JP3038433B2 - Purification method and gene of Yamanoimo nematode mosaic virus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a yam nematode mosaic virus, DNAs and proteins derived from the virus, and a method for diagnosing nematode mosaic disease in a plant of the genus Yam.

[0002]

2. Description of the Related Art Chinese yam is a vegetatively propagated crop, and many strains are infected with the virus even now, and the yield is remarkably reduced. At present, as with other plant viral diseases, there are no effective drugs to eliminate the infectious virus. As a countermeasure, virus-free strains are produced in shoots by shoot apex culture, but reinfection in the field is a problem.

[0003] Neso mosaic disease is one of the viral diseases that occur in Chinese yam, and its causative virus has been reported to be the yam necrotic mosaic virus. This virus is a string of particles about 660 nm long and is aphid-transmitting, so far Carlavir
Thought to be us, but not finalized.

[0004] If there is a means capable of promptly diagnosing necrotic mosaic disease, the damage caused by this virus disease can be greatly reduced. However, although diagnosis by Western blotting using an antiserum against the virus particles is possible, a diagnostic method using genes such as polymerase chain reaction (PCR) has not been established yet. One of the causes is that it is difficult to purify virus particles from diseased leaves due to the fact that no host has been found for this virus other than the plants of the genus Yam, and the presence of slime, etc. unique to the genus Yam. Are listed.

[0005]

As described above, the difficulty in purifying the yam mosaic mosaic virus particles is a major obstacle to establishing an efficient diagnostic method using genes. Therefore, the present invention has as its first object to establish an efficient purification method for the yam nematode mosaic virus particles, and further isolates a viral gene from the virus particles, and based on the sequence, the nematode mosaic disease The second objective is to establish a rapid diagnosis method for

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor first found that when purifying the yam mosquito mosaic virus particles, the treatment with driserase was carried out at a stage where the purification proceeded to some extent. By conducting an organic solvent treatment at the final stage of virus purification, it was found that virus particles could be efficiently purified. Further, a virus gene was isolated from the purified virus particles, and the nucleotide sequence thereof was analyzed. The present invention has been completed based on these findings.

[0007] That is, a first aspect of the present invention is a method for purifying the yam mosaic mosaic virus particles, wherein doriserase is used after centrifugation and / or the organic solvent is used after cesium chloride density gradient centrifugation. It is a method for purifying the yam nematode mosaic virus particles, which is used. Further, the second of the present invention, the following (a)
(C). (a) DNA represented by the nucleotide sequence of SEQ ID NO: 1 (b) represented by a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1, and ( DNA having the same function as the DNA of (a) (c) RNA homologous to the DNA of (a) or (b)

Further, a third aspect of the present invention is the following (a) or
The protein shown in (b). (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) represented by an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 2, and ( a) a protein with a function similar to that of a)

A fourth aspect of the present invention is that a labeled DNA or RNA having a base sequence identical to or complementary to a part of the DNA represented by SEQ ID NO: 1 is extracted from a plant of the genus Yam. A method for diagnosing nematode mosaic disease of a plant of the genus Yam, characterized by determining whether or not the plant is infected with the yam nematode mosaic virus based on whether or not it hybridizes with RNA; Two kinds of oligonucleotides represented by the same and complementary base sequences were prepared, and these oligonucleotides and RNA extracted from plants of the genus Yam
Is mixed under conditions that can cause PCR, and the presence or absence of an infection with the wild yam nematode mosaic virus is determined based on whether an amplification product has been produced.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) purification of viral particles of the invention (method for purifying viral particles) first invention, Driselase (registered
(Trademark ) is used after centrifugation, and / or the organic solvent is used after performing cesium chloride density gradient centrifugation. The use of doriserase for the purification of virus particles had already been attempted, but no good effect was observed with the use at that time
(Fujita et al., 1992). However, as in the present invention, the plant-derived mucilage can be effectively removed by performing the treatment with dricerase at a stage after the virus purification to some extent, that is, after the centrifugation.

It is common practice to use an organic solvent for the purification of virus particles. However, in the conventional plant virus purification method, it was usual to treat the organic solvent at an early stage after the grinding, but when this was applied to the potato, the yield of the virus was greatly reduced, which was not suitable. When the organic solvent treatment is performed at the point where the virus purification, which is the stage after the cesium chloride density gradient centrifugation is performed, reaches the final stage, the decrease in the yield of the virus can be suppressed, and the degree of purification of the virus can be increased. As the organic solvent to be used, those conventionally used for the purification of virus particles may be used, and for example, chloroform, carbon tetrachloride and the like can be used.

(2) Second Invention (Nucleic Acid) The nucleic acid of the present invention includes the following nucleic acids (a) to (c). (a) DNA represented by the nucleotide sequence of SEQ ID NO: 1 (b) represented by a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1, and ( DNA having the same function as the DNA of (a) (c) RNA homologous to the DNA of (a) or (b)

Here, "having the same function as the DNA of (a)" means that it can be used for producing a virus-resistant plant, for example, like the DNA of (a). Also,
"RNA that is homologous to the DNA of (a) or (b)" means (a) or (b)
In which the thymine in the DNA is replaced with uracil. The DNA shown in (a) relates to the nucleotide sequence of the 3′-terminal region of the wild yam nematode mosaic virus genome, which comprises a part of the viral replication enzyme gene (nuclear inclusion body b gene) and the viral coat protein gene. Includes whole and 3 'untranslated regions. As shown in SEQ ID NO: 1, the nucleotide sequence of this DNA has an incomplete but large open reading frame (ORF) that starts with the TTT open reading frame at positions 2 to 4 and ends with the TAG at positions 1673 to 1675. Have.

The DNA shown in (a) can be obtained, for example, by the following method. First, virus particles are isolated and purified from a mosaic symptom. Purification of virus particles may be performed according to a conventional method, but is preferably performed according to the above-described purification method of the present invention. Next, RNA is extracted from the virus particles. RNA can be extracted according to a conventional method. CDNA is prepared from the extracted RNA, and this c
Using the DNA as a template, a specific DNA fragment is amplified according to the 3′RACE method. Thereby, the DNA of the present invention can be prepared. In addition, plasmid pGEMCYV containing the DNA shown in (a)
To FERM P-17000 at Biotechnology and Industrial Technology Research Institute
(Deposit date: September 21, 1998).

The DNA shown in (b) is, for example, the DNA shown in (a)
Is used as a template, and PCR is performed using primers that are not completely identical or complementary to the DNA used as the template. RNA shown in (c) is, for example, (a) or
It can be synthesized using RNA complementary to the DNA of (b) as a template and RNA polymerase. To date, there have been many reports of introducing virus-derived genes such as virus coat protein genes and virus replication enzyme genes into plants to produce virus-resistant plants, so the DNA of the present invention was introduced into plants. By doing so, it is possible to breed a plant resistant to the nematode mosaic disease of the genus Yam.

(3) Protein The protein of the present invention includes the following proteins (a) and (b). (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) represented by an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 2, and ( A protein having the same function as the protein of (a) Here, "having the same function as the protein of (a)" means, for example, that it can be used for the production of a virus antibody similarly to the protein of (a). is there.

The protein of (a) contains a part of the virus replication enzyme of the yam nematode mosaic virus and a viral coat protein. The amino acid sequence of this virus is
It has a part homologous to the cordy virus belonging to the Potyviridae family. The protein of the present invention can be produced by introducing a vector containing the gene or the like of the present invention into Escherichia coli or the like and expressing it. By immunizing a rabbit or the like using the protein of the present invention or a part thereof as an antigen, a highly specific virus antibody can be produced, and can be used for diagnosis of necrotic mosaic disease.

(4) Method for Diagnosing Necrotic Mosaic Disease One of the methods for diagnosing necrotic mosaic disease of the present invention is a method for diagnosing necrotic mosaic disease which is the same or complementary to a part of the DNA represented by the nucleotide sequence of SEQ ID NO: It is characterized in that the presence or absence of the infection of the yam nematode mosaic virus is determined based on whether or not the labeled DNA or RNA having the sequence hybridizes with the RNA extracted from the plant of the genus Yam.

Here, the length of the DNA or RNA to be labeled is
Although it is not particularly limited as long as it functions as a probe, 70
About 0 to 800 b is preferable. The means for labeling DNA or the like is not particularly limited, and a general method such as dicoxigenin labeling can be used. RNA extraction from a plant of the genus Yam can be performed according to a conventional method. Hybridization can also be performed according to a conventional method.

Another method for diagnosing nematode mosaic disease of the present invention is a method for diagnosing DNA represented by the nucleotide sequence of SEQ ID NO: 1.
To produce two types of oligonucleotides represented by the same and complementary nucleotide sequence as a part of the above, and mixed these oligonucleotides and RNA extracted from the plants of the genus Yam under the conditions where PCR can occur, the amplification product It is characterized by judging the presence or absence of the infection with the yam mosaic mosaic virus based on whether or not the virus has occurred.

The length of the oligonucleotide to be prepared is not particularly limited as long as it functions as a primer for PCR, but is preferably about 20 to 25 b. In addition, "PCR
`` Conditions that can occur '' refers to conditions under which an amplified fragment can be obtained by PCR if the oligonucleotide and RNA extracted from the plant of the genus Yam can be hybridized. The term refers to conditions under which an oligonucleotide is made to coexist with a polymerase, dNTPs, salts, and the like, and heating is repeated at a constant cycle.

[0022]

[Example] [Example 1] Purification of virus particles from potato 800 ml of 0.2 M citrate buffer (10 mM DIECA, 5 mM EDTA, 0.4% L-ascorbic acid, 0.5% thioglycol) was added to 100 g of mosaic symptom. Acid, 2% PVP, pH 5.8) and crushed with a mixer for 3-6 minutes. Crush liquid at 50 ° C, 7-8
After processing for 1 minute and quenching, the mixture was filtered through a double gauze, and centrifuged at 4000 g for 10 minutes at 4 ° C. 1% (v / v) 1M calcium chloride solution and 5% (v / v) 0.2M disodium hydrogen phosphate were added to the obtained supernatant, and the mixture was stirred for 20 minutes at 4 ° C., and 6000 g for 10 minutes. 4 ℃
And centrifuged. 5% (v / v) TritonX-100 was added to the obtained supernatant, followed by stirring at 4 ° C. for 2 hours, and centrifugation at 90,000 g for 90 minutes at 4 ° C. 100 ml of 0.1 M phosphate buffer (0.1%
Thioglycolic acid, pH 6.0), resuspend with a Teflon homogenizer while cooling on ice, add 1% (w / v) doriserase, stir at 28 ° C for 2-3 hours, 6000 g, 10 minutes, 4 hours ° C
And centrifuged. The resulting supernatant was washed with 0.2% (w / v) sucrose containing 0.2%.
Layer on top of 05M borate buffer, 90,000g, 2 hours, 4
Centrifuged at ° C. 8 ml of a 0.05M borate buffer was added to the obtained precipitate, and after resuspension with a Teflon homogenizer while cooling with ice, the mixture was centrifuged at 6000 g for 10 minutes at 4 ° C. The obtained supernatant is
Overlay on top of a 0.05 M borate buffer containing 20% (w / v) sucrose and cesium chloride with a density gradient of 0-30% (w / v), 180,000 g for 3 hours at 4 ° C. Centrifuge. A virus-containing fraction was collected, dialyzed overnight against a 0.01 M borate buffer, added with an equal volume of chloroform: carbon tetrachloride (1: 1), stirred for 15 minutes, and then subjected to microcentrifugation at 10,000 rpm for 5 minutes. Centrifuge. The obtained aqueous layer was used as a purified virus solution. When the virus concentration was calculated with the extinction coefficient at 260 nm being approximately E ^0.1% = 2.5, it was estimated that about 40 μg of virus particles were obtained. When rabbits were immunized with the purified virus, the resulting antisera could be used for diagnosis by Western blot, tissue print immunoblot, and immunoelectron microscopy.

[Example 2] Extraction of viral genomic RNA 2 μl per 100 μl of purified virus solution containing 13.2 μg of virus particles
l of Proteinase K (14.4mg / ml: Boehringer Mannheim
) And 10 μl of a 10% sodium dodecyl sulfate solution, and reacted at 37 ° C. for 1 hour. Then add sterile water and add 2
The volume was adjusted to 00 µl, 200 µl of a TE-saturated phenol: chloroform (1: 1) mixed solution was added, and the mixture was vigorously stirred for 5 minutes, and then centrifuged at 15,000 rpm for 5 minutes with a microcentrifuge. The obtained aqueous layer was transferred to a new microtube, and the same operation of TE-saturated phenol: chloroform treatment and centrifugation was repeated again three times. An equal volume of chloroform: isoamyl alcohol (24: 1) was added to the obtained aqueous layer, and the mixture was vigorously stirred for 5 minutes, followed by centrifugation at 15,000 rpm for 5 minutes with a microcentrifuge. Transfer the resulting aqueous layer to a new microtube and add 1/10
After adding 3 M sodium acetate and 2.5 volumes of ethanol, the mixture was allowed to stand at −30 ° C. overnight, and then centrifuged at 15,000 rpm for 10 minutes. The obtained precipitate was washed with 80% ethanol, air-dried, and dissolved in 10 μl of sterilized water to obtain a virus genomic RNA solution.

Example 3 Synthesis and Cloning of cDNA Carlavirus was expected to be the causative virus of potato nematode mosaic disease.
We tried the 3'RACE method using degenerate primers prepared for rlavirus. I tried several primer combinations, but in most cases the expected size of PC
No R product was obtained and only one test yielded a PCR product of the expected size. However, when the base sequence was determined by cloning this PCR product, the sequence of the degenerate primer was not found in this amplified fragment,
It turned out that it was an unexpected amplification product by only the adapter primer. As will be described later, it was confirmed that this cDNA clone was derived from a virus that infects yam. Hereinafter, the production of this clone will be described.

For cDNA synthesis, an RNA PCR Kit (TaKaRa
And TaKaRa Ex Taq (TaKaRa). 1 μl viral genomic RNA, 1 μl -strand primer (10 μM: Oligo (dT) primer with adapter sequence; LIFE TECHNOLO
(GIES), 2 μl of sterilized water, mixed with mineral oil, kept at 70 ° C. for 10 minutes, and then placed on ice for 1 minute. 4 μl of MgCl ₂ Solution, 2 μl of 10 × PC
R Buffer II, 2 μl each of dGTP, dATP, dTTP, dCTP, 1 μl
After adding and mixing 1 μl of Reverse Transcriptase, the mixture was incubated at 42 ° C. for 60 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes to obtain a reverse transcription reaction product. Next, 5 μl of 10 × Ex
Taq Buffer, 4μl dNTP Mixture (2.5mM each), 33μl
l sterile water, 1 μl + strand primer (10 μM: Carlavirus
Degenerate primer), 1 μl -strand primer (10 μM: primer with adapter sequence only; LIFE TEC
HNOLOGIES), 5 μl of the reverse transcription reaction product was mixed, mineral oil was added, and the mixture was incubated at 94 ° C. for 1 minute, and 1 μl of TaK
aRa Ex Taq was added and PCR was performed. PCR denature: 98 ° C, 20
Annealing and extension reaction: 35 cycles at 68 ° C. for 5 minutes. The obtained PCR product was subjected to 1% agarose gel electrophoresis, and an amplified DNA of about 1.9 kbp was cut out and W
izard PCR Preps DNA Purification System (Promega
Was purified. Purified DNA is transferred to plasmid vector pGE
After ligation to MT (Promega), E. coli JM109 was transformed and cloned. This clone is called pG
Named EMCYV.

Example 4 Analysis of Nucleotide Sequence A probe was prepared from pGEMCYV, genomic RNA extracted from the purified virus, and total R of the potato leaf of nematode mosaic disease.
When Northern hybridization was performed using NA, a clear reaction was observed.
Since it was confirmed that the clone was derived from a virus that infects yam, the nucleotide sequence of this cDNA was determined and analyzed.

The nucleotide sequence was determined by the dideoxy method. Reaction was performed in Thermo Sequenase fluorescent lab
elled primer cycle sequencing kit (Amersham),
Primer is M13 Reverse Dye Primer or -21M13 D
Using ye Primer (PERKIN ELMER), 373 DNA
The nucleotide sequence was determined by Sequencer (PERKIN ELMER).

An open reading frame (OR) encoding a protein from the base sequence determined in this manner
F) Searched. Then, for the amino acid sequence of the ORF translated using the nucleotide sequence and the general genetic code, DDB
Analysis such as homology search with gene or protein data registered in gene databases represented by J, GenBank, and EMBL, and comparison of gene structure were performed.
As a result, it was revealed that the cDNA clone pGEMCYV was a clone derived from a virus belonging to Macluravirus, a candidate of a new genus of the Potyviridae family. Furthermore, pGEMCY
The amino acid sequence of the viral coat protein core region deduced from the nucleotide sequence of V is Narc, another Macluravirus.
The amino acid sequences of the core region of the coat protein of issus latent virus and Maclura mosaic virus (only these two species are Macluravirus species candidates at this time) are 56% and 47%, respectively.
Showed that the virus from which the clone was derived was a new species of Macluravirus.

Example 5 Virus Detection by Northern Hybridization [0029] Total RNA was extracted from the leaves (0.2 g) of Chinese yam by a method using phenol / SDS and LiCl (Verwoerd et al., 1989). The obtained total RNA was dissolved in sterile water, and 10 μg of the total RNA was subjected to formaldehyde-modified 1.2% agarose gel electrophoresis. After the electrophoresis, the RNA was transferred to a nylon membrane (Amersham Hy
bond-N +) and immobilized on the membrane by UV crosslinking.
DIG Easy Hyb (Boehringer Mannheim)
Prehybridization at 65 ° C for 1 hour, followed by DIG Easy Hyb
And hybridization was carried out at 65 ° C. overnight. As the virus probe, RNA expressed by digoxigenin (DIG) labeling of a DNA represented by the nucleotide sequence of Nos. 399 to 1155 of SEQ ID NO: 1 with a transcription vector was used. After hybridization, the membrane is 0.3M NaCl-0.
Wash twice with 03M citric acid solution (2xSSC), then 1μg / ml
The cells were treated with 2 × SSC containing RNase A, and washed twice with 2 × SSC again. After washing the membrane, DIG Wash and Block Buffe
r Set and DIG Luminescent Detection Kit (Boehring
(Er Mannheim), and the signals were detected by exposing to an X-ray film. The results were as shown in FIG. Total extracted from virus-free strains
For RNA, no signal was observed, whereas for total RNA extracted from general cultivated strains and genomic RNA extracted from purified virus, a clear signal was observed at about 10 kbase. From this, the virus infection of the general cultivation strain was confirmed.

Example 6 Detection of Virus by RT-PCR Total RNA was extracted from leaf pieces (0.2 g) of Chinese yam by a method using phenol / SDS and LiCl (Verwoerd et al., 1989). The obtained tolal RNA was dissolved in 30 μl of sterilized water, and reverse transcription PCR (RT-PCR) was performed using this as a template. RT-PCR
Was performed using RNAPCR Kit (TaKaRa). -Total RNA solution in reaction solution containing strand primers and reverse transcriptase
0.5 μl was added and the reaction was performed at 42 ° C. for 15 minutes; denaturation: 99 ° C. for 5 minutes; quenching: 5 ° C. for 5 minutes. To the obtained reaction solution, a PCR reaction solution containing a + strand primer and DNA synthase was added, and denaturation: 95 ° C, 1 minute; annealing and extension reaction: 58 ° C, 1 minute
For 35 minutes, 35 cycles of PCR were performed. The primer sequence used was such that the -strand primer was 5'-CCAGAGACACCACGATGTT
The A-3 ′ and + chain primers are 5′-CCAACAAAGAAACCATCTGT-3 ′, which are sequences corresponding to positions 1197 to 1216 and 836 to 855 of SEQ ID NO: 1, respectively. PCR products were analyzed by 1.2% agarose gel electrophoresis. The results were as shown in FIG. No amplification products were observed in RT-PCR performed using total RNA extracted from virus-free strains, whereas RT-PCR performed using total RNA extracted from general cultivated strains and genomic RNA extracted from purified viruses.
In PCR, a clear specific amplification product was observed around 380 bp. This size agrees very well with the theoretical size of the amplification product (381 bp) predicted from the gene sequence of the potato nematode mosaic disease virus, confirming the virus infection of common cultivated strains. .

[0031]

Industrial Applicability The present invention provides a rapid and efficient method for diagnosing nematode mosaic disease in a plant of the genus Yam.

[0032]

[Sequence List] SEQUENCE LISTENING <110> Aomori prefecture <120> Yamanoimo-eso-mozaiku-uirus no seiseihou oyobi sonoidenshi <130> P98-0418 <130> 2 <210> 1 <211> 1906 <212> DNA <213> unknown <400> g ttt gac att tta tac agc ata cgt tcc aat ttc ttt tgc gaa gag 46 Phe Asp Ile Leu Tyr Ser Ile Arg Ser Asn Phe Phe Cys Glu Glu 1 5 10 15 gat agg cag gat gca aaa act gcc atg gca cac atg tat cgc gag ttt 94 Asp Arg Gln Asp Ala Lys Thr Ala Met Ala His Met Tyr Arg Glu Phe 20 25 30 gtt tac aca ccc atc cac acc ata acg ggg cag gtt ctt gtc aag aag 142 Val Tyr Thr Pro Ile His Thr Ile Thr Gly Gln Val Leu Val Lys Lys 35 40 45 ctt gga aat aac agt gga cag cca agc act gtt gtt gat aac acg ctt 190 Leu Gly Asn Asn Ser Gly Gln Pro Ser Thr Val Val Asp Asn Thr Leu 50 55 60 atc ctg atg ctg tca ttt cta tac gca tac ata cgg aaa aca aat gat 238 Ile Leu Met Leu Ser Phe Leu Tyr Ala Tyr Ile Arg Lys Thr Asn Asp 65 70 75 aga aca tgt gct caa att aac cag cgt ttc aaa ttt gtg tgt aat gt 286 Arg Thr Cys Ala Gln Ile Asn Gln Arg Phe Lys Phe Val Cys Asn Gly 80 85 90 95 gat gac aac aag tac tca gtt tca cct gaa ttc cac gaa gaa ttc agc 334 Asp Asp Asn Lys Tyr Ser Val Ser Pro Glu Phe His Glu Glu Phe Ser 100 105 110 gga gat ttt tca agg gag atc gct gaa cta ggt ttg aca tac gaa ttc 382 Gly Asp Phe Ser Arg Glu Ile Ala Glu Leu Gly Leu Thr Tyr Glu Phe 115 120 125 gat gac ttg aca gct gac ata acg caa aac ccg tac atg agt cta gtg 430 Asp Asp Leu Thr Ala Asp Ile Thr Gln Asn Pro Tyr Met Ser Leu Val 130 135 140 atg gta cga acg cct ggt ggc att ggt ttc caa ctc aat cct gag aga 478 Met Val Arg Thr Pro Gly Gly Ile Gly Phe Gln Leu Asn Pro Glu Arg 145 150 155 att att gct att gtt cag tgg att aaa cgg ggt gat gtt ctc caa gca 526 Ile Ile Ala Ile Val Gln trp Ile Lys Arg Gly Asp Val Leu Gln Ala 160 165 170 175 tcg caa gca gca ttt gca gct atg atc gaa gca ttc aat gat ccg tgg 574 Ser Gln Ala Ala Phe Ala Ala Met Ile Glu Ala Phe Asn Asp Pro trp 180 185 190 tta ttc gga gtt ttg cac ttg tt ctt ctt tgc caa tac 622 Leu Phe Gly Val Leu His Leu Tyr Phe Val trp Leu Leu Cys Gln Tyr 195 200 205 agg gaa gaa att agg tac gct atg gat cat gac ctt gga gct gtt tgt 670 Arg Glu Glu Ile Arg Tyr Ala Met Asp His Asp Leu Gly Ala Val Cys 210 215 220 tac atg gat gcc tac caa gtt tat gct ttg cat tac gac acg agg gat 718 Tyr Met Asp Ala Tyr Gln Val Tyr Ala Leu His Tyr Asp Thr Arg Asp 225 230 235 gac gtt aat gat ttg caa att gat tca gcc agt tgc gag att gag aaa 766 Asp Val Asn Asp Leu Gln Ile Asp Ser Ala Ser Cys Glu Ile Glu Lys 240 245 250 255 act gtg gtt gtt gct ggc tca aca cag cac atc agt ctt caa atg gac 814 Thr Val Val Val Ala Gly Ser Thr Gln His Ile Ser Leu Gln Met Asp 260 265 270 ctg agc act cca gtg caa ttg cca aca aag aaa cca tct gtg gaa gac 862 Leu Ser Thr Pro Val Gln Leu Pro Thr Lys Lys Pro Ser Val Glu Asp 275 280 285 aaa gga aaa gct ctt gcc aca cct tca aca aca gct aca ttg cca gat 910 Lys Gly Lys Ala Leu Ala Thr Pro Ser Thr Thr Ala Thr Leu Pro Asp 290 295 300 tca agc cag ggg cac caa cca gtt gaa caca aac aat aac cca gac 958 Ser Ser Gln Gly His Gln Pro Val Glu Glu Pro Asn Asn Asn Pro Asp 305 310 315 aca gtg gat gaa gag gac att gaa tgg cga atc cca gca ata caa aaa 1006 Thr Val Asp Glu Glu Asp Ile Glu trp Arg Ile Pro Ala Ile Gln Lys 320 325 330 335 ggt ttt ggc cac tac aaa atc cca aag gtc aaa gga aag aga ata tgg 1054 Gly Phe Gly His Tyr Lys Ile Pro Lys Val Lys Gly Lys Arg Ile trp 340 345 350 aac cca aag att ttg aag aaa atc gca cat gag caa ttc acg acg aca 1102 Asn Pro Lys Ile Leu Lys Lys Ile Ala His Glu Gln Phe Thr Thr Thr 355 360 365 tcg cag atg gtg aca aca gat aaa ttg gag aaa tgg act gag gag gtc 1150 Ser Gln Met Val Thr Thr Asp Lys Leu Glu Lys trp Thr Glu Glu Val 370 375 380 aaa aga gat ctt gtg gtg aca aat gaa aca gat ttc cag ata tgc tta 1198 Lys Arg Asp Leu Val Val Thr Asn Glu Thr Asp Phe Gln Ile Cys Leu 385 390 395 aca tcg tgg tgt ctc tgg tgt gct aac aat ggt aca tca tct gag tta 1246 Thr Ser trp Cys Leu trp Cys Ala Asn Asn Gly Thr Ser Ser Glu Leu 400 405 410 415 gac gca tca caa ttt a tg gaa gtc cat gca aac gga cag ata atg ggg 1294 Asp Ala Ser Gln Phe Met Glu Val His Ala Asn Gly Gln Ile Met Gly 420 425 430 att cca att caa att ttt gtt gaa ccc gca atc cag cac gga gga ctg 1342 Ile Gln Ile Phe Val Glu Pro Ala Ile Gln His Gly Gly Leu 435 440 445 cgt aaa gtg atg aga cat ttc agt gga atc acg agc aaa atg ttg tct 1390 Arg Lys Val Met Arg His Phe Ser Gly Ile Thr Ser Lys Met Leu Ser 450 455 460 gaa ggg gga aaa atg aca gcg tgg ggc agg aaa agg ggt ttc acc caa 1438 Glu Gly Gly Lys Met Thr Ala trp Gly Arg Lys Arg Gly Phe Thr Gln 465 470 475 aga tca atg ata cca tat gcc ttt gat gtc cca act gat acg 1486 Arg Ser Met Ile Pro Tyr Ala Phe Asp Phe Phe Val Pro Thr Asp Thr 480 485 490 495 acg cca aaa aca atc agg gag cag ctg agt caa agc aag gcc gca gct 1534 Thr Pro Lys Thr Ile Arg Glu Gln Leu Ser Gln Ser Lys Ala Ala Ala 500 505 510 att ggg cgt ggc gtt caa agg gtg atg ctt ctt gat ggg aaa gtt cac 1582 Ile Gly Arg Gly Val Gln Arg Val Met Leu Leu Asp Gly Lys Val His 515 520 520 g gg agc cgc aca agt tac gag aga cac acg gat aac gac cag gac gag 1630 Gly Ser Arg Thr Ser Tyr Glu Arg His Thr Asp Asn Asp Gln Asp Glu 530 535 540 tat gaa cat gga ggg gcg gaa gat cag cgc cca gct ttac 1672 Tyr Glu His Gly Gly Ala Glu Asp Gln Arg Pro Ala Leu Tyr 545 550 555 tagtaaggtt tgttttgcaa caaccattac tactagtatt agaagaatta agtttaatt 1732 ctttcacaat cttcactttt aacttttgcg tttatgagtt aaatgggagt ttttgtggg 1792 ttttcttcca atgctcagca accctcaacg cactgcattt cgttttcttc tttaagctt 1852 agctggacgc tcagctggtt tcgcatagcg aaacttatcc tgttccatag aagg 1906 <210> 2 <211> 557 <212> PRT <213> unknown <400> Phe Asp Ile Leu Tyr Ser Ile Arg Ser Asn Phe Phe Cys Glu Glu Asp 1 5 10 15 Arg Gln Asp Ala Lys Thr Ala Met Ala His Met Tyr Arg Glu Phe Val 20 25 30 Tyr Thr Pro Ile His Thr Ile Thr Gly Gln Val Leu Val Lys Lys Leu 35 40 45 Gly Asn Asn Ser Gly Gln Pro Ser Thr Val Val Asp Asn Thr Leu Ile 50 55 60 Leu Met Leu Ser Phe Leu Tyr Ala Tyr Ile Arg Lys Thr Asn Asp Arg 65 70 75 80 Thr Cys Ala Gl n Ile Asn Gln Arg Phe Lys Phe Val Cys Asn Gly Asp 85 90 95 Asp Asn Lys Tyr Ser Val Ser Pro Glu Phe His Glu Glu Phe Ser Gly 100 105 110 Asp Phe Ser Arg Glu Ile Ala Glu Leu Gly Leu Thr Tyr Glu Phe Asp 115 120 125 Asp Leu Thr Ala Asp Ile Thr Gln Asn Pro Tyr Met Ser Leu Val Met 130 135 140 Val Arg Thr Pro Gly Gly Ile Gly Phe Gln Leu Asn Pro Glu Arg Ile 145 150 155 160 Ile Ala Ile Val Gln trp Ile Lys Arg Gly Asp Val Leu Gln Ala Ser 165 170 175 Gln Ala Ala Phe Ala Ala Met Ile Glu Ala Phe Asn Asp Pro trp Leu 180 185 190 Phe Gly Val Leu His Leu Tyr Phe Val trp Leu Leu Cys Gln Tyr Arg 195 200 205 Glu Glu Ile Arg Tyr Ala Met Asp His Asp Leu Gly Ala Val Cys Tyr 210 215 220 Met Asp Ala Tyr Gln Val Tyr Ala Leu His Tyr Asp Thr Arg Asp Asp 225 230 235 240 Val Asn Asp Leu Gln Ile Asp Ser Ala Ser Cys Glu Ile Glu Lys Thr 245 250 255 Val Val Val Ala Gly Ser Thr Gln His Ile Ser Leu Gln Met Asp Leu 260 265 270 Ser Thr Pro Val Gln Leu Pro Thr Lys Lys Lys Pro Ser Val Glu Asp Lys 275 280 285 Gly Lys Ala Leu Ala Thr Pro Ser Thr Thr Ala Thr Leu Pro Asp Ser 290 295 300 Ser Gln Gly His Gln Pro Val Glu Glu Pro Asn Asn Asn Pro Asp Thr 305 310 315 320 Val Asp Glu Glu Asp Ile Glu trp Arg Ile Pro Ala Ile Gln Lys Gly 325 330 335 Phe Gly His Tyr Lys Ile Pro Lys Val Lys Gly Lys Arg Ile trp Asn 340 345 350 Pro Lys Ile Leu Lys Lys Ile Ala His Glu Gln Phe Thr Thr Thr Ser 355 360 365 Gln Met Val Thr Thr Asp Lys Leu Glu Lys trp Thr Glu Glu Val Lys 370 375 380 Arg Asp Leu Val Val Thr Asn Glu Thr Asp Phe Gln Ile Cys Leu Thr 385 390 395 400 Ser trp Cys Leu trp Cys Ala Asn Asn Gly Thr Ser Ser Glu Leu Asp 405 410 415 Ala Ser Gln Phe Met Glu Val His Ala Asn Gly Gln Ile Met Gly Ile 420 425 430 Pro Ile Gln Ile Phe Val Glu Pro Ala Ile Gln His Gly Gly Leu Arg 435 440 445 Lys Val Met Arg His Phe Ser Gly Ile Thr Ser Lys Met Leu Ser Glu 450 455 460 Gly Gly Lys Met Thr Ala trp Gly Arg Lys Arg Gly Phe Thr Gln Arg 465 470 475 480 Ser Met Ile Pro Tyr Ala Phe Asp Phe Phe Val Pro Thr Asp Thr Thr 485 490 495 Pro Lys Thr Ile ArgGlu Gln Leu Ser Gln Ser Lys Ala Ala Ala Ile 500 505 510 Gly Arg Gly Val Gln Arg Val Met Leu Leu Asp Gly Lys Val His Gly 515 520 525 Ser Arg Thr Ser Tyr Glu Arg His Thr Asp Asn Asp Gln Asp Glu Tyr 530 535 540 Glu His Gly Gly Ala Glu Asp Gln Arg Pro Ala Leu Tyr 545 550 555

[Brief description of the drawings]

FIG. 1 is a photograph showing the results of Northern hybridization using a piece of potato leaf as a sample.

FIG. 2 is a photograph showing the results of RT-PCR using a potato leaf piece as a sample.

Continuation of the front page (56) References Bulletin of the Japanese Society of Plant Pathology, 44 (1), 1-5 (1978) Bulletin of the Japanese Society of Plant Pathology, 61 (1), 41-43 (1995) Virol. Methods, 8 (4), 321-329 (1984), National Institute of Prevention and Health, Alumni Association, “Revised Second Edition, Experimental Virus Studies,” Maruzen Co., Ltd. (published June 1, 1973), p. 379- 382 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N ⁷ /00-7/08 C12N 15/00-15/90 GenBank / EMBL / DDBJ WPI (DIALOG) BIOSIS (DIALOG) CA ( STN) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A nucleic acid represented by the following (a) or (b): (a) DNA represented by the nucleotide sequence of SEQ ID NO: 1 (b) RNA homologous to DNA of (a) 2. Whether or not a labeled DNA or RNA having a nucleotide sequence identical or complementary to a part of the DNA represented by the nucleotide sequence of SEQ ID NO: 1 hybridizes with RNA extracted from a plant of the genus Yam. A method for diagnosing nematode mosaic disease in a plant of the genus Dioscorea, comprising determining the presence or absence of infection with a yam nematode mosaic virus. 3. Two kinds of oligonucleotides represented by a base sequence identical and complementary to a part of the DNA represented by SEQ ID NO: 1 are prepared, and these oligonucleotides and a plant of the genus Yam Diagnosis of nematode mosaic disease in a plant of the genus Yam, which is characterized by mixing the extracted RNA with a condition under which a polymerase chain reaction can occur and judging the presence or absence of an A. niger nematode mosaic virus depending on whether an amplification product has been produced. Method.