WO2017131079A1 - Method for producing virus-free plant body - Google Patents

Method for producing virus-free plant body Download PDF

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WO2017131079A1
WO2017131079A1 PCT/JP2017/002698 JP2017002698W WO2017131079A1 WO 2017131079 A1 WO2017131079 A1 WO 2017131079A1 JP 2017002698 W JP2017002698 W JP 2017002698W WO 2017131079 A1 WO2017131079 A1 WO 2017131079A1
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virus
plant
shoot
tissue piece
solution
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PCT/JP2017/002698
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French (fr)
Japanese (ja)
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税 増田
葉子 長谷部
周平 羽城
寿 安枝
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国立大学法人北海道大学
味の素株式会社
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Priority to JP2017563811A priority Critical patent/JP6915870B2/en
Publication of WO2017131079A1 publication Critical patent/WO2017131079A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a method for producing a virus-free plant body, particularly a virus-free plant body from a plant species in which it is difficult to remove the plant virus by normal shoot apical culture.
  • Patent Document 1 A large number of plant viruses that adversely affect yield, quality, etc. by infecting agricultural crops are known.
  • a technique for obtaining a so-called virus-free plant body free from such virus infection there is a shoot tip culture in which the shoot tip is cultured aseptically (for example, Patent Document 1).
  • the shoot apex consists of a hemispherical apical meristem (also called a meristem) and several leaf primordia differentiated therefrom.
  • the shoot apical culture is aseptically thinned to contain only the apical meristem. It is a technique for producing a virus-free plant by cutting out and regenerating the plant from there.
  • cutting only the apical meristem from the shoot apex means cutting a small tissue piece of about 0.1 to 1 mm, which requires high skill of the operator.
  • the possibility of virus removal increases as the shoot tip tissue piece to be cut out becomes smaller, there also arises a problem that it takes a long time for the subsequent regeneration and cultivation of the plant body.
  • it is difficult to say that conventional shoot tip culture is technically and economically efficient.
  • An object of the present invention is to provide a method for producing a virus-free plant that can be applied to plant species and virus species that are difficult to remove by general shoot apical culture.
  • the present inventor has found that in the shoot tip culture, the production efficiency of the virus-free plant is improved by immersing the tissue piece of the shoot tip in a solution containing an interfering nucleic acid capable of suppressing the growth of the virus, etc.
  • the following inventions were completed.
  • a virus-free method comprising a step of immersing a shoot apical tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus, and a step of regenerating a plant body from the shoot apical tissue piece collected from the solution A method for producing a plant body.
  • the production method according to (1) further comprising a step of centrifuging the solution soaked with the shoot apical tissue piece at a centrifugal acceleration of 6000 ⁇ g or less.
  • interfering nucleic acid is siRNA.
  • a method for removing a plant virus from a shoot tip tissue piece comprising a step of immersing the shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus.
  • a virus-free plant can be produced from a plant species in which virus removal by general shoot tip culture has been difficult.
  • the shoot tip tissue piece in the shoot tip culture is larger than usual, in particular, it can be made into a tissue piece containing not only the apical meristem but also the first leaf primordia, greatly increasing the time required for plant regeneration and subsequent cultivation. Can be shortened.
  • FIG. 2 is an enlarged view of a part of the photograph of FIG. 2 is a photograph showing the results of non-denaturing PAGE electrophoresis of dsRNA prepared as an interfering nucleic acid for the ⁇ -glucuronidase gene and the coat protein gene of Alexis virus and siRNA prepared by treating these with Dicer. It is a photograph showing the result of RT-PCR detection of the presence of Alexis virus after 20 days in culture of Spanish garlic shoot apex treated with water (negative control) or GUS-siRNA (siRNA control).
  • the present invention relates to a virus comprising a step of immersing a shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus, and a step of regenerating a plant body from the shoot tip tissue piece recovered from the solution.
  • the present invention relates to a method for producing a free plant.
  • the present invention can also be expressed as a method for removing plant viruses from shoot tip tissue pieces, which includes a step of immersing the shoot tip tissue pieces in a solution containing an interfering nucleic acid capable of suppressing the growth of plant viruses.
  • the present invention can be applied to any plant species for which production of virus-free plants by shoot tip culture is desired. However, it is preferable to produce virus-free plants by general shoot tip culture. Applied to different plant species.
  • the virus to be removed is not particularly limited, but preferably the present invention is applied to a virus that easily reaches the shoot apex, particularly the apical meristem.
  • Examples of combinations of plant species and virus species to which the present invention is suitably applied include Alexi virus against garlic, asparagus virus 2 (AV2) against asparagus, potato virus S (PVS) against potato, and the like. Viroids that easily penetrate into the shoot apical meristem are also included. These are examples of combinations of plant species and virus species for which it was difficult to obtain virus-free plants by general shoot apical culture.
  • the interfering nucleic acid that can be used in the present invention is a nucleic acid that can suppress the expression in a host cell of a gene involved in the growth of a virus to be removed, and preferably induces RNA interference (RNAi).
  • RNAi RNA interference
  • a particularly preferred interfering nucleic acid is siRNA.
  • Interfering nucleic acid can be prepared based on the nucleic acid sequence of a target gene, that is, a gene involved in virus growth.
  • the target gene in the present invention is not limited to a specific gene, but a gene encoding a protein involved in virus growth in the host, that is, adsorption to the surface of the host cell, entry into the host cell, unshelling, virus It is preferably a gene encoding a protein involved in each step of genome replication and protein synthesis, association, maturation, and extracellular release.
  • a gene encoding an enzyme involved in virus genome replication, a virus coat Examples thereof include a gene encoding a protein, a transfer protein gene, and an RNA silencing suppressor gene.
  • a preferred target gene is a gene that is highly expressed in replication of an enzyme involved in replication or virus. Moreover, in order to deal with more types of viruses, it is preferable to select a gene having a conserved base sequence between different virus genera or species as a target.
  • SiRNA which is a representative example of interfering nucleic acid
  • dsRNA complementary RNA strands
  • ShRNA which is a kind of interfering nucleic acid, is synthesized and used in the form of single-stranded RNA that can take a hairpin loop structure.
  • shRNA forms a double-stranded RNA strand, that is, dsRNA, and then is recognized and cleaved by Dicer in the host cell to become a corresponding siRNA.
  • interfering nucleic acids particularly interfering nucleic acids in the form of dsRNA
  • the number of bases of the interfering nucleic acid is not particularly limited as long as RNA interference can be induced.
  • the length is 15 to 45 bases, preferably 19 to 40 bases, more preferably 21 to 30 bases.
  • Interfering nucleic acids are also formed by the addition of one or several (eg, 1, 2 or 3) bases at the 5 ′ end and / or 3 ′ end of one or both of the sense strand and the antisense strand. May have an overhang arrangement.
  • the interfering nucleic acid in the present invention includes the following DNA and RNA encoded by the DNA.
  • DNA comprising a base sequence having at least about 80% or more, preferably at least about 85% or more, more preferably at least about 90% or more, and most preferably at least about 95% or more identity with the base sequence of the target gene
  • a DNA having an RNA interference ability with respect to a target gene A DNA having an RNA interference ability with respect to a target gene.
  • RNA which is an interfering nucleic acid used in the present invention, is a nucleotide subjected to modification such as 2′O-methylation, 2′-Fation, and 4′-thiolation to improve stability against degradation by nuclease.
  • a derivative may be included.
  • Nucleotide analogs include, for example, 5-position modified uridine or cytidine, such as 5- (2-amino) propyluridine, 5-bromouridine, etc .; 8-position modified adenosine or guanosine, such as 8-bromoguanosine; Mention may be made, for example, of 7-deaza-adenosine; O- or N-alkylated nucleotides, such as N6-methyladenosine.
  • interfering nucleic acids of the invention are chimeric RNAs in which a portion of the ribonucleotides that make up the RNA strand are replaced with corresponding deoxyribonucleotides or nucleotide analogs.
  • the DNA may be in the form of an expression vector prepared according to a general genetic engineering method.
  • an expression vector may further contain any functional base sequence that regulates transcriptional expression, such as a promoter sequence, an operator sequence, a ribosome binding site, an enhancer, and the like. These functional base sequences can be operably linked to the interfering nucleic acid.
  • the present invention includes a step of immersing the shoot apical tissue piece in a solution containing an interfering nucleic acid.
  • the solution may be an aqueous solution containing only the interfering nucleic acid, or may be an appropriate mixed solution (for example, a buffer) containing other components as long as it does not interfere with the function of the interfering nucleic acid.
  • the concentration of the interfering nucleic acid in the solution can be appropriately set, and is, for example, 1 to 10 ⁇ g / 50 ⁇ L, preferably 10 ⁇ g / 50 ⁇ L.
  • Soaking the shoot apical tissue piece in solution means that at least a part, preferably all, of the shoot apex tissue piece is covered with the solution.
  • the tissue piece is submerged.
  • the dipping time is sufficient for a short time, for example, about 10 seconds to several minutes.
  • a preferred embodiment of the present invention includes a step of centrifuging a solution soaked with shoot apical tissue pieces at a centrifugal acceleration of 6000 ⁇ g or less.
  • the centrifugal acceleration is preferably 100 to 6000 ⁇ g, more preferably 500 to 6000 ⁇ g, and still more preferably 2000 to 6000 ⁇ g.
  • the time for centrifugation is sufficient for a so-called spin down operation, for example, about 3 to 10 seconds.
  • Such centrifugal treatment can be performed by a desktop microcentrifuge (for example, Chibitan manufactured by Merck Millipore, or a small microcentrifuge manufactured by ASONE).
  • a desktop microcentrifuge for example, Chibitan manufactured by Merck Millipore, or a small microcentrifuge manufactured by ASONE.
  • the shoot apical tissue piece soaked in the solution may be a tissue containing at least a part of the apical meristem, but is preferably a tissue piece containing the first leaf primordium (the leaf primordium closest to the apical meristem).
  • first leaf primordium the leaf primordium closest to the apical meristem.
  • many viruses are likely to reach the leaf primordium, and thus it is difficult to use the tissue pieces including the leaf primordium in shoot tip culture to obtain virus-free plants, but they contain interfering nucleic acids.
  • the present invention utilizing a solution eliminates such problems and makes it possible to utilize a shoot apical tissue piece containing apical meristem and first leaf primordia.
  • a plant In shoot apical culture, a plant can be regenerated in a shorter time by using a tissue piece containing apical meristem and leaf primordia than using a tissue piece containing only apical meristem.
  • the method of the present invention can significantly reduce the time required for regeneration of a plant body by utilizing a shoot apical tissue piece containing the apical meristem and the first leaf primordia. For example, when garlic is used as a target, it takes about 3 years to produce a virus-free plant by cultivating the shoot apex according to a general method, grow it in the field, and ship the bulb. By applying this method, it is possible to greatly shorten to about one year.
  • the conditions related to shoot tip culture other than the step of immersing the shoot tip tissue piece in a solution containing an interfering nucleic acid and the centrifugation step are general conditions accumulated so far for each plant species.
  • a general reagent such as a sodium hypochlorite solution (antiformin solution).
  • washing with a detergent or ethanol solution Soaking may be performed.
  • the medium used for regeneration of the shoot apical tissue piece may be a common medium such as Murashige-Skoog medium, and may be solid or liquid. Furthermore, conditions such as temperature, illuminance, day length, etc. may be set to conditions suitable for each plant species.
  • % indicates the solvent concentration by volume and the others by weight unless otherwise specified. Further, the mixing ratio of the solvents indicates a volume ratio unless otherwise specified.
  • the deaeration treatment was performed by reducing the pressure for 10 minutes with a vacuum pump, and the mixture was allowed to stand overnight at 4 ° C.
  • the FAA fixative was removed, 50% ethanol was added and left on ice for 15 minutes.
  • the operation of exchanging the liquid was also performed with 70% ethanol and 90% ethanol (when 90% ethanol was treated, it was performed at room temperature).
  • the operation of removing the liquid, adding 100% ethanol and allowing to stand on ice for 30 minutes was performed twice at room temperature. The liquid was removed, 100% ethanol and 100% xylene were added at 1: 1, and the mixture was allowed to stand at room temperature for 30 minutes.
  • skim milk-PBST 10 ⁇ PBS: 25 mL, skim milk: 10 g, Tween-20: 125 ⁇ L prepared to a total volume of 250 mL with sterile water
  • PBS-T Primary antibody GMb-920-802 specific to Alexis virus diluted 1000-3000 times with PBS-T (10 ⁇ PBS: 60 mL, Tween-20: 300 ⁇ L prepared in sterile water to a total volume of 600 mL) (Aomori) (Sold from the Prefectural Industrial Technology Center Vegetable Research Institute) was dropped onto a slide glass and allowed to react at room temperature for 2 hours or at 4 ° C overnight.
  • the plate was washed 3 times with PBS-T for 5 minutes.
  • the secondary antibody Goat AP-rabbit-anti-IgG
  • the plate was washed 3 times with PBS-T for 5 minutes.
  • Detection method of Alexis virus using RT-PCR 0.05 to 0.1 g of plant tissue is put in a mortar, TE saturated phenol and nucleic acid extraction buffer (25 mM Tris-HCl (pH 7.5), 25 mM MgCl 2 ) , 25 mM KCl, 1% SDS) was added in units of 500 ⁇ L and ground with a pestle. They were collected in a tube, mixed by vortex for 15 seconds, and centrifuged at 12000 rpm for 3 minutes at room temperature. 300 ⁇ L of the upper layer was transferred to a new 1.5 mL tube, and 150 ⁇ L of phenol and chloroform were added.
  • TE saturated phenol and nucleic acid extraction buffer 25 mM Tris-HCl (pH 7.5), 25 mM MgCl 2 ) , 25 mM KCl, 1% SDS
  • the mixture was centrifuged at 14,000 rpm for 20 minutes at 4 ° C., and the supernatant was discarded. 500 ⁇ L of 80% ethanol was added and cooled and centrifuged at 4 ° C., 14000 rpm for 5 minutes. The supernatant was discarded, allowed to stand at room temperature, dried, and then suspended in 30-50 ⁇ L of sterile water.
  • RT-PCR reaction was performed using TaKaRa One Step RNA PCR Kit (AMV).
  • a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 was synthesized with reference to Chen et al. (Archives of virology, 2004, 149, 435 pages) for detection of Alexis virus.
  • PCR after heat denaturation at 50 ° C. for 15 minutes and 94 ° C. for 2 minutes, a cycle of “94 ° C. for 30 seconds, 57 ° C. for 30 seconds, 72 ° C. for 1 minute” was performed 35 times.
  • the reaction was performed at -1 ° C for 1 minute, and the size of the amplified fragment was confirmed by agarose gel electrophoresis.
  • the size of the amplified fragment when Alexis virus is present is approximately 750 bp.
  • Example> Treatment of siRNA to garlic shoot apical tissue infected with Alexis virus a) Selection of materials Prepare garlic (Allium sativum) from Spain and Tokoro-cho, Hokkaido, blocking time for shoot apex tissue for 30 minutes, As a result of performing the immunohistochemical method with a staining time of 35 minutes, all garlics were stained in the shoot apical tissue and the first leaf primordium, that is, Alexis virus was observed. The staining results for Spanish garlic are shown in FIGS. b) Preparation of shoot tip tissue pieces The garlic bulbs from Spain and Tokoro-cho were divided into scale pieces and the scale skins were removed. After soaking in tap water for 10 minutes, the skin was removed.
  • the tap water was changed, and an appropriate amount of neutral detergent was put in the tap water.
  • the solution was stirred until it foamed and allowed to stand for 20 minutes. Thereafter, the scales washed with water were put into a nonwoven fabric and sealed with a heat sealer.
  • An 80% ethanol solution was prepared in a 500 mL measuring cup, immersed in garlic and allowed to stand for 1 minute. The 80% ethanol solution was discarded and garlic was immersed in 500 mL of 10% antiformin solution and allowed to stand for 20 minutes. When appropriate, the cup was rotated horizontally to allow the solution to spread through the measuring cup. Thereafter, the measuring cup was transferred into a clean bench and washed three times or more with sterilized water.
  • a razor both Seikan, Feather Safety Razor Co., Ltd.
  • a 1 mm tissue piece containing the apical meristem and the first leaf primordium was taken out, and then placed in an MS medium having the following composition. I transplanted it quickly.
  • MS medium KNO 3 1900 mg NH 4 NO 3 : 1650 mg CaCl 2 ⁇ 2H 2 O: 440 mg MgSO 4 ⁇ 7H 2 O: 370 mg KH 2 PO 4 : 170 mg Na 2 -EDTA: 37.3 mg FeSO 4 ⁇ 7H 2 O: 27.8mg MnSO 4 .4H 2 O: 22.3 mg ZnSO 4 ⁇ 7H 2 O: 8.6 mg H 3 BO 3: 6.2mg KI: 0.83mg Na 2 MoO 4 ⁇ 2H 2 O : 0.25mg CuSO 4 ⁇ 5H 2 O: 0.025mg CoCl 2 ⁇ 6H 2 O: 0.025mg Total 4600mg In addition, the following reagents were added at the time of preparation.
  • thiamine hydrochloride solution 200 ⁇ L myo-inositol: 100mg Sucrose: 30g Agar (for plant medium): 7g After adjusting to pH 5.8 with 1M KOH, adjusted to 1000 mL.
  • dsRNA corresponding to the base sequence of about 700 bp in the coat protein (CP) gene of Alexi virus
  • the CP gene cloned on pGEM vector Promega
  • PCR was carried out using two primer sets, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10.
  • Primestar HS DNA Polymerase (manufactured by Takara Bio Inc.) was used as the polymerase.
  • PCR conditions after heat denaturation at 94 ° C. for 1 minute, a cycle of “98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C.
  • Allexi-dsRNA was allowed to act on Allexi-dsRNA to prepare Allexi-siRNA.
  • the obtained sample was mixed with Novex Hi-Density TBE Sample Buffer (manufactured by Life Technologies), and the result of non-denaturing PAGE electrophoresis (100 V, 35 minutes) with Novex TBE Gels 6% (manufactured by Life Technologies) is shown in FIG. Show. Allexi-siRNA produced in this way acts to suppress the expression of Alexis virus CP.
  • siRNA having no virus-suppressing effect siRNA (GUS-siRNA) against ⁇ -glucuronidase (GUS) gene is used as a template, and GUS gene cloned on pBI121 (manufactured by Clontech) is used as a template. It was prepared in the same manner as described above except that SEQ ID NO: 4 and two primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 were used.
  • Tissue pieces were ground 20 days after the start of the culture, and it was confirmed by RT-PCR method whether or not Alexis virus remained on the tissue pieces.
  • ribavirin known as a potent antiviral agent and also used for shoot tip culture
  • the virus-removing effect of ribavirin was evaluated in garlic infected with Alexis virus.
  • Five pieces of tissue derived from Spanish garlic prepared in b) of the above example were cultured aseptically for 2 weeks each in MS medium and 5 in MS medium containing 12 mg / L ribavirin.
  • the culture conditions were a temperature of 25 ° C. and illumination for 12 hours. After culturing, each piece of tissue was ground, and it was confirmed by RT-PCR method whether Alexi virus remained on the piece of tissue. The result is shown in FIG. It was confirmed that Alexis virus was not removed even by treatment with ribavirin at a high concentration of 12 mg / L.
  • SEQ ID NO: 1 base sequence of Alexis virus detection primer
  • SEQ ID NO: 2 base sequence of Alexis virus detection primer
  • SEQ ID NO: 3 base sequence of siRNA preparation primer for GUS gene
  • SEQ ID NO: 4 for siRNA preparation of GUS gene
  • Primer base sequence SEQ ID NO: 5: base sequence of siRNA preparation primer for GUS gene
  • SEQ ID NO: 6 base sequence of siRNA preparation primer for GUS gene
  • SEQ ID NO: 7 base sequence of siRNA preparation primer for CP gene of Alexis virus
  • SEQ ID NO: 8 nucleotide sequence of primer for siRNA preparation against CP gene of Alexis virus
  • SEQ ID NO: 9 nucleotide sequence of primer for siRNA preparation against CP gene of Alexis virus
  • SEQ ID NO: 10 Alexi genus Virus of CP of siRNA fabrication primers for gene sequences
  • SEQ ID NO: 11 nucleotide sequence of the dsRNA for CP gene Arekishi genus virus

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Abstract

[Problem] To provide a method for producing a virus-free plant body, which can be applied to plant species from which viruses can be hardly removed in a generally-employed apical meristem culture procedure and virus species which can be hardly removed in a generally-employed apical meristem culture procedure. [Solution] The present invention relates to a method for producing a virus-free plant body, comprising the steps of: immersing a bud tissue piece in a solution containing an interfering nucleic acid capable of inhibiting the proliferation of a plant virus; and regenerating the plant body from the bud tissue piece collected from the solution. According to the present invention, it becomes possible to produce a virus-free plant body from a plant species from which a virus can be hardly removed by a generally-employed apical meristem culture procedure. It also becomes possible to greatly reduce the time required for the regeneration of the plant body.

Description

ウイルスフリー植物体の製造方法Method for producing virus-free plant
　本発明は、ウイルスフリー植物体、特に通常の茎頂培養では植物ウイルスの除去が困難な植物種からのウイルスフリー植物体の製造方法に関する。 The present invention relates to a method for producing a virus-free plant body, particularly a virus-free plant body from a plant species in which it is difficult to remove the plant virus by normal shoot apical culture.
　農作物に感染することで、収量や品質などに悪影響を及ぼす植物ウイルスが多数知られている。このようなウイルスの感染がない、いわゆるウイルスフリー植物体を得る技術として、茎頂を無菌的に培養する茎頂培養がある（例えば特許文献１）。 A large number of plant viruses that adversely affect yield, quality, etc. by infecting agricultural crops are known. As a technique for obtaining a so-called virus-free plant body free from such virus infection, there is a shoot tip culture in which the shoot tip is cultured aseptically (for example, Patent Document 1).
　茎頂は、半球形の頂端分裂組織（成長点（ｍｅｒｉｓｔｅｍ）とも呼ばれる）及びそれから分化した数枚の葉原基からなる。茎頂培養は、植物体がウイルスに感染した場合でも茎頂、特に頂端分裂組織にはウイルスが到達しないことがあることを利用して、頂端分裂組織のみを含むよう茎頂を無菌的に薄く切り出し、そこから植物体を再生させることでウイルスフリー植物体を製造する技術である。 The shoot apex consists of a hemispherical apical meristem (also called a meristem) and several leaf primordia differentiated therefrom. By using the fact that the virus may not reach the shoot apex, especially the apical meristem, even if the plant body is infected with virus, the shoot apical culture is aseptically thinned to contain only the apical meristem. It is a technique for producing a virus-free plant by cutting out and regenerating the plant from there.
　しかし、茎頂から頂端分裂組織のみを切り出すことは、０．１～１ｍｍ程度という微小な組織片の切り出しを意味し、これは作業者の高度な熟練を必要とする。また、切り出す茎頂組織片が小さいほどウイルス除去の可能性は高まるものの、その後の植物体の再生及び栽培に長期間を要するという問題も生じる。このように、従来の茎頂培養は、技術的にも経済的にも効率的であるとは言い難い。さらに、特に頂端分裂組織までウイルスが到達し易い植物種やウイルス種については、一般的な茎頂培養によってウイルスフリー植物体を製造することは困難である。 However, cutting only the apical meristem from the shoot apex means cutting a small tissue piece of about 0.1 to 1 mm, which requires high skill of the operator. Moreover, although the possibility of virus removal increases as the shoot tip tissue piece to be cut out becomes smaller, there also arises a problem that it takes a long time for the subsequent regeneration and cultivation of the plant body. As described above, it is difficult to say that conventional shoot tip culture is technically and economically efficient. Furthermore, it is difficult to produce a virus-free plant by general shoot apical culture for plant species and virus species that can easily reach the apical meristem.
ＷＯ９９／１２４１２WO99 / 12412
　本発明は、一般的な茎頂培養ではウイルス除去が困難である植物種やウイルス種についても適用可能な、ウイルスフリー植物体の製造方法を提供することを目的とする。 An object of the present invention is to provide a method for producing a virus-free plant that can be applied to plant species and virus species that are difficult to remove by general shoot apical culture.
　本発明者は、茎頂培養において、茎頂の組織片をウイルスの増殖等を抑制することのできる干渉性核酸を含む溶液に浸すことでウイルスフリー植物体の製造効率が向上することを見出し、以下の各発明を完成した。 The present inventor has found that in the shoot tip culture, the production efficiency of the virus-free plant is improved by immersing the tissue piece of the shoot tip in a solution containing an interfering nucleic acid capable of suppressing the growth of the virus, etc. The following inventions were completed.
（１）植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程、及び該溶液より回収した茎頂組織片から植物体を再生させる工程を含む、ウイルスフリー植物体の製造方法。
（２）茎頂組織片を浸した溶液を６０００×ｇ以下の遠心加速度で遠心処理する工程をさらに含む、（１）に記載の製造方法。
（３）茎頂組織片が頂端分裂組織及び第１葉原基を含む、（１）又は（２）に記載の製造方法。
（４）干渉性核酸がｓｉＲＮＡである、（１）～（３）のいずれかに記載の製造方法。
（５）植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程を含む、茎頂組織片からの植物ウイルスの除去方法。 (1) A virus-free method comprising a step of immersing a shoot apical tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus, and a step of regenerating a plant body from the shoot apical tissue piece collected from the solution A method for producing a plant body.
(2) The production method according to (1), further comprising a step of centrifuging the solution soaked with the shoot apical tissue piece at a centrifugal acceleration of 6000 × g or less.
(3) The production method according to (1) or (2), wherein the shoot apical tissue piece includes apical meristem and the first leaf primordium.
(4) The production method according to any one of (1) to (3), wherein the interfering nucleic acid is siRNA.
(5) A method for removing a plant virus from a shoot tip tissue piece, comprising a step of immersing the shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus.
　本発明により、一般的な茎頂培養によるウイルス除去が困難であった植物種からも、ウイルスフリー植物体を製造することができる。また、茎頂培養における茎頂組織片を通常より大きく、特に頂端分裂組織だけでなく第１葉原基を含む組織片とすることができるため、植物体の再生及びその後の栽培に要する時間を大幅に短縮することができる。 According to the present invention, a virus-free plant can be produced from a plant species in which virus removal by general shoot tip culture has been difficult. In addition, since the shoot tip tissue piece in the shoot tip culture is larger than usual, in particular, it can be made into a tissue piece containing not only the apical meristem but also the first leaf primordia, greatly increasing the time required for plant regeneration and subsequent cultivation. Can be shortened.
スペイン産ニンニクにおけるアレキシ属ウイルスの頂端分裂組織での存在を示す、免疫組織化学染色の写真である。It is a photograph of immunohistochemical staining showing the presence of Alexi virus in the apical meristem in Spanish garlic. 図１の写真の一部を拡大したものである。FIG. 2 is an enlarged view of a part of the photograph of FIG. β－グルクロニダーゼ遺伝子及びアレキシ属ウイルスのコートタンパク質遺伝子に対する干渉性核酸として作製したｄｓＲＮＡと、これらをＤｉｃｅｒで処理して作製したｓｉＲＮＡの、非変性ＰＡＧＥ電気泳動の結果を示す写真である。2 is a photograph showing the results of non-denaturing PAGE electrophoresis of dsRNA prepared as an interfering nucleic acid for the β-glucuronidase gene and the coat protein gene of Alexis virus and siRNA prepared by treating these with Dicer. 水（ネガティブコントロール）又はＧＵＳ－ｓｉＲＮＡ（ｓｉＲＮＡコントロール）で処理したスペイン産ニンニク茎頂の、培養２０日後におけるアレキシ属ウイルスの存在をＲＴ－ＰＣＲ法で検出した結果を示す写真である。It is a photograph showing the result of RT-PCR detection of the presence of Alexis virus after 20 days in culture of Spanish garlic shoot apex treated with water (negative control) or GUS-siRNA (siRNA control). 水（ネガティブコントロール）又は本発明のＡｌｌｅｘｉ－ｓｉＲＮＡで処理した常呂町産ニンニク茎頂の、培養２０日後におけるアレキシ属ウイルスの存在をＲＴ－ＰＣＲ法で検出した結果を示す写真である。It is a photograph showing the results of detecting the presence of Alexis virus by RT-PCR in the garlic shoots of Tokorocho treated with water (negative control) or Allexi-siRNA of the present invention after 20 days of culture. 水（ネガティブコントロール）又は本発明のＡｌｌｅｘｉ－ｓｉＲＮＡで処理した常呂町産ニンニク茎頂を培養して鉢上げした後の、アレキシ属ウイルスの存在をＲＴ－ＰＣＲ法で検出した結果を示す写真である。It is a photograph showing the result of detecting the presence of Alexis virus by RT-PCR method after culturing and potting garlic shoots from Tokorocho treated with water (negative control) or Allexi-siRNA of the present invention. . リバビリンで処理したスペイン産ニンニク茎頂の、培養２週間後におけるアレキシ属ウイルスの存在をＲＴ－ＰＣＲ法で検出した結果を示す写真である。It is a photograph which shows the result of having detected the presence of the Alexis genus virus by the RT-PCR method in the garlic shoot tip produced in Spain treated with ribavirin after 2 weeks of culture.
　本発明は、植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程、及び該溶液より回収した茎頂組織片から植物体を再生させる工程を含む、ウイルスフリー植物体の製造方法に関する。本発明は、植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程を含む、茎頂組織片からの植物ウイルスの除去方法として表すこともできる。 The present invention relates to a virus comprising a step of immersing a shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus, and a step of regenerating a plant body from the shoot tip tissue piece recovered from the solution. The present invention relates to a method for producing a free plant. The present invention can also be expressed as a method for removing plant viruses from shoot tip tissue pieces, which includes a step of immersing the shoot tip tissue pieces in a solution containing an interfering nucleic acid capable of suppressing the growth of plant viruses.
　本発明は、茎頂培養によるウイルスフリー植物体の製造が望まれる植物種であればいずれにも適用可能であるが、好ましくは一般的な茎頂培養ではウイルスフリー植物体の製造が困難であった植物種に対して適用される。また、除去対象とされるウイルスについても特に制限はないが、好ましくは、本発明は茎頂、特に頂端分裂組織にまで到達し易いウイルスに対して適用される。 The present invention can be applied to any plant species for which production of virus-free plants by shoot tip culture is desired. However, it is preferable to produce virus-free plants by general shoot tip culture. Applied to different plant species. The virus to be removed is not particularly limited, but preferably the present invention is applied to a virus that easily reaches the shoot apex, particularly the apical meristem.
　本発明が好適に適用される植物種とウイルス種の組合せとしては、ニンニクに対するアレキシ属ウイルス、アスパラガスに対するアスパラガスウイルス２（ＡＶ２）、ジャガイモに対するジャガイモウイルスＳ（ＰＶＳ）などを挙げることができる。また、茎頂分裂組織に容易に侵入するウイロイドも含まれる。これらは、一般的な茎頂培養ではウイルスフリー植物体を得ることが困難であった植物種とウイルス種の組合せの例である。 Examples of combinations of plant species and virus species to which the present invention is suitably applied include Alexi virus against garlic, asparagus virus 2 (AV2) against asparagus, potato virus S (PVS) against potato, and the like. Viroids that easily penetrate into the shoot apical meristem are also included. These are examples of combinations of plant species and virus species for which it was difficult to obtain virus-free plants by general shoot apical culture.
　本発明で利用可能な干渉性核酸は、除去対象とされるウイルスの増殖に関与する遺伝子の宿主細胞内における発現を抑制することができる核酸であり、好ましくはＲＮＡ干渉（ＲＮＡｉ）を誘導することができる核酸、典型的にはｓｉＲＮＡ、ｓｈＲＮＡ、ｍｉＲＮＡ又はこれらをコードするＤＮＡである。特に好ましい干渉性核酸は、ｓｉＲＮＡである。 The interfering nucleic acid that can be used in the present invention is a nucleic acid that can suppress the expression in a host cell of a gene involved in the growth of a virus to be removed, and preferably induces RNA interference (RNAi). A nucleic acid capable of binding, typically siRNA, shRNA, miRNA or DNA encoding them. A particularly preferred interfering nucleic acid is siRNA.
　干渉性核酸は、標的遺伝子、すなわちウイルスの増殖に関与する遺伝子の核酸配列に基づいて作成することができる。本発明における標的遺伝子は、特定の遺伝子に限定されるものではなく、宿主におけるウイルスの増殖に関与するタンパク質をコードする遺伝子、すなわち宿主細胞表面への吸着、宿主細胞内への侵入、脱殻、ウイルスゲノム複製及びタンパク質合成、会合、成熟、細胞外への放出の各工程に関与するタンパク質をコードする遺伝子であることが好ましく、例えば、ウイルスのゲノム複製に関与する酵素をコードする遺伝子、ウイルスのコートタンパク質をコードする遺伝子、移行タンパク質遺伝子、ＲＮＡサイレンシングサプレッサー遺伝子等を挙げることができる。好ましい標的遺伝子は、複製に関与する酵素又はウイルスの複製において発現量の高い遺伝子である。また、より多くの種類のウイルスに対応するため、異なるウイルス属又は種の間で塩基配列が保存されている遺伝子を標的として選択することが好ましい。 Interfering nucleic acid can be prepared based on the nucleic acid sequence of a target gene, that is, a gene involved in virus growth. The target gene in the present invention is not limited to a specific gene, but a gene encoding a protein involved in virus growth in the host, that is, adsorption to the surface of the host cell, entry into the host cell, unshelling, virus It is preferably a gene encoding a protein involved in each step of genome replication and protein synthesis, association, maturation, and extracellular release. For example, a gene encoding an enzyme involved in virus genome replication, a virus coat Examples thereof include a gene encoding a protein, a transfer protein gene, and an RNA silencing suppressor gene. A preferred target gene is a gene that is highly expressed in replication of an enzyme involved in replication or virus. Moreover, in order to deal with more types of viruses, it is preferable to select a gene having a conserved base sequence between different virus genera or species as a target.
　干渉性核酸の代表例であるｓｉＲＮＡは、通常、相互に相補的な二本のＲＮＡ鎖（ｄｓＲＮＡ）の形態で合成され、使用される。また干渉性核酸の一種であるｓｈＲＮＡは、ヘアピンループ構造を取ることができる一本鎖ＲＮＡの形態で合成され、使用される。ｓｈＲＮＡは、二本鎖構造のＲＮＡ鎖すなわちｄｓＲＮＡを形成した後、宿主細胞内のＤｉｃｅｒにより認識、切断されて、対応するｓｉＲＮＡとなる。 SiRNA, which is a representative example of interfering nucleic acid, is usually synthesized and used in the form of two complementary RNA strands (dsRNA). ShRNA, which is a kind of interfering nucleic acid, is synthesized and used in the form of single-stranded RNA that can take a hairpin loop structure. shRNA forms a double-stranded RNA strand, that is, dsRNA, and then is recognized and cleaved by Dicer in the host cell to become a corresponding siRNA.
　このような干渉性核酸、特にｄｓＲＮＡの形態の干渉性核酸は、当業者に公知の方法によって作製することができ、特別な方法は特に必要とされない。干渉性核酸の塩基数は、ＲＮＡ干渉を誘導できる限り特に制限はないが、例えば、１５～４５塩基の長さ、好ましくは１９～４０塩基の長さ、より好ましくは２１～３０塩基の長さであることができる。また、干渉性核酸は、センス鎖およびアンチセンス鎖の一方または双方の５’末端および／または３’末端において、１または数個（例として１、２または３個）の塩基の付加により形成されるオーバーハング配列を有していてもよい。 Such interfering nucleic acids, particularly interfering nucleic acids in the form of dsRNA, can be prepared by methods known to those skilled in the art, and no special method is particularly required. The number of bases of the interfering nucleic acid is not particularly limited as long as RNA interference can be induced. For example, the length is 15 to 45 bases, preferably 19 to 40 bases, more preferably 21 to 30 bases. Can be. Interfering nucleic acids are also formed by the addition of one or several (eg, 1, 2 or 3) bases at the 5 ′ end and / or 3 ′ end of one or both of the sense strand and the antisense strand. May have an overhang arrangement.
　さらに、本発明における干渉性核酸は、以下のＤＮＡ、及び該ＤＮＡによりコードされるＲＮＡを包含する。
（１）標的遺伝子の塩基配列と少なくとも約８０％以上、好ましくは少なくとも約８５％以上、より好ましくは少なくとも約９０％以上、最も好ましくは少なくとも約９５％以上の同一性を有する塩基配列を含むＤＮＡであって、標的遺伝子に対してＲＮＡ干渉能を持つＤＮＡ。
（２）標的遺伝子の塩基配列において１または複数の塩基を付加し、欠失させまたは置換した塩基配列を含むＤＮＡであって、標的遺伝子に対してＲＮＡ干渉能を持つＤＮＡ。
（３）標的遺伝子の塩基配列を含むＤＮＡとストリンジェントな条件でハイブリダイズするＤＮＡであって、標的遺伝子に対してＲＮＡ干渉能を持つＤＮＡ。 Furthermore, the interfering nucleic acid in the present invention includes the following DNA and RNA encoded by the DNA.
(1) DNA comprising a base sequence having at least about 80% or more, preferably at least about 85% or more, more preferably at least about 90% or more, and most preferably at least about 95% or more identity with the base sequence of the target gene A DNA having an RNA interference ability with respect to a target gene.
(2) DNA having a base sequence in which one or more bases are added, deleted or substituted in the base sequence of the target gene, and having RNA interference ability with respect to the target gene.
(3) DNA that hybridizes under stringent conditions with DNA containing the base sequence of the target gene and has RNA interference ability with respect to the target gene.
　本発明において用いられる干渉性核酸であるＲＮＡは、ヌクレアーゼによる分解に対する安定性を向上させるための、２’Ｏ－メチル化、２’－Ｆ化、４’－チオ化などの修飾を受けたヌクレオチド誘導体を含むものでもよい。ヌクレオチド類似体としては、例えば、５位修飾ウリジンまたはシチジン、例えば５‐（２‐アミノ）プロピルウリジン、５‐ブロモウリジンなど；８位修飾アデノシンまたはグアノシン、例えば８‐ブロモグアノシンなど；デアザヌクレオチド、例えば７－デアザ－アデノシンなど；Ｏ－またはＮ－アルキル化ヌクレオチド、例えばＮ６－メチルアデノシンなどを挙げることができる。また、ＲＮＡ鎖を構成するリボヌクレオチドの一部が、対応するデオキシリボヌクレオチドまたはヌクレオチド類似体に置き換えられたキメラＲＮＡもまた、本発明の干渉性核酸の範囲内にある。 RNA, which is an interfering nucleic acid used in the present invention, is a nucleotide subjected to modification such as 2′O-methylation, 2′-Fation, and 4′-thiolation to improve stability against degradation by nuclease. A derivative may be included. Nucleotide analogs include, for example, 5-position modified uridine or cytidine, such as 5- (2-amino) propyluridine, 5-bromouridine, etc .; 8-position modified adenosine or guanosine, such as 8-bromoguanosine; Mention may be made, for example, of 7-deaza-adenosine; O- or N-alkylated nucleotides, such as N6-methyladenosine. Also within the scope of the interfering nucleic acids of the invention are chimeric RNAs in which a portion of the ribonucleotides that make up the RNA strand are replaced with corresponding deoxyribonucleotides or nucleotide analogs.
　干渉性核酸がＤＮＡである場合、かかるＤＮＡは、一般的な遺伝子工学的方法に従って作成される発現ベクターの形態であってもよい。かかる発現ベクターは、転写発現を調節する任意の機能性塩基配列、例えばプロモーター配列、オペレーター配列、リボソーム結合部位、エンハンサーなどをさらに含んでいてもよい。これらの機能性塩基配列は、上記干渉性核酸と機能的に連結され得る。 When the interfering nucleic acid is DNA, the DNA may be in the form of an expression vector prepared according to a general genetic engineering method. Such an expression vector may further contain any functional base sequence that regulates transcriptional expression, such as a promoter sequence, an operator sequence, a ribosome binding site, an enhancer, and the like. These functional base sequences can be operably linked to the interfering nucleic acid.
　本発明は、干渉性核酸を含む溶液に茎頂組織片を浸す工程を含む。溶液は、干渉性核酸のみを含む水溶液であってもよく、干渉性核酸の機能を妨げない限り他の成分を含む適当な混合液（例えば緩衝液）であってもよい。溶液における干渉性核酸の濃度は適宜設定することができ、例えば１～１０μｇ／５０μＬ、好ましくは１０μｇ／５０μＬである。 The present invention includes a step of immersing the shoot apical tissue piece in a solution containing an interfering nucleic acid. The solution may be an aqueous solution containing only the interfering nucleic acid, or may be an appropriate mixed solution (for example, a buffer) containing other components as long as it does not interfere with the function of the interfering nucleic acid. The concentration of the interfering nucleic acid in the solution can be appropriately set, and is, for example, 1 to 10 μg / 50 μL, preferably 10 μg / 50 μL.
　茎頂組織片を溶液に浸すとは、茎頂組織片の少なくとも一部、好ましくは全部が溶液で覆われるようにすることをいい、典型的な態様は、適当な容器中の溶液に茎頂組織片を沈めるものである。浸す時間は短時間で足り、例えば１０秒～数分程度でよい。 Soaking the shoot apical tissue piece in solution means that at least a part, preferably all, of the shoot apex tissue piece is covered with the solution. The tissue piece is submerged. The dipping time is sufficient for a short time, for example, about 10 seconds to several minutes.
　本発明の好ましい態様は、茎頂組織片を浸した溶液を６０００×ｇ以下の遠心加速度で遠心処理する工程を含む。遠心加速度は、好ましくは１００～６０００×ｇ、より好ましくは５００～６０００×ｇ、さらに好ましくは２０００～６０００×ｇである。遠心処理の時間は、いわゆるスピンダウン操作に用いる程度の短時間で足り、例えば３～１０秒間程度でよい。かかる遠心処理は、卓上型の微量用遠心機（例えば、メルクミリポア社のＣｈｉｂｉｔａｎ、アズワン社の小型微量遠心機）などによって行うことができる。全く意外なことに、かかる穏やかな遠心処理工程を加えることによって、干渉性核酸によるウイルスの除去効率及び植物体の再生率を大きく高めることができる。 A preferred embodiment of the present invention includes a step of centrifuging a solution soaked with shoot apical tissue pieces at a centrifugal acceleration of 6000 × g or less. The centrifugal acceleration is preferably 100 to 6000 × g, more preferably 500 to 6000 × g, and still more preferably 2000 to 6000 × g. The time for centrifugation is sufficient for a so-called spin down operation, for example, about 3 to 10 seconds. Such centrifugal treatment can be performed by a desktop microcentrifuge (for example, Chibitan manufactured by Merck Millipore, or a small microcentrifuge manufactured by ASONE). Surprisingly, by adding such a gentle centrifugation step, the virus removal efficiency by the interfering nucleic acid and the regeneration rate of the plant body can be greatly increased.
　溶液に浸す茎頂組織片は、少なくとも頂端分裂組織の一部を含む組織であればよいが、さらに第１葉原基（頂端分裂組織に最も近い葉原基）を含む組織片であることが好ましい。一般に、多くのウイルスは葉原基にまで到達しやすく、したがってウイルスフリー植物体を得るための茎頂培養では葉原基を含めた組織片を利用することは困難であったが、干渉性核酸を含む溶液を利用する本発明はかかる問題を解消し、頂端分裂組織及び第１葉原基を含む茎頂組織片を利用することを可能にする。 The shoot apical tissue piece soaked in the solution may be a tissue containing at least a part of the apical meristem, but is preferably a tissue piece containing the first leaf primordium (the leaf primordium closest to the apical meristem). In general, many viruses are likely to reach the leaf primordium, and thus it is difficult to use the tissue pieces including the leaf primordium in shoot tip culture to obtain virus-free plants, but they contain interfering nucleic acids. The present invention utilizing a solution eliminates such problems and makes it possible to utilize a shoot apical tissue piece containing apical meristem and first leaf primordia.
　茎頂培養において、頂端分裂組織のみを含む組織片を用いるよりも頂端分裂組織及び葉原基を含む組織片を用いる方が、より短時間で植物体を再生させることができる。本発明の方法は、頂端分裂組織及び第１葉原基を含む茎頂組織片を利用することで、植物体の再生に要する時間を大幅に短縮することができる。例えばニンニクを対象とした場合、一般的な方法による茎頂培養でウイルスフリー植物を製造し、圃場で栽培して鱗茎を出荷するまでに要する時間はおおよそ３年であるのに対して、本発明の方法を適用することでおおよそ１年へと大幅に短縮することが可能となる。 In shoot apical culture, a plant can be regenerated in a shorter time by using a tissue piece containing apical meristem and leaf primordia than using a tissue piece containing only apical meristem. The method of the present invention can significantly reduce the time required for regeneration of a plant body by utilizing a shoot apical tissue piece containing the apical meristem and the first leaf primordia. For example, when garlic is used as a target, it takes about 3 years to produce a virus-free plant by cultivating the shoot apex according to a general method, grow it in the field, and ship the bulb. By applying this method, it is possible to greatly shorten to about one year.
　本発明では、干渉性核酸を含む溶液に茎頂組織片を浸す工程及び遠心処理工程を行うこと以外の茎頂培養に関する条件は、植物種毎にこれまで蓄積された一般的な条件であればよい。例えば、茎頂組織を採取する植物の消毒は、次亜塩素酸ナトリウム溶液（アンチホルミン溶液）などの一般的な試薬を用いて行えばよく、消毒を助けるために、洗剤での洗浄やエタノール溶液への浸漬を行ってもよい。 In the present invention, the conditions related to shoot tip culture other than the step of immersing the shoot tip tissue piece in a solution containing an interfering nucleic acid and the centrifugation step are general conditions accumulated so far for each plant species. Good. For example, disinfection of a plant from which the shoot apex tissue is collected may be performed using a general reagent such as a sodium hypochlorite solution (antiformin solution). To aid disinfection, washing with a detergent or ethanol solution Soaking may be performed.
　また、茎頂組織片の再生に用いる培地も、例えばムラシゲ・スクーグ培地などの一般的な培地であればよく、また固体でも液体でもよい。さらに、温度、照度、日長などの諸条件も、植物種ごとに適した条件に設定すればよい。 The medium used for regeneration of the shoot apical tissue piece may be a common medium such as Murashige-Skoog medium, and may be solid or liquid. Furthermore, conditions such as temperature, illuminance, day length, etc. may be set to conditions suitable for each plant species.
　以下、実施例により本発明を説明する。なお、実施例中、「％」は、特記しない限り、溶媒濃度は容量％、その他は重量％を示す。また、溶媒の混合比は、特記しない限り、容量比を示す。 Hereinafter, the present invention will be described with reference to examples. In the examples, “%” indicates the solvent concentration by volume and the others by weight unless otherwise specified. Further, the mixing ratio of the solvents indicates a volume ratio unless otherwise specified.
＜試験法＞
１）植物組織の免疫組織化学法
　免疫組織化学法を、文献（Ｔ．Ｍｏｃｈｉｚｕｋｉら、Ｊ　Ｇｅｎ．Ｐｌａｎｔ　Ｐａｔｈｏｌ．、２００４年、第７０巻、第３６３ページ）を参考に、以下の通り実施した。 <Test method>
1) Immunohistochemical method of plant tissue The immunohistochemical method was performed as follows with reference to literature (T. Mochizuki et al., J Gen. Plant Pathol. 2004, 70, 363).
ａ）パラフィン切片の調製
植物体から２ｍｍ角程度の組織を切り出し、パラフィン切片を作成するため、ＦＡＡ固定液（エタノール：５０ｍＬ、酢酸：５ｍＬ、ホルムアルデヒド溶液：５ｍＬ、水：４０ｍＬの混合液）に移した。 a) Preparation of paraffin section To cut out a 2 mm square tissue from the plant body and prepare a paraffin section, transfer it to a FAA fixative (ethanol: 50 mL, acetic acid: 5 mL, formaldehyde solution: 5 mL, water: 40 mL). did.
真空ポンプで１０分間減圧して脱気処理を行い、４℃で一晩静置した。ＦＡＡ固定液を取り除き、５０％エタノールを加えて氷上で１５分間静置した。同様に液を入れ替える作業を７０％エタノールと９０％エタノールでも行った（９０％エタノールを処理するときは室温で行った）。次に、液を取り除き、１００％エタノールを加えて氷上で３０分静置する作業を室温で２回行った。液を取り除き、１００％エタノールと１００％キシレンを１：１で加え、室温で３０分間静置した。さらに、液を取り除き、１００％キシレンを加え室温で３０分静置する作業を室温で２回行った。次に、オーブン内で１００％キシレンと等量の溶解パラフィンを加えてよく混合し、キシレンが完全に蒸発するまで６０℃で静置した。キシレンが完全に蒸発したら、試料を冷蔵庫に入れて、パラフィンを固まらせた。固まったパラフィンを四角形に切り出し、ロータリーミクロトームで準薄切片（厚さ２０μｍ）を切り出した。ＭＡＳコートスライドグラスに蒸留水を適量滴下し、パラフィンリボンを乗せ、３７℃のオーブンで一晩静置した。 The deaeration treatment was performed by reducing the pressure for 10 minutes with a vacuum pump, and the mixture was allowed to stand overnight at 4 ° C. The FAA fixative was removed, 50% ethanol was added and left on ice for 15 minutes. Similarly, the operation of exchanging the liquid was also performed with 70% ethanol and 90% ethanol (when 90% ethanol was treated, it was performed at room temperature). Next, the operation of removing the liquid, adding 100% ethanol and allowing to stand on ice for 30 minutes was performed twice at room temperature. The liquid was removed, 100% ethanol and 100% xylene were added at 1: 1, and the mixture was allowed to stand at room temperature for 30 minutes. Further, the operation of removing the liquid, adding 100% xylene and allowing to stand at room temperature for 30 minutes was performed twice at room temperature. Next, 100% xylene and an equivalent amount of dissolved paraffin were added in the oven, mixed well, and allowed to stand at 60 ° C. until the xylene was completely evaporated. Once the xylene had completely evaporated, the sample was placed in the refrigerator to allow the paraffin to harden. The solid paraffin was cut into a square, and a semi-thin section (thickness 20 μm) was cut with a rotary microtome. An appropriate amount of distilled water was dropped onto the MAS-coated slide glass, a paraffin ribbon was placed on the slide glass, and the plate was allowed to stand overnight in an oven at 37 ° C.
　静置後のスライドグラスを１００％キシレンに室温で１５分間浸し、同様にキシレンを入れ替えて、もう一度１５分間浸して脱パラフィン処理した。次に１００％エタノール：１００％キシレン＝１：１の液に５分間浸し、その後、１００％エタノールに５分間浸し、１００％エタノールを入れ替えてもう一度１００％エタノールに５分間浸した。液からスライドグラスを取り出し、室温で静置し、風乾させた。次に、９０％エタノール、７０％エタノール、５０％エタノール、３０％エタノール、蒸留水の順番に２分間ずつ室温で浸すことで、試料を調製した。 The slide glass after standing was soaked in 100% xylene at room temperature for 15 minutes, xylene was replaced in the same manner, and soaked again for 15 minutes for deparaffinization. Next, it was immersed in a solution of 100% ethanol: 100% xylene = 1: 1 for 5 minutes, then immersed in 100% ethanol for 5 minutes, replaced with 100% ethanol and immersed again in 100% ethanol for 5 minutes. A slide glass was taken out from the solution, allowed to stand at room temperature, and air-dried. Next, a sample was prepared by immersing in 90% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and distilled water in this order for 2 minutes at room temperature.
ｂ）抗体反応
ａ）の試料に、４％スキムミルク－ＰＢＳＴ（１０×ＰＢＳ：２５ｍＬ、スキムミルク：１０ｇ、Ｔｗｅｅｎ－２０：１２５μＬを滅菌水で総量２５０ｍＬに調製したもの）を加え、３０分間室温でブロッキングを行った。ＰＢＳ－Ｔ（１０×ＰＢＳ：６０ｍＬ、Ｔｗｅｅｎ－２０：３００μＬを滅菌水で総量６００ｍＬに調製したもの）で、１０００～３０００倍に希釈したアレキシ属ウイルス特異的な一次抗体ＧＭｂ－９２０－８０２（青森県産業技術センター野菜研究所から分譲）をスライドグラスに滴下し、室温で２時間、あるいは４℃で一晩反応させた。その後、ＰＢＳ－Ｔで５分間ずつ３回洗浄した。次に、二次抗体（Ｇｏａｔ　ＡＰ－ｒａｂｂｉｔ－ａｎｔｉ－ＩｇＧ）をＰＢＳ－Ｔで２０００倍に希釈し、室温で２時間、あるいは４℃で一晩反応させた。その後、ＰＢＳ－Ｔで５分間ずつ３回洗浄した。 b) 4% skim milk-PBST (10 × PBS: 25 mL, skim milk: 10 g, Tween-20: 125 μL prepared to a total volume of 250 mL with sterile water) was added to the sample of the antibody reaction a), and blocked at room temperature for 30 minutes. Went. Primary antibody GMb-920-802 specific to Alexis virus diluted 1000-3000 times with PBS-T (10 × PBS: 60 mL, Tween-20: 300 μL prepared in sterile water to a total volume of 600 mL) (Aomori) (Sold from the Prefectural Industrial Technology Center Vegetable Research Institute) was dropped onto a slide glass and allowed to react at room temperature for 2 hours or at 4 ° C overnight. Thereafter, the plate was washed 3 times with PBS-T for 5 minutes. Next, the secondary antibody (Goat AP-rabbit-anti-IgG) was diluted 2000-fold with PBS-T and reacted at room temperature for 2 hours or overnight at 4 ° C. Thereafter, the plate was washed 3 times with PBS-T for 5 minutes.
　洗浄後、１００ｍＭのＡＰ　ｂｕｆｆｅｒ（ｐＨ８．２）（Ｔｒｉｓ－ＨＣｌ　ｐＨ８．２：１２．１１４ｇ、ＮａＣｌ：５．８４４ｇ、ＭｇＣｌ２・６Ｈ２Ｏ：１．０１６５ｇを滅菌水で１００ｍＬに調製したもの）で５分間、平衡化した。ＶＥＣＴＯＲ　ＢＬＵＥ（フナコシ株式会社）で反応液を作成し、３００～４００μＬを滴下して３０分静置した。発色停止液（１Ｍ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）：４０ｍＬ及び０．５Ｍ　ＥＤＴＡ（ｐＨ８．０）：３６０ｍＬの混合液）に浸し、蒸留水で３回洗浄した。コントラストを強くするため、１００％エタノールに１分程度浸漬した。風乾させ、７０％グリセロールで封入した。 After washing, 100 mM AP buffer (pH 8.2) (Tris-HCl pH 8.2: 12.114 g, NaCl: 5.844 g, MgCl 2 · 6H 2 O: 1.0165 g prepared to 100 mL with sterile water) Equilibrated for 5 minutes. A reaction solution was prepared with VECTOR BLUE (Funakoshi Co., Ltd.), and 300 to 400 μL was dropped and allowed to stand for 30 minutes. The plate was immersed in a color stop solution (1M Tris-HCl (pH 8.0): 40 mL and 0.5 M EDTA (pH 8.0): 360 mL) and washed 3 times with distilled water. In order to increase the contrast, the film was immersed in 100% ethanol for about 1 minute. Air dried and encapsulated with 70% glycerol.
２）ＲＴ－ＰＣＲを用いたアレキシ属ウイルスの検出方法
　乳鉢に植物組織０．０５～０．１ｇを入れ、ＴＥ飽和フェノールと核酸抽出緩衝液（２５ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、２５ｍＭ　ＭｇＣｌ２、２５ｍＭ　ＫＣｌ、１％ＳＤＳ）を５００μＬずつ加えて、乳棒で磨砕した。チューブに回収し、ボルテックスで１５秒混合し、室温で１２０００ｒｐｍ、３分間遠心した。上層３００μＬを新しい１．５ｍＬチューブに移し、フェノールとクロロホルムを１５０μＬずつ入れた。これをボルテックスで混合し、室温で１４０００ｒｐｍ、５分間遠心した。さらに、上清２４０μＬを新しい１．５ｍＬチューブに移し、フェノールとクロロホルムを１２０μＬずつ入れた。これをボルテックスで混合し、室温で１４０００ｒｐｍ、５分間遠心した。最後に上清を２００μＬ回収し、ここに３Ｍ酢酸ナトリウム水溶液を１／１０量（２０μＬ）、－３０℃で冷やしておいた９９％エタノールを３倍量（６００μＬ）加え、ボルテックスで混合した。４℃で１４０００ｒｐｍ、２０分間冷却遠心し、上清を捨てた。５００μＬの８０％エタノールを加え、４℃で１４０００ｒｐｍ、５分間冷却遠心した。上清を捨て、室温で静置して乾燥させた後、３０～５０μＬの滅菌水で懸濁した。 2) Detection method of Alexis virus using RT-PCR 0.05 to 0.1 g of plant tissue is put in a mortar, TE saturated phenol and nucleic acid extraction buffer (25 mM Tris-HCl (pH 7.5), 25 mM MgCl 2 ) , 25 mM KCl, 1% SDS) was added in units of 500 μL and ground with a pestle. They were collected in a tube, mixed by vortex for 15 seconds, and centrifuged at 12000 rpm for 3 minutes at room temperature. 300 μL of the upper layer was transferred to a new 1.5 mL tube, and 150 μL of phenol and chloroform were added. This was mixed by vortexing and centrifuged at 14,000 rpm for 5 minutes at room temperature. Further, 240 μL of the supernatant was transferred to a new 1.5 mL tube, and 120 μL each of phenol and chloroform was added. This was mixed by vortexing and centrifuged at 14,000 rpm for 5 minutes at room temperature. Finally, 200 μL of the supernatant was collected, and 1/10 volume (20 μL) of 3M aqueous sodium acetate solution and 3 times volume (600 μL) of 99% ethanol cooled at −30 ° C. were added and mixed by vortexing. The mixture was centrifuged at 14,000 rpm for 20 minutes at 4 ° C., and the supernatant was discarded. 500 μL of 80% ethanol was added and cooled and centrifuged at 4 ° C., 14000 rpm for 5 minutes. The supernatant was discarded, allowed to stand at room temperature, dried, and then suspended in 30-50 μL of sterile water.
　ＴａＫａＲａ　Ｏｎｅ　Ｓｔｅｐ　ＲＮＡ　ＰＣＲ　Ｋｉｔ（ＡＭＶ）を使用してＲＴ－ＰＣＲ反応を行った。アレキシ属ウイルスの検出用に、Ｃｈｅｎら（Ａｒｃｈｉｖｅｓ　ｏｆ　ｖｉｒｏｌｏｇｙ、２００４年、第１４９巻、第４３５ページ）を参考に、配列番号１、配列番号２に示す塩基配列からなるプライマーセットを合成した。ＰＣＲは、５０℃－１５分、９４℃－２分での熱変性後、「９４℃－３０秒、５７℃－３０秒、７２℃－１分」のサイクルを３５回実施し、最後に７２℃－１分反応させ、アガロースゲル電気泳動で増幅断片のサイズを確認した。アレキシ属ウイルスが存在する時の増幅断片のサイズは、約７５０ｂｐとなる。 RT-PCR reaction was performed using TaKaRa One Step RNA PCR Kit (AMV). A primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 was synthesized with reference to Chen et al. (Archives of virology, 2004, 149, 435 pages) for detection of Alexis virus. In PCR, after heat denaturation at 50 ° C. for 15 minutes and 94 ° C. for 2 minutes, a cycle of “94 ° C. for 30 seconds, 57 ° C. for 30 seconds, 72 ° C. for 1 minute” was performed 35 times. The reaction was performed at -1 ° C for 1 minute, and the size of the amplified fragment was confirmed by agarose gel electrophoresis. The size of the amplified fragment when Alexis virus is present is approximately 750 bp.
＜実施例＞アレキシ属ウイルスに感染したニンニク茎頂組織へのｓｉＲＮＡ処理
ａ）材料の選択
　スペイン産及び北海道常呂町産のニンニク（Ａｌｌｉｕｍ　ｓａｔｉｖｕｍ）を用意し、茎頂組織についてブロッキング時間を３０分、染色時間を３５分とした前記免疫組織化学法を行った結果、いずれのニンニクについても茎頂組織及び第１葉原基に染色、すなわちアレキシ属ウイルスが観察された。スペイン産ニンニクにおける染色結果を図１及び図２に示す。
ｂ）茎頂組織片の調製
　スペイン産及び常呂町産のニンニクの鱗茎を鱗片に分け、それぞれの鱗片の皮を取り除いた。水道水に１０分間浸けた後、薄皮を取り除いた。水道水を交換し、その中に中性洗剤を適量入れ、泡立つまでこの溶液を撹拌し、２０分静置した。その後、水洗いした鱗片を不織布に入れ、ヒートシーラーで封をした。８０％エタノール溶液を５００ｍＬ計量カップに用意し、ニンニクを浸して１分静置した。８０％エタノール溶液を捨て、１０％アンチホルミン溶液５００ｍＬにニンニクを浸し、２０分静置した。適時カップを水平に回転させ、溶液が計量カップ内に行き渡るようにした。その後、クリーンベンチ内に計量カップを移し、滅菌水で３回以上洗浄した。エタノール消毒したゴム板上でカミソリ（青函両刃、フェザー安全剃刀（株））を用いて、頂端分裂組織及び第１葉原基を含む１ｍｍの組織片を取り出した後、下記の組成からなるＭＳ培地にすばやく移植した。 <Example> Treatment of siRNA to garlic shoot apical tissue infected with Alexis virus a) Selection of materials Prepare garlic (Allium sativum) from Spain and Tokoro-cho, Hokkaido, blocking time for shoot apex tissue for 30 minutes, As a result of performing the immunohistochemical method with a staining time of 35 minutes, all garlics were stained in the shoot apical tissue and the first leaf primordium, that is, Alexis virus was observed. The staining results for Spanish garlic are shown in FIGS.
b) Preparation of shoot tip tissue pieces The garlic bulbs from Spain and Tokoro-cho were divided into scale pieces and the scale skins were removed. After soaking in tap water for 10 minutes, the skin was removed. The tap water was changed, and an appropriate amount of neutral detergent was put in the tap water. The solution was stirred until it foamed and allowed to stand for 20 minutes. Thereafter, the scales washed with water were put into a nonwoven fabric and sealed with a heat sealer. An 80% ethanol solution was prepared in a 500 mL measuring cup, immersed in garlic and allowed to stand for 1 minute. The 80% ethanol solution was discarded and garlic was immersed in 500 mL of 10% antiformin solution and allowed to stand for 20 minutes. When appropriate, the cup was rotated horizontally to allow the solution to spread through the measuring cup. Thereafter, the measuring cup was transferred into a clean bench and washed three times or more with sterilized water. Using a razor (both Seikan, Feather Safety Razor Co., Ltd.) on a rubber plate sterilized with ethanol, a 1 mm tissue piece containing the apical meristem and the first leaf primordium was taken out, and then placed in an MS medium having the following composition. I transplanted it quickly.
ＭＳ培地
ＫＮＯ３：                          １９００ｍｇ
ＮＨ４ＮＯ３：                    １６５０ｍｇ
ＣａＣｌ２・２Ｈ２Ｏ：      　４４０ｍｇ
ＭｇＳＯ４・７Ｈ２Ｏ：      　３７０ｍｇ
ＫＨ２ＰＯ４：                    　１７０ｍｇ
Ｎａ２－ＥＤＴＡ：            　　３７．３ｍｇ
ＦｅＳＯ４・７Ｈ２Ｏ：      　　２７．８ｍｇ
ＭｎＳＯ４・４Ｈ２Ｏ：      　　２２．３ｍｇ
ＺｎＳＯ４・７Ｈ２Ｏ：      　　　８．６ｍｇ
Ｈ３ＢＯ３：                       　　　６．２ｍｇ
ＫＩ：                                　　　０．８３ｍｇ
Ｎａ２ＭｏＯ４・２Ｈ２Ｏ： 　　　０．２５ｍｇ
ＣｕＳＯ４・５Ｈ２Ｏ：      　　　０．０２５ｍｇ
ＣｏＣｌ２・６Ｈ２Ｏ：      　　　０．０２５ｍｇ
合計                     　　　　４６００ｍｇ
さらに以下の試薬を作成時に加えた。
２ｍｇ／ｍＬ塩酸チアミン溶液：　２００μＬ
ｍｙｏ－ｉｎｏｓｉｔｏｌ：　　　１００ｍｇ
Ｓｕｃｒｏｓｅ　：　　　　　　　　３０ｇ
Ａｇａｒ（植物培地用）：　　　　　　 ７ｇ
１Ｍ　ＫＯＨでｐＨ５．８に調整した後、１０００ｍＬに合わせたもの。 MS medium KNO 3 : 1900 mg
NH 4 NO 3 : 1650 mg
CaCl 2 · 2H 2 O: 440 mg
MgSO 4 · 7H 2 O: 370 mg
KH 2 PO 4 : 170 mg
Na 2 -EDTA: 37.3 mg
FeSO 4 · 7H 2 O: 27.8mg
MnSO 4 .4H 2 O: 22.3 mg
ZnSO 4 · 7H 2 O: 8.6 mg
H 3 BO 3: 6.2mg
KI: 0.83mg
Na 2 MoO 4 · 2H 2 O : 0.25mg
CuSO 4 · 5H 2 O: 0.025mg
CoCl 2 · 6H 2 O: 0.025mg
Total 4600mg
In addition, the following reagents were added at the time of preparation.
2 mg / mL thiamine hydrochloride solution: 200 μL
myo-inositol: 100mg
Sucrose: 30g
Agar (for plant medium): 7g
After adjusting to pH 5.8 with 1M KOH, adjusted to 1000 mL.
ｃ）ｓｉＲＮＡの作製
アレキシ属ウイルスのコートプロテイン（ＣＰ）遺伝子中の約７００ｂｐの塩基配列に対応するｄｓＲＮＡを作製するため、ｐＧＥＭベクター（プロメガ製）上にクローニングされた前記ＣＰ遺伝子を鋳型とし、配列番号７と配列番号８、及び配列番号９と配列番号１０の２組のプライマーセットを用いて、ＰＣＲを行った。ポリメレースとしては、ＰｒｉｍｅＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ製）を使用した。ＰＣＲ条件としては、９４℃－１分での熱変性後、「９８℃－１０秒、５５℃－１５秒、７２℃－１分」のサイクルを３５回実施した。各々のＰＣＲ産物を、ＭｉｎＥｌｕｔｅ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（キアゲン製）で精製し、Ｔ７　ＲｉｂｏＭＡＸ　Ｅｘｐｒｅｓｓ　ＲＮＡｉ　Ｓｙｓｔｅｍ（プロメガ製）に供し、Ａｌｌｅｘｉ－ｄｓＲＮＡ（配列番号１１）を作製した。 c) Preparation of siRNA In order to prepare dsRNA corresponding to the base sequence of about 700 bp in the coat protein (CP) gene of Alexi virus, the CP gene cloned on pGEM vector (Promega) was used as a template, PCR was carried out using two primer sets, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10. Primestar HS DNA Polymerase (manufactured by Takara Bio Inc.) was used as the polymerase. As PCR conditions, after heat denaturation at 94 ° C. for 1 minute, a cycle of “98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1 minute” was performed 35 times. Each PCR product was purified with MinElute PCR Purification Kit (Qiagen) and subjected to T7 RiboMAX Express RNAi System (Promega) to produce Allexi-dsRNA (SEQ ID NO: 11).
更に、Ａｌｌｅｘｉ－ｄｓＲＮＡにＰｏｗｅｒＣｕｔＤｉｃｅｒ（サーモサイエンティフィック製）を作用させて、Ａｌｌｅｘｉ－ｓｉＲＮＡを作製した。得られたサンプルを、Ｎｏｖｅｘ　Ｈｉ－Ｄｅｎｓｉｔｙ　ＴＢＥ　Ｓａｍｐｌｅ　Ｂｕｆｆｅｒ（ライフテクノロジーズ製）と混ぜ、Ｎｏｖｅｘ　ＴＢＥ　Ｇｅｌｓ　６％（ライフテクノロジーズ製）にて非変性ＰＡＧＥ電気泳動（１００Ｖ，３５分）した結果を図３に示す。この様にして作製されたＡｌｌｅｘｉ－ｓｉＲＮＡは、アレキシ属ウイルスのＣＰの発現に対して抑制的に作用する。 Further, PowerCutDicer (manufactured by Thermo Scientific) was allowed to act on Allexi-dsRNA to prepare Allexi-siRNA. The obtained sample was mixed with Novex Hi-Density TBE Sample Buffer (manufactured by Life Technologies), and the result of non-denaturing PAGE electrophoresis (100 V, 35 minutes) with Novex TBE Gels 6% (manufactured by Life Technologies) is shown in FIG. Show. Allexi-siRNA produced in this way acts to suppress the expression of Alexis virus CP.
　また、ウイルス抑制効果のないｓｉＲＮＡとして、β－グルクロニダーゼ（β－ｇｌｕｃｕｒｏｎｉｄａｓｅ、ＧＵＳ）遺伝子に対するｓｉＲＮＡ（ＧＵＳ－ｓｉＲＮＡ）を、ｐＢＩ１２１（クロンテック製）上にクローニングされたＧＵＳ遺伝子を鋳型とし、配列番号３と配列番号４、及び配列番号５と配列番号６の２組のプライマーセットを用いた点以外は上記と同様にして作成した。 In addition, as siRNA having no virus-suppressing effect, siRNA (GUS-siRNA) against β-glucuronidase (GUS) gene is used as a template, and GUS gene cloned on pBI121 (manufactured by Clontech) is used as a template. It was prepared in the same manner as described above except that SEQ ID NO: 4 and two primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 were used.
ｄ）ｓｉＲＮＡ処理
　ｂ）で調製した組織片（常呂町産ニンニクはｎ＝５、スペイン産ニンニクはｎ＝８）を、ｃ）で作製したＡｌｌｅｘｉ－ｓｉＲＮＡ又はＧＵＳ－ｓｉＲＮＡを１０μｇ／５０μＬの濃度で含む水溶液に浸漬した後、速やかに卓上遠心機（２０００×ｇ）で３～５秒間、遠心処理を行った。その後すぐにＭＳ固形培地に組織片を移植し、無菌で培養した。培養条件は、温度２５℃、１２時間照明下とした。 d) siRNA treatment The tissue pieces prepared in b) (n = 5 for Tokorocho garlic, n = 8 for Spanish garlic) and Allexi-siRNA or GUS-siRNA prepared in c) at a concentration of 10 μg / 50 μL After being immersed in the aqueous solution containing the solution, it was immediately centrifuged for 3 to 5 seconds using a tabletop centrifuge (2000 × g). Immediately thereafter, the tissue pieces were transplanted into MS solid medium and cultured aseptically. The culture conditions were a temperature of 25 ° C. and illumination for 12 hours.
培養開始から２０日後に組織片を磨砕し、ＲＴ－ＰＣＲ法でアレキシ属ウイルスが組織片に残っているか確認した。なお、コントロールとしてｓｉＲＮＡに代えて組織片を水で処理したもの（常呂町産ニンニクはｎ＝４、スペイン産ニンニクはｎ＝８）を用意した。 Tissue pieces were ground 20 days after the start of the culture, and it was confirmed by RT-PCR method whether or not Alexis virus remained on the tissue pieces. In addition, instead of siRNA, a tissue piece treated with water (n = 4 for Tokoro-cho garlic and n = 8 for Spanish garlic) was prepared as a control.
　その結果、いずれの産地のニンニクでもコントロールは全ての個体でアレキシ属ウイルスが検出される一方、Ａｌｌｅｘｉ－ｓｉＲＮＡ処理区は常呂町産ニンニクで約６割（処理後ウイルス検出数／処理数　２／５）、スペイン産ニンニクで約４割（処理後ウイルス検出数／処理数　５／８）のウイルス感染率の低下が見られ（培養２０日後）、ウイルスフリー化が明瞭に促進された。また、ウイルス抑制能のないｓｉＲＮＡであるＧＵＳ－ｓｉＲＮＡ処理区では、全ての個体でアレキシ属ウイルスが検出された。代表的な検出結果として、培養２０日後の検出結果を図４（スペイン産ニンニクをＧＵＳ－ｓｉＲＮＡで処理したもの）及び図５（常呂町産ニンニクをＡｌｌｅｘｉ－ｓｉＲＮＡで処理したもの）に示す。 As a result, in all garlics in all production areas, Alexis virus was detected in all individuals, while the Allexi-siRNA treatment group was about 60% of the garlic produced in Tokoro Town (number of detected viruses / number of treatments 2/5) ), About 40% of the Spanish garlic (number of virus detected after treatment / number of treatment 5/8) decreased (after 20 days of culture), and virus-free was clearly promoted. In addition, in the GUS-siRNA treatment group, which is an siRNA having no virus-suppressing ability, Alexis virus was detected in all individuals. As typical detection results, the detection results after 20 days of culture are shown in FIG. 4 (Spanish garlic treated with GUS-siRNA) and FIG. 5 (Tokoro garlic garlic treated with Allexi-siRNA).
　培養２０日目でサンプリングした後も、常呂町産のニンニクは引き続き栽培を続けて、鉢上げまで行った。その後に葉の一部を採取し、アレキシ属ウイルスの検出を行った。その結果、培養２０日目の結果と同様の感染状況が確認され、茎頂培養時に除去されたウイルスはその後再生した植物体においても復活しないことが確認された（図６）。 Even after sampling on the 20th day of cultivation, the garlic from Tokoro continued to be cultivated until the pot was raised. After that, a part of the leaf was collected, and Alexis virus was detected. As a result, an infection situation similar to the result on the 20th day of culture was confirmed, and it was confirmed that the virus removed at the time of shoot tip culture did not recover even in the regenerated plant body (FIG. 6).
＜比較例＞
　強力な抗ウイルス剤として知られ、茎頂培養にも使用されるリバビリンのウイルス除去効果を、アレキシ属ウイルスに感染したニンニクにおいて評価した。上記実施例のｂ）で調製したスペイン産ニンニク由来の組織片５つをＭＳ培地で、５つを１２ｍｇ／Ｌのリバビリンを含むＭＳ培地でそれぞれ２週間、無菌で培養した。培養条件は、温度２５℃、１２時間照明下とした。培養後、各組織片を磨砕し、ＲＴ－ＰＣＲ法でアレキシ属ウイルスが組織片に残っているか確認した。その結果を図７に示す。アレキシ属ウイルスは１２ｍｇ／Ｌという高濃度のリバビリン処理によっても除去されないことが確認された。 <Comparative example>
The virus-removing effect of ribavirin, known as a potent antiviral agent and also used for shoot tip culture, was evaluated in garlic infected with Alexis virus. Five pieces of tissue derived from Spanish garlic prepared in b) of the above example were cultured aseptically for 2 weeks each in MS medium and 5 in MS medium containing 12 mg / L ribavirin. The culture conditions were a temperature of 25 ° C. and illumination for 12 hours. After culturing, each piece of tissue was ground, and it was confirmed by RT-PCR method whether Alexi virus remained on the piece of tissue. The result is shown in FIG. It was confirmed that Alexis virus was not removed even by treatment with ribavirin at a high concentration of 12 mg / L.
＜配列表の説明＞
配列番号１：アレキシ属ウイルス検出用プライマーの塩基配列
配列番号２：アレキシ属ウイルス検出用プライマーの塩基配列
配列番号３：ＧＵＳ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号４：ＧＵＳ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号５：ＧＵＳ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号６：ＧＵＳ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号７：アレキシ属ウイルスのＣＰ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号８：アレキシ属ウイルスのＣＰ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号９：アレキシ属ウイルスのＣＰ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号１０：アレキシ属ウイルスのＣＰ遺伝子に対するｓｉＲＮＡ作製用プライマーの塩基配列
配列番号１１：アレキシ属ウイルスのＣＰ遺伝子に対するｄｓＲＮＡの塩基配列 <Explanation of Sequence Listing>
SEQ ID NO: 1: base sequence of Alexis virus detection primer SEQ ID NO: 2: base sequence of Alexis virus detection primer SEQ ID NO: 3: base sequence of siRNA preparation primer for GUS gene SEQ ID NO: 4: for siRNA preparation of GUS gene Primer base sequence SEQ ID NO: 5: base sequence of siRNA preparation primer for GUS gene SEQ ID NO: 6: base sequence of siRNA preparation primer for GUS gene SEQ ID NO: 7: base sequence of siRNA preparation primer for CP gene of Alexis virus SEQ ID NO: 8: nucleotide sequence of primer for siRNA preparation against CP gene of Alexis virus SEQ ID NO: 9: nucleotide sequence of primer for siRNA preparation against CP gene of Alexis virus SEQ ID NO: 10: Alexi genus Virus of CP of siRNA fabrication primers for gene sequences SEQ ID NO: 11: nucleotide sequence of the dsRNA for CP gene Arekishi genus virus

Claims (5)

  1. 　植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程、及び該溶液より回収した茎頂組織片から植物体を再生させる工程を含む、ウイルスフリー植物体の製造方法。 A virus-free plant comprising a step of immersing a shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of plant virus, and a step of regenerating the plant body from the shoot tip tissue piece collected from the solution. Production method.
  2. 　茎頂組織片を浸した溶液を６０００×ｇ以下の遠心加速度で遠心処理する工程をさらに含む、請求項１に記載の製造方法。 The method according to claim 1, further comprising a step of centrifuging the solution soaked with the shoot apical tissue piece at a centrifugal acceleration of 6000 xg or less.
  3. 　茎頂組織片が頂端分裂組織及び第１葉原基を含む、請求項１又は２に記載の製造方法。 The production method according to claim 1 or 2, wherein the shoot apical tissue piece contains apical meristem and first leaf primordium.
  4. 　干渉性核酸がｓｉＲＮＡである、請求項１～３のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 3, wherein the interfering nucleic acid is siRNA.
  5. 　植物ウイルスの増殖を抑制することができる干渉性核酸を含む溶液に茎頂組織片を浸す工程を含む、茎頂組織片からの植物ウイルスの除去方法。
          A method for removing a plant virus from a shoot tip tissue piece, comprising a step of immersing the shoot tip tissue piece in a solution containing an interfering nucleic acid capable of suppressing the growth of a plant virus.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05260971A (en) * 1990-10-05 1993-10-12 Hoechst Ag Virus-resistant plant and its production
WO1997017429A1 (en) * 1995-11-09 1997-05-15 Oglevee, Ltd. Method for the commercial production of transgenic plants
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JP2002503487A (en) * 1998-02-19 2002-02-05 コットン，インコーポレイテッド Method of creating transgenic plant using shoot tip
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JP2006141274A (en) * 2004-11-19 2006-06-08 Oita General Service Kk Method for culturing shoot apex

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* Cited by examiner, † Cited by third party
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JPH05260971A (en) * 1990-10-05 1993-10-12 Hoechst Ag Virus-resistant plant and its production
WO1997017429A1 (en) * 1995-11-09 1997-05-15 Oglevee, Ltd. Method for the commercial production of transgenic plants
JP2002503487A (en) * 1998-02-19 2002-02-05 コットン，インコーポレイテッド Method of creating transgenic plant using shoot tip
WO2000078128A1 (en) * 1999-06-22 2000-12-28 Wakunaga Pharmaceutical Co., Ltd. Method for constructing virus-free plant
JP2005510455A (en) * 2001-05-04 2005-04-21 テクノロジー・インテグラル・エルティーディー Antiviral agent
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