Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters pigment production in the plant
Reexamination Certificate
1995-06-07
2001-07-31
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters pigment production in the plant
C800S287000, C800S278000, C435S410000, C536S023500, C536S023600, C536S024100
Reexamination Certificate
active
06268546
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates to methods of using in vitro constructed DNA transcription or expression cassettes capable of directing ovary-tissue transcription of a DNA sequence of interest in plants to produce ovary-derived cells having an altered phenotype, and to methods of providing for or modifying existing color in various plant tissues or parts. The invention is exemplified by methods of using ovary tissue promoters for altering the color phenotype of cotton fibers, and cotton fibers produced by the method.
2. Background
In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells, due not only to a lack of suitable vector systems but also as a result of the different goals involved. For many applications, it is desirable to be able to control gene expression at a particular stage in the growth of a plant or in a particular plant part. For this purpose, regulatory sequences are required which afford the desired initiation of transcription in the appropriate cell types and/or at the appropriate time in the plant's development without having serious detrimental effects on plant development and productivity. It is therefore of interest to be able to isolate sequences which can be used to provide the desired regulation of transcription in a plant cell during the growing cycle of the host plant.
One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in ovary tissue, i.e. cotton fiber cells, so as to provide for altered or improved aspects of the mature cell type. In order to effect the desired phenotypic changes, transcription initiation regions capable of initiating transcription only in early ovary development are used. These transcription initiation regions are active prior to the onset of pollination and are less active or inactive, before fruit enlargement, tissue maturation, or the like occur.
Relevant Literature
A class of fruit-specific promoters expressed at or during anthesis through fruit development, at least until the beginning of ripening, is discussed in European Application 88.906296.4, the disclosure of which is hereby incorporated by reference. cDNA clones that are preferentially expressed in cotton fiber have been isolated. One of the clones isolated corresponds to mRNA and protein that are highest during the late primary cell wall and early secondary cell wall synthesis stages. John Crow
PNAS
(1992) 89:5769-5773. cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Mansson et al.,
Mol. Gen. Genet.
(1985) 200:356-361: Slater et al.,
Plant Mol. Biol.
(1985) 5:137-147). These studies have focused primarily on mRNAs which accumulate during fruit ripening. One of the proteins encoded-by the ripening-specific cDNAs has been identified as polygalacturonase (Slater et al.,
Plant Mol. Biol.
(1985) 5:137-147). A cDNA clone which encodes tomato polygalacturonase has been sequenced (Grierson et al.,
Nucleic Acids Research
(1986) 14:8395-8603). Improvements in aspects of tomato fruit storage and handling through transcriptional manipulation of expression of the polygalacturonase gene have been reported (Sheehy et al.,
Proc. Natl. Acad. Sci. USA
(1988) 85:8805-8809; Smith et al.,
Nature
(1988) 334:724-726).
Mature plastid mRNA for psbA (one of the components of photosystem II) reaches its highest level late in fruit development, whereas after the onset of ripening, plastid mRNAs for other components of photosystem I and II decline to nondetectable levels in chromoplasts (Piechulla et al.,
Plant Molec. Biol.
(1986) 7:367-376). Recently, cDNA clones representing genes apparently involved in tomato pollen (McCormick et al.,
Tomato Biotechnology
(1987) Alan R. Liss, Inc., NY) and pistil (Gasser et al.,
Plant Cell
(1989), 1:15-24) interactions have also been isolated and characterized.
Other studies have focused on genes inducibly regulated, e.g. genes encoding serine proteinase inhibitors, which are expressed in response to wounding in tomato (Graham et al.,
J. Biol. Chem.
(1985) 260:6555-6560: Graham et al.,
J. Biol. Chem.
(1985) 260:6561-6554) and on mRNAs correlated with ethylene synthesis in ripening fruit and leaves after wounding (Smith et al.,
Planta
(1986) 168:94-100). Accumulation of a metallocarboxypeptidase inhibitor protein has been reported in leaves of wounded potato plants (Graham et al.,
Biochem & BioPhys. Res Comm.
(1981) 101:1164-1170).
Genes which are expressed preferentially in plant seed tissues, such as in embryos or seed coats, have also been reported. See, for example, European Patent Application 87306739.1 (published as 0 255 378 on Feb. 3, 1988) and Kridl et al. (Seed Science Research (1991) 1:209-219).
Agrobacterium-mediated cotton transformation is described in Umbeck, U.S. Pat. Nos. 5,004,863 and 5,159,135 and cotton transformation by particle bombardment is reported in WO 92/15675, published Sept. 17, 1992. Transformation of Brassica has been described by Radke et al. (Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992) 11:499-505.
Transformation of cultivated tomato is described by McCormick et al.,
Plant Cell Reports
(1986) 5:81-89 and Fillatti et al.,
Bio/Technology
(1987) 5:726-730.
SUMMARY OF THE INVENTION
Novel DNA constructs and methods for their use are described which are capable of directing transcription of a gene of interest in ovary tissue, particularly early in fruit development. The novel constructs include a vector comprising a transcriptional and translational initiation region obtainable from a gene expressed in ovary tissue and methods of using constructs including the vector for altering fruit phenotype. The fruit may be edible or non-edible. The method includes transfecting a host plant cell of interest with a transcription or expression cassette comprising a promoter which is active in ovary cells prior to, and during, the pollination stage of the fruit, then generating a plant, which is grown to produce fruit having the desired phenotype. Constructs and methods of the subject invention thus find use in modulation of endogenous fruit products, as well as production of exogenous products and in modifying the phenotype of fruit and fruit products. The constructs also find use as molecular probes. In particular, constructs and methods for use in gene expression in cotton embryo tissues are considered herein. By these methods, novel cotton plants and cotton plant parts, such as modified cotton fibers, may be obtained.
Also provided in the instant application are constructs and methods of use relating to modification of color phenotype in plant tissues. Such constructs contain sequences for expression of genes involved in the production of colored compounds, such as melanin or indigo, and also contain sequences which provide for targeting of the gene products to particular locations in the plant cell, such as plastid organelles, or vacuoles. Plastid targeting is of particular interest for expression of genes involved in aromatic amino acid biosynthesis pathways, while vacuolar targeting is of particular interest where the precursors required in synthesis of the pigment are present in vacuoles. Production of melanin, for example, may be enhanced by vacuolar targeting in plant tissues which accumulate tyrosine in vacuoles. Transcriptional initiation regions for expression of color-related genes will be selected on the basis of the tissue for which color modification is desired.
REFERENCES:
patent: 4752301 (1988-06-01), Koch
patent: 4801540 (1989-01-01), Hiatt
patent: 4943674 (1990-07-01), Houck
patent: 5005863 (1991-04-01), Umbeck
patent: 5159135 (1992-10-01), Umbeck
patent: 5360726 (1994-11-01), Raikhel
patent: 5487991 (1996-01-01), Vandekerckhove et al.
patent: 5495070 (1996-02-01), John
patent: 5500365 (1996-03-01), Fischhof
McBride Kevin
Stalker David
Calgene LLC
Hauda Karen M.
Rae-Venter Barbara
Rae-Venter Law GroupPC
Wahlsten Jennifer
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