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
1997-06-20
2003-10-28
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
C800S295000, C800S298000, C800S306000, C435S069100, C435S320100, C435S410000, C536S023600
Reexamination Certificate
active
06639130
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to plant sterol biosynthetic enzymes, genes, and their uses.
Plant sterols belong to a large group of secondary compounds known as terpenes or isoprenoids. Sterol biosynthesis in plants generally involves a series of different enzymatic steps in the isoprenoid pathway that result in the formation of a variety of sterol end products (Benveniste
Ann. Rev. Biochem
. 37:275, 1986). Although such sterol compounds have been identified in higher plants, their function in plant growth and development is poorly understood.
One such plant sterol, brassinolide, that belongs to a class of sterols referred to as brassinosterioids (BR), was first discovered in the pollen of
Brassica napus
(Grove et. al.,
Nature
281: 216, 1979). Brassinosteroids are growth-promoting natural products having structural similarities to animal steroid hormones. The wide distribution of brassinosteroids in the plant kingdom, their effect on cell proliferation and elongation, and their interactions with other plant hormones (e.g., cytokinins), have indicated that these compounds are plant-growth regulators. Brassinosteroids are thought to promote hypocotyl elongation, leaf unrolling, and xylem differentiation. In addition, such compounds are also believed to be involved in de-etiolation of cotyledons, root elongation, radial growth, and anthocyanin formation.
The function of plant sterol growth regulators, such as BR, in relationship to other classes of plant growth regulators such as auxin, gibberellin, abscisic acid, and cytokinin, during plant development also needs to be evaluated. For example, the growth regulator, cytokinin, is known to affect a variety of developmental processes including photomorphogenesis, chloroplast biogenesis and maintenance, apical dominance, and senescence. In addition, this growth regulator is thought to antagonize BR's ability to promote hypocotyl elongation and cotyledon de-etiolation.
SUMMARY OF THE INVENTION
In general, the invention features a substantially pure plant C-14 sterol reductase polypeptide. Preferably, the C-14 sterol reductase polypeptide includes an amino acid sequence substantially identical to the sequence shown in
FIG. 14
(SEQ ID NO: 1); and is from a dicot (for example, a crucifer or a solanaceous plant), monocot, gymnosperm, or an alga.
In related aspects, the invention features purified DNA that includes a sequence encoding a C-14 sterol reductase polypeptide (for example, a sequence substantially identical to the DNA sequence shown in
FIG. 14
; SEQ ID NO: 2; or a DNA sequence that encodes a C-14 sterol reductase polypeptide which has an amino acid sequence substantially identical to that shown in
FIG. 14
; SEQ ID NO: 1). The invention also features a vector and a cell, each of which includes purified DNA encoding a C-14 sterol reductase polypeptide; and a method of producing a recombinant C-14 sterol reductase polypeptide involving providing a cell (for example, a plant cell) transformed with purified DNA encoding a C-14 sterol reductase polypeptide positioned for expression in the cell, culturing the transformed cell under conditions for expressing the DNA, and isolating the recombinant C-14 sterol reductase polypeptide. The invention further features recombinant C-14 sterol reductase produced by such expression of a purified DNA, and an isolated antibody that specifically recognizes and binds a plant C-14 sterol reductase polypeptide.
In addition, the invention features nucleotide sequences that hybridize to a C-14 sterol reductase gene (including the coding sequence of such a gene and its complement) and that encode a C-14 sterol reductase polypeptide. Furthermore, the invention includes oligonucleotide probes that detect a C-14 sterol reductase gene or functional equivalents thereof in a plant (for example, dicots (such as solanaceous and cruciferous plants), monocots, gymnosperms, and algae). Such probes are useful to isolate DNA sequences that encode C-14 sterol reductases from other plants. In one particular example, oligonucleotides may be designed based on a C-14 sterol reductase sequence disclosed herein and used as hybridization probes or as primers in polymerase chain reactions (PCR). Conserved regions in the C-14 sterol reductase gene are useful in the design of such primers to facilitate the recovery of C-14 sterol reductases from other related and unrelated plants.
In yet other related aspects, the invention features a transgenic plant (or seeds or cells thereof) containing DNA encoding a C-14 sterol reductase polypeptide integrated into the genome of the plant, where the DNA is expressed in the transgenic plant, resulting in the production of a C-14 sterol reductase polypeptide.
In still another aspect, the invention features a method for reducing the level of a plant C-14 sterol reductase polypeptide in a transgenic plant cell. This method generally involves expressing in the transgenic plant cell an antisense C-14 sterol reductase polypeptide nucleic acid sequence. In general, such an antisense C-14 sterol reductase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell and is based on the nucleotide sequence that is shown in
FIG. 14
(SEQ ID NO: 2) or FIG.
15
. (SEQ ID NO: 3). In preferred embodiments, the plant cell expressing an antisense C-14 sterol reductase nucleic acid sequence is a dicot (for example, crucifer), monocot, gymnosperm, or algal cell. In yet other preferred embodiments, the method involves growing a transgenic plant from the transgenic plant cell, whereby the level of the C-14 sterol reductase polypeptide is reduced in the transgenic plant.
In other related aspects, the invention features a plant cell expressing an antisense C-14 sterol reductase nucleic acid sequence and a plant expression vector that includes an antisense C-14 sterol reductase nucleic acid sequence, where the antisense sequence is operably linked to an expression control region.
In another aspect, the invention features a method for increasing the level of a C-14 sterol reductase in a transgenic plant cell. This method involves expressing in the transgenic plant cell a C-14 sterol reductase polypeptide nucleic acid sequence. Preferably, the method utilizes a C-14 sterol reductase nucleic acid sequence that is substantially identical to the nucleotide sequence that is shown
FIG. 14
(SEQ ID NO: 2). In preferred embodiments, the plant cell expressing a C-14 sterol reductase polypeptide nucleic acid sequence is a dicot (for example, a crucifer), monocot, gymnosperm, or algal cell.
In another aspect, the invention features a transgenic plant having a knockout mutation in DNA encoding a plant C-14 sterol reductase polypeptide. Such knockout genes are constructed according to conventional methods (e.g., Lee et al.
Plant Cell
2: 415, 1990; Miao and Lam,
Plant J
. 7: 359, 1995).
By “plant C-14 sterol reductase” is meant an amino acid sequence that catalyzes the reduction of any sterol precursor having a C14=C15 double bond, for example, as described by Benveniste,
Annu. Rev. Biochem
. 37: 275, 1986. Preferably, such a polypeptide has an amino acid sequence which is at least 30%, preferably 40%, and most preferably 50% or even 80-95% identical to the amino acid sequence of the C-14 sterol reductase polypeptide shown in
FIG. 14
(SEQ ID NO: 1). The length of comparison of amino acid sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.
By “polypeptide” or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By a “substantially identical” polypeptide sequence is meant an amino acid sequence that differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitu
Jang Jyan-Chyun
Sheen Jen
Bui Phuong T.
Clark & Elbing LLP
Kallis Russell
The General Hospital Corporation
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