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
1999-01-20
2001-07-24
Nelson, Amy J. (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
C435S320100, C435S455000, C435S468000, C536S023200
Reexamination Certificate
active
06265639
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences which code for sesquiterpene synthases (cyclases) from Grand Fir (
Abies grandis
), and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant sesquiterpene synthases and their mutants.
BACKGROUND OF THE INVENTION
Conifer oleoresin is a mixture of turpentine and rosin that functions in insect defense and in wound sealing (Johnson, M. & Croteau, R. (1987) in
Ecology and Metabolism of Plant Lipids
(Fuller, G. & Nes, W. D., eds) pp 76-91, ACS Symposium Series 325, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1993) in
Bioactive Volatile Compounds from Plants
(Teranishi, R., et al., eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.). Turpentine is composed of monoterpene (C
10
) and sesquiterpene (C
15
) olefins, while rosin is composed of diterpene (C
20
) resin acids (FIG.
1
). The volatile turpentine fraction of conifer oleoresin, which may consist of up to 30 different monoterpenes (Lewinsohn, E., et al., (1993)
Phytochem. Anal.
4, 220-225) and an even larger number of sesquiterpenes (See Example 1, herein) furnishes a solvent for the diterpene resin acids which, upon stem wounding, harden to form a mechanical barrier thereby sealing the wound site (Johnson, M. A. & Croteau, R. (1987) in
Ecology and Metabolism of Plant Lipids
eds. Fuller, G. & Nes, W. D. (Am. Chem. Soc., Washington, D.C.), ACS Symp. Series 325, pp. 67-91).
Grand fir (
Abies grandis
) has been developed as a model system for the study of both constitutive and wound-induced oleoresin formation (oleoresinosis). The composition of the monoterpene olefin and the diterpene resin acid fractions of grand fir oleoresin has been defined (Lewinsohn, E., et al., (1993)
Phylochem. Anal.
4, 220-225), and the induced biosynthesis of these natural products upon stem wounding has been described in detail (Gijzen, M., et al., (1993) in
Bioactive Volatile Compounds from Plants
(Teranishi, R., et al.. eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.; Lewinsohn, E., et al., (1992) in
Regulation of Isopentenoid Metabolism
(Nes, W. D., et al., eds) pp 8-17, ACS Symposium Series 497, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1992)
Arch. Biochem. Biophys.
294, 670-674; Funk, C., et al., (1994)
Plant Physiol.
106, 999-1005). The time-course of induction, after wounding, of the monoterpene synthases involved in turpentine formation has been analyzed by immunoblotting techniques and the process of induced oleoresinosis was thus shown to involve de nova synthesis of these enzymes (Gijzen, M., et al., (1992)
Arch. Biochem. Biophys.
294, 670-674). The cDNA sequence of abietadiene synthase, a diterpene cyclase from grand fir that is involved in resin acid biosynthesis (LaFever, R. E., et al., (1994)
Arch. Biochem. Biophys.
313, 139-149)) has been reported (Stofer Vogel, B., et al., (1996)
J. Biol. Chem.
271, 23262-23268), and several cDNA clones encoding monoterpene synthases from this conifer species have recently become available (Bohlmann, J., et al., (1997)
J. Biol. Chem.
272, 21784-21792).
In comparison with the monoterpenes and diterpenes of conifer oleoresin, the sesquiterpenes of conifer turpentine have received relatively little experimental attention because they constitute less than 10% of the oleoresin. The relatively low concentrations of sesquiterpenes in conifer oleoresin may, however, belie their biological significance. Sesquiterpenoid phytoalexins, i.e., antibiotic compounds, are well known in angiosperm species (Threlfall, D. R. & Whitehead, I. M. (1991) in
Ecological Chemistry and Biochemistry of Plant Terpenoids
(Harborne, J B. & Tomas-Barberan, F. A., eds) pp 159-208, Clarendon Press, Oxford, UK), suggesting that the sesquiterpenes of conifer oleoresin may play a similar role in antibiosis.
A conifer oleoresin sesquiterpene that has been relatively well-characterized is juvabione. Juvabione is the methylester of todomatuic acid, an oxygenated derivative of the sesquiterpene olefin bisabolene (FIG.
2
). The conifer sesquiterpene juvabione resembles insect juvenile hormones and, thus, can disrupt insect development and reproduction at metamorphosis and diapause (Bowers, W. S., et al., (1976)
Science
193, 542-547; Bowers, W. S. (1991) in
Herbivores: Their Interaction with Secondary Plant Metabeolites
, Vol. I, G. A. Rosenthal and M. R. Berenbaum, eds. (Acad. Press, San Diego), pp. 431-456). Juvabione is sometimes referred to as “paper factor” because its presence in paper made from trees of the genus Abies inhibits maturation of insect larvae reared in contact with the paper (Slama, K. & Williams, C. M. (1965)
Proc. Natl. Acad. Sci. USA
54, 411-414; Slama, K. & Williams, C. M. (1966)
Nature
210, 329-330; Bowers, W. S., et al., (1966)
Science
154, 1020-1021). Accumulation of todomatuic acid, the precursor of juvabione, in grand fir after insect feeding suggests that biosynthesis of the juvenile hormone analogue is induced de novo in response to insect attack (Puritch, G. S. & Nijholt, W. W. (1974)
Can. J. Bot.
52, 585-587).
Only a single sesquiterpene synthase, E-&bgr;-farnesene synthase, from a gymnosperm source, maritime pine (
Pinits pinaister
), has been reported (Salin, F., et al., (1995)
J. Plant Physiol.
146, 203-209). In contrast, several sesquiterpene synthases from angiosperms have been described (Dehal, S. S. & Croteau, R. (1988)
Arch. Biochem. Biophys.
261, 346-356; Munck, S. L. & Croteau, R. (1990)
Arch. Biochem. Biophys.
282, 58-64; Belingheri, L., et al., (1992)
Plant Sci.
84, 129-136), and a number of genes encoding sesquiterpene synthases involved in phytoalexin biosynthesis in angiosperms have been isolated (Facchini, P. J. & Chappell, J. (1992)
Proc. Natl. Acad. Sci. USA
89, 11088-11092; Back, K. & Chappell, J. (1995)
J. Biol. Chem.
270, 7375-7381; Chen, X.-Y., et al., (1995)
Arch. Biochem. Biophys.
324, 255-266).
SUMMARY OF THE INVENTION
In accordance with the foregoing, the present invention relates to isolated nucleic acids that encode gymnosperm sesquiterpene synthases, to isolated, recombinant gymnosperm sesquiterpene synthases, to replicable, recombinant expression vectors that include a nucleic acid sequence that encodes a gymnosperm sesquiterpene synthase, to cells that have been transformed, transfected or otherwise manipulated to include one or more nucleic acids of the present invention, and to methods for imparting or enhancing the production of a gymnosperm sesquiterpene synthase in a host cell including the step of introducing into the host cell an expression vector of the present invention under conditions enabling expression of the sesquiterpene synthase protein in the host cell.
Representative cDNAs encoding the gymnosperm sesquiterpene synthases E-&agr;-bisabolene synthase, &dgr;-selinene synthase and &ggr;-humulene synthase have been isolated from Grand Fir (
Abies grandis
) and sequenced, and the corresponding amino acid sequences have been deduced. Accordingly, in preferred embodiments, the present invention relates to isolated DNA sequences which code for the expression of E-&agr;-bisabolene synthase, such as the sequence designated SEQ ID No:12 which encodes E-&agr;-bisabolene synthase (SEQ ID No:13) from Grand Fir (
Abies grandis
), for the expression of &dgr;-selinene synthase, such as the sequence designated SEQ ID No:19, which encodes &dgr;-selinene synthase (SEQ ID No:20) from Grand Fir (
Abies grandis
), and for the expression of &ggr;-synthase, such as the sequence designated SEQ ID No:23, which encodes the &ggr;-synthase (SEQ ID No:24) from Grand Fir (Abies grandis).
In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a E-&agr;-bisabolene synthase, &dgr;-selinene synthase or &ggr;-synthase. The present invention is also directed to a base sequence sufficiently complementary to at least
Bohlmann Jörg
Crock John E.
Croteau Rodney Bruce
Jetter Reinhard
Steele Christopher L.
Christensen O'Connor Johnson & Kindness PLLC
Nelson Amy J.
Washington State University Foundation
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