Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-11-30
1997-04-08
Fox, David T.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 691, 435 701, 4351723, 4352351, 4353201, 536 2372, 800205, 800DIG43, 800DIG44, C12N 1540, C12N 1562, C12N 1582, C12N 1583
Patent
active
056186991
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to introduction of a foreign gene into a plant using a plant virus vector, improvement of plant properties and expression of a foreign gene throughout a whole plant, and a process for simply and efficiently obtaining a foreign gene product.
BACKGROUND OF THE INVENTION
It is known that plant viruses represented by tobamovirus, after infecting a plant, proliferate in the plant and rapidly spread systemically throughout the plant while producing viral coat protein in large amounts.
So far, some gene engineering systems for these plant viruses have been constructed and some attempts have been carried out to introduce a foreign gene into a plant using said systems. For example, there is a process wherein a coat protein gene is replaced with a foreign gene (Takamatsu et al, EMBO J., 6:307-311 (1987)), and a process wherein a coat protein gene and a foreign gene are directly joined so as to produce a fused protein (Takamatsu et al, FEBS Lett., 269:73-76 (1990 )).
However, all of the plant viruses used in these known processes have a drawback in that they do not spread systemically in a plant (i.e., they do not have systemic infectivity), and thus, it is impossible to introduce a useful property and to produce a useful protein throughout a whole plant.
For a plant virus to exhibit systemic infectivity, particle formation with wild type coat protein is essential (Saito et al, Virology, 176:329 (1990)). As to existing plant virus vectors, since the coat protein is not produced (replacement-type vector), or the coat protein is in the form of a fused protein, resulting in a big change in properties (direct-joining type vector); then particles cannot be formed and systemic infectivity is not exhibited.
In addition, where existing plant virus vectors are used to express a foreign gene, it is very difficult to isolate and purify the foreign gene product from the plant into which the foreign gene was introduced.
The reason is that to establish (1) simple apparatus and economy, (2) high recovery, (3) high purity and (4) good reproduction, which are the goals of isolation and purification, a combination of operations, such as differential precipitation, desalting, concentration, and various chromatographies is essential; however, a series of these operations usually takes one-half to one month to complete, and they are often time- and labor-consuming. Even assuming these operations are simple and rapid, a plant includes proteins whose properties, such as molecular weight, isoelectric point, affinity to a solvent, are similar to those of the foreign gene product; and therefore it is difficult to prevent loss of the foreign gene product during the isolation and purification process. Thus, high recovery of the foreign gene product is not expected.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide plant virus vectors having the ability to systemically infect a whole plant; plasmids, which are transcribed to provide said plant virus vectors; and a process for expressing a foreign gene throughout a whole plant by inoculating said plant virus vector into a plant.
In addition, a process for simply and efficiently obtaining a foreign gene product produced in a plant is provided.
To accomplish the above-mentioned purposes, the present inventors use plant virus vector wherein a foreign gene is linked downstream of a coat protein gene of a plant virus via a nucleotide sequence that causes readthrough (Skuzeski et al, J. Mol. Biol., 218:365-373 (1991)); a plasmid that is transcribed to provide said plant virus vector; and infection throughout a whole plant (systemic infectivity) by inoculating said plant virus vector into a plant so as to express the foreign gene throughout the whole plant. It was also found that introduction of a useful property into a whole plant and the production of a useful protein in a whole plant are possible, and thus, the present invention has been achieved.
In addition the present inventors found that when a foreign gene prod
REFERENCES:
Skuzeski et al. 1991. J. Mol. Biol. 218:365-373.
Takamatsu et al. 1983. Nucleic Acids Research II(11): 3767-3778.
Ugaki et al. 1991. J. Gen. Virol. 72:1487-95.
Solis et al. 1990. Virology 177:553-558.
Isomura et al. 1991. J. Gen. Virol. 72:2247-2249.
Alonso et al. J. Gen. Virol. 72: 2875-2884.
Takamatsu et al, The EMBO Journal vol. 6, No. 2, pp. 307-311 (1987).
Saito et al, Virology 176, pp. 329-336 (1990).
Meshi et al, Proc. Natl. Acad. Sci. USA, vol. 83, pp. 5043-5047 (1986).
Ahlquist et al, Mol. Cell. Biol. vol. 4 No. 12, pp. 2876-2882 (1984).
Rosa, Cell. vol. 16 pp. 815-825 (1979).
Joshi et al, "Strategies for Expression of Foreign Genes in Plants--Potential Use of Engineered Viruses", FEBS Letter, 281(1,2):1-8 (1991).
Takamatsu et al, "Production of Enkephalin in Tobacco Protoplasts Using Tobacco Mosaic Virus RNA Vector", FEBS Letter, 269(1):73-76 (1990).
Skuzeski et al. 1990. Plant Mol. Biol. 15: 65-79.
Hamamoto Hiroshi
Hashida Eiji
Matsunaga Yuji
Nakagawa Noriaki
Nakanishi Noriyuki
Fox David T.
Kanebo Limited
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