Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-08-10
2002-02-12
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S295000
Reexamination Certificate
active
06346656
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to DNA fragments that confer resistance to porphyrin-accumulating type herbicides on plant and algal cells, plasmids and microorganisms that contain these DNA fragments, methods for conferring resistance to porphyrin-accumulating type herbicides onto plant and algal cells by using these DNA fragments, and plants and algae into which these DNA fragments have been introduced for the purpose of conferring resistance to such herbicides thereon.
2. Description of Related Art
A group of widely-known compounds used as active ingredients of some varieties of commercially- and otherwise-available herbicides exhibit herbicidal activity in the presence of light, but exhibit no herbicidal activity in darkness. This has led to their common designation as light-dependent or porphyric herbicides. It has recently been shown that these herbicides induce high levels of porphyrin accumulation in plants and algae, and thus they are now designated as “porphyrin-accumulating type herbicides” [Zoku, Iyakuhin-no-Kaihatsu, (translation: “The Development of Medical Drug Products; continuation”) vol. 18; Development of Agricultural Chemicals II, chapter 16, section 16-1, Hajime Iwamura, Tamio Ueno & Katsuzo Kamoshita, eds., Hirokawa Shoten, Tokyo, pubs.) or simply “porphyric herbicides”. It was reported by Matringe, M., Camadro, J. M., Labbe, P. & Scalla, R. (
Biochem J
. 260:231 (1989)) and by Matringe, M., Camadro, J. M., Labbe, P. & Scalla, R. (
FEBS Lett
. 245:35 (1989)) that porphyrin-accumulating type herbicides (referred to below also as porphyric herbicides) inhibit isolated protopor-phyrinogen oxidase (referred to below as “protox”).
Since most crop plants do not exhibit resistance to these porphyric herbicides, it is not possible to use these herbicides on farmland when such crops are under cultivation. If it were possible to develop crops resistant to porphyric herbicides, such herbicides could be used on these crops. This would make crop management easier, and increase the value of these herbicides in, agricultural applications. For this reason, it is desirable to develop a method for conferring resistance to porphyrin-accumulating type herbicides upon crop plants.
SUMMARY OF THE INVENTION
With this goal in mind, the present inventors have investigated a mutant strain, designated RS-3, of the unicellular green alga
Chlamydomonas reinhardtii
which displays specific resistance to porphyric herbicides. Wild-type strains of this alga are normally highly sensitive to porphyric herbicides. The present inventors have discovered that inhibition by porphyric herbicides of protox activity in chloroplast fragments isolated from the RS-3 strain of
Chlamydomonas reinhardtii
was significantly lower than in chloroplast fragments from the wild-type strain. The inventors therefore constructed a genomic DNA library from total nuclear DNA isolated from the RS-3 mutant strain, and succeeded in isolating clones that contain a gene responsible for resistance to porphyric herbicides. Thus, the inventors were able to obtain DNA fragments that can confer resistance to porphyrin-accumulating type herbicides onto plant and algal cells.
Accordingly, it is an object of the present invention to provide an isolated, purified DNA fragment that confers resistance to porphyrin-accumulating type herbicides when expressed in plant or algal cells, plasmids and microorganisms containing said DNA fragment. A DNA fragment according to the present invention preferably has a nucleotide sequence of one or more portions of DNA comprising the genome of an alga, or has a nucleotide sequence highly homologous to the nucleotide sequence of DNA comprising one or more portions of the genome of an alga.
Additional objects of the present invention are a method for conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragment into said plant or algal cells, wherein said DNA fragment is expressed; and plants or algae into which said DNA fragment has been introduced, wherein said DNA fragment is expressed, thereby conferring herbicide resistance upon said plants or algae.
Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga
Chlamydomonas reinhardtii
that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, and XhoI, and having a restriction site map as shown in FIG.
1
(
a
);
c) having a molecular size of approximately 3.4 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga
Chlamydomonas reinhardtii
that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI and HindIII, and having a restriction site map as shown in FIG.
1
(
b
);
c) having a molecular size of approximately 9.9 kB; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga
Chlamydomonas reinhardtii
that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, and KpnI, and having a restriction site map as shown in FIG.
1
(
c
);
c) having a molecular size of approximately 10.0 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga
Chlamydomonas reinhardtii
that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, BamHI, SalI, HindIII, and KpnI, and having a restriction site map as shown in FIG.
1
(
d
);
c) having a molecular size of approximately 13.8 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
Further objects of the present invention are to provide plasmids and microorganisms containing any of the foregoing DNA fragments or biologically functional equivalents thereof, a method of conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragments or biologically functional equivalents thereof into plant or algal cells in a functionally operable manner so that said DNA fragments or biologically functional equivalents thereof are expressed in said plant or algal cells, and the expression of the DNA fragment confers resistance to porphyrin-accumulating type herbicides upon the transformed plant or algal cells. It is preferred that cells cultured in vitro that have been transformed by the DNA fragments of the invention in a functionally operable manner are resistant to a porphyrin-accumulating type herbicide at a conc
Boynton John
Gillham Nicholas W.
Harris Elizabeth H.
Sato Ryo
Benzion Gary
Birch & Stewart Kolasch & Birch, LLP
Kruse David H
Sumitomo Chemical Company Ltd.
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