KR20050011288A - Cationic Oligopeptide-mediated RNA Delivery into Plants - Google Patents

Abstract

PURPOSE: A cationic oligopeptide-mediated RNA delivery into plants is provided, thereby conveniently delivering the target RNA into the plants, easily carrying out post-transcriptional gene silencing(PTGS) to rapidly identifying the function of a gene in plants, and enabling expression of a target protein in plants without limitation of distinct transcription regulating mechanism in plants. CONSTITUTION: The cationic oligopeptide-mediated RNA delivery into plants comprises the steps of: binding a target RNA to the cationic oligopeptide as a carrier through static electrical interaction by reacting the target RNA with plants in a solution selected from phosphate buffer solution(PBS), MS medium, water and physiological saline solution at 4 deg. C and pH 4 to 10 for 15 minutes to 1 hour; and transmitting the aqueous phase of the cationic oligopeptide-bound RNA into the plants, wherein the RNA is double-stranded RNA or single-stranded RNA; the oligopeptide contains 6- to 30-mers of lysine peptide or arginine peptide; and the transmission is carried out by dipping plants in medium containing the cationic oligopeptide-mediated RNA or injecting the cationic oligopeptide-mediated RNA into plants.

Description

RNA Delivery Technique to Plants Using Cationic Oligopeptides {Cationic Oligopeptide-mediated RNA Delivery into Plants}

The present invention relates to a technique for transferring RNA to plants using cationic oligopeptides, and more specifically, using negative cationic oligopeptides as carriers to bind negatively charged RNAs to be introduced into the carriers through electrostatic interactions. The present invention relates to a new type of RNA delivery technique that includes permeation of plant tissues in aqueous solution.

In the last few decades when plants have been able to transform, in addition to the acquisition of useful transgenic plants through the introduction of useful foreign genes, the sequencing of specific genes by introducing the nucleotide sequences of specific genes into the plants in the antisense direction ( A method of inducing silencing has been developed and used. In particular, in the silencing experiment, it was observed that mRNA was degraded in the cytoplasm after transcription, which has been regarded as a sign of post-transcriptional gene silencing (hereinafter abbreviated as 'PTGS').

PTGS, which was initially regarded as a bizarre phenomenon that only confined to petunias and a few other plant species, is now a focus of interest in molecular biology. PTGS mechanisms in plants, collectively known as RNA silencing, and RNA interference in animal systems or protozoa are known as double stranded RNA. And share a number of factors necessary to degrade host mRNA having the same sequence. Thus, PTGS in plants can be regarded as functionally identical to RNA i in animals, and genetic and biochemical analysis of PTGS indicates that RNA i is highly accurate with the original PTGS model (Dougherty et al., 1994). Prove it.

When plants detect viral RNA, they begin PTGS to selectively degrade foreign viral RNA as a defense against it. Thus, although PTGS was first discovered in the transformation system, it can be assumed that the biological role of PTGS is the plant virus-defense strategy. However, RNA silencing appears to be involved in the normal development and physiological processes of cells as well as acting as an intracellular defense mechanism against viral RNA. RNA silencing has been used to study the gene function of C. elegans (Fraser et al., 2000; Gonczey et al., 2000), Drosophila (Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999), nematode (nematode) Fire et al., 1998; Montgomery et al., 1998), trypanosomes (Ngo et al., 1998), mammals (Wianny and Goetz, 2000), and many organisms, including plants (Chuang and Meyerowitz, 2000; Waterhouse et al., 1998). It is known to be effective.

Double-stranded RNA formed intracellularly or delivered from outside the cell plays a central role in inducing degradation of the target mRNA. According to Hammond et al. (2000; Nykanen et al., 2001; Hutvagner and Zarmore, 2002), double-stranded RNA is referred to as small interference RNA (hereinafter abbreviated as 'siRNA') of 21 to 23 nucleotides in length. The siRNAs that are cleaved and thus formed are attached to an RNA-induced silencing complex (hereinafter abbreviated as 'RISC') that recognizes and degrades mRNA. The siRNA is then amplified by RNA-dependent RNA polymerase (abbreviated as 'RdRP') to maintain a molar ratio far exceeding the amount of target mRNA (Nishikura, 2001). It is also speculated that even double-stranded RNAs of different origins share this mechanism. As a way to more conveniently investigate gene silencing induced by double-stranded RNA in plants, the present inventors have studied a method for effectively delivering in vitro synthesized double-stranded RNA to plant cells.

Introduction of double-stranded RNA in animal systems is possible either by injection (Fire et al., 1998) or by culturing the cells in a medium comprising double-stranded RNA (Timmons and Fire, 1998; Timmons et al., 2001). However, the direct introduction of double stranded RNA in plants has not yet been extensively attempted. Co-suppression or PTGS mechanisms can be used to introduce vectors that form double-stranded RNA for target genes by Agrobacterium-mediated transformation (Chuang and Meyerowitz, 2000; Waterhouse et al., 1998). Robert et al., 1989; Liu et al., 2002; Dalmay et al., 2000), using infiltration (Schiff and Kumar, 2002) or infecting plants with viral vectors that produce double stranded RNA. Most studies have been conducted by transforming plants with additional copies of the associated genes using the method. Viral vectors include a variety of vectors based on TMV, PVX, CaMV, TRV and Gemini viruses. Some viruses induce silencing because they express genes that interfere with the plant's gene-silencing mechanism. It may be more efficient than others. In addition, Johansen and Carrington (2001) developed an Agrobacterium-mediated transient expression system that allows for rapid study of gene silencing in intact tissues without generating transformed plants. did.

However, RNA transfer to plants using viruses is not only difficult to distinguish between traits / symptoms caused by the pathogenicity of viruses used as hosts and traits or symptoms caused by silencing of specific RNAs, Due to mobility through action, there is a disadvantage that it is difficult to control the site where the traits or symptoms appear. In addition, the method using Agrobacterium also has a disadvantage that takes a huge time to prepare and transform the appropriate vector. Therefore, there is an urgent need to develop an RNA delivery technique that can introduce foreign double-stranded RNA into the plant in a short time efficiently and easily to perform PTGS in the plant more easily and thereby to identify the gene function of the plant more quickly. Is required.

In recent years, by introducing a foreign gene into a plant by a recombinant DNA method, it is possible to give a plant a new trait that could not be introduced into the plant by conventional breeding. Plant transformation by recombinant DNA method Until now, a method of inserting a foreign gene of interest into a host DNA using a plant expression vector made by modifying Agrobacterium Ti-plasmid (Ti-plasmid) (Hernalsteens et al., 1980, Nature 287, 654-656), and when using vectors that do not contain T-DNA sites, electroporation, microparticle bombardment, and polyethylene glycol precipitation Polyethylene glycol-mediated uptake is used to introduce recombinant DNA into plants. However, the expression of the target gene in the plant using the plasmid vector as described above is essential for entry into the nucleus of the DNA, and must be able to stably settle and express on the chromosome of the host even after entry. In contrast, expression of a target gene using whole RNA or mRNA is more effective because it does not require entry into the nucleus and translates the target protein directly in the cytoplasm. Therefore, there is a need for the development of an easier and simpler RNA delivery technique that can introduce the foreign mRNA into the plant effectively in a short time.

Accordingly, the present inventors use cationic oligopeptides as carriers to bind negatively charged RNAs to be introduced into the carriers through electrostatic interactions and then permeate the plant tissues in an aqueous solution, thereby not only at the cellular level but also in whole plant tissues. It is possible to target the present invention has been completed to enable the efficient delivery of RNA without going through a tissue culture step.

The present invention has been made to solve the above-mentioned conventional problems, the object of the present invention is to introduce a conventional RNA introduction technique by introducing into the plant by electrostatic interaction with the negatively charged RNA using a cationic oligopeptide as a carrier It provides a new way of delivering RNA to plants, which makes delivering RNA to plants much easier and simpler than their counterparts.

Figure 1 (a) shows a GUS gene and NPTII gene 35S promoter complex of a double-stranded RNA and a 12-mer arginine peptide synthesized in vitro using beta-glucuronidase (GUS: β-glucuronidase) DNA as a template After treatment to overexpressed tobacco suspension cells (tobacco suspension cells) under the control of the abbreviated as reverse transcription polymerase chain reaction (RT-PCR) using RNA isolated therefrom ) Is the result of

Figure 1 (b) shows the results confirmed by the northern blot (problem) of the double-stranded RNA labeled with radioisotope to the total RNA isolated from the same sample as in Figure 1 (a).

Figure 2 (a) shows the total RNA isolated from the leaves of arabidopsis (arabidopsis thaliana) overexpressed by the GFP (green fluorescent protein) gene by the regulation of the 35S promoter to the 12-mer arginine peptide Combined and treated in onion roots and observed by confocal microscopy,

Figure 2 (b) is observed only by confocal fluorescence microscopy after treating only the whole RNA isolated from the leaves of the Arabidopsis, which GFP gene is overexpressed by the control of the 35S promoter.

FIG. 3 (a) shows a methyl guanine cap introduced at the 5 ′ end of mRNA transcribed in vitro using GFP DNA as a template, followed by onion root using a 12-mer arginine peptide. After treatment with, the expression level of GFP mRNA was observed by confocal fluorescence microscopy,

FIG. 3 (b) shows the expression level of GFP mRNA after confocal fluorescence microscopy after treating mRNA in vitro transcribed in onion using GFP DNA as a template using 12-mer arginine peptide.

FIG. 3 (c) shows the expression of GFP mRNA after confocal fluorescence microscopy after only the mRNA transcribed in vitro with GFP DNA was treated in onion roots alone.

In order to achieve the above object, the RNA transfer technique to the plant using the cationic oligopeptide according to the present invention electrostatically charged RNA to be introduced into the carrier using the cationic oligopeptide as a carrier (carrier) After binding through interaction, characterized in that it comprises a direct introduction to the plant in an aqueous solution.

In this case, the RNA that can be introduced into the plant can be either double-stranded RNA or single-stranded RNA, and in the case of double-stranded RNA, the synthesized in vitro is used, and in the case of the single-stranded RNA, mRNA of the target gene is in vitro It is preferable to use mRNA synthesized in or separated from in vivo.

In this method, the cationic oligopeptide is a positively charged 6 to 30-mer lysine peptide or an arginine peptide, and particularly oligolysine or oligoarginine having a length of hexamer, hexamer, 12-mer and 15-mer is preferred. Other peptides or peptide analogs other than those described above can also be used as long as they can penetrate the cell membrane.

Cationic oligopeptides are a modified form of the protein transduction domain (hereinafter abbreviated as 'PTD') of various proteins, including human immunodeficiency virus (HIV) -tat transcription factor (ref). Likewise, PTD contains a lot of positively charged amino groups and can pass through cell membranes (Futaki et al., 2001).

The combination and length of the cationic amino acids constituting the cationic peptide do not significantly affect the efficiency of the RNA delivery technique of the present invention. In particular, the efficiency of the peptide having a length of 6 to 30 dimers was very constant, and when the sum of the positively charged arginine and lysine of the total amino acids is about 90% or more, it is thought to act as an efficient delivery medium. Animal cell transformation using such oligopeptides is already well known, but there is no known literature that suggests that it can be used in plant systems to penetrate nucleic acids such as RNA into plant cells. .

The RNA / peptide complexes presented in the present invention are bound by the electrostatic interaction between the positive charge of amino acids and the negative charge of RNA. Suitable reaction solutions for such binding include general PBS (phosphate buffer, pH 7.4), MS medium and the like can be used, and even purified water or brine at seawater concentration can be used. The pH of the reaction solution does not require a rigorous pH as binding is seen in various pH ranges, about pH 4-10. The reaction temperature is suitably carried out at about 4 ℃, and left for about 15 minutes to about 1 hour, it is confirmed that the RNA and peptide complex is formed. Preferably, the reaction is performed for 1 hour to improve the stability of the complex.

The RNA / peptide complex thus formed can be introduced directly into the cultured cells of plants as well as tissues such as seeds, roots, stems, bulbs in an aqueous solution. This is a very simple method made by adding a solution containing the complex to a cell culture medium, a germination medium or a breeding medium, and culturing cells, germinating seeds or breeding young plants in the medium. In other words, the RNA delivery technique of the present invention is a technique that can be carried out not only at the cellular level but also to whole plant tissues, and can efficiently deliver RNA without going through the tissue culture step. Although not demonstrated by any theoretical basis, this introduction allows the plant itself to absorb an aqueous solution containing RNA / peptide complexes, using natural absorption phenomena such as osmotic action of plant roots, and then cationic in the absorbed aqueous solution. Nuclear orientation of the oligopeptides is thought to induce cytoplasm and even the nucleus through the cell wall and plasma membrane of each plant cell. Alternatively, a method of injecting an aqueous solution containing the RNA / peptide complex by direct injection into the tissue of the plant to which the target RNA is to be introduced may be used.

Using this method, it is easier and more convenient to introduce double-stranded RNA into plants without requiring a long time vector work, so that PTGS, which is part of in-planta gene silencing, can be easily performed. . As a result, it is possible to quickly identify the gene function of the plant, to assist in the development of new trait crops through the interference of specific genes, and to provide a technology based on functional genetics.

In addition, when mRNA is introduced into the plant in this manner, it can be directly translated into the target protein in the cytoplasm without entering the intracellular nucleus, thereby expressing more effectively than the introduction of DNA into the plant. This direct expression in the cytoplasm is not only dependent on the cell division cycle, but also means that the target gene can be expressed in cells that do not divide, which may induce target gene expression in a wider variety of plant tissues. will be. In addition, since it is possible to synthesize a large amount of target mRNA involved in the transcription of a specific protein in the whole RNA in vitro, it can be used for the study of the function of the target gene in a plant, without any modification to the host gene. The ability to express target genes could solve the problem of genetically modified crops (GMOs). In addition, if the whole RNA isolated from in vivo by electrostatic randomly coupled to the cationic peptide and delivered, it may be applicable to the study of target genes that overexpress or tissue-specific expression at a specific developmental stage.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1> Delivery of double stranded RNA / peptide

Under the control of the 35S promoter, it was transformed by Agrobacterium carrying a pBI121 plasmid containing a beta-glucuronidase (GUS: β-glucuronidase) gene and a neomycin phosphotransferase II (NPTII). Tobacco suspension cells were obtained from shoots of tobacco plants overexpressed with NPTII and stored at 25 ° C. in MS medium containing 50 mg / L of kanamycin. Separately, double-stranded RNA was synthesized in vitro using GUS DNA as a template.

As the oligopeptide, 12-molecular arginine peptide (manufactured by Peptron, Korea) powder was dissolved in PBS (phosphate buffered saline, pH 7.4), and then mixed with 5 μg, 10 μg and 20 μg of the prepared GUS double-stranded RNA, respectively. 1 hour on ice to obtain a GUS double stranded RNA / arginine peptide complex. The complex thus obtained was treated and delivered to the prepared tobacco suspension cell line at 25 ° C. for 1 hour.

To confirm selective expression inhibition of gene expression, total RNA was isolated from the tobacco suspension cultured cell line using TRI reagent (manufactured by Molecualr Probes Diagnostics, USA). The result of performing reverse transcriptase polymerase chain reaction (RT-PCR) of the GUS and NPTII genes on the isolated whole RNA is shown in FIG. 1 (a). As shown, GUS mRNA was not detected, whereas NPTII mRNA was GUS. Normal expression was observed regardless of the introduction of double stranded RNA. Therefore, it can be seen that the GUS mRNA in the tobacco suspension cultured cell line generated PTGS as part of in planar gene silencing by the double-stranded GUS RNA introduced into the cell. As evidence to support this, although not shown in the figure, the results of the RNase protection assay (RNase protection assay) revealed a small interfering RNA (siRNA) having 21-23 base pairs from cells treated with double-stranded RNA of GUS DNA. Could. In another method, the whole RNA was isolated from the same sample, and a northern blot was performed using a double-stranded RNA labeled with radioisotopes as a probe. As shown in FIG. While not detected, it was confirmed that NPTII mRNA was normally expressed.

Taken together, these results suggest that 12-mer cationic arginine peptides can be used to induce PTGS by effectively delivering double-stranded RNA to plant cells. In addition, in the above experiment, it was observed that the GUS gene in the tobacco suspension cultured cell line maintained silencing for at least 3 weeks.

<Comparative example 1> Delivery only of double stranded RNA

Except for using only GUS double-stranded RNA in place of the GUS double-stranded RNA / arginine peptide complex, the experiment was carried out under the same conditions as in Example 1, although not shown in the figure, the RNase protection assay (RNase protection assay) As a result, no siRNA could be found.

<Example 2> Delivery of whole RNA / peptide in vivo

Only total RNA was selectively isolated from the leaves of Arabidopsis thalamus expressing green fluorescent protein (GFP) by the regulation of the 35S promoter. The isolated total RNA contained mRNAs of a large amount of GFP gene overexpressed as a result of the regulation of the 35S promoter. An aqueous solution containing the complex after electrostatic binding of 10 µg of the total RNA to the 12-mer arginine peptide After immersing the onion root in about 1 hour, the complex was washed with water and incubated for 2 days. Some root tissues were taken and observed with a confocal fluorescence microscope (Zeiss, Germany, Axiovert2) are shown in Figure 2 (a), it was confirmed green fluorescence as shown. In other words, it can be seen that the GFP mRNA contained in the whole RNA in vivo was delivered to onion root cells by the arginine peptide and expressed directly in the cytoplasm.

<Comparative Example 2> Delivery of only RNA isolated whole in vivo

The experiment was conducted under the same conditions as in Example 2, except that only total RNA was used instead of the GFP RNA / arginine peptide complex. Specifically, onion roots were added to an aqueous solution containing only total RNA isolated from the leaves of Arabidopsis. After immersion for about 1 hour, some root tissues were taken and observed by confocal fluorescence microscopy. As shown in FIG. 2 (b), green fluorescence could not be confirmed.

Example 3 Mass Synthesized GFP mRNA / Capping / Peptide Delivery

After a large amount of mRNA was synthesized in vitro using GFP DNA as a template, methylguanine cap, which is known to play an important role in the initiation of eukaryotic transcription, was introduced. 6 μg of the synthesized mRNA was electrostatically bound to 6 μg of a 12-mer arginine peptide, and the onion roots were immersed in an aqueous solution containing the complex for about 1 hour, washed with water, and incubated. After 2 days, the root tissue was taken and observed with a confocal fluorescence microscope as shown in Figure 3 (a), it was confirmed as shown green fluorescence.

Comparative Example 3 Mass Synthesized GFP mRNA / Peptide Delivery

Except for omitting the capping process of the GFP mRNA 5 'end was tested under the same conditions as in Example 3, as shown in Figure 3 (b) it can be confirmed that the green fluorescence is expressed lower than the panel (a). there was.

<Comparative Example 4> Delivery of only massively synthesized GFP mRNA

The whole mRNA was used instead of the GFP mRNA / arginine peptide complex, and the experiment was performed under the same conditions as in Example 3 except that the capping process was omitted. As shown in FIG. Did.

These studies have been carried out on tobacco suspension cell lines, Arabidopsis leaf and onion roots, and most of the plant cells and tissues are expected to penetrate RNA.

According to the novel RNA transfer technique according to the present invention, it is much easier and simpler than a plant transformation technique or a conventional RNA transfer technique which introduces foreign DNA into a plant, resulting in RNA transfer to the plant. It's fairly efficient at identifying. The introduction of double-stranded RNA into the plant with this technique facilitates the PTGS mechanism, enabling the rapid identification of the plant's gene function and the development of new trait crops through the interference of specific genes. It is thought to provide a technology based on functional genetics. In addition, when mRNA is introduced into the plant by using this technique, the desired protein can be expressed directly in the cytoplasm without being involved in the intrinsic transcriptional control mechanisms in the plant cell, so that the cells are not dependent on the cell division cycle and do not divide. It is thought that the expression of the target gene can be induced to induce the expression of the target gene in various plant tissues.

Claims (7)

RNA transfer technology to plants using cationic oligopeptides, which comprises using cationic oligopeptides as carriers and incorporating RNA to be introduced into the carriers through electrostatic interaction and then permeating the plant tissues in aqueous solution . The method of claim 1, RNA delivery to the plant using a cationic oligopeptide, characterized in that the RNA to be delivered to the double-stranded RNA or single-stranded RNA. The method of claim 1, Oligopeptide is an RNA delivery technique to a plant using a cationic oligopeptide, characterized in that it comprises a 6 to 30 lysine peptide or arginine peptide. The method of claim 1, Oligopeptide is an RNA delivery technique using a cationic oligopeptide, characterized in that it comprises a hexamer, hexamer, 12-mer and 15-mer lysine peptide or arginine peptide. The method of claim 1, Cationic oligopeptide, characterized in that the attachment of the target RNA to the oligopeptide is made by reacting the phosphate buffer solution (PBS), MS medium, water or saline solution in the pH range of 4 to 10 at 4 ℃ for 15 minutes to 1 hour. RNA transfer method to the plant using. The method of claim 1, An RNA transfer technique to a plant using a cationic oligopeptide, characterized in that the plant comprises a cultured cell, seed, root, stem or bulb tissue of the plant. The method of claim 1, A technique for delivering RNA to plants using cationic oligopeptides, wherein permeation into plant tissues is by permeation through culture or by injection.