Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2000-03-01
2004-08-24
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C800S298000, C435S320100, C435S419000, C435S468000, C435S252300, C435S471000, C536S023600
Reexamination Certificate
active
06781035
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to new embryo-specific genes useful in improving agronomically important plants.
BACKGROUND OF THE INVENTION
Embryogenesis in higher plants is a critical stage of the plant life cycle in which the primary organs are established. Embryo development can be separated into two main phases: the early phase in which the primary body organization of the embryo is laid down and the late phase which involves maturation, desiccation and dormancy. In the early phase, the symmetry of the embryo changes from radial to bilateral, giving rise to a hypocotyl with a shoot meristem surrounded by the two cotyledonary primordia at the apical pole and a root meristem at the basal pole. In the late phase, during maturation the embryo achieves its maximum size and the seed accumulates storage proteins and lipids. Maturation is ended by the desiccation stage in which the seed water content decreases rapidly and the embryo passes into metabolic quiescent state. Dormancy ends with seed germination, and development continues from the shoot and the root meristem regions.
The precise regulatory mechanisms which control cell and organ differentiation during the initial phase of embryogenesis are largely unknown. The plant hormone abscisic acid (ABA) is thought to play a role during late embryogenesis, mainly in the maturation stage by inhibiting germination during embryogenesis (Black, M. (1991). In
Abscisic Acid: Physiology and Biochemistry
, W. J. Davies and H. G. Jones, eds. (Oxford: Bios Scientific Publishers Ltd.), pp. 99-124) Koornneef, M., and Karssen, C. M. (1994). In
Arabidopsis
, E. M. Meyerowitz and C. R. Sommerville, eds. (Cold Spring Harbor: Cold Spring Harbor Laboratory Press), pp. 313-334). Mutations which effect seed development and are ABA insensitive have been identified in Arabidopsis and maize. The ABA insensitive (abi3) mutant of Arabidopsis and the viviparous1 (vp1) mutant of maize are detected mainly during late embryogenesis (McCarty, et al., (1989)
Plant Cell
1, 523-532 and Parcy et al., (1994)
Plant Cell
6, 1567-1582). Both the VP1 gene and the ABI3 genes have been isolated and were found to share conserved regions (Giraudat, J. (1995)
Current Opinion in Cell Biology
7:232-238 and McCarty, D. R. (1995).
Annu. Rev. Plant Physiol. Plant Mol. Biol
. 46:71-93). The VP1 gene has been shown to function as a transcription activator (McCarty, et al., (1991)
Cell
66:895-906). It has been suggested that ABI3 has a similar function.
Another class of embryo defective mutants involves three genes: LEAFY COTYLEDON1 and 2 (LEC1, LEC2) and FUSCA3 (FUS3). These genes are thought to play a central role in late embryogenesis (Baumlein, et al. (1994)
Plant J
. 6:379-387; Meinke, D. W. (1992)
Science
258:1647-1650; Meinke et al.,
Plant Cell
6:1049-1064; West et al., (1994)
Plant Cell
6:1731-1745). Like the abi3 mutant, leafy cotyledon-type mutants are defective in late embryogenesis. In these mutants, seed morphology is altered, the shoot meristem is activated early, storage proteins are lacking and developing cotyledons accumulate anthocyanin. As with abi3 mutants, they are desiccation intolerant and therefore die during late embryogenesis. Nevertheless, the immature mutants embryos can be rescued to give rise to mature and fertile plants. However, unlike abi3 when the immature mutants germinate they exhibit trichomes on the adaxial surface of the cotyledon. Trichomes are normally present only on leaves, stems and sepals, not cotyledons. Therefore, it is thought that the leafy cotyledon type genes have a role in specifying cotyledon identity during embryo development.
Among the above mutants, the lec1 mutant exhibits the most extreme phenotype during embryogenesis. For example, the maturation and postgermination programs are active simultaneously in the lec1 mutant (West et al., 1994), suggesting a critical role for LEC1 in gene regulation during late embryogenesis.
In spite of the recent progress in defining the genetic control of embryo development, further progress is required in the identification and analysis of genes expressed specifically in the embryo and seed. Characterization of such genes would allow for the genetic engineering plants with a variety of desirable traits. For instance, modulation of the expression of genes which control embryo development may be used to alter traits such as accumulation of storage proteins in leaves and cotyledons. Alternatively, promoters from embryo or seed-specific genes can be used to direct expression of desirable heterologous genes to the embryo or seed. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the isolation and characterization of LEC1 genes. The invention provides isolated nucleic acid molecules comprising a LEC1 polynucleotide sequence which is at least 68% identical to the B domain of SEQ ID NO:2.
The invention also provides expression cassettes comprising a promoter operably linked to a heterologous polynucleotide sequence or complement thereof, encoding a LEC1 polypeptide comprising a sequence which is at least 68% identical to the B domain of SEQ ID NO:2. In some embodiments, the polynucleotide sequence is heterologous to any element in the expression cassette. In a preferred embodiment, the B domain comprises a polypeptide between about amino acid residue 28 and amino acid residue 117 of SEQ ID NO:2. In a more preferred embodiment, the B domain comprises a polypeptide sequence with an amino terminus at amino acid residues 28-35 and a carboxy terminus at amino acid residues 103-117 of SEQ ID NO:2.
In particularly preferred embodiments, the LEC1 polypeptide is shown in SEQ ID NO:20 or 22. Such LEC1 polypeptides can be encoded by the polynucleotide sequences shown in SEQ ID NO:19 or SEQ ID NO:21, respectively. In another embodiment the LEC1 polypeptide is a fusion between two or more LEC1 polypeptides of polypeptide subsequences.
The expression cassette comprises a promoter operably linked to the LEC1 polynucleotide or its complement. For example, the promoter can be a constitutive promoter. Alternatively, the promoter can be a promoter from a LEC1 gene. For instance, the LEC1 promoter can be from about nucleotide 1 to about nucleotide 1998 of SEQ ID NO:3. In one embodiment, the heterologous polynucleotide can be linked to the promoter in the antisense orientation. In another embodiment, the promoter is SEQ ID NO:23. The promoter can further comprise SEQ ID NO:24.
In another embodiment, the invention provides an expression cassette comprising a promoter operably linked to a heterologous polynucleotide sequence, or complement thereof, encoding a LEC1 polypeptide comprising a subsequence at least 90% identical to the A or C domain of a LEC1 polypeptide. The polynucleotide sequence can be heterologous to any element in the expression cassette. Such expression cassettes can encode fusions of two or more LEC1 polypeptides or polypeptide subsequences.
The invention also provides for an expression cassette for the expression of heterologous polypeptides in a plant. The expression cassette comprises a LEC1 promoter operably linked to a heterologous polynucleotide. In some embodiments, the LEC1 promoter is at least 70% identical to SEQ ID NO:23. In some embodiments, the expression cassette promoter comprises a promoter at least 70% identical to SEQ ID NO:24. Preferably, the promoter comprises the sequence displayed in SEQ ID NO:24.
The invention also provides an isolated nucleic acid or complement thereof, encoding a LEC1 polypeptide comprising a subsequence at least 68% identical to the B domain of SEQ ID NO:2, with the proviso that the nucleic acid is not clone MNJ7. In a preferred embodiment, the B domain comprises a polypeptide sequence with an amino terminus at amino acids 28-35 and a carboxy terminus at amino acids 103-117 of SEQ ID NO:2. In another embodiment, the LEC1 polypeptide is shown in SEQ ID NO:20 or SEQ ID NO:22. Such LEC1 polype
Bui Anhthu
Fischer Robert L.
Goldberg Robert B.
Harada John
Kwong Raymond
Collins Cynthia
Fox David T.
The Regents of the University of California
LandOfFree
Leafy cotyledon1 genes and their uses does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Leafy cotyledon1 genes and their uses, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Leafy cotyledon1 genes and their uses will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3298715