Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1996-07-05
2001-01-09
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S410000, C435S415000, C435S416000, C435S419000, C435S426000, C435S428000, C435S069100, C435S252300, C800S278000, C800S287000, C536S023600, C536S024100
Reexamination Certificate
active
06171864
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the calcium-binding proteins, calreticulin and calnexin, and more specifically, to their genomic structure, coding regions, and promoter sequences.
BACKGROUND OF THE INVENTION
Plants are subject to assault from a variety of diseases that affect growth, flowering, fruiting, and ultimately yield or quality of the plant and plant product. Diseases in plants are caused by sources as diverse as insects, fungi, molds, nematodes, and viruses. Counteractive measures to diseases have been implemented. The use of pesticides, insecticides, fungicides, hormone treatment, and other treatments are widely relied upon. These measures are not always effective or resistance to chemicals develops. Moreover, social acceptance of chemical treatments has waned over the years. Genetic breeding to confer resistance is an environmentally friendly alternative, but labor intensive and difficult to move resistance genes between species of plants. Other mechanisms to confer protection against plant diseases is desirable.
Various genes conferring protection for plants against diseases and insects have been identified. Many of these genes have been cloned as well. Introduction of these genes into plants and control of their expression is important for improving crop development and food production. In addition, transgenic resistance reduces the need for chemicals, which is beneficial for the environment and reduces labor and costs. For effective resistance, expression of the resistance genes in appropriate tissues is critical to their function. As such, tissue-specific promoters and accessory gene products that may increase expression are needed.
One organelle which is particularly important for the expression of genes in higher plant cells is the endoplasmic reticulum (ER). Briefly, the ER comprises the initial site of the protein secretory pathway, the site of the majority of fatty acid modification and triacylglycerol biosynthesis, and the site of an intracellular store of reversibly bound calcium, which is involved in various aspects of plant signal transduction. In developing seed storage tissue, the ER is the primary site of seed storage protein synthesis, folding and assembly. These processes require an array of chaperones, some of which (e.g., BiP, PDI, and GRP94) have been cloned. Calreticulin, which has been cloned as a cDNA from various animals and a few plants (e.g., barley (Chen et al.,
Plant Cell
6:835, 1994), Arabadopsis (Benedetti and Turner,
Plant Physiol.
109:338, 1995), and corn (Napier et al.,
J. Exp. Bot.
46:1603, 1995)), is the primary calcium binding protein of the ER and may have a role in protein folding, assembly, and signal transduction. Calnexin is another calcium binding, chaperone protein in the ER, which has been cloned as a cDNA from various animals and from Arabidopsis.
The present invention discloses novel compositions for calreticulin and calnexin proteins, genomic sequences, and promoters, use of these proteins and sequences in controlling expression of resistance genes, and further, provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides nucleic acid molecules which encode calreticulin and calnexin proteins, as well as promoter regions of the calreticulin and calnexin genes. More specifically, within one aspect of the present invention isolated nucleic acid molecules encoding calreticulin are provided, comprising a nucleic acid sequence that encodes SEQ ID NO:2, or a variant thereof. Within one embodiment, the sequence is SEQ ID NO:1.
Within other aspects of the present invention, isolated nucleic acid molecules are provided which encode calnexin, comprising a nucleic acid sequence that encodes SEQ ID NO:6, or a variant thereof. Within one embodiment, the sequence is SEQ ID NO:5.
Within other related aspects, isolated nucleic acid molecules are provided which encode soluble calnexin (e g., calnexin which lacks a transmembrane and C-terminal domain, as well as optionally, a signal peptide).
Also provided by the present invention are polypeptides encoded by the afore-mentioned nucleic acid sequences, vectors which comprise such sequences, and host cells which contains these vectors. Within one embodiment, the vector is an expression vector such as a binary
Agrobacterium tumefaciens
plasmid vector.
Within other aspects of the present invention, isolated nucleic acid molecules are provided, comprising a castor calreticulin promoter. Within one embodiment, such molecules comprise the nucleic acid sequence of SEQ ID NO:4, or a variant (including portions) thereof which has calreticulin-promoter activity.
Within yet other aspects, isolated nucleic acid molecules are provided, comprising a castor calnexin promoter. Within one embodiment, such molecules comprise the nucleic acid sequence of SEQ ID NO:8, or a variant (including portions) thereof which has calnexin-promoter activity.
Within other aspects, vectors are provided which contain one of the above-described calreticulin or calnexin promoters. Within a particularly preferred embodiment, such vectors further comprise a nucleic sequence encoding a foreign gene operably linked to said promoter. Representative examples of such foreign genes include genes which encode proteins, antisense genes and ribozyme genes. Within one embodiment the foreign gene confers resistance to a disease selected from the group consisting of Sclerotinia, sunflower head moth, canola flea beetle and soybean cyst nematode. Within other related aspects, host cells containing one of the above-described vectors are provided. Representative examples of suitable host cells include plant cells from soybean, canola, sunflower, or alfalfa.
Within further aspects of the invention, methods are provided for producing a foreign gene product, comprising the steps of (a) introducing a vector as described above into a host cell, wherein the vector contains a foreign gene in an expressible form, and (b) growing the host cell under conditions wherein the foreign gene is expressed. As noted above, representative examples of suitable host cells include plant cells such as those obtained from soybean, canola, sunflower, or alfalfa.
Within yet other aspects of the invention methods are provided for producing a plant which expresses a foreign gene, comprising the steps of (a) introducing a vector as described above into an embryogenic plant cell, wherein the vector contains a foreign gene in an expressible form, and (b) producing a plant from the embryogenic plant cell, wherein the plant expresses the foreign gene. Also provided are plants which can be made containing the nucleic acid molecules, or vectors, described herein.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.
REFERENCES:
patent: 5097025 (1992-03-01), Benfey et al.
patent: 5426097 (1995-06-01), Stern et al.
patent: 5591716 (1997-01-01), Siebert et al.
Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, pp. 9.49-9.51, 1989.
George et al. Macromolecular Sequencing and Synthesis: Selected Methods and Applications, ed. DH Schlesinger, Alan R. Liss, Inc., NY, pp. 127-149, 1988.
Chen et al. Identification and characterization of cDNA clones encoding plant calreticulin in barley. The Plant Cell. vol. 6, pp. 835-843, Jun. 1994.
Denecke et al. The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo. The Plant Cell. vol. 7, pp. 391-406, Apr. 1995.
Napier et al. Calcium-binding protein—maize. EMBL Accession No. S49818, Mar. 5, 1995.
Kwiatkowski et al., “Cloning of two cDNAs encoding calnexin-like and calreticulin-like proteins from maize (Zea mays) leaves: identification of potential calcium-binding domains,”Gene16
Coughlan Sean J.
Winfrey, Jr. Ron J.
Kemmerer Elizabeth
Pioneer Hi-Bred International , Inc.
Seed IP Law Group PLLC
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