Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1998-02-19
2003-04-08
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C800S298000, C800S286000, C536S024100, C435S320100
Reexamination Certificate
active
06545201
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, 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 nucleic acids of the invention may also comprise expression cassettes containing a plant promoter operably linked to the LEC1 polynucleotide. In some embodiments, the promoter is from a LEC1 gene, for instance, as shown in SEQ. ID. No. 3. The LEC1 polynucleotide may be linked to the promoter in a sense or antisense orientation.
The invention also provides transgenic plants comprising an expression cassette containing a plant promoter operably linked to a heterologous LEC1 polynucleotide. The LEC1 may encode a LEC1 polypeptide or may be linked to the promoter in an antisense orientation. The plant promoter may be from any number of sources, including a LEC1 gene, such a as that shown in SEQ. ID. No. 3 or SEQ. ID. No. 4. The transgenic plant can be any desired plant but is often a member of the genus Brassica.
Methods of modulating seed development in a plants are also provided. The methods comprise introducing into a plant an expression cassette containing a plant promoter operably linked to a heterologous LEC1 polynucleotide. The LEC1 may encode a LEC1 polypeptide or may be linked to the promoter in an antisense orientation. The expression cassette can be introduced into the plant by any number of means known in the art, including through a sexual cross.
The invention further provides expression cassettes containing promoter sequences from LEC1 genes. The promoters of the invention can be characterized by their ability to specifically hybridizes to a polynucleotide sequence consisting of nucleotides 1 to −1998 of SEQ ID No:3. The promoters of the invention can be operably linked to a variety of nucleic acids, whose expression is to be targeted to embryos or seeds. Transgenic plants comprising the expression cassettes are also provided.
The promoters of the invention can be used in methods of targeting expression of a desired polynucleotide to seeds. The methods comprise introducing into a plant an expression cassette containing a LEC1 promoter operably linked to a heterologous polynucleotide sequence.
DEFINITIONS
The phrase “nucleic acid” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not alter expression of a polypeptide encoded by that nucleic acid.
The phrase “polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands as either individual single strands or in the duplex. It includes, but is not limited to, self-replicating plasmids, chromosomal sequences, and infectious polymers of DNA or RNA.
The phrase “nucleic acid sequence encoding” refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and th
Fischer Robert L.
Goldberg Robert B.
Harada John J.
Lotan Tamar
Ohto Masa-aki
Collins Cynthia
McElwain Elizabeth F.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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