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Method of introducing nucleic acid into plant cells UPDATE

外国特許コード F190009909
整理番号 07999-US-2
掲載日 2019年8月26日
出願国 アメリカ合衆国
出願番号 201314381045
公報番号 20150218569
出願日 平成25年2月27日(2013.2.27)
公報発行日 平成27年8月6日(2015.8.6)
国際出願番号 JP2013056062
国際公開番号 WO2013129698
国際出願日 平成25年2月27日(2013.2.27)
国際公開日 平成25年9月6日(2013.9.6)
優先権データ
  • 特願2012-040622 (2012.2.27) JP
  • 201261691833 (2012.8.22) US
  • 2013JP56062 (2013.2.27) WO
発明の名称 (英語) Method of introducing nucleic acid into plant cells UPDATE
発明の概要(英語) The object of the present invention is to provide a method of introducing a nucleic acid into plant cells, which is simple and widely applicable to various types of plant cells and nucleic acids. The present invention relates to a method of introducing a nucleic acid into a target plant cell, comprising a step of forming a complex by bringing a carrier peptide, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence, into contact with a nucleic acid and a step of bringing the obtained complex into contact with a target plant cell.
従来技術、競合技術の概要(英語) BACKGROUND ART
Providing useful traits that cannot be provided by cross breeding to a plant by introducing foreign genes to plant cells is extremely meaningful in future improvement of crops and development of agriculture. Furthermore, in order to establish a substance production method by plants using carbon dioxide as a raw material, as a novel substance production method alternating a petroleum-dependent substance production method, it is indispensable to use a plant genetic recombination technique.
As a method of introducing a gene into a plant cell, an Agrobacterium method using infection with a soil bacterium, i.e., Agrobacterium, a particle-gun method and a viral vector method are known. The Agrobacterium method is the most frequently used in introducing a gene into a plant cell. However, there are many economically important plants to which a gene cannot be introduced by the Agrobacterium method, at present. The particle-gun method has been much more generally used compared to the Agrobacterium method; however, device cost is high. In addition to this problem, there is a risk of damaging a gene and transformation efficiency is low. The viral vector method is advantageous compared to the Agrobacterium method since transformation efficiency and gene expression efficiency are high; however, the method has a problem in that the size of the gene to be introduced is limited and viral infectious ability is low. In the circumstances, it has been desired to develop a novel method of introducing a gene, which is simple and widely applicable to all types of plants and genes.
It is known that a cell-penetrating peptide (CPP) has a function of transporting a complex comprising the peptide and another substance (for example, a protein and a nucleic acid) through biomembrane of mammalian and human cell strains. However, use of CPP in plant cells is limited. Unlike animal cells, plant cells have double barriers, i.e., cell wall and cell membrane, for preventing internalization of a complex comprising CPP. In the meantime, it is disclosed that a polycationic peptide concentrates negatively charged DNA by ionic interaction to form a complex that can be used for gene delivery, and that such a complex is useful for introducing a gene into animal cells (Patent Document 1: WO2011/006133). It has been also reported that a polycationic peptide was used for introducing a gene into a plant protoplast (Patent Document 2: JP Patent Publication (Kokai) No. 7-213285). However, the method employs a protoplast without a cell wall. In addition, it cannot be said that gene introduction efficiency into a plant cell is sufficient, in this method.
特許請求の範囲(英語) [claim1]
1. A method of introducing a nucleic acid into a target plant cell, comprising a step of forming a complex by bringing a carrier peptide, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence, into contact with a nucleic acid and a step of bringing the obtained complex into contact with a target plant cell.

[claim2]
2. The method according to claim 1, wherein the number of amine groups from the carrier peptide/the number of phosphate groups from the nucleic acid is 2 or less.

[claim3]
3. The method according to claim 1, wherein the complex has an average hydrodynamic diameter of 150 to 500 nm.

[claim4]
4. The method according to claim 1, wherein the cell-penetrating sequence is BP100 or Tat2.

[claim5]
5. The method according to claim 1, wherein the polycationic sequence comprises at least three amino acid residues selected from lysine (K), arginine (R) and histidine (H).

[claim6]
6. The method according to claim 5, wherein the polycationic sequence comprises 3 to 20 repeat sequences of KH or a sequence of 3 to 20 consecutive Rs.

[claim7]
7. The method according to claim 1, wherein the step of forming a complex is performed in the presence of the carrier peptide comprising an organelle transit sequence and a polycationic sequence.

[claim8]
8. The method according to claim 1, wherein the carrier peptide further comprises an organelle transit sequence.

[claim9]
9. The method according to claim 1, wherein incubation time for bringing the complex into contact with a target plant cell is 5 to 150 hours.

[claim10]
10. A carrier peptide for introducing a nucleic acid into a target plant cell, comprising a cell-penetrating sequence and a polycationic sequence.

[claim11]
11. The carrier peptide according to claim 10, wherein the cell-penetrating sequence is BP100 or Tat2.

[claim12]
12. The carrier peptide according to claim 10, wherein the polycationic sequence comprises at least three amino acid residues selected from lysine (K), arginine (R) and histidine (H).

[claim13]
13. The carrier peptide according to claim 10, wherein the polycationic sequence comprises 3 to 20 repeat sequences of KH or a sequence of 3 to 20 consecutive Rs.

[claim14]
14. The carrier peptide according to claim 10, further comprising an organelle transit sequence.

[claim15]
15. A complex for introducing a nucleic acid into a target plant cell, comprising the carrier peptide according to claim 10 and a nucleic acid.

[claim16]
16. A complex for introducing a nucleic acid into a target plant cell, comprising the carrier peptide according to claim 10, a carrier peptide comprising an organelle transit sequence and a polycationic sequence, and a nucleic acid.

[claim17]
17. The complex according to claim 15, wherein the number of amine groups from the carrier peptide/the number of phosphate groups from the nucleic acid is 2 or less.

[claim18]
18. The complex according to claim 15, wherein the complex has an average hydrodynamic diameter of 150 to 300 nm.

[claim19]
19. A kit for introducing a nucleic acid into a target plant cell, which comprises a carrier peptide comprising a cell-penetrating sequence and a polycationic sequence.

[claim20]
20. The kit according to claim 19, which further comprises a carrier peptide comprising an organelle transit sequence and a polycationic sequence.

[claim21]
21. The kit according to claim 19, wherein the cell-penetrating sequence is BP100 or Tat2.

[claim22]
22. The kit according to claim 19, wherein the polycationic sequence comprises at least three amino acid residues selected from lysine (K), arginine (R) and histidine (H).

[claim23]
23. The kit according to claim 19, wherein the polycationic sequence comprises 3 to 20 repeat sequences of KH or a sequence of 3 to 20 consecutive Rs.
  • 発明者/出願人(英語)
  • Numata Keiji
  • Yoshizumi Takeshi
  • Kodama Yutaka
  • Ohtani Misato
  • Demura Taku
  • RIKEN
国際特許分類(IPC)

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