JP2002541764A - Materials and methods for modifying isoprenoid content, composition and metabolism - Google Patents

Abstract

  (57) [Summary] Newly isolated polynucleotides and polypeptides related to the plant isoprenoid biosynthetic pathway are provided, along with constructs containing such sequences. Also provided is a method for altering the content, composition, and metabolism of a polypeptide involved in an isoprenoid biosynthetic pathway in a target organism, wherein the method incorporates one or more polynucleotides of the invention into the genome of the target organism. Such alterations in the content, composition and metabolism of the polypeptide result in alterations in the content, composition and metabolism of the isoprenoid of the target organism.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD OF THE INVENTION The present invention relates to materials and methods for altering isoprenoid content, composition and metabolism in plants and other organisms. More particularly, the invention relates to polypeptides involved in the synthesis of isoprenoid compounds, such as terpenoids and steroid compounds, polynucleotides encoding such polypeptides, expression of such polypeptides and altering the composition and / or expression levels of such polypeptides And thereby altering isoprenoid content, composition and metabolism.

BACKGROUND OF THE INVENTION [0002] Isoprenoids form a large class of naturally occurring compounds, of which 20,000
These different compounds have been described. Isoprenoids include vitamins A, D, E and K, which were initially recognized as fatty substances essential for normal animal growth, and a number of biological pigments. In plants, isoprenoid compounds, including terpenoids and steroids, contain hormones such as gibberellic acid and abscisic acid, pigments, electron carriers, membrane components (phytosterols), plant toxins, antibiotics, flavors such as menthol, and vitamins. It includes high molecular compounds such as rubber and gutta percha.

An isoprenoid compound or a prenylated lipid is mevalonate-5-diphosphate
One or more basic isoprenoid skeletons (C₅From)
Be composed. The aforementioned isopentenyl diphosphate ("active isoprene" or "IPP
)) And its isomer, dimethylallyl diphosphate, to form geranyl diphosphate (C ₁₀ ) Are produced by condensation in the “head-to-tail” direction. Further C₅Units are combined
By doing so, farnesyl diphosphate (C_Fifteen) Is generated. Head-to-tail direction
Or further elongation by condensation in the "tail-to-tail" direction,
With C₂₀, C₃₀And C₄₀Compound. Basics of isoprene compounds
A schematic diagram of a typical biosynthetic pathway is shown in FIG.

[0004] IPPs are the branch points of a wide variety of biologically significant molecules, including isoprenoids, carotenoids, and various sterols of different eukaryotes (fungal sterins, phytosterols and zosterols). In animals, cholesterol is a precursor of some hormones and bile acids. Fungal ergosterol and mammalian cholesterol are synthesized from IPP via squalene oxide and lanosterol, and higher plant sterols such as campesterol and sitosterol are synthesized by cyclization of squalene oxide to cycloartenol and more by plant-specific enzymes. Is done.

[0005] Plant cells comprise an interesting and diverse subclass of isoprenoids, often called terpenoids, which have one or more rings. Plant terpenes fall into several classes, including sesquiterpenes and mono-, di-, triterpenes, etc. (Bohlmann et al., Proc. Natl. Acad. Sci. USA 95:
4126-4133, 1998). Terpenoids are synthesized by combining isoprene units synthesized from acetic acid (C 5 _{H 8).} Terpenoids, isoprene (C 5 _{H 8)} compounds containing isopentenyl diphosphate and active isoprene, menthol, camphor, and monoterpenes (C _{10 H 16)} compound containing geraniol derived the pinene and citronellal, zingiberene, ubiquinone , Plastoquinone,
Sesquiterpene (C ₁₅ H ₂₄ ) compounds, including farnesol, derived from abscisic acid and risitine, and diterpenes (C ₂₀ H ₃₂ ), such as geranylgeraniol, derived from phytol, kaurene, gibberellic acid, and fusicoccin Compound and steroid (sterio)
ds) and triterpenes including squalene from which saponins are derived (C ₃₀ H ₄₈ )
Compounds include tetraterpenes (C ₄₀ H ₆₄ ) containing phytoene and carotene and polyterpenes (C ₅ H ₈ ) _n containing rubber and gutta percha.

[0006] Synthetic enzymes that make terpenes have a common evolutionary origin and are thought to lack strong similarity to other enzymes (except prenyltransferase). Most synthases are capable of synthesizing a variety of end products from a single substrate. This can partially explain the great diversity of terpenoid compounds found in plants (Mitc
Hell-Olds et al., Trends in Plant Science 3
(9): 362-365, 1998). Complex terpenoid mixtures have a diversity and synergistic effect that slow herbivore and pathogen acquisition of resistance,
It is believed to be an important plant defense compound (Langenheim J, J
. Chem. Ecol. 20: 1223-1280, 1994).

[0007] Plant terpenoids have many known medicinal properties, and some plant isoprenoid compounds are administered as drugs. For example, taxol, which has proven to be effective in treating cancer, is derived from terpenoid compounds. It has been suggested that isoprenoids in foods suppress the mevalonate pathway and act on cancer and cardiovascular diseases (Els
on CE, J.M. Nutr. 125 (6 Suppl): 1666S-1672
S, 1995). Farnesol, the last precursor common to all branches of the mevalonate pathway, has been shown to inhibit calcium channels in muscle cells (Roullette J-B, J. Biol. Chem. 51: 32240).
-32246, 1997).

[0008] The isoprenoid derivatives ubiquinone and plastoquinone function as electron carriers in ATP production in mitochondria and chloroplast. In most mammalian tissues, ubiquinone (also called coenzyme Q) has 10 isoprene units. Plastquinone is the plant equivalent of ubiquinone. In their role as electron carriers, ubiquinone and plastoquinone accept one or two electrons and one or two protons to be reduced.

A surprising role for the isoprenyl intermediate has been discovered in studies of proteins involved in human cancer and known to bind to membranes via covalently bound isoprenyl lipids. This protein, called the RAS protein, is the product of a mutant version of a normal protein and a number of related GTP-binding proteins. The normal protein and many of its related GTP-binding proteins are known to those skilled in the art of signal transduction to be activated by neurotransmitters, hormones, growth factors and other extracellular signals. .

[0010] Quantitative and qualitative alteration of plant terpenoid content is known to be induced by external factors such as herbivorous attack and wounding (Bohlmann J.
Proc. Natl. Acad. Sci. USA 95: 6756-676.
1, 1998). Cellular terpenoid synthesis is also induced by pathogen infection. Even agricultural pests are repelled by pine oil terpene compounds. Monoterpenes,
Karen, limonene and saimen stop onion flies (Ntiamoah
Ya, Entom. Exp. et Appl. 79: 219-226,199
6).

[0011] The chemical diversity of isoprenoids is well known and many metabolic pathways have been tentatively identified, but few genes encoding enzymes responsible for the synthesis of isoprenoid compounds have been identified. Thus, the present invention provides novel polynucleotides encoding polypeptides involved in isoprenoid biosynthesis and methods for altering the expression and composition of such polypeptides, thereby altering isoprenoid content, composition and metabolism The purpose is to provide.

[0012] The sequencing of genomes or parts of genomes of numerous biological materials, including humans, animals, microorganisms and various plant varieties, has been performed on a large scale and is still ongoing. Polynucleotides identified using sequencing techniques may be partial or full-length genes,
It may include an open reading frame or a portion of an open reading frame that encodes a polypeptide. The putative polypeptide is determined from the polynucleotide sequence. Sequence data for polynucleotides is valuable and valuable information.

[0013] Polynucleotides can be analyzed for novelty by comparing the identified sequences to sequences published in various known databases, such as EMBL. Newly identified polynucleotides and putative polypeptides are compared to polynucleotides and polypeptides contained in databases to ascertain homology with known polynucleotides and polypeptides. In this way, the degree of similarity, identity or homology between polynucleotides and polypeptides having unknown functions is determined for polynucleotides and polypeptides having known functions.

[0014] US Patent No. 5,589,619 increases the amount of squalene and sterol accumulation in higher plants by altering the copy number of a gene encoding a polypeptide having HMG-CoA reductase activity. And materials and methods for doing so. Genetic material, including polynucleotides, polypeptides, DNA molecules, etc., that are involved in HMG-CoA reductase activity is disclosed, as well as methods for transforming plant cells to produce transgenic plants.

US Pat. No. 5,689,047 discloses a method for utilizing a stilbene synthase gene from grape in a vector and a transformed microorganism,
Disclosed are transformed plant cells and plants.

US Pat. No. 5,753,507 discloses a method for utilizing a plant polynucleotide encoding geraniol / nerol 10-hydroxylase (G ₁₀ H) as a probe with a full or partial polynucleotide. , G ₁₀ H, and methods for increasing the levels of terpenoid indole alkaloids and ibidoid insect pheromones produced by plants.

US Pat. No. 5,429,939, US Pat. No. 5,444,166, US Pat. No. 5,460,949, US Pat. No. 5,470,832
No. 5,474,925, U.S. Pat.
No. 70, US Pat. No. 5,521,078, US Pat. No. 5,545
No. 5,816,816, U.S. Pat. No. 5,547,856, U.S. Pat.
No. 69,832, U.S. Pat. No. 5,580,963, U.S. Pat.
No. 5,597,718; US Pat. No. 5,670,349; US Pat. No. 5,674,485; US Pat. No. 5,684,238; US Pat. No. 5,689. No. 5,056, U.S. Pat. No. 5,691,147;
U.S. Pat. Nos. 5,693,476 and 5,443,978 disclose isoprenoid compounds or related compounds or methods utilizing such compounds. The above-cited U.S. patent specifications are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION Briefly, the present invention provides an isolated polynucleotide encoding a polypeptide involved in isoprenoid production and modification. A gene construct comprising such a sequence or a method of using the gene construct is provided, together with transgenic cells and plants that incorporate the gene construct and exhibit modified isoprenoid content, composition and metabolism.

In a first aspect, the invention relates to isolated polynucleotide sequences identified as SEQ ID NOs: 1-53 and 78-164 in the accompanying sequence listing, variants of these sequences, Extended sequences including the sequences shown in ID Nos. 1 to 53 and 78 to 164, their variants, and the sequences shown in SEQ ID Nos. 1 to 53 and 78 to 164; Corresponding probes and primers, their variants, and SEQ ID Nos. 1 to 53 and 78
A polynucleotide comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOs: 164 to 164 and presented as SEQ ID NOS: 1 to 53 and 78 to 164 The present invention provides extended sequences that include portions of the sequences described herein, all of which are collectively referred to herein as "polynucleotides of the invention."

The present invention provides isolated polypeptide sequences identified as SEQ ID NOs: 165-304 in the accompanying sequence listings, polypeptide variants of these sequences, said isolated polypeptide sequences and A polypeptide comprising at least a specified number of consecutive residues of any of the polypeptides identified as SEQ ID NOs: 165-304; Also provided is a polypeptide comprising a portion of the sequence presented as number 304.

[0021] The polynucleotide sequences identified as SEQ ID NOs: 1-53 and 78-164 are derived from plant sources, especially Eu.
from Calyptus grandis and Pinus radiata. The polynucleotides of the present invention are primarily "partial" sequences in that they do not refer to a full-length gene encoding a full-length polypeptide. Such subsequences can be extended by analyzing and sequencing various DNA libraries using primers and / or probes and well-known hybridization and / or PCR techniques. Thus, the sequence identification numbers 1 to 53 and 78 to 1
The subsequence identified as No. 64 is an open reading frame encoding a polypeptide.
It can be extended until a reading frame, a gene capable of expressing a full-length polynucleotide and / or polypeptide or another useful portion of the genome is identified.
Such extended sequences, including full-length polynucleotides and genes, may be those wherein the extended sequence is one identified sequence of SEQ ID NOs: 1-53 and 78-164 or a variant thereof or an identified contiguous sequence. A sequence identified as one of SEQ ID NOs: 1-53 and 78-164, or a variant or sequence ID NOs.
A portion of the sequence identified as No. 3 and one of Nos. 78-164, or a variant thereof, is described as "corresponding to". Similarly, the RNA sequence, reverse sequence, complementary sequence, antisense sequence and the like corresponding to the polynucleotide of the present invention are determined using the cDNA sequences identified as SEQ ID Nos. 1 to 53 and 78 to 164. In a cut procedure (routinely)
You can get it for sure.

The polynucleotides identified as SEQ ID NOs: 1-53 and 78-164 include an open reading frame (ORF) or a partial open reading frame encoding a polypeptide. further,
The open reading frame encoding the polypeptide may be identifiable among the extended or full-length sequences corresponding to the sequences presented as positions 1-53 and 78-164. Open reading frames can be identified using techniques well known to those skilled in the art. These techniques include, for example, analysis of the location of known start and end codons, identification of the most likely reading frame based on codon frequency, and the like. Suitable tools and software for ORF analysis are available, for example, on the Internet at http: // www. ncbi. nlm. n
ih. gov / gorf / gorf. available at html. Once the partial open reading frame has been identified, the polynucleotide is ligated to the region of the partial open reading frame using techniques well known to those of skill in the art until the complete open reading frame polynucleotide can be identified. Can be extended. Thus, open reading frames encoding polynucleotides and polypeptides can be identified using the polynucleotides of the present invention.

Once an open reading frame has been identified in a polynucleotide of the present invention, the open reading frame can be isolated and / or synthesized. An expressible DNA construct comprising the open reading frame and an appropriate promoter, initiator, terminator and the like well known to those skilled in the art can be constructed. Such a DNA construct is introduced into a host cell to express the polypeptide encoded in the open reading frame.
Suitable host cells include various prokaryotic and eukaryotic cells, including plant cells.

[0024] The polypeptides encoded by the polynucleotides of the present invention can be expressed and used in various assays to determine their biological activity. Such polypeptides can be used to generate antibodies, to isolate proteins or other compounds with which they interact, and to quantitatively determine the level of interacting proteins or other compounds.

The present invention provides a method for altering the content and composition of polynucleotides and / or polypeptides of an organism, according to one embodiment, comprising the steps of providing a gene construct comprising one or more polynucleotides of the present invention. Methods involving stable integration into the genome of an organism are also contemplated. In one embodiment, the target organism is a plant, preferably a woody plant, more preferably a pine or Eucalyptus woody plant, most preferably Eucalyptus grandis or Pinus radi.
ata. In a related aspect, a method of producing an organism having an altered genotype or phenotype, comprising transforming a host cell with a gene construct of the present invention to prepare a transgenic cell, comprising growing and regenerating. Methods are provided that include culturing the transgenic cells under conditions that contribute. As a result of altering the level or content of the polynucleotide or polypeptide of the present invention, an organism having an altered genotype or phenotype, together with its constituents (such as seeds) and the progeny of such organism, It is intended and included in the present invention.

The isolated polynucleotides of the present invention are useful for genomic mapping, physical mapping, and positional cloning of genes. Furthermore, sequence identification numbers 1 to 5
The polynucleotide sequences identified as # 3 and # 78-164 can be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have a sequence that is substantially complementary to the polynucleotide of interest over a portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may have any DNA and RNA sequences with sufficiently similar DNA and RNA sequences in cells using techniques well known to those of skill in the art, such as slot blot DNA hybridization techniques. The presence of a gene in an organism can be detected and used to examine the expression pattern. Oligonucleotide primers designed using the polynucleotide of the present invention can be used for PCR amplification. Oligonucleotide probes and primers designed using the polynucleotides of the present invention are available from Synteni (Palo, CA).
It can also be used in connection with various microarray technologies, including the microarray technology used by Alto).

The polynucleotides of the present invention are also used to tag or identify an organism or its propagation material. Such tagging can be achieved, for example, by stably introducing into a living organism a non-destructive, non-functional, heterologous polynucleotide identifier which comprises one polynucleotide of the present invention.

[0028] The polynucleotides of the present invention encode polypeptides that are active in the isoprenoid biosynthetic pathway. Polynucleotides related to isoprenoid metabolism have been isolated from pine and eucalyptus and putatively (puta) by DNA and protein similarity searches.
tvery) identified. The characteristics of various isoprenoid compounds are well understood and have useful properties. The methods of the invention relating to altering the polynucleotide and / or polypeptide content and composition of an organism and altering the isoprenoid content, composition and metabolism of an organism are applicable to a wide range of activities. The novel materials and methods of the present invention provide fermentation involving isoprenoids in forestry and agriculture for the manipulation of isoprenoid metabolism, in medicine for therapeutic effects including direct application to diseased organisms or indirect application by transgenic organisms. And in the chemical processing industry there are many other applications, some of which have many potential applications, some of which are described in the above cited references. In plant applications, manipulation of the isoprenoid pathway or isoprenoid composition involves:
For example, it affects plant development, insect resistance and the value of extracts (such as pinene, myrcene). In foods, various isoprenoids affect the nutritional quality and pharmacological properties of the ingested material, such as the cholesterol or phytosterol composition of foods derived from human or animal edible animals and plants. In addition, the isoprenoid pathway may be derived from plant pigments such as vitamin A, E and K, phytol chains of carotene and chlorophyll, natural oils, lemon oil, many essential oils such as eucalyptus and musk fragrance essence, insects controlling metamorphosis. It regulates the production of juvenile hormone, dolichol, which is used as a fat-soluble carrier for the synthesis of complex polysaccharides, and ubiquinone and plastoquinone, which are electron carriers for mitochondria and chloroplasts. The diverse roles of isoprenoids make these compounds and the polynucleotides that encode them attractive targets for biotechnological applications in a variety of fields.

[0029] Briefly, the invention provides an isolated polynucleotide encoding a polypeptide involved in isoprenoid synthesis. The polynucleotides and polypeptides of the present invention have been shown to be similar to polypeptides known to be involved in isoprenoid synthesis, as shown in Table 1 below.

[0030]

[Table 1] (Continuation of Table 1) (Continuation of Table 1) (Continuation of Table 1) (Continuation of Table 1)

In one embodiment, the isolated polynucleotide comprises (a) SEQ ID NO: 1
(B) the sequence listed in SEQ ID Nos. 1 to 53 and 78 to 164, and the complement of the sequence listed in SEQ ID Nos. 1 to 53 and 78 to 164 (com
(c) sequence identification numbers 1 to 53 and 78 to 16
A reverse complement of the sequence listed in No. 4;
(D) reverse sequences of the sequences listed in SEQ ID Nos. 1 to 53 and 78 to 164 and (e) (a) to (d)
) Or a particular region of the sequences of (a) to (d) and a group consisting of sequences having any of the following 40%, 60%, 75% or 90% identity as defined herein: Containing the sequence.

In a further aspect, there is provided an isolated polypeptide encoded by a polynucleotide of the invention. In one embodiment, such a polypeptide is an amino acid sequence encoded by a polynucleotide of the invention, including a polynucleotide comprising a sequence presented in the group consisting of SEQ ID NOs: 1-53 and 78-164. Together with a polypeptide comprising the amino acid sequence listed under SEQ ID Nos. 165-304.

[0033] In another aspect, the invention relates to a transgenic composition comprising a polynucleotide construct of the invention, alone or together with one or more additional polynucleotides, or together with one or more known polynucleotides, comprising such a construct. Provide with cells.

In a related aspect, the invention relates to a 5 ′ to 3 ′ orientation of a gene promoter sequence and an open gene encoding at least a functional portion of an enzyme encoded by a polynucleotide of the invention or a variant thereof. A gene construct comprising a reading frame and a gene termination sequence is provided. The open
The reading frames may be arranged in a sense direction or an antisense direction. There is also provided a gene construct comprising a non-coding region or a nucleotide sequence complementary to the non-coding region of a gene encoding an enzyme encoded by the polynucleotide, together with a gene promoter sequence and a gene terminator sequence. A polynucleotide sequence in the direction 5 to 3 ｀, and (1)
A polynucleotide comprising the polynucleotide of the present invention or (2) a polynucleotide comprising at least one of the polynucleotides comprising a non-coding region of a gene encoding a polypeptide comprising an isoprenoid biosynthesis pathway comprising a polynucleotide of the invention A gene construct comprising the sequence is also contemplated. The gene construct may further include a marker for identifying a transformed cell.

In a further aspect, there is provided a transgenic host cell, such as a transgenic plant cell comprising a genetic construct of the present invention, together with a plant comprising such a transgenic cell and the fruits, seeds and progeny of such a plant. Other useful host cells include bacterial cells, insect cells, yeast cells and mammalian cells.

[0036] In yet another aspect, there is provided a method of modifying the isoprenoid content, composition and metabolism of an organism, comprising stably incorporating the gene construct of the present invention into the genome of said organism. In a preferred embodiment, the target organism is a plant, wherein the plant is preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany species, preferably from pine and eucalyptus species. More preferably, it is selected from the group
Most preferably, it is selected from the group consisting of yptus grandis and Pinus radiata. In a related aspect, a method of producing an organism having an altered isoprenoid content, comprising transforming a host cell with a gene construct of the present invention to provide a transgenic cell and growing and regenerating the transgenic cell Provided is a method comprising culturing under conditions conducive to:

In a still further aspect, the present invention provides a method for modifying a polypeptide activity in a target organism, such as a plant, comprising stably incorporating the gene construct of the present invention into the genome of the organism. provide. In a preferred embodiment, the target organism is a plant, and the plant is preferably a woody plant selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany species; Woody plants selected from the group consisting of eucalyptus seeds are more preferred, and Eucalyptus grandis and Pinus radi
Most preferably, it is a woody plant selected from the group consisting of ata.

In yet a further aspect, the invention provides a method of altering one or more of the content, composition, and metabolism of an isoprenoid compound in an organism by administering to the organism an isolated polypeptide of the invention. In applications where the organism is a plant, administration of the polypeptide may be by local means, such as by topical application such as spraying. In applications where the organism is a mammal, administration of the polypeptide can be injection, delivery intradermally,
Systemic means such as oral delivery, nasal or respiratory tract delivery and the like are also possible.

With reference to the following detailed description, the above and further features of the invention and the manner in which it is obtained will become apparent, and the invention is best understood.

DETAILED DESCRIPTION As noted above, isoprenoids are important components in a variety of eukaryotic functions. Early parts of the isoprenoid metabolic pathway, especially isopentenyl diphosphate (
IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and alteration of isoprenoid content, composition and metabolism leading to the production of squalene
It has a profound effect on the synthesis of isoprenoid compounds derived from these two precursors. Inhibiting one or more of the downstream steps diverging from isopentenyl diphosphate and squalene also has a significant effect on the pool of isopentenyl diphosphate and squalene available for terpene or steroid synthesis. Therefore, the isoprenoid biosynthesis pathway, especially the early part of the pathway (IPP => GPP =>
Modification of the synthesis, content, composition and metabolism of any single enzyme (FPP => squalene) affects the content, composition and metabolism of terpene and steroid compounds.

Using the methods and materials of the present invention, the isoprenoid content of a plant can be modified by incorporating a sense or antisense copy of a polynucleotide encoding a polypeptide involved in isoprenoid synthesis into the genome of the target organism. Further, the copy number and combination of polynucleotides encoding different enzymes of the isoprenoid biosynthetic pathway alter the relative amount of isoprenoid synthesized, thereby having an altered composition of isoprenoid and / or an altered metabolic system Manipulated to produce biological material. Similarly, whether for direct application in a target organism or for production of a polypeptide for another use, modification of the isoprenoid composition can
As is apparent from the references cited above and incorporated herein by reference, it is advantageous for various applications.

According to one embodiment, the present invention provides an isolated polynucleotide that encodes or partially encodes a polypeptide similar to a polypeptide known to be involved in isoprenoid synthesis and modification. The polynucleotides of the present invention have been isolated from Eucalyptus and pine species, but may alternatively be isolated from other plant material sources and synthesized using conventional synthetic techniques. Specifically, the isolated polynucleotides of the present invention have SEQ ID NOs: 1-53 and 78-16.
A polynucleotide identified as No. 4; a complement of the sequence identified as SEQ ID Nos. 1-53 and 78-164; and a polynucleotide identified as SEQ ID Nos. 1-53 and 78-164. And the reverse complement of the sequences identified as SEQ ID NOs: 1-53 and 78-164, and at least a specified number of contiguous residues of any of the above polynucleotides (X-mer), a polynucleotide complementary to any of the above polynucleotides, an antisense sequence corresponding to any of the above polynucleotides, and a mutant of any of the above polynucleotides.

As shown in Table 1, the isolated polynucleotides listed as SEQ ID NOs: 1-53 and 78-164 have sequence similarities to polypeptides known to be involved in the isoprenoid biosynthetic pathway. Encode or partially encode a proven polypeptide. More specifically, the isolated polynucleotide listed in the first column of Table 1 was identified in the same row of Table 1 (in alignment).
Encode or partially encode the polypeptide listed in the second column. The predicted amino acid sequences corresponding to the polynucleotides listed as SEQ ID Nos. 1 to 53 and 78 to 164 based on the information available at the time of this application are as shown in Table 1 and have SEQ ID Nos. 165 to 304. Provided to the turn.

As used herein, the term “polynucleotide” refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, and
And HnRNA and mRNA in both corresponding sense and antisense RNA molecules.
RNA molecules containing NA molecules, including cDNA, genomic DNA, and recombinant DNA, as well as fully or partially synthesized polynucleotides. HnRNA contains introns and generally corresponds one-to-one with DNA molecules. mRNA corresponds to HnRNA and DNA molecules with introns removed. A polynucleotide comprises the entire gene or a portion thereof. An operable antisense polynucleotide includes fragments of the corresponding polynucleotide, and the definition of "polynucleotide" includes all such operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known to those of skill in the art, for example, Robinso
n-Benion et al., Methods in Enzymol. 254 (23
): 363-375, 1995 and Kawasaki et al., Artific. Or
gans 20 (8): 833-848, 1996. Polynucleotides of the invention also include polynucleotides that encode a polypeptide that differs from the disclosed sequences but is the same as the polypeptide encoded by the polynucleotides of the invention as a result of the degeneracy of the genetic code.

The definitions of the terms “complement”, “reverse complement”, and “reverse sequence” as used herein are best illustrated in the following examples. For the sequence 5'AGGACC3 ', the complement, reverse complement and reverse sequence are as follows. Complement 3'TCCTGG5 'Reverse complement 3'GGTCCT5' Reverse sequence 5'CCAGGA3 '

The identification of genomic DNA and heterologous DNA can be accomplished by standard DNA / DNA hybridization techniques, under appropriate stringent conditions, using all or part of the cDNA sequence as a probe to screen an appropriate library. This is achieved by using As an alternative, PCR techniques using oligonucleotide primers designed based on known genomic DNA, cDNA and protein sequences have been
It can be used to amplify and identify NA and cDNA sequences. Synthetic DNAs corresponding to the identified sequences and mutants are produced by ordinary synthetic methods. All of the polynucleotides described herein are isolated and purified under conditions commonly used in the field of the present invention.

In another aspect, the invention provides an isolated polypeptide encoded or partially encoded by a polynucleotide as described above. As used herein, the term "polypeptide" includes amino acid chains of any length, including full length, with amino acid residues covalently linked. The term "polynucleotide-encoded polypeptide" as used herein includes polypeptides encoded by a polynucleotide provided herein, including an isolated DNA sequence or variant. In one embodiment, a polypeptide of the invention comprises an amino acid sequence selected from the group consisting of the sequences provided in SEQ ID NOs: 165-304 and variants of such sequences. According to another embodiment, a polypeptide of the invention comprises at least the specified number of contiguous residues (x-mers) of any of the sequences provided in SEQ ID NOs: 165-304.

The polypeptide of the present invention can be produced by a recombinant method by inserting a DNA sequence encoding the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art of the present invention can be used. Expression can be achieved in any suitable host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes the recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast and higher eukaryotic cells. The host cells used are preferably Escherichia coli, insects, yeasts or mammalian cell lines such as COS and CHO. DNA sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.

In a related aspect, the polypeptide is at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of the sequences provided in SEQ ID NOs: 165-304 and variants thereof. Provided. As used herein, a “functional portion” of a polypeptide is an essential active site that affects the function of the polypeptide, eg, the portion of a molecule capable of binding one or more reactants. Means the part containing The active site may be composed of discrete portions present on one or more polypeptide chains and generally exhibits high binding affinity.

The functional portion of the polypeptide may be obtained by first digesting the polypeptide chemically or enzymatically to prepare fragments of the polypeptide, or by analyzing the mutations in the polynucleotide encoding the polypeptide. And subsequent expression of the resulting mutant polypeptide. The polypeptide fragment or mutant polypeptide is tested to determine which portions retain biological activity, for example, using the representative assays provided below.

The functional part containing the active site may be composed of separate parts present on one or more polypeptide chains, and generally shows a high binding affinity. The term "polynucleotide-encoded polypeptide" includes polypeptides encoded by polynucleotides, including partially isolated polynucleotides of the present invention.

[0052] Portions and other variants of the polypeptides of the present invention can also be produced by artificial synthesis or recombinant means. Synthetic polypeptides having less than about 100, generally less than about 50, amino acids can be synthesized using techniques well known to those of ordinary skill in the art of the invention. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, in which amino acids are successively added to a growing chain of amino acids. It is possible to Merrifield, J.M. Am
. Chem. Soc. 85: 2149-2146, 1963. The automated polypeptide synthesizer is available from Perkin Elmer / Applied Bio.
systems, Inc. (Foster City, CA) and are commercially available and can be operated according to the manufacturer's instructions. Variants of the native polypeptide can be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-directed mutagenesis (Kunkel, T., Proc. Natl. Acad. Sci. .USA
82: 488-492, 1985). Fragments of the DNA sequence can also be removed using standard techniques for preparing truncated polypeptides.

Generally, the polypeptides disclosed herein are isolated and prepared in a sufficiently pure form. Preferably, the polypeptide is at least about 80% pure, more preferably at least 90% pure, and most preferably at least 99% pure. In certain preferred embodiments, described below, the isolated polypeptide is incorporated into a pharmaceutical composition or vaccine for the treatment of a dermatological disorder.

As used herein, the term “variant” includes a nucleotide or amino acid sequence that differs from a specifically identified sequence, wherein one or more nucleotides or amino acid sequences have been deleted, substituted. Or the one added. A variant may be a variant of a naturally occurring allele or a variant that does not occur in nature. The variant sequence of the polynucleotide is preferably 40% or more, more preferably 60% or more.
As described above, even more preferably 75% or more, most preferably 90% or more refers to those showing identity to the sequence of the present invention. The variant sequence of the polypeptide is preferably 50%
Above, more preferably 75% or more, even more preferably 90% or more, and most preferably 95% or more. The percentage of identity is
As described below, two sequences to be compared are aligned (aligned), and the number of identical residues in the aligned portion is determined.
Divide by the total number of residues in the sequence (of the query) and multiply the result by 100.

Using publicly available computer algorithms, alignments between polynucleotide and polypeptide sequences can also be made, and the percentage of identical nucleotides in a particular portion determined relative to another polynucleotide. You can also. Representative algorithms for alignment and polynucleotide sequence similarity identification are the BLASTN and FASTA algorithms. Polynucleotides can also be analyzed using the BLASTX algorithm, which compares the six translational conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage of polypeptide sequence identity can be determined using the BLASTP algorithm. The BLASTN, BLASTX and B
The LASTP program is an NCBI anonymous FTP server (ftp: // ncb)
i. nlm. nih. gov) on / blast / executables /. BLASTN algorithm version 2.0.4 (February 24, 1998) and version 2.0.6 (19
(September 16, 1998) is preferably used for the determination of a polynucleotide variant according to the invention. The BLASTP algorithm, set with the following parameter values, is preferably used for the determination of a polypeptide variant according to the invention. BLASTN, B
The BLAST family of algorithms, including LASTP and BLASTX,
The URL of the CBI website (http: //www.ncbi.nlm.
nih. gov / BLAST / newblast. html) and Altsch
ul et al., Nucleic Acids Res. 25: 3389-3402
, 1997 publication.

The computer algorithm FASTA is available on the ftp site ftp: // ftp. virginia. Available at edu / pub / fasta /. Version 2.0.4, February 1996, set to the default parameter values described in the instructions and distributed with the program, is preferably used for the determination of variants according to the invention. The use of the FASTA algorithm is described in Pearson WR and Lipman DJ Proc. Natl. Acad
. Sci. USA 85: 2444-2448, 1988, and Pearson.
WR Methods in Enzymol. 183: 63-98, 1990
Is described.

The following running parameters were used to determine the E value (E val) of the polynucleotide sequence.
es) and to determine alignment and similarity using BLASTN, which contribute to the percent identity. The Unix running command is "bla
stall -p blastn -d embldb -e 10 G0
−E0 −r 1 −v 30 −b 30 −i queryseq
o results, parameters are: -p program name [string]; -d database [string]; -e expected value (E) [real number]; -G cost to open gap (zero is default (Invoke action) [integer];-Cost of expanding E gap (zero invokes default action) [integer]; -r Nucleotide match reward (blastn only) [integer]; (One-l
number of line descriptions (V) [integer]; -b number of alignments to display (B) [integer]; -i query file [F
ile In]; and -o BLAST report output file [F
ile Out] arbitrary.

[0058] "blastall p blastp d swissprotdb e
10 G0E 0 v 30 b 30 i queryseq res
Preferably, the running parameter "ults;" is used to determine alignments and similarities using BLASTP, which contribute to the E value and percent identity of the polypeptide sequence. The parameter is "-p program name [character string]
-D database [string]; -e expected value (E) [real number]
-G the cost of opening the gap (zero invokes the default action) [integer]; -E the cost of expanding the gap (zero invokes the default action) [integer]; -r Reward (blastn only) [Integer]; -v One-line explanation (one-lin
e description) (V) [integer]; -b number of alignments to display (B) [integer]; -i query file [Fil
e In]; and -o BLAST report output file [Fil
e Out] Arbitrary ". A "hit" to one or more database sequences by a query sequence, generated by BLASTN, FASTA, BLASTP or a similar algorithm, is identified by alignment of similar parts of the sequence. A hit to the database sequence generally means that it only overlaps part of the sequence length of the query sequence.

The BLASTN, FASTA and BLASTP algorithms also calculate the “expected” (E) value of the alignment. The E-value indicates the number of hits that can be "expected" to be discovered over a certain number of adjacent sequences by chance when searching a certain size database. The E value is used as a threshold of significance to determine whether a hit to a database, such as the preferred EMBL database, represents true similarity. For example, an E value of 0.1 assigned to a given polynucleotide hit is the EMBL
In a database of database size, it is interpreted that 0.1 matches can be expected by chance over aligned parts of sequences with similar scores. According to this criterion, a portion where the polynucleotide sequence is aligned and matched has a probability of being 90% identical. For sequences that have an E value of less than 0.01 across aligned matches, the probability of finding a match by chance in the EMBL database using the BLASTN or FASTA algorithm is less than 1%.

According to one embodiment, for each of the polynucleotides and polypeptides of the invention, the “variant” polynucleotides and polypeptides have a greater number of nucleic acids or amino acids than each of the polynucleotides or polypeptides of the invention. Has the same or less sequence and 0.01% as compared to the polynucleotide or polypeptide of the present invention.
It is preferable to include an array having an E value of less than. That is, the mutant polynucleotide or polypeptide has a 99% or higher probability of being identical to the polynucleotide or polypeptide of the present invention, and has an E value of 0.01 using the BLASTN, FASTA or BLASTP algorithm with the above parameter settings. Refers to any of the following sequences: According to a preferred embodiment, the mutant polynucleotide is defined as having an E-value of 0.5 using the BLASTN or FASTA algorithm with the above parameter settings.
01 or less, and the same as the polynucleotide of the present invention with a probability of 99% or more,
A sequence having a number of nucleic acids equal to or less than the number of nucleic acids of the polynucleotide of the present invention. Similarly, according to a preferred embodiment, a mutant polypeptide is defined as having an E value of 0.01 or less using the BLASTP algorithm with the above parameter settings and a 99%
A sequence having the same or lower number of amino acids than the polypeptide of the present invention, which is the same as the polypeptide of the present invention at the above probability.

As an alternative, the variant polynucleotide or polypeptide of the invention may be determined as described below, preferably at least 40%, more preferably at least 60%, even more preferably at least 75%, most preferably Refers to those showing 90% or more identity as the polynucleotide or polypeptide sequence of the present invention. Percent identity was determined by setting BLASTN, FASTA or BLAST as set in the running parameters above.
Sequence alignment using one of the algorithms of P, identifying the number of identical nucleic acids or amino acids over the aligned portion, and determining the number of identical nucleic acids or amino acids in the polynucleotide or polypeptide of the invention. Determined by dividing by the total number of nucleic acids or amino acids and multiplying by 100 to calculate the percent identity. For example, a polynucleotide of the invention having 220 nucleic acids will hit over 23 nucleotides upon alignment with a polynucleotide in the EMBL database having 520 nucleic acids and the BLASTN algorithm using the above parameters. The 23 nucleotide hit contains 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the polynucleotides of the present invention to hits in the EMBL database will be 21/220 times 100, which is 9.5%. Thus, the polynucleotide in the EMBL database of this example is not a variant of the polynucleotide of the present invention.

As an alternative, the variant polynucleotide of the present invention may comprise a polynucleotide sequence listed under SEQ ID Nos. 1-53 and 78-164, or the complement, reverse sequence or reverse sequence of these sequences. Hybridizes with the complement under stringent conditions. As used herein, “stringent conditions” refers to 6XS
Pre-wash with a solution of SC and 0.2% SDS, 65 ° C., overnight
), Hybridized in 6X SSC and 0.2% SDS, then 65 ° C, 1X
Wash twice with SSC and 0.1% SDS for 30 minutes each, 65 ° C, 0.2X SS
Washing twice with C and 0.1% SDS for 30 minutes each.

The present invention also includes polynucleotides that differ from the disclosed sequences but that encode polypeptides that have the same enzymatic activity as the polypeptide encoded by the polynucleotides of the present invention as a result of the degeneracy of the genetic code. . Accordingly, as a result of conservative substitutions, polynucleotides comprising the polynucleotide sequences listed as SEQ ID NOs: 1-53 and 78-164, or the complements, reverse sequences, or sequences that differ from the reverse complements of these sequences Nucleotides are contemplated and encompassed by the present invention. Furthermore, the polynucleotide sequences listed at positions 1-53 and 78-164 as a result of deletions and / or insertions that total less than 10% of the previous sequence length,
Alternatively, polynucleotides comprising the complement of these sequences, the reverse sequence or a sequence different from the reverse complement are contemplated by and encompassed by the present invention. Similarly, a polypeptide comprising a sequence that differs from the polypeptide sequences listed in SEQ ID NOs: 165-304 as a result of amino acid substitutions, insertions and / or deletions that total less than 10% of the total sequence length, It is contemplated and encompassed by the present invention provided that the variant polypeptide has activity in the isoprenoid biosynthetic pathway.

The polynucleotides of the present invention may be isolated from various libraries or synthesized using techniques well known to those skilled in the art. The polynucleotide of the present invention may be synthesized using, for example, an automatic oligonucleotide synthesizer (eg, Beckman Oligo 1000M DNA synthesizer) to obtain a polynucleotide segment of 50 or more nucleic acids. A plurality of such polynucleotide segments can be joined using standard DNA manipulation techniques well known in the field of molecular biology. One common and representative polynucleotide synthesis technique is the synthesis of a single-stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridization of the segment with the complementary 85 nucleic acid segments to provide a 5 nucleotide overnucleotide. Is to create a hang. The next segment is synthesized in a similar manner to have a 5 nucleotide overhang on the opposite strand. This "sticky" end ensures proper bonding when the two parts hybridize. In this way, the complete polynucleotide of the present invention can be synthesized in vitro.

[0065] Some of the polynucleotides identified as Nos. 1-53 and Nos. 78-164 do not represent the complete coding portion of a gene encoding a naturally occurring polypeptide, " Partial "arrays. The partial polynucleotide sequences disclosed herein can be modified in various ways, for example, by screening a DNA expression library using a hybridization probe based on the polynucleotide of the present invention or by PCR amplification with primers based on the polynucleotide of the present invention. It can be used to obtain corresponding full-length genes of species and organisms.
In this way, using methods well known to those skilled in the art, the polynucleotide of the present invention is:
It is possible to identify the genomic DNA corresponding to the complete gene, including the promoter and enhancer regions, extending upstream and downstream of the corresponding mRNA. The present invention provides for a sequence identified in positions 1-53 and 78-164 or one variant of the specified sequence, encoding a functional polypeptide, including a full-length gene. Including isolated polynucleotides. Such extended polynucleotides may be from about 50 to about 4,000 nucleic acids or base pairs in length, but have about 4,000 or less nucleic acids or base pairs in length. Preferably, it has a length of about 3,000 nucleic acids or base pairs, and even more preferably has a length of about 2,000 nucleic acids or base pairs. Under certain circumstances, extended polynucleotides of the invention may have less than about 1,800 nucleic acids or base pairs, but preferably have less than about 1,600 nucleic acids or base pairs, and More preferably, it has less than 1,400 nucleic acids or base pairs, even more preferably less than about 1,200 nucleic acids or base pairs, and more preferably has less than about 1,000 nucleic acids or base pairs. Most preferred.

The polynucleotide of the present invention can be any of the polynucleotides identified as Nos. 1-53 and Nos. 78-164, the complement, reverse sequence or reverse complement of such sequences, or variants thereof. Or a polynucleotide comprising a polynucleotide (x-mer) comprising at least a specified number of contiguous residues. Similarly,
The polypeptide of the present invention is a polypeptide comprising at least a specified number of contiguous residues (x-mer), either of the polypeptides identified as SEQ ID NOs: 165-304 or variants thereof. And polypeptides comprising: As used herein, the term “x-mer” refers to the first value for a particular value of “x”.
At least a specified number ("x") of any of the polynucleotides identified as Nos. 53-78 and 164-78 or the polypeptides identified as SEQ ID Nos. 165-304; Refers to the containing sequence. According to a preferred embodiment, the value of x is preferably 20 or more, more preferably 40 or more,
It is more preferably 60 or more, and most preferably 80 or more. Accordingly, the polynucleotide of the present invention is a 20-mer of the polynucleotide or the polypeptide identified as SEQ ID NO: 1 to 53 and 78 to 304 and a mutant thereof,
40mer, 60mer, 80mer, 100mer, 120mer, 150mer, 180mer
Mer, 220 mer, 250 mer or 300 mer, 400 mer, 500 mer or 6
And polynucleotides or polypeptides that contain a thiomer.

Polynucleotide probes and primers complementary to and / or corresponding to Nos. 1-53 and Nos. 78-164 and variants of these sequences are also included in the invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. An "oligonucleotide" as used herein is a relatively short segment of a polynucleotide sequence, generally
And between 60 and 60 nucleotides, including both the probes used for hybridization assays and the primers used for DNA amplification by the polymerase chain reaction.

One oligonucleotide probe or primer is described as “corresponding” to a polynucleotide or variant of the present invention that includes one of the sequences set forth as SEQ ID NOs: 1-53 and 78-164. The oligonucleotide probe or primer, or its complement, may be one of the sequences presented as SEQ ID NOs: 1-53 and 78-164, or a variant of one of the specified sequences. It is when included in crabs.

It can be said that two single-stranded sequences are sufficiently complementary that, when compared with an optimal alignment, the nucleotides of one strand having the appropriate nucleotide insertions and / or deletions will be identical to those of the other strand. It is when more than 80%, preferably more than 90% to 95%, and more preferably at least 98% to 100% of the nucleotides in the strand. Alternatively, sufficient complementarity exists when the first DNA strand selectively hybridizes to the second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity are those with salt conditions less than about 1M, more usually less than about 500 mM, and preferably less than about 200 mM. The temperature for hybridization may be 5 ° C, but is generally about 2 ° C.
Above 2 ° C., more preferably above about 30 ° C., most preferably about 37 ° C.
C or more. Longer DNA fragments require higher hybridization temperatures for specific hybridization. Because the stringency of hybridization is influenced by other factors such as probe composition, the presence of organic solvents and the degree of base mismatching, the combination of parameters is an absolute measure of only one parameter. More important than. DNA from a sample or product containing a plant or plant material is either genomic DNA or DNA obtained by preparing cDNA from RNA present in the sample.

In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, mRNA from the expressed gene is detected instead of genomic DNA or cDNA from sample mRNA. In the second case, an RNA probe is used. In addition, artificial analogs of DNA that specifically hybridize to the target sequence are used.

[0071] In certain embodiments, the oligonucleotide probe and / or primer comprises:
Comprising about 6 or more contiguous residues complementary to the polynucleotide sequence of the invention;
More preferably it contains about 10 or more contiguous residues, most preferably it contains about 20 or more contiguous residues. The probes and primers of the invention may be from 8 to 100 base pairs in length, but are preferably about 10 to 50 base pairs in length, and about 15 to 40 base pairs in length. Is more preferred. The probe is a DNA-
Easily selected using procedures well known in the art, taking into account the stringency of DNA hybridization, the temperatures of annealing and melting, the potential for loop formation, and other factors well known to those skilled in the art. it can. Suitable for probe design and especially for P
Tools and software suitable for the design of CR primers are available on the Internet, for example, at http: // www. horizonpress. com / pcr /
Available at A preferred technique for the design of PCR primers is Dief
PCR primer of a Fenbach CW and Dvksler GS: a
Laboratory Manual, CSHL Press, Cold Spring Harbor, NY, 1995.

A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the invention may be provided in kit form. Such kits generally include a plurality of DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. The kit of the present invention includes one or more probes or primers corresponding to the polynucleotide of the present invention, including the polynucleotide sequences identified at Nos. 1 to 53 and Nos. 78 to 164.

In one embodiment useful for high throughput assays, the oligonucleotide probe kits of the present invention include a kit wherein each probe is spatially localized by a pre-determined address on a solid surface. Includes a plurality of probes in an array-like format, immobilized in a spatially addressable location. The array format effectively used in the present invention is described, for example, in US Pat.
No. 0530, the disclosure of which is incorporated herein by reference.

The probes are preferably arranged in an array and can be used to screen for differences in organisms or samples or products containing the genetic material using high throughput screening techniques well known to those skilled in the art. The significance of high-throughput screening includes identifying lots of seed lots and plant seedlings, testing samples or products for unwanted plant material, including plants or samples or plant materials for quarantine purposes, etc. It is obvious for applications such as plant breeding and quality control, such as identifying the product or ascertaining the true origin of the product, including the plant or sample or plant material. Screening for the presence or absence of the polynucleotide of the present invention used as an identifier for tagging a plant is valuable in detecting the flow of genes in plant breeding, the transfer of genes by scattering of pollen, and other amounts at a later date. There is.

In this manner, the oligonucleotide probe kit of the present invention can be used to detect the presence or absence of the polynucleotide of the present invention in different samples or products containing different materials (
Or, in the case of mixtures, relative amounts) can be used for quick and cost-effective testing. Examples of plant species that can be tested using the present invention include forest species, such as pine and eucalyptus species, other woody species, and agricultural and horticultural plants.

Another aspect of the present invention relates to a collection of polynucleotides of the present invention. A collection of a plurality of polynucleotides of the present invention, especially Nos. 1-53 and 7
Polynucleotides identified as Nos. 8 to 164 and variants thereof may be recorded and / or stored on a recording medium and later accessed for analysis, comparison, and the like. Suitable recording media include magnetic media such as a magnetic diskette or magnetic tape, CD-ROM recording media, optical recording media, and the like. Suitable recording media and methods of recording and storing information and accessing information such as polynucleotide sequences recorded on such media are well known to those skilled in the art. The polynucleotide information stored on the recording medium is preferably readable by a computer, and can be used for analyzing and comparing the polynucleotide information.

According to one embodiment, the recording medium is at least 4, preferably at least 10, more preferably at least 15, most preferably at least 20, polynucleotides of the invention. And preferably the polynucleotides identified as Nos. 1 to 53 and Nos. 78 to 164, or variants of these polynucleotides.

In applications where modification of a polypeptide involved in isoprenoid biosynthesis and / or isoprenoid metabolism is desired, the transformation of the gene construct into a target plant can be accomplished by transforming the gene construct into a target plant with an open reading frame in the sense or antisense orientation. Inserted to effect a change in the expression level of the polypeptide relative to expression in a wild-type organism. Transformation with a gene construct that includes an open reading frame in the sense orientation generally results in altered expression of the selected gene and transforms with a gene construct that includes the open reading frame in the antisense orientation. This results in reduced expression of the selected gene. A population of plants transformed with a gene construct comprising an open reading frame of the invention in sense or antisense orientation is screened for increased or decreased expression of the gene in question using techniques well known to those of skill in the art, and A plant having the phenotype is isolated.

As an alternative, expression of genes involved in isoprenoid biosynthesis can be inhibited by inserting portions of the open reading frames of the invention in either sense or antisense orientation. Such portions need not be full length, but preferably contain at least 25 residues, more preferably at least 50, of the polynucleotides of the present invention. Full-length polynucleotides corresponding to longer portions or the entire open reading frame can also be used. The portion of the open reading frame need not be exactly the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene. Thus, sequences from one species may be used to inhibit the expression of genes of a different species.

According to another embodiment, the gene construct of the present invention comprises a polynucleotide comprising a non-coding region of a gene encoding a polypeptide encoded by a polynucleotide of the present invention or a polynucleotide complementary to such a non-coding region. including. Examples of non-coding regions that can be usefully used in such constructs include introns and 5 'non-coding leader sequences. Transformation of target plants with such DNA constructs is described, for example, in Napoli et al., Plant Cell 2: 279-290, 1990 and de Carva.
lho Niebel et al., Plant Cell 7: 347-358, 199.
5, resulting in a reduction in the amount of isoprenoid compounds synthesized by the plant by the process of cosuppression.

Alternatively, regulation can be achieved by inserting an appropriate sequence or subsequence (eg, DNA or RNA) into the ribozyme construct (McIntyre).
CL and Manners JM, Transgenic Res. 5 (4
): 257-262, 1996). A ribozyme is a synthetic RNA molecule that contains a hybridizing region that is complementary to a region containing five or more contiguous nucleotides of an mRNA molecule, each of which is encoded by one of the polynucleotides of the invention in two regions. Ribozymes have very specific endonuclease activity and cleave the mRNA autocatalytically.

[0082] The gene constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the polynucleotide to be transcribed, to control gene expression. The gene promoter sequence is generally located at the 5 'end of the transcribed polynucleotide and is used to initiate transcription of the polynucleotide. The gene promoter sequence is generally located in the 5 'non-coding region of the gene, but may be located downstream of the open reading frame or in an intron (Luehrsen KR, Mol. Gen. Gene.
t. 225: 81-93, 1991), for example, as in plant defense genes (Douglas et al., EMBO J. 10:
1767-1775, 1991). When the construct contains an open reading frame in the sense direction, the gene promoter sequence also initiates translation of the open reading frame. In DNA constructs containing an open reading frame or non-coding region in the antisense orientation, the gene promoter sequence consists solely of a transcription start site having an RNA polymerase binding site.

Various gene promoter sequences that can be effectively used in the DNA construct of the present invention are well known in the field of the present invention. The gene promoter sequence and the gene termination sequence may be endogenous in the target plant host, or may be exogenous when functional in the target host. For example, promoters and termination sequences may be from other plant species, plant viruses, bacterial plasmids, and the like. Preferably, the gene promoter and termination sequence are common to those of the polynucleotide to be introduced.

Factors affecting promoter selection include the desired tissue specificity of the construct and the timing of transcription and translation. For example, constitutive promoters, such as the 35S cauliflower mosaic virus (CaMV35S) promoter, include enhancers such as Kozak sequences or omega enhancers and Agrobacterium tumefaciens.
iens) with or without a terminator of the nopaline synthase gene of the bacterium. Using a tissue-specific promoter will produce the desired sense or antisense RNA only in the tissue of interest. In gene constructs using an inducible gene promoter sequence, the rate of RNA polymerase binding and transcription initiation can be varied by external stimuli such as light, heat, anaerobic stress, altered nutritional conditions, and the like. Temporally regulated promoters are used to influence variations in the rate of RNA polymerase binding and transcription initiation at specific times in the development of transformed cells. It is preferred to use an original promoter from the enzyme gene in question or a gene from a gene that is expressed only in a particular tissue of the transformed organism, such as eucalyptus or pine. Other examples of gene promoters that can be effectively used in the present invention include mannopine synthase (mas), octopine synthase (ocs), and Chua et al.
Science 244: 174181, 1989.

The gene termination sequence located 3 ′ to the transcribed polynucleotide may be derived from the same gene as the gene promoter sequence, or may be derived from a different gene. Many gene termination sequences well known to those skilled in the art, such as the 3 'end of the Agrobacterium tumefaciens nopaline synthase gene, can be usefully employed in the present invention. However, preferred gene terminator sequences are those derived from the native enzyme gene or the target species to be transformed.

[0086] The gene constructs of the present invention may also include a selectable marker that is effective in target cells, such as plant cells, so that transformed cells containing the constructs of the present invention can be detected. Such markers are well known to those of skill in the art and typically have resistance to one or more toxins. One example of such a marker is the NPTII gene whose expression confers kanamycin or hygromycin resistance, which is normally toxic to plant cells at moderate concentrations (Rogers et al., Weissbach A and Wei).
ssbach H Editing, Methods for Plant Molecul
ar Biology, Academic Press Inc. San Diego, CA, 1988). Alternatively, the presence of the desired construct in the transformed cells can be determined by other techniques well known in the art, such as Southern and Western blotting. A transcription start site is added to a gene construct when the sequence to be transcribed lacks the transcription start site.

Techniques for operatively linking the components of the gene constructs of the present invention are well known to those of skill in the art and are described, for example, in Sambrook et al., (Molecular
cloning: a laboratory manual, CSHL Pr
ess, Cold Spring Harbor, NY, 1989).
And the use of synthetic linkers containing one or more restriction endonuclease sites as described in The DNA constructs of the present invention may be, for example, vectors comprising one or more replication systems, such as E. coli, which can be cloned from the resulting construct after each operation and sequenced to determine the accuracy of the operation. May be connected.

The genetic constructs of the present invention include monocotyledons (eg, grass, corn, cereals, oats, wheat, barley) and dicots (eg, Arabidopsis, tobacco, legumes, alfalfa, oak, eucalyptus, Maple) and gymnosperm (
For example, Scots pine (Aronen, Finnish Forest Re
s. Papers, vol. 595, 1996), white spruce (Elli)
s et al., Biotechnology 11: 84-89, 1993), and larch (Huang et al., In vitro Cell 27: 201-207, 1).
991) may be used to transform various plants. In a preferred embodiment, the DNA construct is used to transform a "woody plant", defined herein as a tree or shrub whose stems survive for multiple years and increase in diameter each year with the addition of wood tissue. Can be The target plant is preferably selected from the group comprising eucalyptus and pine species, most preferably Eucalyptus grandi
s and Pinus radiata. The DN of the present invention
Other species usefully transformed with the A construct include Pinus banks
iana, Pinus brutia, Pinus caribaea, Pin
us clausa, Pinus control, Pinus culture
eri, Pinus echinata, Pinus eldarica, Pi
nus ellioti, Pinus jeffreyi, Pinus lam
bertiana, Pinus monticola, Pinus nigra
, Pinus palustrus, Pinus pinaster, Pinu
s ponderosa, Pinus resinosa, Pinus rig
ida, Pinus serotina, Pinus strobus, Pin
us sylvestris, Pinus taeda, Pinus virg
pine like A. iniana and Abies amabilis, Abies b
alsamea, Abies concolor, Abies grandis
, Abies lasiocarpa, Abies magnificica, Ab
ises procera, Champaecyparis lawsoniona
, Chamaecyparis notokatensis, Chamaecy
paris thyoides, Huniperus virginiana,
Larix decade, Larix laricina, Larix l
eptolepis, Larix ocidentalis, Larixs
iberica, Liboedrus decurrens, Picea a
ties, Picea engelmanni, Picea glauca, P
icea mariana, Picea pungens, Picea rub
ens, Picea sitchensis, Pseudotsuga men
ziesii, Sequoia gigantea, Sequoia semp
ervirens, Taxodium distichum, Tsuga ca
nadensis, Tsuga heterophylla, Tsuga me
rtensiana, Thuja ocidentalis, Thujap
licata and other gymnosperms, Eucalyptus alba, Eu
calyptus bancroftii, Eucalyptus botyr
oides, Eucalyptus bridgesiana, Eucalyp
tus calophylla, Eucalyptus camaldulen
sis, Eucalyptus citriodora, Eucalyptus
cladocalyx, Eucalyptus coccifera, Euc
ayptus curtisii, Eucalyptus dalrympl
eana, Eucalyptus degrupta, Eucalyptus
delagensis, Eucalyptus diversity
, Eucalyptus dunnii, Eucalyptus fififo
lia, Eucalyptus globulus, Eucalyptus g
omphocephala, Eucalyptus gunnii, Eucal
yptus henryi, Eucalyptus laevopinea, E
eucryptus macarthurii, Eucalyptus mac
lorhyncha, Eucalyptus maculata, Eucally
ptus marginata, Eucalyptus megacarpa,
Eucalyptus melliodora, Eucalyptus nic
holii, Eucalyptus nitrens, Eucalyptus n
ova-anglica, Eucalyptus oblikua, Eucal
yptus obtusiflora, Eucalyptus oredes
, Eucalyptus pauciflora, Eucalyptus po
lybractea, Eucalyptus regnans, Eucalyp
tus resinifera, Eucalyptus robusta, Eu
calyptus rudis, Eucalyptus saligna, Eu
calyptus sideroxylon, Eucalyptus stua
rtiana, Eucalyptus teleticornis, Eucal
yptus torelliana, Eucalyptus urnigera
, Eucalyptus urophylla, Eucalyptus vim
inalis, Eucalyptus viridis, Eucalyptus
and eucalyptus such as Eucalyptus youumani.

Techniques for stably incorporating a gene construct into the genome of a target plant are well known to those skilled in the art,
Electroporation method, protoplast fusion method, reproductive organ injection method, immature embryo injection method, high velocity projectile introduction method (high velocity projectile method)
introduction) and others similar to these. The choice of technique will depend on the target plant to be transformed. For example, dicotyledonous plants and some monocotyledonous and gymnospermous plants are, for example, Bevan (Nucleic Acids)
Res. 12: 8711-8721, 1984). Targets for introduction of the gene constructs of the present invention include tissues such as leaf tissue, dispersed cells, protoplasts, seeds, embryos, meristem areas, cotyledons, hypocotyls, and the like. A preferred method of transforming eucalyptus and pine is pollen (eg, Ar
onen, Finnish Forest Res. Papers, Vol.
595: 53, 1996) or a biolistic method using readily reproducible embryonic tissue.

Once the cells have been transformed, cells having the DNA construct of the present invention integrated into the genome can be selected by a marker such as the kanamycin resistance marker described above. Transgenic cells can be prepared using techniques well known to those skilled in the art.
The whole plant is cultured in a suitable medium for regeneration. In the case of protoplasts, the cell wall is regenerated under appropriate osmotic conditions. In the case of seeds or embryos, a suitable germination or callus induction medium is used. For explants, an appropriate regeneration medium is used. Plant regeneration is well established for many species. For a review of forest tree regeneration, see Dunstan et al., "Somatic embryogenesis."
s in woody plants, "(Torpe TA Editing, Inv
intro embryogenesis of plants, Current
Plant Science and Biotechnology in
Agriculture 20 (12): 471-540, 1995). A specific protocol for spruce regeneration is described by Roberts S
omatic embryogenesis of spruce (Reden
Baugh K Editing, Synseded: applications of s
synthetic seed to crop improvement, CR
C Press: Chapter 23, pages 427-449, 1993).
The resulting transformed plants may be propagated sexually or asexually to obtain transgenic plants of the next generation or later using techniques well known in the art.

As described above, RNA production in a target cell can be controlled by selection of a promoter sequence or by selection of a functional copy number or integration site of a polynucleotide incorporated into the genome of the target organism. The target plant is transformed with two or more constructs of the invention, modifies the activity of two or more isoprenoid-metabolizing enzymes, affects the activity of the enzyme in two or more tissues, or the enzyme at the time of two or more expression May affect activity. Similarly, a gene construct can contain two or more open genes encoding enzymes encoded by the polynucleotides of the present invention.
2 of the gene encoding the enzyme to contain the reading frame or
It may be constructed to include the above non-coding region. The polynucleotides of the present invention may also be used in combination with other known sequences that encode enzymes involved in isoprenoid synthesis.

In addition, the polynucleotides of the present invention have particular application as non-destructive tags for living organisms, especially plants. The gene construct containing the polynucleotide of the present invention can be stably introduced into an organism as a heterologous, non-functional, non-destructive tag. Then, at a later date, it is possible to identify the origin or source of the organism by determining whether the tag is present in a sample of the material. Organisms other than plants, including commercially valuable animals, fish, bacteria and yeast, may be tagged with the polynucleotides of the present invention.

[0093] Detection of the tag can be accomplished using a variety of conventional techniques, and generally involves the use of nucleic acid probes. Sensitivity in assaying for the presence of a probe is described by Horn et al., Nuclee.
ic Acids Research 25 (23): 4842-4848, 1
As described in US Pat.
A molecule can be detected.

The following examples are given by way of illustration and not by way of limitation.

Example 1 Isolation and Characterization of cDNA Clones from Pinus radiata and Eucalyptus grandis c of Pinus radiata and Eucalyptus grandis
A DNA expression library was constructed and screened as follows. mRNA can be obtained from Chang et al. (Plant Molecular Biology Repo).
rter 11: 113-116, 1993) with slight modifications. Specifically, the sample is CPC-R
NAXB (100 mM Tris-Cl, pH 8, 0, 25 mM EDT
A, 2.0 M NaCl, 2% CTAB, 2% PVP and 0.05% spermidine trihydrochloride), and chloroform: isoamyl alcohol was added in 24 hours.
: 1 mixture. The mRNA is precipitated with ethanol and the total RNA preparation is prepared using the poly (A) Quick mRNA Isolation Kit (Stratagen).
e, La Jolla, CA). A cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis using a ZAP Express cDNA synthesis kit (Stratagene) according to the manufacturer's protocol, and then inserting a cDNA clone into lambda ZAP. Obtained cD
NA was determined using 1 μl of sample DNA in a 5 μl ligation mix,
Packaging was performed using Gigapack II packaging extract (Stratagene). Mass Excision of Library (Mass Excision)
Are XL1-Blue MRF ｀ cells and XLOLR cells (Stratagen).
e) was used with ExAssist helper phage (Stratagene). The excised phagemid was NZY broth (Gibco BR).
L, Gaithersburg, MD) and plated on LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG).

[0096] Of the colonies plated and picked for DNA minipreps, the majority contained inserts suitable for sequencing. Positive clones were cultured in NZY broth with kanamycin, and the cDNA was prepared using REAL DNA minipreps (Qia
gen, Venlo, The Netherlands). 1% agarose gel was used to screen for chromosomal contamination in the sequencing template. The dye terminator array is used for liquid sample processing (liquid ha
ndling) with a Biomek2000 robot (Beckman Coul)
ter Inc, Fullerton, CA) and a 9700 PCR machine (Perkin Elmer / Applied Bios) for DNA amplification.
system (Foster City, Calif.) according to the manufacturer's protocol.

The polynucleotide of the positive clone was obtained from Perkin Elmer / Appli.
It was obtained using a Prism377 sequencer from ed Biosystems Division. cDNA clones were first sequenced from the 5 'end and, optionally, also from the 3' end. For some clones,
The internal sequence was obtained from the subcloned fragment. Subcloning was performed using standard procedures of restriction site mapping and subcloning into pBluescript IISK + vector and other standard sequencing vectors.

The determined cDNA sequence including the polynucleotide of the present invention has a known sequence (k
The current sequence is compared with known sequences in the alignment. Specifically, the polynucleotides identified as SEQ ID NOs: 1-53 are the same as those in the EMBL database at the end of August 1998, Un
ix running command to "blastall -p blastn -de
mbldb-e10-G0-E0-r1-v30-b
BLAS set to "30-i queryseq results"
Comparisons were made using TN algorithm version 2.0.4 [Feb. 24, 1998]. Polynucleotides identified as SEQ ID NOs: 78-164 were compared to polynucleotides from the EMBL database at the end of May 1999, and Unix
Change the running command to "blastall -p blastn -d emb
ldb-e10-G0-E0-r1-v30-b30
BLASTN set to "-i queryseq results"
Comparisons were made using algorithm version 2.0.6 [September 16, 1998]. Multiple alignments of redundant sequences were used to generate reliable consensus sequences. Isolated polynucleotides of the present invention identified as SEQ ID NOs: 1-53 and 78-164 based on similarity to known sequences from other plant species are set forth in Table 1. Was presumably identified as encoding a polypeptide similar to the polypeptide.

[0099] The isolated cDNA sequences were compared to sequences in the EMBL database using the computer algorithm BLASTN. The corresponding predicted polypeptide sequence was determined and the sequence in the SwissProt database and computer algorithm BLA
Comparisons were made using STP. D provided in SEQ ID NOs: 78-164
Comparison of the NA sequence with the EMBL DNA database (using BLASTN) and the amino acid sequence provided in SEQ ID NOs: 165-304 (BLASTN)
Comparisons with sequences in the SwissProt database (using P) were made in May 1999. The translation results (six-frame translations) of the six reading frames of the polynucleotides of SEQ ID NOs: 78 to 164 are also EMBL.
The translation results of the six reading frames of the polynucleotides in the database were compared and aligned using the TBLASTX program.

BLASTN Polynucleotide Analysis Sequence ID Nos. 1, 2, 4-6, 8-12, 15, 19, 21-23, 27-33, No. 35, No. 37-42, No. 44, No. 46-52, No. 78-80, No. 82, No. 83, No. 86, No. 89-92, No. 96-100, 104th to 113th, 115th, 117th, 120th, 122th to 130th,
The cDNA sequences of positions 132 to 136, 138 to 158, 160, 163 and 164 are less than 40% of the sequences of the EMBL database using the BLASTN computer algorithm in the above-described determination method. Was determined to have only one identity. Sequence identification numbers 3, 7, 14, and 18
No. 20, No. 25, No. 34, No. 36, No. 53, No. 84, No. 85, No. 87, No. 88, No. 101, No. 114, No. 116, The cDNA sequences at positions 118, 119, 131, 137, 159, 161 and 162 were sequenced from the EMBL database using the BLASTN computer algorithm by the determination method described above. And less than 60% identity. Sequence identification numbers No. 16, 17, No. 26, No. 43, No. 45
Nos. 93, 94 and 121 were determined to have less than 75% identity to sequences in the EMBL database using the BLASTN computer algorithm in the manner described above. . The cDNA sequences of SEQ ID Nos. 13, 24, 95, 102 and 103 are less than 90% less than the sequences in the EMBL database using the BLASTN computer algorithm in the manner described above. It was decided that there was only identity.

BLASTP Amino Acid Analysis Sequence ID Nos. 194-200, 202, 216, 223, 230, 235, 239, 240, 243, 250 No. 255, No. 259, No. 260, No. 263, No. 270, No. 272, No. 274, No. 278, No. 291, No. 292, No. 293, No. 296, No. The predicted amino acid sequences at positions 303 and 304 were determined to have less than 50% identity to the sequences in the SwissProt database using the BLASTP computer algorithm by the determination method described above. Sequence identification numbers 166, 168 to 177, 179, 183 to 188,
192nd, 203th to 205th, 207th, 209th to 213th,
No. 218, No. 219, No. 221, No. 224, No. 225, No. 227 to No. 229, No. 231, No. 232, No. 234, No. 237, No. 242,
No. 244, No. 245, No. 251, No. 253, No. 262, No. 267,
No. 268, No. 269, No. 273, No. 276, No. 277, No. 279,
No. 281, No. 282, No. 284, No. 286, No. 289, No. 290,
No. 294, No. 295, No. 297, No. 298, No. 299, No. 300,
The predicted amino acid sequences at positions 301 and 302 were determined to have less than 75% identity to the sequences in the SwissProt database using the BLASTP computer algorithm by the determination method described above. Sequence identification numbers 165, 167, 178, 182, 189 to 191, 193, 201, 206, 208, 214, 215, 215 No. 217, No. 220, No. 222, No. 226, No. 233, No. 238, No. 241, No. 246-250, No. 254, No. 256, No. 257, No. 258, No. The predicted amino acid sequences at positions 261, 264, 265, 266, 275, 280, 283, 285, and 288 were obtained from the SwissProt database using the determination method described above. Sequence and the BLAS
It was determined that there was less than 90% identity using the TP computer algorithm. The predicted amino acid sequences of SEQ ID NOs: 180, 181, and 271 were determined to have less than 95% identity to the sequences in the SwissProt database using the BLASTP computer algorithm by the determination method described above. Was done.

TBLASTX Analysis The translation results of the six reading frames of the polynucleotide sequence of SEQ ID NOs: 78 to 164 are based on the translation results of the six reading frames of the polynucleotide in the EMBL database and the Unix running command “blastall-p blastn”.
-Dembdb-e10-G0-E0-v30-b3
With the running parameter setting of “0-i queryseq results”, the version 2.0.6 of the TBLASTX program [September 1998
And the alignment was carried out. Sequence identification number 82,
No. 83, No. 90, No. 107 to No. 113, No. 115, No. 120, No. 1
22nd, 124th to 126th, 129th, 134th to 136th, 1st
No. 42 to 144, No. 146 to 149, No. 152, No. 153, No. 1
The translation results of the polynucleotides of Nos. 55 to 158 and 164 are determined by comparing the translation results of the oligonucleotides in the EMBL database with TBLA as described above.
It was determined that there was less than 50% identity using the STX computer algorithm. Sequence identification numbers 79, 81, 84 to 89, 91,
No. 92, No. 96 to No. 101, No. 103, No. 105, No. 114, No. 1
Nos. 16 to 118, No. 123, No. 131, No. 132, No. 137 to 1
The translation results of the 41st, 145th, 150th, 154th, and 160th to 162nd polynucleotides are calculated using the translation results of the oligonucleotides in the EMBL database and the TBLASTX computer algorithm according to the above-described determination method. It was determined that there was less than% identity. Sequence identification numbers 78 and 8
No. 0, No. 93, No. 95, No. 102, No. 104, No. 106, No. 119, No. 121, No. 127, No. 128, No. 130, No. 133, No. 151 , 159 and 163, the translation results of the oligonucleotides in the EMBL database and TBLASTX as described above.
Using a computer algorithm, it was determined that there was less than 90% identity. The translation result of the polynucleotide of SEQ ID NO: 94 was determined to be less than 95% identical to the translation result of the oligonucleotide in the EMBL database using the TBLASTX computer algorithm as described above.

Example 2 O-methyltransferase (O-methyltr) for modifying lignin biosynthesis
Transformation of tobacco plants with the Pinus radiata OMT gene Sense and antisense constructs containing polynucleotides containing the coding region of OMT (SEQ ID NO: 54) from Pinus radiata were constructed and published methods (An G et al., "Binary Vectors,"
Editing in Gelvin SB and Schilperroot RA, Pla
nt Molecular Biology Manual, Kluwer A
Acadobacterium tumefaciens by direct transformation using Cademic Publishers, Dordrecht, 1988). General methods for plant transformation are described in Horsch et al., Science 22.
7: 1229-1231, 1985. Construction of sense DNA is performed by first cloning the PBK-CMV cDNA insert into the pART7 vector. The pART7 vector contains the 35S-insert-O
The CS3｀UTR construct was cut with the restriction enzyme NotI to clone it into the plant expression vector pART27 (Gleave A, Plant).
Mol. Biol. 20: 1203-1207, 1992). The presence and integrity of the transgenic construct was confirmed by restriction enzyme digestion and DNA sequencing.

[0104] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were obtained from Horsch et al., Science 227: 12291231,19.
Transformations were performed with the sense and antisense OMT constructs described above using 85 methods. Five independent transformed plant strains were established for the OMT sense construct, and eight independent transformed plant strains were established for the OMT antisense construct. Transformed plants containing the appropriate gene construct were identified using Southern blotting. In the column labeled “Southern” in Table 2,
“+” Indicates that the transformed plant was confirmed to be an independent transformed strain.

Expression of Pine OMT in Transformed Plants Total RNA was isolated from each independent transformed plant strain transformed with the OMT sense and antisense constructs described above. RNA samples
To determine the expression level of the foreign gene in each transformant, it was analyzed by Northern blotting.

In Table 1, the data shown in the column labeled “Northern” is the OMT
It was shown that the transformed plant strain containing the sense and antisense constructs expressed higher levels than the Northern blot background. RNA
OMT expression in the second sense plant strain was not measured because the sample showed signs of degradation. Hybridization with RNA samples from control plants transformed with an empty vector (a vector without any insertion) could not be detected.

Modification of OMT Enzyme Activity in Transformed Plants The total activity of the OMT enzyme encoded by the Pine OMT gene and the endogenous tobacco OMT gene was determined using the OMT sense and antisense constructs, respectively. Of the transformed plant strains were analyzed. Crude protein extracts were prepared from each transformed plant and were prepared using the method of Zhang et al.
ysiol. , 113: 65-74, 1997). In Table 2, "
The data contained in the column labeled "Enzymes" show that transformed plant strains containing the OMT sense construct generally have increased OMT enzyme activity and up to 199% of control plants transformed with empty vectors. However, transformed plant strains containing the OMT antisense construct generally have reduced OMT enzyme activity and have a minimum of 3
It shows that it is 5%. OMT enzyme activity was not estimated in the third sense plant strain.

Effect of Pine OMT on Lignin Concentration in Transformed Plants OMT is an enzyme involved in lignin biosynthesis. Lignin concentration in transformed tobacco plants was determined by thioglycolic acid extraction (Freudenberg et al., C.I.
onstitution and Biosynthesis of Light
in, Springer-Verlag, Berlin, 1968). Briefly, whole 38 day old tobacco plants were frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. One plant strain of a control experiment transformed with an empty vector, the five independent transformed plant strains containing the OMT sense construct, and the eight independent transformed plants containing the OMT antisense construct 100 mg of the frozen powder from the strain was individually extracted with methanol, then with 10% thioglycol, and finally dissolved in 1 M NaOH. The last extract was assayed by absorbance at 280 nm. The data shown in the column labeled "TGA" in Table 2 shows that the transformed plant strains containing the sense and antisense OMT gene constructs were all less than the control plants transformed with the empty vector. Lignin levels were significantly reduced.

[0109]

[Table 2]

These data demonstrate that the polynucleotides and encoding polypeptides identified from the isolated cDNA obtained as in Example 1 are assembled into a DNA construct and used to transform plants. Prove to In addition, the data indicate that transformed plants containing the genetic construct show variability in the level of expression and activity of such enzymes and that the engineered either by sense or antisense expression of a gene encoding an enzyme such as OMT. It also demonstrates that alterations in the metabolism of the enzyme affect the concentration of the end product, such as the lignin concentration in transformed plants.

Example 3 4-Coumarate CoA Ligase (4-Co) to Modify Lignin Biosynthesis
Umarate: Use of CoA ligase, 4CL) gene Transformation of tobacco plant with Pinus radiata 4CL gene Sense and antisense constructs containing a polynucleotide containing the coding region of Pinus radiata-derived 4CL (SEQ ID NO: 55) include:
It was inserted into Agrobacterium tumefaciens strain LBA4301 by the direct transformation method described in Example 2. The presence and integrity of the transgenic construct was confirmed by restriction enzyme digestion and DNA sequencing.

[0112] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described above. 5 for 4CL sense construct
Two independent transformed plant lines were established and eight independent transformed plant lines for the antisense construct were established. Transformed plants containing the appropriate lignin gene construct were identified using Southern blotting. A "+" in the column labeled "Southern" in Table 3 indicates that the listed transformed plant strains were confirmed to be independent transformants.

Expression of Pine 4CL in Transformed Plants Total RNA was isolated from each independently transformed plant strain created with the 4CL sense and antisense constructs. RNA samples were analyzed by Northern blot to determine the expression level of the foreign gene in each transformant. The data shown in the column labeled "Northern" in Table 3 indicate that the transformed plant lines containing the sense and antisense constructs for 4CL all had higher levels of expression compared to the Northern blot background. Is shown. 4CL expression in the first antisense plant strain was not measured because RNA was not available at the time of the experiment. Hybridization with RNA samples from control plants transformed with the empty vector could not be detected.

Modification of 4CL Enzyme Activity in Transformed Plants In transformed tobacco plants, the total activity of the 4CL enzyme encoded by the Pinus 4CL gene and the endogenous tobacco 4CL gene was generated with the 4CL sense and antisense constructs. Each transformed plant strain analyzed was analyzed. A crude protein extract was prepared from each transformed plant, and Zhang
Et al., Plant Physiol. , 113: 65-74, 1997. The data contained in the column labeled "Enzyme" in Table 2 shows that the transformed plant strains containing the 4CL sense construct had up to 258% of the 4CL enzyme construct as compared to control plants transformed with the empty vector. Activity rises and 4CL
Transformed plant strains containing the antisense construct show reduced 4CL enzyme activity to a minimum of 59%.

Effect of Pine 4CL on Lignin Concentration in Transformed Plants Lignin concentration in samples of transformed plant material was determined as described in Example 2. The data shown in the column labeled "TGA" in Table 3 shows that the transformed plant strains containing the sense and antisense 4CL gene constructs were all less than the control plants transformed with the empty vector. Indicates that there is a significantly reduced lignin level. These data demonstrate that the polynucleotide identified from the isolated cDNA obtained in Example 1 can be assembled into a DNA construct and used to transform plants. Transformed plants containing such a genetic construct will exhibit altered levels of enzyme expression and activity. The metabolism of the biosynthetic pathway involving the enzyme is also affected.

[0116]

[Table 3]

Example 4 Transformation of Tobacco Using the Lignin Biosynthesis Gene of the Present Invention Coumarate 3-hydroxylase from Eucalyptus grandis (c
oumarate 3-hydroxylase, C3H, SEQ ID NO: 56)
, Ferulate-5-hydroxylase
e, F5H, SEQ ID NO: 57), cinnamoyl-CoA reductase (cinn
amoyl-CoA reductase, CCR, SEQ ID NO: 58) and coniferyl glucose transferase (coniferyl glucosyl trase).
ansferase, CGT, SEQ ID NO: 59) and Pinus radia
phenylalanine ammonia-lyase (phenylalanine)
ammonium-lyase, PAL, SEQ ID NOs: 60 and 61), cinnamate 4-hydroxylase, C
4H, SEQ ID NOs: 62 and 63), phenolase (PN), PN
L, SEQ ID NO: 64) and sense and antisense constructs containing a polynucleotide containing the coding region of laccase (LAC, SEQ ID NO: 65) were obtained by the above-described direct transformation method using Agrobacterium tumefaciens LBA4301. Inserted into strain. The presence and integrity of the transgenic construct was confirmed by restriction enzyme digestion and DNA sequencing.

[0118] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described in Example 2. For each of the sense constructs and antisense constructs listed in the next paragraph,
Up to two independent transformed plant lines have been established. Transformed plants containing the appropriate lignin gene construct were identified using Southern blotting. It was confirmed that all the transformed plant strains analyzed were independent transformants. This demonstrates that all procedures can be started from the isolated cDNA obtained in Example 1 to produce a transgenic plant having the expressed novel gene.

Example 5 Manipulation of Lignin Content in Transformed Plants Determination of Foreign Gene Expression by Northern Blot Method From each of the independent transformed plant strains described in Example 4, total RNA was isolated. RNA samples were analyzed by Northern blot to determine the expression level of the foreign gene in each transformant. The column labeled "Northern" in Table 4 shows the expression level of the foreign gene in all plant strains tested compared to the Northern blot background. Hybridization with RNA samples from control plants transformed with the empty vector could not be detected.

Determination of Lignin Concentration in Transformed Plants A control plant strain transformed with the empty vector and one for each sense construct and each antisense construct described in Example 5
Lignin concentrations in up to two independent transformants were determined as described in Example 3. The column labeled "TGA" in Table 3 shows the lignin extractable with thioglycolic acid for all tested plant strains as the average percentage of TGA extractable lignin in the transformed plants relative to the control plants. Shown. Mutation widths are shown in parentheses.

[0121]

[Table 4]

All the transformed plant lines containing the lignin biosynthesis gene construct in sense and antisense orientation showed significantly reduced lignin levels compared to the control plant lines transformed with empty vector. The most dramatic effect on lignin concentration was seen in F5H antisense plants, which accounted for only 35% of the lignin content of control plants, and PAL antisense plants, which only accounted for 37% of the lignin content of control plants. These data indicate that the concentration of a polynucleotide such as lignin as determined by the TGA assay was the same as that obtained in Example 1 by the conventional antisense method using the lignin biosynthesis gene of the present invention and the overexpression of the sense strand. It clearly shows that it is possible to start directly from the isolated cDNA and start the whole procedure.

Example 6 Modification of Lignin Enzyme Activity in Transformed Plants The activity and substrate specificity of the selected lignin biosynthetic enzymes was measured using the Pinus rad
Traits including sense and antisense constructs for PAL (SEQ ID NO: 60), PNL (SEQ ID NO: 64) and LAC (SEQ ID NO: 65) from Iata and CGT (SEQ ID NO: 59) from Eucalyptus grandis Assayed on crude extracts from converted tobacco plants.

[0124] Enzyme assays were performed using PAL (Southerton SG and Deverall B).
J, Plant Path. 39: 223-230, 1990), CGT (Ve
Ilekoop P et al., FEBS Lett. , 330: 36-40, 1993.
), PNL (Espin CJ et al., Phytochemistry 44:17).
-22, 1997) and LAC (Bao W et al., Science, 260: 67).
2-674, 1993). The data listed in the column labeled "Enzyme" in Table 5 shows the average enzymatic activity obtained by repeating the measurement for all the plant strains tested, and the enzymatic activity in the control plants transformed with empty vectors Expressed as a percentage. Mutation widths are shown in parentheses.

[0125]

[Table 5]

All transformed plant strains except the PNL antisense transformed plant strain show a mean lignin enzyme activity that is significantly lower than that obtained in control plants transformed with empty vector. Was. The most dramatic effect on lignin enzyme activity is
A PAL antisense-transformed plant strain in which the PAL activity of all strains was reduced, and L which showed 14% of the LAC activity of the control plant transformed with the empty vector
It was found in AC antisense transformed plant strains. These results were obtained by starting the entire procedure from the polynucleotide encoding the enzyme of interest isolated from the cDNA described in Example 1 and transforming the plant with the polynucleotide encoding the enzyme of interest. Prove that the enzyme activity can be modified.

Example 7 Functional Identification of Lignin Biosynthesis Gene PAL (SEQ ID NO: 61), OMT (from Pinus radiata)
SEQ ID NO: 54), 4CL (SEQ ID NOs: 55 and 66) and POX (SEQ ID NO: 67), and OMT (SEQ ID NOs: 68 and 69), CCR (SEQ ID NOs: 70-72) from Eucalyptus grandis, A sense construct comprising a polynucleotide comprising the coding regions of CGT (SEQ ID NOS: 59 and 73) and POX (SEQ ID NOs: 74 and 75) was inserted into the commercially available protein expression vector pProEX-HT (Gibco BRL). Was done. The obtained construct was used for Escherichia coli XL1-Blue (Stratage).
ne), the production of the recombinant protein was induced by the addition of IPTG. Ni column chromatography (Janknecht) for pine OMT and 4CL constructs and Eucalyptus OMT and POX constructs
R et al., Proc. Natl. Acad. Sci. , 88: 8972-8976.
, 1991). Enzymatic assays of each of the purified proteins conclusively demonstrated that the tested genes had the expected substrate specificity and enzymatic activity.

The 4CL gene of Pinus radiata (SEQ ID NO: 55) and Pin
The data of two representative enzyme assay experiments showing confirmation of the enzymatic activity of the OMT gene of S. radiata (SEQ ID NO: 54) are shown in Table 6. For the 4CL enzyme, one unit equals the amount of protein required to convert the substrate to product at a rate of 0.1 absorbance units per minute. For the OMT enzyme, one unit equals the amount of protein required to convert 1 picomole of substrate per minute to product.

[0129]

[Table 6]

The data presented in Table 6 demonstrated that both the purified 4CL enzyme and the purified OMT enzyme showed high activity in enzyme assays, confirming the identity of the 4CL and OMT genes described in this application. . Crude protein preparations from E. coli transformed with the empty vector did not show the activity of either 4CL or OMT. this is,
Starting from all the procedures from the isolated cDNA obtained in Example 1, it is shown that the function of the isolated novel cDNA having only the putative function can be confirmed.

Example 8 Demonstration of Presence / Absence of Unique Sequence Identifier in Plants Transgenic tobacco plants were created using a unique identifier sequence not found in tobacco. The inserted unique identifier sequence is Pinusra
SEQ ID NO: 76 and Eucalyptus g isolated from S. diata
SEQ ID NO: 77 isolated from Randis. The unique identifier sequence can be obtained using published methods (An G et al., "Binary Vectors,"
Edited by Gelvin SB and Schilperroot RA, Plant M
olecular Biology Manual, Kluer Acade
Agrobacterium tumefaciens strain LAB4301 (gifted by Dr. C. Kado of Davis, CA, California) by direct transformation using mic Publishers, Dordrecht, 1988). The presence and integrity of the unique identifier sequence in the Agrobacterium transgenic construct was confirmed by restriction enzyme digestion and DNA sequencing.

[0132] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were obtained from Horsch et al., Science, 227: 1229-1231,19.
Transformation was performed using 85 methods. For each unique sequence identifier used, three independent transformed plant lines were established. The two plant strains of the empty vector control experiment did not insert anything lacking the unique sequence identifier (e
mpty) was established using a gene transfer vector.

To test for the presence of the sequence identifier in the plant genome, the uniqueness of the sequence identifier was tested using Southern blot analysis. When a sequence identifier is unique and valid as a tag, it should clearly be absent in untagged plants and clearly present in tagged plants. In this example, it is expected that no unique identifier will be present in the control plants transformed with the empty vector. The unique identifier is expected to be present in transgenic plants transformed with the unique identifier.

Genomic DNA was obtained from Murray MG and Thompson WF, Nucle.
ic Acids Research 8: 4321-4325, prepared using the 1980 cetyltrimethylammonium bromide (CTAB) extraction method from control plants transformed with empty vectors and plants transformed with unique identifiers. In the case of a plant transformed with the pine genus unique sequence identifier (SEQ ID NO: 76), the Eucalyptus genus unique sequence identifier (
In the case of a plant transformed with SEQ ID NO: 77), the DNA was digested with the restriction enzyme XbaI.
The sample was digested. DNA fragments obtained by the restriction enzyme digestion were separated on a 1% agarose gel. The left panel of FIG. 2 and the right panel of FIG. 2 show the DNA fragment patterns of DNA samples from experiments of the genus Pinus and Eucalyptus, respectively.

After the step of agarose gel electrophoresis, the DNA sample was
n. Mol. Bio. 98: 503-517, using a Hybond-N + brand nylon membrane (Amersham Li).
fe Science, Little Chalfont, Buckingha
mshire, UK). The nylon membrane is reacted with the radioactively labeled probe for the unique sequence identifier identified above, and subjected to high stringency (final wash: 0.5X sodium salt citrate buffer (SSC) plus 0.1% dodecyl sulfate). Sodium (SDS), 15 minutes, 65 ° C). Hybridization of the probe with a complementary sequence in a genomic DNA sample was detected using autoradiography.

The results are shown in FIGS. FIG. 3 (corresponding to the left panel of FIG. 2) shows a hybridization pattern detected by Southern blot analysis using a probe derived from a pine genus sequence identifier (SEQ ID NO: 76). Lanes A and B contain DNA samples from control plants transformed with the empty vector, and lanes C through E contain DNA from plants transformed with SEQ ID NO: 76. Lanes A and B
Has no hybridization and SEQ ID NO: 76 indicates that it is absent in control plants transformed with the empty vector. That is, the sequence identification number 7
No. 6 is a unique tag suitable for marking tobacco plants without ambiguity. Lanes C through E show strong hybridization, and plants that have received SEQ ID NO: 76 by transformation are clearly and unambiguously identified with SEQ ID NO: 76.
Indicates that the tag was tagged with the unique sequence included in the number.

FIG. 4 (corresponding to the right panel in FIG. 2) shows the Eucalyptus sequence identifier (sequence identification number 7).
7 shows a hybridization pattern detected by Southern blot analysis using a probe derived from No. 7). Lanes A and B contain DNA samples from control plants transformed with the empty vector, and lanes C through E contain DNA from plants transformed with SEQ ID NO: 77. Lanes A and B show no hybridization, indicating that SEQ ID NO: 77 is absent from control plants transformed with the empty vector. That is, SEQ ID NO: 77 is a unique tag suitable for marking tobacco plants without ambiguity. Lane C
E to E show strong hybridization, and the plants receiving SEQ ID NO: 77 by transformation were clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO: 77 Indicates that

The above data clearly demonstrate that the sequences disclosed herein are useful for the purpose of unambiguously tagging transgenic material. A unique sequence was selected from a number of potential tabs, indicating that it was not present in the genome of the organism being tagged. The tag is inserted into the genome of the organism to be tagged;
Well-established DNA detection methods were used to unambiguously detect unique sequence identifiers used as tags.

Because of the sequence-specific detection methods used in this example, users of the invention disclosed herein are useful for tagging any organism in the disclosed list. Probability of finding sequence identifiers and proving that tagged organisms could acquire certain tags only by intentionally adding unique sequences to the genome of the tagged organism With both a clear way to do it. If the user of the present invention keeps the exact sequence of tags used for an organism secret, any issues regarding the origin and history of the organism may be unclear using the tag detection techniques disclosed in this example. Can be solved without.

[0140] SEQ ID NOs: 1-304 are provided in the attached Sequence Listing. The nucleotide sequence symbols used in the attached sequence listings, including the symbol "n", are the same as those in WIPO Standard ST. 25 (1998), Appendi
x2, Table 1.

All documents cited herein, including patent documents and non-patent documents, are incorporated in the text as a whole so as to include all cited matters without omission. In this specification, the invention has been described in connection with several preferred embodiments,
While many details have been set forth for purposes of illustration, the invention is capable of further embodiments and the details described therein are without departing from the basic principles of the invention. It will be apparent to those skilled in the art that they can be widely varied.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a basic biosynthetic pathway of an isoprenoid compound.

FIG. 2 shows a genomic DNA sample derived from a tobacco plant produced in a tagging experiment using a unique sequence identifier derived from pine (left panel) and a tobacco plant produced in a tagging experiment using a unique sequence identifier derived from Eucalyptus. The derived genomic DNA sample (right panel) is shown. In both panels, lanes A and B contain DNA samples from control plants transformed with empty vectors, and lanes C through E
Contains DNA samples from plants transformed with the unique sequence identifier.

FIG. 3 shows detection of Pine genus unique sequence identifiers in transformed tobacco plants. Lanes A and B show hybridization of the probe from SEQ ID NO: 402. Lanes C-E show the hybridization of the probe with the genomic DNA of tobacco plants containing 1-3 copies of the Pine unique sequence identifier.

FIG. 4 shows detection of Eucalyptus unique sequence identifiers in transformed tobacco plants. Lanes A and B show hybridization between the probe from SEQ ID NO: 403 and the genomic DNA of a tobacco plant lacking a Eucalyptus unique sequence identifier (a control plant transformed with an empty vector). Lanes C-E show hybridization with tobacco plant genomic DNA containing 1-2 copies of the Eucalyptus unique sequence identifier.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12Q 1/68 A 9/00 C12N 15/00 ZNAA C12Q 1/68 5/00 AC ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2B030 AA02 AA03 AB03 AD08 CA06 CA17 CA19 CB02 CD03 CD07 CD10 4B024 AA01 AA08 BA07 CA02 DA01 EA04 GA11 4B050 CC07 DD13 LL01 4B063 QA01 QA20 QQ09 QQ42 QR08 QR42 QR56 QS25 QS34 QX02 4B065 AA89X AA89Y AB01 BA02 CA02 CA08 CA27 CA43 CA53

Claims (29)

[Claims] (1) Sequence identification numbers 1 to 53 and 78 to 16
The sequence listed in No. 4; (2) the complement of the sequence listed in SEQ ID Nos. 1 to 53 and 78 to 164; and (3) the sequence ID Nos. 1 to 53 and 78. To the reverse complement of the sequences listed in SEQ ID Nos. 1 to 53 and 78 to 164; (5)
) Sequence identification numbers 1, 2, 4 to 6, 8 to 12, 19, 21 to 23, 28 to 33, 35, 37 to 42,
No. 44, No. 46-52, No. 78-80, No. 82, No. 83, No. 86, No. 89-92, No. 96-100, No. 104-113, No. 115, No. 117, No. 120, No. 122-130, No. 132-136, No. 138-158, No. 160, No. 163 and No. 164
A polynucleotide sequence having a percent identity greater than or equal to 40% when compared to a reference sequence selected from the polynucleotide sequences listed above, wherein the percent identity is determined by comparing the sequence to the reference sequence. B set to the described parameter value
Aligning using the LASTN algorithm version 2.0.4, identifying the number of identical nucleic acids over the aligned portion of the sequence and the reference sequence, determining the number of identical nucleic acids as the total number of nucleic acids in the comparison sequence A sequence comprising a polynucleotide sequence having a percent identity of 40% or more, determined by dividing by a number and multiplying by 100; (6) sequence identification numbers 3, 7, 14, and 18; No. 20, No. 25, No. 34, No. 36, No. 53, No. 84, No. 85, No. 87,
Polynucleotide sequences listed at positions 88, 101, 114, 116, 118, 119, 131, 137, 159, 161 and 162 A sequence comprising a polynucleotide sequence having a percent identity of 60% or more as described in (5) above, when compared with a comparison sequence selected from (7);
Sequence identification numbers 16th, 17th, 26th, 43rd, 45th, 93rd,
A sequence comprising a polynucleotide sequence having a percent identity of 75% or more as described in (5) above, when compared to a reference sequence selected from the polynucleotide sequences listed at No. 94 and No. 121; 8) Sequence ID Nos. 13, 24, 9
Includes polynucleotide sequences having a percent identity of 90% or more as described in (5) above when compared to a reference sequence selected from the polynucleotide sequences listed under Nos. 5, 102 and 103. A sequence, (9) a sequence containing a polynucleotide sequence hybridizing with a polynucleotide containing the sequence listed in (1) to (8) above, and (10) a sequence containing a polynucleotide sequence hybridizing with the polynucleotide containing the sequence listed in (1) to (8) above. A sequence comprising a polynucleotide sequence which is a 100-mer of the sequence; (11) a sequence comprising a polynucleotide sequence which is a 40-mer of the sequences listed in (1) to (8) above; And (13) one or more conservative substitutions from the sequences listed in (1) to (12) above, including a polynucleotide sequence that is a 20-mer of the sequences listed in (8). Comprising a polynucleotide sequence selected from the group consisting of a sequence comprising a polynucleotide sequence that do not differ or isolated polynucleotide. 2. An isolated oligonucleotide probe or primer comprising 10 or more contiguous residues complementary to 10 contiguous residues of the nucleotide sequence recited in claim 1. 3. A gene construct comprising the polynucleotide according to claim 1. 4. A transgenic cell comprising the gene construct according to claim 3. 5. The transgenic cell according to claim 4, wherein said cell is selected from one of a bacterial cell, an insect cell, a yeast cell, a mammalian cell and a plant cell. 6. In the direction from 5% to 3%, (a) a gene promoter sequence;
(B) a polynucleotide comprising the nucleotide sequence according to claim 1, which encodes at least a functional part of an enzyme having activity in the isoprenoid biosynthetic pathway; and (2) an enzyme having activity in the isoprenoid biosynthetic pathway. A polynucleotide sequence comprising at least one of the polynucleotide comprising the nucleotide sequence of claim 1 comprising a non-coding region of the encoding polynucleotide; and (c)
A gene construct comprising a gene termination sequence. 7. The construct of claim 6, wherein the polynucleotides are arranged in a sense orientation. 8. The polynucleotide of claim 6, wherein the polynucleotides are arranged in an antisense orientation.
The construct according to 1. 9. The construct according to claim 6, wherein the gene promoter sequence and the gene termination sequence function in a plant host. 10. A transgenic cell comprising the construct according to claim 6. 11. The transgenic cell of claim 10, wherein the polynucleotides are arranged in a sense orientation. 12. The polynucleotide of claim 1, wherein the polynucleotides are arranged in an antisense orientation.
The transgenic cell according to 0. 13. The transgenic cell according to claim 10, wherein the cell is selected from one of a bacterial cell, an insect cell, a yeast cell, a mammalian cell and a plant cell. 14. A plant, or a fruit, seed or progeny thereof, comprising the transgenic cell according to claim 9. 15. The plant according to claim 14, wherein the plant is a woody plant. 16. The plant according to claim 15, wherein the plant is selected from the group consisting of eucalyptus and pine species. 17. The content of an enzyme involved in an isoprenoid biosynthesis pathway of an organism,
Methods for altering one or more of composition and metabolism. 18. The method according to claim 17, wherein the organism is a plant. 19. A method for modifying one or more of the content, composition, and metabolism of an isoprenoid compound of an organism, the method comprising stably incorporating the construct of claim 6 into the genome of the organism. Way to do. 20. The method according to claim 19, wherein the organism is a plant. 21. A method for producing an organism having one or more of an altered isoprenoid content, an altered isoprenoid composition, and an altered isoprenoid metabolism, comprising: (a) providing a transgenic host cell. Transforming a host cell with the construct according to claim 3 to obtain a transgenic host cell under conditions conducive to growth and regeneration.
A way to create living things. 22. The method according to claim 21, wherein the organism is a plant and the host cell is a plant cell. 23. Encoded by the polynucleotide of claim 1,
An isolated polypeptide. 24. The polypeptide according to claim 23, which has an enzymatic activity in a plant isoprenoid biosynthesis pathway. 25. An isolated polypeptide comprising an amino acid sequence expressed from a polynucleotide that hybridizes under stringent conditions to the nucleotide sequences listed as SEQ ID NOs: 1-53 and 78-164. . 26. (1) SEQ ID Nos. 165 to 286 and 288
And (2) SEQ ID Nos. 194 to 200
No. 202, No. 216, No. 223, No. 230, No. 235, No. 239
No. 240, No. 243, No. 250, No. 255, No. 259, No. 260
No., No. 263, No. 270, No. 272, No. 274, No. 278, No. 291
No. 292, No. 293, No. 296, No. 303 and No. 304 comprising a polypeptide sequence having at least 50% identity to a reference sequence selected from the polypeptide sequences listed in Nos. , (3) SEQ ID Nos. 166 and 168
Nos. 177, 179, 183 to 188, 192, 203
To 205, 207, 209 to 213, 218, 219
No. 221, No. 224, No. 225, Nos. 227 to 229, No. 231
No., No. 232, No. 234, No. 237, No. 242, No. 244, No. 245
No. 251, No. 253, No. 262, No. 267, No. 268, No. 269
No., No. 273, No. 276, No. 277, No. 279, No. 281, No. 282
No., No. 284, No. 286, No. 289, No. 290, No. 294, No. 295
And a sequence comprising a polypeptide sequence having at least 75% identity to a reference sequence selected from the polypeptide sequences listed under Nos. 297-302;
4) Sequence identification numbers 165, 167, 178, 182, 189
191st, 193rd, 201st, 206th, 208th, 214th
No., No. 215, No. 217, No. 220, No. 222, No. 226, No. 233
No. 238, No. 241, No. 246 to No. 250, No. 254, No. 256
No. 258, No. 261, No. 264, No. 265, No. 266, No. 275
No. 280, No. 283, No. 285 and No. 288, a sequence comprising a polypeptide sequence having at least 90% identity to a reference sequence selected from the polypeptide sequences listed in Nos. Sequence identification numbers 180, 181, and 27
At least 95% of the reference sequence selected from the polypeptide sequences listed under No. 1
(6) a sequence containing a polypeptide sequence having the identity of
And (7) a sequence comprising a polypeptide sequence which is a 100-mer of the sequence having at least 100 residues and a sequence having at least 40 residues as listed in (1) to (5) above. A sequence comprising a 40-mer polypeptide sequence;
8) An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of a sequence comprising a polypeptide sequence that is a 20-mer of the sequence having at least 20 residues listed in (1) to (5) above. peptide. 27. A method for altering one or more of the content, composition, and metabolism of an isoprenoid compound of an organism, the method comprising administering the isolated polypeptide of claim 26. Way to do. 28. The method according to claim 27, wherein the method of administering the polypeptide isolated from the organism is a local. 29. The method of claim 27, wherein the organism is a mammal and the method of administering the isolated polypeptide is systemic.