CA2474906C - Brassica pyruvate dehydrogenase kinase gene - Google Patents

Abstract

The isolation, purification, characterization and use of a mitochondrial pyruvate dehydrogenase kinase (PDHK) gene from Brassica spp.. Methods of regulating fatty acid synthesis, seed oil content, seed size/weight, flowering time, vegetative growth, respiration rate and generation time using the gene and to tissues and plants transformed with the gene. Transgenic plants, plant tissues and plant seeds having a genome containing an introduced Brassica DNA, characterized in that the sequence has been introduced in an anti-sense or sense orientation, and a method of producing such plants and plant seeds.

Description

BRASSICA PYRUVATE DEHYDROGENASE KINASE GENE
TECHNICAL FIELD
[0001] This invention relates to plant genes useful for the genetic manipulation of plant characteristics. More specifically, the invention relates to the identification, isolation, and introduction of genes of Brassica PDHK sequences.
BACKGROUND

[0002] As described in FIG. 1 of PCT International Patent Application PCT/CA98/00096 to Zou and Taylor, (PCT International Publication WO 9835044 published August 13, 1998, acetyl-CoA plays a central role in mitochondria) respiration and plastidial fatty acid biosynthesis. The pyruvate dehydrogenase complex (PDC) oxidatively decarboxylates pyruvate to yield acetyl-CoA.

[0003] Plants have both mitochondria) and plastidial isoforms of the PDC (see also United States Patent 6,265,636, to Randall et al (July 24, 2001 ). The mitochondria) pyruvate dehydrogenase complex plays a key role in the regulation of acetyl-CoA
generation and availability of acetyl moieties for various catabolic and anabolic reactions in plant cells. The mitochondria) PDC is negatively regulated by phosphorylation of the E 1 a subunit by pyruvate dehydrogenase kinase (PDHK), and positively regulated by dephosphorylation of the PDC by pyruvate dehydrogenase phosphatase (PDCP).
Mitochondrially-generated acetyl moieties can find their way into the respiratory tricarboxylic acid (TCA; Krebs) cycle, but also into the plastid compartment where ultimately, acetate units are used by the enzymes of the fatty acid synthesis (FAS) pathway to synthesize fatty acids. These are eventually incorporated into membrane and also storage glycerolipids.

[0004] Zou and Taylor also disclose the identification, isolation and characterization of the pyruvate dehydrogenase kinase (PDHK) (gene and cDNA) sequence from the model plant system Arabidopsis thaliana and the utilization of this sequence in the genetic manipulation of plants. Also disclosed is a vector containing the fizll-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in an anti-sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants. Zou and Taylor also provided a method to construct a vector containing the fixll-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in a sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants. Also disclosed were methods for modifying Arabidopsis and other plants to change their seed oil content, average seed weight or size, respiration rate during development, vegetative growth characteristics, flowering time or patterns of generative growth, and the period required to reach seed maturity.

[0005] As disclosed in, for example, Zou and Taylor, respiration, which involves the consumption of 02 and the catabolism of sugar or other substrates to produce COZ, plays a central role in the process of plant growth in providing reducing equivalents, a source of energy and an array of intermediates (carbon skeletons) as the building blocks for many essential biosynthetic processes. The intermediate products of respiration are necessary for growth in meristematic tissues, maintenance of existing phytomass, uptake of nutrients, and intra- and inter-cellular transport of organic and inorganic materials. Respiration is important to both anabolic and catabolic phases of metabolism.

[0006] The pyruvate dehydrogenase complex (PDC) is a particularly important site for regulation of plant respiration. Modification of PDC activity through manipulation of PDHK expression can result in a change in the production or availability of mitochondrially-generated acetyl-CoA or a change in the respiration rate.
These changes may in turn affect seed oil content, average seed weight or size, respiration rate during development, vegetative growth characteristics, flowering time or patterns of generative growth, and the period required to reach seed maturity.

[0007] Many examples exist of successful modifications to plant metabolism that have been achieved by genetic engineering to transfer new genes or to alter the expression of existing genes, in plants. It is now routinely possible to introduce genes into many plant species of agronomic significance to improve crop performance (e.g., seed oil or tuber starch content/composition; meal improvement; herbicide, disease or insect resistance; heavy metal tolerance; etc.) (Somerville, 1993; Kishore and Somerville, 1993; MacKenzie and Jain, 1997).

[0008] The Brassica genus includes Arabidopsis thaliana. The Brassicaceae family is comprised of a large and diverse group of plant species which are economically very important throughout the world. Three diploid Brassica species (B. rapa, B.

oleracea and B. nigra) have hybridized in different combinations to give rise to the three amphidiploid species (B. napes, B. juncea, and B. carinata). Other Brassica species include B. oleifera, B. balearica, B. cretica, B. elongate, B. tourneforii, and B. biennis.
B. napes and B. rapa have been improved through breeding programs and are now cultivated as canola crops.

[0009] It would be an improvement in the art to isolate and sequence the PDHK gene from various useful species of plants of the Brassicaceae.
SUMMARY OF THE INVENTION

[0010] The invention involves the isolation, and characterization of PDHK
(gene and cDNA) sequences from Brassica species and the utilization of these sequences in the genetic manipulation of plants.

[0011] The invention also discloses a vector containing the full-length PDHK
sequence or a significant portion of PDHK sequences from the Brassicaceae, in an anti-sense orientation under control of either a constitutive or a seed-specific promoter, for re-introduction into Brassica species or for introduction into other plants.

[0012] The invention further describes a method to construct a vector containing the fill-length PDHK sequence or a significant portion of the PDHK
sequence from Brassica species, in a sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Brassica or for introduction into other plants.

[0013] The invention also provides methods of modifying Brassica and other plants to change their seed oil content, average seed weight or size, respiration rate during development, vegetative growth characteristics, flowering time or patterns of generative growth, and the period required to reach seed maturity.

[0014] According to one aspect of the present invention, there is disclosed isolated and purified deoxyribonucleic acid (DNA) of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, and/or SEQ ID N0:4. In this aspect, SEQ ID NO:1 is the nucleotide sequence and the corresponding amino acid sequence (SEQ ID NO:S) of the Brassica napes PDHK cDNA. SEQ ID N0:2 is the nucleotide sequence and its corresponding amino acid sequence (SEQ ID N0:6) of the Brassica rapa PDHK cDNA. SEQ ID
N0:3 is the nucleotide sequence and the corresponding amino acid sequence (SEQ
ID
N0:7) of the Brassica oleracea PDHK cDNA. SEQ ID N0:4 is the nucleotide sequence and the corresponding amino acid sequence (SEQ ID N0:8) of the Brassica carinata PDHK cDNA.

[0015] In yet another aspect of the invention, there is described a vector containing one or more of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, or SEQ ID
N0:4, or a part thereof, for introduction of the gene, in an anti-sense orientation into a plant cell, and a method for preparing a vector containing one or more of SEQ
ID NO:1, SEQ ID N0:2, SEQ ID N0:3, or SEQ ID N0:4, or a part thereof, for introduction of the gene in a sense orientation, into a plant cell.

[0016] The invention also relates to transgenic plants and plant seeds having a genome containing an introduced DNA sequence of one or more of SEQ ID NO:1, SEQ
ID N0:2, SEQ ID N0:3, or SEQ ID N0:4, or a part thereof, and a method of producing such plants and plant seeds.

[0017] The invention also relates to substantially homologous DNA sequences from plants with deduced amino acid sequences of 25% or greater identity, and 50% or greater similarity, isolated and/or characterized by known methods using the sequence information of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, or SEQ ID N0:4, as will be appreciated by persons skilled in the art, and to parts of reduced length that are still able to function as inhibitors of gene expression by use in an anti-sense or co-suppression (Jorgensen and Napoli 1994) application. It will be appreciated by persons skilled in the art that small changes in the identities of nucleotides in a specific gene sequence may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g., anti-sense or co-suppression), partial sequences often work as effectively as full length versions. The ways in which the gene sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. All such variations of the genes are therefore claimed as part of the present invention.

[0018] Stated more generally, the present invention relates to the isolation, purification and characterization of a mitochondrial pyruvate dehydrogenase kinase (PDHK) genes from the Brassicaceae (specifically Brassica napus, B. rapa, B.
oleracea, and B. carinata) and identifies its utility in regulating fatty acid synthesis, seed oil content, seed size/weight, flowering time, vegetative growth, respiration rate and generation time.

-4a_ According to a first aspect of the invention, there is provided a process for modulating mitochondrially generated acetyl-CoA or respiration rate in a transgenic plant, the process comprising:
cloning a gene encoding a Brassica pyruvate dehydrogenase kinase protein into a vector, said gene having a sequence selected from the group consisting of SEQ ID No:1, SEQ ID NQ:2, SEQ ID N0:3 and SEQ ID NO: 4.
positioning the gene in an anti-sense orientation; and transforming a plant with the vector to produce the plant having modulated mitochondrially generated acetyl-CoA or respiration rate.
According to a Second aspect of the invention, there is provided a process for modulating mitochondrially generated acetyl-CoA or respiration rate in a transgenic plant, the pnacess comprising:
cloning a gene encoding a Brassica pyruvate dehydrogenase kinase protein into a vector, the gene having a sequence selected from the group consisting of SEQ ID N0:1, SEQ ID N0:2, SEQ ID NO:3 and SEQ ID N0:4; and transforming the vector into a plant, thereby reducing production of the Brassica pyruvate dehydrogenase kinase protein in the transgenic plant and producing a plant having modulated mitochondrially generated acetyl-CoA dr respiration rate.
According to a third aspect of the invention, there is provided an isolated, purified or recombinant nucleic acid encoding a Brassica pyruvate dehydrogenase kinase (PDHK) protein, said nucleic acid selected from the group consisting of SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3 and SEQ ID NQ: 4. ' According to a fourth aspect of the invention, there is provided the use of the isolated, purified ar recombinant nucleic acid as described above fpr modulating mitochondrially generated acetyl-CoA or respiration rate in a plant.
According to a fifth aspect of the invention, there is provided the use of the isolated, purified or recombinant nucleic acid as described above for modulating seed oil content or weight in a plant.

-4h-According to a sixth aspect of the invention, there is provided the use of the isolated, purified or recombinant nucleic acid as described above for modulating flowering dr generation time in a plant.
According to a seventh aspect of the invention, there is provided a modified cell comprising the isolated, purified or recombinant nucleic acid as described above.
According to an eighth aspect of the invention, there is provided a method of transforming a plant, comprising introducing the isolated, purified or recombinant nucleic acid as described above into the plant.
t 0 According to a ninth aspect of the invention, there is provided a process for producing a genetically transformed plant seed, said process comprising transforming a plant seed by introducing the isolated, purified or recombinant nucleic acid as described above into the plant seed.
According to a tenth aspect of the invention, there is provided a vector for transforming plant cells comprising the isolated, purified or recombinant nucleic acid as described above.
According to an eleventh aspect of the invention, there is provided the use of the vector as described above for modulating ~mitochondrially generated acetyl-CoA or respiration rate in a plant.
:20 According to a twelfth aspect of the ;invention, there is provided the use of the vector as described above for modulating seed oil content or weight in a plant.
According to a thirteenth 2~spect of the invention, there is provided the use of the vector as described above for modulating flowering or generation time in a plant.
According to a fourteenth aspect of the invention, there is provided 8~
process of producing a transgenic plant, said process comprising introducing the isolated, purified or recombinant nucleic acid sequence as described above into a genoma of a plant thus producing a transgenic plant.

-4c-According to a fifteenth aspect of the invention, there is provided a method of changing the oil or biopolymer content of a plant, plant storage organ or plant seed, said process comprising:
introducipg a sense or anti-sense nucleic acid eonStruct into a plant S transformation vector to produce s modified plant transformation vector, wherein said sense or anti-sense nucleic staid construct comprises the isolated, purified or recombinant nucleic acid sequence as described above;
transforming said plant, plant storage organ or plant seed's genome with said modified plant transformation vector; and growing said plant, plant storage organ or plant seed and extracting said oil or biapoiymer, According to a sixteenth aspect of the invention, there is provided an isolated polypeptide, comprising a BrassiG$ pyruvate dehydrogenase kinase (PDHK) protein, said polypeptide selected from the group Consisting of SEGI ID NQ:S, SEQ ID
N(~:6, SEQ ID N4:7 and SEQ ID N0:8.
According to a seventeenth aspect of the invention, there is provided an isolated, purified or recombinant nucleic acid selected from the group consisting of SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID NQ:14 and SEQ I~ N0:15.
H

[0019] The PDHK of the invention is useful in manipulating PDC activity and the respiration rate in plants. For example, transforming plants with a construct containing the partial PDHK gene in an anti-sense orientation controlled by a constitutive promoter can result in increased mitochondrial PDC activity, an increased production or availability of mitochondrially-generated acetyl-CoA, and hence, an increased respiration rate.

[0020] Additionally, over-expressing the full-length PDHK in a sense orientation may reduce the activity of mitochondrial PDC, resulting in decreased respiratory rates in tissues, such as leaves or tubers, to decrease maintenance respiration and thereby increase the accumulation of biomass.

(0021] Some of the manipulations and deliverables which are possible using the PDHK gene or a part thereof, include, but are not limited to, the following: seeds with increased or decreased fatty acid and oil content; plants exhibiting early or delayed flowering times (measured in terms of days after planting or sowing seed);
plants with increased or decreased vegetative growth (biomass); plants with root systems better able to withstand low soil temperatures or frost; plants with tissues exhibiting higher or lower rates of respiration; plants exhibiting an enhanced capacity to accumulate storage compounds in other storage organs (e.g., tubers); plants exhibiting an enhanced capacity to accumulate biopolymers which rely on acetyl moieties as precursors, such a polyhydroxyalkanoic acids or polyhydroxybutyric acids (Padgette et al., 1997).

[0022] In another exemplary embodiment, the invention discloses a genetically transformed plant having a means for modulating mitochondrially generated acetyl-CoA
and/or respiration rate in the genetically transformed plant, wherein the means is operatively linked to a promoter.

[0023] In a further exemplary embodiment, the invention involves a process for modulating mitochondrially generated acetyl-CoA and/or respiration rate in a transgenic plant. The process includes cloning a gene encoding a Brassica pyruvate dehydrogenase kinase protein into a vector, positioning the gene in an anti-sense orientation, and transforming a plant with the vector.

[0024] An additional process for modulating mitochondrially generated acetyl-CoA and/or respiration rate is disclosed in an additional exemplary embodiment. The process includes cloning a gene encoding a Brassica pyruvate dehydrogenase kinase protein into a vector, reducing production of the Brassica pyruvate dehydrogenase kinase protein, and transforming the vector into a plant.
BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1. Oil content (% of seed dry weight) of T3 seed from B. napes cv NEX710 transgenic lines transformed with the B. napes Phaseolin:Antisense PDHK
construct (R) compared to the oil content of seeds from their corresponding sibling null (S) control plants. Each value represents the average of three determinations.

[0026] FIG. 2. Fatty acid composition of T3 seed oil from B. napes cv NEX710 transgenic lines transfornied with the B. napes Phaseolin:Antisense PDHK
construct (R) compared to the fatty acid composition of seed oil from their corresponding sibling null (S) control plants. % Sats = % of total saturated fatty acids (hatched bars); % Mono = % of total monounsaturated fatty acids (black bars);
% Poly =
total polyunsaturated fatty acids (speckled bars). Each value represents the average of three determinations.

[0027] FIG. 3. Oil content (mg total fatty acids/100 seeds) of T3 seed from B.
napes cv NEX710 transgenic lines transformed with the B. napes Phaseolin:Antisense PDHK construct (R; hatched bars) compared to the oil content of seeds from their corresponding sibling null (S; black bars) or non-transformed (NT-Con; grey bar) control plants. Each value represents the average of three determinations.

[0028) FIG. 4. Average 100-seed weight (mg) of T3 seed from B. napes cv NEX710 transgenic lines transformed with the B. napes Phaseolin:Antisense PDHK
construct (R; hatched bars) compared to the oil content of seeds from their corresponding sibling null (S; black bars) or non-transformed (NT-Con; grey bar) control plants. Each value represents the average of three determinations.

[0029] FIG. 5. Transgenic Nex710 UBP:a/sBnapusPDHK line 20-9 and its vector-only control on July 18th. The transgenic line (left hand side) is fully bolted and is ready to flower (floral buds are well-developed), while the control line (right hand side) bears several leaves only and did not flower until August 1 st, 14 days later.

[0030] FIG. 6. Transgenic Nex710 UBP:a/sBnapusPDHK lines 20-7 and 32-2 9 (right hand side (rhs)) and vector-only controls (left hand side (lhs)) on August 1 lth.
The controls are in full bloom, while the transgenic lines are well into developing siliques, and have completed the flowering stage.

BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The best modes for carrying out the invention are apparent from PCT/CA98/00096 (PCT International Publication W098~.35044), and from the following description of the results of tests and experiments that have been carned out by the inventors. Related technology is disclosed in U.S. Patent 6,265,636 to Randall et al.

[0032] All plant cells undergo mitochondria) respiration and this ubiquitous process is affected by the activity of the PDC and its regulators PDHK and PDCP.
Manipulation of PDHK activity through silencing mechanisms (e.g. anti-sense RNA
technology) using plant transformation can affect, e.g., PDH activity, mitochondria) respiration, seed oil content, flowering time, and growth rate.

[0033] A number of ways exist by which genes and gene constructs can be introduced into plants, and a combination of plant transformation and tissue culture techniques have been successfully integrated into effective strategies for creating transgenic crop plants. These methods, which can be used in the present invention, have been extensively reviewed elsewhere (Potrykus, 1991; Vasil, 1994; Walden and Wingender, 1995; Songstad et al., 1995), and are well known to persons skilled in the art. For example, one skilled in the art will certainly be aware that these methods include Agrobacterium-mediated transformation by vacuum infiltration (Bechtold et al., 1993) or wound inoculation (Katavic et al., 1994), Agrobacterium Ti-plasmid-mediated transformation (e.g., hypocotyl (De Block et al., 1989) or cotyledonary petiole (Moloney et al, 1989) wound infection), particle bombardment/biolistic methods (Sanford et al., 1987; Nehra et al., 1994; Becker et al., 1994) or polyethylene glycol-assisted protoplast transformation (Rhodes et al., 1988; Shimamoto et al., 1989) methods.

[0034] As will also be apparent to persons skilled in the art, and as extensively reviewed elsewhere (Meyer, 1995; Datla et al., 1997), it is possible to utilize plant promoters to direct any intended up- or down-regulation of transgene expression using constitutive promoters (e.g., those based on CaMV35S), or by using promoters which can target gene expression to particular cells, tissues (e.g., napin promoter for expression of transgenes in developing seed cotyledons), organs (e.g., roots), to a particular developmental stage, or in response to a particular external stimulus (e.g., heat shock).

[0035] Particularly preferred plants for modification according to the present invention include borage (Borago spp.), Canola (B. napus, B. rapa, or B.
juncea), castor (Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays), cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oenothera spp., olive (Olea spp.), palm (Elaeis spp.), peanut (Arachis spp.), high erucic rapeseed (B.
napus) germplasm, safflower (Carthamus spp.), soybean (Glycine and Soja spp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Yernonia spp., wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.), oat (Avena spp.) sorghum (Sorghum spp.), rye (Secale spp.) or other members of the Gramineae.

[0036] Methods of modulating PDHK content and composition in a plant is described in U.S. Patent 6,265,636 B1 to Randall et al. (see, e.g., columns 26 through 30 and 37 through 38).

[0037] The invention is fi.~rther described by use of the following exemplary embodiments.
EXAMPLE I

[0038] The PDHK gene was cloned from Brassica napus (cv. Quantum) (SEQ
ID NO:1 ) by Reverse Transcription - Polymerase Chain Reaction (RT-PCR) amplification. Total RNA was extracted from young leaves (Wang and Vodkin, 1994) and cDNA produced by reverse transcription (Life Technologies, Inc., 2002, M-MLV
Reverse Transcriptase page 16-25). Using this cDNA and several pairs of degenerate primers (SEQ ID N0:9 and SEQ ID NO:10) designed from conserved segments of known PDHK amino-acid sequences from Arabidopsis (CAA07447) and corn (AF038585), a fragment of about 1 kb was amplified by the Polymerase Chain Reaction (PCR). The fragment was cloned into the TOPO cloning vector (pCR TOPO 2.1, Invitrogen) and fizlly sequenced in both orientations (DNA lab, PBI/NRC). DNA
sequence analysis revealed that this amplicon shared a high degree of homology with other known mtPDHK genes.

[0039] The missing termini of the gene were subsequently amplified using a 3' and 5' Rapid Amplification cDNA Ends (RACE) kit (Life Technologies, Inc., 2002, 3' RACE system and 5' RACE system pages 21-25). The full-length gene was produced by PCR using Vent DNA polymerase (New England Biolabs) and gene specific primers (SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, and SEQ ID NO:15) designed from the DNA sequence information provided by the RACE-generated fragments. These primers encompassed each end of the gene, i.e., the start and stop codons. At this stage, restriction sites were also added by PCR for subsequent anti-sense insertion of the PDHK gene into expression vectors such as pSE 129A bearing the napin promoter (PBI/NRC) or pBBV-PHAS with the phaseolin promoter (courtesy of DowAgro Science). Orientation of the inserted gene was verified by restriction digestions and DNA sequencing.

[0040] DNA sequence analyses showed that the B. napus PDHK gene has an 1104 by long open reading frame (386 AA). It was analyzed with respect to other PDHK sequences (GenBank) available and amino-acid comparison revealed 93% and 71 % identity with Arabidopsis and corn sequences respectively. All DNA
analyses (sequence alignments, primer design, etc.) were performed using the DNASTAR
LasergeneTM software package.
EXAMPLE II

[0041] The same approach employed for cDNA cloning and sequence analysis of PDHK from Brassica napus as described in Example I was followed for the cloning and sequence analysis of the B. rapa PDHK gene (SEQ ID N0:2).
EXAMPLE III

[0042] The same approach employed for cDNA cloning and sequence analysis of PDHK from Brassica napus as described in Example I was followed for the cloning and sequence analysis of the B. oleracea PDHK gene (SEQ ID N0:3).
EXAMPLE IV

[0043] The same approach employed for cDNA cloning and sequence analysis of PDHK from Brassica napus as described in Example I was followed for the cloning and sequence analysis of the B. carinata PDHK gene (SEQ ID N0:4).
EXAMPLES V-VIII

[0044] The same approach employed for cDNA cloning and sequence analysis of PDHK from Brassica napus as described in Example I is followed for the cloning and sequence analysis of PDHK gene from B. nigra, B. juncea, B. oleifera, B.
balearica, B.
cretica, B. elongata, B. tourneforii, and B. biennis.
EXAMPLE IX

[0045] The oil content of a plant (e.g., borage (Borago spp.), Canola (B.
napes, B. rapa or B. juncea), castor (Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays), cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oenothera spp., olive (Olea spp.), palm (Elaeis spp.), peanut (Arachis spp.), high erucic rapeseed (B. napes) germplasm, safflower (Carthamus spp.), soybean (Glycine and Soja spp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Yernonia spp., wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.), oat (Avena spp.) sorghum (Sorghum spp.), rye (Secale spp.) or other members of the Gramineae)) is modified by first introducing an anti-sense nucleic acid construct into a plant transformation vector (e.g., one including a plant promoter) to produce a suitable plant transformation vector by means known to those of skill in the art (see, e.g., columns 26 to 30 of U.S.
Patent 6,265,636 to Randall et al.) The anti-sense nucleic acid construct includes recombinant nucleic acid sequence encoding Brassica PDHK (e.g., the nucleic acid sequence of SEQ
ID NO:1, SEQ ID N0:2, SEQ ID N0:3, or SEQ ID N0:4). The plant's genome is thus transformed (see, e.g., columns 33 through 37 of U.S. Patent 6,265,636) with the modified plant transformation vector. The plant seed is grown, and oil is extracted from the resulting plant seed.
EXAMPLE X

[0046] Brassica napes (Bn) PDHK in an anti-sense orientation modulates seed oil content and weight in B. napes strain Nex 710. The full length clone of the PDHK
gene (SEQ ID NO:1) was inserted in an anti-sense orientation behind a phaseolin (PHAS) promoter (See, U.S. Pat. 5,504,200) in BBV-PHAS vector (Dow AgroSciences) and mobilized into E. coli (DH Sa) and Agrobacterium (GV3101 pMP90).

[0047] Two PmeI restriction (NEB) sites were added to the PDHK gene (SEQ
ID NO:1 ) for cloning. The PDHK gene (SEQ ID NO:1 ) was inserted in anti-sense (a/s) orientation behind the PHAS promoter to form Bn PDHK-PHAS. The full length B.
napes (Bn) PDHK gene (SEQ ID NO:1) was obtained by RT-PCR and RACE as previously described herein. Forward primer GTTTAAACATGGCGGTGAAGAAGG
(SEQ ID N0:16) and reverse primer GTTTAAACTCATGGCAAAGGCTCC (SEQ ID
N0:17) were designed to add a PmeI restriction site on each end of the PDHK
gene.
The fizll length Bn PDHK gene was amplified using PCR (with Vent polymerase, NEB) with primers (SEQ ID N0:16 and SEQ ID N0:17) that added the PmeI restriction sites.
The amplified PCR product (amplicon) was run on a gel and cleaned from the agarose gel using a Qiagen kit. The cleaned PCR product was cloned into a TOPO cloning vector as previously described herein, transformed into E. coli, grown on media containing Ampicillin and the TOPO cloning vector including the PDHK gene was obtained using DNA preps (Qiagen).

[0048] The obtained TOPO cloning vector including the PDHK gene and the pBBV-PHAS-iaaH vector were digested with PmeI available from NEB. The digested pBBV-PHAS-iaaH vector was desphosphorylated, and the digested products were cleaned by running the products on a gel and purifying. The obtained PHDK gene and digested pBBV-PHAS-iaaH were blunt ligated with T4 ligase available from NEB.
The ligated vector, including the PHDK gene, was electroporated into E coli strain cells available from BRL in a ligation mixture, and the transformed cells were grown on media containing Spectinomycin. DNA preps (Qiagen) were performed on cells containing the vector to obtain Bn PDHK-PHAS. The orientation of the PDHK gene in the vector was checked by XhoI digestion since sense and anti-sense inserts of the PDHK gene result in different digestion patterns.

[0049] Agrobacterium (strain GV 3101, pMP90) was transformed by electroporation with an anti-sense (a/s) PDHK insert-containing DNA prep. The transformed Agrobacterium cells were grown on spectinomycin containing media, and DNA preps were collected and sequenced to check the size and orientation of the PHDK
gene insert. T'he presence of the PHDK insert gene and its actual orientation were checked with several rounds of sequencing before plants were transformed.

[0050] B. napus line Nex 710 (Dow Agrosciences) was chosen as an elite line and used for transformation. About 7,000 B. napus explants (hypocotyls) were inoculated with the Agrobacterium containing the a/s PHAS: Bn PDHK construct.
About 6,500 of the transformed explants formed a callus, and most of the explants formed shoots (transformed or not). After transfer of the explants in 3 different medics having gradually increasing levels of heribicide (L-PPT; 0.8-10 mg/1) for selection, 77 shoot explants were allowed to root in rooting media. Most of the explants formed roots and were PCR screened for the PDHK gene and the selection marker. 55 transformed plants were positive for PAT genes. More PCR reactions were preformed to check for the presence of the marker gene in the plant and the absence of a region of the vector that should not be inserted in the plant, i.e., outside the T-DNA borders, as negative controls.

[0051] Southern analyses using the PAT gene as a probe confirmed the transformation events, gave better estimates of the success of transformation (about 0.8%), and indicated the number of copies of the transgene that were inserted.

52 plants had one or several inserts, and 42 plants were retained for having 1 or 2 copies of the transgene.
[0052] Leaf painting with 3 mg/L Liberty was performed on Tl plantlets and resistant plants per line were selected. The first generation of transgenic lines was harvested and seeds were analyzed by gas chromatography (GC) for oil content.
Several lines looked promising for having up to a 10% increase of seed oil. At this stage, the populations were still genetically segregating (the transgene insertion is hemizygous) and, therefore, the data on oil content represents an average value for each population of segregating genotype. 10 plants of each of the 42 lines that produced seeds were seeded in greenhouse for production of the T2 generation.

[0053] 42 lines were selected based on the number of inserted transgenes (1 or 2 copies of the transgene) for further generations. 10 plants per line were seeded, and the TZ seeds were analyzed by GC for oil content. Several promising lines were identified and exhibited increased oil content. For instance, 16 lines had a 17% increase of oil content and 1 line had a 36% increase of oil content. Other promising cell lines had increased seed weight. For instance, 38 lines had a 16% increase in seed weight and 1 line had an 80% increase in seed weight. Other lines had increased total oil content on a per seed basis. 32 lines had a 27% increase in total oil content per seed and 1 line had a 78% increase of total oil per seed as compared to the control non-transformed wild type or the respective sibling null lines.

[0054] T3 seeds (9 transgenic lines, 10 plants each, plus vector-only, nulls and wild type control lines) were harvested and analyzed for oil content, composition and average seed weight. The T3 data are presented in FIGS. 1-4.
EXAMPLE XI

[0055] Utility of Bn PDHK (SEQ ID NO:1) in modulating flowering (generation) time. The Bn PDHK gene (SEQ ID NO:1) was inserted in an anti-sense orientation behind the Ubiquitin (UBQ) gene promoter (See, U.S. Pat.
6,054,574) in the pDAB4016M vector (Dow AgroSciences). The Bn PDHK gene was obtained by amplifying the full length Bn PDHK gene (obtained by RT-PCR, reverse transcription PCR, and RACE as previously described herein) with a forward primer CGTACGATGGCGGTGAAGAAGGCTA (SEQ ID N0:18) and a reverse primer GGTGACCTCATGGCAAAGGCTCCT (SEQ ID N0:19) designed to add two restriction sites, BstE II and BsiW I, at each end of the Bn PDHK gene. The amplification was performed by PCR using Vent polymerase available from NEB.
The PCR product was run on an agarose gel and cleaned using a Qiagen kit. The cleaned PCR product was cloned into a TOPO cloning vector as previously described herein and the TOPO cloning vector including the Bn PDHK gene was transformed into a cell. The transformed cell was grown on media including Ampicillin and the TOPO cloning vector including the Bn PDHK gene was obtained from the cells using DNA preps from Qiagen.

[0056] The TOPO preps and pDAB4016M vector (courtesy of Dow Agrosciences) were digested with BstE II and BsiW I restriction enzymes available from NEB. The digested pDAB4016M vector was dephosphorylated, and the digested products were cleaned by running on a gel and using a Qiagen kit. The digested pDAB4016M vector and the digested TOPO preps including the Bn PDHK gene were blunt ligated with T4 ligase available from NEB. E. coli strain DH 20B cells, available from BRL, were ligated with a ligation mixture including the ligated vector.
The transformed cells were grown on media including Erythromycin and DNA preps (Qiagen) were performed to obtain the a/s Bn PDHK-DAB vector.

[0057] Agrobacterium strain GV 3101 pMP90 was transformed by electroporation with the a/s insert-containing DNA prep. The Agrobacterium cells were grown on media including Erythromycin, and DNA preps were performed to obtain the a/s insert-containing vector. The a/s insert-containing vectors were sequenced to check the size and orientation of the insert. The presence of the PDHK, the PAT
marker gene and the Ubiquitin promoter was verified by PCR performed on DNA obtained from transformed Agrobacterium DNA.

[0058] 9,000 explants (hypocotyls from the B. napus Nex 710 line) were transformed with Agrobacterium containing the a/s Bn PDHK-DAB vector (5,000 explants) or the pDAB vector as a control (4,000 explants). The transformed explants were selected on a LPPT-containing medium as known in the art, and the transformed explants were screened by PCR for the presence of the PDHK insert, the PAT
gene and the UBQ promoter. 60 plants were analyzed by Southern blots (using a probe for the PAT marker gene) to check the number of transgenes that were inserted. 'The transformation rate was determined to be 0.9% and 37 plants with one or two copies of the insert were retained for further generations.

[0059] T, seeds were harvested and seeded in a greenhouse. The resulting plantlets were leaf painted with Liberty herbicide to detect null-sib lines.
34 lines were retained (8-10 plants each) and 17 lines having a corresponding null-sib line were obtained as control, the rest of the lines had vector-only as a control.

[0060] Most of the transformed B. napus Nex 710 plants displayed a significant early flowering phenotype in the TZ generation as compared to the controls.
FIG. 5 shows a transgenic plant with anti-sense PDHK flowering 2 weeks earlier than the control. Thus, the transformed plant exhibits a 15% shorter cycle when assuming an average cycle of 100 days. A demonstration of early flowering by down-regulating PDHK gene in B. napus Nex 710, a double haploid (DH) line which is known for uniform maturity, demonstrates the utility of manipulating the expression of the PDHK
gene in eliciting early flowering.

(0061] Although described with the use of particular exemplary examples and embodiments, the scope of the invention is to be determined by the appended claims.

REFERENCES:
Bechtold, N., Ellis, J. and Pelletier, G. ( 1993) In plants Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci.
Ser.
III Sci. Vie, 316: 1194-1199.
Becker, D., Brettschneider, R. and Lorz, H. ( 1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J. 5: 299-307.
Datla, R., Anderson, J.W. and Selvaraj, G. (1997) Plant promoters for transgene expression. Biotechnology Annual Review 3: 269-296.
De Block, M., De Brouwer, D. and Terming P. ( 1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91: 694-701.
Jorgensen, R.A. and Napoli, C.A. (1994) Genetic engineering of novel plant phenotypes. U.S. Patent No. 5283184.
Katavic, V., Haughn, G.W., Reed, D., Martin, M. and Kunst, L. (1994) In plants transformation ofArabidopsis thaliana. Mol. Gen. Genet. 245: 363-370.
Kishore G.M. and Somerville, C.R. ( 1993) Genetic engineering of commercially useful biosynthetic pathways in transgenic plants. Current Opinion in Biotechnology. 4: 152-158.
MacKenzie, S.L. and Jain, R.K. (1997) Improvement of oils crops via biotechnology.
Recent Res. Dev. In Oil Chem. 1: 149-158.
Meyer, P. ( 1995) Understanding and controlling transgene expression. Trends in Biotechnology, 13: 332-337.
Moloney, M.M., Walker, J.M. and Sharma, K.K. (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep. 8: 238-242.
Nehra, N.S., Chibbar, R.N., Leung, N., Caswell, K., Mallard, C., Steinhauer, L. Bags, M.
and Kartha K.K. (1994) Self fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant J. 5: 285-297.
Padgette, S.R., Gruys, K.J., Mitsky, T.A., Tran, M., Taylor, N.B., Slater, S.C. and Kishore, G.M. (199?) Strategies for production of polyhydroxyalkanoate polymers in plants. Plant Physiol. Suppl., 114: 3 (abstract 10003).
Potrykus, I. ( 1991 ) Gene transfer to plants: Assessment of published approaches and results. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225.

Rhodes, C.A., Pierce, D.A., Mettler, LJ., Mascarenhas, D. and Detmer, J.J. ( 1988) Genetically transformed maize plants from protoplasts. Science 240: 204-207.
Sanford, J.C., Klein, T.M., Wolf, E.D. and Allen, N. (1987) Delivery of substances into cells and tissues using a particle bombardment process. J. Part. Sci. Technol.
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27-37.
Shimamoto, K., Terada, R., Izawa, T. and Fujimoto, H. (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274-276.
Somerville, C.R. ( 1993) Future prospects for genetic modification of the composition of edible oils from higher plants. Am. J. Clin. Nutr. 58 (2 Suppl.): 2705-2755.
Songstad, D.D., Somers, D.A. and Griesbach, R.J. (1995) Advances in alternative DNA
delivery techniques. Plant Cell, Tissue and Organ Culture 40: 1-15.
Vasil, LK. ( 1994) Molecular improvement of cereals. Plant Mol. Biol. 25: 925-937.
Walden, R. and Wingender, R. (1995) Gene-transfer and plant regeneration techniques.
Trends in Biotechnology 13: 324-331.
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132-145.

SEQUENCE LISTING
(1) GF,~1)RALiNFUItMATION:
(i) APPI~CANT: NATIONAT., RESEARC~I COUNCIL CANADA
Z(?U, rITAO
TAYLOR, DAV)I? C.
MARELL1A. ELITABETIi FRANCE
(ii) ~ OF INVENTION: BRASSICA PYRU'VA2'E Dl~IYDROGENASE
HINASE GENE
(iii) NUMBER OF SEQUENCES: 19 (iv) CORL~SPONI~NGE ADDRESS:
(A) ADDRESSEE: Natipnal Rcsrarch Council Canada {B) SCREET: 1200 Montreal Road, M-58, EQ 06B
(C) CTTY: Ottawa (D) S'CATE: Ontario (>;) COUNTRY: Canada (F~ ZIP: K1A ORb (v) C011BnTT13R READABLE FORM:
(A) MEDIUM TYNE: kloppy disk (B) CQA~fPLrTER: ISM PC compatible (C) ~E1ZATIN(3 S'Y'ST'EM:1'C-I70SIM5-DOS
(I7) 50ItI"WARB: Patentln Release #I Ø Version #1.30 (vi) CUR12ENT APPLTCATTON DATA:
(A) APPt,TCA'I'TON NCIIvJBER: CA 2.474,906 (B) FILING DATE: 2004-AUG-1 B
(viii) ATTORNEY/AGENT )~RMAT10N:
(A) NAME: Margaret Mcl~ay (C} RF.~FELtENCC)yJDOCCI~ET NUMBER: 3015~5442 (ix) TE'LECOMMUNTCATION INFORMATION:
(A) TBi~PHONE: 613-991-6853 (B) TI~~Ek'A1~: 613-952-6082 (2) LNIxORbSATTON FOR SEQ ID NO;1:
(i) SEQUk~ICE CHARACTERISTICS:
(A) I~:NGTT3: 1104 bast pairs (B) TY. PI;: nucleic acid (C) SZ'RANDEDNESS: singe (D) T(7POLOGY: linear (ii) MOLF~ULE TYPE: DNA (genomic) (xi) SEQ'UENCE DESCRIPTION: SEQ ID NO;1;
ATGGCGG TGA AGAAGGCTAG CGAGATCrTTT TCGAAGAGCT TGATCGAGGA CGTTCACA(~'A 60 TGGG(~AT~"sCA TGAAC3CAGAC GGGCGTGAGC CTCAGGTACA TGATC3GAGTT CGICrTTCCACT 1~

CCCACTGAGA GAAACCTTCT GATCTCGGCG CAGTI'z'CITC ACAAGGAGCT180 TGCGATTCGG

AT~CGA,GGC GTGCGATCGA ACTCGAGAGG GTGCCZTATG GCCTCTC~'GA240 GAAACCTGCC

GTCI'I'GAAGG TAAGACrATTG GTATGTGGAG TCATTCAGGG ACATGAGAGC300 GTTTCCTGAG

ATCAAGGATA CT'GCTGATGA GAAAGAGTTC ACACAGATGA TCAA(rGCTGT360 TAAAGTAAGG

CACAACA,ACG TGGTTGCCAT GATGOCTCTG GGTGTGAACC AGCTGAAGAA420 AGGAATGAAA

CTCTACGAAA AGCT'2 GATCrA GATTCATCAG T'~'I'C1"TGATC 480 GCTI'CTAC'Cf O'r'CTCGTATA

GGGATCCGTA TGCTTATCGG GCAGCATGTT GAGThGCATA ATCCAAACCC540 ACCACTTCAC

TGCTAGTGAA

GATGCAA,GGT CGATTTGTTT CAGAGAGTAT GG7~: TCtGCTC CGGAGATAAA660 CATATATGGC

GATCCAA.GTT TCACTTTTGC GTATGT';i'CCG ACCCATTTGC ATC'rTATGGT720 GTATGAGTTA

GTCAAGA,ACT CTGTCCGTGC TGTCCAAGAG CGGTTTGTTG ACTCTGATAG780 GGTTGCACCA

CCAATCCGTA TCATTGTTGC TGATGGA.ATC GAAGATGTTA CAATAAAGGT840 CTCAG,t~TGAA

GGTGGAGGTA TACCGAGAAG CGGTCTGCCT AAAATATT'CA CTTACCTGTA900 CAGCACTGCA

AGAAACC',CAC TT'GAAGAAGATGTGGACTTG GGAACCGCTG ATGT1'CCCCT960 GACTATGGCT

GGTTATGGTT ATGGTGTGCC TATTAGTCGC '~'TGTATGCTC C1CTATTTTGG1020 TGGAGATTTG

TCGTCTTGGA

(2) iNFU~2MATION FOR SEQ lD N0:2:
(i) SEQL'fENCE CHARACTPRISTICS:
(A) T~NGTH: 1 I04 base pairs (B) TYIyE: nucleic acid (C) S7t R.ArTDEbN~SS: single (D) T~~POLOGY: linear (ii} MOI_.I~GULE TYPE: bNA (genonuc}
(xi) SEQ1JE.NCE DE,SCA~IPTION: SEQ ID N0:2:

CGTTCACAGA

rGGGGATGCA TGAAGCAGAC GGOCGTGAGC GTCAGGTACA TGATGGAGTT120 CGGTTCCACT

CCCACTG.AGA GAAACCT1'CT GATCTCGGGG CAGTTT'CTTC ACAAGGAGCTI80 TCCGATTCGG

GAAACC'I'GCC

GTGTTGA,AGG TGAGGGATTG GTATGTGGAG TCATTCAGrCri3 ACATGAGAGC300 GTTTCCTGAG

ATCAAGGATA CTGCTGATGA GA.AAGAGTTC ACTCAGATGA TTAAGGCTGT360 TA,AAGTAA,GG

CACAAC.AACG TGGTTCCCAT GATGC:~C'1'CTG GGTGTG,A.ACC 420 AGCTGAAGAA AGGAATGAAA

CTCTACCiAAA AGCTTGATOA GATTCATCAG TITCITGATC GCTTCTACIT480 GTCTCGTATA

GGGATCCGTA'1'GCTTATCGG GCAGCATGTT GAGTTGCATA ATCCAAACCC540 A,CCAGTTCAC

ACA('rTC~I~C"rTT ACATACACAC CAAGATGTCC CCTATGGAGG 600 TGGCAAGGAA TGCTAGTGAA

GATGCAAGGT CGATTTGTTT CAGAGAGTAT GGTI'CI'GCTC CGGAGATAAAG60 CATATATGGC

GATCCAAGTT CCACTYTTCC GTATGTTCCG ACCCATTTGC ATCTTATGGT72,0 GTATGAGT'TA

GTCAAC3AACT GTCTCCGTGC TGTCCAAGAG CGGTTTGT><'G ACTCTGA'r'AG780 G(3TTGCACCA

GCAATC(:GTA, TCATTGTTGC TGATGGAATC GAAGATGTTA CAATAAAGGT840 CTCAGATGAA

GGTGGAiiGTA TACCGAGAAG CGGTLTCCCT AAAATATTCA CTTACCTCTA900 CAGCACTGCA

GACTATGGCT

GGTT.A.TC~GTT ATGOTGTGCC TATTAGTCGC TTG2'ATGCTC GCTATTTTGG1020 TGGAGATrTG

TCGTCTTGGA

GACTCGC:AGG A.GCCTTTGCC ATGA 1104 (2) l:NrrORMATTON FOR SEQ >iD Np:3:

(i) SEQTJINNCE CHARACT"F~2TSTICS:

(A) z.ENGTH: I 104 base pairs (B) TYPE: nucleic acid (C) STRAIVDEDNESS: single (1~)'1'QPQLOGY: linear (ii) 1VI01'_~3C~(1LE TYPE: 3~NA (gcnomio) (xl) SEQCl'RN'CE DESCRI)?'1'ION: 5EQ ID N0:3:

ATGGCG(iTGA AGAAGGCTAG CGAGATGTTT TCGAAGAGCT TGATCGAGGA60 CGTTCACAGA

TGGGGA'1CGCA TGAAGCAGAC GGGCGTGAGC CTCAGGTACA TGATGGAGTT120 CGGTTCCACT

CCCAGTC~AGA GGAACCTCCT GATCI'CGGCG CAGTTx'CTTC ACAAGGAGC'T180 TCCGATTCGG

ATCGCGAGGC GTGCGATCGA ACTCGAGACG CTGCCTTATG GCCTCTCY'GA240 GAAACCTGCC

GTTTCCTGAG

ATCAAGtyATA CTGCTGAXGA GAAAGAGTTC ACACAGATGA TTAAGGCTGT360 TAAAGTAAGG

AGGAATGAAA

CTCTACGAAA AACTCGATGA GATTCATCAG TTTCTTGATC C~TACTT 480 GTCACGTATA

GGGATCf;QTA TGCTTATCGG GCAGCATGTT GAGTTGCATA ATCCAAACCC540 ACCACTTCAC

ACTGTGC'rGTT ACATACACAC CAAGATGTCT CCTATGGAGG TGGCAAGGAA600 TGCYAGTGAA

GATGCAAGGT CGATT tG~''rT CASAGAGTAT GC1TTCTGCTC CGGAGATAAAb60 C~vtTATATGGC

GATCC.AAGTT TCACCTITCG GTATGTACCA ACCCA'ITI'GC ATCTTATGGT720 GTATGAGCTA

GTCAAGAACT CTGTAGGTGC TGTCC;AAGAG CGATTTCr'TTG ATTCTGATAG780 GGTTGCACCA

CCAATCC~TA TCATTGTTGC TGATGGAATC GAAGATC"TTA CAATAAAGC3'T840 CTCAGATGAA

GGTGGA(sGTA TACC:GAGAAG GGGTCTCiCCC AAAATATTCA CTTACC'TSTA900 CAGCACTGCA

AGAAAC(~CGC TTGAAGAAGA TGTGGACTTG GGAACAC3CTG ATC"TACxCGT960 GACW'ATG(~CT

GGTTATCiGTT ATGGTCTCiCC YATTAGTCGC TTG'1'ATGCTC GATAC'~TTGG1020 TGGAGATTTG

CAGATCATAT CCATGGAAGG ATACGdGACT GATGCTTACT TGCAC''TTGTC1080 TCC"TCiTGGA

GACTCCit.'AAG AGCCJCITTGCC ATGA 1104 (2} 1NFORIViATIC)N FOrt SBQ 1p N0:4:
(i) SL?Q1JENC~ CNARACT~TS'~ICS:
(A) LENGTH: 1104 base pairs (B} TYPE: nucleic acid (G~ ST'RANDEDNESS: smile (D) TOPOLOGY: linear (ii) MOLECL3L~ TYPE: DNA (genomic) (xi) SEQUENCIr IaPSCRIP'I FON: SEQ ID N0:4:

ATGGCGCrTGA AGAACrGCI'AG CGAGATGTTT'1'CGAAGAGCT'TGATCGAGGA60 CGTTCACAGA

TGOGGA7.'GCA TGAAGCAGAC CrGGCC"TGAGC GTCACiGTACA TGATGGAGTT120 CGGTTCCACT

CCCACTCaACIA GGAACCTGCT GATCTCGGCG C:AGTTTCTTC ACAAGGAGCT180 TCCGATTCCiG

ATCGCGAGGC GTGCGATCGA ACTCGAGACG CTGC:CTTA'.I'G GCCTCTC:TGA240 GAAACC.'TGCC

GTCTTGAAGG TAAGAGATTG GTATGTGGAG TCATTCACiGCr' ACATGAGAGC300 GTTTCC)'GAG

ATCAAGCiATA CTGCTGATGA GAAAGACirTTC ACACAGATGA TTAAGGCTGT360 TAAAGTAAGCi CACAACAACG TGGTTCCCAT GATGGGTCTG GGTGTrAACC AC~CTGAAGAA420 AGOAATOAAA

C"fCTACGAAA AACTCGATGA GATTCATCAG'ITIZ"1'hGATC GCTTCTACTT480 GTCACGTATA

GGGATCC:GTA TGCITATCGG GCACiCATGTT GAGTTGCATA ATCCAAACCC540 ACCAC'TTCAC

ACTGTGC~G'~T ACATACACAC CAAGATGTCT CCAATGGAGG TGGCAACyGAA600 TGCTAGTGAA

GATGCAAGGT CGATTTGTTT CCGAGAGTAT GGTTC.'TGCTC CGGACirATAAA660 CATATATGGC

GATCCAAGTT'~CACCTT 1'CC GTATC'TACCA ACCCATTTC~C ATCTTATGGT720 GTATGAGCTA

GTCA,AGAACT CTCTACGTGC TGTCCAAGAG CGG'T~TGTTG ACTCTGATAG780 C~TTGCACCA

CCAATCC'GTA TCATTGTTGC TGATGGAATC GAAGATGTTA CAATAAAGGT&40 CT'CAGATGAA

GGTGGACiGTA TACCGAGAAG CGGCCTGCCC AAAATAT'>i'CA CTTACCTCTA900 CAGCAGTGCA

AGAAAC(:CGC TTGAAGAAGA TGTGGACTTG GGAACAGCTG ATGTACCCGT960 GACTATGGCT

TGGAGATTT'G

CAGATCA,TAT CCATGGAAGG ATACGGGACT GATGCTTACT TGCACTTATC1080 TCGTGTTGGA

GACTCGC:AGG AGCCTTTGCC ATGA 1104 (2) 1NFO1tIvfATION FOIL SEQ TD N0:5:

(1) S)3QiJENCE CHARACTfiRiSTTCS:

(A) LENGTI-1: 367 amino acids (B) TYPE: amino acid (C) S'TRA1~TDBDNESS:

(D) TOl'C7LOGY: linear (il) MOLECULE TYPE: protein (xi) SEQ'(rENCE DFSCR1P'iION: S1~Q 1D N0:5:

Met Ala Va1 Lys Lys Ala Ser Glu Met Phe Ser Lys Ser >;.cu Tle GIu Asp Val His Arg Trp Gly Cys Met Llrs Gln Thr Gly Val Ser I~eu Arg Tyr Met Met G1u Phe Gly Ser Thr Pro Thr G1u Arg Asn Leu Leu Ile Ser A1a nln Phe Leu His Lys Glu Leu Pro lle Arg lle Ala Arg Arg Ala 11e t'rlu Leu Glu Thr Leu Pro Tyr Gly Leu Ser Glu ~,ys Pro Ala Val Txu Lys Val Arg Asp'lYp Tyr Val Glu Ser Phe Arg Asp Met Arg Ala Phe Pro GIu Tle Lys Asp Thr ALa Asp Glu Lys Glu Phe Thr Gln Met Tle l,ys Ala Va1 Lys Val Arg His Asn Asn Val Val Pro Met Met 1.15 120 125 A1a T.eu Gly Val Asn GIn Leu Lys Lys Gly Met Lys Leu Tyr Glu Lys Leu Asp G!u Ile His Gln Phe Leu Asp Arg 1?he Tyr Leu Ser Arg Tle Gly Tle dug Met Leu Ile Giy Gln His Ya! G!u Leu T-lis Asn Pro Asn 165 !70 175 Pro Pro Len His Thr Val Gly Tyr Be His Thr Lys Met Ser Pro Met (31u Val Ala Arg Asu Ala Ser Glu Asp Ala Arg Ser )de Gds P'he Arg 195 2110 2(15 Gla Tyr Gly Ser Ata pro Glu Yle Asn Ile Tyr Gly Asp Fro Ser Fhe 21(1 215 220 2hr Pbe Prn Tyr Val Pro Thr ~s L.eu His Lcu Met Vai Tyr Cllu X,eu Val Lys AsB Ser Len Arg Ala Val Cllr GIu Arg Phe Val Asp Ser Asp Arg VaI Ala Pro Pro Ile Arg >le pe val Ala Asp C'rly Tle Crlu Asp 2~p 2~5 2'70 Val T1a lte Lys Val Ser Asp Glu Gly Gly Gly Ile Fro A,~g S~ Gly i~eu Fro Lys Ile >Phe Thr Tyr T.w Tyr Ser T'hr Ala Arg Asn Pro Leu Cllu C3lu Asp Val Asp Leu Gly Thr AIa Asp Vat Pr8 Y.eu Thr Met A1a Gly Tyr Gly Tyr City Leu Pro Ile Ser Arg Leu Tyr AIa Arg'I'yr Phe Gly Gly Asp J. eu Gln rie Ife Ser Met Glu Gly Tyr GIy Thr Asp Ala Tyr Leu ~s Leu Ser Arg Len Gly Asp Su Gln Glu Pro Leu Pro (2) T~ORMATION FOR SFQ'!ID N4:6:
(7 SEQiJEhTCE CHARACT13RISZTCS:
(A) L~TGTH: 367 amino acids (B) TYPE: ataiuD acid (G~ SI'RAhIDEDNFSS:
(D) TOPOLOGY: lirsrar (ii) MOLECU1,~ TYPE: protein (xi) SPQ'UFN(''E DESCR>PTTON: S1;Q >D NO_6:
Met Ala Val Lys Lys Ala Ser Cilu Met Phe Ser Lys Ser Leu lle Glu Asp Val DTs Arg'I~p Gly Cys Met Lys G1n Tht c~ly Va1 Ser Len Arg Tyr Met Met Cilu F'he Cxly Ser Thr Pro Thr Gtu Arg Asn Len Leu nc Ser AIa Gln Phe I,eu His Lys Glu Leu Pro lle Arg lle Als Arg Arg Ala lle Glu Leu Glu Thr Leu 1'ro Tyr Gly Leu Sea Glu Lys Pro Ala 'Val Lcu Lvs V al Arg Asp 'I~p Tyr Val Glu Ser Phe Arg Asp Met Arg Ala Phe Pro Glu Ile Lys Asp Thr Ala Asp Glu Lys Glu Phe Thr Gln Met lle Lya Ala Val Lys Val Arg His Asn Asn VaI Yal Pro Met Met 1 l:i 120 125 Ala Leu GIy Val Asn Gln Leu Lys Lys Gly Met Lys Leu Tyr Glu Lys Leu Asp Glu lle His Gln Phe Leu Asp Axg Phe Tys Leu Ser Arg Ile 145 150 15S 1b0 Gly lle Ark; Met Leu Tfe Gly Gln ?<Tis Val GIu Leu I-hs Asn Pro Asn Pro Pro Leu His Tttx Yal Gly Tyr lle ~Tis Thr Lys Met Ser ??ro Met l80 185 190 Glu V al Ala Arg Asn Ala Ser Glu Asp Ala Arg Ser De Cys Phe Arg 19:5 200 205 GIu Tyr Gl,y Ser Ala Pro Glu 11e Asn Ile Tyr Gly Asp Pro Ser Ser 210 215 22.0 'T'hr Phe Pro 'I yr Val pro Thr This Leu His Lau Met V al Tyr Glu Ixu Val Lys Assn Ser Leu Arg Ala Va1 Gln Glu Arg Phe Val Asp Ser Asp 245 ?SO 255 Arg Val Ala Pro )Pro Ile Arg Ile IIe Val Ala Asp Gly Tle Glu Asp Val Thr lle Lys Val Ser Asp GIu Gly Gly Gly Ilt pro Arg Sex Gly ?7:i 280 285 Leu Pro Lys 17e >'Ite Thr Tyr Leu Tyr Ser Thr Ala Arg Asn Pco Leu Glu Glu A.sp Va1 Asp Leu Cly '1'hr Ala Asp VaI Pro Leu Thr Met Ala Gly Tyr GIy Tyr Gly Leu Pro >le Ser Arg Leu Tyr Ala Arg Tyr Phe Gly Gly As;p r.eu GFn Ile Ile Ser Met Glu Gly Tyr Gly Thr Asp Ala Tyr Leu >=li;~ Leu Ser Arg Leu Gly Asp Ser Gln Glu 1?ro Leu Pro 3:55 360 365 (2) INFUF;MATION FOR SLQ )D NO!1:
(1) SEQUBNCE CHARACTERIS'~CS:
(A) 1~NGTH: 367 amino acids (B) 7l'YPB; ammo acid (C) STRANDmNESS:
(D) 7nOPOL.OGY: linear (i!~ MOLFCrJLB TY)?'E: protein (xi) SEQiJENCE D~SCRiPTION: SFQ )m N0:7:
Met Alai Val Lys Lys Ala Sea Glu Met Phe Ser Lys Ser Lcu Tle Glu 1 5 i0 15 Asp Val Ids Arg np Gly Cys Met Lys Girt T'hr Gly Val Ser Lea Axg Tyr Met Nlet Glu P'he Gly Ser Thr >?~ro Thr Glu Arg Asn Leu Leu Ile Ser Ala Gln Phe Leu T~.s ~,ys G1u Leu Pro Ile Arg lle Ala Arg Arg Ala Ile c3lu Leu Glu T'hr Leu Pro Tyr Gly Leu Ser Glu Lys Pro Ala Vat Leu Lys Val Arg Asp Trp 75~r Val Glu Ser Phe Arg ~aa Met Arg Ala Phe Pm Glu lle Lys Asp Thr Ala Asp Glu Lys Glu Phe "1'ltr Gln 1Cb lay 110 Met Ile :Lys Aia'Val Lys Val Arg FTs Asn Asn val vat Pro Met Met L!5 12a 125 Ala T~eu Gly Val Asn Gln Leu Lys Lys Gly Met Lys Leu Tyr Glu Lys Leu Asp. Glu Ile His Gln Phe Lcu Asp Arg plte Tyr Leu 5er Axg lle Gly Ile Arg Met Leu lle Gly Gla His Val Glu r.eu His Asn Pro Asn Pm Pro :Cxtt T~'rs '>('ftr Val GPy Tyr lle I~'ts '1'ht z.ys Met Ser Pro Met Glu Val Ala Arg Asn Ala Ser G1u Asp Ala Arg Sea Ile CSrs Phe Arg 195 Z00 ?05 Glu Tyr Gty Sea Ala Pro Glu lle Asn Ile Tyr Gly Asp Pro Ser Phe 21 Ci 215 220 Tlu Phe Pm Tyr Val Pro Thr FFts Leu Iits Leu Met Val Tyr Glu Leu Val Lys Asa Sea Leu Arg Ala VaI Gln Glu Arg Phe Vat Asp Ser Asp Arg Val Ala Pro Pro Ile Arg )Qe I1e Yal Ala A,sp Gly lae Glu Asp Val I~r lle Lys Va1 Ser Asp G1u Gly Gly G1y rie pro Arg Ser Gly :r75 280 285 Leu Pro Lys Ile Phe The Tyr Leu Tyr Ser Thr Ala Arg Asn Pto Leu Glu G1u .Asp Vel Asp Leu Gly Thr Ala Asp Val Pro Va1 Thr Met Ala Gly Tyr Gly Tyr Gly Leu Pro lle Ser Asg I,eu Tyr Ala Arg Tyr Plae Gly Gly Asp Left Gtn Tle Tle Sex Met GIu G1y'lyr Oly 3'hr Asp Ala 'err T~u a~u LCLt Ser Arg Leu Giy Asp Ser Gln Glu Prp Leu Pr4 :355 360 365 (2) IINFOItMA'1ZON FOR SDQ 1D N0:8:
(i) SEQUENCE C~IAItAGTEI~SITCS:
(A) )L~1(i I'H: 367 amino acids (B) T3.'PE: amino acid (G~ S'I'RANDEDNE,SS:
(D) TCJPULt'7GY: >i~
(u~ MOI~3C1JLE TYPE: protein (x~~ sl~aQtIENCE brTON: SEQ lb NG:B:
Met Ala VaI Lys Lys Ala Ser Glu Met Phe Ser Lys Ser Leu 11e Glu Asp Val )<Fs Arg Trp Gly Cys Met Lys Gln Thr Gly Val Ser Leu Arg Tyr Met Met Glu Phe Gly Ser Thr Pro Thr Glu Arg Asn Leu Leu Ile Ser Ala Gln Phe Leu T~is Lys Gtu Leu pro rie Arg Tle Ala Arg Arg Ala Ile Glu Ixu Glu Thr TJeu PFO'xyr Gly Leu Ser Glu Lys Pro A1a ~s 7a 75 so Val Leu L,ys Val Arg Asp Trp Tyr Val Glu Ser P'he Arg Asp Met Arg Ala Phe Pro Olu lye Lys Asp Thr Ala Asp Glu Lys Glu 1?he'I~r Gln Met )!le Lys AIa Yal Lys Val Arg Kis Asn Asn Val Val Pro Met Met 1.15 120 1?5 Al$ Leu Gly V al Asn Gln Leu Lys Lys Oily Met Lys Leu Tyc Crlu Lys 13a 135 140 Leu Asp Glu Ile H3s Gln Phe Phe Asp Arg Phe Tyr Leu Ser Arg lle Gly lle Asg l4Jet Leu he G1y GIn His Val Glu Leu His Asn Pro Asn Pro Pro Txu 1~hs Tbr Val GIy Tyr )le His Thr Lys Met Ser Pro Met Glu Val Ala Axg Asn A18 Ser Glu Asp Ala Arg Scr T1c Gars phe Arg a95 200 205 Glu Tyr Gty Ser Ala Pro Glu lle Asn Ile Tyr Gly Asp Pro Sex 1?be Thr P)ze 3'ro Tyr Val Pro Ttu T3is Leu iiis Leu Met Val Tyr Crlu Leu z25 23a 235 z4a Val Lys nsn Ser Leu Arg Ala Val Gln Glu Axg Phe Val Asp Ser Asp Arg Val ~41a Pro Pro Ile Arg Ile Ile Va! Ala Asp Gty lle Glu Asp 260 265 27(?
val Thr TIe Lys Val Ser Asp Glu CTIy Cily Oily ne lyro Arg Ser Cily 2.75 280 285 Leu Pro I_lrs Ite phe Thr Tyr reu Tyr Ser T!?r Ala Arg Asn Pro l.eu Glu Glu Asp Val Asp Leu Gly'1'hr Ata Asp Va! Pro Va1 Thr Met A1a Gly Tyr Cily Tyr Gly Leu Pro lle Ser Arg Ixu Tyr Ala Arg Tyr Pht Crly Crly Asp Leu aln lle Ile Ser Met Glu Gly Tyr Gly 1'hr Asp Aia 'l~r Txu llis reu Ser Arg Leu Clly Asp Ser Gln Glu Pro Leu Pro .355 360 365 (2) JNFpRNlATTQN FQR 5EQ 1D NO_9:
(i) SEQUENCE CHA12ACTERISTTCS:
(A) LFNCTH: 27 base pairs (~) T~i'P,~: nucleic acid (C) STRANbEbNESS: single (D) TC>PO)LOO'Y': liar (ii) MOI,HCU~T TXIt~: other nucleic acid (A) Dk:SCRIPTI~DN: ldeSC - "Primer"

(xi) S>3QUJRNCB DESCRIPTION: SEQ )D N0:9:

(2) 1NFORl~IATIDN FOR SEQ 1D NO:10:
(i) SEQ~f.7ENCE CLIARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (G) S'~.ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLIxC'(3LI? TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "Primer"
(xi) SEQK7ENCE DBSCRIP'I'ION: SEQ )D NO:10:
TGCTCTAGAT YANGG~ChARG GYTCYTS 27 (2) TNFORPvIATION FDR SEQ ID NO: I I
(i) SEQ>TENCE CHARACTF~S'1~CS:
(A} I:FNGTH: 21 base pairs (B) TYPE: nucleic acid (C) S'1'RANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLaECLIL.E TYPE: other nucleic acid (A) D>rSCRIPTION: Idesc = ..Primer,.
(xi) SEQ1JENCE D)?SCRIPI'TON: SEQ B7 NO:11:
CTTTCTTC:AG CTGGTTCACA C 21 (2) TNFORArfATION FOR SfiQ ID N0:12:
(i) SEQLTENCE CHARACTERrSTTCS:
(A) T.IENG'I ~T: 29 base pairs (B) TYPE: nucleic acid (C) S'.CItANDEDNESS: Single (D) TnPOLOGY: linear (ii) MOLI~~iJL,E TYPE: other nucleic acid (A) D:ESCRIPT~ON: Idesc = "Primer"
(xi) SEQUENCE DESCRIPTION: SEQ m NO: i2:
GACTCCACAT ACCAATCTCx' TACCTTCAA 29 (2) TNPORMAT10N FOR SEQ m N0:13:
(i) SEQUENCE CI-IA,ItACTF.ItISTICS:
(A) I 1~~1GTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDI~NESS: single (D) T'OT'OI,OCrY: linear (ii) MOLECULE TYPE: other nucleic acid (A) I;~ESCRIPTION: /desc='~~riuner"
(xi) 5EQ'U1NCL DFSC12IPTION: SEQ ID N0.13:
CATAAGCiCAG CGTCTCGAGT TCG 23 (2) TNFpRMATION Felt SEQ ID N0:14:
(i) Sl?Qr~N'CE CTi'A)~ACI"ERIS'ITCS:
(A) LBNGTH: 25 base pairs (I#) TYPE: nucleic acid (C) S'.I'RAIVpEDN~SS: single (D) T'~POLOGY: lines ~9~
(ii) MOL:ECrng z"Y'~: other nucleic acid (A) D13SCR1PT'ION: /desc = "Primer'.
(xi) S)JQ17ENCE D1:SC1~TPTTON: SEQ 1D N0:14:
AGATGTGGAC TTGGGAACCG CfGAT 2.5 (2) I1V'EOTthd.4TION xOlt SEQ Ib N0:15:
(i) SEQhrENCE CHARACTERISTICS:
(A) i.7~tG"TH: 2$ base p8irs (B) T'~CPE: nucleic acid (C) S7.'RANDEDNESS: single (D) TI7POLOGY: linear (ii) MOlr13C1CIt~'I'Yl'E: other nucleic acid (A) DIESCRIPTION: /desc = "Primer"
(xi) SEQ1UPNCE DIaSCRIhL'TON: Sl?Q ID NO:1S:
GTi'ATGG'L'GT GCCTATTAGT CGGTTGTA 2$
(2) INFORI~fATION FOR SEQ II7 N0:16:
(~) sEQuENC~ cl~~AcTI~IS'rlcs:
(A) I~INGTfr: 24 base pairs (B) T"5.'PL: nucleic acid (C) S'1'RANDEDNESS: single (D) TOpOL,OGY: linear (ii) MOL~1C Q"L.E TYP13: other nucleic acid (A) D1~CRIPTION: ldesc = "Primer"
(xi) SEQILa)=NCE D)rSCILTPTION: SlrQ lb NO:16:
GTTTAAA(:AT GGCGGTGAAG AAGG ?q.

(2) INFORPrIATION FOR SEQ iD M0.17.
(i} SRQCJENCE CXP~RACT'FTtIS'ITCS:
(A) LiENO'1'~-T: 2A base pairs (B) 1"Y'PE: nucleic acid (C) S'.l'RANDEDNF.SS: single (D)'TOPOLOGY: linear {ii) MOLFCI11.,~ TYPE: other nucleic acid (A) DESC)2TP'T10N: Idesc = .'Primer'.
(xi) S13Q1:JF.NCE DESCR'1P'I'ION: SEQ 1D NO:17:

(2) TN'FOItIHIATION FOR SEQ 1o NO: I8:
(i} SEQI:fENCE CHARACT&RIS"I7CS:
(A) Ll-~TGT~I: 2S base pairs (,»} T'~(PE: nucleic acid (C) S7:'ItANDI?DNESS; single (D) TOPC3LOGY: linear (ii} MOLJ~CUL.E'TYPE: nthcr nucleic acid (A) D:ESCLZrPTION: Idesc = ")?rimer"
{xi) SIrQIJ)~NCE DESCRIP'TTON: SEQ ~ N0:18: , CGTACOA'TaG CC3GTGAAGAA GGGTA 25 (2) INk'O>,thsATIpN FOR S1;Q TI7 N0:19:
(i} SEQIJ'ENCE CHARACTERISTICS:
(A) LFJNGTH: 24 base pairs (H) T'!~'F'E: nucleic acid (C) 57'RAT~)~N13SS; single (D) TOPOLOGY: linear (ii) MOil3CLTLE TYPE: other nucleic acid (A) DIESCRIpTION: Idesc = "Primer' {xi) S)EQiJENCE DESCRIPTION: SPQ ID N0:19:
CDOTGACCI'CA TGGCAAAGGC'x'CCT 24

Claims (41)

1. A process for modulating mitochondrially generated acetyl-CoA
or respiration rate in a transgenic plant, the process comprising:
cloning a gene encoding a Brassica pyruvate dehydragenase kinase protein into a vector, said gene having a sequence selected from the group consisting of SEQ ID No:1, SEQ ID NO:2, SEQ ID NO:3 end SEQ ID NO: 4.
positioning the gene in an anti-sense orientation; and transforming a plant with the vector to produce the plant having modulated mitochondrially generated acetyl-CoA or respiration rate.

2. The process according to claim 1, further comprising linking a promoter to the gene.

3. The process according to claim 2 wherein the promoter is a ubiquitin gene promoter or a phaseolin promoter.

4. The process according to claim 1 wherein the plant is a member of the Gramineae.

5. The process according to claim 1 wherein the plant is selected from the group consisting of borage, Canola, castor, cocoa bean, corn, cotton, Crambe spp., Cuphea spp., flax, Lesquerella and Limnanthes spp., Linola, nasturtium, Oenothera spp., olive, palm, peanut, rapeseed, safflower, soybean, sunflower, tobacco, Vemonia spp., wheat, barley, rice, oat, sorghum and rye.

6. The process according to claim 4 or 5 wherein the plant is Canola.

7. A process for modulating mitochandrially generated acetyl-CoA
or respiration rate in a transgenic plant, the process comprising:
cloning a gene encoding a Brassica pyruvate dehydrogenase kinase protein into a vector, the gene having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; and transforming the vector into a plant, thereby reducing production of the Brassica pyruvate dehydrogenase kinase protein in the transgenic plant and producing a plant having modulated mitochondrially generated acetyl-CoA or respiration rate.

8. The process according to claim 7 wherein the plant is a member of the Gramineae.

9. The process according to claim 7 wherein the plant is selected from the group consisting of borage, Canola, castor, cocoa bean, corn, cotton, Crambe spp., Cuphea spp., flax, Lesquerella and Limnanthes spp., Linola, nasturtium, Oenothera spp., olive, palm, peanut, rapeseed, safflower, soybean, sunflower, tobacco, Vemonie spp., wheat, barley, rice, oat, sorghum and rye.

10. The process according to claim 8 or 9 wherein the plant is Canola.

11. the process according to claim 7 wherein the step for reducing production of the Brassica pyruvate dehydrogenase kinase protein comprises positioning the gene encoding the Brassica pyruvate dehydrogenase kinase protein in an anti-sense orientation in the vector.

12. An isolated, purified or recombinant nucleic acid encoding a Brassica pyruvate dehydrogenase kinase (PDHK) protein, said nucleic acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID
NO:4.

13. The isolated, purified or recombinant nucleic acid of claim 12 comprising the nucleic acid sequence of SEQ ID NO:1.

14. The isolated, purified or recombinant nucleic acid of claim 12, comprising the nucleic acid sequence of SEQ ID NO:2.

15. The isolated, purified or recombinant nucleic acid of claim 12, comprising the nucleic acid sequence of SEQ ID NO:3.

16. The isolated, purified or recombinant nucleic acid of claim 12, comprising the nucleic acid sequence of SEQ ID NO:4.

17. Use of the isolated, purified or recombinant nucleic acid of any one of Claims 12-16 for modulating mitochondrially generated acetyl-CoA or respiration rate in a plant.

18. Use of the isolated, purified ar recombinant nucleic acid of any one of claims 12-16 for modulating seed oil content or weight in a plant.

19. Use of the isolated, purified or recombinant nucleic acid of any one of claims 12-16 for modulating flowering or generation time in a plant.

20. A modified cell comprising the isolated, purified or recombinant nucleic acid of any one of claims 12-16.

21. The cell of claim 20 wherein the isolated, purified or recombinant nucleic acid is arranged to be expressed in an anti-sense direction.

22. The cell of claim 20 or 21 wherein the cell is E. coli.

23. The cell according to claim 20 or 21 wherein the cell is an Agrobacterium cell.

24. The cell of claim 20 or 21 wherein the cell is a B. napus cell.

25. A method of transforming a plant, comprising introducing the isolated, purified or recombinant nucleic acid of any one of claims 12-16 into the plant.

26. A process for producing a genetically transformed plant seed, said process comprising transforming a plant seed by introducing the isolated, purified or recombinant nucleic acid of any one of claims 12-16 into the plant seed.

27. A vector for transforming plant cells comprising the isolated, purified or recombinant nucleic acid of any one of claims 12-16.

28. The vector of claim 27, wherein the isolated, purified or recombinant nucleic acid is oriented in an anti-sense orientation.

29. Use of the vector of claim 27 or 28 for modulating mitochondrially generated acetyl-CoA or respiration rate in a plant.

30. Use of the vector of claim 27 or 28 for modulating seed oil content or weight in a plant.

31. Use of the vector of claim 27 or 28 for modulating flowering or generation time in a plant.

32. A process of producing a transgenic plant, said process comprising introducing the isolated, purified or recombinant nucleic acid sequence of any one of claims 12-16 into a genome of a plant thus producing a transgenic plant.

33. The process of claim 32 wherein the isolated, purified or recombinant nucleic acid sequence is oriented in an anti-sense orientation in the genome.

34. The process according to Claim 32 or 33 wherein the plant is a member of the Gramineae.

35. The process according to claim 32 or 33 wherein the plant is selected from the group consisting of borage, Canola, castor, cocoa bean, corn, cotton, Crambe spp., Cuphea spp., flax, Lesquerella and Limnanthes spp., Linola.
nasturtium, Oenothera spp., olive, palm, peanut, rapeseed, safflower, soybean, sunflower, tobacco, Vemonia spp., wheat, barley, rice, oat, sorghum and rye.

36. A method of changing the oil or biopolymer content of a plant, plant storage organ or plant seed, said process comprising:
introducing a sense or anti-sense nucleic acid construct into a plant transformation vector to produce a modified plant transformation vector, wherein said sense or anti-sense nucleic acid construct comprises the isolated, purified or recombinant nucleic acid sequence of any one of claims 12-16;
transforming said plant, plant storage organ or plant seed's genome with said modified plant transformation vector; and growing said plant, plant storage organ or plant seed and extracting said oil or biopolymer.

37. An isolated polypeptide, comprising a Brassica pyruvate dehydrogenase kinase (PDHK) protein, said polypeptide selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

38. The isolated polypeptide of claim 37, comprising the sequence of SEQ ID NO:6.

39. The isolated polypeptide of claim 37, comprising the sequence of SEQ ID NO:7.

40, The isolated polypeptide of Claim 37, comprising the sequence of SEQ ID NO:8.

41. An isolated, purified or recombinant nucleic acid selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14 and SEQ ID NO:15.