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
2000-04-25
2002-05-14
Nelson, Amy J. (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, C435S320100, C435S252330, C435S418000, C536S023600
Reexamination Certificate
active
06388174
ABSTRACT:
TECHNICAL FIELD
The present invention relates to genes which respectively encode the a-subunits of the two isozymes (isoenzymes) of rice anthranilate synthase, as well as to DNAs relating to said genes. Specifically, the present invention relates to two novel DNA segments respectively encoding such proteins which are respectively the &agr;-subunits of the two isozymes, i.e. the first isozyme and the second isozyme, of anthranilate synthase participating in the biosynthesis of tryptophan in rice plants.
Another aspect of the present invention relates to a novel DNA which encodes a novel protein having the activity of the &agr;-subunit protein of the first isozyme of anthranilate synthase. The present invention also relates to a novel recombinant vector in which said novel DNA has been inserted. Further,
Escherichia coli
, plants and seeds which have been transformed with said novel DNA are embraced within the scope of the present invention.
Another aspect of the present invention relates to a method of increasing the tryptophan content of a plant by the use of the novel DNA of the present invention. Further, the present invention relates to a method of selecting a transformed plant cell containing the novel DNA of the present invention, and to a method of producing a transformed plant containing the novel DNA.
Another aspect of the present invention relates to a DNA which acts as a promoter for the rice anthranilate synthase gene.
BACKGROUND ART
Grains such as rice, maize and wheat are important nutrient source for humans and domestic animals. However, their nutritive value is low because they contain only a small amount of tryptophan, which is one of the essential amino acids. Thus, a need exists for a new plant variety capable of producing grain which has a high nutritive value with a high tryptophan content.
In the tryptophan biosynthetic pathway in a plant, anthranilic acid is biosynthesized from chorismic acid. It is known that anthranilate synthase (hereinafter sometimes referred to as ASA) catalyzes the formation of anthranilic acid and tryptophan is formed from anthranilic acid via indole through six-step enzyme reaction [Seikagaku Jikken Koza (Lectures on Experiments in Biochemistry), vol. 11, pp. 652-653 (1976) published by Tokyo Kagaku Dojin].
Plant anthranilate synthase enzymes so far known consist of plural subunits. For example, anthranilate synthase of Arabidopsis (Japanese name: shiroinunazuna, scientific name:
Arabidopsis thaliana
) is known to consist of two kinds of isozymes, the first isozyme and the second isozyme, each of which is a dimer consisting of the &agr;-subunit and the &bgr;-subunit. The gene encoding the &agr;-subunit of the first isozyme of Arabidopsis anthranilate synthase (abbreviated as ASA1) as well as the gene encoding the &agr;-subunit of the second isozyme of Arabidopsis anthranilate synthase (abbreviated as ASA2) (the genes are referred to as asa1 and asa2, respectively) have been isolated, and their DNA sequences have been determined [The Plant Cell, vol. 4, pp. 721-733 (1992)].
On the other hand, we, the present inventors already took an interest in the &agr;-subunit of anthranilate synthase as expected to have a functional domain which plays an important role in the regulation of tryptophan biosynthesis in rice, and we made studies in 1996 to isolate a gene encoding the anthranilate synthase protein, for the purpose of obtaining information about the biosynthetic regulation mechanism of tryptophan and a phytohormone IAA. According to the abstract of the report of these studies, we, the present inventors extracted mRNA and genomic DNA from rice (Norin No.8) as explant, and we prepared a cDNA library and a genomic DNA library, subjected these libraries to the genomic Southern analysis and made screening of the libraries with using cDNA fragments of the Arabidopsis asa gene as the probes, and thereby we obtained DNA which is supposed to be corresponding to the asa gene of rice anthranilate synthase [Ikushu (Breeding), vol. 46, suppl. vol. 2, p. 28 (1996)]. Although it is reported in this abstract that a DNA fragment corresponding to the ASA gene of rice anthranilate synthase was obtained, specific techniques used for obtaining said DNA fragment are not disclosed there, and it is reported that the nucleotide sequence of said DNA fragment has not been determined yet. The abstract of the above report also refers to the presence of two kinds of DNAs which are supposed to be corresponding to the gene for encoding the rice anthranilate synthase [Ikushu (Breeding), vol. 46, suppl. vol. 2, p. 28 (1996)].
There has also been a report on that DNA of the ASA gene encoding the &agr;-subunit of the first isozyme of Arabidopsis ASA and also a DNA fragment as obtained by modifying said DNA are introduced into a tobacco plant with the expression of the function of said gene in tobacco [Massachusetts Institute of Technology, Cambridge, Mass. (1993)].
However, so far as the present inventors are aware of, no report has been made on the analysis of the amino acid sequence of a protein which is the &agr;-subunit of rice ASA isozyme, and on the method which was actually used for obtaining a gene encoding the &agr;-subunit of rice ASA isozyme. The promoter sequence relating to the expression of said gene is not known, either. Further, there has been no report on the utilization of a gene encoding the rice ASA.
One object of the present invention is to obtain from a rice plant a novel gene relating to the rice ASA, specifically, a new DNA for encoding the &agr;-subunit of the first isozyme of the rice ASA. Another object of the present invention is to determine the nucleotide sequence of this DNA.
Another object of the present invention is to provide a novel DNA capable of encoding a novel protein having the activity of the &agr;-subunit of the first isozyme of rice ASA. A further object of the present invention is to transform useful plants such as maize, rice, soybean, wheat, barley, tomato and potato, with said novel DNA, and to provide novel useful transformant plants capable of producing seeds which have a high tryptophan content. A yet further object of the present invention is to construct a novel DNA sequence capable of encoding a protein having the activity of the &agr;-subunit of the first isozyme of rice ASA, and to provide an efficient method for obtaining cells and plants as transformed with said novel DNA.
The other objects of the present invention will be clear from by the descriptions below.
DISCLOSURE OF THE INVENTION
In order to accomplish the above-described objects, the present inventors have made a series of studies. First, a study has been made for obtaining such genes which respectively encode the &agr;-subunits of the two ASA isozymes from rice. In this study, we, the inventors have extracted a total RNA from tissue of an explant of rice such as disrupted green stem and leaf by a known technique for the gene engineering, and we have isolated mRNAs from the extracted total RNAs by a conventional method, and have successfully obtained cDNAs of rice from the mRNAs with using a commercially available cDNA synthesis kit. It has been found through trials and errors that recombinant vectors can be constructed by ligating the above cDNAs into such a phage vector (available from STRATAGENE) as prepared by treating the end of an EcoRI-cleaved fragment of &lgr; gt11 phage vector with an alkaline phosphatase derived from calf small intestines, and that replicable recombinant &lgr; phages can be constructed by packaging the obtained recombinant vectors in a &lgr; phage.
Further, it has been found that a lot of recombinant &lgr; phages can be obtained in the form of a large number of plaques, by incubation of
Escherichia coli
Y1088 as infected with the above recombinant &lgr; phages, and that a group of recombinant &lgr; phages present in the resultant plaques comprises various phages each containing the rice-derived cDNA and can be utilized as a rice cDNA library.
On the other hand, we, the pres
Hasegawa Hisakazu
Terakawa Teruhiko
Tozawa Yuzuru
Wakasa Kyo
Hokko Chemical Industry Co. Ltd.
Kruse David H
Larson & Taylor PLC
Nelson Amy J.
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