgttacgagc tcttccgaga gcttttggcg agggagaatt ttgcattctc tcctcctctg 360 aaaatggagc ggctgaatta tactccccca atccaaattc ctgtcacaga gcaaacttat 420 atagacttac ttacagctct ttggcagaag gcctacctag atatccagaa atatttgggt 480 acggcacttc attcatccaa acactcattg gctcatatat tagaaaaatc gagcgaggtg 540 ttttcttcct gcgtccagtc tttcctatgg gcgatagtct tggtgtggac ttacgcactt 600 tgggttttct gctactggac ggtagaggtt ataataacca ataccatgct tattatagca 660 gtaggattgc taatgttttg caccgtattt atggtaaaag ctttacactg gctttttgga 720 gacttcttca attggactgt ggtccctgtt ttcaggtgcc tagtttacct atggaagctc 780 cgatcgctga aggtacttgc gggcaagact cagatgatga aggagaagat gacgaagggg 840 tttgggagct atgatatgat gatgtcaccc cctaaaagta gtgttttaga agtgctacat 900 gatgatgagc aacattgcgg ctatgcaagc tgcatattgt tagctgatgg cactgtgggg 960 ttgttgactt ctcaacacgt agcagaggat gcttattgga ttaaatctca taaaactgga 1020 aacaagataa agttttcaga atttaagcct ctgatcaaat cccaaaaggg agatataaca 1080 atattagtgg gaccccctaa ttggcaagga cttttaggct gctcagcagt tctttatgtt 1140 acaatgaaaa acttgtcagt tagcgacgct cgtatctatt accgcaagga aggtgattgg 1200 tactctggag tggctaaatt aatgggtcct aatttgtaca attttgtgga tgttttgtca 1260 aacacagaac ccggttatag tggaacccct tacttcaatg gaaacaaaat agttggggtt 1320 cacaccggtg gcgcctcgaa tactaattgc aatgttatgg ctgccattcc acatattgaa 1380 ggcctgactg ccagcaagta catctatgaa accacagctc ccaaaggaaa aatttttgat 1440 tttgatgatg agttgtggaa tgaattgatg gaagatttta gcgtggcaga gtctcaatat 1500 ttgtacaata aaatcaaaaa cgtcgaacct gagtgctcta tgaacctcac catgagaggg 1560 gatcagcata ctgaacaata cagagtgaac gatgcattct ctccagaatg cacaataaat 1620 atgaagctga gagaccctaa taatggaaga gaagcgagaa ttagatcacc tgaagagcac 1680 aaaggttgtt ttccagatag agcttgggtg gattgcaatc atgatttatc caaagaagaa 1740 atagaaaaat ttttaagtca taatctggga atcgacgctg tgataatccc ccggcacaag 1800 caattcttat gcaaagattg tgatgtagat ctgagagaaa gtttaggtga ttggttgagc 1860 acaaaaatca aaccaccagt ccagtcagaa gattggtcag gtgccgaatt tcatgatgct 1920 cccactgaac tcaaaaccac caaagaagag tggttagatg aaatctcctt tgaaacatcc 1980 aagaaagcca atttaaacgg ggagcgaggc accgacctcg taaaaaccgg cgaaggctgc 2040 acccaaaaga acaagagcga cgatggagac actgtccaga gagtgataga agctctcgtc 2100 gccaagatga atgtaagcca actggaggag agagtagtgg aagtggtatc aaagaagata 2160 tccacccctc cagtgaagaa acaaaggcgc agaggaaagc gtggaggagg gaacaagcaa 2220 acagcttcaa ccacttcttc agctcccaat acaactggga aatacctgcc gcctcaaaag 2280 agatcccagg cttcgaagcc tgcggcaaaa tccccaaata ctaccatcca aagcaaaaac 2340 aacaaggaga atgggagag aaaatcgtca cccaacaccc agaaatgggt gagaaaatca 2400 aaggatttgg gtggccagag ttcggcaagg aagctgaact gaaatccttg cagctacaag 2460 ctgcgaggtg gctcaagcgt gctgagtctg ctaaaatccc ctcagcggag gagcgggagc 2520 acgtaattca gaaaacagta gaggcataca aaaatgttaa atcaaattgc cctgctgtca 2580 caaggctgaa cgagttgagt tgggaccaat ttcaaaaatc ttttcaacca gccgttcact 2640 ccctagaact agacgctgga attggcgttc cttatatagc atatggtctc cccactcaca 2700 gagggtgggt tgagaatcat gagcgccaac tcttgccaat cttggctcag ttgacttatg 2760 accgtttaca gaagatgtca aaagttaact ttgagaactt gaaggcagaa gagttagtac 2820 agcgtggatt atgtgaccct attagggtat ttgttaaggg agaaccacat aaacaatcaa 2880 aacttgatga aggccgctac cgcctcatca tgtcagtgag ccttgttgat caactggtag 2940 cccgggtttt attccaagaa cagaacaaac tggagattaa tctctggaaa gctataccaa 3000 gcaaacccgg aatggggctg tctactgact cccaagtgat agagttcatc aattctctgt 3060 cccacctcgt gcaaatacca gttgaagact tggtatacaa ttgggaaaag cacgtggtgc 3120 ccactgactg ctctggtttt gactggagtg tttctcactg gctattgacg gacgaaatgg 3180 aggttcgtaa ccgtctgaca cgcaacaaca ataccctcac aagaaagctg agggattgtt 3240 ggttaaaatg tttatccaat agtgtgttgg ccttttctga tggaagcctg tatgcacagc 3300 gtgttcctgg agttcaaaag tctggaagct ataatacaag ctcgactaac tcccgaattc 3360 gtgttatgtg tgcttattat gcaggtgctt cctgggccat agcaatggga gatgacgctt 3420 tggaagcagt ggacactgac ctatcagtgt ataaaaatat aggtttaaaa gtcgaggttt 3480 caggacaact ggaattctgc tctcatatct ttgagaagcc tgacctcgcc attccggtaa 3540 atgtaggaaa aatgttgtac aaattgatat atggctacaa cccggaatgt ggatcgattc 3600 aggttctcaa gaattatatc gacgcttgca cttcagtgtt aaatgaattg cgtcatgatc 3660 ctgaattagt tcagcttctc tactcgtggc ttctccatcc agtgttgcca caaaagtaag 3720 cgagtaagag aagcagacag atagccggta tcagttgttg caatagccgg agtcttagtc 3780 aacgtacaat cgtgaaaatt gattataaat ttctcgccgg atttaccgcg ggtttcgtga 3840 catcaattcc aatatcagtt tttgcgattt atatcatcta cctcaagatt tctgcccacg 3900 ttagatcaat agttaatgaa tacggtcgtg gttagaaaca atggaagaag gagaaggaac 3960 aggcgcccta ttcggcgggc tcaacgccgc aacccagtgg ttgtggtcca acccgctagg 4020 cagccacaac gcagaagacg acgaagaaga aaccgtagac gcgctgctag aggaagcaca 4080 gctggaagac gagggtctag cgagacattc atattttcaa aagacaacct caagggcagt 4140 gactcaggac gtatcacgtt cgggccgtct ttatcagact gcccagcttt cagctctgga 4200 attctcaagg cctaccatga atataaaatc acaatggtta agttggagtt catctccgag 4260 gccgcttcca cctcctcagg gtcgatcgct tacgagcttg atccccattg caaagcctct 4320 gagcttgggt cctacatcaa taaatttgga atcaccaaga gtggacaaag gaccttctca 4380 gctcggttta ttaacggcat cgagtggcac tcctcagatg aggaccaatt caggatacta 4440 tacaaaggaa acggaaactc cgcaattgcg gggtcgttcc gcatcaccat caagtgccaa 4500 actcagaatg cgaaataggt agatgacagt tccccccccg gaccagcccc gacaccacct 4560 cctccacctc ctccagcacc ctccccagaa ccacaacctt gcaagaaatt taggttttgg 4620 gggtatgaag gtgttcctca gaacaagata ataacagacac agaataatag aaatatagat 4680 gtacgtgctt tgaattatgt taagttctgg aaatgggaag atgataattg gagtgaagtc 4740 aatatgcaag ccaactactc caccaataat tcgcaatatg ctgaacctta tatggtgatc 4800 cctgcatcta aaggcaagtt ccacgtctat ctcgagtgtg atggacagat ggctgtcaag 4860 agtgtaggtg gaaaagcgga taatacctgg agaggtttga tagcgtatga cacttctcga 4920 agaatgtgga atgtcggaaa ttacaaagga tgtgtaatag aaaattataa aatgagtagc 4980 acctttgtga atggtcaccc tgatgtagaa ttaaatgatt gtaagtttga caaagccagg 5040 ggagtagaag ccgactggta tgcctcattt caattaactt gtgatgacga tgagggctct 5100 tggctccttt acgcacctcc aatcccaaag gacagtttgt ataattacac tgtctcttat 5160 ggtgagtaca cagaaaatat gtgtgaatgg ggtgccgtct caatatctat agatgaggac 5220 aacggctcta ctgggaacga agtctctgac tactgtcgaa gggggtatat gcgacagtct 5280 ctgccagaag gaaaactgga acaacaaccc tacgtggaag acgtagataa aataaactcc 5340 tggaaggaaa accagtctga aacctcaagt gattcggagg atgttgcttt cgcaaataaa 5400 atgcgtggta agcttcctaa ccaaacaaag ctgcctccga aaggcttcct atcgcgttta 5460 aaaccttctg aaaaggagga aatagtacag gccaagccaa cacaagtcac tgatgacgat 5520 gtgttgcgtg gaatgaaagc tgttggtgtt tttgccaacc ctagctacgt ccaccctgtc 5580 cgcgaaaaac tcgaggatat tgagagacaa gaacttatga aagaactcga gaaggaccta 5640 caagaaataa atcgcttgga acccccggat gaaatctcag ctgaagagaa catcccggac 5700 tttgtggaac cggtcttacc ggttcctgac ccggattggt tcaatgaaca cactgcaaaa 5760 acaataagcg tgatagaaca tccgtgggat ttcccgccta ttgaggatac ttcgaaaaag 5820 aaattaagag gtactctttc tcaagcagga ggttccataa gtggtaagtc aagtcttggg 5880 ggaggaactt tgcgcagatc tacagaagat gtttggagag aaacactgaa atctaaactc 5940 tcaacatcag acaggaacag atatgaaaga atcgttaaag gacaaggcaa aactgttgct 6000 gaccaattcc tgagagatcg cgctgcgtag acgtatatca accgccgtct ataaagatac 6060 ggttttcacc aaatgtgggt gcgttcacat ttcctagctt ctggatttga agcaaactcg 6120 ggccgcagag tataaatgtc ggaacgaaag cgaaagcgag taggcaccag tgttttacgt 6180 gggtattccc acggcactgt ggtgt 6205 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F1 primer sequence <400> 2 cctaatctca gcagagcagt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R1 primer sequence <400> 3 gactctgcca cgctaaaatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F2 primer sequence <400> 4 ccggttatag tggaacccct 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R2 primer sequence <400> 5 tagggagtga acggctggt 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F3 primer sequence <400> 6 gtccagagag tgatagaagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R3 primer sequence <400> 7 acgtgggcag aaatcttgag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F4 primer sequence <400> 8 aagctcgact aactcccga 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R4 primer sequence <400> 9 gagtttggca cttgatggtg 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F5 primer sequence <400> 10 gttggagttc atctccgag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R5 primer sequence <400> 11 ccacttatgg aacctcctg 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F6 primer sequence <400> 12 agtgattcgg aggatgttgc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R6 primer sequence <400> 13 ccgagtttgc ttcaaatcca g 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F0 primer sequence <400> 14 gtatatcaac cgccgtctat aa 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F1 primer sequence <400> 15 cctagcttct ggatttgaag c 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R1 primer sequence <400> 16 ggaaattgaa tcccccgagt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R2 primer sequence <400> 17 gccaaaagct ctcggaagag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-F0 primer sequence <400> 18 acagaacccg gttatagtgg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_like-R0 primer sequence <400> 19 tcttctgact ggactggtg 19 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_CP-F1 primer sequence <400> 20 gacctcgcca ttccggta 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpCSV_CP-R1 primer sequence <400> 21 tggtggagta gttggcttgc 20

Claims (8)

White clover mottle virus gene consisting of the nucleotide sequence shown in SEQ ID NO: 1. A recombinant vector comprising the white clover mottle virus gene of claim 1. A host cell transformed with the recombinant vector of claim 2. A composition for diagnosing a white shamroot mutilation virus infection comprising the agent for detecting the white shamrock mottle virus gene of claim 1. The composition according to claim 4, wherein the agent for detecting the viral gene is a primer or a probe that specifically binds to the white clover mottle virus gene. 6. The method according to claim 5, wherein the primer is a primer set for detecting infection with a white clover mottle virus comprising the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19. Composition. 6. The method according to claim 5, wherein the primer is a primer set for detecting infection with a white clover mottle virus comprising the nucleotide sequence of SEQ ID NO: 20 and SEQ ID NO: 21 Composition. And detecting a white shamrock mite virus gene consisting of the nucleotide sequence shown in SEQ ID NO: 1. 2. A method for diagnosing a white shamrock mite virus infection in a plant, comprising:

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