JP3116083B2 - Coat protein gene and 3'-terminal untranslated region of sweet potato latent virus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene for latent sweet potato virus. More specifically, the present invention relates to a coat protein gene and a 3'-terminal untranslated region among the above-mentioned sweet potato latent virus genes.

[0002]

2. Description of the Related Art Heretofore, a virus disease has occurred in sweet potato, which has become a problem nationwide. As its pathogens, there are many viruses, such as a mottled mosaic spot mottle mosaic virus and an unidentified virus belonging to the group of potyviruses of leaf mosaic disease (Potyvirus), and the sweet potato latent virus (SPLV) is one of them. .

[0003] This virus is classified into potato Y
It belongs to Potyviridae represented by virus. The virus is morphologically a string of particles about 750 nm long. The components of the virus are RNA, which is the body of the gene, and one type of coat protein surrounding it.

[0004] The sweet potato is a vegetatively replenishing cultivation system and the virus is transmitted by aphids (Usugi et al. , Annals of the Phytopathologi).
calSociety of Japan, 57: 512-521, 1991)
Once this virus has occurred in sweet potato, its control has been difficult.

[0005]

Generally, in order to know the relationship between a certain plant virus and another virus, the base sequence of the 3'-terminal untranslated region and the coat protein gene of the virus is determined. It is useful to compare with other viruses.

[0006] When a gene of a virus, such as a viral coat protein gene, is introduced into the genome of a plant and transformed, the transformed plant becomes resistant to the virus. Has been known for many combinations of plant viruses and their hosts.

[0007] In order to carry out such transformation, it is necessary to clone the coat protein gene of the virus to be controlled and determine its nucleotide sequence. For SPLV isolated in Taiwan (SPLV Taiwan strain), studies on genomic RNA have been conducted, and coat protein genes,
And the nucleotide sequence of the 3'-terminal untranslated region has been elucidated (Colinet, Gen Bank database, accession numb
er X84012). On the other hand, in Japan, SPLV genome R
Research on NA has not yet been performed.

Analysis of affinity between Japanese SPLV and other viruses including foreign SPLV and cDNAs thereof
In order to use E. coli for plant transformation, it is necessary to use SPL in Japan.
It is necessary to determine the nucleotide sequences of the 3 'untranslated region of V and the coat protein gene.

[0009]

Means for Solving the Problems In order to satisfy the above-mentioned needs, the inventors of the present invention have conducted intensive studies and have conducted SPL studies.
The present invention was completed by performing cDNA cloning of V-RNA and determining a part of its nucleotide sequence.

[0010] That is, a first aspect of the present invention relates to a base sequence encoding a coat protein of SPLV consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or a gene consisting of a base sequence complementary thereto. is there.

[0011] A second aspect of the present invention is a gene for the 3'-terminal untranslated region of SPLV consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing or a nucleotide sequence complementary thereto. Further, a third aspect of the present invention is a gene comprising the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a nucleotide sequence complementary thereto, including the first and second aspects described above. I do.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present invention provides a gene consisting of a base sequence encoding a coat protein of SPLV consisting of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing or a base sequence complementary thereto, and a base sequence consisting of SEQ ID NO: 2 in the sequence listing Alternatively, the present invention provides a 3′-terminal untranslated region gene comprising a nucleotide sequence complementary thereto.

The above gene can be obtained from the sweet potato latent virus (SPLV) as follows. In the present invention, there are several methods for preparing SPLV cDNA. These methods are classified into a method using the genetic information shown in FIG. 1 and a method not using it.

As a method of using the above-mentioned gene, FIG.
Create an appropriate probe based on the genetic information shown in
Method of preparing cDNA library from viral RNA and obtaining from this cDNA library, reverse transcription PCR
There is a method of directly obtaining from viral RNA using a method.

As a method not using the information shown in FIG. 1, for example, the following method used by the inventors of the present invention can be cited. Virus particles were purified from morning glory leaves or tobacco leaves ( Nicotiana benthamiana ) infected with SPLV, and the virus RN was purified from the virus particles.
Extract A.

The separation and purification of RNA from SPLV are performed by protease treatment, protein removal treatment, phenol extraction,
It can be carried out by appropriately combining methods such as ethanol precipitation.

Using the thus obtained RNA as a template and appropriate primers, reverse transcription followed by Polymerization
Viral cDNA can be obtained by ase chain reaction (RT-PCR). Suitable primers include Colinet et al . (Colinet et al ., Phytopathology, 84: 6
5-69, 1994) can be suitably used. S described above
The method for preparing the PLV cDNA may be modified as necessary.

The SP of the present invention obtained by the above means
Using the base sequence of LV cDNA, plants
A genetic diagnosis can be made as to whether or not the subject is infected. Furthermore, SPLV resistance can be imparted by creating a transformed plant in which the SPLV coat protein gene or the 3 ′ terminal untranslated region gene has been introduced into the plant.

[0019]

Embodiments of the present invention will be described below in more detail. Unless otherwise described, the procedure of the operation is described by Maniatis et al. (Molecular Cloning: A Laboratory Manual, 1982,
Cold Spring Harbor Laboratory, New York).

Example 1 cDNA of SPLV-RNA
Cloning (1) Preparation of SPLV-RNA Usugi et al . (Usugi et al ., Annals of the Phytopathologi)
cal Society of Japan, 57: 512-521, 1991) was isolated from sweet potato in Japan and confirmed to be SPLV from the reaction specificity against SPLV antiserum,
Asagao leaves or tobacco leaves ( Nicotian
a benthamiana ) and used as material for virus purification.

Two to three weeks after SPLV inoculation, the infected leaves were collected and ground using a 0.1 M citrate buffer to obtain a ground solution. Butanol was added to the milled liquid, and
Centrifuged for hours. The obtained precipitate was suspended in a 0.1 M citrate buffer and centrifuged by cesium chloride equilibrium density gradient centrifugation (5500
(0 rpm, 3.5 hours) to obtain purified virus particles.

[0022] Protease K and SD
S (sodium dodecyl sulfate) was added to dissociate the coat protein from the RNA. Thereafter, the product was purified using phenol and centrifuged at 15,000 rpm for 10 minutes. Add 3 to this supernatant
RNA was precipitated by adding a double volume of ethanol to obtain viral RNA.

(2) Preparation of cDNA Library Using the RNA obtained in the above (1) as a template, a viral cDNA was synthesized as follows. First, since viruses belonging to the potyvirus group are known to have a poly A sequence at the 3 'end of RNA, reverse transcriptase, RNase H and DNA are used with oligo dT as a primer.
Double-stranded DNA was synthesized using polymerase I, and both ends were blunted using T4 DNA polymerase. This series of steps was performed using a commercially available cDNA synthesis kit (Amersham) according to the instructions.

The blunt-ended cDNA was inserted into the EcoRV site of phagemid vector-pBluescript (manufactured by Strategene). This reaction was performed using a ligation kit (manufactured by Takara Shuzo) according to the method described in the kit instructions. This reaction product was used for E. coli JM109,
Alternatively, it was introduced into XL1-Blue to obtain a cDNA library. However, from this library, SPLV cD
A clone containing NA was not obtained.

(Example 2) SPL by RT-PCR method
CDNA cloning of V-RNA Therefore, Colinet et al. (Colinet et al. , Phytopatholog
y, 84: 65-69, 1994), followed by reverse transcription using SPLV RNA extracted from purified virus particles as a template using synthetic primers common to potyviruses.
cDNA synthesis was attempted by polymerase chain reaction (RT-PCR).

As a result, an amplified fragment of about 1.3 kbp was obtained. This cDNA fragment was ligated to the phagemid vector pBluescr.
After ligation to ipt, this was introduced into E. coli JM109 or XL1-Blue. In this way, a clone containing a cDNA of about 1.3 kb derived from SPLV was obtained.

Approximately 1.3 kb cDNA obtained as described above
Were determined by the dideoxy method according to a conventional method. However, about 1.3k obtained by this method
The cDNA of b contained only about half of the coat protein gene and did not contain the 3'-terminal untranslated region.

Example 3 SPL by RT-PCR
Cloning of the entire coat protein gene of V and the 3′-end untranslated region In order to clone the entire coat protein gene of SPLV and the 3′-end untranslated region, RT-PCR was performed using the following sequences as primers: .

In order to achieve the above object, a 30-base sequence at the 5 'end of the SPLV cDNA determined in Example 2 was used as a 5' primer (FIG. 4). 3 '
As the primer on the side, oligo dT was used in the same manner as in Example 1.
Was used. RT-PCR was performed using the above primers and SPLV-RNA as a template under the same conditions as in Example 2.

As a result, an amplified fragment of about 2.0 kb was obtained. This cDNA fragment was ligated to the phagemid vector pBluescript in the same manner as in Example 2, and this was introduced into E. coli JM109 or XL1-Blue. Thus, the entire coat protein gene of SPLV and 3 ′
A clone containing a cDNA of about 2.0 kb including a terminal untranslated region was obtained.

The nucleotide sequence of the cDNA of about 2.0 kb was determined by the dideoxy method described above. As a result, the gene encoding the coat protein and the untranslated region following the gene were found. In addition, a DAG sequence essential for aphid transmission was found in the amino acid sequence of the coat protein (FIG. 1).

Example 4 SPLV Taiwan Stock (Colinet, G)
en Bank database, accession number X84012) (1) Coat protein gene In the present invention, the nucleotide sequence of the SPLV coat protein gene identified was compared with a known sequence of the SPLV Taiwan strain (Colinet et al., 1994). did. As a result, the homology between the two was 82%.

(2) 3′-terminal untranslated region According to the present invention, the nucleotide sequence of the 3′-terminal untranslated region was compared with that of SPLV Taiwan strain. As a result, the homology of both was 94%. As described above, the SPLV coat protein gene and the nucleotide sequence of the 3′-terminal untranslated region revealed in the present invention are similar to the SPLV Taiwan strain, but clearly different.

[0034]

Industrial Applicability According to the present invention, there are provided a coat protein gene and a 3'-terminal untranslated region gene of a sweet potato latent virus which are clearly different from SPLV having a known sequence. Already in plant viruses, many combinations of viruses and plants (reviews: Beachy (1990), Tepfer (1993))
By introducing a coat protein gene into a plant,
It has been reported that virus resistant plants can be created. Therefore, it is possible to create a sweet potato latent virus-resistant plant using genetic engineering techniques.

Further, there has been reported an example in which cDNA of the 3'-terminal untranslated region of a plant virus was introduced into a plant by transformation to obtain a virus-resistant plant (Zaccomer et al .,
Gene, 136: 87-94, 1995), it is possible to create a sweet potato latent virus resistant plant using the cDNA of the 3'-terminal untranslated region of the sweet potato latent virus.

[0036]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 293 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Biological name: Sweet potato latent virus Sequence: Ala Asp Asp Thr Ile Leu Asp Ala Gly Lys Val Asp Thr Lys Arg Asn 1 5 10 15 Arg Asn Thr Gly Glu Thr Ser Gln Gln Ser Ser Thr Ala Pro Gln Pro 20 25 30 Arg Val Glu Asp Lys Gly Lys Glu Ile Ala Pro Val Gly Gly Gly Glu 35 40 45 Val Ala Arg Thr Ser Glu Leu Asp Arg Asp Ile Asn Thr Gly Thr Leu 50 55 60 Gly Thr Phe Gln Val Pro Arg Leu Arg Ala Ile Pro Thr Lys Ile Arg 65 70 75 80 Leu Pro Leu Val Lys Ser Gly Ala Ala Leu Asn Leu Asp His Leu Leu 85 90 95 Val Tyr Lys Pro Ser Gln Leu Asp Ile Thr Asn Ala Lys Ala Thr Arg 100 105 110 Ser Gln Phe Asn Gln Trp Tyr Glu Gly Val Lys Asn Ala Tyr Glu Val 115 120 125 Asp Asp Gln Gln Met Ser Ile Leu Met Asn Gly Leu Val Val Trp Cys 130 135 140 Ile Glu Asn Gly Thr Ser Pro Asn Ile Asn Gly Asp Trp Val Met Met 145 150 155 160 Asp Gly Asp Thr Gln Val Ser Tyr Pro Ile Lys Pro Leu Ile Asp Phe 165 170 175 Ala Ala Pro Thr Phe Arg Gln Ile Met Lys His Phe Ser Asp Val Ala 180 185 190 Glu Ala Tyr Ile Gln Met Arg Asn Ala Glu Gln Pro Tyr Met Pro Arg 195 200 205 Tyr Gly Leu Gln Arg Asn Leu Thr Asp Met Ser Leu Ala Arg Tyr Ala 210 215 220 Phe Asp Phe Tyr Glu Val Thr Ser Arg Thr Pro Ile Arg Ala Lys Glu 225 230 235 240 Ala Tyr Phe Gln Met Lys Ala Ala Ala Leu Thr Asn Thr His His Arg 245 250 255 Leu Phe Gly Leu Asp Gly Asn Val Ser Thr Thr Glu Glu Asn Thr Glu 260 265 270 270 Arg His Thr Ala Thr Asp Val Asp Arg Asn Ile His Thr Thr Leu Leu Gly 275 280 285 Met Arg Gly Ile His 290 293

SEQ ID NO: 2 Sequence length: 197 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: cDNA Origin: Organism: Sweet potato latent virus Sequence: TAGTGTGCGT ACCTACTTAT AAAGTTATTT ATATTTAAGT ATGTATGTTC GTCGAAGGGG 60 AATCTATTTG TGGAGCGTGG TGAGGGCCTC CCTCGAGCAA AACATATAGT GTGATCCTTT 120 CTATTATATT TCAAGAATTT TATAACATTA GAGAGGCACT GCCTCCAGTC ATAAACTTTT 180 TGTCTTATAC CAGAGAC 197

SEQ ID NO: 3 Sequence length: 1166 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: cDNA Origin: Organism: Sweet potato latent virus Sequence: GCG GAT GAC ACT ATC CTT GAT GCT GGG
AAG GTA GAT ACG AAA AGG AAC 48 CGC AAT ACA GGC GAG ACA TCA CAG CAA
TCC AGC ACA GCA CCA CAA CCC 96 AGA GTT GAG GAT AAG GGT AAG GAG ATA
GCC CCA GTT GGA GGA GGA GAA 144 GTT GCC CGC ACA TCC GAG CTT GAT AGG
GAC ATA AAC ACT GGT ACT CTT 192 GGA ACG TTC CAG GTA CCT CGA CTG CGT
GCT ATA CCC ACA AAA ATT CGC 240 TTA CCA CTA GTT AAG TCT GGA GCA GCG
TTG AAT CTC GAT CAT TTA CTG 288 GTG TAT AAG CCG TCG CAG CTG GAT ATT
ACG AAT GCT AAA GCC ACT AGG 336 AGT CAG TTC AAC CAA TGG TAC GAA GGT
GTG AAG AAT GCA TAC GAA GTG 384 GAT GAC CAG CAG ATG TCG ATC TTG ATG
AAT GGA CTT GTA GTG TGG TGT 432 ATA GAA AAT GGA ACA TCC CCA AAT ATC
AAT GGC GAT TGG GTG ATG ATG 480 GAT GGA GAC ACG CAA GTA TCA TAC CCG
ATA AAG CCC CTT ATA GAT TTT 528 GCG GCA CCA ACA TTC AGG CAG ATA ATG
AAG CAC TTT AGT GAT GTC GCA 576 GAA GCC TAC ATC CAA ATG CGC AAT GCA
GAA CAA CCG TAC ATG CCA AGG 624 TAT GGT TTA CAG CGA AAC TTA ACG GAT
ATG TCT TTG GCT CGT TAC GCG 672 TTT GAT TTC TAT GAA GTT ACA TCA CGC
ACG CCC ATC CGG GCC AAG GAA 720 GCA TAC TTC CAG ATG AAA GCT GCA GCG
CTC ACA AAT ACA CAT CAT CGG 768 CTG TTC GGT TTG GAT GGA AAT GTC TCT
ACC ACT GAA GAA AAC ACC GAG 816 CGG CAC ACT GCA ACG GAT GTG GAC CGG
AAC ATA CAC ACA CTA CTT GGA 864 ATG CGT GGC ATC CAT TAGTGTGCGT ACCTA
CTTAT AAAGTATTATT ATATTTAAGT 919 AGTTATGTTTC GTCGAAGGGGG AATCTATTTG TGG
AGCGTGG TGAGGGCCTC CCTCGAGCAA 979 AACATATAGT GTGATCCTTT CTATTATATTTCA
AGAATTTATAACATTA GAGAGGCACT 1039 GCCTCCAGTC ATAAACTTTTT TGTCTTATAC CAG
AGAC 1166

[Brief description of the drawings]

FIG. 1. c derived from RNA of latent sweet potato virus
BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the base sequence of DNA and the amino acid sequence which it codes.

FIG. 2 c derived from RNA of sweet potato latent virus
It is a figure which shows the base sequence of DNA.

FIG. 3 is a view showing the nucleotide sequence of cDNA derived from the 3 ′ terminal untranslated region of a sweet potato latent virus.

FIG. 4 is a view showing primers used for cloning.

──────────────────────────────────────────────────続 き Continued on the front page (56) Reference Ann. Phytopath. SoC. Japan, 57, p. 512-521, 1991 Phytopathology, 84, p. 65-69, 1993 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 GENBANK / EMBL / DDBJ / GENESEQ BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A nucleotide sequence encoding a coat protein of a sweet potato latent virus comprising the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or a gene comprising a nucleotide sequence complementary thereto. 2. The nucleotide sequence according to SEQ ID NO: 2 in the sequence listing,
Alternatively, a 3'-terminal untranslated region gene of a sweet potato latent virus having a nucleotide sequence complementary thereto. 3. The nucleotide sequence according to SEQ ID NO: 3 in the sequence listing,
Or a sweet potato submarine consisting of a complementary nucleotide sequence
Viral coat protein and 3'-terminal untranslated region
Gene.