NZ554114A - Plants with an increased ratio of oleosin to TAG synthesising enzymes - Google Patents

Abstract

Disclosed is a construct which includes: a) at least one promoter; b) a nucleic acid molecule encoding a single oleosin protein; and c) at least one nucleic acid molecule encoding an triacylglycerol biosynthetic enzyme (TAG synthesising enzyme); wherein the promoter(s) are operatively linked to the nucleic acid molecules so as to cause expression of the oleosin and at least one TAG synthesising enzyme. The construct can be used to produce plants with an increased ratio of oleosin to TAG synthesising enzymes. These plants are useful as animal feed.

Description

554114 PATENTS FORM NO. 5 NEW ZEALAND PATENTS ACT, 1953 No: 554114 Date: 23 April 2007 COMPLETE SPECIFICATION IMPROVEMENTS IN AND RELATING TO OIL PRODUCTION We, AGRESEARCH LIMITED, a New Zealand company of East Street, Ruakura Campus, Hamilton, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 554114 IMPROVEMENTS IN AND RELATING TO OIL PRODUCTION STATEMENT OF CORRESPONDING APPLICATIONS This application is based on the provisional specification filed in relation to New Zealand Patent Application Number 554114, the entire contents of which are 5 incorporated herein by reference.

TECHNICAL FIELD The invention relates to improvements in and relating to oil production in plants and seeds. In particular, this invention relates to methods, including the use of constructs for increasing the ratio of oleosin/polyoleosin to triacylglycerol (TAG) in the seeds of a 10 plant and the ratio of oleosin/polyoleosin to TAG in the vegetative portions of a plant.

The invention also relates to methods for selecting plants capable of generating new varieties which have increased oleosin/polyoleosin content or a high oleosin/polyoleosin relative to TAG ratio. In addition the invention also relates to methods for generating plants, or plant cells which have increased oleosin/polyoleosin 15 content or a high oleosin/polyoleosin relative to TAG ratio.

BACKGROUND ART Triacylglycerol Most plants synthesise and store significant amounts of triacylglycerol (TAG) only in developing seeds and pollen cells where it is subsequently utilised to provide 20 catabolizable energy during germination and pollen tube growth. Dicotyledonous plants can accumulate up to approximately 60% of their seed weight as TAG. Ordinarily, this level is considerably lower in the monocotyledonous seeds where the main form of energy storage is carbohydrates (e.g. starch).

The only committed step in TAG biosynthesis is the last one, i.e. the addition of a third 25 fatty acid to an existing diacylglycerol, thus generating TAG. In plants this step is predominantly (but not exclusively) performed by one of three TAG synthesising enzymes including: acyl CoA: diacylglycerol acyltransferase (DGAT1); an unrelated acyl CoA: diacylglycerol acyl transferase (DGAT2); and phosphatidylcholine-sterol O-acyltransferase (PDAT). 1 A 554114 Oleosin Oleosins are specific plant proteins usually found only in seeds and pollen. Their function is to stop oil bodies coalescing during seed and pollen maturation. In nature, TAG produced in seeds and pollen form micelles encapsulated by a spherical 5 phospholipid monolayer embedded with one or several species of oleosin proteins. Including caoleosin (calcium binding) and steroleosin (sterol binding) (Tzen et al., 2003) Oil bodies in fruit tissues (such as olives and avocados) do not contain oleosins.

The properties of the major oleosins is relatively conserved between plants and is characterised by the following: • 15-25kDa protein corresponding to approximately 140-230 amino acid residues.

• The protein sequence can be divided almost equally along its length into 4 parts which correspond to a N-terminal hydrophilic region, two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region.

• The topology of oleosin is attributed to its physical properties which includes a folded hydrophobic core flanked by^ hydrophilic domains. This arrangement confers an amphipathic nature to oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer (Tzen et al., 1992) while the flanking hydrophilic domains are exposed to the aqueous environment of the cytoplasm.

Polvoleosin Polyoleosin is the head to tail fusion of two or more oleosin units (Roberts et al., 2008). Altering the number of oleosin units enables the properties (thermal stability and degradation rate) of the oil bodies to be tailored. Expression of polyoleosin in planta leads to incorporation of the polyoleosin units to the oil bodies as per single oleosin units (Scott et al., 2007).

Oil Bodies An oil body that is produced in seed or pollen consists of a droplet of TAG surrounded by a monolayer of phospholipid which is in turn surrounded by a layer of oleosin proteins. Where the hydrophobic acyl moiteies of the phospholipids interact with the 2 554114 encapsulated TAG and the hydrophilic head groups face the cytoplasm and the oleosins are orientated with their central hydrophobic amino acid domains protruding through the phospholipid monolayer and into the TAG core of the oil body. Oil bodies are naturally produced in the seeds and pollen of many plants. Oil bodies can also be 5 generated artificially by combining oleosins, triacylglycerides and phospholipids (Peng et al., 2004).

The size and number of oil bodies depends on the ratio of oleosin/polyoleosin to TAG within the plant cell (Siloto et al., 2006).

Biohydrogenation It has been demonstrated that the lipid profile of ruminant animal feed in turn influences the lipid profile of meat and dairy products (Demeyer and Doreau, 1999). Different plants have different lipid profiles; by selectively feeding animals only plants with the desired lipid profile it is possible to positively influence the lipid profile of downstream meat and dairy products. In ruminants the final lipid make up of the meat 15 and milk is not only influenced by the dietary lipids but is also heavily influenced by biohydrogenation (Jenkins and McGuire 2006; Firkins et al., 2006; Lock and Bauman, 2004). Biohydrogenation is the hydrogenation of non-reduced compounds (such as unsaturated fats) by the biota present in the rumen. Biohydrogenation can be prevented/delayed by encapsulating the lipids in a protein or proteins that provide 20 resistance to microbial degradation (Jenkins and Bridges 2007).

Oleosin/polyoleosin in planta The inventors have found that oil bodies with an oleosin/polyoleosin outer layer can protect, or at least delay, degradation and/or biohydrogenation, of TAG, within the stomach and/or rumen of an animal, allowing the intact individual lipids from the TAG 25 to be absorbed by the animal in the intestine. Therefore, it would be useful in terms of dietary intake of an animal if there could be provided a means for increasing the oleosin/polyoleosin content within seeds so as to increase the protection provided to the oil bodies.

The existence of diacylglycerides as membranes within the vegetative portions of 30 plants (e.g. the leaves) has been documented (Mekhedov et al., 2000).

It would therefore be useful if plants could be produced which contain oleosin/polyoleosin in their vegetative portions in addition to the seeds of said plants. 3 554114 These plants or their genetic sequences could then be used to develop plants which have oleosin/polyoleosin and TAG produced in the vegetative portions of a plant which would be useful in terms of dietary intake. In particular, it would be useful to identify plants that produce high levels of oleosin/polyoleosin in their vegetative portions as 5 these can be further manipulated to also produce DGAT1 or other TAG synthesising enzymes so as to simultaneously produce a high level of oleosin/polyoleosin and TAG in the vegetative portion of the plant to generate oil bodies encapsulated with oleosin/polyoleosin.

It is an object of the present invention to address the foregoing problems or at least to 1 o provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the 15 cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be 20 attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is 25 used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to a first aspect of the present invention there is provided a construct which 30 includes: at least one promoter; 4 554114 a nucleic acid molecule encoding a single oleosin protein; a at least one nucleic acid molecule encoding an triacylglycerol biosynthetic enzyme (TAG synthesising enzyme); wherein the promoter(s) are operatively linked to the nucleic acid molecules so as to 5 cause expression of the oleosin and at least one TAG synthesising enzyme.

It will be appreciated that the TAG synthesising enzymes may be any enzyme required to add the third fatty acid to an existing diacylglycerol and generate TAG within a plant and plant cell. By way of example only, the enzymes may preferably be; acyl CoA: diacylglycerol acyltransferase (DGAT1); acyl CoA: diacylglycerol acyl 10 transferase (DGAT2); or phospholipid : diacylglycerol acyltransferase (PDAT).

Preferably, there may be at least one TAG synthesising enzyme within the construct. However, in some other embodiments, there may be more than one enzyme within the construct.

It will be appreciated that the oleosin gene and/or the TAG synthesising enzymes may come from a number of plant species. For example, the plant species may be either monocots, dicots or gymnosperms. Non-limiting examples of monocots, include; grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley. While non-limiting examples of dicotyledons, include: Arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus, poplar, hybrid aspen, and gymnosperms, for example, pine tree.

In some preferred embodiments, the nucleic acid molecule encoding the single oleosin protein may have a nucleotide sequence selected from: a) SEQ ID NO's 1, 3, 5, 7 or 9, or other nucleic acid molecules encoding an 25 oleosin gene; b) a complement of a sequence in a); INTELLECTUAL PROPERTY OFFICE OF N.Z. 21 OCT 2009 jjR EC EIV E D 554114 c) a functional fragment or variant of a sequence in a) or b); or d) a homolog or an ortholog of a sequence in a), b), or c).

In some preferred embodiments, the nucleic acid molecule encoding a TAG synthesising enzyme may have a nucleotide sequence selected from: a) SEQ ID NO's 11, 13 or 15 or other nucleic acid molecules encoding TAG synthesising enzymes; b) a complement of a sequence in a); c) a functional fragment or variant of a sequence in a) or b); d) a homolog or an ortholog of a sequence in a), b), or c); or e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) ord).

Also disclosed is use of at least one construct which includes a nucleic acid molecule encoding oleosin/polyoleosin to increase the ratio of oleosin/polyoleosin to TAG within a seed.

According to a third aspect of the present invention there is provided a use of at least one construct substantially as described above to produce single oleosin and TAG synthesising enzymes in the vegetative portions of a plant.

Also disclosed is a method of increasing the ratio of oleosin/polyoleosin to triacylglycerol (TAG) within a plant seed comprising a step of manipulating the genome 20 of a plant such that the seeds of said plant express more oleosin/polyoleosin than a corresponding wild-type seed.

According to a fourth aspect of the present invention there is provided a method of producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, comprising the step of manipulating the genome of a plant so as to express 25 more single oleosin and TAG synthesising enzyme in the vegetative portions than a corresponding wild-type plant.

According to a fifth aspect of the present invention there is provided a method of INTELLECTUAL PROPERTY OFFICE OF N.Z. 21 OCT 2009 received! RECEIVED at IPONZ on 20 November 2009 554114 producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, including the steps of: (a) genetically manipulating a plant to produce single oleosin in the vegetative portion of the plant; and (b) genetically manipulating a plant to produce TAG synthesising enzyme in the vegetative portion of the plant.

In some embodiments, there may be a further step (c) which is the step of: measuring oleosin and TAG in manipulated plants to find plants with a higher oleosin to TAG ratio in the vegetative portion of the plant.

According to a further aspect of the present invention there is provided a method of producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, including the steps of: (a) genetically manipulating a first plant to produce single oleosin in the vegetative portion of the plant; (b) genetically manipulating a second plant to produce TAG synthesising enzyme in the vegetative portion of the plant; and (c) crossing the first and second plants to produce a plant expressing single oleosin and TAG synthesising enzyme within the vegetative portions of the plant.

In some embodiments, there may be a further step of measuring the levels of: (i) single oleosin in the first plant; and (ii) TAG in the vegetative portions of the second plant; to find a pair of plants for crossing, wherein the first plant produces sufficient levels of single oleosin relative to the amount of TAG synthesising enzyme produced in the 25 second plant such that the plant produced crossing in step (c) produces oil bodies encapsulating TAG which are capable of protecting and/or delaying degradation or biohydrogenation of the TAG within the stomach or rumen of an animal.

The TAG synthesising enzymes may be any enzyme required to add the third fatty 7 RECEIVED at IPONZ on 20 November 2009 554114 acid to an existing diacylglycerol and generate TAG within a plant and plant cell. By way of example only, the enzymes may be selected from one of the following; acyl CoA: diacylglycerol acyltransferase (DGAT1); acyl CoA: diacylglycerol acyl transferase (DGAT2); or phospholipid: diacylglycerol acyltransferase (PDAT).

Preferably, there may be at least one TAG synthesising enzyme within the construct. However, in some other embodiments, there may be more than one enzyme present within the construct.

It will be appreciated that the term 'vegetative portions' of the plant, can include; the shoots, leaves, fruit, bark, pods, roots, nodules, stems and the like, including parts 10 thereof. Preferably, the vegetative portions of the plant may be the leaves.

Also disclosed is a seed of a plant which has been manipulated to express more oleosin/polyoleosin than a corresponding wild-type seed.

Also disclosed is a plant or part thereof which has been manipulated to express more oleosin/polyoleosin than a corresponding wiid-type plant or part thereof.

Also disclosed is a plant cell which has been manipulated to produce more oleosin/polyoleosin than a corresponding wild-type plant cell.

According to a further aspect of the present invention there is provided a leaf of a plant which expresses single oleosin and a TAG synthesising enzyme.

According to a further aspect of the present invention, there is provided a plant or part 20 thereof which has been manipulated to express both single oleosin and a TAG synthesising enzyme, by transforming the plant with a construct of the invention.

According to a further aspect of the present invention, there is provided a plant or part thereof which has been manipulated to produce both single oleosin and TAG synthesising enzyme, by transforming the plant with a construct of the invention.

According to yet another aspect of the present invention there is provided an animal feed which includes seeds and/or vegetative portions of plants, which have been manipulated to have elevated levels of single oleosin and TAG compared to the corresponding wild type seed or plant. 8 (followed by page 8a) RECEIVED at IPONZ on 20 November 2009 554114 According to a further aspect of the present invention there is provided an animal feed which includes seeds which have been manipulated to have elevated single oleosin and TAG synthesising enzyme content compared to the corresponding wild type seeds.

According to a further aspect of the present invention there is provided an animal feed which includes a vegetative portion of a plant which has been manipulated to have elevated single oleosin and TAG synthesising enzyme content compared to the vegetative portion of a corresponding wild type plants. 8a (followed by page 9) 554114 Preferably, there is a high ratio of single oleosin to TAG in said animal feed.

Also disclosed is an animal feed which includes seeds which have been manipulated to have elevated oleosin/polyoleosin content compared to the corresponding wild type seeds.

Also disclosed is an animal feed which includes a vegetative portion of a plant which has been manipulated to have elevated oleosin/polyoleosin to TAG content compared to the vegetative portion of a corresponding wild type plant.

Also disclosed is an animal feed which includes a seed and vegetative portion of a plant wherein the collective ratio of oleosin/polyoleosin to TAG is capable of protecting 10 oil bodies in the rumen of an animal.

Also disclosed is a plant which has been manipulated to produce TAG synthetic enzymes in the vegetative portions of the plants.

Also disclosed is a plant or plant cell which has been transformed with a construct which includes: - at least one promoter; - a nucleic acid molecule encoding at least one TAG synthesising enzyme; wherein said promoter is operatively linked to said nucleic acid molecule so as to cause expression of said TAG synthesising enzyme.

Also disclosed is a plant or plant cell transformed by a construct substantially as 20 described above wherein the nucleic acid molecule ensuring a TAG synthesising enzyme comprising an antisense sequence.

Also disclosed is the use of information regarding the oleosin/polyoleosin and/or TAG content in plants or seeds in a selection process for generating new plants.

Also disclosed is the use of antisense sequence to an RNA molecule obtained from 25 TAG synthesising enzyme nucleic acid molecule to reduce TAG levels with a cell or in planta.

The selection process may involve identifying plants or seeds which have a 9 2 1 OCT 2009 554114 high oleosin/polyoleosin content and/or a low TAG content.

The generation of new plants can be by either molecular genetic techniques or by traditional selection breeding techniques.

Also disclosed is a plant or plant cell which produces more or less TAG than a 5 corresponding wild-type plant or cell.

The manipulation of the genome may in some preferred embodiments be undertaken by molecular genetic techniques.

Manipulation of the genome may be undertaken using traditional selective breeding techniques.

Also disclosed is the use of nucleotide sequence information for TAG synthesising enzyme to generate plantibodies or other molecules which can inhibit the plant metabolic pathways which are involved in the synthesis of TAG synthesising enzyme. It should be appreciated that the generation of plantibodies is well known in the art, (refer Jobling SA et al 2003).

The term "comprising" as used in this specification means 'consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

The term "single oleosin" refers to a single oleosin monomer, and not a multimer or a head to tail fusion of two or more oleosin units, such as a polyoleosin (Roberts et al., 2008).

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene or nucleic acid molecule of interest. In 25 general a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto. 21 OCT 2009 (followed by page 10a) .received 554114 The term "wild type' or grammatical variations thereof, refers to the genome of a plant and/or seed as found in nature.

The term 'seed' may refer to the seed as a whole or a cell within the seed.

The term 'vector' as used herein encompasses both cloning and expression vectors. 5 Vectors are often recombinant molecules containing nucleic acid molecules from several sources.

The cloning vector selected may depend on the host and host cell as used. In general the vector may: • self replicate; 'OTE&WEAOFTZPERTV"| 2 I OCT 2009 received 10a (followed by page 11) 554114 • include a marker gene for selection of transformed cells; • have a nucleic acid sequence capable of being cleaved by an endonuclease Some examples of suitable vectors may include: • General cloning - pGEMT • Binary plant transformation vectors - pRD410, pHZbar, pKR10, pGREEN, pBin19.

An "expression vector" refers to a cloning vector which also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene 1 o of interest, so that the gene product of the gene can be expressed.

The term "promoter" refers to a sequence of DNA to which RNA polymerase (an enzyme) will bind and initiate transcription of a nucleic acid molecule of interest (a gene) to produce mRNA. Preferably, a promoter may be a plant promoter. Even more preferably, the plant promoter may be any suitable promoter for the plant of 15 interest and which operates within (i.e. the promoter) the portion of the plant that expression is required in. For example, a seed specific promoter, or a vegetative specific promoter, may be used dependent on where you want expression to occur.

The promoter molecule may be an RNA, cRNA, genomic DNA or cDNA molecule, and may be single- or doublestranded. The promoter molecule may also optionally 20 comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.

The term 'antisense' includes a nucleic acid molecule which has a nucleotide sequence which can bind (i.e. is complementary) to a TAG synthesising enzyme RNA molecule; as well as RNAi molecules; siRNA molecules and the like; which can inhibit 25 transcription of RNA molecules encoding the TAG synthesising enzyme. The generation of RNAi molecules, siRNA molecules, based on nucleotide sequence information for a gene product, such as a protein or enzyme, is well known to those skilled in the art and a matter of routine experimentation, (refer Small I, 2007; and Mansoos S. et al 2006) The term "nucleic acid molecule" as used herein may be an RNA, cRNA, genomic DNA or cDNA molecule, and may be single- or doublestranded. The nucleic acid 11 554114 molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.

The term "expression" as used herein broadly refers to the process by which a nucleic acid molecule is converted by transcription and then translated into a protein/enzyme.

The term 'fragment nucleic acid molecule' as used herein refers to a nucleic acid molecule which represents a portion of the nucleic acid molecule of the present invention and is therefore less than full length and comprises at least a minimum sequence capable of hybridising stringently with a nucleic acid molecule of the present invention (or a sequence complementary thereto).

A "fragment" of a polypeptide of the present invention is a portion of the polypeptide that is less than full length. Preferably the polypeptide fragment has at least approximately 60% identity to a polypeptide of the present invention, more preferably at least approximately an 80% identity, and most preferably at least approximately a 90% identity. Preferably the fragment has size of at least 10 amino acids, more 15 preferably at least 15 amino acids, and most preferably at least 20 amino acids.

By "functionally active" in relation to a nucleic acid it is meant that the fragment or variant (such as analogue, derivative or mutant) encodes a polypeptide capable of being used to produce the required protein.

The term 'functional' refers to either: a nucleic acid molecule which encodes a 20 polypeptide capable of acting as an oleosin/polyoleosin or TAG synthesising enzyme The term "genome" refers to the DNA or set of chromosomes or genes that make up an organism, and is passed to the organism's offspring.

The term "variant" as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantially 60% or greater 25 homology with the nucleotide or amino acid sequence of the Figures, preferably 75% homology and most preferably 90-95% homology to the sequences of the present invention. - as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides) or BLAST X (nucleotides). The variant may result from modification of the native nucleotide or amino acid sequence by such modifications as insertion, 12 554114 substitution or deletion of one or more nucleotides or amino acids or it may be a naturally-occurring variant. The term "variant" also includes homologous sequences which hybridise to the sequences of the invention under standard hybridisation conditions defined as 2 x SCC at 55°C, or preferably under stringent hybridisation 5 conditions defined as 2 x SSC at 65°C or very stringent hybridisation conditions defined as 0.1 x SSC at 65°C, provided that the variant is capable of substantially performing the equivalent biological function of oleosin/polyoleosin; or TAG synthesising enzyme; as would be required to perform the present invention. Where such a variant is desired, the nucleotide sequence of the native DNA is altered 10 appropriately. This alteration can be effected by synthesis of the DNA or by modification of the native DNA, for example, by site-specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed, using techniques standard in the art.

It will be appreciated that the term 'manipulated', 'manipulation' or grammatical variations thereof, refers to the alteration of genetic information in a plant or part thereof, by a number of suitable genetic techniques, including, but not limited to: - introducing a nucleic acid molecule of interest to a cell; or - mutagenesis techniques; and/or - traditional plant breeding techniques (unless specifically excluded); or a combination thereof. For examples, see Lanfranco, 2003; Tadege et al., 2005.

The term "introducing" (or grammatical variants thereof) when used in the context of inserting a nucleic acid molecule into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation or transfer of a nucleic acid 25 molecule into a eukaryotic or prokaryotic cell where the nucleic acid molecule may be incorporated into the genome of the cell (e.g. chromosome, plasmid, plastid, mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g. transfected mRNA). 13 554114 The term "nucleic acid molecule" as used herein may be an RNA, cRNA, genomic DNA or cDNA molecule, and may be single-, double-, or triple- stranded. The nucleic acid molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.

The term "transformation" as used herein refers to a process by which the genetic material carried by an individual cell is altered by incorporation of exogenous DNA into its genome.

The term "ortholog", "orthologous gene", or "orthologous polypeptide" refers to a functionally equivalent yet distinct corresponding nucleotide or amino acid sequence 10 that may be derived from another plant. In general, an ortholog may have a substantially identical nucleotide or amino acid sequence to the sequences of the present invention as set forth in the sequence listing. The term 'homolog' refers to a related gene from a different but related species.

The compositions, methods and uses disclosed herein may have a number of 15 advantages over the prior art which include one or more of the following: - Increasing the number of oil bodies within a seed and/or the vegetative portions of a plant; - Increasing the amount of oleosin/polyoleosin produced by a seed and/or vegetative portion of a plant; Providing seeds or vegetative portions of a plant which have an increased number of oil bodies therein; Providing plants which produce oleosin/polyoleosin in the vegetative portions therein; Providing constructs and methods for producing oleosin/polyoleosin and/or TAG in plant a, Providing a mechanism to produce more stable oil bodies containing oleosin/polyoleosin in seeds; Providing a mechanism to produce oil bodies containing oleosin/polyoleosin in ,NTELL|CTUAL propertyI OFF/CE OF N.Z. I 2 I OCT 2009 receiv e d 14 554114 leaves.

- Providing plants which produce elevated levels of TAG synthesising enzyme in the vegetative portion of a plant.

- Providing plants which produce elevated levels of TAG in the vegetative 5 portions of the plant.

- Providing an animal feed which has an elevated oleosin/polyoleosin to TAG ratio.

- Providing a plant or plant cell which produces elevated or decreased levels of TAG.

BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 shows a coomassie gel and Immunoblot of five Brassica oleraceae 15 samples which have had oil body preps extracted from seed. All the samples were of 35S-white clover oleosin monomer; Figure 2 shows an Immunoblot of oil body preparations from seeds from independent Arabidopsis thaliana transformants containing 35S-sesame seed oleosin/polyoleosin; Figure 3 shows a DGAT1 nucleotide and translated amino acid sequence; Figure 4 shows a DGAT2 nucleotide and translated amino acid sequence; Figure 5 shows a PDAT nucleotide and translated amino acid sequence; Figure 6 shows an SDS-PAGE/Coomassie gel (top panel) and Immunoblot (bottom panel) of 3.5 day old ryegrass leaves over expressing 25 Arabidopsis thaliana DGAT1. AtDS = Developing Seeds from Arabidopsis thaliana Col; FN0107 line = ryegrass transgenic control containing 35S::GFP. Arrows indicate immunoreactive band of the expected size (59kDa). The indicated band in the AtDS control lane also correlated with the only immunoreactive band from microsomal 554114 preparations from the same sample. DGAT1 would be expected to be in the microsomal preparation as it is localised within the ER.; Figure 7 shows a Nile Red (top panel) staining of ryegrass plants over expressing Arabidopsis thaliana DGAT1 and protein extracts (bottom 5 panel) showing oil in water emusion from the two plants over expressing Arabidopsis thaliana DGAT 1; Figure 8 shows a SDS-PAGE/immunoblot of Arabidopsis thaliana leaves overexpressing sesame seed oleosin under the control of the CaMV35s promoter. pRShl = control plant transformed with empty vector, PSP1 1 o lines = transgenic plants over expressing sesame seed oleosin under the control of the CaMV35s promoter. Arrow indicates immunoreactive band of the expected size; shows an Influence of additional oleosin/polyoleosin accumulation on the thermal stability of oil bodies from Arabidopsis thaliana; shows an over accumulation of oleosin in oil bodies (OBs) increases stability in rumen fluid. The top panel shows SDS-PAGE/lmmunoblot of oil body extracts from transgenic Arabidopsis seeds over expressing a synthetic sesame seed oleosin nucleotide sequence (wild type plants showed no immunoreactive band at the appropriate size). Bottom panel 20 shows the influence of additional oleosin on OB stability in rumen fluid where OB was measured by turbidity (OD6oo); Figure 11 shows an expression of oleosin/polyoleosin and production of AOBs. -ve = negative control, pET29 vector; 1* - 6* = relevant oleosin/polyoleosin (in pET29 expression vector); Ct = pET28 vector 25 expressing 50kDa 7WC protein. Although 70pg of insoluble protein was used in the preparation of the AOBs, the proportion of each oleosin/polyoleosin within that 70|jg decreases with increasing repeat number (See AOB Coomassie and immunoblot panels); Figure 9 Figure 10 Figure 12. Stability of emulsions prepared with different oleosin/polyoleosin then 30 incubated at 90°C. Protein for AOBs sourced from: -ve = negative control, pET29 vector; Ct = pET28 vector expressing unrelated 50kDa 16 554114 Figure 13 Figure 14 7WC protein; 1 * - 6* = relevant oleosin/polyoleosin (in pET29 expression vector). shows a graph of estimated relative emulsion turbidity when the emulsions were prepared with different oleosin/polyoleosin then incubated at 90°C. Protein for AOBs sourced from: -ve = negative control, pET29 vector; Ct = pET28 vector expressing unrelated 50kDa 7WC protein; 1 * - 6* = relevant oleosin/polyoleosin (in pET29 expression vector). The negative control (-ve, ), control (Ct, ) and 6* oleosin/polyoleosin ( ) samples failed to produce a stable emulsion, with either poor emulsification, or breakdown of the emulsion occurring within 10min at 90°C. All of the emulsions prepared with oleosin/polyoleosin showed a decrease in opacity after 5min incubation at 90°C. The oleosin/polyoleosin emulsions with highest stability - 2* and 3* - then appeared to stabilse, while the oleosin/polyoleosin emulsions with medium stability - 1*, 4* and 5* - stabilised after 10min.

By 20min all of the emulsions had begun to dissipate, with the oleosin/polyoleosin emulsions with high stability dissipating slowest; shows a microscopic analysis of AOB prepared with different oleosin/polyoleosin and incubated at 90°C; Figure 15 shows a Mean diameter of AOBs prepared with different oleosin/polyoleosin after incubation at 90°C for 0, 20 and 60min; Figure 16 shows a Microscopic analysis of AOBs prepared with varying amounts of oil and oleosin repeats; Figure 17 shows a Heat stability at 90°C of AOBs prepared with varying amounts 25 of oil and oleosin repeats; Figure 18 shows a Possible incomplete inclusion of polyoleosin in AOBs causes instability in 4*, 5* and 6* oleosin/polyoleosin caused by insufficient disruption provided by sonication to allow the 6* oleosin/polyoleosin 17 554114 peptide fully denature and assemble with the AOB; and Figure 19 shows a Microscopic analysis of AOB stability in rumen fluid. AOB stability decreased as the oleosin/TAG ratio decreased (seen by increase in AOB diameter as AOBs coalesce due to decreased oleosin present on the surface). The greatest AOB stability was shown with the oleosin and 4% TAG sample which only showed signs of aggregation and some coalescence after 60 minutes in rumen fluid. The negative control shows that the oil never forms a true oil body and the oil is coalescing into droplets immediately after sonication; after 60 minutes the sample is virtually clear of oil (panel A) and TAG was only present as large droplets (panel B) indicating no protection was afforded by the addition of a non oleosin control protein.

Figure 20 CaMV35S promoter nucleotide sequence and white clover oleosin nucleotide and translated amino acid sequence.

Figure 21 CaMV35S driving 1 sesame seed oleosin tandem repeat with the UBQ10 intron in the first repeat and translated amino acid sequence.

Figure 22 CaMV35S driving 3 sesame seed oleosin tandem repeats with the UBQ10 intron in the first repeat and translated amino acid sequence.

Figure 23 Arabidopsis oleosin promoter driving 1 sesame seed oleosin tandem repeat 20 with the UBQ10 intron in the first repeat and translated amino acid sequence.

Figure 24 Arabidopsis oleosin promoter driving 3 sesame seed oleosin tandem repeats with the UBQ10 intron in the first repeat and translated amino acid sequence.

BRIEF DESCRIPTION OF SEQUENCE LISTING Sequence Name Corresponding sequence SEQ ID No. 1 CaMV35S driving 1 white clover oleosin tandem repeat (Figure 20) SEQ ID No. 2 Translated amino acid sequence of SEQ ID No. 1 (Figure 20) SEQ ID No. 3 CaMV35S driving 1 sesame seed oleosin tandem repeats with the UBQ10 intron in the first repeat (Figure 21) 18 554114 SEQ ID No. 4 Translated amino acid sequence of SEQ ID No. 3 (Figure 21) SEQ ID No. 5 CaMV35S driving 3 sesame seed oleosin tandem repeats with the UBQ10 intron in the first repeat (Figure 22) SEQ ID No.6 Translated amino acid sequence of SEQ ID No. 3 (Figure 22) SEQ ID No. 7 Arabidopsis oleosin seed promoter driving 1 sesame seed oleosin repeat with the UBQ10 intron in the first repeat (Figure 23) SEQ ID No. 8 Translated amino acid sequence of SEQ ID No. 7 (Figure 23) SEQ ID No. 9 Arabidopsis oleosin seed promoter driving 3 sesame seed oleosin tandem repeats with the UBQ10 intron in the first repeat (Figure 24) SEQ ID No. 10 Translated amino acid sequence of SEQ ID No. 9 (Figure 24) SEQ ID No. 11 DGAT1 nucleotide sequence (Figure 3) SEQ ID No. 12 DGAT1 amino acid sequence (Figure 3) SEQ ID No. 13 DGAT2 nucleotide sequence (Figure 4) SEQ ID No. 14 DGAT2 amino acid sequence (Figure 4) SEQ ID No. 15 PDAT nucleotide sequence (Figure 5) SEQ ID No. 16 PDAT amino acid sequence (Figure 5) BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1: WHITE CLOVER OLEOSIN CONSTRUCTION Generation of white clover oleosin plant binary vectors We have cloned a white clover oleosin cDNA and placed this under the control of the 5 constitutive promoter CaMV35s in a plant binary vector for plant transformation (Scott et al., 2007) Figure 20. We have cloned a white clover oleosin cDNA and placed this under the control of the Arabidopsis thaliana oleosin promoter in a plant binary vector for plant transformation (Scott et al., 2007). It should be noted that the oleosin sequence used is for example only. Any oleosin sequence or combinations of 10 oleosins, steroleosins and caoleosins and oleosin linking sequences could be used.

EXAMPLE 2: SYNTHETIC SESAME SEED OLEOSIN AND POLYOLEOSIN CONSTRUCTION 19 554114 We designed a synthetic sesame seed oleosin either as a single unit or as tandem repeats for expression in both £ coli as well as plants (e.g., Arabidopsis and Lotus). The original sesame seed oleosin nucleotide sequence and translated peptide sequence are from a sesame seed oleosin, GenBank clone AF091840. The codons 5 were optimised for both £ coli and Arabidopsis expression. Each repeat used randomised degenerate codons to code for the specific amino acid sequence thus ensuring that the repeats will not be rearranged by non rec" bacteria such as Agrobacterium rhizogenes. The construct was designed so that it can be relatively simply sublconed from the original backbone (pUC57) into both pET29a and various 10 plant binary vectors. In order to allow simple restriction digestion and re-ligation to reduce the number of repeats as well as to enable us to paste in future peptides between the repeats we engineered restriction sites between them.

We have synthesised a nucleotide sequence encoding for a sesame seed oleosin and polyoleosin and placed these under the control of the constitutive promoter CaMV35s 15 in a plant binary vector for plant transformation (Scott et al., 2007) Figures 21-22. We have a nucleotide sequence encoding for a sesame seed oleosin and polyoleosin and placed these under the control of the Arabidopsis thaliana oleosin promoter in a plant binary vector for plant transformation (Scott et al., 2007) Figures 23-24.

The design allows various numbers of tandem repeats to be easily transferred into 20 pET29a and to perform simple digestions on the original clone to remove different numbers of inserts then transfer to binary vectors. This included a Ncol site on each end of the sesame oleosin/polyoleosin to place it into pET29a which gives the peptide an N-terminai S*Tag and thrombin cleavage site and a C-terminal His«Tag. For transfer to plant binary vectors we designed an attL1 site to the 5'end and an attL2 site 25 to the 3'end, these are compatible for with our GATEWAY constructs built in house.

Codon analysis We compared the codon usage by both E. coli and Arabidopsis. From this we were able to identify codons that were not suitable for use in this construct; these included the following: Arg AGG lie ATA Arg AGA Leu CTA Arg CGG stop TAG Arg CGA Arg CGC 554114 These codons were removed from the codon table; the remaining codons were placed in a table which randomised the possibilities still available for each amino acid. Thus, while the codon usage was randomised the peptide sequence for the sesame seed oleosin was conserved. The randomisation was repeated six times, one for each 5 oleosin repeat. An alignment of these sequences showed that the homology dropped to approximately 75% between each repeat and the drop in homology was generally distributed evenly across the whole sequence.

Amino E.coli A.thaliana Acid Acid Codon frequency freque Gly GGG 0.16 0.15 Gly GGA 0.12 0.37 Gly GGT 0.34 0.34 Gly GGC 0.38 0.14 Glu GAG 0.32 0.48 Glu GAA 0.68 0.52 Asp GAT 0.63 0.68 Asp GAC 0.37 0.32 Val GTG 0.36 0.26 Val GTA 0.16 0.15 Val GTT 0.26 0.41 Val GTC 0.22 0.19 Ala GCG 0.34 0.14 Ala GCA 0.22 0.27 Ala GCT 0.17 0.44 Ala GCC 0.27 0.16 Arg ■H ■I 0.20 Arg AGA iM 0.35 Ser AGT 0.16 0.16 Ser AGC 0.27 0.13 Lys AAG 0.24 0.52 Lys AAA 0.76 0.48 Asn AAT 0.47 0.52 Asn AAC 0.53 0.48 40 Met ATG 1.00 1.00 lie ATA ■ 0.24 lie ATT 0.50 0.41 He ATC 0.41 0.35 45 Thr ACG 0.27 0.15 Thr ACA 0.15 0.30 Thr ACT 0.17 0.34 Thr ACC 0.41 0.20 21 554114 Trp TGG 1.00 1.00 End TGA 0.32 0.43 Cys TGT 0.44 0.60 Cys TGC 0.56 0.40 End tag 0.O8 0.20 End TAA 0.60 0.36 Tyr TAT 0.58 0.52 Tyr TAC 0.42 0.48 Leu TTG 0.13 0.22 Leu TTA 0.13 0.14 Phe TTT 0.58 0.51 Phe TTC 0.42 0.49 Ser TCG 0.15 0.10 Ser TCA 0.13 0.20 Ser TCT 0.15 0.28 Ser TCC 0.15 0.13 Arg CGG 0.11 m Arg CGA ■ 0.12 Arg CGT 0.36 0.17 Arg CC-C 0.37 ■ Gin CAG 0.66 0.44 Gin CAA 0.34 0.56 His CAT 0.58 0.61 His CAC 0.42 0.39 Leu CTG 0.48 0.11 Leu CTA HH 0.11 Leu CTT 0.11 0.26 Leu CTC 0.10 0.17 Pro CCG 0.50 0.18 Pro CCA 0.20 0.33 Pro CCT 0.17 0.38 40 Pro ccc 0.13 0.11 Table 1: Comparison of E. coli and A. thaliana codon usage.

Each number represents the proportion that codon is used to code for the respective amino acid. Codons in grey were not used in the polyoleosin construct since they coded for the respective amino acid less than 10% of the time in either organism. 45 Codons in bold and underlined were removed to raise the GC content and to remove cryptic splice sites and mRNA degradation signals (ATTTA). 22 554114 Selection and location of restriction sites between oleosin repeats Restriction sites were inserted as linkers between the repeats. The sites were chosen to allow the subcloning strategy detailed below; they also allowed for the generation of 8 amino acid linkers between each repeat to allow for free rotation etc. Linkers with 5 undesirable codons were not used.

The multiple cloning sites of both pUC57 and pET29a allowed the design of a sub-cloning strategy using multiple placements of the following restriction sites within the polyoleosin construct.

BstXI Cla I Pstl Nco I Nde I Not I Xho I The randomised oleosin repeats were checked for these sites and alternative codons were then used to eliminate the sites when discovered.

Unique restriction sites We also engineered unique Eco47 III, Dra I, Mlu Nl, Sac I, Sal I, Sea I, Hpa I, Alw44l sites between different repeats. These have been included to allow future additions of 15 peptides between the repeats.

Not I sites Not I sites flank the ORF of the complete clone. This is to allow sub-cloning into pART binary vectors if necessary.

Optimisation for: Improving Translation Efficiency; increasing RNA stability; 20 Correct Splicing.

Tetranucleotide stop codon.

Brown et al., (1990) reported that there was an increasing number of reports where the tri-nucleotide stop codons do not signal the termination of protein synthesis; they found that the signals UAA(A/G) and UGA(A/G) are the preferred stop codons in eukaryotes. 25 Hence we have added an A to the 3' end of the second stop codon (TGA) in our construct. mRNA degradation signal 23 554114 Beelman and Parker (1995) reported the degradation signal ATTTA (AUUUA) can destabilize transcripts in plants as well as animals. The proposed construct ORF originally had 7 ATTTA sites. These were predominantly caused by the sequence coding for isoleucine followed by a tyrosine residue. The ATTTA sites were removed 5 by changing the relevant isoleucine codons to ATC. Re-analysis of the splice sites after the removal of the ATTTA sites showed that fewer regions were predicted to be introns (partially determined by the GC content). poly T The original proposed construct ORF would have had 27 TTTT sites and 12 TTTTT 10 sites.

To reduce the number of these regions the phenylalanine codon TTT was removed and replaced by TTC; in one case the site was eliminated by moving the engineered Dral site to the 5' end of the Mlu Nl site. Combined these changes reduced the number of TTTT sites to 14 and the number of TTTTT sites to 1.

Plant Intron Insertion The insertion of a recognised plant intron into an expression construct frequently results in a significantly enhanced expression of the construct in planta\ this is termed Intron Mediated Enhancement (Rose 2004 and references therein). The sequence and position of the intron is important in terms of expression enhancement with the 20 highest gains obtained by placing the Arabidopsis thaliana ubiquitinIO (UBQ10) intron within the first 250 bases or so of the 5' end of the transcript (Rose 2004 and references therein). Rose and Beliakoff (2000) found that utilising a PstI site was a useful way to insert introns. This was achieved by engineering a PstI site to the 5' end of the intron and by modifying the existing 3' end of the intron to contain a PstI site, 25 from this they were able to add or delete functional introns wherever a PstI site existed in the gene or cDNA.

Vector NTI identified approximately 4 PstI sites within the proposed polyoleosin construct with the closest to the 5' end occurring approximately 500 bases downstream. All these sites were eliminated using various combinations of 30 degenerate codons and a new PstI site was engineered at position 300 using the degenerate codons. This places the intron in the first oleosin repeat and therefore enables the generation of all truncated versions with the intron. Using the UBQ10 intron sequence (Norris et al., 1993) we engineered the 3' end to include a Pst1 site. 24 554114 The new polyoleosin construct (containing the intron) was then analysed by NetGene2 (Hebsgaard et al., 1996; Brunak et al., 1991, web site address http://www.cbs.dtu.dk/services/NetGene2/) to confirm that the engineered intron would be predicted to be spliced correctly. This analysis revealed that not only 5 would the UBQ10 be spliced out correctly but there were also high confidence cryptic donor and acceptor sites that would likely result in aberrant splicing. The putative cryptic splice sites were either eliminated where ever possible or reduced in confidence by using alternative redundant codons. The analysis was repeated and showed that the only high confidence donor and acceptor sites remaining were 10 flanking the engineered UBQ10 intron (NetGene 2 results of both the polyoleosin prior to cryptic splice site removal and polyoleosin with intron and cryptic splice sites removed are shown below); it was predicted that the splicing would remove only the intron and would leave the construct in frame. The analysis showed that a number of regions did not appear to be coding regions and as such may be susceptible to some 15 aberrant splicing. To further reduce the possibility of cryptic splicing we then modified the GC content of the construct (see GC content below).

GC content.

Oleosins with optimised and randomised codons; no ATTTA sites or TTT were still found to have relatively low GC content compared to the original sequence (see table). 20 To increase the GC content the additional codons were removed: ATT, AAT, TTA, CTT. This raised the GC content to close to the original content.

The repeats were linked using the previously engineered linking regions. These sequences were modified to remove all but the first Pst I sites in the first oleosin repeat and the removal of an Xho site in the second oleosin repeat. In our construct 25 the ORF has no ATTTA or TTTT sites. Furthermore, when the sequence was re- analysed by NetGene2 the only predicted intron splice site in the ORF was the UBQ10 intron engineered into the PstI site and the % identity of the repeats increased from an average of 74.8% identical to 79.1% identical.

NetGene2 was used to predict the splicing of the proposed construct. The results 30 indicated that the RNA should only be spliced at the acceptor and donor sites of the UBQ10 intron.

Generation of Sesame seed oleosin and polyoleosin plant binary vectors 554114 We have synthesized a sesame seed oleosin/polyoleosin with intron and placed this under the control of the constitutive promoter CaMV35s in a plant binary vector for plant transformation (Figures 21-22). We have synthesized a sesame seed oleosin/polyoleosin with intron and placed this under the control of the Arabadopsis 5 thaliana oleosin/polyoleosin promoter in a plant binary vector for plant transformation (Figures 23-24). It should be noted that the oleosin/polyoleosin sequence used is for example only. Any oleosin/polyoleosin sequence or combinations of oleosin/polyoleosins, steroleosin/polyoleosins and caoleosin/polyoleosins and oleosin/polyoleosin linking sequences could be used. The coding sequence of the 10 complete ORF (after splicing) was then checked against a heptameric repeat of the original oleosin/polyoleosin translated sequence and found to be identical over the oleosin/polyoleosin coding regions.

EXAMPLE 3: ENHANCEMENT OF OLEOSIN AND POLYOLEOSIN PRODUCTION IN THE SEEDS Brassica oleracea plant transformation and analysis The white clover oleosin and polyoleosin constructs were transformed into fast cycling Brassica oleracea plants (using Agrobacterium mediated transformation).

T1 seed was received and 6 seeds per plant line were germinated. Seedlings were tested for Basta resistance, all resistant plants were potted and one susceptible plant 20 was retained per line (used to generate a WT control). Plants were allowed to self pollinate and set seed, then seed was collected and cleaned up.

Crude oil body preps were prepared by grinding 200mg seed with a spatula tip of sand and 750 |jl extraction buffer (10 mM Phosphate buffer, pH 7.5 containing 600 mM sucrose) in a mortar and pestle. A further 750 pi extraction buffer was added and the 25 slurry transferred to an eppendorf tube. The sample was centrifuged 5 min at 14,000rpm then the aqueous layer removed to a new tube using a thin gel loading tip. The oil layer (any material remaining attached to the side of the tube) was resuspended with extraction buffer, and removed to a new tube where the volume was made up to the 500 nl mark on the side of the 2ml eppendorf tube. Samples of 30 aqueous and oil layers were removed and added to an equal volume of 2 x loading buffer, mixed and boiled 5 mins.

Western Blot Analysis was carried out on the T2 seed samples, except that the white clover oleosin antibody was used as primary antibody. This antibody was raised 26 554114 against the N-terminal hydrophilic portion of the white clover oleosin protein. The positive control for these samples was an oil body prep extracted from white clover (Trifolium repens Huia C19739) seed. The negative control was an oil body prep from seed of a Gus-expressing plant (GH#23664 Tube43, containing At Oleosin promoter-5 Gus construct, tested +ve for Gus in seed) (Figure 1).

Construction of synthetic sesame seed oleosin and polyoleosin constructs for prokaryotic and in planta expression, generation of polyclonal antibodies to sesame seed oleosin, culture and induction of expression system, preparation of artificial oil bodies (AOBs), transformation of Arabidopsis thaliana with oleosin and polyoleosin 10 constructs under the control of the CaMV35s construct or the Arabidopsis oleosin seed promoter, transformation of Arabidopsis thaliana var Columbia with oleosin and polyoleosin binary constructs, analyses of T2 seeds for polyoleosin, immunoblot analysis of Arabidopsis thaliana oil bodies containing sesame seed oleosin was performed as described previously (Scott et al., 2007). 25 mg of Arabidopsis seed was ground in 300 |jl extraction buffer (10 mM Phosphate buffer, pH 7.5 containing 600 mM sucrose) using a Wiggenhauser D-130 Homogenizer. Seed was ground until crushed and the sample appeared "creamy" and frothy as starch was released from the seeds. The homogenizer tip was rinsed in 1 ml buffer and this volume was added to the crushed seed. Samples were prepared up to 20 this point in lots of 4, then centrifuged 14,000rpm for 5 mins. A thin gel loading tip was used to gently push the oil layer to the side of the tube, and the aqueous layer removed to a fresh tube. The oil layer was resuspended from the side of the tube using extraction buffer and placed in a fresh 2 ml tube. The final volume was made up to 0.5 ml (as read on the side of the tube) with extraction buffer. Oil body preparation 25 was added to an equal volume of 2 x get loading buffer and boiled 5 mins before loading on to gel.

Samples were run either on duplicate pre-cast NuPAGE Novex 4-12% Bis-Tris Midi Gel (Invitrogen) on the Criterion gel rig system (Bio-Rad), or on hand-cast Tris-HCI gels. One gel was stained by SafeStain (Invitrogen) to show total protein loaded and 30 the other was blotted onto Nitrocellulose membrane using the iBIot system (Invitrogen). In each case, the negative control was a sample extracted from wild type Columbia seed and the positive control was the same extraction method (although grinding was by mortar and pestle) performed on wild type sesame seed. 10|jl of each sample and the negative control were loaded onto the gel, and 5|jI was used for 35 the positive control. 27 554114 Following blotting, the membrane was blocked in a solution of 12.5% skim milk powder in TBST (50 mM Tris pH 7.4, 100 mM NaCI, 0.2 % Tween) for at least 1.5 hours. The membrane was then washed in TBST 3x5 mins before incubating with primary antibody (anti-sesame) at 1/1000 in TBST for 1 hour at room temperature. Following 3 5 further TBST washes, incubation with secondary antibody (anti-rabbit) at 1/5000 was carried out for 1 hour at room temperature. The membrane underwent 3 further washes then the signal was developed using standard chemiluminesence protocol. Figure 2 shows the accumulation of single and tandem repeat sesame seed oleosin units in the seed under the control of the CaMV35S promoter. It can be seen that 10 recombinant oleosin and polyoleosin was found to accumulate in the seeds of Arabidopsis thaliana and was correctly targeted to the oil bodies (Figure 2).

EXAMPLE 4: PRODUCTION OF TRIACYLGLYCEROL IN THE VEGETATIVE PORTIONS OF THE PLANT In most plants (including Lolium perenne) the majority of leaf lipids are attached to a 15 glycerol backbone and exist as diacylglycerols. These are incorporated into lipid bi-layers where they function as membranes of multiple sub-cellular organelles or the as the membrane of the cell itself. The majority of lipid bilayer in the leaf is the chloroplast thylakoid membrane. A smaller amount of leaf lipid exists as epicuticular waxes and an even smaller percentage is present in the form of triacylglycerol (TAG).

Most plants synthesise and store TAG in developing embryos and pollen cells where it is subsequently utilised to provide catabolizable energy during germination and pollen tube growth. Dicotyledonous plants can accumulate up to approximately 60% of their seed weight as TAG. Ordinarily, this level is considerably lower in the monocotyledonous seeds where the main form of energy storage is carbohydrates 25 (e.g., starch)The only committed step in TAG biosynthesis is the last one, i.e., the addition of a third fatty acid to an existing diacylglycerol, thus generating TAG. In plants this step is performed by one of three enzymes including: acyl CoA:diacylglycerol acyltransferase (DGAT1), Figure 3; an unrelated acyl CoA:diacylglycerol acyl transferase (DGAT2), Figure 4; and 30 phospholipid:diacylglycerol acyltransferase (PDAT), Figure 5 (Zou et al., 1999; Bouvier-Nave et al., 2000; Dahlqvist et al., 2000; Lardizabal et al., 2001). Over expression of the transcribed region of any of these genes in the vegetative portions of plants leads to the formation of TAG droplets in the cytoplasm of leaf cells, as demonstrated by the over expression of: Arabidopsis DGAT1 in tobacco by Bouvier-35 Nave et al., (2000); Tung tree DGAT2 in yeast and tobacco by Shockey et al., (2006); 28 554114 Arabidopsis PDAT in Arabidopsis by Stahl et al., (2004) Arabidopsis DGAT1 in Lotus japonicus hairy roots (Bryan et al., 2004).

Production of TAG in Lolium perenne Leaves by Over Expressing Arabidopsis thaliana DGAT 1.

Sub-c!oning Arabidopsis thaliana DGAT1 cDNA and cloning Arabidopsis thaliana DGAT1 genomic transcribed region; transformation of Lolium perenne by microprojectile bombardment of embryogenic callus and microprojectile bombardment of Lolium perenne with the Bio-Rad Particle Delivery System (PDS-1000/He) was performed as per Bryan et al., (2007) Analysis of Lolium perenne Leaves Over Expressing Arabidopsis thaliana DGAT1 by SDS-PAGE/lmmunoblot, Nile Red Staining and FAMES GC/MS.

Total soluble protein from leaves from hygromycin plants were analysed for the presence of the Arabidopsis thaliana DGAT1 recombinant protein using SDS-PAGE/immunoblot. Polyclonal antisera generated against prokaryotically expressed 15 Arabidopsis thaliana DGAT1 identified two plants that accumulated detectable levels of recombinant protein (Figure 6). Ryegrass leaf blade was harvested at 3.5 days after cutting back; pproximately 100 mg fresh leaf were homogenized in 400 ml grinding buffer containing 50 mM Tris-HCI pH8, 5mM EDTA pH8, 0.4% (v/v) b-Mercaptoethanol, and 1mM PMSF using acid sterile sand. Transfer plant slurry to new 20 1.5mL micro-centrifuge tubes, and 150|jl of 4x sample loading buffer (200mM Tris-HCI pH6.8, 40% Glycerol, 4% (w/v) SDS, 4% (v/v) b-Mercaptoethanol, and a spatula tip of Bromophenol blue) were added, then the samples were boiled for 10min. Protein extracts were centrifuged at 4°C, 14,000 rpm for 5 min. Total soluble proteins (TSP) were transferred to the new eppendorf tubes ands ubsequently loaded on SDS-PAGE 25 for further Comassie staining and Immunoblotting. 5|jL of TSP was loaded for Comassie staining and 25|jL. of TSP was loaded for Immunoblotting.

Two ryegrass lines (FU0806 and FU1103) were found to accumulate an immunoreactive band of the expected size (Figure 6); these plants and control were analysed for lipid accumulation by staining with Nile Red which is an excellent vital 30 stain for the detection of intracellular lipid droplets by fluorescence microscopy (Greenspan et al., 1985). It can be seen that the two plants found to accumulate recombinant Arabidopsis thaliana DGAT1 also demonstrated considerably higher accumulation of intracellular lipid droplets and formed an oil in water emulsion upon 29 554114 extraction of soluble proteins (Figure 7). FAMES GC/MS was also used to analyse transgenic ryegrass plants, the results are presented semi-quantitatively in Table 2.

Transgenic Line Total peak area of fatty acids/mg fresh tissue Average peak area of fatty acids/mg fresh tissue % fatty acid content compared to control F0107 (control) - rep 1 287803 303187 100 NF0107 (control) - rep 2 318570 FU0702 - rep 1 352044 342645 113 FU0702 - rep 2 333246 FU0806 - rep 1 370238 432469 143 FU0806 - rep 2 494700 FU01103 - rep 1 406050 421697 139 FU01103 - rep 2 437344 Table 2. Total lipid content (GC/MS trace peak area/mg of fresh weight) of transgenic ryegrass 5 leaves overexpressing Arabidopsis thaliana DGAT 1.

EXAMPLE 5: PRODUCTION OF OLEOSIN/POLYOLEOSIN IN THE LEAVES OF ARABIDOPSIS THALIANA Construction of synthetic sesame seed oleosin construct for prokaryotic and in pianta expression, generation of polyclonal antibodies to sesame seed oleosin, culture and 10 induction of expression system, preparation of artificial oil bodies (AOBs), transformation of Arabidopsis thaliana with polyoleosin constructs under the control of the CaMV35s construct or the Arabidopsis oleosin seed promoter, transformation of Arabidopsis thaliana var Columbia with Polyoleosin binary constructs, analyses of T2 seeds for polyoleosin, immunoblot analysis of Arabidopsis thaliana oil bodies 15 containing sesame seed oleosin was performed as described previously (Scott et al., 2007). Leaves were sampled from plants over expressing the single sesame seed oleosin construct and analysed by SDS-PAGE/immunoblot using the polyclonal anti-sesame seed oleosin antisera. It can be seen that recombinant oleosin was found to 554114 accumulate in the leaves of Arabidopsis thaliana leaves (Figure 8). This used approximately 3 fully mature leaves of Arabidopsis growing in the potted soil or 100 mg of leaves harvested from Arabidopsis seedlings germinated on plant medium containing 1/2x MS with 1 % sucrose (w/v). This was homogenized with 300 ml 5 grinding buffer and added 100 ml. of 4x sample loading buffer to the plant slurry and 7.5|Jl loaded on SDS-PAGE and Comassie stained while 30|jI was loaded on SDS-PAGE and Immunoblotted with the sesame oleosin antibody.

The simultaneous expression and accumulation of oleosin/polyoleosin protein in the same cell (for example leaf cell) will result in the production of triglyceride oil bodies 10 encapsulated by a phospholipid monolayer embedded with oleosin; this has been demonstrated in yeast (Ting et al., 1997) and seeds (Abell et al., 2004).

EXAMPLE 6: INCREASED SEED OIL BODY THERMAL STABILITY DUE TO INCREASED OLEOSIN/POLYOLEOSIN LEVELS Oil body preparations were made from Arabidopsis wild type seeds and seeds 15 accumulating recombinant sesame seed oleosin and polyoleosin were subjected to 90°C for 24hr, it can be seen that the oil body preparations made from seeds accumulating recombinant sesame seed oleosin/polyoleosin were more stable than those made from wild type seeds accumulating wild type levels of oleosin, this is seen by the thicker emulsification layer correlating with increased oleosin and increasing 20 oleosin repeat number (Figure 9). In addition, increasing the number of oleosin units increased the thermal stability.

EXAMPLE 7: INCREASED SEED OIL BODY STABILITY IN RUMEN FLUID DUE TO INCREASED OLEOSIN/POLYOLEOSIN LEVELS Oil bodies were extracted from the seeds of four Arabidopsis lines expressing 25 recombinant sesame seed single oleosin constructs and the level of oleosin expression determined by immunoblot (Figure 10). Oil bodies were extracted using the homogeniser from 25mg seed, in a final volume approximately 500|jL and stored at 4°C overnight. The preparations were re-mixed and an aliquot added to an equal volume (75pL) of rumen fluid. Samples were incubated at 39°C without shaking; 30 samples were vortexed each time before an aliquot was removed at each time point (0, 0.5,1, 2, 4, and 6h). The different lines expressed oleosin at different levels and corresponded to line 61<47<48<65 in ascending order. Oil bodies were extracted from these seeds as well as wild type seeds and incubated with clarified rumen fluid. 31 554114 Aliquots were removed during the digestion incubation and the relative number of intact oil bodies was monitored by measuring turbidity (OD6OO) (Figure 10). It can be seen that increasing amounts of oleosin in the oil bodies increased their stability in rumen fluid where the stability is shown in ascending order from WT=61<<48<47<65.

EXAMPLE 9: INCREASED ARTIFICIAL OIL BODY STABILITY IN RUMEN FLUID DUE TO INCREASED OLEOSIN /POLYOLEOSIN LEVELS Expression of recombinant oleosin/polyoleosin and preparation of Artificial Oil Bodies (AOBs) A fresh 10mL culture (pET29 [Novagen] vector containing a oleosin/polyoleosin 10 sequence, within E. coli BL21 Rosetta-Gami) was grown at 37°C with shaking (180-200rpm) until OD600 « 0.5 - 0.7 (~120min). Recombinant protein expression was induced by the addition of IPTG to 1mM final concentration. The induced culture was incubated at 37°C with shaking (180-200rpm) for a further 2 - 3h. 10OOpL aliquots of the culture were transferred to 1.5mL microfuge tubes and the cells pelleted by 15 centrifugation at 2655xg for 5min at 4°C. The supernatant was discarded and the cell pellet immediately used for subsequent steps or stored at -20°C until required.

Each cell pellet was resuspend in lOOOpL Oilbody Buffer (50mM NaP04 pH8.0, 100mM NaCI) and sonicated (Sonics & Materials Vibra-Ceii VC600, 600W, 20kHz; 1/8" tapered micro-tip probe) off/on ice four times using the following settings -20 Pulse 100, Time 10, Power 4. insoluble protein was pelleted by centrifugation at 20,817*g for 10min at 4°C. The supernatant was discarded and 500|jL Oilbody Buffer was added to the pellet, which was briefly sonicated to resuspend the insoluble protein pellet (Pulse 100, Time 10, Power 4). Small aliquots of the resuspended pellet were removed for protein 25 concentration determination (using Bio-Rad Protein Dye). A maximum of 500pl_ of the resuspended pellet was transferred to a fresh 1,5mL microfuge tube, ensuring that the amount of protein in each tube was the same then made up to 1000pL with Oilbody Buffer.

A 40|jL aliquot of Purified Sesame Oil (ether extracted oil from sesame seed flour) was 30 added to each tube and sonicated off/on ice four times using the following settings -Pulse 100, Time 10, Power 4. The Artificial Oil Bodies (AOBs) that had formed during the sonication of the insoluble protein, comprised mostly of recombinant oleosin or polyoleosin, and oil mix was compacted into a floating layer by centrifugation at 32 554114 ,817xg for 10min at 4°C. The AOBs were recovered by tilting the tube on its side and removing the lower aqueous phase and any pelleted particulates from beneath the AOB layer. The AOBs were resuspended in lOOOpL Oilbody Buffer.

Total protein, pellets and AOBs were analysed by SDS-PAGE/Coomassie and 5 immunoblot, each polyoleosin was expressed, however, the total quantity of recombinant oleosin decreased with an increase in oleosin repeat number; consequently there was proportionally less recombinant oleosin in the AOBs generated using the longer polyoleosin chains (Figure 11).

Analysis of heat stability A 100|jL aliquot of each of the resuspended oleosin/polyoleosin AOBs (containing 70ng total protein), was transferred into a PCR tube. The tubes were incubated at 90°C in a PCR machine without agitation or centrifugation. A time course assessment of heat stability of the AOBs was conducted. Images of the emulsion were captured after 0, 5,10, 15, 20, 30 and 60min incubation (Figure 12). Using these images the 15 relative turbidity (an approximation of emulsion stability) of the emulsions was estimated visually and graphed (Figure 13). Aliquots of the AOBs were also analysed by microscopic examination after 0, 20 and 60min incubation. 1jjI_ aliquots of each sample were pipetted onto a glass slide and photographed for comparison (Figure 14). The diameters of randomly selected micelles were also determined (Figure 15) using 20 imaging software (Olympus Soft Imaging Solutions GmbH, Germany).

For AOBs prepared using single oleosin or 6 oleosin repeats, as the duration of incubation at 90°C increased, so did the mean diameter of the AOBs (Figure 15). Although the diameters of the AOBs prepared with p29 actually decreased, this was likely caused by the larger AOBs having coalesced to form oil micelles large enough to 25 float to the top of the tube. It appears that AOBs prepared using 3* oleosin/polyoleosin are the most stable, as they did not coalesce or aggregate even after incubation at 90°C for 1h. The 6* oleosin/polyoleosin seems to be the most unstable at high temperature and then 1 x oleosin/polyoleosin, in comparison the 2x, 3x, 4x and 5x polyoleosin AOBs showed very little change in diameter over time at 30 90°C (Figure 15). 3x oleosin/polyoleosin appeared to be the most heat stable, as indicated by the diameter of oil bodies which were less than 2.0|jm even after incubation at high 33 554114 temperature for 1h. Mean diameter of AOB prepared using 6* oleosin/polyoleosin after incubation at 90°C for 1 h averaged to 8.2(jm, indicating less stability.

Effects of percentage of oil used during preparation on AOB physical properties Do increased numbers of oleosin units require more oil to form AOBs (i.e. provide the 5 surface area to fit the polyoleosin peptides).

To increase the amount of oil encapsulated by the oleosin/polyoleosin within a micelle, additional oil was added to pre-prepared AOBs and sonicated to incorporate the additional oil. Three 100pL aliquots of AOBs diluted for equal protein concentration were transferred into 1.5mL microfuge tubes, and OpL, 40(jL, and 120pL of purified 10 sesame oil was added to the relevant tubes. This provided final concentrations of 4%, 8% and 16% v/v, respectively. Oilbody Buffer was added to bring the final volume up to lOOOpL. The mix was sonicated four times using the following settings - Pulse 100, Time 10, Power 4. Aliquots were removed for microscopic observation (Figure 16) and heat stability analysis (Figure 17).

The number of oleosin units in a oleosin/polyoleosin peptide influences AOB stability The results from Figures 12-17 indicate that emulsions of AOBs prepared with the 2* and 3x oleosin/polyoleosin are more stable than those prepared with 4* and 5* oleosin/polyoleosin, which are in turn more stable than those prepared with 1 * and 20 6* oleosin/polyoleosin (2* & 3* > 4* & 5* > 1 x & 6x). An explanation for the lower stability of the larger oleosin/polyoleosin is that firstly there is less polyoleosin protein (seen from Figure 11) and secondly the oleosin/polyoleosin may not be fully incorporated into the AOBs thus assuming incorrect topologies in the oil micelle (Figure 18).

It can be expected that with increasing numbers of oleosin units requires more energy to separate the increasing number of hydrophobic regions from one another in an inclusion body. Therefore, not all of the central hydrophobic regions in the larger oleosin/polyoleosin peptides would be available to become incorporated into the oil micelles during the preparation of the AOBs (Figure 18). The greater the ratio of non-30 incorporated hydrophobic regions to incorporated hydrophobic regions, the greater the disruptive tension perpendicular to the micelle surface, compared to the binding tension parallel to the surface. Increasing the amount of sonication energy used to form an emulsion from 6x polyoleosin may offer a means of generating emulsions 34 554114 more stable than those seen thus far with the 3* oleosin/polyoleosin emulsions. The incorporation of the hydrophobic regions into the micelle can also be used to explain why the degree of stabilisation offered by the 1 x oleosin/polyoleosin is not as great as that seen with the majority of the longer oleosin/polyoleosin. The single oleosin unit 5 lacks a secondary anchoring point in the form of another embedded hydophobic region, and the steric hindrance is overcome by the proximity of nearby micelles and is thus unable to prevent coalescence.

Higher ratios of oleosin:TAG increase the stability of AOBs in rumen fluid AOBs with a constant amount of recombinant oleosin were generated with 4, 8 and 10 18% TAG. These were incubated in 1/2x rumen fluid at 39°C and analysed by microscope at various time points. AOB stability decreased as the oleosin/TAG ratio decreased (seen by increase in AOB diameter as AOBs coalesce due to decreased oleosin present on the surface) (Figure 19). The greatest AOB stability was shown with the oleosin and 4% TAG sample which only showed signs of aggregation and 15 some coalescence after 60 minutes in rumen fluid. The negative control shows that the oil never forms a true oil body and the oil is coalescing into droplets immediately after sonication; after 60 minutes the sample is virtually clear of oil (Figure 19, Panel A) and TAG was only present as large droplets (Figure 19, Panel B) indicating no protection was afforded by the addition of a non oleosin control protein.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 554114 REFERENCES Abell BM, Hahn M, Holbrook LA, Moloney MM. (2004). Membrane topology and sequence requirements for oil body targeting of oleosin. Plant J., 37: 461-70 Bouvier-Nave P, Benveniste P, Oelkers P, Sturley SL, Schaller H. (2000) Expression 5 in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase. Eur. J. Biochem. 267, 85-96.

Bryan GT, Burling MF, Trollope AJ, Woodfield DR, Roberts NJ. (2007) Modification of fatty acid biosynthesis. United States Patent 20070118927 Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S. 10 (2000) Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA. 97, 6487-6492.

Firkins JL, Hristov AN, Hall MB, Varga GA, St-Pierre NR. (2006). Integration of ruminal metabolism in dairy cattle. J Dairy Sci. 89 Suppl 1:E31-51. Review. Greenspan P, 15 Mayer EP, Fowler SD. (1985). Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 100(3):965-73. Links Jenkins TC, McGuire MA. (2006). Major advances in nutrition: impact on milk composition. J Dairy Sci. 89(4): 1302-10. Review. Jenkins TC, Bridges WC Jr. (2007). Protection of fatty acids against ruminal biohydrogenation in cattle. Eur. J. Lipid Sci. 20 Technol. 109:778-789.

Jobling SA, Jarman C, Teh MM, Holmberg N, Blake C, Verhoeyen ME., Immunomodulation of enzyme function in plants by single-domain antibody fragments. Nat Biotechnol. 2003 Jan;21(1):77-80.

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Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ. (2001) DGAT2 Is a new diacylglycerol acyltransferase gene family. J.B.C. 276, 38862-38869.

Lock AL, Bauman DE. (2004). Modifying milk fat composition of dairy cows to enhance fatty acids beneficial to human health. Lipids. 39(12): 1197-206. Review. 36 554114 Mansoor S, Amin I, Hussain M, Zafar Y, Briddon RW. Engineering novel traits in plants through RNA interference. Trends Plant Sci. 2006 Nov;11(11):559-65.

Mekhedov S, de llarduya OM, Ohlrogge J. (2000). Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis. Plant Physiol. 122(2):389-5 402).

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Stahl U, Carlsson AS, Lenman M, Dahlqvist A, Huang B, Banas W, Banas A, Stymne 10 S. (2004). Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant Physiology, 135, 1324-1335.

Shockey JM, Gidda SK, Chapital DC, Kuan J-C, Dhanoa PK, Bland JM, Rothstein SJ, Mullen RT, Dyer JM. (2006). Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localised to different subdomains of 15 the endoplasmic reticulum. Plant Cell., 18,2294-2313.

Siloto RM, Findlay K, Lopez-Villalobos A, Yeung EC, Nykiforuk CL, Moloney MM. (2006). The accumulation of oleosin/polyoleosins determines the size of seed oilbodies in Arabidopsis. Plant Cell. 18(8):1961-74.

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Claims (35)

1. A construct which includes: a) at least one promoter; b) a nucleic acid molecule encoding a single oleosin protein; and c) at least one nucleic acid molecule encoding an triacylglycerol biosynthetic enzyme (TAG synthesising enzyme); wherein the promoter(s) are operatively linked to the nucleic acid molecules so as to cause expression of the oleosin and at least one TAG synthesising enzyme .

2. The construct as claimed in claim 1, wherein the nucleic acid molecule encoding the oleosin protein has a nucleotide sequence selected from: a) SEQ ID NO's 1, 3, 5, 7 or 9 or other nucleic acid molecules encoding an oleosin gene; b) a complement of a sequence in a); c) a functional fragment or variant of a sequence in a) or b); or d) a homolog or an ortholog of a sequence in a), b), or c).

3. The construct as claimed in claim 1, wherein the nucleic acid molecule encoding the TAG synthesising enzyme has a nucleotide sequence selected from: a) SEQ ID NO's 11, 13 or 15 other nucleic acid molecules encoding TAG synthesising enzymes; d) a homolog or an ortholog of a sequence in a), b), or c); or e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) or d). b) a complement of a sequence in a); INTEOfSPAKcPROperty UhFICE OF (VJ.2. c) a functional fragment or variant of a sequence in a) or b); 2 1 OCT 2009 39 554114

4. The construct as claimed in any one of the above claims, wherein the TAG synthesising enzymes are any enzyme required to add the third fatty acid to an existing diacylglycerol and generate TAG within a plant or plant cell.

5. The construct as claimed in any one of the above claims, wherein the TAG synthesising enzymes are selected from one of the following; acyl CoA: diacylglycerol acyltransferase (DGAT1); acyl CoA: diacylglycerol acyl transferase (DGAT2); or phospholipid: diacylglycerol acyltransferase (PDAT).

6. The construct as claimed in any one of the above claims, wherein there is at least one TAG synthesising enzyme is present within the construct.

7. The construct as claimed in any one of the above claims, wherein there is more than TAG synthesising enzyme is present within the construct.

8. The construct as claimed in any one of the above claims, wherein the oleosin gene and/or the TAG synthesising enzymes are selected from either monocot or dicot plant species.

9. The construct as claimed in claim 8, wherein the monocot plant species are selected from one of the following; grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley.

10. The construct as claimed in claim 8, wherein the dicotyledons plant species are selected from one of the following: Arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus, poplar, hybrid aspen, gymnosperms, and pine trees.

11. A use of at least one construct to produce single oleosin and TAG synthesising enzymes in the vegetative portions of a plant.

12. The use of at least construct as claimed in claim 11, wherein the construct includes: a) at least one promoter; b) a nucleic acid molecule encoding a single oleosin protein; and c) at least one nucleic acid molecule encoding an triacylglycerol biosynthetic enzyme (TAG synthesising enzyme); 40 554114 wherein the promoter(s) are operatively linked to cause expression of oleosin and TAG synthesising enzyme .

13. The use of a construct as claimed in claim 12, wherein the nucleic acid molecule encoding the oleosin protein has a nucleotide sequence selected from: a) SEQ ID NO's 1, 3, 5, 7 or 9 or other nucleic acid molecules encoding an oleosin gene; b) a complement of a sequence in a); c) a functional fragment or variant of a sequence in a) or b); or d) a homolog or an ortholog of a sequence in a), b), or c).

14. The use of construct as claimed in claim 12, wherein the nucleic acid molecule encoding the TAG synthesising enzyme has a nucleotide sequence selected from: a) SEQ ID NO's 11, 13 or 15 or other nucleic acid molecules synthesising enzymes; b) a complement of a sequence in a); c) a functional fragment or variant of a sequence in a) or b); d) a homolog or an ortholog of a sequence in a), b), or c); or e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) or d).

15. The use of a construct as claimed in any one of claims 11 to 14, wherein the TAG synthesising enzymes is any enzyme required to add the third fatty acid to an existing diacylglycerol and generate TAG within a plant or plant cell.

16. The use of a construct as claimed in any one of claims 11 to 15, wherein the TAG synthesising enzymes are selected from one of the following; acyl CoA: diacylglycerol acyltransferase (DGAT1); acyl CoA: diacylglycerol acyl transferase (DGAT2); or phospholipid: diacylglycerol acyltransferase (PDAT).

17. The use of a construct as claimed in any one of claims 11 to 16, wherein there encoding TAG 2 I OCT 2009 [RECEIVE: 41 554114 ' NTELLECTUrtL PfiOVfcrt I v QF~iOF OF M / 2 i OCT 'MM received is at least one TAG synthesising enzyme is present within the construct.

18. The use of a construct as claimed in any one of claims 11 to 17, wherein there are more than TAG synthesising enzyme is present within the construct.

19. The use of a construct as claimed in any one of claims 11 to 18, wherein the nucleic acid molecule encoding the oleosin and/or the TAG synthesising enzymes are selected from either; monocots, dicots, or gymnosperms plant species.

20. The use of a construct as claimed in claim 19, wherein the monocot plant species are selected from one of the following; grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley.

21. The use of a construct as claimed in claim 19, wherein the dicotyledonous plant species are selected from one of the following; Arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus, poplar, hybrid aspen, gymnosperms, and a pine tree.

22. A method of producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, comprising the step of manipulating the genome of a plant so as to express more single oleosin and TAG synthesising enzyme in the vegetation portions than a corresponding wild-type plant.

23. The method as claimed in claim 22, wherein the step of manipulating the genome does not include traditional selective breeding techniques.

24. A method of producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, including the steps of: a) genetically manipulating the plant to produce single oleosin in the vegetative portion of the plant; and b) genetically manipulating the plant to produce TAG synthesising enzyme in the vegetative portion of the plant.

25. The method as claimed in claim 24, wherein there is a further step (c) which is the step of: measuring oleosin and TAG in manipulated plants to find plants with a higher oleosin to TAG ratio in the vegetative portion of the plant. 42 RECEIVED at IPONZ on 20 November 2009 554114

26. A method of producing single oleosin and TAG synthesising enzyme within vegetative portions of a plant, including the steps of: a) genetically manipulating a first plant to produce single oleosin in the vegetative portion of the plant; b) genetically manipulating a second plant to produce TAG synthesising enzyme in the vegetative portion of the plant; and c) crossing the first and second plants to produce a plant expressing single oleosin and TAG synthesising enzyme within the vegetative portions of the plant.

27. A method as claimed in claim 26, wherein there is a further step of measuring the levels of: i) oleosin in the first plant; and ii) TAG in the vegetative portions of the second plant; to find a pair of plants for crossing, wherein the first plant produces sufficient levels of oleosin relative to the amount of TAG synthesising enzyme produced in the second plant such that the plant produced by crossing in step (c) produces oil bodies encapsulating TAG which are capable of protecting and/or delaying degradation or biohydrogenation of the TAG within the stomach or rumen of an animal.

28. The method as claimed in any one of claims 22 to 27, wherein the vegetative portions of the plant are one of the following; the shoots, leaves, fruit, bark, pods, roots, nodules, flowers, stems and the like, or parts thereof.

29. The method as claimed in any one of claims 22 to 28, wherein the vegetative portions of the plant are the leaves.

30. An animal feed which includes seeds and/or vegetative portions of plants, which have been manipulated to have elevated levels of single oleosin and TAG compared to the corresponding wild type seed or plant.

31. An animal feed which includes seeds which have been manipulated to have elevated single oleosin and TAG synthesising enzyme content compared to the corresponding wild type seeds. 43 RECEIVED at IPONZ on 20 November 2009 554114

32. An animal feed which includes a vegetative portion of a plant which has been manipulated to have elevated single oleosin and TAG synthesising enzyme content compared to the vegetative portion of a corresponding wild type plants.

33. An animal feed as claimed in claim 31 or 32 wherein the collective ratio of single oleosin to TAG produced by the TAG synthesising enzyme, is capable of protecting and/or delaying degradation or biohydrogenation of the TAG in oil bodies in the rumen of an animal.

34. A plant or part thereof which has been manipulated to express both a single oleosin and a TAG synthesising enzyme by transforming the plant with a construct of any one of claims 1 to 10.

35. A plant or part thereof which has been manipulated to produce both a single oleosin and TAG synthesising enzyme by transforming the plant with a construct of any one of claims 1 to 10. 44