JP2000312540A - Plant resistant to beet rizomania - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an improved transformed plant which has good resistance to Beet Necrotic Yellow Vein Virus(BNYVV), by transforming the genome of the plant with a gene originated from the genome of BNYVV or the like in a state expressible in the plant genome. SOLUTION: This transformant is obtained by transforming the genome of a plant such as beet or benthamiana with a DNA corresponding to a gene originated from BNYVV genome or to a part of the gene or the substantially same DNA, preferably a DNA originated from a 4.8 kb RNA (RNA-two molecules) among the genome RNA of the BNYVV. The DNA originated from the RNA-two molecules is preferably a DNA originated from the terminal end side region of the RNA-two molecules. A vector structure which is used for transforming a plant, expressibly contains a DNA originated from a part of a nucleotide sequence of formula I or the substantially same DNA as the DNA, and can impart virus resistance to the BNYVV is prepared. A vector structure pM 27 which contains a DNA of the sequence of formula II is preferably prepared.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to resistance to beet necrotic yellow vein virus (hereinafter referred to as "BNYVV"), which is a virus causing root rot of sugar beet. Related to creating plants. More specifically, it relates to creating BNYVV-resistant plants by transforming plant cells with BNYVV-derived DNA. More specifically, the present invention relates to the production of BNYVV-resistant plants by transforming plant cells with DNA derived from BNYVV RNA-2 molecule. The invention further relates to BN
The present invention relates to creating a BNYVV-resistant plant that can stably transmit YVV-resistant traits to progeny.

[0002]

BACKGROUND OF THE INVENTION The root disease of sugar beet is a viral disease caused by the beet necrotic yellow vein virus (BNYVV). BNYVV can survive in the soil for a long time by lurking in the dormant spores of Polymyxa betae (Polymyxa betae, hereinafter referred to as “Polymixa bacterium”), a kind of fungus. The dormant spores of Polymyxa germinate when the sugar beet is cultivated, producing zoospores. If BNYVV is contained in these zoospores, the virus is carried along with the movement, and BNYVV infects the fine roots of sugar beet, leading to onset.

[0003] Since BNYVV lurks in dormant spores of Polymyxobacteria, it is extremely difficult to completely remove the virus from soil once contaminated with BNYVV.
Known as a typical soil disease. When this virus disease occurs, the sugar content in the roots is remarkably reduced. Therefore, it is regarded as one of the most important diseases in sugar beet cultivation areas around the world including Japan and Europe. In Hokkaido, where sugar beet cultivation is thriving, about 20% of the fields in Hokkaido have been infected with root rot of sugar beet, and there is a concern about the spread of pollution.

[0004] Conventional measures for controlling root rot of sugar beet have been to detect the occurrence of root rot of sugar beet at an early stage, and to suppress the BNYVV infection by improving the cultivation environment and suppressing the activity of polymyxobacteria. Was to do. For example, preventing the spread of polymyxin bacteria from contaminated soil, controlling the growth of polymyxin bacteria possessing BNYVV by adjusting pH, improving soil by improving drainage facilities, sterilizing contaminated soil by chemical treatment and heat treatment, etc. I went. However, none of the methods completely eliminate BNYVV or polymyxobacteria, so the possibility of recurrence was high, and the economic loss due to repeated similar treatment was excessive.

[0005] On the other hand, breeding has been carried out by selecting varieties resistant to root rot of sugar beet, and attempts have been made to introduce varieties resistant to BNYVV. However, none of the varieties had true resistance to BNYVV, so they could not be cultivated in highly diseased fields.

In recent years, in breeding for imparting virus resistance to plants, a breeding method using a genetic recombination technique (hereinafter referred to as “breeding method”) has been proposed.
"Molecular breeding method") (Cell Engineering Separate Volume, Plant Cell Engineering Series 8, 1997, Shujunsha). In conventional breeding methods, crossing with genetically distant varieties in order to introduce superior traits to certain varieties often resulted in sterile individuals, making breeding difficult. Therefore, in the molecular breeding method, not only genes derived from plants but also viruses, bacteria, yeasts, genes derived from animals, etc.
It is characterized by the ability to introduce excellent traits. Similarly, if sugarcane can be genetically introduced with disease resistance, it will not be necessary to take a less reliable method of improving the environment, and it is attracting attention as an innovative method.

[0007] Molecular breeding methods for imparting virus resistance by introducing a viral gene include, for example, a method of introducing a viral coat protein (CP) gene, a method of using a genomic gene of an attenuated virus strain, and a satellite.
Methods using RNA, methods using antisense RNA, methods using viral replication enzymes, methods using ribosome-inactivating proteins, methods using S-adenosylhomocysteine hydroxylase, methods using monoclonal antibodies specific for viruses A method using 2'5 'oligoadenylate synthase and ribonuclease L, a method using a double-stranded RNA degrading enzyme, and the like have been conventionally known. In the present invention, BNYV
By transformation with any of the genes of V, B
Consideration was given to providing resistance to NYVV.

[0008] BNYVV is a virus consisting of a coat protein (CP) and five types of single-stranded RNA molecules, and these single-stranded RNA molecules are RNA-1 (7.1 kN) according to their size.
b), RNA-2 (4.8kb), RNA-3 (1.8kb), RNA-4 (1.5k
b) and RNA-5 (1.4 kb). To transform a plant cell with the BNYVV gene, first,
It is necessary to analyze the structure and function of these genomic RNA genes possessed by V. The present inventors have published in a previously published paper the detailed structure of the genomic RNA molecule of BNYVV (M. Saito, et al., Arch. Vi.
rol., 1996, 141, 2163-2175).

[0009]

DISCLOSURE OF THE INVENTION The present invention relates to an RN other than the CP protein gene and the read-through protein gene.
The present invention relates to breeding of sugar beet by genetic engineering techniques using DNA derived from the A-2 molecule or DNA substantially identical thereto. More specifically, the present invention searches for a DNA derived from an RNA-2 molecule other than the CP protein gene and the read-through protein gene, which may function as a resistance among the genes possessed by BNYVV, and this DNA Another object of the present invention is to breed sugar beet using genetically identical DNA by using gene recombination technology, and to produce a sugar beet root disease resistant crop.

[0010]

Among the conventionally known molecular breeding methods, many methods for imparting virus resistance by transforming with a virus CP protein gene have been reported. Therefore, the present inventors conducted various studies on the beet necrotic vein yellowing virus (BNYVV) genome, and found that the CP protein gene was present in the 5 ′ terminal region in the RNA-2 molecule. (M. Saito, et a
l., Arch. Virol., 1996, 141, 2163-2175). And
We attempted to confer virus resistance to BNYVV by introducing the CP protein gene present on the RNA-2 molecule into plants. However, plants transformed with the CP protein gene were unable to confer virus resistance.

[0011] Further, based on detailed structural analysis of the RNA-2 molecule, DNAs corresponding to various regions in the RNA-2 molecule are further analyzed.
A plurality of types of plants transformed with were prepared. as a result,
BNYVV plant transformed with 3 'terminal region in RNA-2 molecule
The inventors have found that they have virus resistance and completed the present invention.

[0012] Accordingly, the present invention provides a plant derived from beet necrotic vein yellowing virus (BNYVV) by transforming a DNA corresponding to a gene derived from the genome or a partial sequence thereof into a plant genome so as to express the gene. A transformed plant cell imparted with resistance, and a transformed plant having resistance to BNYVV.

In the present invention, the gene derived from the BNYVV genome is preferably 4.8 kb of the genomic RNA of BNYVV.
DNA derived from an RNA molecule (RNA-2 molecule). In the present invention, DNA derived from an RNA-2 molecule is preferably an RNA-2 molecule.
DNA derived from the 3'-terminal region or DNA substantially identical thereto
And more preferably the DNA of the sense strand.

[0014] The term "DNA derived from RNA-2 molecule" used herein refers to a DNA having any one gene or a partial sequence thereof present on the RNA-2 molecule. The DNA substantially identical to the DNA derived from the RNA-2 molecule refers to a substitution, deletion, insertion, or degeneracy or a combination thereof of any one of the genes or a partial sequence thereof present on the RNA-2 molecule. DNA modified by
Say. The RNA-2 molecule used in the present invention or a DNA substantially identical thereto may be any as long as it can introduce BNYVV virus resistance into plants. For example, in the DNA substantially identical to the DNA derived from the RNA-2 molecule used in the present invention, the hydrophilic region of the protein encoded by the gene present on the RNA-2 molecule or a part thereof has hydrophobic amino acids. DN modified nucleic acid sequence to be replaced by
Including A. Generally, the hydrophilic region of a protein is located outside in the higher-order structure of the protein, and is considered to play an important role in processes such as virus infection and transmission.
For this reason, it is considered that by substituting the hydrophilic region with a hydrophobic amino acid, virus infection / transmission can be inhibited. When the protein of interest contains two or more hydrophilic regions, only one of the hydrophilic regions may be modified to be hydrophobic, or two or more hydrophilic regions are simultaneously modified to hydrophobic amino acids. You may.

The 3′-terminal region of the RNA-2 molecule is RNA
-2 means the region excluding CP protein gene,
More preferably, 42k protein, 13k of RNA-2 molecules
Regions encoding proteins, 15K protein and 14K protein (e.g., nucleotides 2130 to 44 of SEQ ID NO: 1)
Region consisting of the 20th base), most preferably RNA-
Regions encoding 13k protein and 15k protein of the two molecules (for example, nucleotides 3284 to 4 of SEQ ID NO: 1)
034 base).

Hereinafter, the present invention will be described in detail. BNYVV genomic RNA-2 molecule It is known that BNYVV, which is the causative virus of radish root disease, has a plurality of virus strains due to the diversity of its RNA molecules. The RNA-2 molecule used in the present invention may be isolated from any virus strain including BNYVV. For example, a virus strain derived from a field isolate can be used, but is not limited thereto.

To prepare a virus solution, BNYVV
Infects plant cells or plants and proliferates. Examples of plant cells or plants that can be used to propagate the virus in this case are cells or plants derived from sugar beet, trunna, benthamiana, macrocarpa, quinoa, and the like.

In order to recover RNA-2 molecules from the propagated virus, RNA is isolated by the SDS-phenol method, and then, for example, by electrophoresis on an agarose gel or polyacrylamide gel, a fraction having a molecular weight of about 4.8 kb. This can be done by separating the minutes. Specific examples of RNA-2 molecule isolation and analysis results are described in SEQ ID NO: 1 and Example 1.

In order to substitute the hydrophilic region of the protein encoded by the RNA-2 molecule with a hydrophobic amino acid, the nucleic acid sequence of the cloned DNA is subjected to site-directed mutagenesis to introduce the hydrophilic region. Can be carried out by introducing a desired mutation into a site corresponding to the above. It is easy for those skilled in the art to estimate the amino acid sequence from the obtained nucleic acid sequence and calculate the hydrophilic region from the estimated amino acid sequence at a location corresponding to the hydrophilic region of the protein. Can be. Examples of hydrophobic amino acids include, for example, Ala, Phe, Trp, Ile, Leu, Pro, Met
Any of hydrophobic amino acids such as When the amino acid is encoded by a plurality of codons, any of the plurality of codons may be used for each amino acid. Examples of the protein having a mutation produced in this manner include the mutant protein (ORF4M, SEQ ID NO: 4) described herein.

Construction of transformation vector and production of transformant
To give viral resistance to BNYVV to manufacturing beet, DNA from the RNA-2 molecules are introduced into a plant transformation plasmid. The plasmid to be used desirably has a promoter sequence operable in transformed plant cells in the upstream portion of the DNA to be introduced, and has a terminator sequence in the downstream portion. As such a plant transformation plasmid, pBI121 or the like can be used.

A plant cell or plant is transformed using the produced transformation vector. In order to transform plant cells, any of electroporation, Agrobacterium, microinjection, polycation, and other methods may be used. In order to culture the cells used for the transformation and the transformed cells, for example, MS medium supplemented with BAP (0.25 mg / L), glucose (9%), agarose (0.6-1.2%), or PPo medium Can be used. The transformed plant cells can be redifferentiated into plants by a method such as agarose bead method and nurse culture method.

A transformed plant can be obtained by redifferentiating transformed plant cells or directly transforming a plant. In order to transform a plant, any of the methods such as the Agrobacterium method and the particle gun method may be used. Even if the transformed plant is grown as it is,
Once the callus is dedifferentiated, the plant may be differentiated again.

By using these methods, BNYVV
A transformed plant having resistance to E. coli can be produced. The transformed plants thus prepared are selected based on the virus resistance to BNYVV as an index.

Hereinafter, examples of the present invention will be described. However, these examples do not limit the scope of the present invention, but serve to explain the present invention more specifically.

[0025]

EXAMPLES Example 1: Sequence and structure of RNA molecule of BNYVV
Field isolates and virus isolates selected by sap inoculation were used as putative and putative functional virus isolates. A local lesion formed on a virus-infected trunchy leaf was ground in a borate buffer, and the precipitate obtained by ultracentrifugation was dissolved in distilled water to obtain a virus solution. The virus solution is further processed by the SDS-phenol method to prepare RNA, and further fractionated by an oligo (dT) cellulose column.

Using a commercially available kit (cDNA synthesis system, Amersham) for the RNA thus obtained,
Gubler and Hoffman's method (Gubler, U., and Hoffman,
BJ, Gene, 1983, 25, 263-269), or
CDNA is synthesized by the RT-PCR method. Gubler and Hoffma
To amplify using the method of n, a 2C primer (5'-CTACCTTCACTCGACAT-3 '(SEQ ID NO: 5)) was used in addition to the oligo dT primer and the random primer. In order to amplify using the RT-PCR method, 2A primer (5′-TTTAAATTCTAACTATTATC-3 ′) is used as an upstream primer.
(SEQ ID NO: 6)) and 2B primer as a downstream primer (5′-ACCCCGTTCCATTTATACCC-3 ′ (SEQ ID NO: 7))
It was used. The amplification product was treated with any of the restriction enzymes DraI / SpeI, SphI or DraI / SphI and cloned into pUC119 treated with the same restriction enzymes.

Next, the target sequence was amplified by performing PCR on the cDNA using specific primers. The conditions of PCR can be optimized by appropriately changing the conditions by those skilled in the art. For example, Taq polymerase (2.5 U
nit / 100μl) upstream and downstream primers (each
0.4 μM), magnesium chloride (1.5 mM), dNTP (0.2 m
In 100 μl of a buffer solution containing M), 35 cycles were performed, with one cycle consisting of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. P
The CR product was cloned into pUC119.

It was then cloned into a plasmid.
RNA? ? The molecules were sequenced using a DNA sequencer according to standard procedures. For the sequenced results,
The position of the putative open reading frame (ORF), the putative amino acid sequence encoded by the ORF, and its characteristics were analyzed using commercially available software.

As a result of sequencing, it was found that the RNA-2 molecule was composed of 4609 bases and had six ORFs (ORF1 to ORF6). BNYVV RNA? ? The nucleotide sequence of cDNA produced from the molecule is described in SEQ ID NO: 1. As a result of detailed analysis of this sequence, ORF1 encodes a coat protein (CP) (21 kDa), and ATP is located almost at the center of the ORF3 protein (42 kDa).
The / GTP binding region and the GXXGXGKS / T helicase motif were present, and the ORF6 protein (14 kDa) was found to be rich in cysteine and had a Zinc finger motif.

Example 2 Production of Transformants Sugar beet is a self-incompatible multiplying crop.
There is a drawback that one generation is long because it is a grade, and it is difficult to use it as a test plant. Therefore, in the present invention, BN
The test was conducted using Nicotiana benthamiana, an experimental plant that is sensitive to YVV and easy to handle.

As a plant transformation vector used for the transformation of the present invention, T-
Plant expression vector pBI12 containing left and right border sequences at both ends of DNA
1 was used. The GUS gene was removed by cutting pBI121 with restriction enzymes BamHI and SacI, and the cDNA of the gene used for transformation was inserted into this BamHI / SacI site. That is, the gene amplified by PCR is treated with BamHI and SacI.
A BamHI / SacI gene fragment was prepared, and the gene fragment was ligated to a BamHI / SacI site of an expression vector. As a result, a recombinant transformation vector containing eight nonstructural protein genes amplified by PCR from a viral RNA cDNA clone was constructed.

In this example, cDNA derived from an RNA-2 molecule other than the CP protein gene and the read-through protein gene was used for transformation for imparting virus resistance to BNYVV. That is, a transformation vector (pM23) containing the full-length ORF3, and a transformation vector (pM2) containing the full-length ORF3 (ORF3M) into which the frameshift mutation was introduced.
4), transformation vector containing the full length ORF4 (pM25), ORF5
Full length transformation vector (pM26), ORF4 full length and ORF5
A transformation vector containing the full length (pM27, SEQ ID NO: 2),
ORF4 (ORF4M) with hydrophilic region replaced with hydrophobic amino acid
Transformation vector containing the full length and ORF5 (pM28, SEQ ID
NO: 3), a transformation vector containing the full length ORF6 (pM21),
And a transformation vector (pM22) containing the full length antisense ORF6 (ORF6M).

The ORF4M molecule herein refers to a 13K protein present on the RNA-2 molecule, which is a hydrophilic region consisting of the sequence of Gly Gly Lys Tyr Arg Asp Gly Thr corresponding to amino acids 51-58, which is composed of Ala Ala Lys Phe Arg Ala A nucleic acid sequence coding for one substituted with a hydrophobic region consisting of ALa Ala. Such a nucleic acid sequence is, for example, SEQ ID NO: 3
There is a sequence described in. In the present invention, Ala or Phe was used as the hydrophobic amino acid, but is not limited thereto. Also, there are four types of codons that encode Ala, Ph
There are two types of codons encoding e, and any of these codons may be used to define the amino acid sequence.

The transformed plant is 0.25% in a culture bottle.
The cells were cultured on MS medium containing gellite. 70% in this medium
Seeds of N. benthamiana sterilized with ethanol and antiformin with an effective chlorine concentration of 1.5% were seeded and placed in a sunlight incubator (23
After culturing for about 1 week at 16 ° C. and illumination for 16 hours, the medium was subcultured and cultured for about 5 weeks to grow a sterile plant.

Agrobacterium containing a cDNA fragment prepared from genomic RNA of BNYVV was isolated from N. cerevisiae by the leaf disk method.
After infecting a leaf piece (5 to 10 mm square) of a sterile benthamiana plant, introducing the BNYVV gene, the transgenic plant was re-differentiated.

DNA is extracted from the leaves of the regenerated plant by the SDS-phenol method, and it is confirmed that the target gene fragment has been introduced into the plant. For plant-derived DNA,
Using the specific primers used for cloning, the viral gene was amplified by PCR, and the presence or absence of a band was examined by agarose gel electrophoresis. As a result, transformants containing the above-mentioned eight nonstructural protein genes could be produced.

Since the transformation vector used to transform N. benthamiana has a kanamycin resistance gene, it is considered that the target gene has been introduced if the plant is kanamycin resistant. Therefore, on an MS medium (containing 0.25% gellite) containing kanamycin (200 μg / ml), sterilized N. benthamia as described above.
Transformants were selected by sowing and culturing na seeds.

Example 3 Assay and Evaluation of Transformed Plant Inbred Next Generation (R1) of the Transformant Produced in Example 2
Generation) was assayed for the degree of virus resistance.

As a transformant, a kanamycin-resistant individual germinated and grown in a kanamycin-containing medium as described in Example 2 was used for the inoculation test. As a control non-transformant, a sterile plant similarly cultured in a kanamycin-free MS medium (containing 0.25% gellite) was used.

The inoculation of the virus and the cultivation of the plants were carried out in a sunlight incubator. Quartz sand (20-30 mesh) was placed in a 30 ml syringe or test tube with a discharge tube, and aseptically cultured tobacco seedlings (about 6 weeks after sowing) were transplanted. A culture solution (improved solution of Hoagland &Arnon's solution, pH 7.0) was perfused daily. The virus was partially purified from trunca-inoculated leaves inoculated with BNYVV N-type lines (containing RNA-1 to RNA-4) and OD26
The adjusted leaves were adjusted to 0 = 0.3, and the developed leaves of the transformant and the non-transformant (both about 7 weeks after seeding) were inoculated with juice. 10 after inoculation
The upper leaves were collected every day, and the presence or absence of virus infection was examined by ELISA.

Eight kinds of transformed N. bent
Among hamiana, in plants into which pM27 and pM28 had been introduced, there was a tendency for some strains to delay the transfer / proliferation of the virus as compared to non-transformants (Table 1).

[0042]

Table 1: Virus detection frequency after virus inoculation Plasmid 10 days after inoculation 20 days after inoculation 30 days after inoculation pM21 70.0% 94.0% 100.0% pM22 36.1% 75.0% 88.9% pM23 55.6% 94.4% 94.4% pM24 33.3 % 80.0% 100.0% pM25 33.3% 91.7% 100.0% pM26 9.1% 68.6% 72.7% pM27 10.0% 23.3% 40.0% pM28 16.1% 16.1% 25.8% Non-converted plant 0.0% 80.0% 100.0% Plants introduced with pM27 and pM28 Was further subjected to an inoculation test. As a result, no virus was detected from the upper leaves of individuals in some of the transformants into which pM27 and pM28 had been introduced (Table 2, plant numbers 1 and 2 of pM27). Migration was slow (Table 2, plant numbers 1-3 of pM28).

[0043]

Table 2: Frequency of virus detection in plants transformed with pM27 and pM28 Plasmid Plant number 10 days after inoculation 20 days after inoculation 30 days after inoculation pM27 1 1/12 2/12 2/12 2 0/12 3/12 3/12 pM28 1 1/12 4/12 7/12 2 0/12 1/12 4/12 3 4/12 6/12 7/12 Non-convertible 3 6/12 12/12 12/12 * The virus infected 12 individual plants. The DNA cloned into pM27 and pM28 is a DNA having the sequence of the 3 ′ terminal region of the RNA-2 molecule, and the detailed sequences are described in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

E. coli NM52 transformed with pM27
The two strains were deposited on April 15, 1999 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit No .: FERM P-1736).
9).

[0045]

The transformation vector pM27 provided by the present invention and
By using pM28, a plant cell or plant can be transformed to produce a plant cell or plant having resistance to BNYVV.

[0046]

[Sequence List] SEQUENCE LISTING <110> Hokkaido Hokkaido Sugar Beat Co. <120> Rhizomania disease resistant plant. <130> 980375 <160> 7 <210> 1 <211> 4609 <212> DNA <213> Beet necrotic yellow vein virus <220> <223> Genome <300> <301> M. Saito, et al. <302> Complete nucleotide sequence of the Japanese isolate S of beet ne crotic yellow vein virus RNA and comparison with Europian isolates. <303> Archieves of Virology <304> 141 <306> 2163-2175 <308> D84411 <309> April 12, 1996 <400> gaccgatcga tgggcccgtg tttcggacgt cgtgagtgtt 240 attaaacaat cgcatgctat ggacttgtcc aaggctgcga atctatctat aattaaaact 300 gctttggcag gattgggctc gggttggact gacaataatc cttttgtgtc cctgactt gactt gactt gactt gctact gc tactcgt gact gactt gact gact gactt taaggta agtactttaa ctgattcagg gttagcagat 480 aacgcatctg ctaatgtgcg tagagatgtg gtgtccggaa ataaagccga atcatccggt 540 aaaactgctg gcactaatga gaattctgct tatacgctta ccgttagtct tgctggtttg 600 gctcaagctc ttaggcttga ggaattaatg tggactcggg ataagtttga ggaccggttg 660 aaattaccat ggacacctgt tcaaggtaga accagtccac ccggacaata gcaattagct 720 gctgctcggg tgacggcaca cattcgagcg gcgaagcggg cactattata tcctggtgat 780 agtcccgagt gggttggttg gaaacatttc tatcctcctc caccatatga tgtgtacgat 840 gtgccaccgc tggatattat taacgccaaa ttggctgctg atgatatcgg cggtcttgtt 900 actcctacac cggcatcctc acatggtctt ccttttgaag tttccgagga agttgagcag 960 gcaaatagga atagtctatg gctaacagtt ggactgctat tagctgcttt ggcagttggg 1020 attggtgtag ctgcttatca taggaagaag ctccaaagta gattacgtga gttgaagttg 1080 ctatggggtt ctactggtgg gtctggtggt ggtggtggtt ttgacaccga gctgtatatg 1140 cgtgctacag atactgttag tttgggaacc actctttcag agcatgctgc ttcagctccg 1200 tcggggttac ggcaccgacc tgctgctact gatagtggac ctcatgaagc gctgccgttc 1260 gaggtgtggg tgtttgataa tctagctgta gtgtatg att cgattggtat gagtgattta 1320 ttttatactg ttagagagtt tgttggggtg ttcaacggtg agtttgaagg gcttatagag 1380 ctgttagagt cacccgatga tgatgatggt gtgtatacga atgctcctag agacactgcc 1440 attgacgcct atgaatctca agaaaactac gaccgtattg atattgaaac tgtcttgatc 1500 gagaggcgta taaacttgaa aaagttgctt cttgaagaag cagagctaga acgacgagag 1560 cgagatatga ctatgattgc tgatgaagaa caaagaacat tgctacatag gttggaaagt 1620 tctagggttg aagcaactca tgcagttgcc aaagccgaag ctgatgctcg ggcagctgtc 1680 gctatggctg ctcttgcttc taaggaagct aatgattacg acagtaagat ggcttttgac 1740 aggtcttgta aagaacagga actgcggttg cgcgaactcg aagtgaatag tatgccgagt 1800 aaaacagaga ggtatgttca cactggtata caaggtggcg cgcaattggc tggagctatg 1860 gctgtcggtg ctatgctgcg acgtggggct ggttcttctt ctcaaaccgt ttctagtggt 1920 gctaatattg gttctcgttc gcagagtttg actcgtggtc gtagtgcatc acaacctttg 1980 tcatcggttg gtggttctac tcgtggggtt aataataata ttagtaatac taatcttgtt 2040 agggctggta atagtgctga agtttctgct ggtagatcta ctaatagtgg taatagtaat 2100 ttttggtcca aattacgtgt tggtgaagga tggtccaagt ac agcgtaga acgggcggcg 2160 acaagggcgc aaagggcaat cgtgcttcca gcgcccccgt ccgctcccgc cggatgactc 2220 aggatgactg gtcacgtacc catcccgacg atattttctc agttattgag aaaacactag 2280 tagaggatgg gtataaatgg aacggggtaa aacccggaca ttgcgattgg ggcaaattga 2340 aggaatctgg tgctattgat aattttaggg gtacattaga aggcgagtta ggtaaaaatt 2400 gtgatttgac ttgtaatgct gctgccgtta aacttgacac attgcaaaag gtgaaaatgt 2460 catcagattg gactgccaga gttggtattg ttttgggtgc tcctggtgtt gggaaatcta 2520 cctcgattaa gaacttatta gacaaatttg gagcaaaaca taaaatggtg ttatgcttac 2580 cttttagtca gttgttagaa ggagtgtttg ctggtcggtt ggacactttt ctggttgatg 2640 atttgttctg taggtccgtg ggatatggaa aatacaacac catgcttgta gatgaggtca 2700 ctcgtgtgca tatgtgtgag attttggtac ttgccggaca tttaggtgtt aagaacgtga 2760 tatgttttgg tgatccggcg caagggttaa attataaggc cggttctgcc gtgaactata 2820 attttccgat tattgctgaa tgttatgcta gtagacggtt cggtaaggcg actgccgatc 2880 tcattaattc cagcaatggt ggtggtaaac ctgtagtcgg taataacgag gtaaaggata 2940 gttggacttt tgaagaacta tgtgggaaga tactagatat gtctactg tt ttggtagcta 3000 cacgcgaaac ccaaaagttt ctattagaag ataatattga gtctattctt tactcggacg 3060 cccacgggca aacatacgac gttgtcacta tcattttgga agacgagttt gatgatgctg 3120 ccatttgcga cccaaatgtt agggctgtct tgttaactag agctcgtaag ggtggtatga 3180 ttaagatggg tcctaacatt gctgccaggt tcaaaaacgg tgattttaat tcacgtggag 3240 ttagtaagtc ttgcaccgga gatacttttt gcgaagatag ataatgtcta gggaaataac 3300 cgctcgaccc aataagaatg tgcctattgt tgttggtgtt tgtgttgtgg ctttctttgt 3360 attgctggcg ttcatgcagc aaaaacataa gacacattct gggggcgatt acggagtccc 3420 aacattttct aacggtggta aatacagaga cggtacaagg tcagctgatt ttaatagtaa 3480 taatcatcgt gcttacgggt gcggtgggtc tgggggtagc gttagtagtc gagtcgggca 3540 gcaacttgtt gtgttagcta ttgtgtctgt gttaatagta tcactgttac aacgattaag 3600 atctccacca gaacatattt gtaatggtgc ttgtggttaa agtagattta tctaatattg 3660 tgttgtatat agttgccggt tgtgttgttg ttagcatgtt gtactcacca tttttcagca 3720 acgatgttaa agcgtccagc tatgcgggag cagtttttaa agggagtggc tgtatcatgg 3780 acaggaattc gtttgctcaa tttgggagtt gcgatattcc aaagcatgta gcc gagtcca 3840 tcactaaggt tgctaccaaa gagcacgatg ctgatataat ggtaaaaaga ggtgaagtga 3900 ccgttcgtgt tgtgactctc accgaaactc ttttcataat attatctaga ttgtttggtt 3960 tggcggtgtt tttgttcatg atatgtttaa tgtctatagt ttggttttgg tgtcatagat 4020 aaattgtggt agaatgagta tggggatggt agatagtttg tgtgtgtttg ttggtcgggt 4080 cataactgag ggatctgaaa gtgttgaggg tgtggaacgg ttttccatta agtttagtga 4140 ctggaaattg ttcaccaccg cggtgtacgt tgaatatcgt cagttaggtg agaaagagtg 4200 tagtttgaag gatgttggta ggttacattt taatatgtca tgtgtgaaat gctgtcaaaa 4260 acttaaatgc aagaaacaaa ataaaaatca tagtaaacac gtccaaaatg gatatttacg 4320 caaggtgcgt aatttttcca ttttaggtgt ttgcggtgat tgttgtgagt cttttacact 4380 tgcggacgaa aaacatcatg ttattgtcga tcctgaggtg taatagggtt tattcaagag 4440 actatgttta atattaataa tcagggccat gccacaggcc tcctattggg ttgttccgaa 4500 ggttgttgtg gtttatattg cttattggta agtgatttga ttaaggttgc agtgtactga 4560 ctgggtgtga attgtaccag tccatgtagg gtctgttttc agtatattg 4609 <210> 2 <211> 739 <212> DNA <213> Beet Necrotic Yellow Vein Virus <220> <223 > Sequence inserted in pM27 <400> 2 atgtctaggg aaataaccgc tcgacccaat aagaatgtgc ctattgttgt tggtgtttgt 60 gttgtggctt tctttgtatt gctggcgttc atgcagcaaa aacataagac acattctggg 120 ggcgattacg gagtcccaac attttctaac ggtggtaaat acagagacgg tacaaggtca 180 gctgatttta atagtaataa tcatcgtgct tacgggtgcg gtgggtctgg gggtagcgtt 240 agtagtcgag tcgggcagca acttgttgtg ttagctattg tgtctgtgtt aatagtatca 300 ctgttacaac gattaagatc tccaccagaa catatttgta atggtgcttg tggttaaagt 360 agatttatct aatattgtgt tgtatatagt tgccggttgt gttgttgtta gcatgttgta 420 ctcaccattt ttcagcaacg atgttaaagc gtccagctat gcgggagcag tttttaaagg 480 gagtggctgt atcatggaca ggaattcgtt tgctcaattt gggagttgcg atattccaaa 540 gcatgtagcc gagtccatca ctaaggttgc taccaaagag cacgatgctg atataatggt 600 aaaaagaggt gaagtgaccg ttcgtgttgt gactctcacc gaaactcttt tcataatatt 660 atctagattg tttggtttgg cggtgttttt gttcatgata tgtttaatgt ctatagtttg 720 gttttggtgt catagataa 739 <210> 3 <211> 739 <212> DNA <213> Beet Necrotic Yellow Vein Virus <220> <221> mutation <222> (151). . (174) <223> Sequence inserted in pM28 <400> 3 atgtctaggg aaataaccgc tcgacccaat aagaatgtgc ctattgttgt tggtgtttgt 60 gttgtggctt tctttgtatt gctggcgttc atgcagcaaa aacataagac acattctggg 120 ggcgattacg gagtcccaac attttctaac gctgctaaat tcagagctgc agcaaggtca 180 gctgatttta atagtaataa tcatcgtgct tacgggtgcg gtgggtctgg gggtagcgtt 240 agtagtcgag tcgggcagca acttgttgtg ttagctattg tgtctgtgtt aatagtatca 300 ctgttacaac gattaagatc tccaccagaa catatttgta atggtgcttg tggttaaagt 360 agatttatct aatattgtgt tgtatatagt tgccggttgt gttgttgtta gcatgttgta 420 ctcaccattt ttcagcaacg atgttaaagc gtccagctat gcgggagcag tttttaaagg 480 gagtggctgt atcatggaca ggaattcgtt tgctcaattt gggagttgcg atattccaaa 540 gcatgtagcc gagtccatca ctaaggttgc taccaaagag cacgatgctg atataatggt 600 aaaaagaggt gaagtgaccg ttcgtgttgt gactctcacc gaaactcttt tcataatatt 660 atctagattg tttggtttgg cggtgttttt gttcatgata tgtttaatgt ctatagtttg 720 gttttggtgt catagataa 739 <210> 4 <211> 119 <212> PRT <213> Beet Necrotic Yellow Vein Virus <220> <221> Modified 13 k protein encoded by pM28 <400> 4 Met Ser Arg Glu Ile Thr Ala Arg Pro Asn Lys Asn Val Pro Ile Val 1 5 10 15 Val Gly Val Cys Val Val Ala Phe Phe Val Leu Leu Ala Phe Met Gln 20 25 30 Gln Lys His Lys Thr His Ser Gly Gly Asp Tyr Gly Val Pro Thr Phe 35 40 45 Ser Asn Ala Ala Lys Phe Arg Ala Ala Ala Arg Ser Ala Asp Phe Asn 50 55 60 Ser Asn Asn His Arg Ala Tyr Gly Cys Gly Gly Ser Gly Gly Ser Val 65 70 75 80 Ser Ser Arg Val Gly Gln Gln Leu Val Val Leu Ala Ile Val Ser Val 85 90 95 Leu Ile Val Ser Leu Leu Gln Arg Leu Arg Ser Pro Pro Glu His Ile 100 105 110 Cys Asn Gly Ala Cys Gly Stop 115 <210> 5 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer for amplification of RNA-2 <400> 5 ctaccttcac? tcgacat 17 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <221> Primer for amplification of RNA-2 <400> 6 tttaaattct? aactattatc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <221> Primer for amplification of RNA-2 <400> 7 accccgttcc atttataccc 20

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Minako Saito, Inventor 4-2-1, Oyajihigashi, Atsubetsu-ku, Sapporo, Hokkaido, Japan (72) Inventor Tadahiko Kiguchi 4-116, 4-4-1, Kuriyamacho, Yubari-gun, Hokkaido ) Inventor Shunzo Kusume 4-116-3-101 Chuo, Kuriyama-cho, Yubari-gun, Hokkaido CD07 CD09 CD13 4B024 AA08 CA03 CA04 CA06 CA11 DA01 EA04 FA02 FA07 GA11 GA17 HA01 4B065 AA89X AA97Y AB01 AC20 BA02 BA25 CA53

Claims (15)

[Claims] 1. The beet necrotic vein yellowing virus (BNYV)
V) Transformation imparting resistance to BNYVV by transforming a DNA corresponding to a genome-derived gene or a partial sequence thereof or a DNA substantially identical thereto into a plant genome so that it can be expressed, plant. 2. A DNA corresponding to a gene derived from the BNYVV genome or a partial sequence thereof is a DNA derived from a 4.8 kb RNA molecule (RNA-2 molecule) among genomic RNAs possessed by BNYVV.
A transformed plant according to claim 1. 3. The DNA derived from an RNA-2 molecule is 3 ′ of the RNA-2 molecule.
The transformed plant according to claim 2, which is DNA derived from a terminal region. 4. The method of claim 1, wherein the substantially identical DNA is derived from an RNA-2 molecule.
DNA encoding a protein in which the hydrophilic region of the protein encoded by the DNA is replaced with a hydrophobic amino acid
The transformed plant according to any one of claims 1 to 3, which is 5. The transformed plant according to claim 1, wherein the plant is sugar beet. 6. The plant of claim 1, wherein the plant is benthamiana.
5. The transformed plant according to any one of 4. 7. A DNA corresponding to a gene derived from the BNYVV genome or a partial sequence thereof or a DNA substantially identical thereto
Is transformed into a plant cell genome so that it can be expressed, thereby imparting resistance to BNYVV. 8. The transformed plant cell according to claim 7, wherein the DNA corresponding to the gene derived from the BNYVV genome or a partial sequence thereof is DNA derived from an RNA-2 molecule. 9. The DNA derived from an RNA-2 molecule is 3 ′ of the RNA-2 molecule.
The transformed plant cell according to claim 8, which is a DNA derived from a terminal region. 10. The method according to claim 10, wherein the substantially identical DNA is located on the RNA-2 molecule.
DNA encoding a protein in which the hydrophilic region of the protein encoded by the DNA is replaced with a hydrophobic amino acid
The transformed plant according to any one of claims 7 to 9, which is 11. The transformed plant cell according to claim 7, wherein the plant cell is derived from sugar beet. 12. The transformed plant cell according to claim 7, wherein the plant cell is derived from tobacco. 13. A plant transformation, which comprises a DNA derived from a part of the nucleotide sequence shown in SEQ ID NO: 1 or a DNA substantially the same as the DNA and expresses BNYVV, thereby conferring virus resistance. Vector construct. 14. The vector construct pM27 of claim 11, comprising the nucleotide sequence set forth in SEQ ID NO: 2 or DNA substantially identical thereto in the sense orientation between the promoter and terminator. 15. The vector construct pM28 of claim 11, comprising the nucleotide sequence set forth in SEQ ID NO: 3 or substantially identical DNA in a sense orientation between the promoter and terminator.