Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-15
2003-03-18
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C435S252200, C435S252300, C435S252330, C435S320100, C435S419000, C435S440000, C530S350000, C530S351000, C536S023200, C536S023600, C800S025000, C800S302000
Reexamination Certificate
active
06534265
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention (Technical Field)
The present invention relates to a novel oryzacystatin protease inhibitor peptide, including a signal peptide component thereof, nucleic acid sequences encoding the protein and signal peptide, incorporation of the oryzacystatin protease inhibitor gene into the genome of a plant and the expression of the inhibitor gene in plants whereby such plants are less susceptible to insect and plant pest damage, and to damage caused by certain plant viruses. The invention is further directed to methods for producing the complete oryzacystatin protease inhibitor peptide, compositions containing the complete oryzacystatin protease inhibitor peptide for use as insecticidal agents, and the use of oryzacystatin protease inhibitor peptide for decreasing insect and virus damage to plants.
2. Background Art
There is significant interest in the use of materials and methods to control insect damage to commercially valuable crop plants that does not require the use of conventional chemical insecticides and chemical fumigation methods. One approach that has been studied employs the use of protease or peptidase inhibitors that are toxic to or substantially inhibit certain insects.
Proteases and peptidases are enzymes that hydrolyze the peptide bonds of proteins or peptides, respectively. These proteases or peptidases (collectively referred to as proteases) play a number of roles in biochemical regulation of organisms, including insects. For example, peptides are generated from proteins by the action of proteases in the gastrointestinal tract of organisms. Through use of these proteases, proteins are broken down into absorbable peptides or individual amino acids, thereby providing sustenance to the organism. Disruption of protease activity can thus have a significant adverse effect on the life cycle of organisms.
There are a number of mechanisms that regulate protease enzymatic activity; one of the most potent and direct mechanisms is through the use of protease inhibitors. Based on the catalytic mechanism employed, there are four classes of protease enzymes, of which one such class is cysteine or thiol proteases. Cysteine proteases are widely distributed, and occur in plants, animals, bacteria and a wide variety of microorganisms. These organisms include specifically plant and animal pests and parasites, as described generally in U.S. Pat. Nos. 5,494,813 and 5,863,775.
Cystatins are well known inhibitors of cysteine proteases. A wide variety of cystatin-type inhibitors have been documented, and these are naturally expressed in a wide variety of plants and animals as part of the regulatory scheme of proteolytic activity. Thus cystatins are naturally found in chickens, including egg whites, U.S. Pat. No. 5,212,297; humans, U.S. Pat. No. 5,919,658; rice and maize, U.S. Pat. No. 5,863,775; and a wide variety of other plants and animals.
Research on the use of cystatins as a means of crop pest control has grown due to the wide spectrum of activity these proteinacious inhibitors possess. The cloning of oryzacystatin-I (OC-I), one of the earliest characterized cystatins of plant origin (Abe et al., Purification of a cysteine proteinase inhibitor from rice,
Oryza sativa. Agric Biol Chem
49: 3349-3350 (1985)), was first published over a decade ago (Abe et al., Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin).
J Biol Chem
262: 16793-16797 (1987); GenBank accession M29259), with other plant cystatins quick to follow, including oryzacystatin-II (OC-II) (Kondo et al., Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases.
J Biol Chem
265: 15832-15837 (1990)), corn cystatin-I (CC-I), corn cystatin-II (CC-II) (Abe et al., Structural organization of the gene encoding corn cystatin.
Biosci Biotech Biochem
60: 11731-1175 (1996)), and a cystatin from sorghum (Li et al., Direct Submission to GenBank, Accession # 1076759, PID g1076759). These cystatins share a high degree of similarity and contain a highly conserved sequence, Gln-Val-Val-Ala-Gly (SEQ ID NO: 7) believed to be the active region of the inhibitor responsible for binding cysteine proteases (Abe et al., The NH
2
-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily.
J Biol Chem
263: 7655-7659 (1988)).
To date, the transformation of plants with OC-I cDNAs has resulted in low or inconsistent protein yields (Masoud et al., Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants.
Plant Mol Biol
21: 655-663 (1993); Irie et al., Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases.
Plant Mol Biol
30: 149-157 (1996)). There are reports that plants transformed with OC-I do not significantly hinder the growth of certain crop pests, such as the Coleoptera,
Phaedon cochleariae
, and may actually cause the pests (
Psylliodes chrysocephala
L. and
Ceutorhynchus assililis
) to thrive.
Low protein yields of OC-I occurred even when the cDNA was inserted back into the source plant,
Oryza sativa
L. japonica, for the purpose of enhancing the effectiveness of the innate OC-I against pests (Irie et al., supra). Thus it has not heretofore been possible to express consistent and high protein yields of OC-I using cDNA transformation methods with the heretofore-identified gene segments.
A variety of different methods have been attempted to employ transformation schemes that result in commercially and agriculturally viable expression of useful levels of OC-I. The earliest report was by Masoud et al. (Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgentic tobacco plants.
Plant Mol Biol
21: 655-663 (1993)). Attempts have been made to stabilize OC-I in plants through such approaches as engineering fusion proteins using genes from cowpea trypsin inhibitor (CPTI) (Urwin et al., Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs.
Planta
204: 472-479 (1998), beta-glucuronidase (Hosoyama et al., Introduction of a chimeric gene encoding an oryzacystatin-&bgr;-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants.
Plant Cell Reports
15: 174-177 (1995)),
Bacillus thuringiensis
Cry 3A (Klypina et al., A chimeric oryzacystatin-I/
Bacillus thruingiensis
Cry3A gene which is expressed at high levels in transgenic plants, Abst. 330,
ASPP
p. 84 (1998)) and the 10 kDa zein seed storage protein from maize (Kikuta-Oshima et al., A 10 kDa zein/oryzacystatin-I protease inhibitor chimeric gene designed for the stabilization of proteins for the purpose of plant pest control is expressed in transgenic plants. Abst. 340,
ASPP
, p. 86 (1998)). Although these strategies seem to improve the stability of OC-I accumulation in transgenic plants, it is unclear whether the resulting fusion proteins retain any cystatin activity.
It has been heretofore universally known that oryzacystatins, unlike the maize cystatins, have no N-terminal leader sequences, and therefore probably remain cystostolic after translation. This assumption was based, in significant part, upon a protein produced by the OC-I gene reported by Abe et al. (1987) and Kondo et al. (Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.
Gene
81: 259-265 (1989)).
Since its discovery, OC-I has been investigated for use as a crop pest deterrent primarily because of its function as an insect digestive system protease inhibitor (see, e.g., U.S. Pat. Nos. 5,863,775 and 5,494,813, Irie et al., supra.). The belief that OC-I is a cytosolic protein was considered important in the work of Urwin et al. (Engineered oryzacystatin-I expressed in transgenic having roots confers resistance to
Globodera pallida. Plant J
8: 121-131 (1995)), since nematodes appear to feed only on the cytosol. This belief was supported by the report that OC-I did not appear to have a functional signa
Kemp John D.
Randall Jennifer J.
Womack James S.
New Mexico State University Technology Transfer Corporation
Peacock Deborah A.
Slusher Stephen A.
Taylor Janell E.
Zitomer Stephanie W.
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