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
2001-03-21
2004-05-04
Mehta, Ashwin (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...
C435S320100, C435S419000, C435S468000, C536S024100, C800S288000, C800S298000
Reexamination Certificate
active
06730826
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a trichome specific regulatory sequence such as a promoter, and heterologous constructs comprising the trichome specific regulatory sequence and DNAs encoding a single or multiple polypeptide of interest. The present invention is also related to transgenic expression of a polypeptide(s) of interest in the trichome.
BACKGROUND OF THE INVENTION
Most surfaces of most plants are said to be pubescent, or bearing of trichomes or hairs. The morphology of these structures can vary greatly with tissues and species. Indeed, the botanical literature is said to contain more than 300 descriptions (uniseriate, capitate-sessile, etc.) to characterize various morphological types. These characteristics have often been used in plant classification. Functionally, trichomes may be simple hairs which deter herbivores, guide the path of pollinators or affect photosynthesis, leaf temperature, or water loss through increased light reflectance as in desert species. Or, they may be more specialized tissues (glandular secreting trichomes) whose principal function(s) may be to produce pest- or pollinator-interactive chemicals which are stored or volatilized at the plant surface. It has been suggested that in some desert species the principal role of glandular secreting trichomes is to produce such high levels of exudate that it forms a continuous layer on the plant surface. This layer may increase light reflectance and thereby reduce leaf temperature.
It is the glandular, merocrine, secreting trichome type which often produce and accumulate terpenoid oils that is the focus of this application. Other secretory tissues occurring on or in plants include salt glands, salt hairs, nectary and slime glands, resin ducts, and osmophores (perhaps responsible for fragrances in flowers). Useful modification might be genetic manipulation of an enzyme to alter the chemical nature of exudate for the purpose of enhancing disease resistance, enhancing attractiveness to pollinators, or to expand the metabolism of secretory cells to include synthesis of compounds or intermediates not normally formed. It is important to point out that the potential of secreting trichomes to accumulate exudate is highly significant in certain plants where, under optimal conditions, secreted products can reach a level of 10 to 30% of the dry weight of leaves. Glandular trichome exudates usually contain terpenes and essential oils, as well as lipophilic components not easily stored in large amounts within the cell. Therefore, if it were desirable to manipulate plants to produce or overproduce a compound in this class of biochemicals, the secreting trichome system, which amasses secretions outside of gland cells, may be more amenable to overproduction than one requiring intracellular storage.
Esau,
Plant Anatomy,
John Wiley & Sons, N.Y. has defined trichomes as “epidermal appendages of diverse form, structure and functions . . . represented by protective, supporting, and glandular hairs, by scales, by various papillae, and by absorbing hairs of roots.” This definition suggests a close relationship between trichomes and root hairs. From this viewpoint, aerial trichomes and subterranean “trichomes” could be said to cover or nearly cover the surfaces of most plants. However, while trichomes and root hairs may resemble each other in morphology, their genetic determinants may differ. Both trichomes and root hairs develop projections from protodermal cells. Glandular structures of trichomes arise from a series of anticlinal and periclinal divisions to form supporting auxiliary cells and glands as in the case of Cannabis. The appearance of glands atop supporting cells and the occurrence of exudate around gland cells has suggested to most observers that secretions are produced in gland cells and not by other epidermal or subepidermal cells.
Hutvagner et al.,
Gene
188 (1997) 247-252 describes the potato P450 gene. The function, tissue specificity and promoter of this potato gene is not presented or discussed in the paper.
Clark et al., Plant
Molecular Biology
33 (1997) 875-885 describes another P450 gene isolated
Nepeta racemosa
that hybridizes to RNA from trichomes but not that prepared from leaves stripped of trichomes. This gene shows 36 and 45 to 50% homology to 1-31, protein and DNA levels, respectively. No promoter is described in this paper.
Wagner,
Plant Physiol.
96 (1991) 675-679 provides some brief background on plant glanded trichomes.
In order to produce genetically engineered polypeptides in trichomes there is a need to make a trichome specific regulatory sequence that allows for targeting of gene expression specifically in trichome glands.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an isolated nucleic acid molecule comprising:
a polynucleotide set forth in SEQ ID NO:1;
a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:1, wherein said variant or fragment has trichome specific transcriptional regulatory activity; or
a polynucleotide sequence which hybridizes to the polynucleotide of SEQ ID NO:1 under 5×SSC and 42° C. wash conditions.
The nucleic acid may be a polynucleotide comprising the sequence set forth in SEQ ID NO:1. The nucleic acid may further comprise a polynucleotide encoding a polypeptide. The polypeptide may be a disease- or pest-resistance conferring protein. The polypeptide may be a pharmaceutical compound. The polypeptide may function to alter horticultural or food plants. The polypeptide may also be a phytotoxic protein, or a protein highly sensitive to modification during isolation from plant tissue homogenates. Any polypeptide at all can be used with the trichome specific regulatory sequence of the invention. The trichome specific regulatory sequence also encompasses a promoter sequence.
Another object of the invention is to provide a composition comprising a polypeptide made from expression of the above-mentioned nucleic acid, and a trichome gland exudate.
The present invention also relates to a vector comprising a replicable vector and the above-mentioned polynucleotide inserted into said vector.
The vector may be preferably a cloning or expression vector, or a plant vector, preferably a binary vector.
The present invention also relates to a cell containing the above-mentioned vector. Preferably, the cell is a plant cell.
The present invention also provides for a plant comprising the above-mentioned nucleic acid.
The present invention is also related to a method for expressing a polypeptide comprising constructing a vector comprising a trichome specific regulatory sequence and a nucleic acid encoding a polypeptide, and inserting said vector into a plant, wherein said polypeptide is expressed trichome specifically.
REFERENCES:
Kim et al, “A 20 nucleotide upstream element is essential for thenopaline synthase (nos) promoter activity”, 1994, Plant Molecular Biology vol. 24, pp. 105-117.*
Hsu et al, “Analysis of promoter activity of cotton lipid transfer protein gene LTP6 in transgenic tobacco plants”, 1999, Plant Science vol. 143, pp. 63-70.*
Vaeck et al, “transgenic plants protected from insect attack”, 1987, Nature vol. 328, pp. 33-37.*
Düring et al, Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum, 1990, Plant Molecular Biology, vol. 15, pp. 281-293.*
Del Sorbo et al, “Functional Expression of the Gene cu, Encoding the phytotoxic Hydrophobin Cerato-ulmin, Enables Ophiostoma quercus, a Nonpathogen on Elm, to Cause Symptoms of Dutch Elm Disease”, 2000, MPMI vol. 13 No. 1, pp. 43-53.*
Clark et al., “Spatially distinct expression of two new cytochrome P450s in leaves ofNepeta racemosa:identification of a trichome-specific isoform”, Plant Molecular Biology 33, 1997, pp. 875-885.
G. Hutvagner, et al., “Isolation and sequence analysis of a cDNA and a related gene for cytochrome P450 proteins fromSolanum chacoense”,GENE 188 (1997), pp. 247-252.
Gan Susheng
Wagner George J.
Wang Erming
Wang Rui
McDermott & Will & Emery
Mehta Ashwin
University of Kentucky Research Foundation
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