Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1999-01-11
2001-07-24
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S069100, C435S419000, C435S468000, C435S471000, C435S320100, C536S023600, C800S266000, C800S298000
Reexamination Certificate
active
06265637
ABSTRACT:
This invention relates to the genetic control of flowering in plants and the cloning and expression of genes involved therein. More particularly, the invention relates to the cloning and expression of the Late Elongated Hypocotyl (LHY) gene of
Arabidopsis thaliana
, and homologues from other species, and manipulation and use of the gene in plants.
BACKGROUND OF THE INVENTION
Efficient flowering in plants is important, particularly when the intended product is the flower or the seed produced therefrom. One aspect of this is the timing of flowering: advancing or retarding the onset of flowering can be useful to farmers and seed producers. An understanding of the genetic mechanisms which influence flowering provides a means for altering the flowering characteristics of the target plant. Species for which flowering is important to crop production are numerous, all crops which are grown from seed, with important examples being the cereals, rice and maize, probably the most agronomically important in warmer climatic zones, and wheat, barley, oats and rye in more temperate climates. Important seed products are oil seed rape and canola, sugar beet, maize, sunflower, soybean and sorghum. Many crops which are harvested for their roots or leaves are, of course, grown annually from seed and the production of seed of any kind is very dependent upon the ability of the plant to flower, to be pollinated and to set seed. Delaying flowering is important in increasing the yield of plants from which the roots or leaves are harvested. In horticulture, control of the timing of flowering is important. Horticultural plants whose flowering may be controlled include lettuce, endive, spinach and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums.
Arabidopsis thaliana
is a facultative long day plant, flowering early under long days and late under short days. Because it has a small, well-characterized genome, is relatively easily transformed and regenerated and has a rapid growing cycle, Arabidopsis is an ideal model plant in which to study flowering and its control.
We have discovered that one of the genes required for this response to photoperiod is the Late Elongated Hypocotyl or LHY gene. We have found that plants carrying dominant gain of function mutations of the LHY gene flower later than their wild-types under long days but earlier than their wild-types under short days. We have now cloned and sequenced the LHY gene, which is provided herein, and demonstrated that the mutation causes the gene to be transcribed at higher levels than the wild-type gene. This suggests that increased expression of LHY delays flowering under long days.
BRIEF DESCRIPTION OF THE INVENTION
According to a first aspect of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with LHY function. Those skilled in the art will appreciate that “LHY function” may be used to refer to the ability to influence the timing of flowering phenotypically like the LHY gene of
Arabidopsis thaliana
(the timing being substantially unaffected by vernalisation). LHY mutants exhibit delayed flowering under long days, the timing of flowering being substantially unaffected by vernalisation. Also provided is a nucleotide sequence comprising the 5′ non-coding region of a gene encoding a polypeptide with LHY function, preferably including substantially the whole promoter region of the gene, which gene may have the sequence of a LHY gene of
Arabidopsis thaliana.
Further aspects based on the promoter region are disclosed below. However, discussion of mutation and manipulation of nucleic acid according to the invention encoding a LHY qene product (e.g. to make mutants etc., transform cells and plants and so on) applies mutatis mutandis to promoter nucleic acid according to the present invention.
Nucleic acid according to the present invention may have the sequence of a LHY gene of
Arabidopsis thaliana
, including coding and/or non-coding regions, or be a mutant, variant, derivative or allele of the sequence provided. Preferred mutants, variants, derivatives and alleles are those which encode a protein which retains a functional characteristic of the protein encoded by the wild-type gene, especially the ability to repress or delay flowering, for example by means of the regulation of other genes, as discussed herein.
A mutant, variant, derivative or allele in accordance with the present invention may have the ability to affect a physical characteristic of a plant, particularly a flowering characteristic. In various embodiments a mutant, variant, derivative or allele represses flowering compared with wild-type on expression in a plant, e.g. compared with the effect obtained using a gene sequence encoding the polypeptide of
FIG. 1
(SEQ ID NO:2). “Repression” of flowering delays, retards, inhibits or slows it down. In other embodiments, a mutant, variant, derivative or allele promotes flowering compared with wild-type on expression in a plant, e.g. compared with the effect obtained using a gene sequence encoding the polypeptide of
FIG. 1
(SEQ ID NO:2). “Promotion” of flowering advances, accelerates or brings it forward in time. Comparison of effect on flowering or other characteristic may be performed in
Arabidopsis thaliana
, although nucleic acid according to the present invention may be used in the production of a wide variety of plants and for influencing a characteristic thereof.
As discussed further below, over-expression of nucleic acid according to the present invention may delay flowering while under expression may promote flowering in a transgenic plant.
Changes to a sequence, to produce a mutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included, including changes to the non-coding regions such as the promoter or to binding sites for factors influencing regulation of gene expression.
A preferred nucleic acid sequence for a LHY gene is shown in
FIG. 1
(SEQ ID NO:1), along with the predicted amino acid sequence of a polypeptide which has LHY function. Preferred nucleic acid according to the present invention encodes the amino acid sequence encoded by the sequence of nucleotides shown in
FIG. 1
(SEQ ID NO:2).
A mutant, allele, variant or derivative amino acid sequence in accordance with the present invention may include within the sequence shown in
FIG. 1
(SEQ ID NO:2), a single amino acid change with respect to the sequence shown in
FIG. 1
(SEQ ID NO:2), or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the amino acid sequence shown in
FIG. 1
(SEQ ID NO:2), a mutant, allele, variant or derivative amino acid sequence may include additional amino acids at the C-terminus and/or N-terminus.
A sequence related to a sequence specifically disclosed herein shares homology with that sequence. Homology may be at the nucleotide sequence and/or amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares homology with the coding sequence (SEQ ID NO:1) or the sequence encoded by the nucleotide sequence of
FIG. 1
(SEQ ID NO:2), preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for “conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine
Coupland George M.
Schaffer Robert J.
Benzion Gary
Mehta Ashwin
Nixon & Vanderhye P.C.
Plant Bioscience Limited
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