Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2000-01-27
2003-06-03
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S419000, C435S320100, C435S468000, C435S471000, C536S023600
Reexamination Certificate
active
06573430
ABSTRACT:
The present invention relates to genetic control of flowering and is based on the cloning of the cen gene of Antirrhinum and the tfl1 gene of Arabidopsis.
BACKGROUND OF THE INVENTION
There are three main types of meristem involved in ariel plant development; vegetative, inflorescence and floral. The apical meristem in many species, such as
Antirrhinum majus
, first undergoes a vegetative phase whereby cells set aside from the apex become leaf primordia with an axillary vegetative meristem (Coen, 1991). Upon floral induction, the apical meristem is converted to an inflorescence meristem. The traits commonly associated with the inflorescence are the modification of leaf organs and a change in internode length. The inflorescence of Antirrhinum is a raceme or spike, with the apical meristem growing indeterminately. Floral meristem arise in the axils of modified leaves and are determinate, producing four whorls or rings of floral organ primordia. Thus the apical meristem goes through two distinct identities, vegetative and then inflorescence. In species which produce terminal flowers, the apical meristem is determinate and eventually adopts a third identity, that of a floral meristem. A key developmental question has been to understand how the identity of the apical meristem is controlled.
The centroradialis (cen) mutant of Antirrhinum was first described in Gatersleben, Germany (Kuckuck and Schick, 1930; Stubbe, 1966). The cen mutant produces a number of axillary flowers before the apical meristem is converted to a floral meristem. Thus in cen plants, the apical meristem goes through three distinct identities; vegetative, inflorescence and then floral. The wild-type role of cen is therefore to prevent the apical meristem from switching to a floral fate.
Cen mutants of Antirrhinum may differ from wild type in several respects. Mutants produce a terminal flower, converting the inflorescence from indeterminate to determinate. Consequently, the architecture is changed to a shorter, more bushy plant, as shoots cannot grow indefinitely. About 10 axillary flowers are made below the terminal flower. The terminal floral meristem is developmentally more advanced than the axillary flowers below it. Unlike axillary flowers, organ numbers and their arrangement (phyllotaxy) are very variable in terminal flowers. The terminal flower is usually radially symmetrical, with all petals resembling the ventral (lowest) petal of axillary flowers.
A similar mutant to cen, terminal floweri (tfl1), has been described in Arabidopsis (Shannon and Meeks-Wagner, 1991; Alvarez et al., 1992). In addition to affecting meristem identity, tfl1 mutations also result in early flowering. Therefore, the normal role of the tfl1 gene is to inhibit flowering as well as preventing the apical meristem from switching to a floral fate.
In Arabidopsis, tfl1 mutants have two key features distinguishing from wild type: bolting early and the apical meristem eventually acquiring floral identity, leading to the production of a terminal flower (FIG.
1
). Typically, about half the normal number of rosette leaves are produced before bolting and about 1-5 peripheral flowers are made before the inflorescence apical meristem finally acquires floral identity. The structure of the terminal flower is often different to the wild-type. Wild-type flowers consist of 4 whorls of organs; 4 sepals outermost, 4 petals, 6 stamens and a central whorl of 2 unlimited carpels. In the terminal flower of tfl1 mutants in Arabidopsis, numbers of organs often vary and they may arise in a spiral, unlike the whorled arrangement of wild-type. Mosaic organs, composed of two types of floral organ, can also be found. All of these phenotypic effects, except for a marked change in flowering time, are also seen in cen mutants of Antirrhinum.
Both these genes therefore play key roles in apical meristem identity.
To delineate the action of cen and the molecular pathway by which it acts, a transposon-mutagenesis programme was set up to isolate the gene. In 1992, three new alleles of cen (cen-663, cen-665 and cen-666) were successfully isolated and a transposon linked to the cen phenotype in one allele was identified. Early in 1994, the flanking DNA of this transposon insertion was used to reveal that the cen locus had been cloned, allowing isolation of the cen cDNA and characterisation of its expression. CEN has similarly to a class of animal lipid-binding proteins and is expressed in the shoot apex.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is based on cloning of the cen gene from Antirrhinum and a homologue from Arabidopsis, tfl1. See also Bradley et al.,
Nature
1996, Vol. 379, 791-797 (cen) and Bradley, Carpenter and Coen, “Conserved control of inflorescence architecture in Arabidopsis and Antirrhinum”, submitted.
DETAILED DESCRIPTION OF THE INVENTION
According to an aspect of the present invention there is provided a nucleic acid isolate comprising a nucleotide sequence encoding a polypeptide with cen, tfl1 or indeterminacy function. Those skilled in the art will appreciate that the terms “cen function”, “tfl1 function” and “indeterminacy function” refer to the ability to influence the timing of flowering and/or the prevention of meristems switching to a floral fate phenotypically like the respective cen or tfl1 gene of Antirrhinum or Arabidopsis. “Indeterminacy function” refers to ability to keep the meristem growth indeterminantly. Certain embodiments of the present invention may have ability to complement a cen or tfl1 mutation in Antirrhinum or Arabidopsis.
Nucleic acid according to various aspects of the present invention may have the sequence of a cen or tfl1 gene or be a mutant, variant, derivative or allele of the sequence provided. Preferred mutants, variants, derivatives and alleles are those which encode a product (nucleic acid molecule or polypeptide) which retains a functional characteristic of the product encoded by the wild-type gene, especially, as for cen, the ability to inhibit apical meristem from switching to a floral fate and/or, as for tfl1, the additional ability to inhibit/delay flowering. Other preferred mutants, variants, derivatives and alleles encode a product which promote flowering compared to wild-type or a gene with the sequence provided and/or promote switching of apical meristems to a floral fate. 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, which may lead to the addition, insertion, deletion or substitution of one or more amino acids in an encoded polypeptide product. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.
In a preferred embodiment of the present invention a nucleic acid molecule comprises a nucleotide sequence which encodes an amino acid sequence shown in FIG.
4
(
a
). The nucleotide sequence may comprise an encoding sequence shown in FIG.
4
(
a
) or may be a mutant, variant, derivative or allele thereof encoding the same amino acid sequence.
In a further embodiment, a preferred nucleic acid molecule according to the present invention comprises a nucleotide sequence encoding an amino acid sequence shown in FIG.
6
(
a
) or may be a mutant, variant, derivative or allele thereof encoding the same amino acid sequence.
Sequences comprising changes to or differences from the sequences shown in the figures may also be employed in the present invention, as discussed herein.
The present invention also provides a vector which comprises nucleic acid with any of the provided sequences, preferably a vector from which a product polypeptide or nucleic acid molecule encoded by the nucleic acid sequence can be expressed. The vector is preferably suitable for transformation into a plant cell. The invention further encompasses a host cell transformed with such a vector, especially a plant cell. Thus, a host cell, such as a plant cell, comprising nucleic acid according to the present invention is provided. Within the
Bradley Desmond J.
Carpenter Rosemary
Coen Enrico S.
Mehta Ashwin
Nelson Amy J.
Nixon & Vanderhye PC
Plant Bioscience Limited
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