WO2013062069A1 - ジェミニウイルス複製阻害剤 - Google Patents
ジェミニウイルス複製阻害剤 Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
- A01N55/02—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur containing metal atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/12011—Geminiviridae
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/12011—Geminiviridae
- C12N2750/12061—Methods of inactivation or attenuation
- C12N2750/12062—Methods of inactivation or attenuation by genetic engineering
Definitions
- the present invention relates to effective infection control means for plant viruses. More specifically, the present invention relates to a replication inhibitor for plant viruses belonging to the genus Masterovirus included in the plant virus Gemini virus, and a plant having resistance to infection by plant viruses belonging to the genus Masterovirus.
- a zinc finger is a DNA-binding motif together with a helix-turn-helix motif and a leucine zipper motif.It has two cysteines on the amino-terminal side and two histidines on the carboxyl-terminal side, and zinc ( It has a three-dimensional structure coordinated with Zn). Since zinc fingers have a very strong binding force to DNA, artificial DNA binding proteins that bind strongly to DNA using this motif (hereinafter referred to as ⁇ AZP '' in this specification) AZP designed to be able to recognize a specific base sequence using a recognition code table (NondegenerategnRecognition Code ⁇ ⁇ Table) has been reported (Japanese Patent Publication No. 2004-519211) ; Biochemistry, 41, pp.7074-7081, 2002).
- the zinc finger motif can recognize and bind 3 bp or 4 bp, and the length of the base sequence to be specifically bound can be adjusted by connecting the zinc fingers with a peptide linker.
- the fourth recognition base sequence of the zinc finger motif is an antisense strand and overlaps with the first recognition base sequence of the next zinc finger motif, so it recognizes and binds the base sequence of 3N + 1bp for every N zinc finger motifs. (See Figure 1).
- WDV wheat dwarf virus
- ⁇ WDV '' WDV wheat dwarf virus
- Geminivirus is a general term for viruses having one or two single-stranded circular DNAs that infect plants, and includes a variety of plant viruses, including Begomovirus, Topokvirus, Kurtovirus, and Mastere. It is roughly divided into four types of virus genus.
- viruses belonging to the genus Begomovirus include tomato yellow leaf curl virus (Tomato Yellow Curl Virus: ⁇ TYLCV), potato yellow mosaic virus (Potato Yellow Mosaic Virus: ⁇ ⁇ PYMV) and bean golden mosaic virus ( Bean Golden Mosaic Virus: BGMV) and other viruses belonging to the genus Mastelevirus include the above-mentioned WDV, maize streak virus (MSV), Miscanthus streak virus (Miscanthas Streak) Virus: MiSV), tobacco yellow dwarf virus (Tobacco Yellow Dwarf Virus: TYDV), chloris strite mosaic virus (Chloris Straite Mosaic Virus: CSMV), and the like.
- the virus belonging to the genus Topovirus is Tomato Pseudo-curly Top Virus (TPCTV), and the virus belonging to the genus Kurtovirus is Beet Mild Curly. Top Virus: BMCTV) (see Fig. 3).
- the geminivirus When entering the plant, the geminivirus is first converted into double-stranded circular DNA by the plant's endogenous factors. Next, the virus-derived replication protein (Rep) binds to the Rep binding site upstream of the stem loop of the Intergenic Region (IR). Rep is a multifunctional protein that binds to the Rep binding site, nicks the 9 base sequence of the loop portion of the stem loop, and then covalently binds to the 5 ′ end of the nicked DNA. Subsequently, DNA synthesis was started from the 3 ′ end using the ⁇ strand as a template, and when a single copy of the genome was synthesized, the newly created 9 base sequence was nicked again by Rep.
- Rep virus-derived replication protein
- An object of the present invention is to provide effective infection control means against geminivirus. More specifically, the present invention provides a drug that inhibits replication of plant viruses belonging to the genus Masterovirus included in Geminivirus, a plant having resistance to plant viruses belonging to the genus Masterovirus, and the like. It is a problem.
- the present inventor paid attention to this stem loop portion and conducted earnest research to provide means capable of commonly inhibiting the replication of various viruses belonging to Geminivirus.
- Rep that can cleave only single-stranded DNA by stabilizing the double-stranded structure of viral DNA by specifically binding AZP to the DNA of the stem-loop part and inhibiting the structural change to the stem-loop. It was found that the cleavage of viral DNA can be inhibited.
- the present inventor has also confirmed that this virus replication inhibitory action actually functions in plants. This technique is extremely useful for providing a general-purpose virus replication inhibitor for viruses belonging to the genus Begomovirus, for example, by utilizing a stem loop portion that is particularly highly conserved in the genus Begomovirus of Geminivirus. .
- the present inventors applied a similar technique using a stem loop portion and its peripheral region conserved in a virus belonging to the genus Masterovirus of geminivirus, thereby causing wheat atrophy disease.
- the present inventors have found that a general-purpose virus replication inhibitor can be provided for viruses belonging to the genus Masterovirus including viruses (WDV) and the like, and have completed the present invention.
- WDV viruses
- a replication inhibitor for viruses belonging to the genus Masterovirus of the Geminiviridae family which is specific to the full length DNA of the stem loop region of the virus or one or more partial DNAs selected from the full length DNA
- a replication inhibitor is provided that includes a zinc finger protein that is capable of binding, and is capable of inhibiting the formation of a stem-loop structure.
- the replication inhibitor comprising a single zinc finger protein capable of binding to one partial DNA selected from the full-length DNA of a stem loop region of a virus belonging to the genus Masterovirus; A single DNA that can bind to a continuous DNA consisting of one partial DNA selected from the full length DNA of the stem loop region of a virus belonging to the genus Televirus and one DNA selected from the peripheral region that binds to the DNA.
- the above-mentioned replication inhibitor containing a zinc finger protein; and two zinc finger proteins that can bind to two or more partial DNAs selected from DNA consisting of a stem loop region and a peripheral region that binds to the stem loop region.
- the above-described replication inhibitor containing the zinc finger protein bound thereto is provided.
- the replication inhibitor wherein the zinc finger protein is a zinc finger protein comprising 8 to 13, preferably 9 to 12 zinc finger domains.
- the present invention also provides a nucleic acid encoding the zinc finger protein and a replication inhibitor for geminivirus, the replication inhibitor including the nucleic acid encoding the zinc finger protein.
- the replication inhibitor described above wherein the virus belonging to the genus Masterovirus is wheat dwarf virus (WDV).
- WDV wheat dwarf virus
- an antiviral agent against a virus belonging to the genus Masterovirus comprising the above zinc finger protein or a nucleic acid encoding the above zinc finger protein; the above zinc finger protein or the above zinc finger protein
- An agent for preventing infection of a virus belonging to the genus Masterovirus, which contains a nucleic acid encoding the above; a pesticide for controlling infection by a virus belonging to the genus Masterovirus, which encodes the above zinc finger protein or the above zinc finger protein A pesticide containing the nucleic acid is provided.
- a method for preventing viral infection belonging to the genus Masterovirus of a plant wherein a preventive effective amount of the zinc finger protein or the nucleic acid encoding the zinc finger protein
- a method comprising controlling a virus infection belonging to the genus Mastelevirus, the method comprising applying a control effective amount of the zinc finger protein or the nucleic acid encoding the zinc finger protein to a plant.
- a method is provided.
- a plant having resistance to a virus belonging to the genus Masterovirus which is a genetically modified plant capable of expressing the zinc finger protein;
- a transformed plant into which a gene encoding the above zinc finger protein is introduced ;
- a method for allowing a plant to acquire resistance to a virus belonging to the genus Mastelevirus, the gene encoding the above zinc finger protein A method comprising the step of transforming a plant with is provided.
- a recombinant vector comprising a nucleic acid encoding the above zinc finger protein, and the above recombinant vector used for transforming a plant into a plant having resistance to a virus belonging to the genus Masterovirus.
- a plant viral vector or the like can be used as the vector.
- the replication inhibitor of the present invention targets the stem loop region conserved in viruses belonging to the genus Masterovirus of the Geminiviridae family, it is common to infection by various viruses belonging to the genus Masterovirus. It can act as a replication inhibitor. Therefore, the replication inhibitor of the present invention exhibits high effectiveness not only for WDV infection, which is a typical virus belonging to the virus belonging to the genus Masterovirus, but also for other viruses belonging to the genus Masterovirus. Therefore, it is extremely useful as a control means for various viruses belonging to the genus Masterovirus.
- 35S Cauliflower mosaic virus-derived promoter
- NLS nuclear localization signal
- ⁇ 5: 5'-leader sequence for increasing translation efficiency
- NOST terminator
- TYLCV3 / 4/6 ⁇ all TYLCVs bind to common nucleotide sequences AZP (recognition sequence is 5'-GGCCATCCGTATAATATTACCGGATGGCCGC-3 '). It is the figure which showed the preparation method of the APZ expression plasmid for transformation.
- FIG. 3 is a view showing the structure of an inserted gene and a PCR primer set for detecting a kanamycin resistance gene and an AZP gene for transformant T1. It is the figure which showed the result of having detected the kanamycin resistance gene and the AZP gene about transformant T1.
- Lanes 1 to 4 show the results of PCR using DNA extracted from each T1 plant, N using DNA extracted from wild-type tomatoes, and P using the binary vector used for transformation. It is the figure which showed the primer set for confirming that it was inserted in the structure of the insertion gene in the whole area of an AZP expression cassette, and a genome. It is the figure which showed the result of having confirmed AZP gene insertion in T2 plant obtained by introduce
- lanes 1 to 8 show the results of PCR using DNA extracted from each T1 plant, and P shows the binary vector used for transformation.
- Lanes 1 to 18 show the results of PCR using DNA extracted from T2 plants from a specific transformant T1
- N is DNA extracted from wild-type tomatoes
- P is a binary vector used for transformation.
- AZP was detected by Western blotting using anti-HA antibody from the leaf extract of the T2 plant shown in FIG. The lane numbers in the figure correspond to those in FIG.
- Lanes 1 to 16 show the results of PCR using DNA extracted from T3 plants from a specific T2 line. This T2 plant in which the AZP insertion gene was confirmed in all T3 individuals was selected as homozygous. It is the figure which showed the result of having confirmed the expression of AZP in the T3 plant obtained by introduce
- the replication inhibitor of the present invention is a replication inhibitor for viruses belonging to the genus Masterovirus of the Geminiviridae, and is a full-length DNA of the stem loop region of the virus or one or more partial DNAs selected from the full-length DNA It contains a zinc finger protein that can specifically bind to and is capable of inhibiting the formation of a stem-loop structure.
- the term “geminivirus” refers to a DNA virus that infects a plant and that has one or two single-stranded circular DNAs. , Chemistry and Biology, 41, pp.311-317, 2003, etc. Geminiviruses are divided into the following four genera, depending on the genome structure, host range, and type of vector insect: Mastrevirus genus, Kurtovirus genus, Topocuvirus genus, and Begomovirus genus Among them, the replication inhibitor of the present invention can specifically target any virus belonging to the genus Mastrevirus. The genomic organization of viruses belonging to each genus is specifically shown in FIG. 2 of the above-mentioned publication (Chemistry and Biology, 41, pp.311-317, 2003).
- geminiviruses include not only known geminiviruses, but also unknown geminiviruses and new geminiviruses in which known geminiviruses are mutated.
- viruses belonging to the genus Mastrevirus such as MSV (Maize streak virus), WDV (Wheat dwarf virus), BeYDV (Bean yellow dwarf virus), BCTV (Beet cury top virus), etc.
- viruses belonging to the genus Kurtovirus viruses belonging to topok virus such as TPCTV (Tomato pseudo-cury top virus), BGMV (Bean golden mosaic virus), ACMV (African cassava mosaic virus), SLCV (Squash leaf curl virus), TGMV Viruses belonging to the genus Begomovirus such as (Tomato golden mosaic virus) and TYLCV (Tomato Yellow Leaf Curl Virus) can be mentioned, but are not limited thereto.
- the replication inhibitor of the present invention is provided as a replication inhibitor for viruses belonging to the genus Mastrevirus such as MSV (Maize streak virus), WDV (Wheat dwarf virus), BeYDV (Bean yellow dwarf virus), etc. Of these, WDV is a particularly preferred target.
- viruses belonging to the genus Begomovirus have a highly conserved stem loop region that serves as a binding site for replication inhibitors.
- Viruses belonging to the genus Begomo virus include, for example, TYLCCNV, TYLCGV, TYLCMalV, TYLCSV, TYLCTHV, TYLCV, ACMV, BGMV, CaLCuV, ToCMoV, TGMV, ToGMoV, ToMHV, ToMoV, ToMoV, RMToLC, RMToLC , CLCuAV, ClCuGV, CLCuKV, CLCuMV, CLCuRV), East African cassava mosaic (EACMCV, EACMMV, EACMV, EACMZV), potato yellowing mosaic (PYMPV, PYMTV, PYMV), pumpkin cigar (SLCCNV, CYSLCV, CYSLCV, CYSLCV (SPLCGV)
- TYLCCNV TYLCCNV
- TYLCGV TY
- FIG. 3 shows the inclusion relationship between geminivirus and TYLCV and WDV.
- the replication inhibitor of the present invention comprises a zinc finger protein that can specifically bind to the full length DNA of a stem loop region of a virus belonging to the genus Mastrevirus or one or more partial DNAs selected from the full length DNA, In addition, it has the effect of inhibiting the formation of a stem loop structure.
- ⁇ stem loop region '' of geminivirus for example, taking TYLCV belonging to the genus Begomovirus as an example, the stem loop region is composed of two stem regions (regions each consisting of 11 bases) that bind complementarily to each other, and between them A 33-base region consisting of a loop region (region consisting of 11 bases) that exists and forms a loop.
- TYLCV Although various strains are known for TYLCV, the base sequence of the stem loop region is highly conserved in all TYLCV.
- Fig. 4 shows the stem loop region of TYLCV. This stem loop region is highly conserved in other viruses belonging to Begomovirus, for example, there is a stem loop region consisting of 34 bases on the CR (common region) of both DNAs of BGMV, Its base sequence is extremely homologous to the base sequence of the stem loop region of other viruses belonging to Begomovirus.
- the base sequence of the stem loop region is ⁇ highly conserved '' means that the homology of the base sequence to be compared is 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably Means 97% or more, particularly preferably 99% or more.
- the stem loop region is highly conserved.
- FIG. 5 shows the homology of the stem loop region for several viruses included in the geminivirus.
- the replication inhibitor of the present invention specifically binds to the full length DNA of the stem loop region conserved in viruses belonging to the genus Mastrevirus as described above, or one or more partial DNAs selected from the full length DNA, As a result of specific binding, it can be designed to inhibit the formation of stem-loop structures.
- the replication inhibitor of the present invention is located upstream and / or downstream of the stem loop region DNA. It can also be designed to specifically bind to the DNA of the flanking region to be bound, and such an embodiment is a preferred embodiment in the present invention.
- One particularly preferred embodiment is: (a) one partial DNA selected from the full-length DNA of the stem loop region of a virus belonging to the genus Masterovirus, and one DNA selected from the flanking region that binds to the DNA.
- a replication inhibitor comprising a single zinc finger protein capable of binding to continuous DNA consisting of
- AZP-11 and 12 are disclosed in Example 1 of the present specification (FIG. 30).
- AZP-13 is disclosed in Example 1 of the present specification (FIG. 30).
- two or more zinc finger proteins that can bind to two or more partial DNAs selected from DNA consisting of a stem loop region and a peripheral region (flanking region) that binds to the stem loop region are bound by a linker.
- the above-mentioned replication inhibitors comprising a zinc finger protein.
- the inhibitor of the present invention binds to the selected DNA to stabilize the double-stranded structure of the viral DNA, but as appropriate based on the stem loop region and, if necessary, the base sequence of the surrounding region. It is possible to design a zinc finger protein that inhibits the formation of a stem loop structure of a virus belonging to the genus Masterovirus.
- the zinc finger domain contained in the zinc finger protein can be designed so that a specific base sequence can be recognized using a recognition code table (Nondegenerate Recognition Code Table).
- a zinc finger domain means a domain constituting a DNA binding site present in a zinc finger protein, and may be simply referred to as a “finger”.
- zinc finger proteins typically have about 2, 3, 4, 6, or 10 zinc finger domains.
- the design method of a zinc finger protein that recognizes and specifically binds to a recognition code table and a specific base sequence is described, for example, in JP-T-2004-519211. The entire disclosure of the above patent publication is included in the disclosure of this specification by reference. Biochemistry, 41, pp.7074-7081, 2002, and the like can also be referred to.
- the base sequence information of the stem loop region of the genomic DNA of a virus belonging to the genus Mastrevirus can be easily obtained, and one skilled in the art can select at least one or two or more selected from the full length DNA or full length DNA of the stem loop region. It is possible to easily design and produce a zinc finger protein that can specifically bind to the partial DNA of
- a method for designing a replication inhibitor targeting only TYLCV is shown in Reference Example 1 of the Examples. It is only necessary to design a zinc finger protein that can bind to DNA containing the full length or almost full length of stem loop region DNA (33 bases) that is well conserved among TYLCV, and one type selected from such zinc finger proteins It is possible to inhibit the replication of all TYLCV by using the zinc finger protein of the present invention as a replication inhibitor of the present invention. As such a zinc finger protein, for example, a zinc finger protein containing 10 zinc finger domains can be designed. It will be readily understood by those skilled in the art that the above method can be appropriately applied to the design of replication inhibitors targeting the geminivirus family other than TYLCV.
- a method for designing replication inhibitors targeting various geminiviruses including viruses belonging to the genus Begomovirus in addition to TYLCV is also shown in Reference Example 2 of the Examples.
- linker in addition to a peptide linker having 1 to 40 amino acid residues, preferably 1 to 20, more preferably about 1 to 10, a synthetic linker such as an alkylene chain or a polyethylene glycol chain, a sugar chain, etc. May be used.
- a synthetic linker such as an alkylene chain or a polyethylene glycol chain, a sugar chain, etc. May be used.
- examples of replication inhibitors targeting only TYLCV and examples of replication inhibitors targeting various geminiviruses including viruses belonging to the genus Begomovirus are shown in FIG.
- the upper side is an example in which only TYLCV is targeted
- the lower side is an example in which various diverse geminiviruses including viruses belonging to the genus Begomovirus are targeted.
- a protein having substantially the same replication inhibitory activity as the protein consisting of the amino acid sequence specified by SEQ ID NO: 1 or 2 can also be used as a replication inhibitor.
- an amino acid having 70%, preferably 80%, more preferably 90% or more homology with the amino acid sequence specified by SEQ ID NO: 1 or 2, and the amino acid specified by SEQ ID NO: 1 or 2 A protein having a replication inhibitory action substantially similar to a protein comprising a sequence can also be used as a replication inhibitor.
- a nucleic acid containing DNA encoding the protein (b) or (c) can be used.
- DNA encoding the protein represented by (b) or (c) above for example, DNA capable of hybridizing under stringent conditions to the DNA specified by the nucleotide sequence shown in SEQ ID NO: 3 or 4 Etc. are included.
- DNA for example, a DNA is used as a probe, and a colony or plaque-derived DNA or DNA fragment-immobilized filter is used in a colony hybridization method, plaque hybridization method, or Southern blot hybridization method.
- a 0.1 to 2 ⁇ SSC solution (1 ⁇ SSC solution contains 150 ⁇ mM sodium chloride and 15 ⁇ mM sodium citrate) is used. Examples thereof include DNA that can be identified by washing the filter under the condition of ° C.
- a DNA having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more with respect to the base sequence of DNA used as a probe is preferable. Can be used.
- the design of a replication inhibitor for viruses belonging to the genus Mastrevirus can be carried out as follows.
- a typical example of a virus belonging to the genus Mastrevirus is WDV.
- WDV the mutant shown in FIG. 29 is known, but the stem loop region is highly conserved, and further downstream of the stem region. And the surrounding region (flanking region) that binds upstream is also highly conserved (for the WDV genome sequence and variants, Plant Pathology, 57, pp. 838-841, 2008; Plant Pathology, 58, pp. 1161 -1169, 2009; Virus genes, 34, ppo.359-366, 2007, etc.).
- the number of bases in the peripheral region to be considered in the design of the replication inhibitor of the present invention is, for example, 200 or less, preferably 100 or less, more preferably 50 or less, particularly preferably 30 or less from the end of the stem region. Furthermore, these regions are also conserved in other viruses belonging to the genus Mastrevirus, and the homology of this region is usually 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more. Particularly preferably, it is 95% or more.
- a zinc finger protein that specifically binds to the stem loop region of WDV is used, or it binds to a part of the stem loop region of WDV and upstream of the stem region of WDV and A zinc finger protein that specifically binds to DNA bound downstream can be used.
- FIG. 30 shows a target site of the replication inhibitor of the present invention for a virus belonging to the genus Mastrevirus containing WDV.
- a zinc finger protein that specifically binds to the antisense strand may be used.
- Example 1 of the present specification AZP11 and AZP13 are specifically disclosed as zinc finger proteins that recognize the sense strand, and AZP12 is specifically exemplified as a zinc finger protein designed based on the sequence on the antisense strand side. Is disclosed. Further, the design methods of AZP11, AZP12, and AZP13 are shown in FIG. 31, FIG. 32, FIG. And 33, respectively, and the amino acid sequences of AZP11, AZP12, and AZP13 are shown in SEQ ID NOs: 5, 6, and 7, respectively. .
- deletion, substitution and / or selection or addition of 1 to several amino acids preferably about 1 to 5 amino acids in the amino acid sequence specified by SEQ ID NO: 5, 6, or 7
- a replication inhibitor consisting of an amino acid sequence having a replication inhibitory action substantially similar to a protein consisting of the amino acid sequence specified by SEQ ID NO: 5, 6, or 7 against viruses belonging to the genus Mastrevirus Proteins can also be used.
- a protein having substantially the same replication inhibitory activity as the protein consisting of the amino acid sequence specified by 7 can also be used as the replication inhibitor of the present invention.
- nucleic acids used for preparing a replication inhibitor for viruses belonging to the genus Mastrevirus include, for example, DNAs encoding the above AZP11, AZP12, and AZP13 (base sequences shown in SEQ ID NOs: 8, 9, and 10 in the sequence listing, respectively)
- a nucleic acid containing DNA encoding the protein (d) or (e) can be used.
- the DNA encoding the protein represented by (d) or (e) above for example, hybridizes under stringent conditions to the DNA specified by the nucleotide sequences shown in SEQ ID NOs: 8, 9, and 10. DNA that can be used.
- DNA for example, a DNA is used as a probe, and a colony or plaque-derived DNA or DNA fragment-immobilized filter is used in a colony hybridization method, plaque hybridization method, or Southern blot hybridization method.
- a 0.1 to 2 ⁇ SSC solution (1 ⁇ SSC solution contains 150 ⁇ mM sodium chloride and 15 ⁇ mM sodium citrate) is used. Examples thereof include DNA that can be identified by washing the filter under the condition of ° C.
- a DNA having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more with respect to the base sequence of DNA used as a probe is preferable. Can be used.
- the replication inhibitor of the present invention is provided in the form of the above zinc finger protein or a nucleic acid encoding the zinc finger protein.
- Infection control against the virus to which it belongs can be performed.
- the application method of the replication inhibitor of this invention is not specifically limited, For example, it can prepare as an agrochemical composition using the additive for a formulation well-known in this industry. Agrochemical compositions containing proteins or nucleic acids as active ingredients are known in the art, and agrochemical compositions can be prepared using any suitable means.
- a method of transiently transforming a plant by introducing the nucleic acid into a plant cell using a vector such as a plasmid incorporating the nucleic acid a method of incorporating the nucleic acid into a plant genome using a vector, etc.
- a vector such as a plasmid incorporating the nucleic acid
- a method of incorporating the nucleic acid into a plant genome using a vector etc.
- Vectors that can be used in the method of the present invention include viral vectors that infect plants.
- the form of the agricultural chemical composition is not particularly limited, and any form may be adopted as long as it is a form that can be used in the industry.
- a composition in the form of an emulsion, solution, oil, water solvent, wettable powder, flowable, powder, fine granule, granule, aerosol, fumigant, or paste can be used.
- a method for producing the composition for agricultural chemicals is not particularly limited, and any method available to those skilled in the art can be appropriately employed.
- other agrochemical active ingredients such as other antiviral agents, insecticides, fungicides, insecticide fungicides, herbicides and the like can be added to the agrochemical composition.
- the present invention provides a transformed plant capable of expressing the replication inhibitor.
- the plant to be transformed in the present invention is not particularly limited, in addition to the whole plant body, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, Any of a xylem, a vascular bundle, a fence-like tissue, a spongy tissue, etc.) or a plant cultured cell may be used.
- the type of plant is not particularly limited, and any plant can be targeted, but it is preferable to target a plant species that is infected with a virus belonging to the genus Mastrevirus.
- mallow such as okra
- red crustaceae beet, spinach, etc.
- cruciferous such as turnip, cauliflower, broccoli, cabbage, komatsuna, stock, Japanese radish, duckweed, Chinese cabbage, wasabi, etc.
- Iridaceae Iris, Gladiolus, Freesia, etc.
- Pinaceae Statices, etc.
- Gramineae Rhice, Sheep, Corn, Wheat, etc.
- Siwa Tobacco Saintpaulia, etc.
- Argiaceae Udo, etc.
- Walnut family such as walnut
- mulberry family such as fig, mulberry, hop
- Lamiaceae Iceland Poppy, etc.
- Scorpionaceae Antirrhinum majus
- Primula Cicumamen, Primula, etc.
- Taro Konjac, Taro, etc.
- Cactaceae Cactus, etc.
- Perilla Salvia, Perilla, etc.
- Glyceraceae such as begonia
- Ginger such as ginger and agar
- Lilyaceae such as lotus root
- Violet such as
- Rose family (apricot, strawberry, plum, sweet cherry, plum, pear, rose, loquat, peach, snow willow Apples, pears, etc.), convolvulaceae (morning glory, sweet potatoes, etc.), sorghum (geraniums, etc.), vines (grape, etc.), beechs (chestnuts, etc.), buttons (buttons, peonies, etc.), matabidae (kiwi) Fruits, etc.), legumes (adzuki bean, green beans, green beans, green beans, peas, sweet peas, broad beans, soybeans, groundnuts, etc.), citrus (citrus, etc.), yams (such as yams), saxifragaceae (such as cymbidium), liliaceae (E.g.
- tomato, pepper, tobacco, pumpkin, manioc, sweet potato, cotton, melon, potato, soybean, wine, corn, wheat, rice, sugar cane, bean, watermelon, okra, cassava and the like can be mentioned.
- a more preferred plant is wheat or rice, and a particularly preferred plant is wheat.
- plant sources to be transformed include protoplasts, seeds, seedlings, seedlings, callus, cultured cells, and plant bodies, but are not particularly limited. Depending on the type of target plant, those skilled in the art can perform transformation by selecting an appropriate site.
- the type of vector used for transformation is not particularly limited, but the vector preferably includes a promoter and / or enhancer sequence for expressing the gene encoding the zinc finger protein.
- the promoter and enhancer sequence are not particularly limited as long as the gene can be expressed in plant cells, and any promoter and enhancer sequence can be used.
- promoters derived from plants, plant viruses, or bacteria, including genes expressed in plant cells such as Agrobacterium or Rhizobium can be used.
- promoters include, for example, a promoter derived from Agrobacterium tumefaciens T-DNA, Smas promoter, cinnamon alcohol dehydrogenase promoter, NOS promoter, ribulose diphosphate carboxylase oxygenase (Rubisco) promoter, GRP1-8 promoter, cauliflower mosaic virus (CaMV) ) -Derived 35S promoter and plant-derived actin and histone promoters / enhancers can be used, but are not limited thereto.
- the vector can contain sequences encoding various antibiotic resistance genes and other marker genes as selection marker genes.
- marker genes include, for example, anti-spectinomycin gene, streptomycin resistance gene, kanamycin resistance gene, geneticin resistance gene, hygromycin resistance gene, resistance gene for herbicide that inhibits acetolactate synthase (ALS), glutamine synthesis
- examples include, but are not limited to, resistance genes to herbicides that inhibit enzymes (eg, the bar gene), ⁇ -glucuronidase genes, luciferase genes, and the like.
- poly (A) + sequences can be derived from various plant genes or T-DNA, but are not limited thereto.
- Another sequence useful for expressing a gene at a high level for example, an intron sequence of a specific gene, a 5 ′ untranslated region sequence, or the like may be introduced into the vector.
- NLS nuclear localization signal
- Vectors useful for gene expression in higher plants are well known in the art, and any vector can be used.
- a vector derived from Agrobacterium tumefaciens Ti plasmid as a vector that can incorporate a part of vector DNA into the genome of a host plant when the vector is introduced into a plant cell KYLX6 derived from Ti pbasmid, pKYLX7, pBI101, pBH2113 , PBI121 and the like, but are not limited thereto.
- Expression vectors are known methods for introducing foreign genes into plant cells, such as particle gun method, electroporation method, polyethylene glycol (PEG) method, calcium phosphate method, DEAE dextran method, microinjection, lipofection method, and agro It can be introduced into a desired plant cell using a microorganism-mediated transfection method such as a bacterial method.
- particle gun method, electroporation method, polyethylene glycol method, Agrobacterium method and the like are preferable, but Agrobacterium method can be used particularly preferably (Methods Mol. Biol, 82, pp.259- 266, 1998).
- genetic recombination can be performed efficiently by using a two-component vector (binary vector) method.
- the desired plant can be transformed to express the replication inhibitor of the present invention by appropriately modifying or modifying the type of vector, the sequence to be introduced into the vector, the transformation method, etc. .
- AZP-2 TYPV-specific AZP
- genes with linked zinc fingers were synthesized by PCR, and each gene was cloned into the BamH I / Hind III site of pET-21a (Novagen) of the E. coli expression vector.
- pET-TYLCV-3, pET-TYLCV-4, and pET-TYLCV-5 were obtained.
- the three-finger AZP genes in pET-TYLCV-3 and pET-TYLCV-4 were amplified by PCR and ligated to finally obtain pET-TYLCV3 / 4.
- a zinc finger gene recognizing 5′-TATA-3 ′ was prepared, and pET-TYLCV6 was prepared by ligating with a 3-finger AZP gene in pET-TYLCV5 by the method described above. Finally, by recognizing and linking the 6-finger AZP gene and the 4-finger AZP gene from pE-TYLCV3 / 4 and pET-TYLCV6 by PCR, 31 bases of the 33 bases forming the stem loop region sequence are recognized. An AZP-2 expression plasmid (pET-TYLCV3 / 4/6) was prepared.
- a Geminivirus general-purpose AZP (hereinafter referred to as “AZP-3”) was prepared according to the scheme shown in FIG.
- a precursor plasmid (pET-MCS) was prepared in order to incorporate two AZP genes and linker peptide genes that recognize two regions conserved in geminiviruses within the stem loop region.
- a 6-finger AZP gene that recognizes the longer region conserved by geminivirus was amplified by PCR from pET-TYLCV3 / 4 and cloned into pET-MCS to prepare pET-TYLCV3 / 4-MCS.
- a 3-finger AZP gene that recognizes the shorter region conserved in Geminivirus is amplified from pET-TYLCV5 by PCR and cloned into pET-TYLCV3 / 4-MCS, so that it has 6 amino acids as a linker peptide
- a plasmid (pET-TYLCV3 / 4-MCS-TYLCV5) expressing AZP-3 was prepared.
- Each AZP was basically purified by the same method. Add 10 ml of lysis buffer (100 mM Trs-HCl, 100 mM NaCl, 0.1 mM ZnCl 2 , 5 mM DTT, pH 8.0) to E. coli stored at -80 ° C, and freeze and thaw 3 times to remove the E. coli cell wall. Made it fragile. Next, the E. coli was crushed using an ultrasonic crusher, and then the supernatant containing the target protein was recovered by centrifugation.
- lysis buffer 100 mM Trs-HCl, 100 mM NaCl, 0.1 mM ZnCl 2 , 5 mM DTT, pH 8.0
- the supernatant was applied to the cation exchange resin Biorex-70 (Bio-Rad) to adsorb the target protein to the resin, and then washed with a wash buffer (50 mM Trs-HCl, 50 mM NaCl, 0.1 mM ZnCl 2 , Wash thoroughly with 0.2 mM DTT, pH 8.0).
- a wash buffer 50 mM Trs-HCl, 50 mM NaCl, 0.1 mM ZnCl 2 , Wash thoroughly with 0.2 mM DTT, pH 8.0.
- an elution buffer 50 mM Trs-HCl, 300 mM NaCl, 0.1 mM ZnCl 2 , 0.2 mM DTT, pH 8.0).
- RepN has a DNA binding ability at the N-terminal region (191 amino acid residues) of the viral replication protein Rep.
- RepN was prepared by the following method for use in inhibition experiments of Rep binding to direct repeats by AZP.
- the RepN gene was amplified from the TYLCV genome by PCR using the TYLCV genome recovered from the infected tomato and cloned into the BamH I / Hind III site of pET-21a in the same manner as in AZP.
- a plasmid for RepN expression (pET-RepN) was prepared by confirming the base sequence of the obtained plasmid.
- RepN protein expression and purification RepN expression was carried out in the same manner as in the case of AZP expression to obtain a sufficient amount of expression.
- the obtained E. coli was stored at -80 ° C until protein purification.
- RepN was purified in the same manner as in AZP. In ion exchange chromatography using Biorex-70, high purity RepN could be obtained by elution with elution buffer (50 mM Tris-HCl, 250 mM NaCl, 0.2 mM DTT, pH 8.0).
- GST-AZP fusion protein AZP was used as a fusion with glutathione S-transferase (GST), the GST-AZP gene was placed downstream of the T7 promoter, and an expression vector was introduced into E. coli.
- the E. coli was cultured in 120 mL of LB-Amp liquid medium until the OD 600 was 0.65 to 0.75. After culturing, IPTG was added to a final concentration of 1 mM, and further cultured for 3 hours to induce and express GST-AZP protein.
- the induced Escherichia coli was collected by centrifugation and stored at -80 ° C.
- the GST-AZP protein was purified by the same method as the GST-Rep protein.
- Reference example 2 Materials and methods (1) Preparation of AZP transformed tomato (a) Preparation of AZP plant-use stable expression vector The gene encoding AFP-2 was inserted into the plant genome by the Agrobacterium method. For protoplast experiments, pUC35SO-TYLCV3 / 4/6 was prepared from pUC35SO-MCS by the method of FIG. 14, and a region containing 35S promoter-AZP gene-NOS terminator was excised from this plasmid with EcoR I and Hind III. The fragment was purified on an agarose gel and then cloned into the EcoR I / Hind III site of the binary plasmid pBI121 to obtain pBI-OTYLCV3 / 4/6. The nucleotide sequence was confirmed to be correct by sequencing. The same operation was performed for AZP-3.
- the cotyledons of Micro-Tom were cut out with a razor and cut into two near half from the tip. These cotyledon sections were immersed in the above-mentioned Agrobacterium suspension and allowed to stand for 10 minutes for infection. Absorb excess suspension on sterilized Kim towel, coexisting medium (1 ⁇ MS medium, 30 g / L sucrose, 3 g / L gelrite, 1.5 mg / L t-zeatin, 40 ⁇ M acetosyringone, 0.1% Leave the leaves on MES, pH 5.7). The lid was sealed with surgical tape, and shielded from light with aluminum foil and cultured at 25 ° C.
- callus induction medium (1 ⁇ MS medium, 3 g / L gelrite, t-zeatin 1.5 mg / L, Kan 100 mg / L, Augmentin 667 mg / L, 0.1% MES, Moved to pH (5.7). Callus was formed from some cotyledon sections infected in about 2 weeks, and some formed shoots.
- the individuals that did not root within 2 weeks on the first rooting medium (plate) were cut into thin cuts, transplanted to a new rooting medium, and induced rooting again.
- Individuals whose rooting was seen in the plant box rooting medium were planted in soil to produce fruit and to obtain seeds.
- the humidity was gradually lowered to acclimatize. Specifically, moistened soil was put in a plant box, rooted individuals were planted in it, and the humidity was initially lowered to a high humidity, and the lid was gradually loosened to lower the humidity. Plants that had been fully acclimatized over about one month in the plant box were planted in pots and grown.
- a primer set for amplifying a kanamycin resistance gene (NPT2 gene) and a primer set for amplifying a region including an artificial transcription gene and a NOS terminator were designed and used.
- Virus infection experiment (a) Preparation of plasmid for virus infection Virus infection was performed using the infectivity of Agrobacterium. In order to introduce a viral genome copy having two origins of replication into a binary plasmid, target plasmids were prepared in two stages as shown below for two types, TYLCV and TYLCV-mild. TYLCV-mild differs from TYLCV in the direct repeats sequence to which Rep binds, and was used to investigate the versatility against geminivirus.
- a DNA fragment of 0.5 copies including the replication origin was amplified by PCR from the viral genomic DNA of TYLCV and cloned into the EcoR I / Hind III site of the binary plasmid pBI121 to obtain pBI-TYLCV (0.5).
- the nucleotide sequence was confirmed to be correct by sequencing.
- cloning DNA amplified by PCR when introducing a DNA fragment of one copy of TYLCV it is necessary to confirm the base sequence of the prepared plasmid. However, because the target plasmid contains 1.5 copies of the viral genome, There are overlapping DNA regions, and it is not possible to confirm that the nucleotide sequence is correct by sequencing.
- a DNA fragment of one copy containing the origin of replication was amplified by PCR from the viral genomic DNA of TYLCV and cloned into the Pst I / Hind III site of pBluescript II KS + to obtain pBS-TYLCV. Sequencing confirmed that the entire base sequence was correct.
- a DNA fragment of one copy of the viral genome was excised from pBS-TYLCV with BsrG I and Hind III, purified on an agarose gel, cloned into the BsrG I / Hind III site of pBI-TYLCV (0.5), and finally The target plasmid pBI-TYLCV (1.5) was obtained. The same operation was performed for TYLCV-mild, and finally the target plasmid pBI-TYLCV-mild (1.5) was obtained.
- This suspension was injected into the cotyledons of seedlings about 10 days after sowing to infect them. Periodic observation of plant individuals and detection of viral DNA in leaves were performed after infection. For this purpose, DNA samples were prepared as described above, and viral infection was evaluated at the molecular level by analyzing PCR products obtained using primer sets specific to each TYLCV.
- AZP-transformed tomatoes were introduced into Micro-Tom tomatoes via Agrobacterium, respectively. Genes were introduced by infecting cotyledon sections with Agrobacterium transformed with a binary vector having each AZP expression cassette shown in FIG. Next, callus was induced using a medium containing kanamycin to induce shoots and then roots. Transformants were further selected at the time of induction of rooting by selecting individuals whose roots were deeply extended on the agar medium, and after acclimation, the transformants were obtained by transplanting to soil.
- AZP expression in the transformants of each approach was confirmed by Western blot.
- the AZP expression cassette for each approach was preliminarily attached with an HA epitope tag so that the expression of AZP protein in the transformant could be verified by Western blotting using an anti-HA antibody.
- FIG. 21 it was confirmed that the AZP protein was also strongly expressed in the T2 plant introduced with AFP-2.
- T3 seedlings from each T2 plant it was determined by PCR analysis of T3 seedlings from each T2 plant whether each T2 line obtained from T1 plants in which insertion of one copy of the AZP gene was confirmed was homozygous or heterozygous. If T3 seedlings from each T2 line (about 20 seedlings per line) are extracted from the DNA samples extracted from the leaves, and if all seedlings retain the AZP gene, the parent T2 line Can be determined to be homozygous (if the separation ratio is 1: 3, the parent T2 line is heterozygous). In all T3 plants from the same T2 line, the AZP gene was retained, indicating that this T2 line was homozygous (FIG. 22). It was also confirmed to be homozygous by statistical processing. It was also confirmed by Western blot that AZP was expressed in T3 plants (FIG. 23). For the plants transformed with AFP-3, the same operation was performed on T3 seedlings from each T2 plant, and similar results were obtained.
- TYLCV-mild ⁇ Infection of TYLCV by the agroinoculation method was also confirmed at the molecular level. After the infection was established, leaves were collected at each stage, and TYLCV genomic DNA was detected by PCR in all infected leaves. The same experiment was performed for TYLCV-mild. Unlike TYLCV, the infection was mild. In particular, the symptoms at the early stage of infection are such that the color around the leaves becomes light, and it may be difficult to judge infection from the expression system. Therefore, not only the determination by the expression system but also the accurate determination can be made by identifying at the molecular level that the TYLCV-mild is replicated in the infected individual by the PCR method.
- Example 1 Design of AZP targeting WDV A zinc finger protein that recognizes each of the following two DNA regions was designed based on the recognition code table described in JP-T-2004-519211. a. Upstream stem region and its flanking region b. Stem loop region c. Downstream stem region and its flanking region
- AZP-11 was designed to recognize 10 base pairs shown in Fig. 30 by binding 10 zinc finger domains continuously.
- AZP-12 12 zinc finger domains were designed to be linked sequentially to recognize the 37 base pairs shown in FIG. 30 (however, AZP-12 was designed based on the sequence on the antisense strand side).
- AZP-13 was designed to recognize the 28 base pairs shown in Fig. 30 by linking 9 zinc finger domains sequentially.
- AZP expression plasmid AZP-11 was prepared according to the scheme shown in FIG. First, genes with linked zinc fingers were synthesized by PCR, and each gene was cloned into the BamH I / Hind III site of pET-21a (Novagen) of the E. coli expression vector. As a result, pET-WDV3-1, pET-WDV3-2, and pET-WDV3-3 were obtained. Next, the three-finger AZP genes in pET-WDV3-2 and pET-WDV3-3 were ligated by PCR amplification, and finally pET-WDV6 was obtained.
- a zinc finger gene recognizing 5′-GGGT-3 ′ was prepared, and pET-WDV4 was prepared by ligation with the three-finger AZP gene in pET-WDV3-1 by the method described above. Finally, 6-finger AZP gene and 4-finger AZP gene from pET-WDV4 and pET-WDV6, respectively, are amplified by PCR and linked to recognize 31 consecutive bases including the upstream stem region and its flanking region. A plasmid (pET-WDV10) encoding AZP-11 was prepared.
- AZP-12 was produced with the scheme shown in FIG. First, genes with linked zinc fingers were synthesized by PCR, and each gene was cloned into the BamH I / HindHIII site of pET-21a (Novagen) of the E. coli expression vector. As a result, pET-WDV3-4, pET-WDV3-5, pET-WDV3-6, and pET-WDV3-7 were obtained. Next, the three-finger AZP genes in pET-WDV3-4 and pET-WDV3-5 were amplified and ligated by PCR to finally obtain pET-WDV6-2.
- pET-WDV3-6 and pET-WDV3-7 were amplified by PCR and ligated to finally obtain pET-WDV6-3.
- 6-finger AZP genes from pE-WDV6-2 and pET-WDV6-3 are each amplified by PCR and ligated to recognize 37 consecutive bases including the downstream stem region and its flanking region.
- a plasmid (pET-WDV12) encoding AZP-12 was prepared.
- AZP-13 was produced with the scheme shown in FIG. First, genes with linked zinc fingers were synthesized by PCR, and each gene was cloned into the BamH I / HindHIII site of pET-21a (Novagen) of the E. coli expression vector. As a result, pET-WDV3-8, pET-WDV3-9, and pET-WDV3-10 were obtained. Next, the three-finger AZP genes in pET-WDV3-9 and pET-WDV3-10 were amplified by PCR and ligated to finally obtain pET-WDV6-4. Finally, a 6-finger AZP gene was amplified from pE-WDV6-4 by PCR and ligated to prepare a plasmid (pET-WDV9) encoding AZP-13 that recognizes 28 bases of the stem loop region.
- Example 2 Materials and methods (1) Preparation of stable expression vector for plant of AZP Each gene fragment encoding AZP11 and AZP12 designed in Example 1 was inserted into the enzyme cleavage site in the multicloning site of binary vector pUBIN-ZH2.
- the binary vector pUBIN-ZH2 contains a cauliflower mosaic virus 35S promoter, a nopaline synthase terminator and a T-DNA portion of pPZP202 (P. Hajdukiewicz, Z. Svab, P. Maliga, 1994. Plant Molecular Biology 25: 989-994).
- T1 generation T0 generation individuals in which PCR bands were confirmed were grown and T1 seeds were obtained.
- the obtained T1 seeds were germinated, the genome was extracted from the leaves of the grown individuals, and PCR was performed. The method was carried out in the same manner as described in (3) above.
- Transgene expression was checked by PCR using 1 ⁇ L of the cDNA solution prepared in (5). The number of PCR cycles was 30.
- a primer a primer set that amplifies a region containing AZP was designed and used. In addition, it was confirmed by sequence analysis that the obtained band was an AZP fragment.
- AZP-transformed wheat The AZP gene was introduced into wheat through Agrobacterium.
- a binary vector having an AZP expression cassette was prepared by introducing the AZP gene into the binary vector shown in FIG. 34, and the gene was introduced by infecting wheat seeds with Agrobacterium transformed with this vector.
- the transformed seed was transferred to a pot containing culture soil and grown at 25 ° C. under long-day conditions (16 hours light period, 8 hours dark period).
- T1 seed obtained from the transformed individual was germinated, and it was confirmed by PCR that the grown T1 generation individual had the AZP gene.
- PCR was performed using the PCR primer set shown in FIG. As shown in FIG. 36, since the target fragment was detected in several individuals (Nos. 4, 5, and 7), it was confirmed that the transformation operation was successfully performed. It was confirmed by sequence analysis that the obtained fragment was a ubiquitin promoter and an AZP gene fragment.
- Example 3 Materials and methods (1) Preparation of plasmid for WDV infection Yunnan Kunming type (Accession Number: EU541489) of WDV was used for infection.
- the WDV binary plasmid for infection was prepared in three stages as shown below. First, the DNA fragment of one copy of WDV was cloned into the cloning plasmid pBluescript II KS +.
- a DNA fragment of one copy of WDV was synthesized by reconstitution from a synthetic DNA oligomer by PCR, the DNA end was cleaved with Bsa I and Hind III, and the obtained DNA fragment was then converted into pBluescript II KS + Acc65 I PBS-WDV was obtained by cloning into the / Hind III site. The nucleotide sequence was confirmed to be correct by sequencing.
- a DNA fragment of one copy of the viral genome was excised from pBS-WDV with BsiW I and Hind III, purified on an agarose gel, cloned into the BsiW I / Hind III site of pBI-WDV (0.5), and finally The target plasmid pBI-WDV (1.5) was obtained.
- WDV was inoculated in the same manner as above for each of three randomly selected T1 transformants prepared by introducing AZP11 or AZP12 gene. On the 20th, the leaves were collected, and whether or not WDV genomic DNA was detected was examined by PCR. As shown in FIG. 39, no WDV viral DNA was detected in any transformed wheat, and no viral growth was observed.
- the replication inhibitor of the present invention can exhibit high effectiveness against WDV and other viruses belonging to the genus Masterovirus, it is extremely useful as a control means for various viruses belonging to the genus Masterovirus.
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Abstract
Description
参考例1
1.材料と方法
(1)AZPのデザイン
以下の2種類のDNA領域をそれぞれ認識するジンクフィンガータンパク質(以下、実施例において「AZP」と略す)を特表2004-519211号公報に記載された認識コード表に基づいてデザインした。
a.TYLCVで保存されているステムループ領域
b.ジェミニウイルスで保存されているステムループ領域
図6の上段に示したAZP(TYLCV専用)では10個のジンクフィンガードメインを連続的に結合した。図6の下段に示すAZP(ジェミニウイルス汎用)ではステムループ領域内でジェミニウイルスに保存されている2つの領域を認識する2種のAZPを短いペプチドで連結した。
TYLCV専用AZP(以下、「AZP-2」と呼ぶ)を図7に示すスキームで作製した。まず3個ずつジンクフィンガーを連結した遺伝子をPCRにより合成し、それぞれの遺伝子を大腸菌発現ベクターのpET-21a(Novagen社)のBamH I/Hind IIIサイトにクローニングした後、得られたプラスミドの塩基配列を確認することにより、pET-TYLCV-3、pET-TYLCV-4、及びpET-TYLCV-5を得た。次にpET-TYLCV-3及びpET-TYLCV-4内の3フィンガーAZPの遺伝子をPCRにより増幅して連結し、最終的にpET-TYLCV3/4を得た。5'-TATA-3'を認識するジンクフィンガー遺伝子を作製し、上述した方法でpET-TYLCV5内の3フィンガーAZP遺伝子と連結することによりpET-TYLCV6を作製した。最後に、pE-TYLCV3/4及びpET-TYLCV6からそれぞれ6フィンガーAZP遺伝子及び4フィンガーAZP遺伝子をPCRにより増幅して連結することにより、ステムループ領域の配列を形成する33塩基のうち31塩基を認識するAZP-2発現用プラスミド(pET-TYLCV3/4/6)を作製した。
AZP発現プラスミドで大腸菌BL21(DE3)を形質転換し、得られた形質転換体をアンピリシリンを含むLB培地で37℃で培養し、OD600が0.6-0.7になったときにIPTGを最終濃度1 mMになるように添加し、目的タンパク質の発現を誘導した。さらに3時間培養した後、遠心分離により大腸菌を回収し、タンパク質精製まで-80°Cに保存した。
各AZPは基本的に同じ方法で精製した。-80℃で保存した大腸菌にlysis buffer(100 mM Trs-HCl、100 mM NaCl、0.1 mM ZnCl2、5 mM DTT、pH 8.0)10 mlを加え、凍結及び融解を3回繰り返して大腸菌の細胞壁を壊れやすくした。次に超音波破砕機にかけて大腸菌を破砕した後、遠心分離することにより目的タンパク質を含む上清を回収した。この上清を陽イオン交換樹脂のBiorex-70(Bio-Rad社)にアプライして目的タンパク質を樹脂に吸着させた後、wash buffer(50 mM Trs-HCl、50 mM NaCl、0.1 mM ZnCl2、0.2 mM DTT、pH 8.0)で十分洗浄した。次に、elution buffer(50 mM Trs-HCl、300 mM NaCl、0.1 mM ZnCl2、0.2 mM DTT、pH8.0)で目的タンパク質を溶出させた。目的タンパク質を含むフラクションのみを集め、限外ろ過膜で濃縮後、等量のグリセロールを加えて撹拌した後、-80℃にて保存した。AZP純度はSDS-PAGE上のクマシーブルー染色のバンドの量で判断した。各タンパク質の濃度は、Protein Assay ESL(Roche社)を用いて決定した。
RepNはウイルス複製タンパク質RepのN末領域部(191アミノ酸残基)でDNA結合能を有している。AZPによるRepのdirect repeatsへの結合の阻害実験に用いるためにRepNを以下の方法で調製した。感染したトマトから回収したTYLCVゲノムを用いてRepN遺伝子をPCRによりTYLCVゲノムから増幅し、AZPの場合と同様に、pET-21aのBamH I/Hind IIIサイトにクローニングした。得られたプラスミドの塩基配列を確認することによりRepN発現用のプラスミド(pET-RepN)を作製した。
RepNの発現はAZP発現の場合と同様に行い十分量の発現を得た。得られた大腸菌は、タンパク質精製まで-80°Cに保存した。RepNの精製はAZPの場合と同様に行った。Biorex-70を用いたイオン交換クロマトグラフィーにおいて、elution buffer(50 mM Tris-HCl、250 mM NaCl、0.2 mM DTT、pH8.0)により溶出することにより、純度の高いRepNを得ることができた。
各タンパク質の標的DNA配列への結合能の評価はゲルシフトアッセイにより行った。標的DNA配列を含むDNAオリゴマーを作成し、5'末端を32Pで標識した。次に標識DNAを含むbinding buffer(10 mM Tris-HCl、100 mM NaCl、5 mM MgCl2、0.1 mM ZnCl2、0.05% BSA、10% glycerol、pH7.5)に所定量のタンパク質を添加し、氷上で1時間反応させた。この反応物を6%非変性アクリルアミドゲルにアプライし、4℃で2時間電気泳動した(running buffer:45 mM Tris-borate)。泳動後、ゲルをクロマト紙に載せて乾燥させた。十分乾燥した後にX線フィルムに感光させ、標識DNAのバンドを検出した。遊離DNAとタンパク質とのDNA複合体の量比が1:1になるときのタンパク質濃度が標的DNA配列との解離定数に相当する。そのタンパク質濃度に基づいてAZP及びRepNの結合能の比較を行った。
(a)Rep発現プラスミドの作製-1
切断阻害能の評価には切断活性を有するfull lengthのRepが必要となるので、Rep発現プラスミドの作製を行った。RepN発現プラスミドの作製と同様に、Rep遺伝子はPCRによりTYLCVゲノムから増幅し、pET-21aのBamH I/Hind IIIサイトにクローニングした。得られたプラスミドの塩基配列を確認することによりRep発現用のプラスミド(pET-Rep)を作製した。
Rep単独では、大腸菌破砕後に可溶化の状態で検出できない場合があることから、溶けにくいタンパク質の可溶化を促進し、かつ精製が簡便なglutachione S-transferase(GST)との融合体としてRepを作製した。T7プロモーター及びGST遺伝子を含むDNA領域をGST融合タンパク質発現用のプラスミド(pET-41a, Novagen社)からPCRにより増幅し、pET-RepのBamH I/Sph Iサイトにクローニングした。DNA塩基配列を確認することにより、GST-Repタンパク質発現用のプラスミド(pET-GST-Rep)を作製した。
3種類の大腸菌、BL21(DE3)、Rosetta 2(DE3)pLysS、及びBL21-Codon-Plus(DE3)-RILをそれぞれpET-GST-Repで形質転換し、得られた各クローンをRepNタンパク質発現時と同様に37℃、1 mM IPTGで発現誘導した。それぞれの大腸菌で発現量は同じであったが、大腸菌破砕後のGST-Repの可溶化量はBL21(DE3)において最も高かった。そこでBL21(DE 3)形質転換体を用いて30℃でのタンパク質発現を行った。
大腸菌ペレットをLysis Buffer (4.3 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH7.3, 0.1 mM ZnCl2, 5 mM DTT) 3 mLに懸濁し、ソニケーションを行った。GST- Repタンパク質の可溶化をSDS-PAGEで確認後、遠心分離して上清のみを取り出した。20倍量の1x GST-Bind Wash Bufferであらかじめ洗浄したGST結合レジンを15 mLコニカルに移し、さらに1x GST-Bind Wash Buffer (4.3 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH7.3) 5 mLで洗浄し、400×g、25℃、5 分間遠心して上清を丁寧に取り除いた。この前処理したレジンにソニケーション後のGST- Repタンパク質を含む上清を0.45μmメンブレンフィルターでろ過したものを添加した。4℃で一晩振盪して、レジンにGST-AZPタンパク質を吸着させた。このレジンをカラムに流し、Washing Buffer (4.3 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1 mM ZnCl2)で洗浄後、Elution Buffer (50 mM Tris・HCl, pH8.0, 0.1 mM ZnCl2, 10 mM reduced glutathione)で溶出した。溶出した各フラクションをSDS-PAGEで確認し、GST- Repタンパク質を含むフラクションを集め、限外ろ過膜で全量が300μLになるまで濃縮した。タンパク質濃度は市販のキット(Protein Assay ECL)で決定した。
AZPをglutachione S-transferase(GST)との融合体とし、そのGST-AZP遺伝子をT7プロモーターの下流においた、発現ベクターを大腸菌に導入した。この大腸菌をLB-Amp液体培地120 mLでOD600が0.65~0.75になるまで培養した。培養後, 最終濃度が1 mMとなるようにIPTGを添加し、さらに3 時間培養することにより、GST-AZPタンパク質を誘導発現させた。誘導後の大腸菌を遠心して回収して-80℃に保存した。GST-AZPタンパク質の精製はGST-Repタンパク質の精製と同じ方法で行った。
Repの結合サイトを含む200塩基対からなる標識DNA(5 nM)を含む反応溶液(25 mM Tris-HCl, pH7.5, 75 mM NaCl, 2.5 mM DTT)にGST-AZP(又は性能比較実験のためにGST-RepN若しくはコントロール実験のためGST)を添加して混合し、氷上に30分間静置した。その後、最終濃度が2μM及び5 mMとなるようにGST-Rep及びMgCl2をそれぞれ添加した後、25℃で反応させた。30分後、0.5 M EDTAを2μLを添加して反応を終了させ、フェノール処理及びエタノール沈殿を行った。Loading Buffer (80 % formamide, 10 mM EDTA ) 3μLで溶解して作製したサンプルを8%変性アクリルアミドゲルにおいて電気泳動した。
(1)AZPの標的DNA配列への結合能の評価
精製したAZP及びRepNの標的DNA配列への結合能をゲルシフトアッセイにより評価した。この実験においては、32PでラベルしたDNAに各種濃度でタンパク質を添加し結合反応を行わせた後、遊離DNAとタンパク質とのDNA複合体を非変性ゲル上で分離する。遊離DNAとタンパク質とのDNA複合体のバンドの比が1:1になるタンパク質濃度(解離定数に相当)を求めたところ、TYLCV専用のAZP-2の解離定数は0.3~1 nM(図9)、ジェミニウイルス汎用のAZP-3の解離定数は<10 nM(図10)であることがわかった。一方、RepNの解離定数は30 nMであった(図11)。この実験により、デザインしたAZP-2及びAZP-3の標的DNA配列に対する結合力はいずれもRepNより強いことが確認された。
精製したTYLCV専用のGST-AZP(AZP-2)は図12のレーン4~7に示されているようにRepによる複製起点の切断を効果的に阻害できた。その阻害効果はAZP濃度に依存しており、20μMで完全な阻害が認められた。一方、Repのドミナントネガティブ体であるRepNでは全く切断阻害が認められなかった(図12のレーン8~11)。RepNはDNA結合ドメインを有しており、当然のことながら阻害したいRepとはDNA結合が全く同じである。GSTについてはレーン12に見られるように切断阻害が全く見られないことからも、レーン4~7で確認されたGST-AZPの切断阻害活性はもっぱらAZPに由来するものであることが確認された。また、GST-AZP(AZP-3)についても同様にして切断阻害能を評価した。その結果を図13に示す。
1.材料と方法
(1)AZP形質転換トマトの作製
(a)AZPの植物用安定的発現ベクターの作製
AFP-2をコードする遺伝子の植物ゲノムへの挿入はアグロバクテリア法により行った。プロトプラスト実験用にpUC35SO-TYLCV3/4/6をpUC35SO-MCSから図14の方法で調製し、このプラスミドから35Sプロモーター‐AZP遺伝子‐NOSターミネーターを含む領域をEcoR I及びHind IIIで切り出した。断片をアガロースゲル上で精製した後、バイナリープラスミドpBI121のEcoR I/Hind IIIサイトにクローニングしてpBI-OTYLCV3/4/6を得た。シークエンシングにより塩基配列が正しいことを確認した。AZP-3についても同様の操作を行った。
72穴のプラスチックトレイに栽培土をつめ、如雨露で軽く土を湿らせた後、Micro-Tomの種をひとつずつ蒔き、その上に湿らせた土を軽くかぶせ、全体をサランラップで覆った。このトレイを人工気象室(明期:25°C、16時間;暗期:22°C、8時間)で培養した。発芽が認められた時点でサランラップをはずし、培養を同一条件下で培養を継続した。播種後約2週間経過後に苗を直径12 cmのプラスチックポットに移し、種を回収するまで培養した。
赤く熟したMicro-Tomの実を回収し、赤道線上でナイフで2つに分割し、スパチュラですべての種を50 mlプラスチックチューブに回収した。水で軽く洗った後、1%塩酸水で10分間洗浄し、種の周囲のゼラチン層を溶解させた。次に流水で10分間種を洗浄し、余分な水分をペッパータオルで吸い取った後、室温で2日間風乾させた。乾燥した種は4℃で保存した。
Micro-Tomの種子10~20 粒を10%希釈したハイター(花王)で殺菌した後、滅菌水を用いて4回洗浄した。この種子を播種用培地(1×Murashige-Skoog (MS) 培地 、15 g/L sucrose、3 g/L gelrite)を固めたプラントボックスに播種し、6日間、25℃、16時間日長の条件で生育させた。本葉が数ミリ程度になった個体を形質転換に用いた。
形質転換植物の1~2 cmの葉をマイクロチューブに採取した。これに液体窒素を加えて凍結させ、ホモジナイズペッスルを用いて細かく砕いた。液体窒素が気化した後、200μLのSDS サンプルバッファー(0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.01% BPB, 10% 2-ME])を加え、さらにすりつぶした。95℃で10分間保温したあと、遠心後に上清を新しいマイクロチューブに移した。これを植物抽出タンパク質のサンプルとした。
抽出したタンパク質のうち1μLを12% SDSポリアクリルアミドゲルを用いて電気泳動した。分子量マーカーとしてPerfect Protein Western Marker (Novagen)を同時に泳動した。タンパク質をアクリルアミドゲルからPVDFメンブレンに転写したあと、ポンソーSを用いてタンパク質を確認した。メンブレンをブロッキング液(5% スキムミルク、0.05% tween 20, PBS)で振盪したのち、ペルオキシダーゼ標識-抗HA抗体を反応させた。分子量マーカーに対する抗体としてS-protein HRPも同時に反応させた。ECL化学発光システムを用いてX線フィルムを感光させ、シグナルを検出した。このシグナルのサイズとシグナル強度から、形質転換植物内でAZPが発現しているかどうかを検証した。
(a)ウイルス感染用プラスミドの作製
ウイルス感染はアグロバクテリアの感染力を利用して行った。複製起点を2つ有するウイルスゲノムコピーをバイナリープラスミドに導入するために、TYLCV及びTYLCV-mildの2種類について、目的プラスミドの作製を以下に示す2段階で行った。TYLCV-mildはTYLCVとはRepの結合するdirect repeats配列が異なり、ジェミニウイルスに対する汎用性を調べる目的で用いた。
アグロバクテリア C58C1RifR (GV2260) のコンピテントセルを作製した。このコンピテントセルに、作製したTYLCVゲノムあるいはTYLCV-mildゲノムを1.5コピー有するバイナリーベクターを導入し、アグロイノキュレーション用のアグロバクテリアのグリセロールストックを作成し、-80℃に保存した。野生型トマトに感染する前日に、このグリセロールストックを6 mLのLB 培地 (Kan 100 mg/L、Amp 50 mg/L) に植菌し、30℃で一昼夜培養した。次にアグロバクテリアを集菌し、バッファー1 mLに懸濁させた。この懸濁液を播種後約10日の苗の子葉に注入して、感染させた。感染後定期的に植物個体の観察及び葉の中のウイルスDNAの検出を行った。そのためのDNAサンプルの作製は上述したように行い、それぞれのTYLCVに特異的なプライマーセットを用いて行って得たPCR生成物の解析により、ウイルス感染を分子レベルで評価した。
形質転換体T3からの苗にウイルスバイナリーベクターを保持するアグロバクテリアの懸濁液を注入し、感染症状を経時的に肉眼で確認した。また、感染させたトマトの葉からDNAを抽出し、植物体内でウイルスが増殖しているかどうかを、前項の方法に従ってPCRで検証した。
(1)AZP形質転換トマトの作製
Micro-TomトマトにAZP遺伝子をそれぞれアグロバクテリアを介して導入した。図15に示す各AZP発現カセットを有するバイナリーベクターで形質転換されたアグロバクテリアを子葉切片に感染させて遺伝子を導入した。次にカナマイシンを含む培地を用いてカルスを誘導させ、シュート、次に根を誘導させた。発根が深く寒天培地に伸びている個体を選ぶことにより発根誘導時に形質転換体をさらに選別し、順化後、土に植替えることにより形質転換体を得た。
得られたT1植物におけるAZP遺伝子のコピー数は、T2植物のAZP遺伝子挿入個体の割合を調べ、カイ2乗検定により同定した。すなわち、各T1ラインからT2種子を回収し、それらを播種して得られた各T2個体でのAZP遺伝子の有無をPCRにより同定した。AZP-2を導入して得られたT2植物のうち、各々ひとつのラインを例として図19に示した。図19を例に取ると、PCR法により特定のT1ラインから得られた18個体のT2植物中、13個体がAZP遺伝子を有しており、分離比は13対5となる。もし、このT1ラインがAZP遺伝子1コピーを有しているのであれば、その分離比は3対1となるはずである。そこでカイ2乗検定により1コピーと仮定するとカイの2乗値は0.074であり、P = 0.01の臨界値は6.63であることから、この帰無仮説は棄却されない。他方、2コピー挿入と仮定すると、カイの2乗値は14.2となり、臨界値より大きくなり、この帰無仮説は棄却される。以上の検証結果から、このT1ラインは1コピー挿入体であることがわかる。そのほかのT1個体についても同様にして1コピー挿入体の選別を行った(図20)。
アグロイノキュレーション法によりMicro-Tomトマトを感染させることが可能かどうかを検証した。複数の野生型Micro-TomにTYLCVゲノムを有するアグロバクテリアを注入し、TYLCVの感染を試みた。複数回の試験を行った結果、各回とも高効率で感染させることができた。感染後約10日には若い葉においてTYLCV感染の特徴的な葉の縮退が観察された。さらに成長させた個体ではTYLCV感染の特徴的症状である葉のカーリングや黄色化が明確に観察された。感染した個体では明白な成長の阻害が認められ(図24)、開花は多いものの、結実する確率は著しく低かった。
AZP-2を導入して作製したT1植物のうちの3個体からそれぞれ得られたホモのT2ライン(表1参照)から得られたT3植物に対して上記と同様にしてTYLCVを感染させた。図25に示されるとおり、感染した野生型(図の左側の植物)に見られるような葉の萎縮や黄色化は、形質転換トマトにおいては認められなかった。さらに、感染耐性をPCRにより分子レベルで評価した。図26に示されているように、T3ホモ体でウイルスDNAは検出されなかった。また、ホモ体だけでなく、ヘテロ体でもウイルスDNAは検出されず、ウイルスの増殖は見られなかった。
(1)WDVをターゲットとするAZPのデザイン
以下の2種類のDNA領域をそれぞれ認識するジンクフィンガータンパク質を特表2004-519211号公報に記載された認識コード表に基づいてデザインした。
a.上流側のステム領域とそのフランキング領域
b.ステムループ領域
c.下流側のステム領域とそのフランキング領域
AZP-11を図31に示すスキームで作製した。まず3個ずつジンクフィンガーを連結した遺伝子をPCRにより合成し、それぞれの遺伝子を大腸菌発現ベクターのpET-21a(Novagen社)のBamH I/Hind IIIサイトにクローニングした後、得られたプラスミドの塩基配列を確認することにより、pET-WDV3-1、pET- WDV3-2、及びpET- WDV3-3を得た。次にpET- WDV3-2及びpET- WDV3-3内の3フィンガーAZPの遺伝子をPCRにより増幅して連結し、最終的にpET-WDV6を得た。5'-GGGT-3'を認識するジンクフィンガー遺伝子を作製し、上述した方法でpET-WDV3-1内の3フィンガーAZP遺伝子と連結することによりpET-WDV4を作製した。最後に、pET-WDV4及びpET-WDV6からそれぞれ6フィンガーAZP遺伝子及び4フィンガーAZP遺伝子をPCRにより増幅して連結することにより、上流側のステム領域とそのフランキング領域を含む連続した31塩基を認識するAZP-11をコードするプラスミド(pET-WDV10)を作製した。
1.材料と方法
(1) AZPの植物用安定的発現ベクターの作製
上記実施例1で設計したAZP11及びAZP12をコードする各遺伝子断片をバイナリーベクターpUBIN-ZH2のマルチクローニングサイト中の当該酵素切断部位に挿入した。バイナリーベクターpUBIN-ZH2は、pPZP202(P. Hajdukiewicz, Z. Svab, P. Maliga, 1994. Plant Molecular Biology 25: 989-994)のT-DNA部分に、カリフラワーモザイクウイルス35Sプロモーターとノパリンシンターゼターミネーターとの間にハイグロマイシン耐性遺伝子を導入したカセット、及びトウモロコシユビキチン遺伝子プロモーター (Plant physiology Volume 100, 1992, Pages 1503-1507)とノパリンシンターゼターミネーターとの間にマルチクローニングサイトを持つ遺伝子発現用カセットを組み込んだものである(図34)。このようにしてAZP11及びAZP12を含む2種類の植物用安定的発現ベクターを作製した。
上記(1)で得られた形質転換用ベクターを用いて、凍結融解法(Hofgen et al.(1998) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. Oct 25;16(20):9877)によりアグロバクテリウム(LBA4404株)を形質転換した。さらに、上述の手法にて取得したアグロバクテリウムの形質転換体を用いて、コムギ(品種:ハルヨコイ)の形質転換を実施した。コムギの形質転換には特許第4754968号に記載のインプランタ形質転換法を用いた。
上記(2)で得られた形質転換処理個体を培養土の入ったポットに移し、23℃、長日条件(16時間明期、8時間暗期)にて生育させた。目的の遺伝子が導入されているかどうかをPCRによって確認した。T0世代の形質転換個体が第6葉期まで成長した段階で、約5 mmの本葉1枚を切り取り、CTAB法によってゲノム DNAを抽出した。ゲノム DNA溶液(10ng/1μL )1μLを用いてPCR法により遺伝子導入をチェックした。プライマーとしては、ユビキチンプロモーターとAZPを含む領域が増幅されるプライマーセットを設計して用いた。
PCRのバンドが確認できたT0世代個体を生育し、T1種子を取得した。得られたT1種子を発芽させ、生育した個体の葉よりゲノムを抽出し、PCRを実施した。方法は上記(3)に記載の方法と同一の方法にて実施した。
形質転換植体T1世代の葉(第1~第2葉)をマイクロチューブに採取し、RNeasy Plant Mini Kit(QIAGEN社製)を用いて、total RNAを抽出した。方法はキットのマニュアルに従った。得られたtotal RNA 1ugを用いて、High Capacity RNA-to-cDNA(登録商標) Kit (アプライドバイオシステムズ社製)によりcDNAを合成した。
(5)にて調製したcDNA溶液1μLを用いてPCR法により導入遺伝子発現をチェックした。PCRのサイクル数は30サイクルとした。プライマーとしては、AZPを含む領域が増幅されるプライマーセットを設計して用いた。また、得られたバンドがAZP断片である事を、シークエンス解析により確認した。
(1)AZP形質転換コムギの作製
コムギへのAZP遺伝子導入はそれぞれアグロバクテリアを介して実施した。図34に示すバイナリーベクターにAZP遺伝子を導入してAZP発現カセットを有するバイナリーベクターを調製し、このベクターで形質転換されたアグロバクテリウムをコムギ種子に感染させて遺伝子を導入した。形質転換処理した種子を培養土の入ったポットに移し、25℃、長日条件(16時間明期、8時間暗期)にて生育させた。
形質転換処理した個体から得られた種子(T1種子)を発芽させ、生育したT1世代の個体がAZP遺伝子を有することをPCR法により確認した。ユビキチンプロモーターとAZPの断片を増幅するため、図35に示すPCRプライマーセットを用いてPCRを行った。図36に示すように、いくつかの個体(No. 4, 5, 7)において目的の断片が検出されたことから形質転換操作がうまく行われたことを確認できた。なお、得られた断片がユビキチンプロモーター及び、AZP遺伝子の断片であることをシークエンス解析により確認した。
さらに各形質転換体でのAZP発現をRT-PCRで確認した。プライマーとしては、AZPを含む領域が増幅されるプライマーセットを設計して用いた(図37)。図38に示すように、AZP11及びAZP12を導入したT1個体においてAZPが強く発現されていることが確認できた。なお、得られた断片がAZP遺伝子の断片であることをシークエンス解析により確認した。
1.材料と方法
(1)WDV感染用プラスミドの作製
感染にはWDVのうちYunnnan Kunming型(Accession Number: EU541489)を用いた。感染用のWDVバイナリープラスミドの作製は、以下に示すように3段階でおこなった。
まず、WDVの1コピー分のDNAフラグメントをクローニングプラスミドpBluescript II KS+にクローニングした。すなわち、WDVの1コピー分のDNAフラグメントを合成DNAオリゴマーからPCRにより再構成して合成した後、DNA末端をBsa I及びHind IIIで切断した後、得られたDNAフラグメントをpBluescript II KS+のAcc65 I/Hind IIIサイトにクローニングし、pBS-WDVを得た。シークエンシングにより塩基配列が正しいことを確認した。
アグロバクテリア C58C1RifR (GV2260) のコンピテントセルを作製した。このコンピテントセルに、作製したWDVゲノムを1.5コピー有するバイナリープラスミドを導入し、アグロイノキュレーション用のアグロバクテリアのグリセロールストックを作製し、-80℃に保存した。小麦に感染する前日に、このグリセロールストックを6 mLのLB 培地 (Kan 100 mg/L、Amp 50 mg/L) に植菌し、30℃で一昼夜培養した。次にアグロバクテリアを集菌し、バッファー1 mLに懸濁させた。この懸濁液を播種後約20日の苗の茎に注入して、感染させた。感染後植物個体の葉の中のウイルスDNAの検出を行った。そのためのDNAサンプルの作製は上述したように行い、WDVのYunnnan Kunming型に特異的なプライマーセットを用いて行ったPCR生成物の解析により、ウイルス感染を分子レベルで評価した。
AZP11あるいはAZP12遺伝子を導入して得られたT1形質転換体からの苗にWDVウイルスバイナリープラスミドを保持するアグロバクテリアの懸濁液を注入した。感染させた小麦の葉からDNAを抽出し、植物体内でウイルスが増殖しているかどうかを、前項の方法に従ってPCRで検証した。
(1)WDVバイナリープラスミドの作製およびウイルス感染の確認
アグロイノキュレーション法により小麦を感染させることが可能かどうかを検証した。複数の野生型小麦(品種:「春よ来い」)にWDVゲノムを有するアグロバクテリアを注入し、WDVの感染を試みた。アグロバクテリア注入後20日に若い葉を回収し、DNAを抽出した。このDNAサンプルを用いたPCR法により、アグロバクテリアを注入した野生型小麦個体において、回収した葉でWDVゲノムDNAを検出することができた。その一例を図39に示す。
AZP11又はAZP12遺伝子を導入して作製したT1形質転換体のうち無作為に選んだ3個体それぞれに対して上記と同様にしてWDVを接種し、接種後20日に葉を回収し、WDVゲノムDNAが検出されるかどうかをPCR法により調べた。図39に示されるとおり、すべての形質転換小麦でWDVウイルスDNAは検出されず、ウイルスの増殖は見られなかった。
Claims (13)
- ジェミニウイルス科のマステレウイルス属に属するウイルスに対する複製阻害剤であって、該ウイルスのステムループ領域の全長DNA又は該全長DNAから選ばれる1又は2以上の部分DNAに特異的に結合することができるジンクフィンガータンパク質を含み、かつステムループ構造の形成を阻害することができる複製阻害剤。
- マステレウイルス属に属するウイルスのステムループ領域の全長DNAから選ばれる1個の部分DNAに結合可能な単一のジンクフィンガータンパク質を含む請求項1に記載の複製阻害剤。
- マステレウイルス属に属するウイルスのステムループ領域の全長DNAから選ばれる1個の部分DNAと、該DNAに結合しフランキング領域から選ばれる1個のDNAとからなる連続したDNAに結合可能な単一のジンクフィンガータンパク質を含む請求項1に記載の複製阻害剤。
- 上記ジンクフィンガータンパク質が9個ないし12個のジンクフィンガードメインを含むジンクフィンガータンパク質である請求項1ないし3のいずれか1項に記載の複製阻害剤。
- マステレウイルス属に属するウイルスがコムギ萎縮病ウイルスである請求項1ないし4のいずれか1項に記載の複製阻害剤。
- 請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質をコードする核酸。
- 請求項6に記載の核酸を含む植物形質転換用の組換えベクター。
- 請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質又は該ジンクフィンガータンパク質をコードする核酸を有効成分として含む農薬。
- ジェミニウイルス科のマステレウイルス属に属するウイルスによる植物の感染を予防する方法であって、請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質又は該ジンクフィンガータンパク質をコードする核酸の予防有効量を植物に施用する工程を含む方法。
- ジェミニウイルス科のマステレウイルス属に属するウイルスに対して耐性を有する植物であって、請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質を発現可能な遺伝子組み換え植物。
- 請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質をコードする核酸を導入することにより形質転換された植物。
- ジェミニウイルス科のマステレウイルス属に属するウイルスに対する耐性を植物に獲得させる方法であって、請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質をコードする核酸を該植物に導入して形質転換する工程を含む方法。
- 請求項1ないし5のいずれか1項に記載のジンクフィンガータンパク質をコードする核酸を含む植物形質転換用ベクター。
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