Feedstuffs for Aquaculture Comprising Stearidonic Acid
The present invention relates to feedstuffs for use in aquaculture, as well as methods for producing said feedstuffs. The invention also provides methods for rearing fish and/or crustaceans. In particular, the present invention provides a method of rearing a fish or crustacean, the method comprising feeding the fish or crustacean a feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA).
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This application claims priority from U.S. 60/737,946, the entire contents of which are incorporated herein by reference.FIELD OF THE INVENTION
The present invention relates to feedstuffs for use in aquaculture, as well as methods for producing said feedstuffs. The invention also provides methods for rearing fish and/or crustaceans.BACKGROUND OF THE INVENTION
Global production of farmed fish and crustacea has more than doubled in the last 15 years and its expansion places an increasing demand on global supplies of wild fish harvested to provide protein and oil as ingredients for aquafeeds (Naylor et al., 2000). The supply of seafood from global capture fisheries sources is around 100 million tones per annum (FAO, 2001). This amount has not increased since the mid-1980's and will not increase in the future as most fisheries are at or above sustainable levels of production, and are further subjected to sharp, periodic declines, due to climatic factors such as El Niño (FAO, 2001; Barlow 2000). Fish oil stocks are also under increasing demand not only from aquaculture, but from the agriculture and nutraceutical/biomedical industries.
Replacement oils for the aquaculture industry have been sourced from a variety of commercial terrestrial plant sources including sunflower (Bransden et al, 2003; Bell et al., 1993), canola/rapeseed (Bell et al, 2003; Polvi and Ackman, 1992), olive, palm (Fonseca-Madrigal et al, 2005; Bell et al, 2002) and linseed (Bell et al., 1993; Bell et al., 2004). The inclusion of vegetable oil to replace part or all of the fish oil in fish diets resulted in the same growth rates and feed conversion ratios (Bransden et al., 2003; Polvi and Ackman, 1992; Torstensen et al., 2004; Fonseca-Magrigal et al., 2005; Bell et al., 2002; Bell et al., 2004). However, since these plant oils had essentially no ω3 long-chain (≧C20) polyunsaturated fatty acids (ω3 LC-PUFA) and had high levels of monounsaturated fatty acids (MUFA), ω6 PUFA and low ω3/ω6 ratios, fish fed such diets displayed reduced levels of ω3 LC-PUFA. This is thought to be associated with reduced health benefits to the consumer compared to fish fed a diet high in fish oil containing greater levels of ω3 LC-PUFA (Seierstad et al., 2005). Therefore, raising fish or crustacea on diets high in vegetable oil has the potential to dilute the important cardiovascular and other benefits which are associated with eating fish.Pathways of LC-PUFA Synthesis
Biosynthesis of LC-PUFA from linoleic and α-linolenic fatty acids in organisms such as microalgae, mosses and fungi may occur by a series of alternating oxygen-dependent desaturations and elongation reactions as shown schematically in
Alternative routes have been shown to exist for two sections of the ALA to DHA pathway in some groups of organisms. The conversion of ALA to ETA may be carried out by a combination of a Δ9 elongase and a Δ8 desaturase (the so-called Δ8 desaturation route, see
Besides these desaturase/elongase systems, EPA and DHA can also be synthesized through an anaerobic pathway in a number of organisms such as Shewanella, Mortiella and Schizochytrium (Abbadi et al., 2001). The operons encoding these polyketide synthase (PKS) enzyme complexes have been cloned from some bacteria (Morita et al., 2000; Metz et al., 2001; Tanaka et al., 1999; Yazawa, 1996; Yu et al., 2000; WO 00/42195). The EPA PKS operon isolated from Shewanella spp has been expressed in Synechococcus allowing it to synthesize EPA (Takeyama et al., 1997). The genes encoding these enzymes are arranged in relatively large operons, and their expression in transgenic plants has not been reported. Therefore it remains to be seen if the anaerobic PKS-like system is a possible alternative to the more classic aerobic desaturase/elongase for the transgenic synthesis of LC-PUFA.
The biosynthetic pathways for PUFA are well known (Sargent et al., 2002). Vertebrates lack ω12 and ω15 (ω3) lipid desaturases and cannot produce linoleic acid (18:2ω6, LA) and α-linolenic acid (18:ω3, ALA) from oleic acid (18:ω9, OA) (see
The desaturase enzymes that have been shown to participate in LC-PUFA biosynthesis all belong to the group of so-called “front-end” desaturases which are characterised by the presence of a cytochrome b5 domain at the N-terminus of each protein. The cyt b5 domain presumably acts as a receptor of electrons required for desaturation (Sperling and Heinz, 2001). The enzyme Δ6 desaturase catalyses the desaturation of linoleic acid (LA) to form gamma-linoleic acid (GLA, 18:3ω6) and linolenic acid (ALA) to form stearidonic acid (SDA, 18:4ω3) (
The enzyme Δ5 desaturase catalyses the desaturation of C20 LC-PUFA leading to arachidonic acid (ARA, 20:4 ω6) and EPA (20:5ω3). Genes encoding this enzyme have been isolated from a number of organisms, including algae (Thraustochytrium sp. Qiu et al., 2001), fungi (M. alpine, Pythium irregulare, Michaelson et al., 1998; Hong et al., 2002), Caenorhabditis elegans and mammals. A gene encoding a bifunctional Δ5-/Δ6-desaturase has also been identified from zebrafish (Hasting et al., 2001). The gene encoding this enzyme might represent an ancestral form of the “front-end desaturase” which later duplicated and evolved distinct functions.
The last desaturation step to produce DHA is catalysed by a Δ4 desaturase and a gene encoding this enzyme has been isolated from the freshwater protist species Euglena gracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001; Meyer et al., 2003).Elongases
Several genes encoding PUFA-elongation enzymes have also been isolated (Sayanova and Napier, 2004). The members of this gene family were unrelated to the elongase genes present in higher plants, such as FAE1 of Arabidopsis, that are involved in the extension of saturated and monounsaturated fatty acids. An example of the latter is erucic acid (22:1) in Brassicas. In some protist species, LC-PUFA are synthesized by elongation of linoleic or α-linolenic acid with a C2 unit, before desaturation with Δ8 desaturase (
Transgenic oilseed crops that are engineered to produce major LC-PUFA by the insertion of various genes encoding desaturases and/or elongases have been suggested as a sustainable source of nutritionally important fatty acids. However, the requirement for coordinate expression and activity of five new enzymes encoded by genes from possibly diverse sources has made this goal difficult to achieve and only low yields have generally been obtained (reviewed by Sayanova and Napier, 2004; Drexler et al., 2003; Abbadi et al., 2001).
A gene encoding a Δ6-fatty acid desaturase isolated from borage (Borago officinalis) was expressed in transgenic tobacco and Arabidopsis, resulting in the production of GLA (18:3ω6) and SDA (18:4ω3), the direct precursors for LC-PUFA, in the transgenic plants (Sayanova et al., 1997 and 1999). However, this provides only a single, first step.Feedstuffs for Aquaculture
Research in feedstuffs for aquaculture have largely focused on enriching salmon diets by increasing the dietary supply of ALA (Bell et al., 1993) and EPA/DHA (Harel et al., 2002; Carter et al., 2003).
There is a need for further diets for aquaculture which, upon consumption, enhance the production of omega-3 long chain polyunsaturated fatty acids in aquatic animals.SUMMARY OF THE INVENTION
The present inventors have determined that fish and crustaceans can be produced with appropriate levels of LC-PUFA, such as EPA, DPA and/or DHA, without the need to feed these organisms diets which are rich in LC-PUFA. In particular, the LC-PUFA precursor stearidonic acid (SDA) can be provided to the fish or crustaceans whilst still producing fish or crustaceans with desirable levels of LC-PUFA.
Thus, in a first aspect, the present invention provides a method of rearing a fish or crustacean, the method comprising feeding the fish or crustacean a feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA).
In a preferred embodiment, the lipid comprises a phytosterol.
In a particularly preferred embodiment, at least 1% of the SDA in the feedstuff was obtained from a plant. The plant may be non-transgenic, such as an Echium sp., Oenothera biennis, Borago officinalis or Ribes nigrum, or transgenic. In an embodiment, at least some of the SDA is from oil obtained from seed of the plant.
In a preferred embodiment, the transgenic plant comprises an exogenous nucleic acid encoding a Δ6 desaturase. The transgenic plant may further comprise an exogenous nucleic acid encoding a ω3 desaturase or Δ15 desaturase, which increases the production of ALA in the plant. The transgenic plant may further comprise an exogenous nucleic acid encoding a Δ12 desaturase. Examples of suitable transgenic plants include, but are not limited to, canola, soybean, flax, other oilseed plants, cereals or grain legumes.
In a particularly preferred embodiment, the fish is a salmon.
In one embodiment, the fish or crustacean is fed predominantly the feedstuff over a period of at least 6 weeks, preferably at least 7 weeks and even more preferably at least 12 weeks. In an embodiment, after having been fed the feedstuff for at least 6 weeks, the fish or crustacean has similar weight, specific growth rate, weight gain, total feed consumption, feed efficiency ratio, hepatosomatic index and/or survival when compared with the same species of fish or crustacean fed the same feedstuff but which substantially lacks SDA.
In another embodiment, the fish or crustacean, after having been fed the feedstuff for at least 6 weeks, has higher SDA and/or ETA levels in muscle tissue when compared with the same species of fish or crustacean fed the same feedstuff but which substantially lacks SDA.
In a further embodiment, the fish or crustacean, after having been fed the feedstuff for at least 6 weeks, has lower SFA levels in muscle tissue when compared with the same species of fish or crustacean fed the same feedstuff but which comprises fish oil instead of the plant oil comprising at least 5.5% SDA. In preferred embodiments, the levels of 14:0 and 16:0 are reduced, for example by at least 10% or at least 20%.
In another aspect, the present invention provides a feedstuff for a fish or crustacean, the feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA, 18:4Δ6,9,12,15, ω33). The feedstuff may have any of the characteristics as described herein in the context of the methods.
In a further aspect, the present invention provides a fish or crustacean produced using a method of the invention.
In yet another aspect, the present invention provides a fish, wherein the fatty acid of the white muscle lipid of said fish comprises less than 29.6% SFA and at least 18.3% DHA. In certain embodiments, the white muscle lipid of the fish comprises fatty acid comprising less than 28%, less than 27%, or more preferably less than 26% SFA. In other embodiments, the white muscle lipid of the fish comprises fatty acid comprising at least 19%, at least 20%, at least 21%, or more preferably at least 22% DHA.
In another aspect, the present invention provides a fish, wherein the fatty acid of the red muscle lipid of said fish comprises fatty acid comprising less than 28.2% SFA and at least 9.6% DHA. In certain embodiments, the red muscle lipid of the fish comprises fatty acid comprising less than 27%, less than 26%, or more preferably less than 25% SFA. In other embodiments, the muscle lipid of the fish comprises fatty acid comprising at least 10%, at least 11%, or more preferably at least 12% DHA.
In a further aspect, the present invention provides a fish or crustacean, wherein the fatty acid of the muscle lipid of said fish or crustacean comprises at least 2.7% SDA. In embodiments of this aspect, the muscle lipid of said fish or crustacean comprises at least 3%, at least 3.5%, or more preferably at least 4% SDA.
In a further aspect, the present invention provides a fish, wherein the fatty acid of the white muscle lipid of said fish comprises at least 2.1% SDA. In embodiments of this aspect, the white muscle lipid of said fish comprises at least 2.5%, at least 3%, or more preferably at least 3.5% SDA.
Preferably, a fish of the invention is a salmon.
In yet a further aspect, the present invention provides a method for producing a feedstuff for fish and/or crustaceans, the method comprising admixing oil obtained from a plant with at least one other ingredient, wherein the fatty acid of said oil comprises at least 5.5% (w/w) SDA. In a preferred embodiment, the other ingredient comprises fish meal, a high protein source other than fishmeal, a starch source or a combination of these. Other ingredients may include vitamins, minerals, choline, or pigments such as, for example, carotenoids or carophyll pink.
Preferably, the plant is transgenic.
Preferably, the oil is obtained from the seed of the plant.
In certain embodiments, it is preferred that the fatty acid of said oil comprises at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11.0%, at least 15%, at least 20%, or at least 30% (w/w) SDA.
In another aspect, the present invention provides a method for producing a feedstuff for fish and/or crustaceans, the method comprising admixing a transgenic organism, or extract or portion thereof, with at least one other ingredient, wherein the organism is genetically modified such that it produces SDA and/or produces higher levels of SDA than when compared to a corresponding non-transgenic wild-type organism. The method may comprise the step of extracting the oil from the organism, for example from the seed of a plant. The extraction may comprise physical means such as crushing of seed, chemical means such as extraction with a solvent, heating or other processes, or any combination of these. The oil may be further purified before mixing with other ingredients. The method preferably includes preparation of an extruded product from the mixed ingredients by an extrusion process, suitable for providing to fish or crustacean. The method may comprise the step of analysing the feedstuff, such as for example measuring the level of lipid or the level of SDA in the fatty acid, or other measurements.
Preferably, the organism is a plant or yeast.
In another aspect, the present invention provides a feedstuff produced using a method of the invention. The feedstuff may have the characteristics as described above. Other ingredients that may be included in the feedstuff include fish meal, a high protein source other than fishmeal, a starch source, vitamins, minerals, pigments such as, for example, carotenoids or carophyll pink, or any combination of these. Fishmeal is a preferred protein source for the major carnivorous fish such as salmon, trout, tuna, flatfish, barramundi, particularly for Atlantic salmon. Fishmeal, typically about 65% protein, may be added in an amount from 20 to 700 g per kg dryweight. A high protein source other than fishmeal may be from a plant or animal source such as, for example, wheat or other cereal gluten, soymeal, meal from other legumes, casein, protein concentrates, protein isolates, meat, meat and bone, blood, feathers. These are typically at least 30% protein and may be milled with or without extraction of oil. Starch may be added, typically at 10-150 g/kg, and may be in the form of cereal grain or meal. For crustaceans, krill meal, mussel meal or other similar components may be added at 1-200 g/kg, cholesterol and/or lecithin at 0-100 g/kg. The mixture may comprise a binding agent such as sodium alginate, for example Manucol from Kelco International.
In a further aspect, the present invention provides oil extracted from a fish or crustacean of the invention, comprising SDA, EPA, DPA, DHA or any combinations thereof.
In yet another aspect, the present invention provides a cotton or flax plant capable of producing seed, wherein the oil of said seed comprises fatty acid comprising at least 5.5% SDA on a weight basis.
Furthermore, the present inventors have found that expressing a Δ6 desaturase gene in a fibre producing plant results in surprisingly high levels of Δ6 desaturase PUFA products.
Thus, in a further aspect the present invention provides a cotton or flax plant capable of producing seed, wherein the seed synthesizes GLA that is the product of Δ6-desaturation of LA and/or SDA that is the product of Δ6-desaturation of ALA, and wherein the efficiency of conversion of LA to GLA and/or ALA to SDA in the seed is at least 25%, at least 35%, or at least 45%. For example, at least 25%, preferably at least 45% of the polyunsaturated fatty acid in the cotton or flax seed that has a carbon chain of C18 or longer is desaturated at the Δ6 position.
Preferably, the cotton plant is Gossypium hirstum or Gossypium barbadense.
Preferably, the flax plant is Linum usitatissimum.
Preferably, the fatty acid of the oil comprises at least 8% SDA, or at least 10% SDA, at least 11% SDA, at least 15% SDA, at least 20% SDA, at least 25% SDA, at least 30% SDA, at least 35% SDA, at least 40% SDA, at least 45% SDA or at least 50% SDA.
In one preferred embodiment, the plant comprises a transgenic Δ6 desaturase gene. In another preferred embodiment, the plant comprises a transgenic Δ15 desaturase or ω3 desaturase gene which may be in additional to the transgenic Δ6 desaturase gene. In an embodiment, the protein coding region of said gene is from a plant, microalgal, fungal or vertebrate source.
Also provided is the seed of a plant of the invention, wherein the oil of said seed comprises fatty acid comprising at least 5.5% SDA on a weight basis.
In a further aspect, the present invention provides a method of producing a plant of the invention, comprising the introduction of a Δ6 desaturase gene into a cotton or flax plant cell and the regeneration of a plant therefrom.
In an embodiment, the method comprises the step of determining the fatty acid composition of seedoil obtained from seed of said plant and/or the step of selecting a plant on the basis of its seed oil composition.
In another embodiment, the method further comprises the introduction of a Δ15 desaturase or ω3 desaturase gene into said plant.
In yet a further aspect, the present invention provides a method of producing the seed of the invention, comprising growing said plant and harvesting seed from said plant.
As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
SEQ ID NO:1—Δ6 desaturase from humans (Genbank Accession No: AAD20018).
SEQ ID NO:2—Δ6 desaturase from mouse (Genbank Accession No: NP—62673).
SEQ ID NO:3—Δ6 desaturase from Pythium irregulare (Genbank Accession No: AAL13310).
SEQ ID NO:4—Δ6 desaturase from Borago officinalis (Genbank Accession No: AAD01410).
SEQ ID NO:5—Δ6 desaturase from Anemone leveillei (Genbank Accession No: AAQ10731).
SEQ ID NO:6—Δ6 desaturase from Ceratodon purpureus (Genbank Accession No: CAB94993).
SEQ ID NO:7—Δ6 desaturase from Physcomitrella patens (Genbank Accession No: CAA11033).
SEQ ID NO:8—Δ6 desaturase from Mortierella alpina (Genbank Accession No: BAC82361).
SEQ ID NO:9—Δ6 desaturase from Caenorhabditis elegans (Genbank Accession No: AAC15586).
SEQ ID NO:10—Δ6 desaturase from Echium plantagineum.
SEQ ID NO:11—Δ6 desaturase from Echium gentianoides (Genbank Accession No: AY055117).
SEQ ID NO:12—Δ6 desaturase from Echium pitardii (Genbank Accession No: AY055118).
SEQ ID NO:13—Δ5/Δ6 bifunctional desaturase from Danio rerio (zebrafish).
SEQ ID NO's 14 to 16—Conserved motifs of Echium sp. Δ6 desaturases.
SEQ ID NO's 17 to 22, 30 and 31—Oligonucleotide primers.
SEQ ID NO:23—Linin promoter from Linum usitatissimum.
SEQ ID NO:24—Linin terminator from Linum usitatissimum.
SEQ ID NO:25—cDNA sequence encoding Δ6 desaturase from Echium plantagineum.
SEQ ID NO:26—Δ15 desaturase from Perilla frutescens (Genbank Accession No: AF213482).
SEQ ID NO:27—Δ15 desaturase from Brassica napus (Genbank Accession No: L01418).
SEQ ID NO:28—Δ15 desaturase from Betula pendula (Genbank Accession No: AAN17504).
SEQ ID NO:29—Δ15 desaturase from Arabidposis thaliana (Genbank Accession No: AAC31854).
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, plant biology, molecular genetics, immunology, immunohistochemistry, fatty acid synthesis, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and are incorporated herein by reference.
As used herein, the term “lipid” generally refers to an organic molecule, typically containing a hydrocarbon chain(s), that is insoluble in water but dissolves readily in nonpolar organic solvents. Feedstuffs of the invention are defined herein relative to the composition of their lipid component. This lipid component includes fatty acids (either free or esterified, for example in the form of triacylglycerols), sterols and polar lipids.
As used herein, the term “fatty acids” refers to a large group of organic acids made up of molecules containing a carboxyl group at the end of a hydrocarbon chain; the carbon content may vary from C2 to C34. The fatty acids may be saturated (contain no double bonds in the carbon chain) (SFA), monounsaturated (contain a single double bond in the carbon chain) (MUFA), or polyunsaturated (contain a two, three, four or more double bonds in the carbon chain) (PUFA). Unless stated to the contrary, the fatty acids may be in a free state (non-esterified) or in an esterified form such as part of a triacylglycerol, diacylglyceride, monoacylglyceride, acyl-CoA bound or other bound form, or mixture thereof. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms.
As used herein, the terms “long-chain polyunsaturated fatty acid”, “LC-PUFA” or “C20+ polyunsaturated fatty acid” refer to a fatty acid which comprises at least 20 carbon atoms in its carbon chain and at least three carbon-carbon double bonds. Ordinarily, the number of carbon atoms in the carbon chain of the fatty acids refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes those in side groups. Generally, the long-chain polyunsaturated fatty acid is an ω3 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. Preferably, the long-chain polyunsaturated fatty acid is selected from the group consisting of; eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3) eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA, 22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19, ω3). It would readily be apparent that the LC-PUFA that is in (or limited in amount or even excluded from) a feedstuff of the invention, or produced by a fish or crustacean fed a feedstuff of the invention, may be a mixture of any or all of the above and may include other LC-PUFA or derivatives of any of these LC-PUFA.
Use of the term “fish” includes all vertebrate fish, which may be bony or cartilaginous fish. The present invention may be practiced with any of the considerable variety of fresh, brackish, or salt water fish species including, but not limited to, salmon, trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod, haddock, Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike, pike-perch, yellowtail, tilapia, eel or tropical fish (such as the fresh, brackish, and salt water tropical fish). In an embodiment, the fish is not hybrid striped bass. In a further embodiment, if the fish is hybrid striped bass, the fatty acid of said lipid comprises at least 11.0%, at least 12% or at least 15% (w/w) SDA. In another embodiment, if the fish is hybrid striped bass, the SDA content of the feedstuff is at least 2.1% (w/w). Yet other species with which the present invention can be practiced will be apparent to those skilled in the art, including those species outlined in Table 1. The invention may be practised with any, all, or any combination of the listed fish.
As used herein, the term salmon refers to any species of the Family Salmonidae. Preferably, the salmon is a Salmo sp. or Oncorhynchus sp. More preferably, the salmon is a Salmo sp. Even more preferably, the salmon is Atlantic Salmon (Salmo salar).
In an embodiment, the fish, preferably salmon, is at a “larval” or “juvenile” stage. Fish development recognises 5 periods that occur in the following order: embryonic period; larval period; juvenile period; adult period; senescent period. The larval period occurs once the embryo has hatched and has the ability to feed independently of the egg yolk (or mother in rare cases), organ systems develop morphologically and gain physiological function. The juvenile period is when all organ systems are fully formed and functional (bar the gonads) and fish attain the appearance of miniature adults, the period lasts until the gonads become mature. Once the gonads mature the fish attain the adult period, and then senescence when growth ceases and gonads do not produce gametes (Adapted from Moyle, P. B. & Cech, J. J. 2004. Fishes An Introduction to Ichthyology, 5th Edition, Prentice Hall).
The “crustacean” may be any organism of the subphylum “Crustacea”, and hence the crustacean may be obtained from marine sources and/or freshwater sources. Such crustacea include, but are not limited to, organisms such as krill, clams, shrimp (including prawns), crab, and lobster. Further examples of crustacea that can be reared on feedstuffs of the invention are provided in Table 2. The invention may be practised with any, all, or any combination of the listed crustacea.
For purposes of the present invention, “feedstuffs” include any food or preparation, for fish or crustacean consumption.
The present invention provides a feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA). The invention also provides methods of using said feedstuff for rearing a fish or crustacean.
In embodiments of the invention, the fatty acid of said lipid comprises at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11.0%, at least 15%, at least 20%, or at least 30% (w/w) SDA.
In further embodiments, the fatty acid of said lipid comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or more preferably less than 8% (w/w) total saturated fatty acids (SFA). In particular, the feedstuff comprises reduced levels of 14:0 and/or 16:0 compared to the corresponding feedstuff made with fishoil rather than plant oil comprising at least 5.5% SDA.
Although the level of SDA that may be produced in seedoil of transgenic plants may be in excess of 40% of the fatty acid, the invention may be practised with plant oil that has less SDA, such as for example at least 5.5% SDA. That is, not all of the ALA is converted to SDA in the plant, and the oil may contain both SDA and ALA. Therefore, in yet other embodiments, the fatty acid of said lipid comprises at least 10%, at least 15%, at least 16%, at least 17%, at least 18%, or at least 19% (w/w) α-linolenic acid (ALA 18:3Δ9,12,15, ω3). In an embodiment, the ALA level is in the range 10-45% (w/w).
Preferably, the lipid of the feedstuff comprises phytosterol, which may provide additional benefit. In embodiments of the invention, the lipid comprises at least 0.025%, at least 0.05%, or at least 0.1% (w/w) phytosterol. It may comprise at least 0.2% phytosterol, typically in the range 0.2-0.8% (w/w) phytosterol. The phytosterol may be any plant derived sterol from plants such as, but not limited to, Echium sp., canola, soybean, flax, cereal or grain legume. Examples of phytosterols include, but are not limited to, brassicasterol, campesterol, stigmasterol, β-sitosterol or any combination of these.
In a further embodiment, the lipid is substantially free of cholesterol, which may be advantageous in limiting the cholesterol level in the fish or crustacean that is produced, in particular for fish. As used herein, the term “substantially free of cholesterol” refers to the lipid comprising less than 0.1% (w/w) cholesterol, preferably at an undetectable level. Typically, lipid obtained from plants is substantially free of cholesterol.
In other embodiments, at least 25%, at least 50%, at least 75% or at least 90% of the SDA is esterified in the form of triacylglycerol.
In yet further embodiments, the lipid content of the feedstuff is at least 10, at least 15, at least 20, at least 30, at least 50, at least 100, at least 200, or at least 250 g/kg dry matter. In another embodiment, the lipid content of the feedstuff is no more than 350 g/kg dry matter or any range between these figures.
In other embodiments, the feedstuff comprises at least 0.55, at least 1, at least 2.5, at least 5, at least 7.2, at least 10, at least 12.5, or more preferably at least 14.3 g/kg dry matter of SDA.
In yet another preferred embodiment, the fatty acid of the lipid content of the feedstuff comprises less than 2% EPA and/or DHA, more preferably less than 1% EPA and/or DHA.
The SDA can be from any source. In a preferred embodiment, the SDA is provided in the form of a transgenic organism, or extract or portion thereof, wherein the organism is genetically modified such that it produces SDA and/or produces higher levels of SDA than when compared to a wild-type organism. Preferably, the transgenic organism is a plant or yeast. In a particularly preferred embodiment, the SDA is provided in the form of oil extracted from a plant, especially a transgenic plant. Typically, such oil is extracted from the seed of the plant. However, in some embodiments, the SDA may be obtained from a non-transgenic organism which naturally produces SDA, for example, Echium plantagineum.
Fish and crustaceans can be fed feedstuffs of the present invention in any manner and amount, and according to any feeding schedule employed in fish or crustacean cultivation. Feeding rates typically vary according to abiotic factors, mainly seasonal such as temperature, and biotic, in particular the size of the animal. Juvenile fish are typically fed 5-10% of their body weight per day over about 4-6 feeds per day. Larger fish are typically fed at 2-5% of their body weight per day over about 1-2 feeds per day. Juvenile crustaceans may fed up to 5-10% of their body weight over about 6 feeds per day, while larger crustaceans may be fed a minimum of about 2% of their body weight per day over about 2-3 feeds per day. The fish or crustaceans may be allowed to feed to appetite.
Preferably, the fish or crustaceans are fed at least once per day, more preferably two or more times per day such as, for example, 2-6 or 4-6 times per day. It is preferred that any excess food be removed after the feeding period, e.g., by flushing out of a race-way system, or through removal out of the bottom of the sea-cage. Alternatively, a fish such as catfish can be added to the fish population to consume any excess food.
The benefits increase when fish or crustacean are fed over longer periods of time, for example over at least 6, 7 or 12 weeks. However, it would be appreciated that there is some benefit when the fish or crustacean is provided with the feedstuff over shorter time periods, relative to feeding the fish or crustacean feedstuff containing plant oil not comprising substantial SDA. Feedstuffs other than those described herein may also be used in the time period, however it is preferred that the feedstuff of the invention is used predominantly over the time period if not exclusively.
As used herein, “predominantly” means at least 50% of the time, occasions or in amount, as the context determines.
It is preferable that fish or crustaceans be fed SDA containing feedstuffs as a mixture with other well-known ingredients included in commercial fish or crustaceans food formulations so as to provide a nutritionally balanced complete food, including, but not limited to, plant matter, e.g., flour, meal, starch or cracked or processed grain produced from a crop plant such as wheat or other cereals, alfalfa, corn, oats, potato, rice, soybeans or other legumes; cellulose in a form that may be obtained from wood pulp, grasses, plant leaves, and waste plant matter such as rice or soy bean hulls, or corn cobs; animal matter, e.g., fish or crustacean meal, oil, protein or solubles and extracts, krill, meat meal, bone meal, feather meal, blood meal, or cracklings; algal matter; yeast; bacteria; vitamins, minerals, and amino acids; organic binders or adhesives; and chelating agents and preservatives. A wide variety of formulations are reported in both the patent and scientific literature. Alternatively, SDA is used to supplement other foods, e.g., commercial fish or crustacean foods.
In one embodiment, the feedstuff comprises fishmeal (which may or may not be defatted) but does not comprise, as a separate ingredient, fish oil. Alternatively, the feedstuff may comprise some fishoil as an added separate ingredient. However, the minimum level of SDA in the fatty acid of the total lipid of the feedstuff should remain at least 5.5%.
On a commercial scale feedstuffs may conveniently be provided in the form of pressed or extruded feed pellets.
The components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.
With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Examples of these include Stay C which is a commercial stabilised vitamin C product, trisodium phosphate or Banox E which is an antioxidant. Other such vitamins and minerals may also be added.Desaturases
Organisms useful for producing feedstuffs of the invention typically comprise a gene encoding a Δ6 desaturase, which may be a transgene or an endogenous gene. As used herein, a “Δ6 desaturase” is at least capable of converting ALA to SDA, and/or linoleic acid (LA, 18:2Δ9,12, ω6) to γ-linolenic acid (GLA, 18:2Δ9,12, ω6). Examples of suitable Δ6 desaturases include, but are not limited to, those which comprises (i) an amino acid sequence as provided as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, (ii) an amino acid sequence which is at least 50% identical to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, or (iii) a biologically active fragment of i) or ii). In a further embodiment, the Δ6 desaturase comprises an amino acid sequence which is at least 90% identical to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. In a further embodiment, the Δ6 desaturase is encoded by the protein coding region of one of the Δ6 desaturase genes listed in Table 3 or gene at least 75% identical thereto.
The Δ6 desaturase may also have other activities such as Δ5 desaturase activity. Such enzymes are known in the art as a “Δ5/Δ6 bifunctional desaturase” or a “Δ5/Δ6 desaturase”. These enzymes are at least capable of i) converting ALA to SDA, and ii) converting eicosatetraenoic acid to eicosapentaenoic acid. A gene encoding a bifunctional Δ5-/Δ6-desaturase has been identified from zebrafish (Hasting et al., 2001). The gene encoding this enzyme might represent an ancestral form of the “front-end desaturase” which later duplicated and the copies evolved distinct Δ5- and Δ6-desaturase functions. In one embodiment, the Δ5/Δ6 bifunctional desaturase is naturally produced by a freshwater species of fish. In a particular embodiment, the Δ5/Δ6 bifunctional desaturase comprises
i) an amino acid sequence as provided in SEQ ID NO:13,
ii) an amino acid sequence which is at least 50% identical to SEQ ID NO:13, or
iii) a biologically active fragment of i) or ii).
Organisms useful in producing feedstuffs of the invention generally comprise a gene encoding an “ω3 desaturase”, which may be a transgene or an endogenous gene. As used herein, an “ω3 desaturase” is at least capable of converting LA to ALA and/or GLA to SDA and are therefore able to introduce a desaturation at the third carbon-carbon bond from the co end of the acyl substrate. Such desaturases may also be known in the art as Δ15 desaturases when active on a C18 substrate, for example 18:2 (LA), introducing a desaturation at the fifteenth carbon-carbon bond from the carboxy (Δ) end of the acyl chain. Examples of ω3 desaturase include those described by Pereira et al. (2004), Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al. (1997) or as listed in Table 4. Examples of suitable Δ15 desaturases include, but are not limited to, those which comprise (i) an amino acid sequence as provided in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29, (ii) an amino acid sequence which is at least 50% identical to any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29, or (iii) a biologically active fragment of i) or ii). In a further embodiment, the Δ15 desaturase comprises an amino acid sequence which is at least 90% identical to any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29. In a further embodiment, the Δ15 desaturase has an amino acid sequence according to an Accession No listed in Table 4, or is encoded by the protein coding region of one of the Δ15 desaturase genes listed in Table 4, or a protein or gene at least 75% identical thereto.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence and a sequence defined herein are aligned over their entire length.
The term “polypeptide” is used interchangeably herein with the terms “protein” and “enzyme”.
With regard to the defined polypeptides/enzymes, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
As used herein, the term “biologically active fragment” refers to a portion of the defined polypeptide/enzyme which still maintains desaturase activity. Such biologically active fragments can readily be determined by serial deletions of the full length protein, and testing the activity of the resulting fragment.Cells
Suitable cells for use in feedstuffs of the invention, or which can be used to produce SDA for feedstuffs of the invention, include any cell containing SDA or that can be transformed with a polynucleotide encoding a polypeptide/enzyme described herein, and which is thereby capable of being used for producing SDA. Host cells into which the polynucleotide(s) are introduced can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Such nucleic acid molecule may be related to SDA synthesis, or unrelated. Host cells either can be endogenously (i.e., naturally) capable of producing proteins described herein or can be capable of producing such proteins only after being transformed with at least one nucleic acid molecule.
The cells may be prokaryotic or eukaryotic. Host cells can be any cell capable of producing SDA, and include fungal (including yeast), parasite, arthropod, animal and plant cells. Preferred host cells are yeast and plant cells. In a preferred embodiment, the plant cells are seed cells.
In one embodiment, the cell is an animal cell or an algal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic animals such as fish or crustacea, invertebrates, insects, etc.
The cells may be of an organism suitable for fermentation. Suitable fermenting cells, typically microorganisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting microorganisms include fungal organisms, such as yeast. As used herein, “yeast” includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowia lipolytica.Gene Constructs and Vectors
Transgenic organisms, and/or host cells, producing SDA are typically transformed with a recombinant vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
One type of recombinant vector comprises a nucleic acid molecule which encodes an enzyme useful for the purposes of the invention (such as a polynucleotide encoding a Δ6 desaturase or ω3 desaturase) operatively linked to an expression vector. As indicated above, the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of a desired nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in yeast, animal or plant cells.
In particular, expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of desired nucleic acid molecules. In particular, recombinant molecules include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells. A variety of such transcription control sequences are known to those skilled in the art.
Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.Transgenic Plants and Parts Thereof
The term “plant” as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. In preferred embodiments, plant useful for the production of feedstuffs of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. The plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit. The plants of the invention may be: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), cassava (Manihot esculenta), coconut (Cocos nucifera), olive (Olea europaea), oats, or barley.
In one embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an “oilseed plant” is a plant species used for the commercial production of oils from the seeds of the plant. The oilseed plant may be oil-seed rape (such as canola), maize, sunflower, soybean, sorghum, oil palm or flax (linseed). Furthermore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, mustard, castor bean, sesame, safflower, or nut producing plants. The plant may produce high levels of oil in its fruit, such as olive or coconut.
Examples of cotton of the, and/or useful for, the present invention include any species of Gossypium, including, but not limited to, Gossypium arboreum, Gossypium herbaceum, Gossypium barbadense and Gossypium hirsutum.
When the production of SDA is desired it is preferable that the plant species which is to be transformed has an endogenous ratio of ALA to LA which is at least 1:1, more preferably at least 2:1. Examples include most, if not all, oilseeds such as linseed. This maximizes the amount of ALA substrate available for the production of SDA. This may be achieved by transgenic means; for example by introduction of a Δ15 desaturase gene into the plant to increase the levels of the ALA substrate for conversion into SDA.
The plants produced for use in feedstuffs of the invention may already be transgenic, and/or transformed with additional genes to those described in detail herein.
Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Leguminous plants include beans, peas, soybeans, lupins and the like. Beans include guar, locust bean, fenugreek, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
The term “extract or portion thereof” refers to any part of the plant. “Portion” generally refers to a specific tissue or organ such as a seed or root, whereas an “extract” typically involves the disruption of cell walls and possibly the partial purification of the resulting material. Naturally, the “extract or portion thereof” will comprise SDA. Extracts can be prepared using standard techniques of the art.
Transgenic plants, as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques. This would generally be to cause or enhance production of at least one protein/enzyme defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of said plants such as, for example, cultured tissues, callus, protoplasts. Transformed plants contain genetic material that they did not contain prior to the transformation. The genetic material is preferably stably integrated into the genome of the plant. Such plants are included herein in “transgenic plants”. A “non-transgenic plant” is one which has not been genetically modified with the introduction of genetic material by recombinant DNA techniques. In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype.
Several techniques exist for introducing foreign genetic material into a plant cell. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (see, for example, U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may be transformed using Agrobacterium technology (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135). Electroporation technology has also been used to transform plants (see, for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335). In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during development and/or differentiation using appropriate techniques known to those skilled in the art.
A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Examples of plant promoters include, but are not limited to ribulose-1,6-bisphosphate carboxylase small subunit, beta-conglycinin promoter, phaseolin promoter, high molecular weight glutenin (HMW-GS) promoters, starch biosynthetic gene promoters, ADH promoter, heat-shock promoters and tissue specific promoters. Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency. Typical enhancers include but are not limited to Adh-intron 1 and Adh-intron 6.
Constitutive promoters direct continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these promoters may also be used.
In a particularly preferred embodiment, the promoter directs expression in tissues and organs in which lipid and oil biosynthesis take place, particularly in seed cells such as endosperm cells and cells of the developing embryo. Promoters which are suitable are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980), the linin gene promoter from flax, or the legumin B4 promoter (Baumlein et al., 1992), and promoters which lead to the seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. Notable promoters which are suitable are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Other promoters include those described by Broun et al. (1998) and US 20030159173.
Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress. Other desirable transcription and translation elements that function in plants may be used.
In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S) and the like may be used.EXAMPLES Example 1 Materials and Methods Lipid Extraction and Isolation
Samples were freeze dried and extracted using a modified Bligh and Dyer protocol (Bligh and Dyer, 1959). A single phase extraction, CHCl3/MeOH/H2O, (1:1:0.9, by vol), was used to yield a total lipid extract (TLE).
Lipid classes were analysed by an Iatroscan MK V thin-layer chromatography-flame ionization detector (TLC-FID) analyser (Iatron Laboratories, Japan). Samples were spotted onto silica gel SIII Chromarods (5 μm particles size) and developed in a glass tank lined with pre-extracted filter paper. The solvent system used for the lipid separation was hexane: diethyl ether: acetic acid (60:17:0.1, v/v/v) (Volkman and Nichols, 1991). After development for 25 minutes, the chromarods were oven-dried and analysed immediately to minimise adsorption of atmospheric contaminants. Lipid classes were quantified by DAPA software (Kalamunda, WA, Australia). The FID was calibrated for each compound class: phosphatidylcholine; cholesterol; cholesteryl ester; oleic acid; hydrocarbon (squalene); wax ester (derived from fish oil); triacylglycerol (derived from fish oil); and DAGE (purified from shark liver oil).
An aliquot of the TLE was trans-methylated in methanol:chloroform:hydrochloric acid (10:1:1, v/v/v) for 1 hour at 100° C. After addition of water the mixture was extracted three times with hexane: chloroform (4:1, v/v) to produce fatty acid methyl esters (FAME). FAME were concentrated under nitrogen and treated with N,O-bis(trimethylsilyl)-trifloroacetamide (BSFTA, 50 μl, 60° C., 1 h) to convert hydroxyl groups to their corresponding trimethylsilyl ethers. Samples were made up to a known volume with an internal injection standard (23:0 or 19:0 FAME) and analysed by gas chromatography (GC) using an Agilent Technologies 6890N GC (Palo Alto, Calif., USA) equipped with an HP-5 cross-linked methyl silicone fused silica capillary column (50 m×0.32 mm i.d.), and an FID. Helium was used as the carrier gas. Samples were injected, by a split/splitless injector and an Agilent Technologies 7683 Series auto sampler in splitless mode, at an oven temperature of 50° C. After 1 min the oven temperature was raised to 150° C. at 30° C. min−1, then to 250° C. at 2° C. per min and finally to 300° C. at 5° C. min−1. Peaks were quantified by Agilent Technologies GC ChemStation software (Palo Alto, Calif., USA). Individual components were identified by mass spectral data and by comparing retention time data with those obtained for authentic and laboratory standards. GC results are typically subject to an error of ±5% of individual component area. GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermoquest GCQ GC-mass spectrometer fitted with an on-column injector with Thermoquest Xcalibur software (Austin, Tex., USA). The GC was fitted with a capillary column similar to that described above.
A polar column was used to separate 18:1ω9 and 18:3ω3 which coeluted on the HP5 column. FAME were analysed with a Hewlett Packard 5890 gas chromatograph (GC) equipped with a flame ionisation detector (FID) at 250° C. FAME samples were injected using a split/splitless injector into a polar BPX-70 fused-silica column (50 m×0.32 mm i.d.). The carrier gas was helium. The GC oven temperature was initially held at 45° C. for 2 min after injection and then increased at 30° C./min to 120° C. and at 3° C./min to 240° C., then held isothermal for 10 min.Statistical Analysis
Mean values were reported plus or minus standard error of the mean. Percentage data were arcsin transformed prior to analysis. Normality and homogeneity of variance were confirmed and a comparison between means was achieved by 1-way analysis of variance (ANOVA). Multiple comparisons were achieved by Turkey-Kramer HSD. Significance was accepted as probabilities of 0.05 or less. Statistical analysis was performed using SPSS for windows version 11.Brassica Transformation
Brassica napus (Line BLN 1239) seeds were surface sterilized by soaking them in 70% (v/v) ethanol for 2 min and then rinsed for 10 min in tap water at 55° C. The seeds were sterilized for 20 min in 25% commercial bleach (10 gl−1 sodium hypochlorite) containing 0.1% Tween-20. The seeds were washed thoroughly with sterile distilled H2O, placed on GEM medium in tissue culture jars and kept in the cold room for two days for germination. The jars were transferred to low light (20 μMm2s−1) for about four to six days at 24° C. for growth of the cotyledons. Roots and apices were removed under asceptic conditions. Excised hypocotyl segments (10 mm) were washed with 50 ml CIM medium for about 30 min without agitation in the laminar flow cabinet. The CIM was removed and the segments transferred to a 250 ml flask with 50 ml of CIM, sealed with sterile aluminium foil and shaken for 48 hours at 24° C. under low light (10 μMm2s−1).
Agrobacterium strains containing plasmid transformation vectors were grown in 5 ml of LB media with appropriate antibiotics at 28° C. for about two days, transferred to a 250 ml Erlenmeyer flask with 45 ml of LB without antibiotics and cultured for four hours at 28° C. with shaking. The Agrobacterium cells were pelleted by centrifugation, washed, and gently re-suspended in about 20 ml BM. The optical density at 600 nm of the resultant Agrobacterium suspension was adjusted to 0.2 with BM. The cell suspension was added to the explants which had been drained of the CIM medium, mixed briefly and allowed to stand for 20 min. The Agrobacterium suspension was removed, the hypocotyl explants washed once with 50 ml CIM and co-cultivation continued for 48 hours on an orbital shaker. After this, the medium was slightly milky due to Agrobacterium growth. CIM was removed and the explants washed three times with 50 ml CIM for one minute and then twice for one hour on an orbital shaker at 140×g. Following the washes, 50 ml CIM containing 200 mg/l Timentin® was added and placed on an orbital shaker for 24 hours. Under sterile conditions, the CIM medium was clear at this stage.
Regeneration of transformed shoots on SIM was carried out on a two-stage selection process. Initially, the hygromycin concentration in the SIM medium used was 5 mg/l. After about two weeks, explants with developing calli were transferred to SIM containing 20 mg/l hygromycin. When the regenerating shoots had developed leaves longer than one cm, they were excised carefully and were transferred to SEM with 20 mg/l hygromycin. After two weeks, stems usually had elongated and apices were transferred to RIM containing 10 mg/l hygromycin. Non-elongating shoots were sub-cultured in SEM every two to three weeks until they were long enough to be transferred to RIM. When the roots were about two cm in length, the regenerated plantlets were removed from tissue culture pots and transferred to soil for further growth.Media Recipes
Composition of the tissue culture media used in this procedure is given below. They contained MS salts (Murashige and Skoog, 1962), MS or B5 vitamins (Gamborg et al., 1968), sucrose and MES. The pH was adjusted to 5.8 with KOH prior to sterilization. For solid media, agar was added and then autoclaved. Media containing agar was allowed to cool to below 50° C. and filter-sterilized compounds were added to the melted media before pouring it into either plastic Petri dishes or 250 ml polycarbonate tissue culture jars (Sarstedt, No 75.9922519). The composition of various media with all additives are given below: germination medium (GEM); basal medium (BM); callus-inducing medium (CIM, modified from Radke et al., 1988); washing medium (WM); shoot-inducing medium (SIM, modified from Radke et al., 1988); shoot-elongation medium (SEM) and root-inducing medium (RIM, modified from De Block et al., 1989).
GEM: 1×MS salts, 1×MS vitamins, Sucrose (20 gl−1), MES (500 mgl−1), Agar (8 gl−1), pH to 5.8.
BM: 1×MS salts, 1×B5 vitamins, Sucrose (30 gl−1), MES (500 mgl−1), pH to 5.8.
CIM: 2,4-D (1.0 mgl−1) and Kinetin (1.0 mgl−1) added to BM.
WM: 2,4-D (1.0 mgl−1), Kinetin (1.0 mgl−1) and Timentin® (200 mgl−1) added to BM.
SIM: AgNO3 (500 mgl−1), Zeatin riboside (0.5 mgl−1), BAP (2.0 mgl−1), GA3 (0.01 mgl−1), Timentin® (200 mgl−1), Hygromycin (5 to 30 mgl−1), and Agar (8 gl−1) added to BM.
SEM: 0.5×MS salts, 0.5×B5 vitamins, Sucrose (10 gl−1), MES (500 mgl−1), Timentin® (200 mgl−1), Hygromycin (20 to 30 mgl−1), Agar (8 gl−1), pH to 5.8.
RIM: 0.5×MS salts, 0.5×B5 vitamins, Sucrose (10 gl−1), MES (500 mgl−1), IBA (0.1 mgl−1), Timentin® (200 mgl−1), Hygromycin (20 to 30 mgl−1), Agar (8 gl−1), pH to 5.8.Example 2 Fish Fed with Food Compositions Including Plant-Derived SDA
Stearidonic acid (SDA, 18:4ω3) is an LC-PUFA precursor, derived by desaturation of ALA by Δ6 desaturase (
Oil from a few plant sources such as Echium plantagineum have SDA in the fatty acid profile, up to about 15-20% as a percentage of the fatty acid in the oil. To determine whether SDA-rich oil might serve as an efficient substrate for ω3 LC-PUFA accumulation in fish, a feeding trial was conducted in vivo using salmon (Salmo salar L.). Diets including an equivalent level of canola oil were used as a control source of ALA, as described in Tables 5 and 6.
Four diets were formulated to compare canola oil (CO), two different levels of stearidonic acid oil (100% (SO), 1:1 SO:CO (Mix)), and fish oil (FO) (Tables 5 and 6). Fish meal was defattened three times using a 2:1 mixture of hexane and ethanol (400 ml 100 g−1 fish meal). Soybean (Hamlet Protein A/S, Horsens, Denmark), casein (MP Biomedcals Australasia Pty Ltd, Seven Hills NSW, Australia), wheat gluten (Starch Australasia, Land Cove, NSW, Australia) and BOIIC pre-gelatinised maize starch (Penford Australia Limited, Lane Cove, NSW, Australia) were used. Stearidonic acid rich oil was provided as Crossential SA14 (Croda Chemicals, East Yorkshire, UK). Fish oil was from jack mackerel (Skretting Australia, Cambridge, Tasmania Australia). Stay-C and Rovimix E50 were supplied from Roche Vitamins Australia (Frenchs Forest, NSW, Australia), and the remaining ingredients were supplied by Sigma-Aldrich (Castle Hill, NSW, Australia). Yttrium Oxide was used as a digestibility marker. The diets were manufactured into 3 mm diameter pellets using a California Pellet Mill (CL-2), dried and stored at −5° C.
The feeding experiment was conducted at the School of Aquaculture, University of Tasmania, Launceston, Australia. Atlantic salmon (Salmo salar) parr were obtained from Wayatinah Salmon hatchery (SALTAS, Tasmania, Australia) and randomly stocked into 300 l tanks at 25 fish per tank. They were acclimated for 10 days. The tanks were held at a constant temperature of 15.0° C. and a photoperiod of 16:8 (light:dark). The fish were held in a partial freshwater recirculation system. Water was treated through physical, UV and biofilters, with a continuous replacement of approximately 15% per day. Dissolved oxygen, pH, ammonia, nitrate, nitrite, and chlorine were monitored daily to ensure water quality remained within parameters recommended for Atlantic salmon (Wedemeyer, 1996).
Fish were initially anaesthetized (50 mg benzocaine) and weights and lengths were recorded. Four fish were killed and assessed for initial lipid content and composition. Twenty five fish were randomly allotted into twelve 300 l tanks. Fish weights were not significantly different between tanks (43.6 g±0.7). The four diets were fed in triplicate on a ration of 1.1% body weight per day (% BW d−1), in two equal feeds at 0900 and 1700 hrs by automatic belt feeders. Every three weeks all fish were anaesthetized (50 mg l−1, benzocaine) and weighed. Fish were starved the day prior to measuring. Every 7 days the total feed consumption (kg DM) was estimated from the amount of feed that was not eaten by collection in sediment collectors. The amount of uneaten feed was estimated from the number of uneaten pellets using the average weight of a pellet from each feed (Helland et al., 1996).
Specific growth rates (SGR) were calculated as
SGR (% day−1)=100×(ln(W2/w1))×d−1
where W1 and W2 were the weights (g) at the two times and d was the number of days.
At the end of the experiment fish were starved for one day prior to being anaesthetized (50 mg l−1, benzocaine) and their weight and fork length measured. Three fish per tank were killed by a blow to the head after immersion in anaesthetic. Samples of tissue were dissected with red muscle and white muscle sampled below the dorsal fin. Samples were frozen at −80° C. until analysis.Results
No significant difference was found between fish fed the four diets with respect to initial and final weight, weight gain, specific growth rate (SPR), total feed consumption (FC), feed efficiency ratio (FER), hepatosomatic index (HSI) or survival as determined using ANOVA (Table 7).
After 42 days there was no statistical difference in the composition of flesh lipid with respect to the lipid classes for the different dietary groups, in either red or white muscle (Tables 8 and 9). The predominant lipid class in red muscle was TAG (94.0-96.7%). There was significantly (p>0.02) less TAG in the fed fish (42.0-67.0%) compared to the initial measurement (82.0%) for the white muscle.
For fatty acid composition, there were significantly (p>0.01) higher levels of 18:3ω3 and 18:4ω3, in both white and red muscle tissues, in the fish fed SO than in fish fed the Mix diet. Both 18:3ω3 and 18:4ω3 levels were significantly higher than in the FO and CO fed fish (Tables 8 and 9). There were significantly (p>0.01) higher levels in both muscle tissues of 22:6ω3 and total ω3 in the FO and SO diets compared to the Mix and CO diets. There were significantly (p>0.01) higher levels of 20:5ω3 in the FO and SO fed fish compared to the CO fed fish in both the red and white muscle. The ratio of ω3/ω6 was significantly (p>0.01) lower in the CO and Mix diet fed fish compared to the SO and FO diets.
In both muscle tissues, the FO diet surprisingly provided significantly (p>0.01) higher levels of 14:0, 16:0 and total saturates compared with CO and Mix fed. The FO diet also provided significantly (p>0.01) higher levels of 14:0 in both muscle tissues and 16:0 and total saturates in the red muscle compared with the SO fed fish. In both muscle tissues, FO and SO fed salmon had significantly (p>0.01) lower levels of 18:1 ω9 and total MUFA compared to the fish fed CO and Mix diets. There was significantly (p>0.01) higher levels of 18:2 ω6 and total ω6 in the fish fed CO and Mix diets compared with FO fed fish.
The inclusion of SO at 130 or 65 g/kg of diet for Atlantic salmon parr did not significantly influence growth or feed conversion rates compared to other experimental diets during the 42 day growth trial in freshwater (Table 7). There was little effect between diets in the lipid class profiles (Tables 8 and 9). There was significantly less TAG in the white muscle of the fed fish compared to the diet due to the inclusion of oil in the diet at a level of 130 g/kg compared to the commercial diet (approx. 300 g/kg) they were fed pre-experiment.
Fish muscle FA profiles were closely related to the FA profile of their diet. It has been shown previously for salmon fed using canola, sunflower and linseed oils, i.e. diets rich in ALA and without EPA and DHA, that there was a significant reduction in total ω3 and ω3 LC-PUFA, in particular DHA and EPA (Bransden et al., 2003; Bell et al., 2003; Polvi and Ackman, 1992; Bell et al., 2004). Therefore, minimal conversion to, or negligible accumulation of, LC-PUFA occurred when fish were fed vegetable oil. In those studies growth rates and the health of fish fed vegetable oils were not affected.
In the study described here, Atlantic salmon parr sizes were initially 43.6 g±0.7 g to a final weight of 72.4 g±1.9 g. The fish were at an important stage of the growth. Pre-smoltification Atlantic salmon store FA, in particular ω3 LC-PUFA, prior to the energy requiring transfer to salt water, during which salmon undergo major changes in their lipid metabolism.
The inclusion of SDA at 14.3 or 7.2 g/kg significantly influenced the FA profiles of the salmon (Tables 8 and 9). Fish fed on the diet containing the higher level of SDA had significantly higher levels of EPA, DPA, DHA and total ω3 in the muscle samples than fish fed on the CO diet. In some respects, the fatty acid composition of the fish tissues was improved over that of fish fed the FO diet. For example, the level of saturated fat was reduced. The SO diet was also advantageous for this feature in combination with the high levels of LC-PUFA.
Neither the CO diet nor the SO diet contained EPA or DHA at substantial levels, being <0.7% of the fatty acid present in the lipid, the trace level probably originating with the fishmeal component. Therefore the increased accumulation of EPA; DPA and DHA in the fish tissues must have represented increased biosynthesis of the fatty acids from SDA in the fish.
This experiment showed that high levels of total ω3, DHA and EPA could be maintained in fish such as salmon without their inclusion as dietary FA. This experiment also demonstrated that the levels of fatty acids achieved, as reported in Tables 8 and 9, for example the levels of SDA, EPA, DPA, DHA, total LC-PUFA ω3, or total ω3 PUFA (includes C18 fatty acids), were minimum levels that could be achieved through feeding the fish a diet including plant derived SDA, and that even higher levels could be expected by using diets with even higher levels of SDA and/or longer feeding times.
The conversion of ALA to SDA involves the desaturation at the Δ6 position of the carbon chain with further chain elongation steps, followed by Δ5 desaturation to form EPA. The synthesis of EPA to DHA requires additional chain elongations and also involves the Δ6 desaturation in the conversion of 24:5 ω3 to 24:6 ω3 before chain shortening to DHA (
For feeding of lobsters, prawns or other crustacean with diets high in SDA oil, the following feed compositions can be used (Table 10). Values provided as g/kg dry matter.Example 4 Isolation of a Gene Encoding a Δ6-Desaturase from Echium plantagineuin
Some plant species such as evening primrose (Oenothera biennis), common borage (Borago officinalis), blackcurrant (Ribes nigrum), and some Echium species belonging to the Boragenacae family contain the ω6- and ω3-desaturated C18 fatty acids, γ-linolenic acid (18:3ω6, GLA) and stearidonic acid (18:4ω3, SDA) in their leaf lipids and seed TAG (Guil-Guerrero et al., 2000). GLA and SDA are recognized as beneficial fatty acids in human nutrition. The first step in the synthesis of LC-PUFA is a Δ6-desaturation. GLA is synthesized by a Δ6-desaturase that introduces a double bond into the Δ6-position of LA. The same enzyme is also able to introduce a double bond into Δ6-position of ALA, producing SDA. Δ6-desaturase genes have been cloned from members of the Boraginacae, like borage (Sayanova et al., 1997) and two Echium species (Garcia-Maroto et al., 2002).
Echium plantagineum is a winter annual native to Mediterranean Europe and North Africa. Its seed oil is unusual in that it has a unique ratio of ω3 and ω6 fatty acids and contains high amounts of GLA (9.2%) and SDA (12.9%) (Guil-Guerrero et al., 2000), suggesting the presence of Δ6-desaturase activity involved in desaturation of both ω3 and ω6 fatty acids in seeds of this plant.
Cloning of E. plantagineum EplD6Des Gene
Degenerate primers with built-in XbaI or SacI restriction sites corresponding to N- and C-termini amino acid sequences MANAIKKY (SEQ ID NO:14) and EALNTHG (SEQ ID NO:15) of known Echium pitardii and Echium gentianoides (Garcia-Maroto et al., 2002) L6-desaturases were used for RT-PCR amplification of Δ6-desaturase sequences from E. platangineum using a proofreading DNA polymerase Pfu Turbo® (Stratagene). The 1.35 kb PCR amplification product was inserted into pBluescript SK(+) at the XbaI and SacI sites to generate plasmid pXZP 106. The nucleotide sequence of the insert was determined. It comprised an open reading frame encoding a polypeptide of 438 amino acid residues (SEQ ID NO:10) which had a high degree of homology with other reported Δ6-desaturases from E. gentianoides(SEQ ID NO:11), E. pitardii (SEQ ID NO:12) and Borago officinalis (SEQ ID NO:4). It has a cytochrome b5 domain at the N-terminus, including the HPGG (SEQ ID NO:16) motif in the heme-binding region, as reported for other Δ6- and Δ8-desaturases (Sayanova et al. 1997; Napier et al. 1999). In addition, the E. plantagineum Δ6 desaturase contains three conserved histidine boxes present in majority of the ‘front-end’ desaturases (Napier et al., 1999). Cluster analysis including representative members of Δ6 and Δ8 desaturases showed a clear grouping of the cloned gene with other Δ6 desaturases especially those from Echium species.
Heterologous Expression of E. plantagineum Δ5-Desaturase Gene in Yeast
Expression experiments in yeast were carried out to confirm that the cloned E. platangineum gene (cDNA sequence provided as SEQ ID NO:25) encoded a Δ6-desaturase enzyme. The gene fragment was inserted as an XbaI-SacI fragment into the SmaI-SacI sites of the yeast expression vector pSOS (Stratagene) containing the constitutive ADH1 promoter, resulting in plasmid pXZP271. This was transformed into yeast strain S288Cα by a heat shock method and transformant colonies selected by plating on minimal media plates. For the analysis of enzyme activity, 2 mL yeast clonal cultures were grown to an O.D.600 of 1.0 in yeast minimal medium in the presence of 0.1% NP-40 at 30° C. with shaking. Precursor free-fatty acids, either linoleic or linolenic acid as 25 mM stocks in ethanol, were added so that the final concentration of fatty acid was 0.5 mM. The cultures were transferred to 20° C. and grown for 2-3 days with shaking. Yeast cells were harvested by repeated centrifugation and washing first with 0.1% NP-40, then 0.05% NP-40 and finally with water. Fatty acids were extracted and analyzed. The peak identities of fatty acids were confirmed by GC-MS.
The transgenic yeast cells expressing the Echium EplD6Des were able to convert LA and ALA to GLA and SDA, respectively. Around 2.9% of LA was converted to GLA and 2.3% of ALA was converted to SDA, confirming the Δ6-desaturase activity encoded by the cloned gene.
Functional Expression of E. platangineum Δ6-Desaturase Gene in Transgenic Tobacco
In order to demonstrate that the EplD6Des gene could confer the synthesis of Δ6 desaturated fatty acids in transgenic plants, the gene was expressed in tobacco plants. To do this, the gene fragment was excised from pXZP106 as an XbaI-SacI fragment and cloned into the plant expression vector pBI121 (Clonetech) at the XbaI and SacI sites under the control of a constitutive 35S CaMV promoter, to generate plant expression plasmid pXZP341. This was introduced into Agrobacterium tumefaciens AGL1, and used for transformation of tobacco W38 plant tissue, by selection with kanamycin.
Northern blot hybridization analysis of transformed plants was carried out to detect expression of the introduced gene, and total fatty acids present in leaf lipids of wild-type tobacco W38 and transformed tobacco plants were analysed as described above. Untransformed plants contained appreciable amounts of LA (21% of total fatty acids) and ALA (37% of total fatty acids) in leaf lipids. As expected, neither GLA nor SDA, products of Δ6-desaturation, were detected in the untransformed leaf. Furthermore, transgenic tobacco plants transformed with the pBI121 vector had similar leaf fatty acid composition to the untransformed W38 plants. In contrast, leaves of transgenic tobacco plants expressing the EplD6Des gene showed the presence of additional peaks with retention times corresponding to GLA and SDA. The identity of the GLA and SDA peaks were confirmed by GC-MS. Notably, leaf fatty acids of plants expressing the EplD6Des gene consistently contained approximately a two-fold higher concentration of GLA than SDA even when the total Δ6-desaturated fatty acids amounted up to 30% of total fatty acids in their leaf lipids (Table 11).
Northern analysis of multiple independent transgenic tobacco lines showed variable levels of the EplD6Des transcript which generally correlated with the levels of Δ6-desaturated products synthesized in the plants. For example, transgenic plant ET27-2 which contained low levels of the EplD6Des transcript synthesised only 1.95% of its total leaf lipids as Δ6-desaturated fatty acids. On the other hand, transgenic plant ET27-4 contained significantly higher levels of EplD6Des transcript and also had a much higher proportion (30%) of Δ6-desaturated fatty acids in its leaf lipids.
Analysis of the individual tobacco plants showed that, without exception, GLA was present at a higher concentration than SDA even though a higher concentration of ALA than LA was present in untransformed plants. In contrast, expression of EplD6Des in yeast had resulted in approximately equivalent levels of conversion of LA into GLA and ALA into SDA. Echium plantagineum seeds, on the other hand, contain higher levels of SDA than GLA. EplD6Des probably carries out its desaturation in vivo in Echium plantagineum seeds on LA and ALA esterified to phosphatidyl choline (PC) (Jones and Harwood 1980). In the tobacco leaf assay, the enzyme is most likely desaturating LA and ALA esterified to the chloroplast lipid monogalactosyldiacylglyerol (MGDG) (Browse and Slack, 1981). In the yeast assay, free fatty acid precursors LA and ALA added to the medium most likely enter the acyl-CoA pool and are available to be acted upon by EplD6Des in this form.
In conclusion, the transgenic tobacco plant described herein can be used to produce feedstuffs of the invention.
Functional Expression of E. platangineum Δ6-Desaturase Gene in Transgenic Seed
To show seed-specific expression of the Echium Δ6-desaturase gene, the coding region was inserted into the seed-specific expression cassette as follows. An NcoI-SacI fragment including the Δ6-desaturase coding region was inserted into pXZP6, a pBluescriptSK derivative containing a Nos terminator, resulting in plasmid pXZP157. The SmaI-ApaI fragment containing the coding region and terminator EplD6Des-NosT was cloned into pWVec8-Fpl downstream of the Fpl prompter, resulting in plasmid pXZP345. The plasmid pXZP345 was used for transforming wild type Arabidopsis plants, ecotype Columbia, and transgenic plants selected by hygromycin B selection. The transgenic plants transformed with this gene were designated “DP” plants.
Fatty acid composition analysis of the seed oil from T2 seed from eleven T1 plants transformed with the construct showed the presence of GLA and SDA in all of the lines, with levels of Δ6-desaturation products reaching to at least 11% (Table 12). This demonstrated the efficient Δ6-desaturation of LA and ALA in the seed.Example 5 Transformation of Flax with a Seed-Specific Echium Δ6 Fatty Acid Desaturase Gene Construct
The full protein coding region of the Echium Δ6 fatty acid desaturase gene was PCR amplified with the following primers incorporating an XhoI site at the both ends: Ed6F: 5′-ACTCGAGCCACCATGGCTAATGCAATCAA-3′ (SEQ ID NO:17) and Ed6R: 5′-CCTCGAGCTCAACCATGAGTATTAAGAG-3′ (SEQ ID NO:18). PCR was conducted by heating to 94° C. for 2 min, followed by 30 cycles of 94° C. for 40 sec, 62° C. for 40 sec and 72° C. for 1 min 20 sec. After the last cycle, reactions were incubated for 10 min at 72° C. The PCR fragment was cloned into a pGEMTeasy® vector (Promega) and sequenced to ensure that no PCR-induced errors had been introduced. The insert was then digested with XhoI and inserted into the XhoI site of the binary vector, pWBVec8, in a sense orientation between the promoter derived from a seed-specifically expressed flax 2S storage protein gene, linin, and its polyadenylation site/transcription terminator.
The binary vector, pWBVec8 contained a hygromycin resistance gene as a selectable marker for plant transformation (Wang et al., 1998). The construct, designated pVLin-Ed6 and containing the Echium Δ6 desaturase gene for seed-specific expression was shown schematically in
Approximately 150 hypocotyls were excised from 6-7 day old seedlings of flax cultivar Ward grown in sterile condition on MS media. This cultivar was found to produce the highest transformation efficiency among many flax cultivars, however many other cultivars were also amendable for gene transformation. The hypocotyls were inoculated and co-cultivated with Agrobacterium tumefaciens strain AGL1 harbouring the binary construct pVLin-Ed6 in a similar fashion to that described for Brassica transformation in Example 1. Following a co-cultivation period of 3-4 days at 24° C., the hypocotyls were transferred onto selection medium which was MS medium containing 200 mg/l Cefotaxime, 10 mg/l hygromycin, 1 mg/l BAP (6-benzyl-aminopurine) and 0.1 mg/l NAA (napthaleneacetic acid). Shoot development was initiated after about 2 weeks. Shoots were transferred onto fresh MS medium with the same additives except NAA was reduced to 0.02 mg/l. After 2-3 weeks, healthy green shoots were transferred onto fresh MS media without growth regulators for induction of roots. Rooted shoots were planted in potting mix in glasshouse.
The transgenic nature of regenerated flax plants was confirmed by PCR amplification of part of the Echium Δ6 fatty acid desaturase sequence with the primers Ed6s1, 5′-ACTCTGTTTCTGAGGTGTCCA-3′ (SEQ ID NO:19); and Ed6a1, 5′-CATATTAACCCTAGCCATACACAT-3′ (SEQ ID NO:20). DNA extracted from individual, regenerated flax plants was used as template in PCR reactions using the following amplification conditions: denaturation at 94° C. for 2 min, followed by 30 cycles of 94° C. for 40 sec, 58° C. for 40 sec and 72° C. for 1 min. Seeds set on forty primary transgenic flax plants will be analysed for the presence of SDA and GLA using lipid extraction followed by gas chromatography. It is expected that high levels of SDA will be produced in many of the plants and that SDA levels will be greater than GLA levels.
Seed from the transformed flax plants or extracts such as the oil or the seed meal can be used in feed compositions for use in feeding fish or crustacea.Example 6 Transformation of Cotton with a Seed-Specific Construct Expressing an Echium Δ6 Fatty Acid Desaturase Gene
Cottonseed normally contains only negligible amounts (<0.5% of total fatty acids) of α-linolenic acid (ALA). In order to produce ALA at increased levels in cottonseed oil, cotton (Gossypium hirsutum) was transformed with a seed-specific gene construct expressing a FAD3 gene from Brassica napus (Arondel et al., 1992) (encoded protein amino acid sequence provided as SEQ ID NO:27). The accession number of the cDNA clone of this gene was L01418. The full protein coding region of the B. napus FAD3 gene was amplified by PCR using the primers BnFAD3-S1, 5′-CTCCAGCGATGGTTGTTGCTAT-3′ (SEQ ID NO:21) and BnFAD3-A1, 5′-AATGTCTCTGGTGACGTAGC-3′ (SEQ ID NO:22). The PCR product was cloned into a pGEMTeasy® vector (Promega) and the excised by restriction digest with NotI. The B. napus FAD3 coding sequence was inserted in the sense orientation into the NotI site between the soybean lectin gene promoter and terminator sequences (Cho et al., 1995), to provide a seed-specific expression construct. This vector contained an NPTII gene conferring kanamycin resistance as a selectable marker for plant transformation. This vector was introduced into Agrobacterium and used to transform cotton as described in Liu et al (2002). Independent transgenic plants expressing the FAD3 gene were obtained and lines accumulating ALA retained.
Separate cotton transformation experiments were performed using a similar seed-specific lectin cassette expressing a Δ6 fatty acid desaturase, to convert LA to GLA and ALA to SDA. The full protein-coding region of the Δ6 desaturase from Echium plantagineum (Zhou et al., 2006; SEQ ID NO:25) was amplified by PCR using the following primers incorporating a SmaI site at the 5′ end, and SacI at the 3′ end. Ed6F: 5′-ATCCCCGGGTACCGGTCGCCACCATGGCTAATGCAATCAAGAAGTA-3′ (SEQ ID NO:30) and Ed6R: 5′-TTGGAGCTCAACCATGAGTATTAAGAGCTTC-3′ (SEQ ID NO:31). The PCR fragment was cloned into pGEM-Teasy® vector (Promega) and sequenced to ensure no PCR-induced errors were introduced. The PCR amplified Δ6 desaturase gene was subsequently cloned into the corresponding SmaI/SacI sites in a sense orientation behind the napin (Fpl) promoter and upstream of the nos3′ terminator-polyadenylation signal. Agrobacterium tumefaciens strain AGL1 harbouring the resulted construct, pGNapin-E6D, was used to transform cotton variety Coker315 by the method described by Liu et al. (2002).
Nine fertile independently transformed plants were obtained. The transformed cotton plants were positive for the presence of the transgene, and expression in developing seeds, by PCR and Northern blot analysis of the expressed RNA. 15 individual mature seeds from each of these primary transgenic plants were subjected to the analysis of fatty acid composition using gas chromatography (GC) as described above. Surprisingly high levels of γ-linolenic acid (GLA) were found to accumulate in four transgenic lines, while there was no detectable GLA in the non-transformed control plants. Levels of GLA of greater than 15% were observed in many seeds, and the level reached greater than 25% in some seeds that were likely to be homozygous for the introduced Δ6 desaturase gene. The accumulation of GLA is mainly at the expense of linoleic acid. Indeed, the conversion of LA to GLA (measured as % GLA×100/(% LA+% GLA) in the seedoil) was highly efficient in these cottonseeds relative to seeds of other plants, being greater than 25% in many seed and reaching in excess of 45% in some seed.
Cotton lines containing both genes will be produced by crossing the transformants expressing the FAD3 gene and transformants expressing the Δ6 desaturase gene, to produce lines containing SDA. By the methods described above, oilseed plants such as cotton or flax may be produced which produce at least 5.5% SDA on a weight basis in the fatty acid of the seed oil. Preferably, the level of SDA in the fatty acid is at least 11%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% on a weight basis. The efficiency of conversion of ALA to SDA (measured as % SDA×100/(% ALA+% SDA) in the seedoil) is at least 25% and preferably at least 45%. That is, at least 25%, preferably at least 45% of the polyunsaturated fatty acid in the cotton or flax seed that has a carbon chain of C18 or longer is desaturated at the Δ6 position.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
All publications discussed above are incorporated herein in their entirety.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.REFERENCES
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61. A process for increasing the long-chain polyunsaturated fatty acid (LC-PUFA) content in the white muscle, the red muscle, or the white and red muscle of a fish comprising feeding the fish a feedstuff comprising plant oil whose fatty acids comprise at least 5.5% (w/w) stearidonic acid (SDA), so as to thereby increase the LC-PUFA content of the white muscle, the red muscle, or the white and red muscle of the fish relative to the white muscle, the red muscle, or the white and red muscle of a corresponding fish fed a feedstuff comprising canola oil and whose fatty acids lack at least 5.5% (w/w) SDA, and wherein levels of 14:0 and 16:0 fatty acids in muscle tissue of the fish are reduced by at least 10% relative to a corresponding fish fed a corresponding feedstuff which comprises fish oil instead of the plant oil whose fatty acids comprise at least 5.5% (w/w) SDA.
62. The process of claim 61, wherein the LC-PUFA is DHA.
63. The process of claim 62, wherein the DHA content of the fatty acids of the white muscle lipid of the fish is increased to comprise 18.3% (w/w) DHA.
64. The process of claim 62, wherein the DHA content of the fatty acids of the red muscle lipid of the fish is increased to comprise 9.6% (w/w) DHA.
65. The process of claim 61, wherein the fish is a trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod, haddock, Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike, pike-perch, yellowtail, tilapia, eel or tropical fish.
66. The process of claim 61, wherein the fish is a larval or a juvenile fish.
67. The process of claim 61, wherein the fish is fed the feedstuff for at least 6 weeks.
68. The process of claim 61, wherein the plant oil is from a plant which is canola, soybean, or flax.
69. The process of claim 68, wherein the plant is genetically modified such that the fatty acids of the oil comprise at least 5.5% (w/w) SDA.
70. The process of claim 69, wherein the plant comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
71. The process of claim 61, wherein the feedstuff comprises a transgenic organism, or extract or portion thereof and at least one other ingredient, wherein the organism is genetically modified such that the fatty acids of the organism, or extract or portion thereof comprise at least 5.5% (w/w) SDA.
72. The process of claim 71, wherein the organism is yeast.
73. The process of claim 71, wherein the transgenic organism, or extract or portion thereof comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
74. The process of claim 65, wherein the feedstuff comprises plant oil and at least one other ingredient, wherein the fatty acids of the oil comprise at least 5.5% (w/w) SDA.
75. The process of claim 74, wherein the plant oil is from a plant which is canola, soybean, or flax.
76. The process of claim 65, wherein the feedstuff comprises a transgenic organism, or extract or portion thereof and at least one other ingredient, wherein the organism is genetically modified such that the fatty acids of the organism, or extract or portion thereof comprise at least 5.5% (w/w) SDA.
77. The process of 76, wherein the organism is yeast.
78. The process of claim 76, wherein the transgenic organism, or extract or portion thereof comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
79. The process of claim 76, wherein the at least one other ingredient is fishmeal, a starch source, a vitamin or a mineral.
Filed: Jun 6, 2014
Publication Date: Nov 27, 2014
Applicant: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION (Australian Capital Territory)
Inventors: Matthew Robert Miller (Atawhai), Christopher Guy Carter (Howden), Peter David Nichols (West Hobart), Surinder Pal Singh (Downer), Xue-Rong Zhou (Evatt), Allan Graham Green (Red Hill)
Application Number: 14/298,399
International Classification: A23K 1/18 (20060101); A01K 61/00 (20060101);