JPH10313719A - Method for imparting potato y virus normal group resistance or immunity and potato y virus normal group resistant or immune potato - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the potato excellent in waste and safety provided with immunity by imparting resistance to a potato Y virus normal group by using a specified recombination vector and transforming the potato. SOLUTION: This potato is transformed by using the recombination vector provided with the leader sequence of the RNA 4 of a cucumber mosaic virus on the downstream of a promoter and a gene for coding the coating protein of the amino acid sequence or the like of an expression of a potato Y virus gangrene group and the resistance to the potato Y virus normal group is imparted. Also, the potato produced by the method is provided with the resistance to the potato Y virus normal group and excellent growth characteristics and efficiently controls a virus disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a potato Y virus normal strain (hereinafter referred to as PVY normal strain) for potatoes.
For imparting resistance or immunity to P and P
VY relates to potato resistant or immune resistant.

[0002]

2. Description of the Related Art PVY ordinary lines vary in varieties, but there are two types of potatoes: a leafy leaf symptom associated with chlorotic mottle (a leafy leaf type) and a leafy necrotic symptom (a leafy type). Distributed throughout the world and severely damaged. Damaged strains are 50% severe,
A 20% decrease in sales due to illness. In addition, PVY ordinary system,
Aphids (peach aphids, cotton aphids,
Non-persistent propagation due to tulip beetles and aphids. Among potato varieties, even when inoculated with the PVY normal line, there are varieties that show necrotic spots on the inoculated leaves in the current generation, but are not symptomatic in the next generation and infection is not observed. Breeding may result in potatoes resistant to PVY normal lines. However, it is expected that it will take more than 10 years to produce a resistant variety by this method. If potatoes can be given PVY normal line resistance by genetic recombination technology, it is very advantageous to obtain PVY normal line resistance potatoes in a short period of time.

[0003] Conventionally, as a method for imparting virus resistance to plants, a virus coat protein (hereinafter referred to as a virus coat protein) has been used.
A method of introducing a gene encoding "CP" into a plant is known (JP-A-62-2015).
No. 27, JP-A-62-285791, Plant Cell Engineering Vo
l. 4, No. 2 (1992) pp.101-109, Bio / Technology 8,1
27-134 (1990)). According to this method, the plant is conferred virus resistance, but the plant produces viral CP. When a plant produces a viral CP, amino acids and energy are expended for its production, and the growth of the plant is C
It is worse than those that do not produce P. In addition, when edible plants such as potatoes produce virus CP, changes in taste and safety to health due to CP must also be considered. Therefore, it would be very advantageous if plants were obtained that were resistant to the virus but did not produce CP.

[0004]

Accordingly, an object of the present invention is to provide a method for producing PVY-T without causing a plant to produce PVY-T CP.
It is an object of the present invention to provide a method for imparting resistance or immunity to a common Y line to a plant and a PVY normal line resistant or immunized potato which does not produce CP of PVY-T.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies and found that cucumber mosaic virus (hereinafter referred to as "CM
V) may be transformed with a recombinant vector in which a gene encoding the CP of PVY-T is inserted downstream of the leader sequence of RNA4.
The present inventors have found that a potato having resistance or immunity to a PVY common strain can be obtained without producing CP of PVY-T, and completed the present invention.

That is, the present invention comprises transforming potatoes using a recombinant vector having a leader sequence of RNA4 of cucumber mosaic virus downstream of a promoter and a gene encoding a PVY-T coat protein downstream of the promoter. , A method for imparting resistance or immunity to potatoes to a PVY common strain. Furthermore, the present invention provides a potato produced by the method of the present invention, which has resistance or immunity to a PVY common strain.

Hereinafter, the present invention will be described in detail. CMV RNA inserted into the recombinant vector used in the present invention
The leader sequence of No. 4 is shown in the literature, Nitta et al. (1988) Bulletin of the Japanese Society for Plant Pathology 54: 516-522. It is well known that the leader sequence of a structural protein of a plant virus generally enhances protein expression (DESleat and TMA Wilson, 1992, Plan
t Virus Genomes as Source of Novel functions for G
enetic Engineering, In Genetic Engineering with Pl
ant Viruses, TMA Wilson and JW Davies ed., CR
C Press, pp55-113).

[0008] The base sequence of the sequence used in the following examples is shown in SEQ ID NO: 1 in the sequence listing. R of this CMV
The leader sequence of NA4 is obtained by chemical synthesis or CMV
Can be easily prepared by cutting out from the DNA or amplifying by PCR. Downstream of the CMV RNA4 leader sequence, a gene encoding a PVY-T coat protein is inserted. The amino acid sequence of the coat protein of PVY-T and the nucleotide sequence of the gene encoding it are known, and the amino acid sequence and the nucleotide sequence of the gene used in the following Examples are shown in SEQ ID NO: 2 in the sequence listing. The gene encoding the coat protein can be easily prepared from PVY-T by a conventional method (such as a PCR method).

The gene encoding the leader sequence of RNA4 of CMV and the coat protein of PVY-T is as follows:
Inserted downstream of the promoter. The promoter may be any known promoter capable of effecting transcription in plant cells. Preferred examples include the cauliflower mosaic virus 35S promoter, but are not limited thereto.

The recombinant vector used in the present invention can be based on any vector capable of transforming potato and replicating in potato cells. The vector preferably has an origin of replication that functions in potato cells in addition to the above-mentioned promoter, and preferably has an appropriate selection marker such as a terminator and drug resistance. Such a vector is commercially available, and in the following examples, a plant expression plasmid vector pBI121 commercially available from Clonetech, USA
Is used as a base. FIG. 1 shows an example of the recombinant vector of the present invention produced in the following Examples, in which the leader sequence of RNA4 of CMV and the CP gene of PVY-T were incorporated into pBI121.

It is well-recognized by those skilled in the art that slight substitution, deletion or insertion of DNA generally does not substantially affect its function. Therefore, even if a slight substitution, deletion or insertion occurs in the leader sequence of RNA4 of CMV shown in SEQ ID NO: 1 or the CP or amino acid sequence of PVY-T shown in SEQ ID NO: 2, Potatoes that can be transformed by the methods described herein, thereby conferring potato resistance or immunity to PVY common lines without producing PVY-T CP, are described in this specification. Or "gene encoding a coat protein of potato Y virus nematode lineage" described in the claims of the present invention.

For example, regarding the CP of PVY-T, PVY-T isolated in the Netherlands (Reference: Van Der Vlugt et a)
l., (1989) J. Gen. Virology 70, 229-233) and PVY-T presented at Hokkaido University (literature: Kazuri Oshima et al. (19)
91) The Japanese Society for Plant Pathology 57, 615-622)
CP of PVY-T used in the following examples shown in SEQ ID NO: 2
Differs from the amino acid sequence by 2 or 3 amino acids,
It goes without saying that sequences encoding these sequences are also included in the "gene encoding a coat protein of the potato Y virus nematode lineage" according to the present invention.

[0013] The recombinant vector used in the present invention is obtained by inserting the leader sequence of RNA4 of CMV and the sequence encoding CP of PVY-T into the above-mentioned base vector by using restriction enzymes in a conventional manner. Can be easily prepared. An example of a specific preparation method is described in detail in the following Examples.

As a method for transforming a potato used in the present invention, first, Agrobacterium tumefaciens having a strong infectivity to a plant is transformed with the above-mentioned recombinant vector. Is preferably used to infect potatoes, but the method is not limited thereto. Methods for transformation of Agrobacterium tumefaciens are known per se, and include, for example, freeze-thaw method (Literature: G. An et al., (1988)
Binary Vectors, In Plant MolecularBiology Manual
A3, Kluwer Academic, Dordrecht pp.1-19).

The potato used in the present invention includes:
Although not particularly limited, Maquin, Sac
o and Desiree are preferred. When immunity is given, Desiree is particularly preferred. In the present invention, the term "resistance" refers to the case where, after artificial inoculation of the virus, virus growth was observed in the upper leaves from the inoculated leaves, but the amount of the virus was smaller than that in the non-transformant, or the symptom was reduced. In addition, among those showing such resistance, those showing no virus growth and no symptom even after artificial inoculation are regarded as immunity. As specifically shown in the examples below, transforming potatoes with the recombinant vector of the present invention allows P.
It has become possible to impart resistance or immunity to the VY normal line. Moreover, in the potatoes which have been given resistance or immunity by the method of the present invention, PVY
Transcription of the -T CP gene occurs, but no CP is produced. In addition, the form of the potato produced by the method of the present invention does not change at all in comparison with a normal potato. Hereinafter, the present invention will be described more specifically based on examples.
However, the present invention is not limited to the following examples.

[0016]

【Example】

(1) Isolation of CP gene of PVY-T A tobacco leaf was inoculated with a freeze-dried preparation of a leaf infected with PVY-T (Udagawa / Tomaru, 1972, Journal of the Botanical Society of Japan 38, 210), and allowed to grow. Purified virus was obtained from the systemically infected tobacco leaf ground juice by differential centrifugation. CP and RNA are dissociated by adding 1% final concentration of sodium dodecyl sulfate to the purified virus, and sucrose density gradient centrifugation (0-32.5
% Sucrose, 39000 rpm, 4 hours), PVY-T obtained by precipitating RNA by adding ethanol to a final concentration of 70% and centrifuging.
RNA was obtained. Primer 1 (Table 1) was added to the obtained RNA, and M-MLV reverse transcriptase (BRL, USA) was added.
Using the conditions described in the instructions attached to the product
DNA was prepared.

[0017] Primers 1 and 2 were added to the obtained cDNA.
After the addition of (Table 1), the reaction was performed at 93 ° C. for 1 hour using Taq DNA polymerase (manufactured by Takara Shuzo) in a buffer attached to the instruction sheet.
Cycle at 42 ° C for 1 minute, 72 ° C for 1 minute, 30 minutes
The polymerase chain reaction (PCR) was performed twice to amplify the CP gene. The amplified DNA was cleaved at both ends with the addition of restriction enzymes SstI and XbaI.
After 2% agarose electrophoresis, DEAE cellulose membrane NA45 (Schleicher & Schue, USA)
(manufactured by LL Corporation). PBluescript cleaved from the recovered DNA with restriction enzymes SstI and XbaI
ptII SK (+) (Stratagene, USA) to transform E. coli JM109. The plasmid (pPTCP) was extracted from the obtained colony,
Cleavage with restriction enzymes SstI and XbaI confirmed about 0.8 kb of the PVY-TCP gene.

(2) Determination of base sequence of CP of PVY-T Plasmid (pPTCP) was ligated with 0.4M NaOH (37
(5 ° C., 5 minutes), precipitated with ethanol and dried to dryness, and then primers (M4, RV, Takara Shuzo) with T7 DNA polymerase sequencing kit (Pharmacia)
Was used to determine the nucleotide sequence.

(3) Insertion of CMV RNA4 leader sequence Escherichia coli BW313 transformed with plasmid pPTCP
Was cultured in a liquid, and when the absorbance at 600 nm became 0.4, a helper phage M13K07 was added, and the mixture was cultured overnight in the presence of kanamycin (70 μg / ml). The culture was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was centrifuged. With polyethylene glycol 6000 and NaCl to a final concentration of 4
%, 0.5M, left on ice for 1 hour, and centrifuged at 15000 rpm for 15 minutes to precipitate phage. The recovered phage was TE (10 mM Tr
The resultant was suspended in is-HCl, 1 mM EDTA, pH 8.0), an equal volume of water-saturated phenol / chloroform was added, stirred for 5 minutes, and then centrifuged at 15000 rpm for 15 minutes to precipitate the supernatant with ethanol to obtain a template DNA.

Primer 3 (Table 1) was added to the template DNA, and a part of the leader sequence of CMV RNA4 was introduced using a site-specific recombination kit, Mutan-K (manufactured by Takara Shuzo Co., Ltd.). I got Further pPTC
P (CM) was again used as a template by the above method, and primer 4
The remaining leader sequence was introduced using (Table 1). The nucleotide sequence of the obtained plasmid was determined in the same manner as described above. The determined nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing. This indicates that CMV RNA4 is located upstream of the CP gene of PVY-T.
Plasmid pPTCP (CML) having a leader sequence of
Was obtained.

[0021]

[Table 1]

(4) Construction of Plant Transformation Vector The β-glucuronidase gene of the plant expression vector pBI121 (manufactured by Clonetech, USA) is replaced with the restriction enzyme XbaI.
And SstI, and 0.8% agarose electrophoresis was performed to recover a vector fragment. Recovered pBI121
Derived fragment and pba fully digested with XbaI and SstI
Ligation with PTCP (CML), E. coli JM
109 was transformed to form a colony on an LB plate containing 30 μg / ml kanamycin to obtain a plant transformation vector pBIPT (CML) (FIG. 1).

(5) Transformation of Agrobacterium tumefaciens Agrobacterium tumefaciens LBA440
4 (Hockman et al., Nature 303: 179-183, 1983)
It was transformed with the Y-TCP expression vector pBIPT (CML).

(6) Transformation of potato Agrobacterium tumefaciens LBA4404 (p.
BIPT (CML)) was used to transform potatoes. For transformation, Maquin, Saco (US variety) and Desiree were tested. These virus-free plants were converted to Linsmeier and Skoog (1).
965), aseptically propagated in a medium containing 30 g / l of sucrose and agar (0.8%), and the leaves of the germ-free plant were reduced to a size of 0.6 to 1.0 × 0.5 to 1.0 cm. In the case of a stem, it was cut into 0.5 to 2.0 cm (either with or without knots may be included). Agrobacterium tumefaciens LBA4404 (pBIPT (C
ML)) about 10 8 cells with Linsmeier and
Co-culture was performed at 25 ° C. for 48 hours in a liquid medium containing inorganic salts of Skoog and 30 g / l glucose.

The leaf and stem fragments were washed with sterilized water containing 250 mg / l of cefotaxime (2 to 3 times) to wash out the bacteria, and then placed on a regeneration medium shown in Tables 2 and 3. In the case of the regeneration medium (LSZK100) in Table 3,
About 20 days after the culture, kanamycin-resistant calli appeared from the implanted leaf and stem fragments. As a result of culturing these calli on the medium for 10 to 30 days, leaf stems showing kanamycin resistance appeared. In addition, the regenerated leaf stems were cut and grown on agar (0.8%) containing inorganic salts of Linsmeier and Skoog, 30 g / l sucrose, 250 mg / l cefotaxime and 100 mg / l kanamycin. In addition, the regeneration medium (K
In the case of S1), callus formation and regeneration are LSZK100
Tended to be slower than Table 4 shows the types and numbers of the transformants obtained by the above method.

[0026]

[Table 2]

[0027]

[Table 3]

[0028]

[Table 4]

(7) Assay for PVY normal line resistance of transformants Among the transformants prepared above, Maquin, Sac
For o and Desiree, PVY ordinary lines were artificially inoculated, and the degree of virus preservation was investigated by ELISA.
That is, tobacco BY which is an indicator plant of PVY ordinary line
No. 4 was inoculated with purified virus particles of PVY ordinary strain,
A week later, after confirming the vein vegetation symptom, the infected leaves were sampled, diluted with a PBS-T buffer (phosphate buffer 0.02M) 2 to 10 times the fresh weight, homogenized, and the ground solution thereof was obtained. Was inoculated into each transformant.
The transformants to be tested were picked up and then illuminated for 16 hours at day 23.
C., cultivated in an incubator prepared at 17.degree. C. at night, and artificially inoculated 2 to 3 weeks after raising the pot. The artificial inoculation is performed on the five leaves from the upper leaf to the middle leaf of the test plant.
This was performed by applying a mixture of a 600-mesh carborundum and a predetermined concentration of a virus trituration solution.

After inoculation, the leaves were sampled over time (one week to two months), and the amount of virus in the homogenized trituration solution was quantified by ELISA. ELISA was performed according to a conventional method. That is, an anti-PVY normal-system polyclonal antibody was used as a primary antibody, which was fixed in a well of a microtiter plate, and after blocking, a sample was added to the well and allowed to react. Then, anti-PVY labeled with alkaline phosphatase as a secondary antibody was used. After adding a normal-system polyclonal antibody, reacting and washing, disodium p-nitrophenylphosphate was added as a substrate, and the absorbance at 405 nm was measured. From the above results, as shown in Table 5, resistant and immune strains were obtained among the transformants. Here, a resistant strain is defined as one in which, after the artificial inoculation of the virus, virus growth was observed in the upper leaves from the inoculated leaves, but the virus amount was smaller than that in the non-transformant, or the symptom was reduced. An immune strain is defined as one in which no virus growth and no symptoms were observed after artificial inoculation. Therefore, as a result of this experiment, an immunized individual without onset or preservation was obtained.

[0031]

[Table 5]

As a result of using a polyclonal antibody of the PVY ordinary strain and examining the amount of CP produced by ELISA, no coat protein was detected in any of the resistant or immune transformants of Maquin, Toyoshiro and Saco (ELISA). Is about 10 ng for the coat protein detection. In addition, as a result of investigating the degree of poisoning of the diseased variety, in the case of Maquin, 5 to 10 μg per 1 g of fresh weight was used.
Virus particles were detected.

(8) Confirmation of CP gene by PCR The transformant of Desiree selected in (7) is PVY
For D1 and D10 transformants of Desiree showing immunity to common lines, CP by PCR analysis
The gene was confirmed. First, the method of Dellaporta (Dellaporta, SL et al. Plant Mol. Biol.
p. 1,19- (1983)), total DNA was extracted from the leaves of these two individuals. In addition, PCR analysis was performed by using a primer-1 and a primer-2 shown in Table 1 according to a conventional method. As a result, one band was observed at about 0.8 kb for D1 and D10. This is the control plasmid (pBI
PT (CML)) is the same as the size of the CP gene. FIG. 2 shows the result. Further, such a band was not observed in the non-transformed transformants used as the control, ie, Desiree and the transformant potato into which only the GUS gene was introduced. This result indicates that the CP gene is present in these two individuals.

(9) Morphology of the transformant The result obtained by cultivating the transformant obtained in (7) and its parent variety (non-transformant) in a closed greenhouse prepared at 23 ° C in the day and 17 ° C at night. For all of the above resistant or immune transformants, the morphology and growth characteristics are not different from the parent varieties,
Tuber shape was normal as well as the parent variety.

[0035]

As described in detail above, according to the present invention,
Resistance to PVY normal strains without producing PVY-T CP (virus growth was observed but virus load was less than non-transformants or those with reduced symptoms), or For the first time, potatoes with immunity (ie, neither infectious nor poisoned when inoculated with the PVY common strain) were produced. INDUSTRIAL APPLICABILITY The present invention is effective for controlling a potato virus disease involving the PVY common strain.

[0036]

[Sequence list] SEQ ID NO: 1 Sequence length: 85 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Cucumber mosaic virus coat protein leader sequence Origin: Cucumber mosaic virus sequence TCTAGAGTTA TTGTCTACTG ACTATATAGA GAGTGTGTGT GTGCTGTGTT TTCTCTTTTG 60 TGTCGTAGAA TTGAGTCGAG TCATG 85

SEQ ID NO: 2 Sequence length: 801 Sequence type: number of nucleic acids and protein chains: single strand Topology: linear Sequence type: coat protein gene Origin: PVY-T sequence GGA AAT GAC ACA ATC GAT GCA GGA GGA AGC ACT AAG AAA GAT GTA AAA 48 Gly Asn Asp Thr Ile Asp Ala Gly Gly Ser Thr Lys Lys Asp Val Lys 1 5 10 15 CAA GAG CAA GGT AGC ATT CAA CCA AAT CTC AAC AAG GAA AAG GAA AAG 96 Gln Glu Gln Gly Ser Ile Gln Pro Asn Leu Asn Lys Glu Lys Glu Lys 20 25 30 GAC TTG AAT GTT GGA ACA TCT GGA ACT CAC ACT GTG CCA CGA ATT AAA 144 Asp Leu Asn Val Gly Thr Ser Gly Thr His Thr Val Pro Arg Ile Lys 35 40 45 GCT ATC ACG TCC AAA ATG AGA ATG CCC AAG AGT AAG GGT GCA ACT GTA 192 Ala Ile Thr Ser Lys Met Arg Met Pro Lys Ser Lys Gly Ala Thr Val 50 55 60 CTA AAT TTG GAA CAC TTA CTC GAG TAT GCT CCA CAG CAA ATT GAC ATC 240 Leu Asn Leu Glu His Leu Leu Glu Tyr Ala Pro Gln Gln Ile Asp Ile 65 70 75 80 TCA AAT ACT CGA GCA ACT CAA TCA CAG TTT GAT ACA TGG TAT G AA GCA 288 Ser Asn Thr Arg Ala Thr Gln Ser Gln Phe Asp Thr Trp Tyr Glu Ala 85 90 95 GTA CAA CTT GCA TAC AAC ATA GGA GAA ACT GAA ATG CCA ACT GTG ATG 336 Val Gln Leu Ala Tyr Asn Ile Gly Glu Thr Glu Met Pro Thr Val Met 100 105 110 AAT GGG CTT ATG GTT TGG TGC ATT GAA AAT GGA ACC TCG CCA AAT ATC 384 Asn Gly Leu Met Val Trp Cys Ile Glu Asn Gly Thr Ser Pro Asn Ile 115 120 125 AAT GGA GTT TGG GTT ATG ATG GAT GGA GAT GAA CAA GTC GAA TAC CCA 432 Asn Gly Val Trp Val Met Met Asp Gly Asp Glu Gln Val Glu Tyr Pro 130 135 140 CTG AAA CCA ATC GTT GAG AAT GCA AAA CCA ACA CTT AGG CAA ATC ATG 480 Leu Lys Pro Ile Val Glu Asn Ala Lys Pro Thr Leu Arg Gln Ile Met 145 150 155 160 GCA CAT TTC TCA GAT GTT GCA GAA GCG TAT ATA GAA ATG CGC AAC AAG 528 Ala His Phe Ser Asp Val Ala Glu Ala Tyr Ile Glu Met Arg Asn Lys 165 170 175 AAG GAA CCA TAT ATG CCA CGA TAT GGT TTA GTT CGT AAT CTG CGC GAT 576 Lys Glu Pro Tyr Met Pro Arg Tyr Gly Leu Val Arg Asn Leu Arg Asp 180 185 190 GGA AGT TTG GCT CGC TAT GCT TTT GAC TTT TAT GAA GTT ACA TCA CGG 624 Gly Ser Leu Ala Arg Tyr Ala Phe Asp Phe Tyr Glu Val Thr Ser Arg 195 200 205 ACA CCA GTG AGG GCT AGA GAG GCA CAC ATT CAA ATG AAG GCC GCA GCT 672 Thr Pro Val Arg Ala Arg Glu Ala His Ile Gln Met Lys Ala Ala Ala 210 215 220 TTA AAA TCA GCT CAA TCT CGA CTT TTC GGA TTG GAT GGT GGC ATT AGT 720 Leu Lys Ser Ala Gln Ser Arg Leu Phe Gly Leu Asp Gly Gly Ile Ser 225 230 235 240 ACA CAA GAG GAA AAC ACA GAG AGG CAC ACC ACC GAG GAT GTT TCT CCA 768 Thr Gln Glu Glu Asn Thr Glu Arg His Thr Thr Glu Asp Val Ser Pro 245 250 255 AGT ATG CAT ACT CTA CTT GGA GTG AAG AAC ATG 801 Ser Met His Thr Leu Leu Gly Val Lys Asn Met 260 265

SEQ ID NO: 3 Sequence length: 895 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Transgene Origin: CMV coat protein gene leader sequence and PV
YT coat protein gene sequence TCTAGAGTTTA TTGTCTACTG ACTA TATAGA GAG
TGTGTGT GTGCTGTGTT TTCTCTTTTTG 60 TGTCGTAGAA TTGAGTCGAG TCATGGGAAA TGA
CACAATC GATGCAGGAG GAAGCACTAA 120 GAAAGATGTA AAACAAGAGGC AAGGGTAGCAT TCA
ACCAAAT CTCAACAAGG AAAAGGAAAA 180 GGACTTTGAAT GTTGGAACAT CTGGAACTCA CAC
TGTGCCA CGAATTAAAG CTATCACGTC 240 CAAAATGAGA ATGCCCAAGA GTAAGGGTGC AAC
TGTACTA AATTTGGAAC ACTTACTCGA 300 GTATGCTCCA CAGCAAATTG ACATCTCAAA TAC
TCGAGCA ACTCAATCAC AGTTTGATAC 360 ATGGTATGAA GCAGTACAAC TTGCATACAAAA CAT
AGGAGAA ACTGAAATGC CAACTGTGAT 420 GAATGGGCTT ATGGTTTGGT GCATTGAAAAA TGG
AACCCTCG CCAAATATCA ATGGAGTTTTG 480 GGTTATGAT GATGGAGATG AACAAAGTCGA ATA
CCCACTG AAACCAATCG TTGAGAATGC 540 AAAACCAACA CTTAGGCAAAA TCATGGCACA TTT
CTCAGAT GTTGCAGAAG CGTATATAGA 600 AATGCCGCAAC AAGAAGGAAC CATATATGCC ACG
ATATGGT TTAGTTCGTA ATCTGCGCGA 660 TGGAAGTTTG GCTCGCTATAG CTTTTGACTTTA
TGAAGTT ACATCACGGA CACCAGTGAG 720 GGCTAGAGAG GCACACATTC AAATGAAGGC CGC
AGCTTTA AAATCAGCTC AATCTCGACT 780 TTTCGGATGTG GATGGTGGCA TTAGTACCACA AGA
GGAAAAC ACAGAGAGGC ACACCACCGA 840 GGAGTTTCT CCAAGTATGC ATACTCTACT TGG
AGTGAAG AACATGTGAG AGCTC 895

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing pBIPT (CML).

FIG. 2 is a photograph showing confirmation of a CP gene by PCR.

Claims (4)

[Claims] 1. A potato is transformed using a recombinant vector having a cucumber mosaic virus RNA4 leader sequence downstream of a promoter and a gene encoding a coat protein of a potato Y virus nematode line downstream thereof. For imparting resistance to common potato virus Y strains. 2. A method for imparting immunity to a potato Y virus common strain wherein the potato is Desiree. 3. A potato produced by the method according to claim 1, which has resistance to a common strain of potato Y virus. 4. A potato having immunity against a common strain of potato Y virus produced by the method of claim 2.