Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1997-12-01
2001-11-20
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C435S069100, C435S419000, C435S468000, C800S288000
Reexamination Certificate
active
06320099
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a technology for creating a virus resistant plant expressing an animal cell-derived (2′-5′) oligoadenylate synthetase and an animal cell-derived ribonuclease L using recombinant DNA technology. More specifically, the present invention relates to a method for creating a plant having resistance to viruses, comprising incorporating DNA sequences encoding an animal cell-derived (2′-5′)oligoadenylate synthetase and an animal cell-derived ribonuclease L into a chromosome(s) of the plant and expressing the DNA sequences; and a virus resistant plant obtained by the above method.
BACKGROUND ART
Viruses are one of the major stress sources for plants. It is not seldom that crop-producing districts shift or cultivars are renewed due to damage from viral diseases. At present, there is no effective drug that acts on viruses directly. Thus, plants infected with viruses are subjected to incineration. Although virus resistance is an important goal for breeding, it has been impossible to rear a virus resistant cultivar with conventional breeding technologies such as crossing where a source of a resistance gene cannot be found in wild-type or related species.
As a method for supplementing such conventional breeding technologies, a method have been developed recently in which virus resistance is conferred on plants using recombinant DNA technology. Recombinant DNA technology has made gene transfer possible which goes beyond the deadlock of conventional crossing described above. Furthermore, this technology allows to introduce into existing cultivars a virus resistance gene alone and, thus, it has become possible to save the time required for breeding greatly.
As a method for creating virus resistant plants using recombinant DNA technology, methods of expressing a gene encoding a virus coat protein or a viral replication protein, an antisense gene, and a gene encoding a satellite RNA, and the like have been reported (see, for example, Arch. Virol., 115, 1, 1990). These methods confer resistance to only one kind of virus or its related viruses alone. A method for conferring resistance to various kinds of viruses is still under development.
As a method for conferring resistance to a large number of viruses at the same time, a method using a double-stranded RNA specific ribonuclease (International Publication WO93/20686) and a method using a (2′-5′)oligoadenylate synthetase (Bio/Technology, 11, 1048, 1993) have been reported.
With respect to the method using a (2′-5′)oligoadenylate synthetase, it has been reported that the virus resistance in the resultant transformant plants is extremely weak (The Proceedings of XVIIIth Eucarpia Symposium, Section Ornamentals, 1995). Briefly, a (2′-5′)oligoadenylate synthetase was introduced into a tobacco plant (cv Samsun) and then the tobacco mosaic virus (hereinafter referred to as “TMV”) OM strain was inoculated into the resultant plant expressing the (2′-5′)oligoadenylate synthetase. As a result, no delay in the development of disease symptoms was recognized as compared to controls. Also, there have been many reports that the existence of ribonuclease L-like molecules are not recognized in plant cells (Biochem. Biophys. Res. Commun., 108, 1243, 1982; J. Biol. Chem., 259, 3482, 1984; The Proceedings of XVIIIth Eucarpia Symposium, Section Ornamentals, 1995).
DISCLOSURE OF THE INVENTION
The present inventors have made intensive and extensive researches expecting that, by expressing both an animal cell-derived (2′-5′)oligoadenylate synthetase gene and an animal cell-derived ribonuclease L gene in a plant, virus resistance will be remarkably increased compared to that obtained by the expression of a (2′-5′) oligoadenylate synthetase gene alone. Thus, the present invention has been achieved.
The present invention relates to a method for creating a plant having resistance to RNA viruses, comprising incorporating a DNA sequence encoding an animal cell-derived (2′-5′)oligoadenylate synthetase (hereinafter referred to as “2-5Aase”) and a DNA sequence encoding an animal cell-derived ribonuclease L (hereinafter referred to as “RNaseL”) into a chromosome(s) of the plant and expressing the DNA sequences; and a virus resistant plant created by the above method.
Hereinbelow, the present invention will be described in detail.
(1) DNA Sequences Encoding 2-5Aase and RNaseL, Respectively
The presence of a 2-5Aase/RNaseL system in animal cells has been known as partly contributing to the anti-virus state induced by interferon (Annu. Rev. Biochem., 51, 251, 1982). 2-5Aase protein is a protein having an enzymatic activity to synthesize (2′-5′) oligoadenylate (usually a trimer or tetramer; hereinafter referred to as “2-5A”) from its substrate adenosine triphosphate (ATP) when activated upon recognition of double-stranded RNA. A cDNA encoding 2-5Aase has been cloned from human (EMBO J., 4, 2249, 1985), mouse (J. Biol. Chem., 266, 15293, 1991; Nuc. Acids. Res., 19, 1919, 1991) and rat (EMBL Acc. No. Z18877). This time, the present inventors have succeeded in cloning a cDNA encoding 2-5Aase from bovine. Other DNA sequences encoding 2-5Aase may also be used in the present invention as long as they can provide the above-described 2-5Aase activity. In other words, DNA sequences encoding 2-5Aase cloned from animal species other than those enumerated above, and even the above DNA sequences having replacement, deletion, addition or insertion in the encoded amino acids may also be used in the present invention as long as they can provide the above-described 2-5Aase activity. RNaseL protein is a protein which has an activity to bind with 2-5A and which exhibits RNA degradation activity upon binding with 2-5A. A cDNA encoding RNaseL has been cloned from human (Cell, 72, 753, 1993). Also, the present inventors have succeeded this time in cloning a cDNA encoding RNaseL from bovine. Other DNA sequences encoding RNaseL may also be used in the present invention. DNA sequences encoding RNaseL cloned from animal species other than those mentioned above, and even the above DNA sequences having replacement, deletion, addition or insertion in the encoded amino acids may also be used in the present invention as long as they can provide the above-described RNaseL activity.
In the Examples described below, human- or bovine-derived cDNA clones are used as DNA sequences encoding 2-5Aase and RNaseL. Needless to say, however, DNA sequences encoding 2-5Aase and RNaseL which may be used in the present invention are not limited to these human- or bovine-derived 2-5Aase and RNaseL.
In general, when a DNA sequence is coding for a polypeptide having an amino acid sequence, there exist a plurality of DNA sequences (degenerate isomers) corresponding to the single amino acid sequence because there exist a plurality of genetic codes (codons) corresponding to an amino acid. In the DNA sequences encoding 2-5Aase and RNaseL used in the present invention, it is needless to say that any genetic code may be used as long as it does not alter the amino acid sequence of the polypeptide encoded by the DNA sequence.
(2) Expression of DNA Sequences Encoding 2-5Aase and RNaseL, Respectively
In order for DNA sequences encoding 2-5Aase and RNaseL, respectively, to be expressed in a transgenic plant, at least these DAN sequences must be transcribed into RNAS. When a foreign gene is incorporated in a plant chromosome, it is known that such a gene is incorporated into a transcription region on the chromosome with a certain probability (EMBO J., 6, 3891, 1987). Therefore, it is possible to incorporate a DNA sequence encoding 2-5Aase or RNaseL alone into a plant chromosome and to express it in the plant. However, it is preferable to incorporate such a DNA sequence after ligating thereto appropriate promoter and terminator sequences.
In this case, as a promoter, any promoter which has been known to function in plant cells may be used. Specific examples include a promoter for a
Ishida Isao
Ogawa Toshiya
Yoshioka Masaharu
Foley & Lardner
Kirin Beer Kabushiki Kaisha
McElwain Elizabeth F.
Mehta Ashwin
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