CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/JP2017/001736, filed Jan. 19, 2017, which claims priority from Japanese application JP 2016-009207, filed Jan. 20, 2016.

TECHNICAL FIELD

The present invention relates to a carrier peptide/protein complex comprising a protein of interest to be introduced into a target plant cell and a carrier peptide, a method for producing the complex, a method for introducing a protein of interest into a target plant cell using the complex, and a kit comprising a protein of interest to be introduced into a target plant cell and a carrier peptide.

BACKGROUND ART

Genetic recombination is a plant breeding technique that has been extensively employed to improve crop productivity and enhance adaptability. Examples of properties that can be improved by such technique include the yield, nutritional quality, herbicide resistance, drought resistance, pesticide resistance, and virus resistance. Genetically modified plants can be used as, for example, living factories for production of hormones, vaccines, aromatic chemicals, and colorants.

At present, genetically modified plants are primarily produced via DNA transformation, such asAgrobacteriumtransformation, protoplast transformation, or a microparticle gun etc. Such DNA transformation techniques, however, have some underlying problems, for example, unexpected incorporation of a foreign DNA into the plant nuclear genome or organelle genome that destructs an endogenous gene, or incorporation of antibiotic-resistance caused by pathogenic bacteria in soil that mediates horizontal gene transfer (Non Patent Literature 1). In order to prepare and make use of modified plants with higher safety, accordingly, it is essential to develop a method, which is simple and extensively applicable to various types of plant cells and proteins. As such a technique, in recent years, a non-transgenic direct protein delivery system has drawn attention. A non-transgenic direct protein delivery system in animals has been extensively developed in vitro and in vivo (Non Patent Literatures 2 to 4). In the case of plants, however, development of such technique had been considered difficult because of that plants have thick and strong cell walls, proteins are generally macromolecules, and plant cells have proteolytic activity, and other reasons. So far, accordingly, it has been difficult to safely modify many plants and produce an improved new variety of a plant.

In recent years, genome editing techniques, such as TALEN, the CRISPR/Cas9 system, and ZFN, have also drawn attention. Genome-editing techniques enable genetic modification at the genome level, such as knock-out of any gene in the genome and knock-in of a foreign gene into the genome (Non Patent Literature 5). While such genome editing techniques are excellent in being able to produce plants with an inheritable genetic modification via a single modification treatment, it is difficult to produce plants with a non-inheritable and transient genetic modification. An RNA interference (RNAi) technique such as siRNA is known in the art as another technique to inhibit gene expression (Non Patent Literature 6). Since effects of the RNAi technique are limited to a single to several generations in many cases, it is often convenient in terms of preparing transient genetically modified plants. The RNAi technique, however, utilizes the inhibitory mechanism at the mRNA level, but it is not a modification technique at the genome level unlike the genome editing technique. Accordingly, a novel technique that modifies a particular gene at the genome level and can limit the effects to a single generation is demanded.

CITATION LIST

Non Patent Literature

Non Patent Literature 1: Nielsen K. M. et al., 1998, FEMS Microbiol. Rev., 22, pp. 79-103Non Patent Literature 2: Liang X. et al., 2015, J. Biotechnol., 208, pp. 44-53Non Patent Literature 3: Sarker S. R. et al., 2014, Mol. Pharm., 11, pp. 164-174Non Patent Literature 4: Schwarze S. R. et al., 1999, Science, 285, pp. 1569-1572Non Patent Literature 5: Esvelt K M. et al., 2013, Mol. Syst. Biol., 9, 641Non Patent Literature 6: Mahmood-ur-Rahman et al., Biotechnol. Adv., 26, 3, pp. 202-209

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to provide a method for introducing a protein into a plant, which is simple and extensively applicable to various types of plant cells and proteins, and to provide a method for producing a transient or inheritable genome-modified plant by introducing a protein into a plant.

Solution to Problem

The present inventors constructed a fusion peptide comprising a cell-penetrating sequence and a polycationic sequence in combination as a carrier peptide to deliver a protein of interest into a plant cell. They discovered that a carrier peptide/protein complex which can introduce a protein of ino a plant cell with excellent efficiency can be formed by mixing the carrier peptide with the protein of interest to be introduced into the plant cell, and thus have completed the present invention.

Specifically, the present invention encompasses the following aspects.

(1) A carrier peptide/protein complex comprising:

a carrier peptide comprising a cell-penetrating sequence and a polycationic or polyanionic sequence; and

a protein of interest to be introduced into a target plant cell.

(2) The complex according to (1), wherein the cell-penetrating sequence is KKLFKKILKYL (SEQ ID NO: 1).

(3) The complex according to (1) or (2), wherein the polycationic sequence comprises at least three amino acid residues selected from lysine (K), arginine (R), and histidine (H).

(4) The complex according to (3), wherein the polycationic sequence comprises 3 to 20 KH repeats or a sequence of 3 to 20 contiguous Ks.

(5) The complex according to (1) or (2), wherein the polyanionic sequence comprises at least three amino acid residues selected from aspartic acid (D) and glutamic acid (E).

(6) The complex according to (4), wherein the carrier peptide comprises the amino acid sequence of KKLFKKILKYLKKLFKKILKYLKKKKKKKK (SEQ ID NO: 23) or KKLFKKILKYLKHKHKHKHKHKHKHKHKH (SEQ ID NO: 24).

(7) The complex according to (1) or (2), wherein the complex has an average hydrodynamic diameter of 150 to 700 nm.

(8) The complex according to any of (1) to (7), wherein the protein of interest has a molecular weight of 5 kDa to 200 kDa.

(9) The complex according to any of (1) to (8), wherein the protein of interest is TALEN-L or TALEN-R, ZFN, or Cas9.

(10) A method for producing the carrier peptide/protein complex according to any of (1) to (9) comprising:

a step of mixing a carrier peptide with a protein of interest to form the carrier peptide/protein complex according to any of (1) to (9).

(11) A method for introducing a protein of interest into a target plant cell comprising:

a step of mixing a carrier peptide with protein of interest to form the carrier peptide/protein complex according to any of (1) to (9); and

a step of bringing the resulting complex into contact with the target plant cell.

(12) A method for producing a genome-modified plant cell comprising:

a step of mixing a carrier peptide with a protein of interest to be introduced into a target plant cell to form a carrier peptide/protein complex; and

a step of bringing the resulting complex into contact with the target plant cell,

wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic or polyanionic sequence, and

wherein the protein of interest is TALEN-L or TALEN-R, ZFN, or Cas9.

(13) A method for producing a genome-modified plant comprising:

a step of producing a genome-modified plant from the genome-modified plant cell obtained by the method according to (12).

(14) A genome-modified plant cell obtained by the method according to (12) or a genome-modified plant obtained by the method according to (13).

(15) The method according to any of (10) to (13), wherein the carrier peptide is mixed with the protein of interest at a molar ratio of 2:1 to 25:1 in the step of forming a complex.

(16) The method according to any of (10) to (13) and (15), wherein the target plant cell is obtained from a plant of Gramineae, Brassicaceae, Solanaceae, Leguminosae, or Salicaceae.

(17) An agent for introducing a protein of interest into a target plant cell consisting of a carrier peptide comprising a cell-penetrating sequence and a polycationic or polyanionic sequence.

(18) A kit for introducing a protein of interest into a target plant cell comprising: a protein of interest to be introduced into a target plant cell; and the carrier peptide as defined in any of (1) to (6).

(19) The kit according to (18), wherein the protein of interest is TALEN-L or TALEN-R, ZFN, or Cas9.

This description includes part or all of the content as disclosed in the description and/or drawings of Japanese Patent Application No. 2016-009207, to which the present application claims priority.

Advantageous Effects of Invention

The present invention provides a method for introducing a protein into a plant cell, which is simple and extensively applicable to various types of plant cells and proteins. According to this method of introduction, varieties of many plants can be safely and readily improved. In addition, the present invention can modify a particular gene at the genome level and can limit the gene modification effects to transient effects.

