CA2810336A1 - Gene combinations for producing punicic acid in transgenic plants - Google Patents

Abstract

The present invention relates to transgenic plants with enhanced punicic acid accumulation resulting from overexpression of PgFADX and PgFAD2. Also provided are isolated Punica granalum diacylglycerol acyltransferases: type 1 (PgDGAT1), type 2 (PgDGAT2) and phospholipid:diacylglycerol acyltransferases (PgPDAT1); polynucleotide sequences encoding the DGATs and PDAT enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.

Description

CA PATENT APPLICATION
DOCKET NO. 55326.154 GENE COMBINATIONS FOR PRODUCING PUNICIC ACID IN TRANSGENIC
PLANTS
INVENTORS: WESELAKE, Randall J.; MIETKIEWSKA, Elzbieta ASSIGNEE: THE GOVERNORS OF THE UNIVERSITY OF ALBERTA
FIELD OF THE INVENTION
[0001] The present invention relates to isolated desaturases (FAD2) and fatty acid conjugases (FADX), diacylglycerol acyltransferases: type 1 (DGAT1) and type 2 (DGAT2), and phospholipid:diacylglycerol acyltransferases (FDA Ti) and polynucleotide sequences encoding the DGATs and PDAT1 enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.
BACKGROUND OF THE INVENTION

[0002] Pomegranate (Punica granatum) seed oil has been attracting increasing interest since its main component, punicic acid, may be used as a therapeutic agent in inflammatory diseases and as a dietary agent for chemoprevention of prostate and breast cancer (Kim et al., 2002;
Shyed et al., 2008; Boussetta et aL, 2009). Punicic acid (18:3A9cis' I trans, 13cis.
) is an uncommon form of conjugated linolenic acid. The conjugated double bond in punicic acid is synthesized by a divergent form of the A 12-oleic acid desaturase (fatty acid conjugase, designated here as FADX) which catalyzes the conversion of the Al2 double bond of linoleic acid (18:2A9cis'126s) into two conjugated and trans-cis configurated double bonds at the 11 and 13 positions (Hornung et al., 2002; Iwabuchi et al., 2003). Delta-12 desaturase (PgFAD2, GenBank accession #AY178447) and fatty acid conjugase (PgFADX, GenBank accession #AY178446) involved in the synthesis of punicic acid in pomegranate have been isolated by PCR based cloning. Over-expression of PgFADX in Arab idopsis thaliana resulted in the limited accumulation of punicic acid up to 3.5% accompanied by increased accumulation of oleic acid (Iwabuchi et al., 2003;
Cahoon et al., 2006). These amounts of punicic acid are considerably lower compared to the amounts in seeds of P. granatum (up to 80%).

[0003] Similar problems with low accumulation of other conjugated fatty acids in transgenic plants have been reported. Genes encoding divergent FAD2 enzymes have been expressed transgenically in A. thaliana and oilseed plants, but resulted in relatively low levels of accumulation of the unusual fatty acid, usually <20% compared to the 60-90%
typically found in the source species (Broun and Somerville, 1997; Cahoon et al., 1999; Dyer and Mullen 2007;
Dyer etal., 2008, van Erp et al., 2011). Oleic acid content increased considerably in these transgenic plants, suggesting that production of the unusual fatty acids in some way inhibits the FAD2 desaturase activity (Cahoon etal., 2006; Zhou et al., 2006; Thomaeus etal., 2001). This was ascribed to the competition between the housekeeping FAD2 and the diverged FAD2-like enzymes, and an inhibition of the normal FAD2 by the conjugated acyl residues in the phosphatidylcholine (PC) substrate molecules (Drexler et al., 2003).

[0004] It has been recently demonstrated that levels of unusual/conjugated fatty acids present on PC in transgenic plants were substantially higher than those observed in the source plants, indicating that accumulation of these fatty acids in transgenic plants is primarily limited by their inefficient removal from PC and passage through the Kennedy pathway (Cahoon et al., 2006;
Cahoon etal., 2007; Dyer etal., 2008). The route to a high-level of accumulation of punicic acid in transgenic plants may necessitate the identification and introduction of genes encoding key enzymes such as phospholipases PLA1 and PLA2 (Stahl etal., 1995; Singh eta!,, 2005), diacylglycerol acyltransferase type 1 (DGAT1) and 2 (DGAT2) (Burgal etal., 2008; Li etal., 2010; Shockey et al., 2006), and phospholipid:diacylglycerol acyliransferase (PDAT) (van Erp et al., 2011). Over-expression of DGAT1, DGAT2 and PDAT led to increased levels of other unusual fatty acids such as epoxy and hydroxyl fatty acids in transgenic plants (Li et al., 2010;
Burgal etal., 2008; van Erp et al., 2011).

[0005] Accordingly, there is a need in the art for methods of producing punicic acid from a sustainable source.
SUMMARY OF THE INVENTION

[0006] In general terms, the present invention relates to transgenic plants with enhanced punicic acid accumulation resulting from overexpression of PgFADX and PgFAD2.
The present invention also relates to isolated PgDGAT1, PgDGAT2, and PgPDAT1 genes from Pun/ca granatum, and methods for their use.

[0007] In one aspect, the present invention comprises an isolated polynucleotide sequence encoding a protein or polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, 4 or 6, respective biologically active variants and biologically active portions thereof, with respective sequences having at least 85% identity thereto, and wherein the variants have diacylglycerol acyltransferases type 1 (DGAT I) and type 2 (DGAT2) or phospholipid diacylglycerol acyltransferasc (PDAT1) activity.

[0008] In one embodiment, the polynucleotide encodes a polypeptide having type 1 (DGAT1) and type 2 (DGAT2) activity and comprising the amino acid sequence of SEQ ID
NO: 2 or 4, or an amino acid sequence having DGAT activity and having at least 85% sequence identity therewith.

[0009] In one embodiment, the polynucleotide encodes a polypeptide having PDAT
activity and comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having PDAT activity and having at least 85% sequence identity therewith.

[00010] In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID
NO: 1,3 or 5.

[00011] In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity to one of SEQ ID NO: 2, 4 or 6.

[00012] In one embodiment, the polynucleotide is derived from Pun/ca granatum.

[00013] In another aspect, the invention comprises a recombinant expression vector comprising at least one polynucleotide as described herein, operably linked with transcriptional and translational regulatory regions or sequences to provide for expression of the at least one polynucleotide sequence in a host cell.

[00014] In another aspect, the invention comprises a transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising (a) the above recombinant expression vector, or (b) a recombinant expression vector encoding PgFADX and i PgFAD2.

[00015] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore comprises PgFADX and PgFAD2, and one or more of a DGAT and PDAT1.

[00016] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore comprises a progeny plant generated from the transgenic plant, wherein the progeny plant comprises PgFADX and PgFAD2, and one or more of a DGAT and PDAT.

[00017] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore comprises PgFADX, PgFAD2, PgDGAT1 or PgDGAT2 or both, and PgPDAT1.

[00018] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore is selected from a linseed, rapeseed, canola, peanut, safflower, flax, hemp, camelina, soybean, pea, sunflower, olive, palm, oats, wheat, triticale, barley, corn, thale cress, and legume plant, plant cell, plant seed, callus, plant embryo, or microspore-derived embryo or microspore.

[00019] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore comprises Arabidopsis thaliana.

[00020] In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore comprises Arabidopsis thaliana fad3/fael double mutant.

[00021] In another aspect, the invention comprises a method of increasing punicic acid production in an oilseed plant, comprising the steps of:

a) constructing one or more vectors comprising one or more of the polynucleotides described herein; and b) transforming the one or more vectors into a host cell under conditions sufficient for over-expression of PgFADX and PgFAD2.

[00022] In one embodiment, the host cell is also transformed to over-express one or more of a DGAT and PDAT.

[00023] In one embodiment, the method further comprises generating a transgenic plant from the host cell and obtaining a progeny plant, wherein the progeny plant comprises PgFADX and PgFAD2, and one or more of a DGAT and PDAT encoded by the polynucleotides, and the polynucleotides are over-expressed in the progeny plant.

[00024] In one embodiment, PgFADX, PgFAD2, PgDGAT1 or PgDGAT2 or both, and PgPDAT1 are over-expressed in the plant.

[00025] In one embodiment, the plant comprises Arab idopsis thaliana. In one embodiment, the plant comprises an Arabidopsis thaliana fad3/fae 1 double mutant.

[00026] Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS

[00027] The invention will now be described by way of an exemplary embodiment with reference to the accompanying simplified, diagrammatic, not-to-scale drawings.
In the drawings:

[00028] Figure 1 is a schematic diagram showing the pathway for synthesizing punicic acid.
,=

[00029] Figure 2 is a schematic diagram showing the NCJ and NCJD constructs used to transform A. thaliana fadVael plants.

[00030] Figure 3 shows the fatty acid composition (i.e., proportions of 18:1, 18:2, and punicic acid contributing to the total fatty acid profile) in the seed oils of A.
thalianafad3ffael mutant-T2 (Hi) plants transformed with the NCJ construct to express the PgFADX gene compared to non-transformed plants.

[00031] Figure 4 shows the fatty acid composition (i.e., proportions of 18:1, 18:2, and punicic acid contributing to the total fatty acid profile) in the seed oils of A.
thalianafad3/fael mutant-T2 (Hi) plants transformed with the NCJD construct to express the PgFADX and PgFAD2 genes compared to non-transformed plants.

[00032] Figure 5 shows the fatty acid composition (i.e., proportions of 18:1, 18:2, and punicic acid contributing to the total fatty acid profile) in the seed oils of A.
thallanafad3/fael mutant-T3 (Ho) plants transformed with the NCJ construct to express the PgFADX gene compared to non-transformed and null segregant plants.

[00033] Figure 6 shows the fatty acid composition (i.e., proportions of 18:1, 18:2, and punicic acid within the total fatty acid profile) in the seed oils of A. thaliana fad3/fael mutant-T3 (Ho) plants transformed with the NCJD construct to express the PgFADX and PgFAD2 genes compared to non-transformed plants.

[00034] Figure 7 shows the relative content of punicic acid in phosphatidylcholine (PC) and triacylglycerol (TAG) from P. granatum and A. thaliana seed line NCJD-30-2.
The values represented by the bars are the average SD from analyses of three independent samples.

[00035] Figure 8A shows the PgDGAT1 nucleotide sequence, and Figure 8B shows the PgDGAT1 amino acid sequence.

[00036] Figure 9A shows the PgDGAT2 nucleotide sequence, and Figure 9B shows the PgDGAT2 amino acid sequence.

[00037] Figure 10A shows the PgPDAT1 nucleotide sequence, and Figure 10B shows the PgPDAT1 amino acid sequence.

[00038] Figure 11 shows the relative content of punicic acid in polar lipids (PL) and triacylglycerol (TAG) from yeast cells over-expressing PgFADX, PgFADX PgDGAT1, and PgFADX + PgDGAT2. The values represented by the bars are the average SD from analyses of three independent samples.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[00039] The present invention relates to transgenic plants with enhanced punicic acid accumulation resulting from overexpression of PgFADX and PgFAD2. The present invention also relates to isolated polynucleotides and polypeptides of the PgDGAT1, PgDGAT2, and PgPDAT1 genes from Punica granatum; nucleic acid constructs, recombinant expression vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same. When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.

[00040] To facilitate understanding of the invention, the following definitions are provided.

[00041] A "cDNA" is a polynucleotide which is complementary to a molecule of mRNA. The "cDNA" is formed of a coding sequence flanked by 5' and 3' untranslated sequences.

[00042] A "coding sequence" or "coding region" or "open reading frame (ORF)"
is part of a gene that codes for an amino acid sequence of a polypeptide.

[00043] A "complementary sequence" is a sequence of nucleotides which forms a duplex with another sequence of nucleotides according to Watson-Crick base pairing rules where "A" pairs with "T" and "C" pairs with "G." For example, for the polynucleotide 5'-AATGCCTA-3' the complementary sequence is 5'-TAGGCATT-3'.

[00044] A "construct" is a polynucleotide which is formed by polynucleotide segments =
isolated from a naturally occurring gene or which is chemically synthesized.
The "construct"

which is combined in a manner that otherwise would not exist in nature, is usually made to achieve certain purposes. For instance, the coding region from "gene A" can be combined with an inducible promoter from "gene B" so the expression of the recombinant construct can be induced.

[00045] "Downstream" means on the 3' side of a polynucleotide while "upstream"
means on the 5' side of a polynucleotide.

[00046] "Expression" refers to the transcription of a gene into RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.

[00047] "Gene" means a DNA segment which contributes to phenotype or function, and which may be characterized by sequence, transcription or homology.

[00048] "Isolated" means that a substance or a group of substances is removed from the coexisting materials of its natural state.

[00049] "Nucleic acid" means polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA.

[00050] As used herein, the term "plasmid" means a DNA molecule which is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids used in genetic engineering are known as vectors and are used to multiply or express particular genes. Any plasmid may be used for the present invention provided that the plasmid contains a gene which encodes a PgDGAT1, PgDGAT2, and PgPDAT1, or a variant thereof in an expressible manner. In one embodiment, the plasmid comprises a yeast expression vector. Those skilled in art will recognize that any plasmid in the art may be modified for use in the compositions and methods of the present invention.

[00051] As used herein, the term "regulatory element" includes, but is not limited to, a promoter, enhancer, terminator, and the like which are required for the expression of the encoded PgDGAT1, PgDGAT2, and PgPDAT1, or variant thereof.

[00052] A "polynucleotide" is a linear sequence of ribonucleotides (RNA) or deoxyribonucleotides (DNA) in which the 3' carbon of the pentose sugar of one nucleotide is linked to the 5' carbon of the pentose sugar of another nucleotide. The deoxyribonucleotide bases are abbreviated as "A" deoxyadenine; "C" deoxycytidine; "G"
deoxyguanine; "T"
deoxythymidinc; "I" dcoxyinosine. Some oligonucleotides described herein are produced synthetically and contain different deoxyribonucleotides occupying the same position in the sequence. The blends of deoxyribonucleotides are abbreviated as "W" A or T;
"Y" C or T; "H"
A, C or T; "K" G or T; "D" A, G or T; "B" C, G or T; "N" A, C, G or T.

[00053] A "polypeptide" is a linear sequence of amino acids linked by peptide bonds.
Common amino acids are abbreviated as "A" alanine; "R" arginine; "N"
asparagine; "D" aspartic acid; "C" cysteine; "Q" glutamine; "E" glutamic acid; "G" glycine; "H"
histidine; "I" isoleucine;
"L" leucine; "K" lysine; "M" methionine; "F" phenylalanine; "P" proline; "S"
serine; "T"
threonine; "W" tryptophan; "Y" tyrosine and "V" valine.

[00054] Two polynucleotides or polypeptides are "identical" if the sequence of nucleotides or amino acids, respectively, in the two sequences is the same when aligned for maximum correspondence as described here. Sequence comparisons between two or more polynucleotides or polypeptides can be generally performed by comparing portions of the two sequences over a comparison window which can be from about 20 to about 200 nucleotides or amino acids, or more. The "percentage of sequence identity" may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of a polynucleotide or a polypeptide sequence may include additions (i.e., insertions) or deletions (i.e., gaps) as compared to the reference sequence. The percentage is calculated by determining the positions at which identical nucleotides or identical amino acids are present, dividing by the number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity.
Polynucleotide and polypeptide sequence alignment may be performed by implementing specialized algorithms or by inspection. Examples of sequence comparison and multiple sequence alignment algorithms are: BLAST and ClustalW software. Identity between nucleotide sequences can also be determined by DNA hybridization analysis, wherein the stability of the double-stranded DNA hybrid is dependent on the extent of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid, and can be varied to prevent annealing of sequences having less than a selected degree of homology.
Hybridization methods are described in Ausubel et al. (2000).

[00055] An "oleic acid" is a monounsaturated omega-9 fatty acid which is abbreviated with a lipid number of 18:1 cis-9.

[00056] A "linoleic acid" is an unsaturated omega-6 fatty acid which is abundant in many vegetable oils, and is an essential dietary requirement for all mammals lacking the delta-12 desaturase involved in its synthesis.

[00057] A "punicic acid" is a conjugated linolenic acid isomer containing cis-A9, trans-A11, cis-A13 double bonds in the C15 carbon chain and having the structure below:
HO
.\\N

[00058] A Punica granatum conjugase (FADX) is an enzyme which catalyzes the conversion of the delta-12 double bond of linoleic acid (18:26,96s' 12els) into two conjugated and trans-cis configurated double bonds at the 11 and 13 positions. A "fatty acid conjugase"
(FADX) is an enzyme which utilizes linoleic acid as a substrate for punicic acid synthesis.

[00059] A "PgFAD2" is a gene encoding a FAD2 from Punica granatum (pomegranate).

[00060] A "PgFAD2" refers to a polypeptide from Punica granatum which exhibits enzymatic activity (delta-12 desaturase). A polypeptide having "FAD2 activity"
is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of FAD2.

[00061] A "PgFADX' is a gene encoding a FADX from Pun/ca granatum.

[00062] A "PgFADX" refers to a polypeptide from Pun/ca granatum which exhibits FADX
enzymatic activity. A polypeptide having "FADX activity" is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of FADX.

[00063] A "triacylglycerol" is an ester having three fatty carboxylic acids attached to a single glycerol backbone. It is the main component of vegetable oil and animal fats.
Alternative names include: triglyceride, triacylglyceride, TG and TAG.

[00064] A diacylglycerol acyl transferase (DGAT) is an enzyme of the class EC
2.3.1.20 which catalyzes the reaction: acyl-CoA + sn-1,2-diacylglycerol CoA +
triacylglycerol.
Alternative names include: diacylglycerol 0-acyltransferase, diacylglycerol acyltransferase, diglyceride acyltransferase and acylCoA:diacylglycerol acyltransferase.

[00065] A "PgDGAT" is a gene encoding a DGAT from Pun/ca granatum. Two types of P.granatum DGATs are described here: type 1 (DGAT]) and type 2 (DGAT2).

[00066] A "PgDGAT" refers to a polypeptide from Pun/ca granatum which exhibits DGAT
enzymatic activity. Two types of PgDGATs are described here (for example, type 1: PgDGAT1 or type 2: PgDGAT2) refers to a specific polypeptide which exhibits DGAT
enzyme activity.

[00067] A polypeptide having "DGAT activity" is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of DGAT.

[00068] A "phospholipid:diacylglycerol acyl transferase" (PDAT) is an enzyme of the class EC 2.3.1.158 which catalyzes the reaction: phospholipid + 1,2-diacylglycerol 4-lysophospholipid + TAG.

[00069] A "PgPDAT]" is a gene encoding a PDAT from Punica granatum. A number denoted after PgPDAT (for example, PgDGAT1) refers to a specific gene encoding a PDAT.

[00070] A "PgPDAT1" refers to a polypeptide from Punica granatum which exhibits PDAT
enzymatic activity. A number denoted after PgPDAT (for example, PgPDAT1) refers to a specific polypeptide which exhibits PDAT enzyme activity.

[00071] A polypeptide having "PDAT activity" is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of PDAT.

[00072] A "promoter" is a polynucleotide usually located within 20 to 5000 nucleotides upstream of the initiation of translation site of a gene. The "promoter"
determines the first step of expression by providing a binding site to DNA polymerase to initiate the transcription of a gene. The promoter is said to be "inducible" when the initiation of transcription occurs only when a specific agent or chemical substance is presented to the cell. For instance, the GAL
"promoter" from yeast is "inducible by galactose," meaning that this GAL
promoter allows initiation of transcription and subsequent expression only when galactose is presented to yeast cells.

[00073] "Transformation" means the directed modification of the genome of a cell by external application of a polynucleotide, for instance, a construct. The inserted polynucleotide may or may not integrate with the host cell chromosome. For example, in bacteria, the inserted polynucleotide usually does not integrate with the bacterial genome and might replicate autonomously. In plants, the inserted polynucleotide integrates with the plant chromosome and replicates together with the plant chromatin.

[00074] A "transgenic" organism is the organism that was transformed with an external polynucleotide. The "transgenic" organism encompasses all descendants, hybrids and crosses thereof, whether reproduced sexually or asexually and which continue to harbor the foreign polynucleotide.

[00075] A "vector" is a polynucleotide that is able to replicate autonomously in a host cell and is able to accept other polynucleotides. For autonomous replication, the vector contains an "origin of replication." The vector usually contains a "selectable marker"
that confers the host cell resistance to certain environment and growth conditions. For instance, a vector that is used to transform bacteria usually contains a certain antibiotic "selectable marker" which confers the transformed bacteria resistance to such antibiotic.

[00076] The present invention relates to isolated polynucleotides and polypeptides of the PgDGAT1, PgDGAT2, and PgPDAT1 genes from Punica granatum; nucleic acid constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same. In particular, the invention relates to a method for increasing the production of punicic acid in oilseed plants through the over-expression of PgFADX and PgFAD2, and one or more of PgDGAT1 , PgDGAT2, and PgPDAT1, and a method for the production of oils having an increased content of punicic acid. The invention furthermore relates to the production of transgenic plants, preferably a transgenic oilseed plant, having an increased content of punicic acid.

[00077] In one aspect, the invention provides isolated PgDGAT1, PgDGAT2, and PgPDAT1 polynucleotides, and polypeptides having DGAT or PDAT activity. PgDGAT1, PgDGAT2, and PgPDAT1 polynucleotides include, without limitation (1) single- or double-stranded DNA, such as cDNA or genomic DNA including sense and antisense strands; and (2) RNA, such as mRNA.
PgDGAT1, PgDGAT2, and PgPDAT1 polynucleotides include at least a coding sequence which codes for the amino acid sequence of the specified PgDGAT1, PgDGAT2 and PgPDAT

polypeptide, but may also include 5' or 3' untranslated regions and transcriptional regulatory elements such as promoters and enhancers found upstream or downstream from the transcribed region.

[00078] In one embodiment, the invention provides a PgDGAT I polynucleotide which is a cDNA comprising the nucleotide sequence of 1732 base pairs depicted in SEQ ID
NO: 1 (Figure 8A), and which was isolated from Punica granatum. The cDNA comprises a coding region of 1623 base pairs (107-1729 region/position of SEQ ID NO: 1). The PgDGAT1 encoded by the coding region (SEQ ID NO: 2; Figure 8B) is a 540 amino acid polypeptide.
1000791 In one embodiment, the invention provides a PgDGAT2 polynucleotide which is a cDNA comprising the nucleotide sequence of 1479 base pairs depicted in SEQ ID
NO: 3 (Figure 9A), and which was isolated from Punica granatum. The cDNA comprises a coding region of 1005 base pairs (243-1247 region/position of SEQ ID NO: 3). The PgDGAT2 encoded by the coding region (SEQ ID NO: 4; Figure 9B) is a 334 amino acid polypeptide.
1000801 In one embodiment, the invention provides a PgPDAT1 polynucleotide which is a cDNA comprising the nucleotide sequence of 2743 base pairs depicted in SEQ ID
NO: 5 (Figure 10A), and which was isolated from Punica granatum. The cDNA comprises a coding region of 2052 base pairs (129-2180 region/position of SEQ ID NO: 5). The PgPDAT1 encoded by the coding region (SEQ ID NO: 6; Figure 10B) is a 683 amino acid polypeptide.
[00081] Those skilled in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for identical polypeptides.
Accordingly, the invention includes polynucleotides of SEQ ID NOS: 1, 3, and 5, and variants of polynucleotides .=
encoding the polypeptides of SEQ ID NOS: 2, 4 and 6. In one embodiment, polynucleotides having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequences depicted in SEQ ID
NOS: 1, 3, and 5 are included in the invention. Methods for isolation of such polynucleotides are well known in the art (see for example, Ausubel et al., 2000).
1000821 In one embodiment, the invention provides isolated polynucleotides which encode PgDGAT1, PgDGAT2, and PgPDAT1, or polypeptides having amino acid sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity to the amino acid sequences depicted in SEQ ID NOS: 2, 4, and 6.
1000831 The above described polynucleotides of the invention may be used to express = polypeptides in recombinantly engineered cells including, for example, bacterial, yeast, fungal, mammalian or plant cells. In one embodiment, the invention provides polynucleotide constructs, vectors and cells comprising PgDGAT1, PgDGAT2, and PgPDAT1 polynucleotides.
Those skilled in the art are knowledgeable in the numerous systems available for expression of a polynucleotide. All systems employ a similar approach, whereby an expression construct is assembled to include the protein coding sequence of interest and control sequences such as promoters, enhancers, and terminators, with signal sequences and selectable markers included if desired. Briefly, the expression of isolated polynucleotides encoding polypeptides is typically achieved by operably linking, for example, the DNA or cDNA to a constitutive or inducible promoter, followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA. High level expression of a cloned gene is obtained by constructing expression vectors which contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
Vectors may further comprise transit and targeting sequences, selectable markers, enhancers or operators. Means for preparing vectors are well known in the art. Typical vectors useful for expression of polynucleotides in plants include for example, vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaM-VCN transfer control vector.
Promoters suitable for plant cells include for example, the nopaline synthase, octopine synthase, and mannopine synthase promoters, and the caulimovirus promoters. Seed-specific promoters, such as ACP and napin-derived transcription initiation regions, have been shown to confer preferential expression of a specific gene in plant seed tissue. In one embodiment, the seed-specific napin promoter is preferred.
[00084] Those skilled in the art will appreciate that modifications (i.e., amino acid substitutions, additions, deletions and post-translational modifications) can be made to a polypeptide of the invention without eliminating or diminishing its biological activity.
Conservative amino acid substitutions (i.e., substitution of one amino acid for another amino acid of similar size, charge, polarity and conformation) or substitution of one amino acid for another within the same group (i.e., nonpolar group, polar group, positively charged group, negatively charged group) are unlikely to alter protein function adversely. Some modifications may be made to facilitate the cloning, expression or purification. Variant PgDGAT1, PgDGAT2, and PgPDAT1 polypeptides may be obtained by mutagenesis of the corresponding polynucleotides depicted in SEQ ID NOS: 1, 3 and 5 using techniques known in the art including, for example, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (Ausubel et al., 2000).
[00085] Various methods for transformation or transfection of cells are available. For prokaryotes, lower eukaryotes and animal cells, such methods include for example, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE
dextran, electroporation, biolistics and microinjection. The transfected cells are cultured, and the produced PgDGAT1, PgDGAT2, and PgPDAT1 polypeptides may be isolated and purified from the cells using standard techniques known in the art. Various industrial strains of microorganisms including for example, fungi, such as Mortierella or Traustochytrium; mosses such as Physcomitrella or Ceratodon; algae such as Crypthecodinium or Phaeodactylum; or Aspergillus, Pichia pastoris, Saccharomyces cerevislae may be used to produce PgDGAT1, PgDGAT2, and PgPDAT1 polypeptides. Alternatively, exogenous DNA may be transferred into yeast by electroporation, biolistics, glass bead agitation and spheroplasts.
[00086] Methods for transformation of plant cells include for example, infiltration, electroporation, PEG poration, particle bombardment, Agrobacterium tumefaciens-or Agrobacterium rhizogenes-mediated transformation, direct protoplast transformation, and microinjection. The transformed plant cells, seeds, callus, embryos, microspore-derived embryos, microspores, organs or explants are cultured or cultivated using standard plant tissue culture techniques and growth media to regenerate a whole transgenic plant which possesses the transformed genotype. Transformation may be confirmed by use of a DNA marker gene encoding for an enzyme that confers herbicide tolerance or antibiotic resistance; catalyzes deamination of D-amino acids; or by conducting methods such as PCR or Southern blot hybridization. Transgenic plants may pass polynucleotides encoding PgDGAT1, PgDGAT2, and PgPDAT1 polypeptides to their progeny, or can be further crossbred with other species.
Accordingly, in one embodiment, the invention provides methods for producing transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores comprising PgDGAT1 , PgDGAT2, and PgPDAT1 polynucleotides.
[00087] In one embodiment, the invention provides transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, or microspores, comprising PgDGAT1, PgDGAT2, and PgPDAT1 polynucleotides. Plant species of interest for transformation include, without limitation, oilseeds (for example, the linseed plant, rapeseed or canola, peanut, safflower), flax, hemp, camelina, canola, sunflower, olive, palm, oats, wheat, triticale, barley, corn, thale cress, and legume plants including soybean and pea. In one embodiment, the plant comprises Arabidopsis thaliana. In one embodiment, the plant comprises Arabidopsis thaliana fadVfael double mutant.
[00088] In one embodiment, the invention comprises a method of increasing punicic acid production in an oilseed plant, comprising the steps of:
a) constructing one or more vectors comprising one or more of the polynucleotides claimed herein;
b) transforming the one or more vectors into a host cell under conditions sufficient for over-expression of PgFADX and PgFAD2.
[00089] In one embodiment, the host cell is also transformed to over-express one or more of a DGAT or PDAT.
[00090] The following describes specific examples of embodiments of the present invention.
It will be appreciated by those skilled in the art that the isolated polynucleotide and polypeptides of the PgDGAT1 , PgDGAT2, and PgPDATI genes from Punica granatum have industrial and nutritional applications. The PgDGAT1, PgDGAT2, and PgPDAT1 genes encode PgDGAT1, PgDGAT2, and PgPDAT1, respectively. The DGAT and PDAT polynucleotides and polypeptides may be used in the industrial production of punicic acid using recombinant technology using transformed bacterial, yeast or fungal cells. Transformed cells may be engineered to accumulate punicic acid which may be incorporated into human food and animal feed applications to produce health supplements or to improve the nutritional quality of products.

These examples demonstrate how these genes can be used to produce punicic acid.
[00091] The A. thaliana fad3/fae 1 double mutant is deficient in delta-15-desaturase (FAD3) and fatty acid elongase (FAE1) activity. A. thaliana FAD3 catalyzes the conversion of linoleic acid (18:2A9' 12) into linolenic (8:3A9'12, 15), while FAE1 catalyzes the elongation of oleic acid (18:1A9) mostly into eicosenoic acid (20:1A"). Since oleic acid is a substrate for ongoing desaturation and elongation, consequently, the A, thaliana fad3/fael double mutant has a two-fold higher amount of linoleic acid substrate than is present in a wild type A. thaliana, resulting in a significant increase of punicic acid accumulation in the seed oil.
However, since substrate availability limits punicic acid production in transgenic A. thaliana seeds, additional genes and enzymes are needed to enhance punicic acid accumulation.
[00092] As described in the following Examples, the A. thaliana fad3/fael double mutant was transformed with various expression constructs comprising PgFADX and PgFAD2, as well as with PgDGAT1, and PgDGAT2. Seeds from the treated plants were plated out, with the transformants being selected and transferred to soil to establish primary transgenic plants (Ti) which were grown to maturity. T2 seeds were harvested from the Ti plants, analyzed for the fatty acid composition, and used to establish T2 progeny plants. T3 seeds were harvested and analyzed for the fatty acid composition.
[00093] PgFADX catalyzes the conversion of the delta-12 double bond of linoleic acid into two conjugated and trans-cis configurated double bonds at the 11 and 13 positions. PgFADX is a fatty acid conjugase which utilizes linoleic acid as a substrate for punicic acid synthesis. Over-expression of both PgFADX and PgFAD2 genes in the transformed A. thaliana fad3/fael double mutant restored the relative proportion of C18:1 fatty acids to normal levels and resulted in a two-fold increase in the punicic acid content compared to that which was present in plants over-expressing only PgFADX.
[00094] Punicic acid is synthesized in developing seed tissues and accumulates in the seed storage lipid in the form of triacylglycerol (TAG). TAGs may be synthesized through a combination of DGAT and PDAT activities. DGAT catalyzes the acyl-CoA-dependent synthesis of TAG, whereas PDAT catalyzes the transfer of a fatty acyl chain from sn-2 position of phosphatidylcholine (PC) to the sn-3 position of diacylglycerol DAG, producing TAG, Without being bound by theory, over-expression of PgDGAT1, PgDGAT2, and/or FDA Ti to produce PgDGAT1, PgDGAT2, and/or PDAT1 may facilitate the incorporation of punicic acid into TAGs, further enhancing the punicic acid accumulation in transformed plants carrying both PgFADX and PgFAD2.
[00095] Exemplary embodiments of the present invention are described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. As will be apparent to those skilled in the art, various modifications, adaptations and variations of the specific disclosure herein can be made without departing from the scope of the invention claimed herein.
[00096] Example 1 - Isolation and characterization of fatty acid conjugase (PgFADX) and delta-12 desaturase (PgFAD2) from Punica granatum [00097] Using sequence information available at GenBank (NCBI), PgFADX
(1188bp) and PgFAD2 (1164bp) ORFs were amplified by PCR and cloned into pCR4-TOPO
(Invitrogen).
Sequence analysis confirmed that isolated PgFADX and PgFAD2 were identical with sequences submitted by Iwabuchi et aL, 2003 (AY178446 and AY178447, respectively).
[00098] To confirm the function of PgFADX, the coding region was amplified by PCR with primers 5'-GAAATGGGAGCTGATGGAACAAT-3' (forward Fl, SEQ ID NO: 7) and 5'-TCAGAACTTGCTCTTGAACC-3 (reverse R1, SEQ ID NO: 8); cloned under the control of GAL1 inducible promoter into pYES2.1N5-His-TOPO yeast expression vector (Invitrogen); and expressed in Saccharomyces cerevisiae strain INVScl (Invitrogen).
[00099] Punicic acid was detected (up to 1.2%) only in yeast cells expressing PgFADX after supplementation of the growth media with linoleic acid (18:2 A9.12) at the final concentration of 300 M. The results indicated that isolated PgFADX encodes a functional fatty acid conjugase utilizing linoleic acid as a substrate for punicic acid synthesis (Figure 1).

[000100] Example 2 - Preparation of plant transformation vectors [000101] The binary constructs NCJ and NCJD carrying the cassettes described below were developed in pRD400 vector background (CLONTECH) (Figure 2).
[000102] For NCJ (napin-P/PgFADX/NOS-T), PCR was used to amplify the napin promoter (Josefsson et at, 1987), PgFADX, and Nos terminator (Bevan, 1983) using the primers set out in Table 1. The napin-P/PgFADX/NOS-T expression cassette was cloned in EcoRI and KpnI sites of pRD400 to yield the NCJ construct.
Table 1.
napin forward F2, EcoRI 5'-ATAGAATTCAAGCTTTCTTCATCGGTGAT-3' promoter site is underlined (SEQ ID NO: 9) reverse R2, Smal 51-ATACCC000GTCCGTGTATGTTITTAATC-3' site is underlined (SEQ ID NO: 10) PgFADX forward F3, SmaI 5'-TATCCCGGGATGGGAGCTGATGGAACA-3' is underlined (SEQ ID NO: 11) reverse R3, Not! 5'-CGCGCGGCCGCTCAGAACTTGCTCTTGAAC-3' site is underlined (SEQ ID NO: 12) Nos forward primer F4, 5'-CGCCGGCGGCCGCGATCGTTCAAACATTTGGCA-3' terminator NotI site is (SEQ ID NO: 13) underlined reverse primer R4, 5'-TATGGTACCCGATCTAGTAACATAGATGAC-3' KpnI site is (SEQ ID NO: 14) underlined [000103] For NCJD (napin-P/PgFADX/NOS-T/Napin-P/PgFAD2/NOS-T), PCR was used to amplify the napin promoter, PgFAD2, and NOS terminator using the primers set out in Table 2.
The napin-P/PgFAD2/NOS-T expression cassette was cloned into KpnI and Sall sites of NCJ
(described above) to yield the NCJD construct.
Table 2.
napin forward F5, KpnI 5'-ATAGGTACCAAGCTTTCTTCATCGGTGAT-3' promoter site is underlined (SEQ ID NO: 15) reverse R5, XhoI 5LATACTCGAGGTCCGTGTATG MTIAATCT-3' site is underlined (SEQ ID NO: 16) PgFAD2 forward F6, XhoI 51-TAACTCGAGATGGGAGCCGGTGGAAG-3' is underlined (SEQ ID NO: 17) reverse R6, XbaI 5t-TAITCTAGATCAGAGGITCTTCTTGTAC-3' site is underlined (SEQ ID NO: 18) Nos forward F7, Xbal 5'-TATTCTAGAGATCGTTCAAACATTTGGCAA-3' terminator site is underlined (SEQ ID NO: 19) reverse R7, Sall 5'-ATAGTCGACCGATCTAGTAACATAGATGAC-3' site is underlined (SEQ ID NO: 20) [000104] Example 3 - Production of punicic acid in A. (Indiana seeds [000105] The binary vectors NCJ or NCJD were electroporated into Agrobacterium tumefaciens cell strain GV3101 and introduced into Arabidopsis thaliana fad3/fael (Smith et al., 2003) double mutant background using the floral dip method (Clough and Bent, 1998).
Transgcnic plants were selected and analyzed as described before (Mietkiewska et al., 2007).
[000106] Seeds from twenty-eight independent transgenic lines carrying NCJ
construct in high linoleic acid (52%) A. thaliana fad3ffael mutant background were analyzed.
Results from the best 11 A. thalina T2 lines are shown in Figure 3. Significant changes in fatty acid composition in comparison to the control lines (fad3/fael) were found. Seed specific expression of PgFADX
resulted in an increased proportion of punicic acid (18:36,9z'l1E'13z) from 0%
in the controls up to 11.26% in the best transgenic line NCJ-11. The increased proportion of punicic acid was correlated with concomitant reduction in the proportion of its corresponding precursor 18:2 (reduced by 52%). The production of punicic acid in A. thaliana seeds was accompanied by up to a 43% increase in oleic acid content, indicating that over-expression of PgFADX led to the inhibition of the native FAD2 activity.
[000107] To enhance the accumulation of punicic acid and reduce the effect of native FAD2 inhibition observed in seeds over-expressing PgFADX, a second construct carrying PgFADX and PgFAD2 (NCJD) was developed. The fatty acid compositions of forty-five independent transgenic lines carrying NCJD construct in A. thaliana fad3/fae 1 mutant were determined. The results from the best 17 A. thaliana T2 seeds are shown in Figure 4. Over-expression of PgFADX and PgFAD2 in A. thaliana seeds resulted in higher proportion of punicic acid compared to the seeds over-expressing only PgFADX (Figure 3). In the best transgenic line, NCJD-33, punicic acid content was increased up to 15.24% at the expense of its precursor 18:2 (reduced by 27%). Oleic acid content in NCJD-33 seeds was reduced by 8.5%
compared to not-transformed A. thaliana .fad3/fael mutant. These results indicate that over-expression of PgFAD2 reduced significantly the inhibition effect of native FAD2 desaturase activity observed in the seeds over-expressing only PgFADX.
[000108] Since the preliminary analysis of fatty acid composition was performed on T2 segregating seeds for the presence of the transgene(s), it was proposed that T3 homozygous seeds of A. thaliana might contain higher proportions of punicic acid. Seeds from the T2 lines with the highest proportion of punicic acid were thus sown and grown to obtain the T3 seed generation.
[000109] Seeds from eight T3 homozygous transgenic lines carrying NCJ
construct in high linoleic acid (52%) A. thaliana fad3/fael mutant background were analyzed (Figure 5).
Significant changes in fatty acid composition in comparison to the control lines (fad3/fael) were found. The proportion of punicic acid (18:3A9Z,11E,13Z ) increased from 0% in control lines (fad3/fael) to as high as 11.4% in the best transgenic line, NCJ-11-4. The increased proportion of punicic acid was correlated with concomitant reduction in the proportion of its precursor 18:2 (reduced by 51.3%). The production of punicic acid in A. thaliana seeds was accompanied by up to a 45% increase in oleic acid content, indicating that over-expression of PgFADX led to the inhibition of the native FAD2 activity.
[000110] To enhance the accumulation of punicic acid and reduce the effect of native FAD2 inhibition observed in seeds over-expressing PgFADX, a second construct carrying PgFADX and PgFAD2 (NCJD) was developed. The fatty acid compositions of nine T3 transgenic lines carrying NCJD construct in A. thaliana fad3/fael mutant are shown in Figure 6.
Over-expression of PgFADX and PgFAD2 in A. thaliana seeds resulted in higher proportion of punicic acid compared to the seeds over-expressing only PgFADX (Figure 5). In the best transgenic lines, NCJD-33-2 and 34-3, punicic acid content was increased up to 21% at the expense of its precursor 18:2 (reduced by 28.6%). Oleic acid content in the best transgenic NCJD-33-2 seed line was reduced by 24% compared to non-transformed A. thaliana fad3/fael mutant, indicating that over-expression of PgFAD2 reduced significantly the inhibition effect of native FAD2 activity observed in the seeds over-expressing only PgFADX.
[000111] Example 4 - Fatty acid composition of the selected lipid classes in P. granatum seeds and A. thaliana engineered to synthesize punicic acid in the seed oil [000112] Examination of the fatty acid content of specific lipid classes was performed for the A. thaliana line over-expressing PgFADX+PgFAD2 (NCJD-30-2) with the highest content of punicic acid (21.2%) in the T3 seeds (Figure 7). Total lipids from seeds were extracted and separated as described earlier (Mieticiewska et al., 2011). In transgenic A.
thaliana seeds NCJD-30-2, the punicic acid content of phosphatidylcholine (PC) was 12.5% which was higher than that observed in TAG (6.6%). A different situation was found in P. granatum seeds where the punicic acid content was 60% of the fatty acids in TAG, and only 0.8% of fatty acids in PC. The data indicate that in A. thaliana, an efficient mechanism of trafficking punicic acid from PC to TAG is missing.

[000113] Example 5 - Isolation of strategic genes involved in punicic acid trafficking [000114] Using a degenerate primer RT-PCR approach performed on cDNA amplified from P. granatum seeds, the following genes were isolated:
[000115] a) P. granatum diacylglycerol acyltransferase type I (PgDGAT1):
[000116] Degenerate primers designed in the conserved region included the forward primer (YQDWWNA, SEQ ID NO: 21) and reverse primer (HELCIAVP, SEQ ID NO: 22) which were used to amplify a 190 bp PCR internal fragment of a putative DGATI from P.
granatum showing up to 89% of identity to plant DGAT1 amino acid sequences. The sequence of 190 bp PCR
product was used to design a gene specific primer to amplify the 5' and 3' ends of cDNA using a SMART RACE cDNA Amplification kit (CLONTECH, Palo Alto, CA, USA). Using the sequence information coming from the assembly of the partial sequences, the full length ORF
(1623 bp) of a putative DGAT1 was amplified by PCR using the forward F8 primer (5'-ATGGCGACCTCCGACGGC-3'; SEQ ID NO: 23) and the reverse R8 primer (5'-TTACGGCCGGGAGCCITTT-3'; SEQ ID NO: 24). The PgDGAT1 cDNA (SEQ ID NO: 1;
Figure 8A) encodes a polypeptide of 540 amino acids (SEQ ID NO: 2; Figure 8B) that is most closely related to DGAT1 from Glycine max and Vernica fordii (70% identity to both of them).
The PgDGAT1 sequence was submitted to GenBank and accorded NCBI accession #JQ478414.
[000117] b) P. granatum diacylglycerol acyltransferase type 2 (PgDGAT2):
[000118] Degenerate primers designed in the conserved regions included the forward primer (VPGGVQE, SEQ ID NO: 25) and reverse primer (PMHVVVG, SEQ ID NO: 26) which were used to amplify a 276 bp PCR internal fragment of a putative DGAT2 from P.
granatum showing up to 78% of identity to plant DGAT2 amino acid sequences. A similar approach as above was used to isolate full-length ORF (1005bp) of DGAT2 from pomegranate seeds by PCR using the forward F9 primer (5'-ATGGGAGAGGAGGCGAGC-3', SEQ ID NO: 27) and reverse R9 primer (5'-TCAGAGGATCTTCAGTTCC-3', SEQ ID NO: 28). The P. granatum DGAT2 cDNA (SEQ ID NO: 3) encodes a polypeptide of 334 amino acids (SEQ ID NO: 4) with the highest sequence identity (71%) to DGAT2 from Olea europaea. The sequence of the PgDGAT2 homolog was submitted to GenBank and accorded NCBI accession #.1Q513387.
[000119] c) P. granatum phospholipid:diacylglycerol acyltransferase 1 (PgPDAT1):
[000120] Degenerate primers designed in the conserved regions included the forward primer (LCWVEHM, SEQ ID NO: 29) and reverse primer (TQSGAHV, SEQ ID NO: 30) which were used to amplify a 1.4 kb PCR internal fragment of a putative PDAT from P.
granatum showing up to 82% of identity to plant PDAT homologs. A similar approach as above was used to isolate full-length ORF (2052bp) of PDAT from pomegranate seeds by PCR using the forward F10 primer (5'-ATGGCGTTTCTCTGGCGGA-3', SEQ ID NO: 31) and the reverse R10 primer (5'-CTAGAGTGGCAAGTCAATCC-3', SEQ ID NO: 32). The PgPDAT1 cDNA (SEQ ID NO: 5) encodes a polypeptide of 683 amino acids (SEQ ID NO: 6) with the highest sequence identity (83%) to PDAT1 from Glycine max and Vitis vinifera (82%). The sequence of the PgPDAT1 homolog was submitted to GenBank and accorded NCBI accession OQ513388.
[000121] Example 6 - Functional characterization of PgDGAT1 and PgDGAT2 [000122] To establish the function of PgDGAT1 and PgDGAT2, constructs carrying two genes were developed, namely PgFADX + DGAT1 and PgFADX + DGAT2, in the yeast expression vector pESC-URA (Agilent).
[000123] PgFADX was amplified by PCR using the forward Fl 1 primer (5'-AATAGGATCCGAAAT000AGCTGATGGAACA-3'; BamHI site is underlined; SEQ ID NO:
33) and the reverse R11 primer (5'-TTATGGTACCTCAGAACTTGCTCTTGAAC-3'; KpnI site is underlined; SEQ ID NO: 34), and cloned in BamHI and KpnI sites of pESC-URA
to yield the pEX construct.
[000124] PgDGAT1 was amplified by PCR using the forward F12 primer (5'-GCAGAGCGGCCGCGAAATGGCGACCTCCGACGGC-3'; NotI site is underlined; SEQ ID
NO: 35) and the reverse R12 primer (5'ATATTTAATTAATTACGGCCGGGAGCCTTTT'-3';
Pad site is underlined; SEQ ID NO: 36), and cloned in the NotI and Pad sites of pEX.
[000125] PgDGAT2 was amplified by PCR using the forward F13 primer (5'-GCAGAGCGGCCGCGAAATGGGAGAGGAGGCGAG-3'; NotI site is underlined; SEQ ID
NO: 37) and the reverse R13 primer (5'-ATATTTAATTAATCAGAGGATCTTCAGTTCC-3';
Pad site is underlined; SEQ ID NO: 38); and cloned in NotI and Pad sites of pEX.
[000126] The prepared constructs were transformed into yeast cells H1246 (Sandager et al., 2002). Yeast cultures were grown at 30 C for 48 h. Expression of the recombinant genes was induced using minimal medium containing 2% (w/v) galactose and 1% (w/v) raffinose supplemented with 100 uM of linoleic acid (18:2).
[000127] In yeast cells over-expressing only PgFADX, punicic acid was found only in the polar lipids (PL) fraction where its synthesis occurs (Figure 11). In yeast cells over-expressing PgFADX + PgDGAT1, the punicic acid content of polar lipids was 0.82% and was higher than that observed in triacylglycerol (TAG, 0.13%). Significantly higher accumulation of punicic acid in TAG (0.4%) was found in yeast cells over-expressing PgFADX + PgDGAT2.
[0001281 Without being bound by theory, these results indicate that PgDGAT1 and PgDGAT2 encode enzymes involved in the efficient trafficking of punicic acid from the origin of its synthesis (PL) to the storage lipids (TAG). PgDGAT1 and PgDGAT2 appear to be suitable strategic genes to enhance the accumulation of punicic acid to higher levels than those observed in A. thaliana plants over-expressing only PgFADX+ PgFAD2.
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Claims (20)

1. An isolated polynucleotide sequence encoding a protein or polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, 4 or 6, respective biologically active variants and biologically active portions thereof, with respective sequences having at least 85% identity thereto, and wherein the variants have diacylglycerol acyltransferase type 1 (DGAT1), type 2 (DGAT2) or phospholipid diacylglycerol acyltransferase (PDAT) activity.

2. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having DGAT activity and comprising the amino acid sequence of SEQ ID NO: 2 or 4, or an amino acid sequence having DGAT activity and having at least 85% sequence identity therewith.

3. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having PDAT activity and comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having PDAT activity and having at least 85% sequence identity therewith.

4. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 3 or 5.

5. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one of SEQ ID NO: 2, 4 or 6.

6. The isolated polynucleotide of claim 1, wherein the polynucleotide is derived from Punica granatum.

7. A recombinant expression vector comprising a polynucleotide of claim 1 operably linked with transcriptional and translational regulatory regions or sequences to provide for expression of the at least one polynucleotide sequence in a host cell.

8. A transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising: (a) the recombinant expression vector of claim 7, or (b) a recombinant expression vector encoding PgFADX and PgFAD2,

9. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 8, which comprises PgFADX and PgFAD2.

10. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 9, which further comprises one or more of a PgDGAT and PgPDAT.

11. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 9 or 10, which comprises a progeny plant generated from the transgenic plant.

12. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 10, which comprises PgFADX, PgFAD2, PgDGAT1 or PgDGAT2 or both, and PgPDAT1.

13. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 9, 10 or 11, which is selected from a linseed, rapeseed, canola, peanut, safflower, flax, hemp, camelina, soybean, pea, sunflower, olive, palm, oats, wheat, triticale, barley, corn, thale cress, and legume plant, plant cell, plant seed, callus, plant embryo, or microspore-derived embryo or microspore.

14. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 13, which comprises Arabidopsis thaliana.

15. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 14, which comprises Arabidopsis thaliana fad3/fae 1 double mutant.

16. A method of increasing punicic acid production in an oilseed plant, comprising the steps of:
a) constructing one or more vectors comprising polynucleotides encoding PgFADX
and PgFAD2;
b) transforming the one or more vectors into a host cell under conditions sufficient for over-expression of PgFADX and PgFAD2.

17. The method of claim 16 further comprising the step of constructing one or more vectors comprising one or more of the polynucleotides encoding a PgDGAT or PgPDAT and transforming the one or more vectors into a host cell under conditions sufficient for over-expression of one or more of a PgDGAT or a PgPDAT.

18. The method of claim 16 or 17, further comprising the step of generating a transgenic plant from the host cell and obtaining a progeny plant, wherein the progeny plant comprises PgFADX and PgFAD2, and one or more of a DGAT and PDAT encoded by the polynucleotides, and the polynucleotides are over-expressed in the progeny plant.

19. The method of claim 16 or 17, wherein PgFADX, PgFAD2, PgDGAT1 or PgDGAT2 or both, and PgPDAT1 are over-expressed in the plant.

20. The method of claim 19, wherein the plant comprises Arabidopsis thaliana or an Arabidopsis thaliana fad3/fae 1 double mutant,