Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-08-28
2001-10-30
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S069100, C435S468000, C536S023600, C800S288000, C800S302000
Reexamination Certificate
active
06310275
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an artificially synthesized gene encoding an active protein. More specifically, the present invention relates to an artificially synthesized gene encoding a protein having a trypsin inhibitor activity. The present invention also relates to a plant which expresses a gene encoding a protein having trypsin inhibitor activity, which also confers resistance to lepidopteran insect pests.
2. Description of the Related Art
There exist several protease inhibitors including trypsin inhibitors in Fabaceae plant (Yamamoto et al.
J. Biochem
. 94, pp. 849-863, 1983).
The trypsin inhibitors derived from Fabaceae plant are classified into two groups on the basis of their molecular weight and cystine content. One is the Bowman-Birk type inhibitors which have molecular weights of around 8,000 and are characterized by a high cystine content, namely seven disulfide bridges (Norioka et al.,
J. Biochem
. 94, pp. 589-599). The other is the Kunitz type inhibitors which have molecular weights of around 20,000 and include two disulfide bridges (Shibata et al.,
J. Biochem
. 99: pp. 1147-1155, 1986). The Kunitz type inhibitors are known to exist not only in Fabaceae plant but also in soybean, silk tree, barley, acacia, and rice (Yamamoto et al.
J. Biochem
. 94, pp. 849-863, 1983). Trypsin inhibitors are present in the pancreas and plasma of vertebrates as peptides which can inhibit the activity of the digestive enzyme, trypsin. Although the role of the trypsin inhibitor in a plant is not known very well, it has been reported that a trypsin inhibitor ingested by an insect inhabits growth of the insect (R. Johnson et al.,
Proc. Natl. Acad. Sci. USA
86, pp. 9871-9875, 1989).
A trypsin inhibitor WTI-1b derived from the winged bean (
Psophocarpus tetragonolobus
(L.) DC.) is a Knitz type inhibitor which has molecular weight of 19,200, and the complete amino acid sequence of the purified protein has been identified (Yamamoto et al.
J. Biochem
. 94, pp. 849-863, 1983). A gene encoding the WTI-1b has not been isolated.
SUMMARY OF THE INVENTION
According to one aspect of this invention, an artificially synthesized gene encoding a protein having a trypsin inhibitor activity, which is designed so as to be expressed in rice in a stable manner, includes a base sequence encoding an amino acid sequence from Glu at the 1st position to Ser at the 172th position represented in SEQ ID NO 1 of the Sequence Listing or a base sequence encoding an amino acid sequence in which one or more amino acids are subjected to deletion, substitution, or addition in the amino acid sequence. The gene is designed by the steps of: (a) determining a plurality of amino acid pairs by comparing an amino acid sequence of a trypsin inhibitor with an amino acid sequence of a chymotrypsin inhibitor derived from a plant, wherein each of the amino acid pairs consists of a first amino acid from the amino acid sequence of the trypsin inhibitor and a second amino acid from the amino acid sequence of the chymotrypsin inhibitor; (b) selecting a codon for an amino acid from each of the plurality of amino acid pairs, wherein when the first amino acid and the second amino acid in the amino acid pair are identical to each other, a codon for the second amino acid is selected; and when the first amino acid and the second amino acid in the amino acid pair are different from each other, a codon for the first amino acid is selected so as to be similar to the codon of the second amino acid; (c) obtaining a first base sequence by positioning the selected plurality of codons along the amino acid sequence of the trypsin inhibitor; (d) obtaining a second base sequence by replacing a codon for an amino acid having low codon usage in the first base sequence with a codon for an amino acid having a high codon usage; and (e) obtaining a base sequence of interest by modifying a sequence which causes instability of mRNA in the second base sequence so as not to change an amino acid sequence encoded by the second sequence.
In one embodiment of the present invention, the amino acid sequence of the trypsin inhibitor is an amino acid sequence of a trypsin inhibitor derived from winged bean.
In another embodiment of the present invention, the amino acid sequence of the trypsin inhibitor derived from winged bean is an amino acid sequence of WTI-1b protein.
In still another embodiment of the present invention, the amino acid sequence of the chymotrypsin inhibitor derived from the plant is an amino acid sequence of a chymotrypsin inhibitor derived from winged bean.
In still yet another embodiment of the present invention, the amino acid sequence of the chymotrypsin inhibitor derived from winged bean is an amino acid sequence of WCI-3.
In still yet another embodiment of the present invention, a base sequence encoding a processing region positioned at 5′ side of the gene is further included.
In still yet another embodiment of the present invention, the processing region is an amino acid sequence from Met at the 1st position to Ala at the 24th position represented in SEQ ID NO:3 of the Sequence Listing.
In still yet another embodiment of the present invention, the artificially synthesized gene consists of a base sequence represented by SEQ ID NO:4 of the Sequence Listing.
According to another aspect of this invention, in a plant, an artificially synthesized gene according to claim
1
is introduced. An expression vector comprising (i) an expression cassette containing the artificially synthesized gene which is operably liked to a plant promoter, and (ii) a selectable marker gene is introduced in the plant.
Thus, the invention described herein makes possible the advantages of (1) providing a method for designing a useful gene which is expressed in high efficiency in a useful plant; and (2) providing means for conferring a new character on a useful plant. More specifically, the objective of the present invention is the expression of a trypsin inhibitor in a useful plant such as rice by the genetic engineering. To the extent that the inventor is aware of, prior to the filing of Japanese Patent application NO:9-236332, on which the present application claims priority, there were no reports on examples where an artificially synthesized gene encoding a protein from a Fabaceae plant was highly expressed in higher plant, conferring a new character thereto.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.
REFERENCES:
Johnston et al, Insect Biochem. Molec. Biol. vol. 25, pp. 375-383, 1995.*
Christeller et al, Arch. Insect Biochem. Physiol., vol. 25, pp. 159-173, 1994.*
Rashid et al, Plant Cell Rep., vol. 15, pp. 727-730, 1996.*
Johnson et al, Proc. Natl. Acad. Sci., vol. 86, pp. 9871-9875, 1989.*
Murray et al, Nucl. Acids Res., vol. 17, pp. 477-490, 1989.*
Adang et al, Plant Mol. Biol., vol. 21, pp. 1131-1145, 1993.*
Yamamoto et al, J. Biochem., vol. 94, pp. 849-863, 1983.*
Habu et al, J. Biochem., vol. 111, pp. 249-258, 1992.
Benzion Gary
Director General of Tohoku National Agricultural Experiment Stat
Mehta Ashwin
Townsend and Townsend / and Crew LLP
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