WO2018037635A1 - Vecteur de virus contenant une séquence d'acide nucléique ciblée par le mir398 pour le transfert de gènes - Google Patents

Vecteur de virus contenant une séquence d'acide nucléique ciblée par le mir398 pour le transfert de gènes Download PDF

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WO2018037635A1
WO2018037635A1 PCT/JP2017/018011 JP2017018011W WO2018037635A1 WO 2018037635 A1 WO2018037635 A1 WO 2018037635A1 JP 2017018011 W JP2017018011 W JP 2017018011W WO 2018037635 A1 WO2018037635 A1 WO 2018037635A1
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mir398
nucleic acid
acid sequence
sequence
plant
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PCT/JP2017/018011
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English (en)
Japanese (ja)
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和大 石橋
哲也 中条
学 吉川
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国立研究開発法人農業・食品産業技術総合研究機構
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Publication of WO2018037635A1 publication Critical patent/WO2018037635A1/fr

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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
    • 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 viral vector for gene transfer comprising a miR398 target nucleic acid sequence, And a gene transfer method comprising the step of infecting a plant with the same.
  • Non-Patent Document 1 Non-Patent Document 1
  • the use of a viral vector is one promising alternative technique, but it has been necessary to remove the viral vector after completion of genome editing, which has been a technical obstacle.
  • miR398 is a small RNA that has been shown to be involved in stress response and copper metabolism, but it has been reported as an miRNA whose expression is induced by environmental stress in Arabidopsis and the like and suppresses the target gene. (Non-patent document 2).
  • Examples of the stress that induces the expression of miR398 include sucrose contained in the dedifferentiation / redifferentiation medium of many plants.
  • the purpose of the present invention is to find a plant virus vector that can be easily removed after the completion of gene introduction and can minimize the adverse effects on the plant by the removal operation.
  • the present inventors introduced a genome editing enzyme gene into a plant by using a viral vector for gene introduction containing a miR398 target nucleic acid sequence, which was not conventionally known, and genome editing.
  • a viral vector for gene introduction containing a miR398 target nucleic acid sequence which was not conventionally known, and genome editing.
  • the gist of the present invention is as follows.
  • a viral vector for gene transfer comprising a miR398 target nucleic acid sequence.
  • the virus vector according to [1] which has a miR398 target nucleic acid sequence targeted by miR398 having a sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequences of SEQ ID NOS: 5-26.
  • the miR398 target nucleic acid sequence is complementary to a nucleic acid sequence having at least 80% identity in the region of positions 2 to 17 of the sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NOs: 5 to 26
  • the virus vector according to any one of [1] to [3], which is a target.
  • the viral vector according to any one of [1] to [6] which is a plant viral vector.
  • a gene introduction method comprising a step of infecting a plant with the virus vector according to any one of [1] to [9].
  • the gene introduction method according to [10] further comprising a step of dedifferentiating and redifferentiating the plant.
  • the present invention it is not necessary to perform a special operation for removing the viral vector after completion of gene introduction, and the gene can be introduced into the plant with minimal adverse effects on the plant.
  • Figure 3 shows the design of a viral vector according to one embodiment of the present invention.
  • the present invention provides a viral vector for gene transfer containing a miR398 target nucleic acid sequence, and a gene transfer method comprising a step of infecting a plant with the virus vector.
  • MiR398 is widely preserved in plants regardless of monocotyledonous or dicotyledonous.
  • the miR398 in the present invention may be any plant miR398 that has the same expression format as tobacco and can be regenerated from a virus-infected leaf.
  • Examples of such miR398 include, but are not limited to, miR398 such as tobacco, Arabidopsis, rice, corn, soybean, wheat, potato, melon, apple apple, poplar, and oilseed rape.
  • miR398 in the present invention is tobacco miR398.
  • the sequence of tobacco miR398 is 5'-UGUGUUCUCAGGUCGCCCCUG-3 '(SEQ ID NO: 1) However, additional miR398 sequences available in the present invention are illustrated in Table 1.
  • the miR398 target nucleic acid sequence in the viral vector of the present invention may be complementary to a nucleic acid sequence having at least 70%, preferably 80%, more preferably 90% identity with the miR398 sequence, preferably SEQ ID NO: 1. And at least 80%, preferably 85%, more preferably 90%, most preferably 100% identity in the region of positions 2-17 of a sequence selected from the group consisting of What is necessary is just to be complementary to the nucleic acid sequence to have.
  • the miR398 target nucleic acid sequence in the viral vector of the present invention is: 5′-CAGGGGCGACCCTGAGAACACA-3 ′ (SEQ ID NO: 2) 5′-TGCGGGTGACCCTGGGAAATACATA-3 ′ (SEQ ID NO: 3) 5′-ACTCGGTGACCTGGGACACACT-3 ′ (SEQ ID NO: 4); And a sequence consisting of a sequence having at least 90% identity with them, preferably the sequence of SEQ ID NO: 2, 3 or 4.
  • the virus vector of the present invention may be any plant virus vector, but preferably a solanaceous plant virus vector such as a tomato mosaic virus vector, a tomato bushy stunt virus vector, a tobacco mosaic virus vector, a tobacco stem virus.
  • a vector, a potato X virus vector, a potato Y virus vector and the like can be mentioned, but not limited thereto.
  • the viral vector of the present invention is preferably deficient in the coat protein, which makes it easier to remove.
  • any plant can be used as long as the expression pattern of miR398 is the same as that of tobacco and the plant can be regenerated from infected leaves.
  • tobacco, Arabidopsis thaliana, rice, corn, soybean examples include, but are not limited to, wheat, potato, melon, apple apple, poplar, oilseed rape.
  • the plant into which the gene can be introduced according to the present invention is tobacco.
  • the present invention provides a gene introduction method including a step of infecting a plant with the virus vector.
  • the gene introduction method according to the present invention preferably further comprises a step of dedifferentiating and redifferentiating the plant.
  • the step of infecting a plant with a viral vector and the step of dedifferentiating and redifferentiating the plant may be any step commonly used in the art.
  • An example thereof includes, but is not limited to, a step of inoculating tobacco with a virus, and then a step of forming callus from infected leaves and redifferentiating to obtain shoots.
  • Viral vector design To easily measure the efficiency of gene transfer, coat protein coding region of tomato mosaic virus (ToMV) vector pTLW3 (Kubota et al. J Virol., 2013, 77 (20): 11016-1026) was replaced with the yellow fluorescent protein (YFP) gene or the meganuclease I-SceI gene.
  • the YFP gene was used because it is convenient for visualizing the site of infection, but it does not affect the properties of the viral vector and can be replaced with any sequence.
  • I-SceI is an endonuclease that specifically recognizes an 18-base sequence.
  • Tobacco normally does not express fluorescence, but by using transformed tobacco with a gene cassette in the genome that expresses firefly luciferase when the I-SceI target site is cleaved and subsequently restored to frame when repaired, Visualization of genome editing sites is possible. Furthermore, the miR398 target nucleic acid sequence or Spacer sequence having SEQ ID NO: 2, 3 or 4 was placed immediately after the stop codon of the YFP gene or I-SceI gene. The design of the viral vector is shown in FIG.
  • a primer To86: 5′-TCAACAGAGTAACTGGGT-3 ′ (SEQ ID NO: 27); and To87: 5′-GCAGCTACAGACTTAGA-3 ′ (SEQ ID NO: 27), which uses 100 ng of RNA as a template and targets the ToMV replication protein coding region. 28)
  • PrimeScript One Step RT-PCR Kit Ver. RT-PCR 40 cycles was performed using 2 (Dye Plus) (Takara Bio Inc.).
  • viral RNA prepared by in vitro transcription and RNA extracted from virus-free tobacco leaves were used, respectively.
  • the sample after RT-PCR was subjected to agarose gel electrophoresis, and an image was captured using LAS-3000 (Fuji Film Co., Ltd.).
  • virus was detected in 100% (25/25) sample, whereas in shoots redifferentiated from virus vector-infected leaves having SEQ ID NO: 2, 60% (15/25) sample. Only virus was detected, and the remaining 40% of samples had viral genome accumulation below the detection limit.
  • the gene transfer method according to the present invention does not require mating for removing foreign genes, it can be used for non-recombinant gene introduction into plants that want to avoid mating of vegetatively breeding crops, F1 varieties, fruit trees, etc. . Further, the present invention can be applied even to plants that are difficult to transform. In addition, the present invention provides virus removal after carrying out eradication treatment of the virus by suppressing the expression of endogenous genes using a virus vector or inducing RNA silencing for other viruses already infected. It can also be applied to.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Plant Pathology (AREA)
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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un vecteur de virus destiné au transfert de gènes, ledit vecteur de virus contenant une séquence d'acide nucléique ciblée par le miR398.
PCT/JP2017/018011 2016-08-24 2017-05-12 Vecteur de virus contenant une séquence d'acide nucléique ciblée par le mir398 pour le transfert de gènes WO2018037635A1 (fr)

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JP2016-163824 2016-08-24
JP2016163824A JP2018029521A (ja) 2016-08-24 2016-08-24 ｍｉＲ３９８標的核酸配列を含む、遺伝子導入のためのウイルスベクター

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006073727A2 (fr) * 2004-12-21 2006-07-13 Monsanto Technology, Llc Constructions d'adn recombinant et procedes pour le controle de l'expression genetique
JP2008545406A (ja) * 2005-05-27 2008-12-18 フォンダツィオーネ セントロ サン ラファエレ デル モンテ タボール 遺伝子ベクター
WO2012063817A1 (fr) * 2010-11-09 2012-05-18 独立行政法人産業技術総合研究所 Procédé de production de cellules souches pluripotentes dérivées de monocytes de sang périphérique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006073727A2 (fr) * 2004-12-21 2006-07-13 Monsanto Technology, Llc Constructions d'adn recombinant et procedes pour le controle de l'expression genetique
JP2008545406A (ja) * 2005-05-27 2008-12-18 フォンダツィオーネ セントロ サン ラファエレ デル モンテ タボール 遺伝子ベクター
WO2012063817A1 (fr) * 2010-11-09 2012-05-18 独立行政法人産業技術総合研究所 Procédé de production de cellules souches pluripotentes dérivées de monocytes de sang périphérique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHUJO T. ET AL.: "A removable virus vector suitable for plant genome editing", THE PLANT JOURNAL, vol. 91, no. 3, 21 April 2017 (2017-04-21), pages 558 - 561, XP055604096 *
DUGAS D. V. ET AL.: "Sucrose induction of Arabidopsis miR398 represses two CU /Zn superoxide dismutases", PLANT MOL BIO L, vol. 67, 2008, pages 403 - 417, XP019613450 *
KOMAROVA T.V. ET AL.: "New Viral Vector for Efficient Production of Target Proteins in Plants", BIOCHEMISTRY (MOSCOW, vol. 71, no. 8, 2006, pages 846 - 850, XP019392024, DOI: doi:10.1134/S0006297906080049 *
KUMAR P. ET AL.: "Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences", JOURNAL OF VIROLOGY, vol. 83, no. 20, 2009, pages 10472 - 10479, XP055604092 *
PARIZOTTO E. A. ET AL.: "In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA", GENES & DEVELOPMENT, vol. 18, 2004, pages 2237 - 2242, XP002989682 *

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