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
1998-06-24
2001-05-22
Nelson, Amy J. (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
C435S320100, C536S023600, C800S278000, C800S286000, C800S298000
Reexamination Certificate
active
06235975
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 lec1mutant 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, typically about 630 nucleotides in length, which specifically hybridizes to SEQ ID NO:1 under stringent conditions. The LEC1 polynucleotides of the invention can encode a LEC1 polypeptide of about 210 amino acids, typically as shown in SEQ ID NO:2.
The invention provides an isolated nucleic acid molecule comprising a LEC1 polynucleotide sequence, the polynucleotide sequence defined as follows: the polynucleotide sequence specifically hybridizes to SEQ ID NO:1 under stringent conditions; or the polynucleotide sequence has at least 70% sequence identity to SEQ ID NO:1. In alternative embodiments, the isolated nucleic acid molecule of can have at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity to SEQ ID NO:1; and, the isolated nucleic acid molecule can have the sequence set forth in SEQ ID NO:1.
In different embodiments, the isolated nucleic acid molecule of the invention is a LEC1 polynucleotide between about 100 nucleotides and about 630 nucleotides in length, and, between about 50 and about 210 amino acids in length. The isolated nucleic acid molecule of the invention can encode a LEC1 polypeptide having an amino acid sequence as shown in SEQ ID NO:2.
The invention also provides an isolated LEC1 nucleic acid molecule further comprising an operably linked promoter. In alternative embodiments, the promoter is a constitutive promoter, where the constitutive promoter is a cauliflower mosaic virus (CaMV) 35S transcription initiation region or a 1′- or 2′- promoter derived from T-DNA of
Agrobacterium tumafaciens
, and the promoter is an inducible promoter. The promoter can also be a plant promoter. In various embodiments, the plant promoter is a tissue-specific promoter and is a tissue-specific promoter active in vegetative tissue or reproductive tissue. The plant promoter can be from a LEC1 gene, where LEC1 gene can be as shown in SEQ ID NO:3. In different embodiments, the plant promoter can be from about nucleotide 1 to about nucleotide 1998 of SEQ ID NO:3, or, the promoter can be from the LEC1 gene is as shown in SEQ ID NO:4. The LEC1 polynucleotide can be linked to the promoter in an antisense orientation.
The invention also provides LEC1 polynucleotide further comprising an expression vector, an expression cassette, or a plant virus.
The invention further provides an isolated nucleic acid molecule comprising a LEC1 polynucleotide sequence, wherein the polynucleotide sequence specifically hybridizes to SEQ ID NO:1 under stringent conditions, or (b) the polynucleotide sequence has at least 70% sequence identity to SEQ ID NO:1, and, (ii) wherein the polynucleotide sequence encodes a LEC1 polypeptide of between about 50 and about 210 amino acids. The LEC1 polypeptide can have an amino acid sequence as shown in SEQ ID NO:2. In alternative embodiments, the polynucleotide sequence can have at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity to SEQ ID NO:1; and, the polynucleotide sequence can
Fischer Robert L.
Goldberg Robert B.
Harada John J.
Lotan Tamar
Ohto Masa-aki
Nelson Amy J.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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