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
1999-03-08
2003-05-13
Bui, Phuong T. (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, C800S298000, C800S295000, C800S317000, C435S069100, C435S468000, C435S419000, C435S430000, C536S023100, C536S023200, C536S023600, C536S024100
Reexamination Certificate
active
06563025
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the field of plant genetics. In particular, the invention provides novel selectable markers and promoters for plants.
BACKGROUND OF THE INVENTION
The selection of mutants using cultured plant cells is in principle similar to that done with microorganisms, but in practice is much more difficult. The reasons for the difficulties include the usual clumpy nature of plant cell cultures; single cells or protoplasts usually cannot be easily grown to form clones, cell growth is slow and the cells are usually not monoploid. Despite these problems a large number of successful selection experiments have been carried out to produce mutants of value for producing compounds, for biochemical and molecular biology studies, for markers in genetic experiments and for improving crop plants. Part of the reason for the success is that cell systems allow the screening of millions of cells for the desired trait.
Whether the selected phenotype is under genetic or epigenetic control can most easily be determined by regenerating plants and by following the phenotype in progeny. Genetically controlled phenotypes would be inherited by progeny and would generally be more stable at the cell level in comparison to epigenetically controlled traits. A large number of in vitro selected traits have been shown to be expressed in regenerated plants and to be passed on to progeny.
There are several types of in vitro selection that can be used to obtain cells containing the trait of interest (J. Widholm,
Iowa State J. of Research
, 62: 587-597, 1988). These include selection for growth, selection for valuable compound production, auxotroph selection and resistance selection. Selection for resistance should be the easiest kind of selection to accomplish and from the number of reports in the literature this would appear to be true.
The selection for amino acid analog resistance in plants has been pursued for a number of years. A primary focus of this research has been directed to the enzyme anthranilate synthase (AS). AS catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate),pathway toward the biosynthesis of tryptophan (Trp). As a branchpoint enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis.
Available information indicates that AS plays a key role in regulation of Trp biosynthesis. In plants, bacteria, and fungi, AS activity is regulated by Trp feedback inhibition (Matsui et al.,
J. Bacteriol
, 169: 5330-5332, 1987). In microbes, AS usually consists of two nonidentical subunits, referred to as the alpha subunit (component I) and the beta subunit (component II). component I can convert chorismate to anthranilate in the presence of high levels of ammonia (ammonia-dependent AS activity), whereas-component II is responsible for the use of glutamine (hereinafter referred to as “Gln”) as the amino donor (Hutter et al.,
Annu Rev Microbiol
, 40: 55-77, 1986).
As a means to investigate regulation of the Trp pathway, toxic analogs of Trp have been used in metabolic studies of plant cell cultures and as a tool to select mutants. Many of these studies have been conducted with the growth inhibitor 5-methyltryptophan (5MT). In a number of species including Datura innoxia (hereinafter referred to as
D. innoxia
),
Catharanthus roseus
, and
Solanum tuberosum
, variant cell lines resistant to inhibitory concentrations of 5MT were found to have AS that was less sensitive to feedback inhibition by Trp (Carlson and Widholm,
Physiol Plant
, 44: 251-255, 1978; Scott et al.,
Phytochemistry
, 18: 795-798, 1979; Ranch et al.,
Plant Physiol
, 71: 136-140, 1983). Widholm (
Planta
, 134: 103-108, 1977) described 5MT-resistant carrot cell lines and a potato cell line that were auxin autotrophic.
In addition, 5-methylanthranilate was successfully used to isolate plant auxotrophic mutants defective in three different genes, trp1, trp2, and trp3 (Last and Fink, Science, 240: 305-310, 1988; Last et al.,
Plant Cell
, 3: 345-358, 1991) and mutants of
Chlamydomonas reinhardtii
(Dutcher et al.,
Genetics
, 131: 593-607, 1992). Mutants resistant to 5MT or alpha-methyltryptophan (&agr;MT) were reported in
Arabidopsis thaliana
(hereinafter referred to as
A. thaliana
) (Koornneef and van Loenen Martinet,
Arabidopsis Inf Serv
, 20: 104-108, 1983; Kreps & Town,
Plant Physiol
, 99: 269-275, 1992), maize (Kang & Kameya,
Euphytica
, 69: 95-101, 1993),
Lemna gibba
(Tam et al.,
Plant Physiol
, 107: 77-85, 1995) and
Oryza sativa
(Lee & Kameya,
Theor Appl Genet
, 82: 405-408, 1991). The specificity of selection with these analogs have not been systematically investigated.
A feedback-insensitive AS gene (ASA1 mutant) has been recently obtained by selection of mutagenized Arabidopsis seeds resistant to 6-methylanthranilate (Li & Last,
Plant Physiol
., 110: 51-59, 1996). In addition, &agr;MT resistance led to identification of a mutant in
A. thaliana
with the same amino acid change (Kreps et al.,
Plant Physiol
., 110: 1159-1165, 1996).
One method for the production of trahsgenic plants is to transform plant cells in tissue culture with a plasmid containing a promoter and selectable marker which also contains a gene (“desirable gene”) which would express the desired trait in the regenerated plant. Thus when one selects cells transformed with the selectable marker, many of these cells will also carry the desirable gene that will also be expressed to produce the desired result such as insect resistance, disease resistance, herbicide resistance, changed. starch, drought tolerance, etc. An example is where the nptII (neo) gene is driven by a constitutive promoter, nosP (Vermeulen et al., Plant.
Cell Reports
, 11: 243-247, 1992). Next to this selectable marker gene is a mutant acetolactate synthase gene with its own promoter. This latter gene makes the regenerated plants resistant to certain herbicides.
An AS gene which encodes an enzyme that is highly resistant to an amino acid analog, such as 5MT, would be an ideal selectable marker for the production of transgenic plants as described above. Especially if the promoter which regulates the expression of this enzyme provided for high level expression of the enzyme in tissue culture, and little or no expression in regenerated plants. There has been considerable environmental concern because most selectable markers are constitutively expressed in all tissues of the plant and are not of plant origin. The former concern would be reduced by using such a tissue culture specific promoter while the latter concern would be eliminated by using the plant-derived AS gene as the selectable marker. In fact, the use of a tissue culture specific promoter would even allow one to use selectable markers that are not of plant origin. Traditional selectable markers that are not of plant origin include nptII, which encodes kanamycin resistance.
BRIEF SUMMARY OF THE INVENTION
A first aspect of the present invention is an isolated deoxyribonucleic acid (DNA) molecule comprising a DNA sequence (SEQ ID NO: 4), the ASA2 gene of
Nicotiana tabacum
(hereinafter referred to as
N. tabacum
), and fragments thereof, which encode a feedback-insensitive form of AS. The ASA2 gene product could function as a selectable marker for transforming plant cells.
A second aspect of the present invention is an isolated DNA molecule comprising a DNA promoter sequence, the ASA2 promoter sequence (SEQ ID NO: 14), which is capable of directing tissue culture specific transcription of a downstream structural gene in a plant cell. The functional promoter sequence may be selected from the group consisting of the tobacco ASA2 promoter and DNA sequences which are at least 70 percent homologous to a fragment of the tobacco ASA2 promoter which is from about 150 to about 606, more preferably from about 150 to about 370, and most preferably about 150 bases in length. For constitutive expression of the promote
Brotherton Jeffrey E.
Song Hee-Sook
Widholm Jack M.
Board of Trustees of the University of Illinois
Bui Phuong T.
Ibrahim Medina A.
Wong Wean Khing
LandOfFree
Nucleotide sequences encoding anthranilate synthase does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleotide sequences encoding anthranilate synthase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences encoding anthranilate synthase will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3032557