AU2007334364B2 - Genes encoding a novel type of lysophophatidylcholine acyltransferases and their use to increase triacylglycerol production and/or modify fatty acid composition - Google Patents

Description

GENES ENCODING A NOVEL TYPE OF LYSOPHOPHATID YLCHOLINE

ACYLTRANSFERASES AND THEIR USE TO INCREASE TRIACYLGLYCEROL

PRODUCTION AND/OR MODIFY FATTY ACID COMPOSITION

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application USSN 60/874,497, filed on December 13, 2006, and priority, under the Paris Convention, to U.S.S.N. 11/820,014, filed on June 15, 2007, the contents of the entirety of both of which are incorporated herein by this reference.

TECHNICAL FIELD

The invention relates generally to biotechnology, and, more particularly, to /^-phosphatidylcholine (LPC) acyltransferase, polynucleotides that encode LPC acyltransferases, and associated means and methods.

BACKGROUND

Phosphatidylcholine (PC) serves not only as a major component of cellular membranes, but also as a major source of fatty acyl donors for triacylglycerol biosynthesis in eukaryotic organisms.

, At least three pathways through which PC is generated exist: (i) the CDP-choline pathway where diacylglycerol (DAG) is a direct precursor; (ii) a pathway where CDP-DAG is a direct precursor, involving phosphatidylserine formation and decarboxylation and phosphatidylethanolamine methylation (Zheng and Zou, 2001); and (iii) a pathway with LPC as substrate. The third pathway is exerted by LPC acyltransferases (LPCAT).

LPCAT enzymes catalyze the acylation of LPC molecules to form PC and play a pivotal role in membrane biogenesis. They can also exert a reversible reaction to release the fatty acyl chain esterified to the sn-2 position of PC, thereby contributing to a continuous remodeling of fatty acyl-CoA and PC pools.

The significance of LPCAT in glycerolipid metabolism of eukaryotic systems has been noted for many years. For genetic engineering of plant-based production of very long chain polyunsaturated fatty acid (PUFA), this enzyme is believed to represent a bottleneck for acyl exchange between the fatty acyl elongation and de-saturation systems. In higher plants, the function of this enzyme is largely unknown, but it has been proposed that the enzyme is involved in the selective incorporation of fatty acids into a storage pool.

Although LPCAT relating to the synthesis of surfactant lipid located on the surface of (pulmonary) cells have been reported in mammalian systems (X. Chen et al., PNAS 2006 103:11724-11729; H. Nakanishi et al., JBC 2006 281:20140-20147), an LPC acyltransferase involved in membrane or storage lipid synthesis has not been reported.

Recently, a mitochondrial acyl-CoA independent LPCAT from Saccharomyces cerevisiae has been identified. This enzyme has been shown to function in cardiolipin metabolism (Testet et al. 2005). In addition, Shindou et al. (2007) reported that aceyl-CoA:lyso-PAF (platelet-activating factor) acetyltransferase possesses LPCAT activity.

SUMMARY OF THE INVENTION

Novel types of LPCAT enzymes whose sequences are unrelated to any known LPCAT enzymes have been identified. Known domains for other sn-2 acyl transferases such as the mammalian LPC acyltransferases are not identifiable in the LPC acyltransferase assay disclosed herein.

Previously reported LPCAT enzymes share a substantial sequence homology to glycerol-3-phosphate acyltransferase and lysophosphatidic acyltransferase. In contrast, the LPCAT sequences disclosed herein are unrelated to any known LPCAT sequences, and belong to a new class of LPCAT. Four conserved motifs were identified in this novel class of LPCAT enzymes. The identified motifs are different from previously reported LPCAT, which contain motifs having a high degree of similarity to those in other known acyltransferases employing glycerol-3-phosphate and lysophosphatidic acid as substrates. In contrast, sequence information of the motifs identified herein is novel, and can lead to the identification of new class of LPCAT genes from a broad spectrum of species.

Thus, in certain embodiments, a /yso-phosphatidylcholine acyltransferase gene or class of genes is identified. The LPC acyltransferase gene may be expressed or overexpressed in a cell and used to modify glycerolipid biosynthesis in a cell. Such an LPC acyltransferase gene may be expressed or overexpressed in a cell and used to modulate or enhance production of fatty acids, especially polyunsaturated fatty acids (PUFA) or other unusual fatty acids, and/or to increased oil content in the cell. The LPC acyltransferase gene may be expressed or overexpressed inplanta in order to modify glycerolipid biosynthesis in a plant. In certain embodiments, the LPC acyltransferase gene is expressed or overexpressed, in planta, in order to enhance the production of fatty acids in a plant.

In certain embodiments, a vector is provided having an LPC acyltransferase gene of the invention. The vector may be used to transform a cell, thus producing a recombinant cell having the LPC acyltransferase gene. The cell may comprise, for example, a bacterial cell, a yeast cell, or a plant cell. In certain embodiments, a plant, plant seed or progeny thereof includes a cell having a recombinant LPC acyltransferase gene.

In other embodiments, knock-out mutants disrupted in LPC acyltransferase gene of yeast and plants are identified. In certain embodiments, a recombinant cell expresses an LPC acyltransferase gene and produces an LPC acyltransferase polypeptide that may be isolated or purified from the cell.

In certain embodiments, nucleotide and deduced amino acid sequences associated with an LPC acyltransferase gene are disclosed. The sequence, or a portion thereof, may be used to identify genes from other species that encode polypeptides with LPC acyltransferase activity. In certain embodiments, a process for producing fatty acids includes transforming a cell with an LPC acyltransferase gene. The transformed cell expresses the fatty acid acyltransferase gene and produces fatty acids. The fatty acids may be isolated or purified from the recombinant cell or culture media in which the cell grows, and subsequently incorporated into a composition.

In certain embodiments, a peptide comprising one or more of the four motifs identified herein may be used as an LPC Acyltransferase. Similarly, a nucleotide sequence encoding a peptide comprising one or more of the four motifs may be used as an LPC Acyltransferase.

Provided is an isolated or recombinant nucleic acid molecule encoding an LPC acyltransferase, and a cell transformed with the isolated or recombinant nucleic acid molecule as described herein. Also provided is a process for increasing fatty acid production in a cell, the process comprising: transforming a cell with a nucleic acid molecule encoding an LPC acyltransferase; and, growing the cell under conditions wherein the LPC acyltransferase is expressed. Also provided is a use of an isolated or recombinant nucleic acid molecule encoding an LPC acyltransferase for producing an LPC acyltransferase in a cell. Also provided is a purified or an isolated LPC acyltransferase. LPCAT enzymes play a critical role in remodeling fatty acid and PC pools as well as PC synthesis. The remodeled fatty acyl chains in the form of acyl-CoA or esterified at the sn-2 position of PC can be used for triacylglycerol synthesis. Thus, this novel type of LPCAT isolated from the organisms where very-long-chain polyunsaturated fatty acids (VLCPUFA) are present at a high level can be used to increase the production of VLCPUFA. As well, this novel type of LPCAT isolated from species containing high amount of unusual fatty acids can be used to increase the production of unusual fatty acids. For instance, LPCAT enzymes isolated from castor bean are useful in increasing the production of hydroxyl fatty acids in oil seeds.

The enzyme activity described herein provides support that the motif-based gene searching is a useful approach. BRffiF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of LPCAT activity (nmol/mg-h) of wild type (WT) and YOC 175c mutant yeast strains. FIG. 2 is an alignment of LPCAT sequences from different species that revealing, among other things, four conserved motifs unique for this type of LPCAT enzymes.

FIG. 3 is another alignment of LPCAT sequences from different plant species that revealed four conserved motifs (SEQ ID NOS:81-84).

FIG. 4 depicts that the expression of the TpLCAT in an lpcat mutant was able to complement the sensitivity of the lpcat mutant to Lyso-PAF.

FIG. 5 is a graph showing the expression of TpLPCAT in yeast. LPCAT assays were performed on cell lysates of yeast lpcat mutant strain ByO2431 transformed with TpLPCAT/pYES2.1 and pYes2. l/V5-His-TOPO plasmid only (control) in the presence of ¹⁴C-Lyso-PC and different acyl-CoAs. FIG. 6 comprises TLC plates of LPCAT assays on cell lysates of yeast lpcat mutant strain

ByO2431 transformed with TpLPCAT/pYES2.1 and pYes2.1/V5-His-TOPO plasmid only (control) in the presence of ¹⁴C-Lyso-PC and different acyl-CoAs. 1, 3, 5, 7, 9, 11, and 13-TpLPCAT; 2, 4, 6, 8, 10, 12, and 14 - empty vector.

FIG. 7 shows a LysoPAF sensitivity test of YOR175c mutant, AtLPCATs transformant, wherein A is VO/B Y02431 , B is AtLPCAT 1 /B Y02431 , and C is AtLPC AT2/B Y02431.

FIG. 8 is a graph showing the Lyso-lipid substrate specificity of Arabidopsis LPCATs. FIG. 9 is a graph comparing LPAAT and LPCAT activity of slclΔ, leal A, and congenic WT yeast strain. Cell lysates equivalent to 200μg protein were assayed for acylation of oleoyl-LPA and oleoyl-LPC with [¹⁴C] oleoyl-CoA. The reaction mixture contained 45 μM 18:1-LPA or 18:1-LPC, 18 μM (10 nCi/nmol) 18:l-CoA. The results are presented as a mean of three assays.

FIG. 10 is graph depicting lysophospholipid acyltransferase activity in icalΔ and its congenic WT yeast strains. Microsomal preparations were assayed for acylation of palmitoyl-LPA, LPC, LPE, LPG, LPI, and LPS with [¹⁴C] palmitoyl-CoA. The reaction mixture contained 45 μM lysophospholipid, 27 μM (10 nCi/nmol) 16: 1-CoA and 50 μg protein. The results are presented as a mean of three assays.

FIG. 11 is a graph showing the substrate specificity of LCAl. The assays were performed with 3 μg microsomal protein from leal A harboring an empty vector (VO) and leal A expressing LCAl. The reaction contained 112.5 μM [¹⁴C] palmitoyl-CoA (5.5 nCi/nmol) and 50 μM lysophospholipid substrate (LPA, LPC, LPE, LPG, LPI, and LPS). Reaction was allowed for two minutes with 100 rpm shaking. The results are presented as a mean of three assays.

FIG. 12 shows the lyso-PAF and lyso-PC Sensitivity test icalΔ, WT and lcaldelta over-expressing LCAl. Cells were frown first in SC-URA +2% glucose media overnight then in protein expression induction media for six hours. Cultures were diluted to OD₆₀O value of OD₆oo

0.5, 1, 2, 3, respectively, from which 5 μl were inoculated (from left to right) onto YPD plate containing lyso-PAF or lyso-PC. The plates were incubated at 28°C for 36 hours.

FIG. 13 depicts two graphs showing substrate preference of LCAl. A. Acyl-CoA substrate preference. Assays were performed with 3 μg microsomal protein from IcalΔ harboring an empty vector (VO) and IcalΔ expressing LCAl, with 112.5 μM [¹⁴C] palmitoyl-CoA (1.35 nCi/nmol) and 50 μM acyl-CoA species. B. LPC substrate preference. Assays were performed with 3 μg microsomal protein, 112.5 μM [¹⁴C] palmitoyl-CoA (5.5 nCi/nmol) and 50 μM lysophospholipid species. The values for IcalΔ are not shown. The results are presented as a mean of three assays. FIG. 14 depicts a TLC pattern of choline-containing compounds in the TCA fraction for

PC turnover assessment. Samples were chromatographed and visualized on MERCK® Silica Gel G60 plate as described herein. Lane 1, TCA fraction of WT yeast; lane 2, TCA fraction of IcalΔ yeast; lane 3, [14C] choline; lane 4, palmitoleoyl-LPC; lane 5, GroPC; lane 6, phosphocholine; and lane 7, CDP-choline. Dashed line indicated sample origin. FIG. 15 is a graph illustrating lyso-lipid substrate specificity of AtLPCATs expressed in

IcalΔ. The assays were preformed with 3 μg microsomal protein from IcalΔ harboring an empty vector (VO) and IcalΔ expressing AtLPCATl and AtLPCAT2. The reaction contained 45 μM [¹⁴C] palmitoyl-CoA (5.5 nCi/nmol) and 45 μM Lysophospholipid substrate (LPA, LPC, LPE, LPG, LPI and LPS). The reaction was allowed for ten minutes with 100 rpm shaking. The results were presented as a mean of three assays.

FIG. 16 depicts plates showing LysoPAF Sensitivity test of a yorl75c mutant strain transformed with empty pYES2.1, pYES2. IwAtLPCATl and pYES2.1::LPCAT2. Cells were grown first in SC-URA+2% glucose media overnight then in protein expression induction media for six hours. Cultures were diluted to OD₆₀₀ value of OD_60O =1, 2, 4, respectively, from which 5 μl was inoculated (from left to right) onto YPD plate containing Lyso-PAF or Lyso-PC. The plates were incubated at 28°C for 36 hours.

FIG. 17 is a graph depicting the Acyl-CoA preference assessment of AtLPCATs expressed in IcalΔ. Assays were preformed with 20 μg microsomal protein from IcalΔ harboring an empty vector (VO) and leal A expressing AtLPCATl and AtLPCAT2, with 45 μM [¹⁴C]palmitoyl-LPC (1.35 nCi/nmol) and 45 μM acyl-CoA species.

FIG. 18 is a graph depicting the LPC substrate preference of AtLPCATs expressed in icalΔ. Assays were preformed with 20 μg microsomal protein, 45 μM [¹⁴C]palmitoyl-CoA (5.5 nCi/nmol), 45 μM LPC species. The results were presented as a mean of three assays.

FIG. 19 is a graph depicting the inhibitory effect of Zn²⁺ on AtLPCATs activity. The IcalΔ over-expressing AtLPCATs was used to asses Zn²⁺ effect. The reactions contained 25 μM palmitoyl-LPC (1.35 nCi/nmol), 20 μg microsomal proteins, 0.1 M HEPES (pH 7.4), 25 μM stearyl-CoA and indicated concentration of ZnCl₂. FIG. 20 is a sequence alignment of YOR 175c with its selected human and mouse homologs. Alignment was performed with CLUSTALV from the DNASTAR package run with default multiple alignment parameters (gap opening penalty: 10; gap extension penalty: 10).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferably, the nucleic acid molecule encoding the LPC acyltransferase is derived from yeast, plant, or mammalian species. Yeast species include, for example, species of the genus Saccharomyces, for example, Saccharomyces cerevisiae. Plant species include, for example, species of the family Brassicaceae. Of the family Brassicaceae, species of genus Brassica and genus Arabidopsis are of particular note, for example, Arabidopsis thaliana. Mammalian species include mouse and human.

In particular, provided are a nucleic acid molecule encoding an LPC acyltransferase from S. cerevisiae and two nucleic acid molecules encoding two different isoforms of LPC acyltransferase from A. thaliana. There is also provided the LPC acyltransferases encoded by the herein described nucleic acid molecules. Provided herein is an isolated or recombinant nucleic acid molecule having a nucleotide sequence encoding an LPC acyltransferase such as amino acid sequence comprising SEQ ID NO:2, SEQ ID NO:4, SEQ BD NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO.ll; SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39; SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO: 87, or SEQ ID NO:88. In particular, there is provided an isolated or recombinant nucleic acid molecule having a nucleotide sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ DD NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34. Preferably, the LPC acyltransferase encoded by a nucleic acid molecule comprises an amino acid sequence comprises an amino acid sequence having at least 60% homology to the aforementioned sequences. Homology is more preferably at least 70%, 80%, 90%, or 95%. It will be appreciated that this disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art.

Homologs of the LPC acyltransferase genes described herein obtained from other organisms, for example, plants, may be obtained by screening appropriate libraries that include the homologs, wherein the screening is performed with the nucleotide sequence of the specific LPC acyltransferase genes of the instant invention or portions or probes thereof, or identified by sequence homology search using sequence alignment search programs such as BLAST, FASTA.

Further included are nucleic acid molecules that hybridize to the above disclosed sequences. Hybridization conditions may be stringent in that hybridization will occur if there is at least a 90%, 95% or 97% identity with the nucleic acid molecule that encodes the LPC acyltransferase of the instant invention. The stringent conditions may include those used for known Southern hybridizations such as, for example, incubation overnight at 42°C in a solution having 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in 0. IxSSC at about 65°C. Other known hybridization conditions are well known and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N. Y. (2001), incorporated herein in its entirety by this reference.

Nucleic acid molecules that code for an LPC acyltransferase may be transformed into an organism, for example, a plant. As known in the art, there are a number of ways by which genes and gene constructs can be introduced into organisms, for example, plants, and a combination of transformation and tissue culture techniques have been successfully integrated into effective strategies for creating transgenic organisms, for example, crop plants. These methods, which can be used in the invention, have been described elsewhere (Potrykus, 1991; Vasil, 1994; Walden and Wingender, 1995; Songstad et al., 1995), and are well known to persons skilled in the art. For example, one skilled in the art will certainly be aware that, in addition to Agrobacterium-mediated transformation of Arαbidopsis by vacuum infiltration (Bechtold et al., 1993) or wound inoculation (Katavic et al., 1994), it is equally possible to transform other plant and crop species, using Agrobαcterium Ti-plasmid-mediated transformation {e.g., hypocotyl (DeBlock et al., 1989) or cotyledonary petiole (Moloney et al., 1989) wound infection), particle bombardment/biolistic methods (Sanford et ah, 1987; Nehra et al., 1994; Becker et al., 1994) or polyethylene glycol-assisted, protoplast transformation (Rhodes et al., 1988; Shimamoto et al., 1989) methods.

As will also be apparent to persons skilled in the art, and as described elsewhere (Meyer, 1995; Dada et al., 1997), it is possible to utilize plant promoters to direct any intended up- or down-regulation of transgene expression using constitutive promoters (e.g., those based on

CaMV35S), or by using promoters which can target gene expression to particular cells, tissues

(e.g., napin promoter for expression of transgenes in developing seed cotyledons), organs (e.g., roots), to a particular developmental stage, or in response to a particular external stimulus (e.g., heat shock).

Promoters for use herein may be inducible, constitutive, or tissue-specific or have various combinations of such characteristics. Useful promoters include, but are not limited to constitutive promoters such as carnation etched ring virus (CERV), cauliflower mosaic virus (CaMV) 35S promoter, or more particularly the double enhanced cauliflower mosaic virus promoter, comprising two CaMV 35S promoters in tandem (referred to as a "Double 35S" promoter).

It may be desirable to use a tissue-specific or developmentally regulated promoter instead of a constitutive promoter in certain circumstances. A tissue-specific promoter allows for overexpression in certain tissues without affecting expression in other tissues. By way of illustration, a preferred promoter used in overexpression of enzymes in seed tissue is an ACP promoter as described in PCT International Publication WO 92/18634, published October 29, 1992, the disclosure of which is herein incorporated by reference.

The promoter and termination regulatory regions will be functional in the host plant cell and may be heterologous (that is, not naturally occurring) or homologous (derived from the plant host species) to the plant cell and the gene. Suitable promoters which may be used are described above.

The termination regulatory region may be derived from the 3' region of the gene from which the promoter was obtained or from another gene. Suitable termination regions which may be used are well known in the art and include Agrobacterium tumefaciens nopaline synthase terminator (Tnos), A. tumefaciens mannopine synthase terminator (Tmas) and the CaMV 35S terminator (T35S). Particularly preferred termination regions for use herein include the pea ribulose bisphosphate carboxylase small subunit termination region (TrbcS) or the Tnos termination region. Such gene constructs may suitably be screened for activity by transformation into a host plant via Agrobacterium and screening for increased isoprenoid levels. Suitably, the nucleotide sequences for the genes may be extracted from, for instance, the

GenBank® (a registered trademark of the U.S. Department of Health and Human Services) nucleotide database and searched for restriction enzymes that do not cut. These restriction sites may be added to the genes by conventional methods such as incorporating these sites in PCR primers or by sub-cloning.

Preferably, a DNA construct for use herein is comprised within a vector, most suitably an expression vector adapted for expression in an appropriate host (plant) cell. It will be appreciated that any vector which is capable of producing a plant comprising the introduced DNA sequence will be sufficient. Suitable vectors are well known to those skilled in the art and are described in general technical references such as Pouwels et al., Cloning Vectors, A Laboratory Manual, Elsevier, Amsterdam (1986). Particularly suitable vectors include the Ti plasmid vectors.

Transformation techniques for introducing the DNA constructs into host cells are well known in the art and include such methods as micro-injection, using polyethylene glycol, electroporation, or high velocity ballistic penetration. A preferred method relies on Agrobacterium-mediated transformation. After transformation of the plant cells or plant, those plant cells or plants into which the desired DNA has been incorporated may be selected by such methods as antibiotic resistance, herbicide resistance, tolerance to amino-acid analogues or using phenotypic markers. Various assays may be used to determine whether the plant cell shows an increase in gene expression, for example, Northern blotting or quantitative reverse transcriptase PCR (RT-PCR). Whole transgenic plants may be regenerated from the transformed cell by conventional methods. Such transgenic plants having improved isoprenoid levels may be propagated and self-pollinated to produce homozygous lines. Such plants produce seeds containing the genes for the introduced trait and can be grown to produce plants that will produce the selected phenotype.

Plants that may be modified or used for fatty acid production according to the instant invention include, without limitation, borage (βorago spp.), Canola, castor (Ricinus communis); cocoa bean (Theobroma cacao), corn (Zea mays), cotton {Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oeanothera spp., olive (Olea spp.), palm {.Elaeis spp.), peanut (Arachis spp.), rapeseed, safflower (Carthamus spp.), soybean {Glycine and Soja spp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Vernonia spp,, wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.), oat (Avena spp.) sorghum (Sorghum spp.), rye (Secale spp.) or other members of the Gramineae. It will further be apparent to those of ordinary skill in the art that genomic or sequence libraries of each of these plants may be screened with the nucleotide or amino acid sequences described herein (e.g., for one or more of the hereinafter identified conserved motifs (SEQ ID NO:46 through SEQ ID NO:49) for other sequences that encode or are homologous to sequences associated with the LPC acyltransferase of the instant invention. Plants transformed with a nucleotide sequence of the instant invention that codes for an

LPC acyltransferase may be grown. Seeds of the transgenic plants are harvested and fatty acids of the seeds are extracted. The extracted fatty acids are used for subsequent incorporation into a composition, for example, a pharmaceutical composition, a nutraceutical composition or a food composition. In certain embodiments, a peptide comprising one or more of the four motifs may be used as an LPC Acyltransferase. Similarly, a nucleotide sequence encoding a peptide comprising one or more of the four motifs may be used as an LPC Acyltransferase.

Further described herein is a lyso-PAF sensitivity screen to identify novel LPCAT. This is detailed in FIGs. 12 and 16. For example, a method of screening for an LPCAT, wherein the method comprises expressing a candidate gene in a yeast LPCAT mutant, plating the yeast on to lyso-PAF plates, and detecting yeast colonies showing higher tolerance to the lyso-PAF, wherein the colonies showing higher tolerance indicate that the candidate gene is a LPCAT gene hereof. The candidate gene may be identified by screening a gene to determine the presence of one of more of nucleic acid sequences encoding at least one motif selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, and any combination thereof.

In certain embodiments, other methods of enhancing or altering oil production may also be used with the plant to be transformed (e.g., incorporating, for expression in the plant, a nucleic acid sequence selected from the group consisting of a nucleic acid sequence encoding a peptide having, for example, Brassica pyruvate dehydrogenase kinase activity (see, e.g., U.S. Patent 7,214,859 to Marilla et al. (May 8, 2007), U.S. Patent 6,500,670 to Zou et al. (Dec. 2002), and U.S. Patent 6,256,636 to Randall et al. (July 2001), the contents of the entirety of each of which is incorporated herein by this reference), a nucleic acid sequence encoding a peptide having diacylglycerol acyltransferase activity (see, e.g., U.S. Patent 7,015,373 and U.S. Patent 6,500,670 to Zou et al. (Dec. 2002), the contents of the entirety of each of which is incorporated herein by this reference), a nucleic acid sequence encoding a peptide having glycerol-3-phosphate dehydrogenase activity (see, e.g., U.S. Patent 7,112,724, the contents of the entirety of which is incorporated herein by this reference), and combinations thereof). Also described is a method of transforming a cell or a plant, the method comprising introducing the isolated, purified or recombinant nucleic acid into the cell or plant. A process for producing a genetically transformed plant seed comprises introducing the nucleic acid into the plant seed. Also described is a vector comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ED NO:5, SEQ

ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and/or SEQ ID NO:34.

Also described is a vector comprising a nucleic acid sequence encoding a polypeptide having lyso-phosphatidylcholine acyltransferase activity, wherein the nucleic acid sequence comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34, or a fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO.16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34, or having 90% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ED NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34, wherein the fragment encodes the polypeptide having the lyso-phosphatidylcholine acyltransferase activity.

Also described is a method for increasing fatty acid production in a cell, the method comprising transforming a cell with a nucleic acid molecule encoding a lyso-phosphatidylcholine acyltransferase; and growing the cell under conditions wherein the lyso-phosphatidylcholine acyltransferase is expressed. The method can further comprise isolating the fatty acid. In such a method, the lyso-phosphatidylcholine acyltransferase preferably comprises at least one motif selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and any combination thereof.

Also described is a method of altering oil content in a plant comprising screening for a peptide encoded by a nucleotide sequence for at least one motif selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49; selecting the peptide based upon the presence of at least one of the four motifs; and expressing the nucleotide sequence encoding the peptide in the plant to alter the oil content of the plant.

Also described is a method of changing the oil content of a plant or plant seed, the method comprising introducing a nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 13, SEQ ID NO: 15, SEQ ED NO: 17; SEQ ID NO: 19, SEQ ID NO:21, SEQ LD NO:25, SEQ ID NO:27, SEQ ED NO:29, SEQ ED NO:31, SEQ ED NO:33, SEQ ED NO:35, and an amino acid sequence having at least 60% homology to any thereof having lyso-phosphatidylcholine acyltransferase activity into a plant transformation vector; transforming a genome of a plant or plant seed with the plant transformation vector; expressing the nucleic acid sequence; growing the plant or plant seed; and extracting the oil from the plant seed.

The methods can further comprise incorporating, for expression in the plant, a nucleic acid sequence selected from the group consisting of a nucleic acid sequence encoding a peptide having pyruvate dehydrogenase kinase activity, a nucleic acid sequence encoding a peptide having diacylglycerol acyltransferase activity, a nucleic acid sequence encoding a peptide having glycerol -3 -phosphate dehydrogenase activity, and any combination thereof.

The invention is further described with the aid of the following illustrative Examples.

EXAMPLES

Example 1: Identification of yeast LPC acyltransferase gene

Nucleotide sequences of nucleic acid molecules of the invention were identified through yeast genetic and functional screening. Yeast (S. cerevisiae) LPC acyltransferase gene was identified based on enzyme assays of yeast mutant strains in which the gene, YOR 175c, was knocked out. The enzyme activity was assessed using 14C-Iabeled acyl-CoA and

/vso-phosphatidylcholine. The reaction product of the /ysø-phosphatidylcholine and radio-labeled acyl-CoA was separated through TLC and measured through scintillation counting.

Deletion of the YOR175c gene in yeast resulted in a 90% reduction of LPC acyltransferase activity (FIG. 1). Therefore, YOR175c encodes LPC acyltransferase. Details are given below for the In Vitro Assay protocol for LPCAT (/ysø-phosphatidylcholine (LPC) acyltransferase) activity.

We designate YOR175c as LCAl in following description.

Gene expression vector construction: For TOPO TA-cloning and yeast complementation, Saccharomyces cerevisiae YORl 75c ORF was PCR-amplified with primers FP: 5' GGTGATTCTAGACTGCTGCTGATCGCTT 3' (SEQ ED NO:91) and RP: 5' GCATCTGTCGTTTTTGGAGCTCTACTCTT 3' (SEQ ED NO:92), and cloned into pYES2.1 vector (Invitrogen). Correctly oriented plasmids were identified by DNA sequencing and subsequently introduced into YOR175c mutant yeast strain Y02431. Microsomal preparation: Yeast strains were first grown in 15 ml of SC-Leu-His-Ura medium containing 2% glucose. Protein expression induction was carried out as described in Invitrogen manufacturer manual for yeast expression vector pYES2.1. After 24 hours of growth in SC+ 2% galactose+ 1% raffinose induction conditions, the cells were washed, first with distilled water and then with wall-breaking buffer (50 mM Tris-HCl, 1 mM EDTA, 0.6 M sorbitol, pH 7.4, 1 mM DTT). After centrifugation at 4,000 rpm (Eppendorf Centrifuge 5145C), the cells were resuspended in 1 ml wall-breaking buffer with 10 μl yeast protease cocktail (Sigma), and shaken vigorously in the presence of acid-washed glass beads (diameter 0.5 mm). The resultant homogenate was centrifuged at 12,000 rpm for ten minutes at 4⁰C. The decanted supernatant was further centrifuged at 100,000 Xg for 90-120 minutes at 4°C. The supernatant was discarded, and the pellet was suspended in homogenization buffer containing 20% glycerol and frozen at -80°C until use. Protein concentration was measured using Bio-Rad Protein Assay Kit for final enzyme activity calculation.

In Vitro Assay of LPCAT activity: LPCAT substrate specificity was determined by measuring incorporation of [¹⁴C] lysophosphatidylcholine or [¹⁴C] palmitoyl-CoA into phosphatidylcholine. All assays were performed at least twice. For lysophospholipid substrate specificity assessment, 400 μl HEPES buffer contained 3 μg microsomal protein, 50 μM of lysophospholipid substrates and 112.5 μM [¹⁴C] palmitoyl-CoA (5.5 nCi/nmol). For acyl-CoA substrate selectivity analysis, 400 μl HEPES reaction buffer (pH7.4, 0.1 M) contained 3 μg microsomal protein, 50 μM acyl-CoA and 112.5 μM [¹⁴C] palmitoyl-PC (1.35 nCi/nmol). Reaction was allowed for 2 minutes at 30°C with 100 rpm shaking. The reaction products were extracted with chloroform/methanol (2/1, v/v) and separated with Merck silica G60 TLC plates. Spots corresponding to different phospholipid species products were scraped off and ¹⁴C incorporation were scintillation counted. Different concentrations of ZnCl₂ were added in to reactions for Zn²⁺ inhibitory effect assay.

Table 1

Inhibitory effect of Zn 2+ on LCAl activity

ZnCl₂ concentration LPCAT activity (% control)

0 mM (control) 100 ± 7.9

20 mM 6 ± 2.0

0.1 mM 35 ± 22.4

25 μM 149.7 ± 12.0

10 μM 136.8. ± 3.9

5 μM 98 ± 5.9

Results are expressed as means ± S.D. The leal A over-expressing LCAl was used to assess Zn²⁺ effect. The reactions contained 5.6 μM palmitoyl-LPC (1.35 nCi/nmol), 1.5 μg microsomal proteins, 0.1 M HEPES (pH 7.4), 11.25 μM stearyl-CoA and indicated concentration of ZnCl₂. The reaction was stopped after two minutes by adding 2 ml of chloroform/methanol solution (2:1).

Table 2

Phosphatidylcholine turnover in leal A, slclA and BY4741 (WT) strains

28⁰C 37°C

Chase time (h): 0 1 0 1 lcalA Medium 12.1 ±1.7 16.2 ±1.1 17.4 ±2.7 12.1 ±1.7 13.2 ±3.5 13.3 ±1.5

Intracellular 41.1 ±3.2 47.8 ±2.8 53.6 ±1.5 41.1 ±3.2 67.9 ±2.7 73.5 ±2.5

Membrane 46.8 ±2.8 36.0 ± 2.3 28.9 ±1.5 46.8 ± 2.8 18.9 ± 3.4 13.2 ±2.3

slclA Medium 14.5 ±1.1 16.1 ±1.3 17.2 ±1.6 14.5 ±0.3 18.3 ±2.3 19.7 ±0.7

Intracellular 37.4 ±2.1 40.1 ±2.2 42.1 ±3.3 37.4 ±1.5 49.1 ±1.9 57.4 ±4.1

Membrane 48.1 ±1.7 43.8 ±2.6 40.7 ±1.7 48.1 ±0.9 32.6 ± 2.8 22.9 ±1.3

WT Medium 14.1 ±0.8 16.1 ±2.8 17.1 ±3.1 14.1 ±0.8 14.9 ±1.5 15.3 ±0.5

Intracellular 36.5 ±0.2 37.1 ± 1.6 41.6 ±0.1 36.5 ±0.2 55.9 ±2.1 60.7 ± 4.3

Membrane 49.4 ± 0.8 46.9 ±1.2 41.3 ±3.2 49.4 ± 0.8 29.2 ± 0.6 24.1 ±2.7

Yeast cells were labeled at starting OD600 = 1.5 for five hours in chemically defined synthetic medium containing 0.15 μCi/ml. The cells were then washed twice, cultured in medium containing 10 mM non-radioactive choline at 28°C and 37⁰C. One microliter culture aliquot was removed, separated into three factions then scintillation counted. The data were presented as mean of three analyses.

Lyso-PAF sensitivity: Yeast strains Y02431 over-expressing LCAl or harboring empty vector were first grown in 15 ml of SC-ura medium containing 2% glucose then transferred to SC-ura+2% galactose and 1% raffinose. After 12 hours LCAl expression induction, the culture was diluted to correspond to OD₆₀₀ value of 0.5, 1, 2, 3, 4. Five μl of each dilution was spotted to a YPD plate supplemented with varying concentrations of Lyso-PAF. The plates were incubated at 28°C for twp days.

PC turnover analysis: PC turnover analysis was performed according to previously described method [13] with slight modification. Briefly, Y02431 and BY4741 yeast cells were grown overnight in chemically defined synthetic media without inositol and choline. Yeast at OD₆oo=l-5 were used to inoculate fresh chemically defined synthetic media containing 0.15 μCi/ml [¹⁴C]choline chloride (20 μM). Cells were harvested through centrifugation after 5 hours labeling, washed twice in fresh non-radioactive medium, and then inoculated into in medium containing 10 mM non-radioactive choline. At different time points, 1 ml aliquots were removed and centrifuged. The supernatant was saved as the "medium" fraction. The cell pellet was suspended in 0.5 ml 5% trichloroacetic acid (TCA) and incubated on ice with frequent vortexing. Following centrifugation at 14,000 rpm (Eppendorf), the TCA-containing supernatant was decanted as "intracellular water-soluble fraction", and neutralized by adding 1 M Tris-HCl (pH 8.0) to avoid acid-induced luminescence in scintillation counting. The pellet was saved as the "membrane" fraction. The labeling of each fraction was measured and presented as percentage of total counts in all the three fractions. To confirm that the majority of choline-containing compounds in TCA fraction are glycerophosphorylcholine (GroPC), the fractions from WT and icalΔ yeast cells chased for two hours at 37°C were applied to Merck silica G60 gel and developed in solvent system methanol/0.5% NaCl/NH₃Η₂O (50/50/1, v/v/v)[14]. After drying, choline-containing chemicals on the plate were detected with scanner (Bioscan, Inc.) and only one major [¹⁴C]-labeled spot was clearly detected. The spot was scraped off and re-extracted into distilled water then concentrated with a vacuum refrigerator. The purified TCA fractions were spotted on Merck silica G60 plate with soluble choline-containing compound standards including GroPC, phosphocholine, CDP-choline, [¹⁴C] choline and 16:1-LPC, loaded on parallel lanes. The TLC plate was developed in the above-mentioned solvent system. [¹⁴C] choline and [¹⁴C] choline containing compound in TCA fractions were detected with scanner (Bioscan, Inc.), LPC was stained by iodine exposure, and other choline-containing standards were visualized by spraying molybdenum blue, which is specific to phosphorus present in GroPC, phosphocholine and CDP-choline [15]. Yeast culture: One colony each of wild-type (strain BY4741) and LPCAT mutants (YOR175c deletion strains Y12431, Y02431) are inoculated in 10 ml YPD media and grown overnight. After 24 hours, another 20 ml YPD media is added and growth is continued for another 24 hours.

Protein extraction: Yeast cultures are spun at 2800 rpm at 4⁰C for 20 minutes. The supernatant is discarded and the yeast pellet washed with 10 ml of ice cold IB buffer (80 mM HEPES, 320 mM sucrose, 5 mM EDTA pH 8, 10 mM KCl, 2 mM DTT). The pellets are spun again and re-suspend in 500 μl of IB buffer. Yeast cells are divided and transferred into two tubes appropriate for a mini-bead beater. 0.5 mm cold glass beads are added to fill completely the tube. To break the yeast cell, three 60-second pulses of the mini-bead beater are used. The mixtures are spun again to remove unbroken cells and debris.

Protein assay conditions: A reaction is conducted using the recipe for fatty-CoA substrate specificity, as listed in Table 3.

Table 3

The reaction mixture is allowed to sit in a water bath at 30°C and stirred at 100 rpm for 30 minutes. The reaction is then terminated by adding 2 ml of CH₂Cl₂: Isopropanol (1:2). The mixture is allowed to sit at room temperature for 15-30 minutes with occasional vortexing. Phases are separated by adding 2 ml CH₂Cl₂ followed by 2 ml IM KCl in H₃PO₄. The lower layer is transferred to a clean tube and the upper aqueous phase is backwashed twice with CH₂Cl₂ and centrifuged, saving the organic phase each time. Organic phases are combined and dried under nitrogen. Dried material is taken up in 200 μl CH₂Cl₂: MeOH (2:1) and protein is separated by thin layer chromatography (TLC) using silica G (250 μm) commercial plate. Plates are developed to within 2 cm of top in ethyl acetate:isooctane:acetic acid (45:15:10, V/V/V), then dried and scraped. The phosphatidyl choline region is counted in 4 ml Aquasol-2 by a scintillation counter.

The YOR 175c gene from S. cerevisiae has been identified as encoding an LPC acyltransferase. The coding sequence of this yeast LPC acyltransferase gene is SEQ ID NO:1: ATGTACAATCCTGTGGACGCTGTTTTAACAAAGATAATTACCAACTATGGGATTGATAGT TTTACACTGCGATATGCTATCTGCTTATTGGGATCGTTCCCACTGAATGCTATTTTGAAG AGAATTCCCGAGAAGCGTATAGGTTTAAAATGTTGTTTTATCATTTCTATGTCGATGTTT TACTTATTCGGTGTGCTGAATCTAGTAAGTGGATTCAGGACCCTGTTTATTAGTACCATG TTTACTTACTTGATCTCAAGATTTTACCGTTCCAAGTTTATGCCACACTTGAATTTCATG TTTGTTATGGGTCATTTGGCAATAAATCATATACACGCCCAATTCCTTAACGAACAGACT CAAACTACCGTTGACATTACAAGTTCACAAATGGTTTTAGCCATGAAACTAACTTCTTTT GCATGGTCGTACTATGATGGTTCATGCACTAGCGAAAGCGATTTCAAAGATTTGACTGAG CATCAAAAATCTCGTGCTGTCAGAGGTCATCCACCCTTATTAAAGTTCCTGGCATATGCA TTTTTCTATTCAACGTTGCTAACTGGCCCAAGTTTCGATTATGCCGATTTTGACAGCTGG TTGAATTGTGAGATGTTCCGTGACTTGCCTGAAAGCAAAAAGCCTATGAGAAGACACCAC CCTGGTGAAAGAAGACAGATTCCAAAGAATGGTAAACTTGCATTATGGAAAGTTGTTCAA GGTCTTGCTTGGATGATTTTAAGTACACTAGGAATGAAGCACTTCCCCGTAAAATACGTT TTGGACAAAGATGGCTTCCCAACGAGATCTTTTATATTCAGAATCCATTACTTATTCTTG CTTGGTTTCATCCATAGATTCAAGTACTACGCTGCCTGGACTATTTCGGAAGGATCTTGT ATTTTGTGCGGTTTGGGTTATAATGGTTATGATTCAAAGACACAAAAGATCAGATGGGAT CGTGTCAGAAATATTGACATTTGGACCGTAGAAACGGCGCAGAATACGCGTGAAATGTTG GAAGCATGGAATATGAATACTAACAAGTGGCTAAAATACTCTGTTTATTTACGTGTCACA AAGAAGGGCAAAAAACCTGGTTTCCGCTCAACTTTGTTTACTTTCCTAACTTCCGCATTT TGGCATGGTACCAGACCTGGGTACTATCTGACTTTTGCGACAGGGGCTTTGTACCAAACA TGTGGTAAAATCTACAGACGCAATTTTAGACCAATTTTCTTGCGAGAAGATGGTGTCACT CCTTTGCCTTCTAAAAAAATCTACGATTTAGTTGGCATATATGCAATTAAACTAGCATTT GGTTACATGGTGCAACCATTTATTATCCTTGATTTGAAGCCATCTTTAATGGTATGGGGC TCTGTTTATTTCTATGTTCATATTATTGTTGCTTTCTCATTTTTCCTATTCAGAGGACCA TATGCTAAACAAGTTACTGAATTTTTTAAATCCAAACAACCTAAAGAAATATTCATTAGA AAACAAAAGAAGTTGGAAAAAGATATTTCTGCAAGCTCTCCAAACTTGGGTGGTATATTG AAGGCAAAGATTGAACATGAAAAGGGAAAGACAGCAGAAGAAGAAGAAATGAACTTAGGT ATTCCACCAATTGAGTTAGAAAAGTGGGACAATGCTAAGGAAGATTGGGAAGATTTCTGC AAAGATTACAAAGAATGGAGAAATAAAAATGGTCTTGAAATAGAAGAGGAAAACCTTTCT AAAGCTTTTGAAAGATTCAAGCAGGAATTTTCTAACGCTGCAAGTGGATCAGGTGAACGT GTGAGAAAAATGAGTTTTAGTGGTTACTCACCAAAGCCTATTTCAAAAAAGGAAGAGTAG

The deduced amino acid sequence of the yeast LPC acyltransferase encoded by the gene ID NO:2: MYNPVDAVLTKIITNYGIDSFTLRYAICLLGSFPLNAILKRIPEKRIGLKCCFIISMSMF YLFGVLNLVSGFRTLFISTMFTYLISRFYRSKFMPHLNFMFVMGHLAINHIHAQFLNEQT QTTVDITSSQMVLAMKLTSFAWSYYDGSCTSESDFKDLTEHQKSRAVRGHPPLLKFLAYA FFYSTLLTGPSFDYADFDSWLNCEMFRDLPESKKPMRRHHPGERRQIPKNGKLALWKWQ GLAWMILSTLGMKHFPVKYVLDKDGFPTRSFIFRIHYLFLLGFIHRFKYYAAWTISEGSC ILCGLGYNGYDSKTQKIRWDRVRNIDIWTVETAQNTREMLEAWNMNTNKWLKYSVYLRVT KKGKKPGFRSTLFTFLTSAFWHGTRPGYYLTFATGALYQTCGKIYRRNFRPIFLREDGVT PLPSKKIYDLVGIYAIKLAFGYMVQPFIILDLKPSLMVWGSVYFYVHIIVAFSFFLFRGP YAKQVTEFFKSKQPKEIFIRKQKKLEKDISASSPNLGGILKAKIEHEKGKTAEEEEMNLG IPPIELEKWDNAKEDWEDFCKDYKEWRNKNGLEIEEENLSKAFERFKQEFSNAASGSGER VRKMSFSGYSPKPISKKEE

Deletion of YOR175cp leads to reduced lysophosphatidylcholine acyltransferase (LPCAT) activity: YOR175c is a MBOAT family protein, and was shown to be localized in endoplasm reticulum. In a preliminary experiment, we first examined if disruption of YORl 75c would have any impact on lysophosphatidic acid acyltransferase (LPAAT) and LPCAT activities using both the parental strain and slclΔ mutant as controls. When lysophosphatidic acid (18:1) was supplied as acyl acceptor, the cell lysate of slclΔ mutant had a LPAAT level reduced to 63% of the parental strain, but we detected no significant LPAAT reduction in the yorl75cΔ mutant. In marked contrast, when LPC was provided as acyl acceptor, our in vitro assay showed acyltransferase activity reduction in yorl75cA to a level approximately 28% of the parental strain. The slclΔ displayed no significant decrease in LPCAT activity as compared with WT strain (FIG. 9). We further investigated sn-2 lysophospholipid acyl transferase activity in yorl75cΔ by using microsomal enriched fractions with different lysophospholipid acyl acceptors and palmitoyl-CoA (16:0-CoA). In keeping with the results of total cell lysate, microsomal fractions of the yorl75cΔ strain showed a striking decrease in LPCAT activity. LPE and LPG acyltransferase were also slightly decreased, but to a much lesser degree (FIG. 10).

YOR175cp displays in vitro acyltransferase activity with preference for LPC: Microsomal preparations of icalΔ mutant expressing YORl 75c and icalΔ harboring the empty vector (VO) were used to perform acyltransferase assays with [¹⁴C] palmitoyl-CoA and various lysophospholipids substrates including LPA, LPC, LPE, LPG, LPI and LPS. As shown in FIG. 11, the highest activity was found with LPC as substrate. The activity of LPC acylation was linear at 30°C for 20 minutes, and the conversion of LPC to PC is negligible in the absence of 16:0-CoA (data not shown). Over-expression of YORl 75c also caused substantial increases in the acylation of LPG and LPE. But the rates of LPG and LPE acylation were at a level .

-20-

approximately 60% and 20%, respectively, of the activity registered for LPC. Activities for LPA, LPS and LPI, were all less than 1% of the activity of LPCAT. Thus, YOR175c appeared capable of accepting several major lysophospholipid classes, but under our assay conditions it exhibited the highest activity with LPC. Correlation of YOR175c LPCAT activity with Lyso-PAF sensitivity: Although not an endogenous acyl acceptor, ether-linked glycerolipid, lyso-PAF, can be acylated in yeast, and the reaction was attributed to a LPCAT. When lyso-PAF was used as acyl acceptor, the icalΔ strain had a rate of lyso-PAF acylation reduced to 31.1 % of WT strain. Conversely, over-expression of LCAl resulted in 86.3-fold increase in lyso-PAF acyltransferase activity. It was established previously that high lyso-PAF level exerts toxic effect on yeast cells. Consistent with in vitro results, LCAl mediating Lyso-PAF acylation was also evident in a plate assay (FIG. 12). In our study, both the parental strain and the IcalΔ were capable of tolerating LPC at a level up to 20 μg/ml, but the IcalΔ mutant displayed hypersensitivity to lyso-PAF at a concentration above 5 μg/ml. Moreover, its sensitivity to lyso-PAF was ameliorated by the expression of LCAl. In contrast, slclΔ strain could survive and grew well on lyso-PAF plate without any apparent difference from WT cells, indicating SLCl disruption did not affect lyso-PAF acylation.

Zn²⁺ inhibitory effect on LPCAT activity: Zn²⁺ caused significant reduction of LPCAT activity of LCAl in a range between 0.1 mM to 20 mM (Table 1). Our results also suggested that a lower (10-25 μM) concentration of Zn²⁺ enhanced LPCAT activity. The maximum increase was observed with 25 μM ZnCl₂. We did not detect significant effect of Mg²⁺ on LPCAT activity of LCAl, in a concentration ranging from 5 to 40 μM (data not shown).

Kinetic parameters of , LCAl: Kinetics constants based on Lineweaver-Burk double-reciprocal plot analysis showed that LCAl had an apparent Km for acyl-CoA at 0.89 + 0.25 μM and a Vmax of 524 pmol/min/μg protein. PC molecules are distinguished by fatty acid chain length. As shown in FIG. 13 (in graph A), LCAl exhibited a LPC substrate preference in the order of oleic (18:1)-LPC> stearic (18:0)-LPC> palmitic (16:0)-LPC. The fatty acid substrate specificity of the LCAl was also assessed using acyl-CoA with chain lengths ranging from 14 to 22 carbons. Based on assays using 50 μM acyl-CoAs, LCAl could use a broad range of acyl-CoAs (FIG. 13, in graph B), but it displayed particularly high activities with 16:0-CoA, 18.O-CoA andl8:l-CoA, regardless whether 18:1-LPC or 16:0-LPC was used as acyl acceptor. Interestingly, LCAl could also efficiently mediate LPC acylation using very long chain fatty acyl-CoAs, such as 20:0-CoA and 22:6-CoA. LPC (16:0) at concentrations above 75 μM, and acyl-CoA at higher than 10 μM, exerted inhibitory effects on LPCAT activity (data not shown). LCAl is involved in PC turnover: We studied PC turnover by following an established protocol [13]. We included a slclΔ strain in the PC turnover analysis in order to differentiate the involvement of SLCl and LCAl. The yeast cells were cultured and labeled in chemically defined synthetic medium containing [¹⁴C] choline at 28°C. Because higher growth temperature particularly accelerates the deacylation process [13], [¹⁴C] choline was subsequently chased by 10 mM exogenously added choline at 28°C and 37°C, respectively. The ¹⁴C labels in the membrane fraction, intracellular non-membrane fraction, and in the medium were monitored at different time points. There was no significant difference with regard to the dynamics of membrane-associated labels between slclA and WT. Each lost about 8% at 28°C, and 25% at 37°C of labeling, in the membrane fraction over the course of 2 hours. In contrast, the icalΔ strain lost 18% at 28°C, and 33% at 37°C, over the same period of time. The label was rising in the intracellular, non-membrane fraction, which was suggested to be of mainly glycerophosphorylcholine (GroPC) [13, 24], a product of PC deacylation. We attempted to separate the compounds in TCA fraction on Merck silica G60 plate and found only one [¹⁴C]-choline band detected. We then purified the choline-containing compound in TCA fractions and developed on the same TLC plate with commercial choline-containing chemical standards. The compound clearly showed the same migration rate as GroPC (FIG. 14). Since an increased GroPC level was observed in both 28°C and 37°C, these results suggested that the metabolic impact was independent of PC deacylation, therefore strongly suggesting that IcalΔ was compromised in the reacylation process of the Lands' cycle. That slclΔ had a similar PC turnover rate to that of the WT strain indicated that, although being a major sn-2 acyltransferase, SLCl did not appear to play a significant role in PC turnover.

Example 2: Identification of plant nucleotide sequences encoding LPC acyltransferase The nucleotide sequence of the yeast LPC acyltransferase gene was used to search for homologous sequences using computer programs designed to search for homologous sequences.

For instance, readily commercially available computer programs that may be used for such searches include without limitation, BLASTN, BLASTX and TBLASTX which may be used to search for nucleotide sequences, and BLASTP and TBLASTN which may be used to search for amino acid sequences. Such computer programs are readily accessible at the web-site

WorldWideWeb.ncbi.nlm.nih.gov.

Two plant (A thaliana) homologs were identified through sequence alignment searching using BLAST. The two homologs are cDNA sequences that encode two different isoforms of

LPC acyltransferase. Arabidopsis LPC acyltransferase 1

Nucleotide sequence of Arabidopsis LPC acyltransferase 1 cDNA is SEQ ID NO:3:

1 ACCAACAACC ACACGACACG ACACGACCGA TCTATAGATT CGGCGAGATC 51 AGAAGAAAGC TTCCCGGAGC AACTCGGTCG TTGTGACTCA TTCCGAGTTA

101 AAAAAAACGG GTTTTCGACA CCATGGATAT GAGTTCAATG GCTGGTTCAA

151 TCGGAGTTTC GGTAGCCGTA CTCCGATTCC TCCTCTGTTT CGTTGCCACG

201 ATCCCTGTTT CATTCGCTTG TCGAATCGTC CCGAGTAGAC TCGGTAAACA

251 CTTGTATGCC GCTGCTTCAG GTGCTTTCCT CTCTTACCTC TCCTTTGGCT 301 TCTCCTCCAA CCTTCACTTC CTTGTTCCGA TGACGATCGG ATATGCTTCA

351 ATGGCGATTT ATAGACCCAA GTGTGGAATC ATCACTTTCT TCCTCGGTTT

401 CGCTTATCTT ATTGGCTGTC ATGTGTTTTA TATGAGTGGT GATGCGTGGA

451 AAGAAGGAGG AATCGATTCT ACTGGAGCGT TAATGGTGTT GACGCTGAAA

501 GTCATCTCAT GTTCAATGAA TTACAATGAT GGGATGTTGA AGGAGGAAGG 551 TCTACGTGAA GCTCAGAAGA AAAACAGATT GATTCAGATG CCGTCTTTGA

601 TTGAGTACTT TGGTTACTGC CTTTGTTGTG GTAGCCATTT TGCTGGTCCT

651 GTTTATGAAA TGAAAGATTA TCTTGAATGG ACCGAAGGGA AAGGGATTTG

701 GGATACTACT GAGAAAAGAA AGAAGCCATC GCCTTATGGA GCTACAATCC

751 GAGCTATTTT GCAAGCTGCG ATTTGCATGG CTCTGTATCT CTATTTAGTG 801 CCTCAATATC CGTTAACTCG GTTCACAGAA CCAGTGTATC AAGAATGGGG

851 ATTCTTGAGA AAATTTAGTT ACCAATACAT GGCTGGATTC ACGGCTCGTT

901 GGAAGTATTA CTTCATCTGG TCAATTTCAG AGGCTTCTAT TATCATCTCT

951 GGTTTGGGTT TCAGTGGTTG GACTGATGAT GCTTCACCAA AGCCCAAATG

1001 GGACCGTGCC AAGAACGTAG ATATTCTCGG TGTTGAACTA GCTAAGAGCG 1051 CGGTTCAGAT TCCACTTGTG TGGAACATAC AAGTCAGCAC GTGGCTCCGT

1101 CACTATGTGT ATGAGAGACT TGTGCAGAAC GGAAAGAAAG CGGGTTTCTT

1151 CCAGTTACTA GCTACACAAA CCGTCAGCGC GGTTTGGCAT GGACTGTATC

1201 CTGGATATAT GATGTTCTTT GTTCAGTCAG CTTTGATGAT CGCAGGCTCA

1251 CGGGTTATTT ACCGGTGGCA ACAAGCGATC AGTCCGAAAA TGGCAATGCT 1301 GAGAAATATA ATGGTCTTCA TCAACTTCCT TTACACTGTT TTGGTTCTCA

1351 ACTACTCAGC CGTCGGTTTC ATGGTGTTAA GCTTGCACGA AACACTTACC 1401 GCCTACGGAA GCGTATATTA CATTGGAACA ATCATACCTG TTGGATTGAT 1451 TCTCCTCAGT TACGTTGTGC CTGCAAAACC TTCAAGACCA AAACCGCGTA 1501 AAGAAGAATA AGCAGTTATC TTCTTCTCTT AACGGTAAGT AAGTTTCCCG 1551 CGCTTGCCAG CTTCTTCTTC TTCTTCTGTA ACATTTGGAA ACAAACCGAT 1601 CCGGTTCTTG TTTCTCTCTG ATTTTTTAGC ACCGATATTT TTTTTGTATT

1651 TGTTGCTTAT AAATCTTATT TTTCACACTT CTTTTTTTTA ATTAGTATTG

1701 GATTTGCAAT TATATAGACA ATAAGTATAA ATATGTAACT GTAAATTGCA

1751 AATGGGAAAA AATAGTAGTG TTTATGTTTG

The deduced amino acid sequence of Arabidopsis LPC acyltransferase 1 is SEQ ID NO:4:

1 MDMSSMAGSI GVSVAVLRFL LCFVATIPVS FACRIVPSRL GKHLYAAASG

51 AFLSYLSFGF SSNLHFLVPM TIGYASMAIY RPKCGIITFF LGFAYLIGCH

101 VFYMSGDAWK EGGIDSTGAL MVLTLKVISC SMNYNDGMLK EEGLREAQKK 151 NRLiQMPSLi EYFGYCLCCG SHFAGPVYEM KDYLEWTEGK GIWDTTEKRK

201 KPSPYGATIR AILQAAICMA LYLYLVPQYP LTRFTEPVYQ EWGFLRKFSY 251 QYMAGFTARW KYYFIWSISE ASIIISGLGF SGWTDDASPK PKWDRAKNVD 301 ILGVELAKSA VQIPLVWNIQ VSTWLRHYVY ERLVQNGKKA GFFQLLATQT 351 VSAVWHGLYP GYMMFFVQSA LMIAGSRVIY RWQQAISPKM AMLRNIMVFI 401 NFLYTVLVLN YSAVGFMVLS LHETLTAYGS VYYIGTIIPV GLILLSYWP 451 AKPSRPKPRK EE

Arabidopsis LPC acyltransferase 2

Nucleotide sequence of Arabidopsis LPC acyltransferase 2 cDNA is SEQ ID NO:5: 1 AGATGTCCGA ACTGTGAGAG TCGTCGTCGT CGTCGTAACT CAGTCCGAGT

51 TGACACAATC TTCCACTTCA CGCAAGATAC AACCATGGAA TTGCTTGACA

101 TGAACTCAAT GGCTGCCTCA ATCGGCGTCT CCGTCGCCGT TCTCCGTTTC

151 CTCCTCTGTT TCGTCGCAAC GATACCAATC TCATTTTTAT GGCGATTCAT

201 CCCGAGTCGA CTCGGTAAAC ACATATACTC AGCTGCTTCT GGAGCTTTCC 251 TCTCTTATCT CTCCTTTGGC TTCTCCTCAA ATCTTCACTT CCTTGTCCCA 301 ATGACGATTG GTTACGCTTC AATGGCGATT TATCGACCCT TGTCTGGATT 351 CATTACTTTC TTCCTAGGCT TCGCTTATCT CATTGGCTGT CATGTGTTTT 401 ATATGAGTGG TGATGCTTGG AAAGAAGGAG GAATTGATTC TACTGGAGCT 451 TTGATGGTAT TAACACTGAA AGTGATTTCG TGTTCGATAA ACTACAACGA 501 TGGAATGTTG AAAGAAGAAG GTCTACGTGA GGCTCAGAAG AAGAACCGTT 551 TGATTCAGAT GCCTTCTCTT ATTGAGTACT TTGGTTATTG CCTCTGTTGT 601 GGAAGCCATT TCGCTGGCCC GGTTTTCGAA ATGAAAGATT ATCTCGAATG 651 GACTGAAGAG AAAGGAATTT GGGCTGTTTC TGAAAAAGGA AAGAGACCAT 701 CGCCTTATGG AGCAATGATT CGAGCTGTGT TTCAAGCTGC GATTTGTATG 751 GCTCTCTATC TCTATTTAGT ACCTCAGTTT CCGTTAACTC GGTTCACTGA 801 ACCAGTGTAC CAAGAATGGG GATTCTTGAA GAGATTTGGT TACCAATACA

851 TGGCGGGTTT CACGGCTCGT TGGAAGTATT ACTTTATATG GTCTATCTCA

901 GAGGCTTCTA TTATTATCTC TGGTTTGGGT TTCAGTGGTT GGACTGATGA

951 AACTCAGACA AAGGCTAAAT GGGACCGCGC TAAGAATGTC GATATTTTGG 1001 GGGTTGAGCT TGCCAAGAGT GCGGTTCAGA TTCCGCTTTT CTGGAACATA

1051 CAAGTCAGCA CATGGCTCCG TCACTACGTA TATGAGAGAA TTGTGAAGCC

1101 CGGGAAGAAA GCGGGTTTCT TCCAATTGCT AGCTACGCAA ACCGTCAGTG

1151 CTGTCTGGCA TGGACTGTAT CCTGGATACA TTATATTCTT TGTGCAATCA

1201 GCATTGATGA TCGATGGTTC GAAAGCTATT TACCGGTGGC AACAAGCAAT 1251 ACCTCCGAAA ATGGCAATGC TGAGAAATGT TTTGGTTCTC ATCAATTTCC

1301 TCTACACAGT AGTGGTTCTC AATTACTCAT CCGTCGGTTT CATGGTTTTA

1351 AGCTTGCACG AAACACTAGT CGCCTTCAAG AGTGTATATT ACATTGGAAC

1401 AGTTATACCT ATCGCTGTGC TTCTTCTCAG CTACTTAGTT CCTGTGAAGC

1451 CTGTTAGACC AAAGACCAGA AAAGAAGAAT AATGTTGTCT TTTTAAAAAA 1501 TCAACAACAT TTTGGTTCTT TTCTTTTTTT CCACTTGGAC CGTTTTATGT

1551 AAAACAAGAG AAATCAAGAT TTGAGGTTTT ATTCTTCTTC TCCTTCCCAA

1601 TTTTCGAAAA TGATTTTATT TTTTCTGATA TATATCTAAG CTAGTCCAAA

1651 GTCAACTCG

The deduced amino acid sequence of Arabidopsis LPC acyltransferase 2 is SEQ ID NO:6:

1 MELLDMNSMA ASIGVSVAVL RFLLCFVATI PISFLWRFIP SRLGKHIYSA

51 ASGAFLSYLS FGFSSNLHFL VPMTIGYASM AIYRPLSGFI TFFLGFAYLI

101 GCHVFYMSGD AWKEGGIDST GALMVLTLKV ISCSINYNDG MLKEEGLREA

151 QKKNRLIQMP SLIEYFGYCL CCGSHFAGPV FEMKDYLEWT EEKGIWAVSE 201 KGKRPSPYGA MIRAVFQAAI CMALYLYLVP QFPLTRFTEP VYQEWGFLKR 251 FGYQYMAGFT ARWKYYFIWS ISEASIIISG LGFSGWTDET QTKAKWDRAK 301 NVDILGVELA KSAVQIPLFW NIQVSTWLRH YVYERIVKPG KKAGFFQLLA 351 TQTVSAVWHG LYPGYIIFFV QSALMIDGSK AIYRWQQAIP PKMAMLRNVL 401 VLINFLYTW VLNYSSVGFM VLSLHETLVA FKSVYYIGTV IPIAVLLLSY 451 LVPVKPVRPK TRKEE

AtLPCATl and AtLPCAT2 lysophospholipid acyltransferase activity was in vitro assessed with various lysophospholipid including lysophosphatidic acid ("LPA"),

^phosphatidylcholine ("LPC"), lysophosphatidylethanolamine ("LPE"), lysophosphatidylinositol ("LPI"), lysophosphatidylglycerol ("LPG"), lyso-platelet-activating factor as acyl group acceptor and [¹⁴C]-palmitoyl-CoA as acyl group donor. Results clearly showed that lysophosphatidylcholine and lyso-platelet-activating factor were the most preferred lysophospholipid substrates (FIG. 15). Preference of LPCATl and LPCAT2 towards lyso-platelet-activating factor was also evidenced by lyso-PAF plate test (FIG. 16). Acyl-CoA preference of AtLPCATl and AtLPCAT2 was assessed in vitro with various acyl-CoA species as acyl-group donor and [¹⁶C]-palmitoyl-₁sn2-lysophosphatidylcholine as acyl-group acceptor. AtLPCATl preferred monounsaturated 16:1 and 18:l-acyl-CoA followed by 16:0 and 18:0-acyl-CoA. AtLPCAT2 similarly preferred 16:0, 16:1, 18:0 and 18:l-CoA. Comparatively, both AtLPCATs discriminated against 18:2 acyl-CoA as acyl group donor (FIG. 17).

AtLPCATl and AtLPC AT2 preferences towards LPC species of different chain length were in vitro assessed with lysophosphatidylcholine of various chain lengths as acyl-group acceptor and [¹⁴C]-palmitoyl-CoA as acyl-group donor. AtLPCATl and AtLPCAT2 both preferred 16:0 and 18:l-lysophosphatidylcholine (FIG. 18). Zn²⁺ sensitivity of AtLPCATs was investigated, activities of both AtLPCATs decreased with increasing concentration of Zn²⁺ added into in vitro assay reactions (FIG. 19).

Example 3: Transformation of a plant with LPC acyltransferase gene

Transformation protocol is adapted from that described by Bechtold et al. (1993). Plants are grown in moist soil at a density of 10-12 plants per pot, in 4-inch square pots, and are covered with a nylon screen fixed in place with an elastic band. When the plants reach the stage at which bolts emerge, plants are watered, the bolts and some of the leaves are clipped, and the plants are infiltrated in Agrobacterium suspension as outlined below.

Agrobacterium transformed with the LPC acyltransferase gene of the instant invention is grown in a 25 mL suspension in LB medium containing kanamycin at a concentration of 50 μg/mL. The Agrobacterium is cultured for two to three days. The day before infiltration, this "seed culture" is added to 400 mL of LB medium containing 50 μg/mL kanamycin. When the absorbance at 600 nm is >2.0, the cells are harvested by centrifugation (5,000 times g, ten minutes in a GSA rotor at room temperature) and are re-suspended in 3 volumes of infiltration medium (one times Murashige and Skoog salts, one times, B5 vitamins, 5.0% sucrose, 0.044 μM benzylaminopurine) to an optical density at 600 nm of 0.8. The Agrobacterium suspension is poured into a beaker and the potted plants are inverted into the beaker so that the bolts and entire rosettes are submerged. The beaker is placed into a large Bell jar and a vacuum is drawn using a vacuum pump, until bubbles form on the leaf and stem surfaces and the solution starts to bubble a bit, and the vacuum is rapidly released. The necessary time and pressure vanes from one lab setup to the next; but good infiltration is visibly apparent as uniformly darkened, water-soaked tissue. Pots are removed from the beaker, are laid on their side in a plastic tray and are covered with a plastic dome, to maintain humidity. The following day, the plants are uncovered, set upright and are allowed to grow for approximately four weeks in a growth chamber under continuous light conditions as described by Katavic et al. (1995). When the siliques are mature and dry, seeds are harvested and selected for positive transformants.

Example 4: Selection of putative transformants (transgenic plants) and growth and analysis of transgenic plants

Seeds are harvested from vacuum-infiltration transformation procedures, and are sterilized by treating for one minute in ethanol and five minutes in 50% bleach/0.05% Tween™ 20™ in sterile distilled water. The seeds are rinsed several times with sterile distilled water. Seeds are plated by re-suspending them in sterile 0.1% agarose at room temperature (about 1 mL agarose for every 500-1000 seeds), and applying a volume equivalent to about 2,000-4,000 seeds onto 150x15 mm selection plates (l/2xMurashige and Skoog salts, 0.8% agar, autoclave, cool and add lxB5 vitamins and kanamycin at a final concentration of 50 μg/mL). The plates are dried in a laminar flow hood until seed no longer flows when the plates are tipped. The plates are vernalized for two nights at 4⁰C in the dark, and are moved to a growth chamber (conditions as described by Katavic et al., 1995). After seven to ten days, transformants are clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend over and into the selective medium.

Seedlings are transplanted to soil, plants are grown to maturity and mature seeds (T₂ generation as defined in Katavic et al., 1994) are collected and analyzed. T₂ seeds are propagated. The vegetative growth patterns are monitored by measuring shoot tissue dry weights, and/or by counting the number of rosette leaves present by the time plants began to enter the generative (flower initiation) stage. Floral initiation (beginning of generative phase of growth) is analyzed by recording, on a daily basis, the percentage of plants in which a flower bud first appears and/or the percentage of plants that are bolting (as described by Zhang et al., 1997). Data are reported in terms of percentage of plants flowering/bolting on a given day after planting (d.a.p.). Example 5: Analysis of fatty acids

Cells or plants transformed with the LPC acyltransferase gene of the instant invention are grown to maturity and mature seeds are harvested. Fatty acids are extracted from the cells or plants transformed with the LPC acyltransferase gene. Normal-phase HPLC analysis is used to assay for the production of fatty acids in the transformed cells or plants.

Example 6: Analysis of LPCAT from Various Species

(1) Identification of LPCAT from the alga Thalassiosira pseudonana We made use of the sequence information of LPCAT from S. cerevisiae (SEQ ID NO:1) and identified a sequence coding for LPCAT from the alga T. pseudonana. This algal LPCAT shows 27% identity at the amino acid to the yeast LPCAT which is encoded by YOR175c.

The nucleotide and amino acid sequences of LPCAT from T. pseudonana

(a) The nucleotide sequence of LPCAT from the alga T. pseudonana ATGCGATTGTATTTGCAATTCAACTTATCCATCAATGATTATTGTCACTTCTTC

ACAGTACCATCCTTTGTCAAAGAGGGCGTCGAGTCTCTCTCTGCATCCACCGGACAA GACGTCGAGACTCTCGAGTACCTCCTTGGTATGCTCATCTGCTACCCCCTCGGAATG ATCATGCTCGCTCTACCCTACGGAAAAGTAAAACATCTCTTCTCCTTCATCCTCGGAG CCTTCCTACTTCAATTCACCATTGGTATCCAGTGGATTCATCACTTAATCTCCTCAAT GATTGCCTACGTCATGTTCCTCGTCCTTCCTGCCAAATTTGCCAAAACGGCAGTGCCT GTGTTTGCCATGATCTACATCACCGCGGGACATTTGCATCGTCAATACATCAATTATC TTGGGTGGGATATGGACTTCACGGGGCCTCAGATGGTGCTTACGATGAAACTCTACA TGCTTGCTTACAACCTTGCGGATGGGGACTTGCTCAAGAAGGGAAAGGAGGATAGG GCTGCAAAGAAGTGTGCGGATGTCGCTATTTCGTCTGTTCCCGGAATCATTGAGTAC TTGGGCTACACGTTCTGCTTTGCCAGTGTTTTAGCAGGCCCTGCTTTTGAGTACAAAT TCTACGCCGATGCATGCGACGGATCACTCTTGTACGACAAATCTGGCAAACCCAAAG GAAAGATCCCCAGTCAGGTGTGGCCTACATTGCGTCCTCTTTTTGGAAGTCTCTTGTG TCTCGGCATCTTTGTTGTGGGAACTGGAATGTATCCTCTTTTGGATCCCAACGATCCT CAGAATGCCACTCCTATCCCTCTCACTCCAGAGATGTTGGCCAAACCAGCCTATGCT CGATACGCTTACTCGTGGCTTGCACTCTTTTTCATCCGATTTAAGTATTACTTTGCTTG GATGAACGCCGAAGGAGCAAGCAACATTTGGTATGCTGGATTTGAGGGATTTGATG CCAGCGGCAACCCCAAAGGATGGGAGGTATCCAATAACATTGACGTAATTCAGTTC GAGACTGCACCCAATCTCAAGACTTTGAGTGCTGCTTGGAATAAGAAGACTGCGAAC TGGTTGGCGAAGTATGTGTACATTCGCACGGGTGGTTCTCTCTTTGCGACGTACGGA ATGAGTGCTTTCTGGCATGGCTTCTACCCTGGATACTACCTCTTCTTCATGTCGGTAC CCATGATGGCTTTCTGTGAGAGGATTGGAAGGAAGAAACTTACACCTCGTTTCGGAA ATGGAAAGAAGTGGAGTCCTTATGGCATTGTGTGCATTATCGCCACATCGTTGATGA CGGAATACATGATTCAGCCATTCCAACTACTTGCGTTTGATTGGGCCTGGGAGAACT GGAGCAGCTACTACTTTGCTGGACACATTGTTTGTGTTGTGTTTTACCTCGTTGTGTC CAACATGCCTACACCAAAGACGAAGGAGACTTAA (SEQ ID NO:7)

(b) The amino acid sequence of LPCAT from T. pseudonana MRLYLQFNLSIND YCHFFTVPSFVKEGVESLSASTGQDVETLEYLLGMLICYPLG MIMLALPYGKVKHLFSFILGAFLLQFTIGIQWIHHLISSMIAYVMFLVLPAKFAKTAVPVF AMIYITAGHLHRQYINYLGWDMDFTGPQMVLTMKLYMLAYNLADGDLLKKGKEDRA AKKCADVAISSVPGIIE YLG YTFCFASVLAGPAFEYKFY ADACDGSLL YDKSGKPKGKIP SQVWPTLRPLFGSLLCLGIFVVGTGMYPLLDPNDPQNATPIPLTPEMLAKPAYARYAYS WLALFFIRFKYYFAWMNAEGASNIWYAGFEGFDASGNPKGWEVSNNIDVIQFETAPNL KTLSAAWNKKTANWLAKYVYIRTGGSLFATYGMSAFWHGFYPGYYLFFMSVPMMAFC ERIGRKKLTPRFGNGKKWSPYGIVCIIATSLMTEYMIQPFQLLAFDW A WENWSSYYFAG HIVCVVFYLVVSNMPTPKTKET (SEQ ID NO: 8)

(2) Identification of LPCAT from diverse plant species Taking the same approach as described above, identified were the full-length or partial sequences of LPCAT from various plant species, including apple, barley, Capsicum annuum, castor bean, grapevine, maize, peach, rice, tomato, snapdragon, sorghum, sunflower, vaccinium corymbosum and wheat as well as Arabidopsis.

(a) The partial nucleotide sequence of LPCAT from apple

TCAGGAGGCCCAAATTTCCTTTGTCAAGATTTACTGAGCCCATATACCAAGAA TGGGGGTTTTGGAAACGACTTTTCTACCAGTATATGTCTGGATTCACAGCAAGGTGG AAATATTATTTCATTTGGTCAATATCAGAGGCTTCTATCATTCTTTCTGGCCTCGGTTT CAGTGGCTGGACAGAGTCCTCACCACCAAAACCTCGATGGGATCGTGCAAAAAATG TTGATATTATAGGCGTTGAGTTTGCAAAGAGTTCAGTTCAGTTACCACTTGTTTGGAA CATACAAGTCAGCACCTGGCTTCGCCATTATGTTTATGATAGGCTTGTTAAACCTGG AAAGAAGCCTGGTTTCTTCCAGTTGCTGGCTACACAGACCGTCAGTGCTGTTTGGCA TGGCCTCTATCCTGGCTACATCATATTCTTTGTTCAGTCAGCGTTGATGATTGCTGGA TCAAGAGTGATTTACCGATGGCAGCAAGCTGTACCTCCAACTATGGATGTTGTTAAG AAGATATTGGTGTTCATCAACTTTGCTTACACTGTCTTGGTTCTGAACTACTCCTGTG TTGGTTTCATTGTATTAAGCCTTCGTGAAACACTGGCCTCGTATGGAAGCGTGCATTT C (SEQ ID NO:9)

The partial amino acid sequence of LPCAT from apple

RRPKFPLSRFTEPIYQEWGFWKRLFYQYMSGFTARWKYYFIWSISEASIILSGLGFS GWTESSPPKPRWDRAKNVDIIGVEFAKSSVQLPLVWNIQVSTWLRHYVYDRLVKPGKK PGFFQLLATQTVSAVWHGLYPGYIIFFVQSALMIAGSRVIYRWQQAVPPTMDVVKKILV FINFAYTVLVLNYSCVGFIVLSLRETLASYGSVHF (SEQ ID NO: 10)

(b) The partial amino acid sequence of LPCAT from barley EAAIIISGLGFTGWSDSSPPKAKWDRAINVDILGVELAGSAAQLPLKWNIQVSTWL

RYYVYERLIQKGKKPGFLQLLGTQTVSAIWHGLYPGYMIFFVQSALMINGSKVIYRWQQ AVKQFRPPHYPVFTKLLHTP (SEQ ID NO: 11)

(c) The partial nucleotide sequence of LPCAT from Capsicum annuum GGCACGAGAAACGGTTGGGTTACCAATATATGGCTGGCTTTACTGCCCGGTG

GAAGTATTATTTTATCTGGTCAATCTCTGAAGCTGCTATAATCATATCTGGACTGGGT TTCAGTGGTTGGACAGACTCTTCTCCGCCAAAACCACGTTGGGACCGTGCAAAAAAT GTTGATGTATTGGGTGTTGAGTTAGCAAAGAGCTCGGTTCAGTTGCCTGCTGTCTGG AACATTCAAGTCAGCACATGGCTGCGGCATTATGTATATGAAAGGCTCATACAAAAG GGAAGGAAGCCTGGTTTCTTCCAGTTACTGGCTACCCAAACTGTCAGTGCCGTATGG CATGGATTATATCCTGGGTATATCATATTCTTTGTACAGTCCGCTTTGATGATTGCTG GATCAAGAGTCCTTTACAGATGGCAGCAAGCTGCTAAAGGTTCTATGTTTGAGAAGA TACTGGTAGCAATGAATTTTGCATACACACTGCTGGTTCTAAATTACTCCGCTGTTGG GTTCATGGTATTAAGCCTGCATGAAACTCTTACTGCTTATGGAAGTGTATACTATGTT GGAACAATTATACCAATTGCTCTCATCCTGCTCAGTAAAGTAATTAAGCCTCCAAGA CCCTGCACATCTAAAG (SEQ ID NO: 12)

The partial amino acid sequence of LPCAT from Capsicum annuum

HEKRLGYQYMAGFTARWKYYFIWSISEAAIIISGLGFSGWTDSSPPKPRWDRAKN VDVLGVELAKSSVQLPAVWNIQVSTWLRHYVYERLIQKGRKPGFFQLLATQTVSAVWH GLYPGYIIFFVQSALMIAGSRVLYRWQQAAKGSMFEKILVAMNFAYTLLVLNYSAVGF MVLSLHETLTAYGSVYYVGTIIPIALILLSKVIKPPRPCTSK (SEQ ID NO: 13) (d) The partial nucleotide sequence of LPCAT from castor bean ATTCATTTATACTTGGTGCCCCACTATCCTTTATCCCGGTTCACTGATCCTGTG TACCAAGAATGGGGCTTCTGGAAACGATTAACTTATCAGTATATGTCAGGTTTAACA GCACGTTGGAAATACTACTTCATCTGGTCAATTTCCGAGGCCTCCATTATTATCTCTG GATTGGGTTTCAGTGGTTGGACAGATACTTCTCCACCAAAGCCACAGTGGGATCGCG CTAGAAACGTTGACATTCTAGGTGTTGAGTTTGCAAAGAGTGCAGCTGAGTTGCCAC TTGTGTGGAACATACAAGTCAGCACATGGCTTCGCCACTATGTTTATGATCGACTTGT TCCAAAGGGAAAGAAAGCTGGTTTCCTTCAGTTGTTGGCCACTCAGACTACCAGTGC TGTTTGGCATGGATTATATCCTGGATACATTATATTCTTTGTCCAGTCAGCATTAATG ATTGCAGGTTCGAAAGTCATATACAGATGGCAACAAGCTATACCTTCAAATAAGGCT CTTGAAAAGAAGATACTAGTGTTTATGAACTTTGCTTACACAGTTTTGGTTCTAAATT ACTCCTGTGTTGGTTTCATGGTTTTAAGCTTGCATGAAACGATTGCAGCATATGGAA GTGTATATTTTATTGGCACCATAGTGCCCGTTGTATTTTTCCTCCTTGGCTTCATTATT AAACCAGCAAGGCCTTCCAGGTCTAAACACGGAACGATGAGTGAGGTAGAAACTGT TTTTCTTCTCCTT (SEQ ED NO: 14)

The partial amino acid sequence of LPCAT from castor bean

IHLYLVPHYPLSRFTDPVYQEWGFWKRLTYQYMSGLTARWKYYFIWSISEASIIIS GLGFSGWTDTSPPKPQWDRARNVDILGVEFAKSAAELPLVWNIQVSTWLRHYVYDRLV PKGKKAGFLQLLATQTTSAVWHGLYPGYIIFFVQSALMIAGSKVIYRWQQ AIPSNKALE KKILVFMNFA YTVLVLNYSCVGFMVLSLHETIAA YGSVYFIGTIVPVVFFLLGFIIKPARPS RSKHGTMSEVETVFLLL (SEQ ID NO: 15)

(e) The partial nucleotide sequence of LPCAT from grapevine

CTCGTCCAATCTCCACTTCCTCGTTCCCATGCTTCTTGGCTACGCGGCTATGCT TCTCTGTCGCCGTCGATGCGGTGTGATCACCTTTTTCTTGGGATTCGGCTACCTCATT GGCTGCCATGTATACTACATGAGTGGGGATGCATGGAAGGAAGGGGGTATTGATGC TACTGGAGCTCTAATGGTTTTAACATTGAAAGTCATTTCATGTGCAATGAATTATAAT GATGGATTGTTAAAAGAAGACGGTTTGCGTGAGGCACAGAAGAAAAACCGATTGCT TAAGTTACCATCATTGATCGAGTACTTTGGTTATTGTCTCTGCTGTGGAAGTCACTTT GCTGGACCAGTTTATGAAATAAAGGATTATCTTGAATGGACAGAAAGAAAAGGGAT TTGGGCCAAATCAGAGAAAGGGCCACCACCATCACCTTATGGGGCAACGATTCGAG CTCTTATCCAAGCTGCCTTTTGCATGGGCTTGTATGTGTATCTAGTACCCCATTTTCCC TTGACCATATTTACTGATCCTGTATATCAAGAATGGGGCTTCTGGAAACGGTTGGGA TACCAATATATGTGTGGCTTTACAGCACGCTGGAAATACTATTTCATCTGGTCAATCT CTGAGGCAGCTGTCATTATTTCTGGCCTGGGATTCAGTGGGTGGACAGAATCTTCCC CACCAAAACCAAAATGGGACCGTGCAAAGAATGTTGACATTTTAGGTGTTGAGTTGG CAAAGAGTGCAGTAACACTGCCACTTGTTTGGAACATACAAGTCAGCACCTGGCTAC GTTATTATGTTTATGAGAGGCTCATTCAAAATGGGAAGAAACCTGGTTTCTTCCAGTT GCTGGCTACACAAACTGTCAGTGCTGTTTGGCATGGATTATATCCTGGATACATCAT ATTCTTTGTTCAGTCTGCACTGATG (SEQ ID NO: 16)

The partial amino acid sequence of LPCAT from grapevine

SSNLHFLVPMLLGYAAMLLCRRRCGVITFFLGFGYLIGCHVYYMSGDA WKEGGI DATGALMVLTLKVISCAMNYNDGLLKEDGLREAQKKNRLLKLPSLIEYFGYCLCCGSHF AGPVYEIKD YLEWTERKGIWAKSEKGPPPSPYGATIRALIQAAFCMGLYV YLVPHFPLTI FTDPVYQEWGFWKRLGYQYMCGFT ARWKYYFIWSISEAAVnSGLGFSGWTESSPPKPK WDRAKNVDILGVELAKSAVTLPLVWNIQVSTWLRYYVYERLIQNGKKPGFFQLLATQT VSAVWHGLYPGYIIFFVQSALM (SEQ ID NO: 17)

(f) The partial nucleotide sequence of LPCAT from maize CATTTCGTGTCTCATAAACTACAGTGATGGTATCTTGAAGGAAGAGGGTTTAC GCGATGCTCAGATTAAACACCGATTGACTAAGCTTCCTTCTCTAATTGAATATTTTGG GTACTGTCTCTGTTGTGGGAGCCACTTTGCTGGACCGGTATATGAGATGAAAGATTA TCTTGAATGGACTGAAAGGAAAGGAATATGGGCTAGCCCAACTCCTTCGCCATTGTT ACCTACTTTGCGTGCTCTAGTTCAGGCTGGTATATGCATGGGGTTATATTTATACCTG TCACCTAAATTTCCACTCTCACGGTTTAGTGAGCCCCTATATTATGAATGGGGTTTTT GGCACCGACTCTTCTATCAGTACATGTCAGGCTTTACCGCTCGTTGGAAATATTACTT TATATGGTCAATTTCAGAAGCCTCAATTATCATATCTGGTCTAGGCTTTACTGGTTGG TCGGAATCTTCTCCCCCAAAAGCCAAATGGGATCGTGCAAAAAATGTTGATGTATTA GGTGTTGAATTAGCTGGAAGTTCAGTTCAATTGCCCCTTGTGTGGAATATTCAAGTG AGCACATGGCTACGATACTATGTCTATGAGAGGTTAATTCAGAAAGGAAAGAAACC AGGTTTCCTTCAATTGTTGGGTACACAGACAGTCAGTGCCATCTGGCATGGACTATA TCCTGGATATATCATATTCTTTTTTTCATCAGCATTGATGATNAATGGTTCACGAGTT ATATACAGATGGCAGCAAGCAGCGAGCAGTTCATTCCTGAGCGGTATCCTGGCCCTT CTAATTTTGCTATACATTGCTGGGGCTTACTACTCCTGCATCGGGGTCCAGGTACTGA GCTTCAA (SEQ ID NO: 18) The partial amino acid sequence of LPCAT from maize

ISCLINYSDGELKEEGLRDAQIKHRLTKLPSLffiYFGYCLCCGSHFAGPVYEMKDY LEWTERKGIWASPTPSPLLPTLRALVQAGICMGLYLYLSPKFPLSRFSEPLYYEWGFWHR LFYQYMSGFTARWKYYFIWSISEASIIISGLGFTGWSESSPPKAKWDRAKNVDVLGVELA GSSVQLPLVWNIQVSTWLR YYV YERLIQKGKKPGFLQLLGTQTVSAIWHGL YPGYIIFFF SSALMXNGSRVIYRWQQAASSSFLSGILALLILLYIAGAYYSCIGVQVLSF (SEQ ID NO: 19)

(g) The partial nucleotide sequence of LPCAT from peach

AAATATTATTTCATCTGGTCAATTTCAGAGGCTTCTATCATTCTTTCTGGTTTG GGTTTCACTGGCTGGACAGAATCTTCACCACCAAAGCCGCGATGGGATCGTGCAAAA AATGTTGATATTCTAGGCGTTGAGTTTGCAAAGAGTTCAGTTCAGTTACCACTTGTTT GGAACATACAAGTCAGCACCTGGCTACGTCATTATGTTTATGAAAGGCTTGTTAAAC CTGGCAAGAAGGCTGGTTTCTTCCAGTTGCTGACTACACAGACCGTCAGTGCGGTTT GGCATGGACTCTATCCTGGGTACATCATATTCTTTGTTCAGTCAGCATTGATGATTGC TGGTTCAAGAGTGATTTACAGATGGCAACAAGCTGTACCTCAAAACATGGATGCTGT TAAGAACATACTGGTGTTCATAAACTTTGCTTACACTCTCTTGGTTCTGAACTACTCC TGCGTTGGTTTCATTGTATTAAGCCTTCGTGAAACACTTGCCTCATATGGGAGCGTGC ATTTCATCGGAACCATTCTTCCGATAGCATTGATACTACTGAGTTACGTAATAAAAC CTCCAAGGCCTGCAAGATCAAAGGCTCGGAAGGAAGAGTGAGGTTGTCANCCGCAA CAGCATTTTTAACG (SEQ ID NO:20)

The partial amino acid sequence of LPCAT from peach KYYFIWSISEASIILSGLGFTGWTESSPPKPRWDRAKNVDILGVEFAKSSVQLPLV

WNIQVSTWLRHYVYERLVKPGKKAGFFQLLTTQTVSAVWHGLYPGYIIFFVQSALMIAG SRVIYRWQQA VPQNMDA VKNILVFINFA YTLLVLNYSCVGFIVLSLRETLASYGSVHFIG TILPIALILLSYVIKPPRPARSKARKEE (SEQ ID NO:21)

(h) The full-length or partial amino acid sequence of LPCAT from rice

Sequence 1 (accession number Os02g0676000 (SEQ ID NO:22))

MGLEMEGMAAAIGVSVPVLRFLLCFAATIPTGLMWRAVPGAAGRHLYAGLTGA ALSYLSFGATSNLLFVVPMAFGYLAMLLCRRLAGLVTFLGAFGFLIACHMYYMSGDAW KEGGIDATGALMVLTLKIISCAINYSDGMLKEEGLRDAQKKYRLAKLPSLIEYFGYCLCC GSHFAGPVYEMKDYLEYTERKGLWASPTPSPLLPTLRALVQAGACMGLYLYLSPQFPLS RFSEPLYYEWGFWHRLFYQYMSGFTARWKYYFIWSLSEAAIIISGLGFSGWSDSSPPKAK WDRAKNVDVLGVELATSAVQLPLMWNIQVSTWLRYYVYERLVQKGKKPGFLQLLGTQ TVSAVWHGLYPGYIIFFVQSALMINGSKVIYRWQQAVSNPVFHAILVFVNFSYTLMVLN YSCIGFQVLSFKETLASYQSVYYIGTIVPIVVVLLGYVIKPARPVKPKARKAE

Sequence 2 (accession number EAY87053 (SEQ ID NO:23))

MYYMSGDAWKEGGIDATGALMVLTLKIISCAINYSDGMLKEEGLRDAQKKYRL AKLPSLIEYFGYCLCCGSHFAGPVYEMKD YLE YTERKGLW ASPTPSPLLPTLRALVQAG ACMGLYL YLSPQFPLSRFSEPLYYEWGFWHRLFYQYMSGFTARWKYYFIWSLSEAAIIIS GLGFSGWSDSSPPKAKWDRAKNVDVLGVELATSAVQLPLMWNIQVSTWLRYYVYERL VQKGKKPGFLQLLGTQTVSAVWHGLYPGYIIFFVQSALMINGSKVIYRWQQAVSNPVFH AILVFVNFSYTLMVLNYSCIGFQFVFTMLYTLRFLQVLSFKETLASYQSVYYIGTIVPIVV VLLGYVIKPARPVKPKARKAE

(i) The partial nucleotide sequence of LPCAT from snapdragon GCATTAATTACAACGATGGATTACTTAAAAAGGAAGATCTACGTGAGCCACA AAAGAAAAACCGCTTGCTCAAGATGCCATCATTACTTGAGTACATTGGTTACTGTTT GTGTTGTGGAAGTCACTTTGCTGGTCCTGTGTATGAAATGAAAGATTATCTTGAATG GACTGAGAGGAAAGGGATCTGGCAACATACAACCAAGGGACCGAAACCTTCTCCGT ATTGGGCGACTCTCAGGGCTATTTTGCAAGCTGCCATCTGTATGGGCTTGTATCTATA TCTTGTACCACATTACCCACTTTCCAGATTCACGGAGCCAGAATACCAAGAGTATGG GTTCTGGAAACGGTTAAGtTACCAGTACATGTCAGGCTTCACCGCTCGTTGGAAGTA CTATTTCATTTGGTCTATCTCAGAAGCTTCCATAATTATTTCTGGCCTGGGGTTCAGT GGCTGGACAGATTCTGATCCACCCAAAGCACTGTGGGATCGTGCAAAAAATGTTGAT GTATTAGGTGTTGAGTTGGCAAAGAGTTCTGTGCAGTTACCACTTGTATGGAATATT CAAGTTAGCACCTGGCTTAAACACTATGTCTATGAGAGGCTGGTTCAGAAAGGTAAG AAACCAGGCTTCTTCCAGTTGCTGGCTACCCAGACCGTGAGTGCAGTGTGGCATGGA

TTGTACCCTGGGTACATCATATTCTTT (SEQ ID NO:24)

The partial amino acid sequence of LPCAT from snapdragon

INYNDGLLKKEDLREPQKKNRLLKMPSLLEYIGYCLCCGSHFAGPVYEMKDYLE WTERKGIWQHTTKGPKPSPYW ATLRAILQ AAICMGLYLYLVPHYPLSRFTEPEYQEYGF WKRLSYQYMSGFTARWKYYFIWSISEASIIISGLGFSGWTDSDPPKALWDRAKNVDVLG VELAKSSVQLPLVWNIQVSTWLKHYVYERLVQKGKKPGFFQLLATQTVSAVWHGLYP GYIIFF (SEQ ID NO:25)

(j) The partial nucleotide sequence of LPCAT from sorghum GCACGAGGCTCTCACGGTTTAGTGAGCCCTTATATTATGAATGGGGTTTCTGG

CACCGACTCTTCTATCAGTACATGTCAGGCTTCACTGCTCGTTGGAAATATTACTTTA TATGGTCAATTTCAGAAGCCTCAATTATCATATCTGGTCTGGGCTTTACTGGTTGGTC AGAATCTTCTCCCCCGAAAGCCAAATGGGATCGTGCGAAAAATGTTGATGTATTAGG TGTTGAATTAGCTGGAAGTGCAGTTCAAATTCCCCTTGTGTGGAATATTCAAGTGAG CACATGGTTACGATACTATGTCTATGAGAGGCTAATTCAGAAAGGAAAGAAACCAG GTTTCCTTCAGTTGTTGGGTACACAGACAGTCAGCGCCATCTGGCATGGACTGTATC CTGGATATATCATATTCTTTGTTCAGTCAGCATTGATGATAAATGGTTCACGAGTTAT ATACAGATGGCAGCAAGCAGTGAGCAGTTCATTCCTCCGCGGTATCCTGGCTTTTCT AAATTTTGCTTATACATTGCTGGTGCTTAACTACTCCTGCATCGGGTTCCTGGTACTG AGCTTCAAAGAAACCTTGGCGTCCTACCAGAGCGTATATTATGTTGGCACAATTGTT CCCATTGTGTTTCTCCTGCTGGGCAAT (SEQ ID NO:26)

The partial amino acid sequence of LPCAT from sorghum

TRLSRFSEPLYYEWGFWHRLFYQYMSGFTARWKYYFΓWSISEASIΠSGLGFTGWS ESSPPKAKWDRAKNVDVLGVELAGSAVQIPLVWNIQVSTWLRYYVYERLIQKGKKPGF LQLLGTQTVSAIWHGLYPGYIEFFVQSALMINGSRVIYRWQQA VSSSFLRGILAFLNFA YT LLVLNYSCIGFLVLSFKETLASYQSVYYVGTIVPIVFLLLGN (SEQ ID NO:27)

(k) The partial nucleotide sequence of LPCAT from sunflower GAAAACCGCATACTTAAGTTGCCATCTTTAATCGAGTATGTGGGATATTGCTT

ATGCTGCGGAAGTCACTTTGCTGGTCCGGTTTACGAAATCAAAGATTATTTGGATTG GACCGAAAGAAAGGGGATTTGGACAAAGTCCGAGAAAGGCACACCATCACCATTTT TGCCAACACTACGAGCGATTCTCCAAGCGGGTTTCTGTATGGGTTTGTATTTATATCT ATCGCCTTCGTATCCGCTTTCAAGATTCAGTGAGCCGATATATCAAGAATGGGGATT TGTGAAACGTCTGACCGTCCAATACATGTCGGGCTTCACCGCGCGTTGGAAATACTA TTTCATTTGGTCTATCTCAGAAGCTTCTATCATTATTTCGGGCTTCGGTTTCAGTGGCT GGACTGATTCTTCTCCACCAAAAGCCCGATGGGACCGTGCGAAAAACGTTGACGTTT TGGGTGTTGAGTTTGCAAAGAGTTCAGTTGAGTTACCACTCGTGTGGAATATCCAAG TCAGCACATGGCTTCGTCACTATGTTTATGACAGACTTGTTCAAAAGGGAAAGAAGC CTGGCTTTTTCCAATTGTTAGCAACACAGACTGTTAGCGCTGTCTGGCATGGATTATA TCCTGGGTACTTGATATTCTTTGTTCAATCTGCTTTGATGATTTCCGGGTCAAGAGCC ATTTACAGATGGCAGCAGGCGGTTCCGCCAACCGTTAAGAAGTTTTTGATGCTCATG AACTTTGCTTACACGCTTCTTGTTCTTAACTACTCCTGCATAGGTTTTATGGTATTAA GCCTACACGAAACACTGGCTGCATACGGAAGTGTATACTACGTTGGAAACATCATTC CAGTGGCGT (SEQ ID NO:28)

The partial amino acid sequence of LPCAT from sunflower ENRILKLPSLIEYVGYCLCCGSHFAGPVYEIKD YLD WTERKGIWTKSEKGTPSPFL PTLRAILQAGFCMGLYLYLSPSYPLSRFSEPIYQEWGFVKRLTVQYMSGFTARWKYYFI WSISEASIIISGFGFSGWTDSSPPKARWDRAKNVDVLGVEFAKSSVELPLVWNIQVSTWL RHYVYDRLVQKGKKPGFFQLLATQTVSAVWHGLYPGYLIFFVQSALMISGSRAIYRWQ QAVPPTVKKFLMLMNFAYTLLVLNYSCIGFMVLSLHETLAAYGSVYYVGNIIPVA (SEQ

ID NO:29)

(1) The partial nucleotide sequence of LPCAT from tomato

GGTATGGGGTTGTATCTCTATCTGGTGCCTCAGTTCCCACTTTCCAGGTTCACT GAGTCAGTATACCACGAATGGGGTTTCTTCAAACGACTGGGTTACCAATATATGGCT GGCTTTACTGCCCGGTGG AAATATTATTTTATTTGGTCAATCTCTGAAGCTTCTATAA TCATATCTGGACTGGGTTTCAGTGGTTGGACAAACTCTTCTCCGCCAAAACCACGTT GGGACCGAGCAAAAAATGTTGATGTATTGGGTGTTGAGTTAGCAAAGAGCTCGGTTC AGTTACCACTAGTATGGAACATTCAAGTCAGCACATGGCTGCGGCATTATGTGTATG AAAGGCTCGTACAGAAGGGAAGGAAGCCTGGTTTCTTCCAGTTGCTGGCTACCCAAA CTGTCAGTGCCGTTTGGCATGGATTATATCCTGGATACATCATATTCTTTGTTCAGTC CGCTTTGATGATTGCTGGATCAAGAGTCATTTACAGATGGCAGCAAGCTACAAAAGG TACTATGTTTGAGAAGATACTGATAGCAATGAATTTTGCATACACACTGCTGGTTCT AAACTACTCCGCTGTTGGATTCATGGTATTAAGTCTGCATGAAACTCTTACTGCTTAT GGAAGTGTATACTATATTGGAACAATTGTACCAATTCTTCTCATCCTGCTTAGTAAAG TGATTAAGCCTCCAAGACCTGCGACGTCTAAAGCTAGGAAAGCAGAGTAAATCCAA GTCAGTT (SEQ ID NO:30)

The partial amino acid sequence of LPCAT from tomato

GMGLYLYLVPQFPLSRFTESVYHEWGFFKRLGYQYMAGFTARWKYYFIWSISEA SπiSGLGFSGWTNSSPPKPRWDRAKNVD VLGVELAKSSVQLPLVWNIQVSTWLRHYVY ERLVQKGRKPGFFQLLATQTVSAVWHGLYPGYIIFFVQSALMIAGSRVIYRWQQATKGT MFEKILIAMNFAYTLLVLNYSAVGFMVLSLHETLTAYGSVYYIGTIVPILLILLSKVIKPPR PATSKARKAE (SEQ ID NO:31)

(m) The partial nucleotide sequence of LPCAT from Vaccinium corymbosum

GGGGTTGGGTTACCAGTACATGGCTGGCTTTACAGCACGGTGGAAGTATTATT TCATTTGGTCAATCTCAGAAGCTTCCATCATCATTTCTGGCCTGGGGTTCAGTGGTTG GACAGATTCTTCTCCACCAAAACCAAAATGGGACCGTGCAAAGAATGTAGATATTTT GCGGGTTGAGTTTGCAAAGACTGCAGCTCAGATTCCACTTGCATGGAACATTCAAGT CAGCACCTGGCTACGCCATTATGTTTATGAGAGGCTCGTGCAGAAGGGAAAGAAAC CTGGTTTCTTTCAGTTGTTGGCTACCCAGACTGTCAGTGCTGTTTGGCATGGTTTATA TCCTGGATACATCATATTCTTTGTGCAGTCAGCATTGATGATTGCTGGTTCAAGAGTT ATTTATAGATGGCAGCAAGCTGTTCCTCCTAAAATGGATCTGGTGAAGAAAGTATTC GTACTTTTAAACTTTGCTTACACAGTTCTGGTGTTGAACTACTCCTCTGTCGGTTTCAT GGTACTAAGCCTACATGAAACAATTGTTGCATACGGGAGCGTGTATTCGTTGGAACC ATTGTTCCCATACTTGTAATCCTCCTTGGTTACGTAATT (SEQ ID NO:32)

The partial amino acid sequence of LPCAT from Vaccinium corymbosum

GLGYQYMAGFTARWKYYFIWSISEASIIISGLGFSGWTDSSPPKPKWDRAKNVDI LRVEFAKTAAQIPLAWNIQVSTWLRHYVYERLVQKGKKPGFFQLLATQTVSAVWHGLY PGYIIFFVQSALMIAGSRVIYRWQQAVPPKMDLVKKVFVLLNFAYTVLVLNYSSVGFMV LSLHETIVAYGSVYSLEPLFPYL (SEQ ID NO:33)

(n) The partial nucleotide sequence of LPCAT from wheat CACTTTGCTGGACCAGTATATGAGATGAAAGATTATCTTGAATGGACTGAAA

GGAAAGGAATATGGGCCGGCTCAACTCCTTCACCATTATTACCTACTCTGCGTGCTC TAGTTCAGGCTGGAATATGCATGGGGTTATATTTGTATCTGTCACCTATGTTTCCCCA TTCATAATATAGAGGTTCACTAAATCGTGAAAGGGGTTTCTGGCACCGGCTCTTCTTT CAATACATGTCAGGATTTACTGCTCGATGGAAATACTACTTTATATGGTCAGTCTCA GAAGCTGCAATTATTATATCTGGCCTGGGTTTCACTGGTTGGTCTGATTCTTCTCCCC CAAAAGCCAAATGGGACCGTGCTATAAATGTTGATATTCTGGGCGTCGAGCTAGCTG GAAGTGCAGCTCAATTGCCACTTAAGTGGAATATTCAAGTGAGCACATGGCTAAGAT ACTATGTGTATGAGAGGTTAATTCAGAAAGGGAAGAAGCCTGGTTTCCTTCAGTTGT TGGGTACACAGACAGTCAGTGCTATCTGGCATGGACTGTATCCAGGATATATGTTTT TCTTTGTTCAGTCAGCGTTGATGATAAATGGTTCAAAAGTTATATACAGATGGCAAC AAGCTGTGAGCAATCCAGGCCTCCGCACTATCCTGTCTTTACTAAATTGTGCATACA CCATGATGGTGCTTAACTACTCATGCATTGGCTTCCAGGTACTGAGCTTCCAGGAGA CCTTAGCATCCTACAAGAGCGTGTATTATGTCGGCACAATCGTTCCTATTCTATGTGT CTTGCTGGGCTATGTCGTCAAGCCCACGAGACCTGTGAAGCCGA (SEQ ID NO:34)

The partial amino acid sequence of LPCAT from wheat

HFAGPVYEMKDYLEWTERKGIWAGSTPSPLLPTLRALVQAGICMGLYLYLSPMF

PHS*YRGSLNRERGFWHRLFFQ YMSGFTARWKYYFIWSVSEAAIIISGLGFTGWSDSSPP KAKWDRAINVDILGVELAGSAAQLPLKWNIQVSTWLRYYVYERLIQKGKKPGFLQLLG

TQTVSAIWHGLYPGYMFFFVQSALMINGSKVIYRWQQA VSNPGLRTILSLLNCA YTMM

VLNYSCIGFQ VLSFQETLASYKSVYYVGTIVPILCVLLGYVVKPTRPVKP (SEQ ID NO:35)

(o) The amino acid sequences of LPCAT from A. thaliana Sequence (accession number At 1 g 12640 (SEQ ID NO : 36))

MDMSSMAGSIGVSVAVLRFLLCFVATIPVSFACRIVPSRLGKHLYAAASGAFLSY LSFGFSSNLHFLVPMTIGYASMAIYRPKCGΠTFFLGFA YLIGCHVFYMSGDA WKEGGIDS TGALMVLTLKVISCSMNYNDGMLKEEGLREAQKKNRLIQMPSLIEYFGYCLCCGSHFAG PVYEMKD YLEWTEGKGIWDTTEKRKKPSPYGATIRAILQ AAICMALYLYLVPQ YPLTRF TEPVYQEWGFLRKFSYQYMAGFTARWKYYFIWSISEASIIISGLGFSGWTDDASPKPKW DRAKNVDILGVELAKSAVQIPLVWNIQVSTWLRHYVYERLVQNGKKAGFFQLLATQTV SAVWHGLYPGYMMFFVQSALMIAGSRVIYRWQQAISPKMAMLRNIMVFINFLYTVLVL NYSAVGFMVLSLHETLTAYGSVYYIGTIIPVGLILLSYVVPAKPSRPKPRKEE

Sequence (accession number Atlg63050 (SEQ ID NO:37))

MELLDMNSMAASIGVSVAVLRFLLCFVATIPISFLWRFIPSRLGKHIYSAASGAFLS YLSFGFSSNLHFLVPMTIGYASMAIYRPLSGFITFFLGFAYLIGCHVFYMSGDAWKEGGID STGALMVLTLKVISCSINYNDGMLKEEGLREAQKKNRLIQMPSLIEYFGYCLCCGSHFAG PVFEMKD YLEWTEEKGIW A VSEKGKRPSPYGAMIRAVFQAAICMALYLYLVPQFPLTRF TEPVYQEWGFLKRFGYQYMAGFTARWKYYFIWSISEASIIISGLGFSGWTDETQTKAKW DRAKNVDILGVELAKSAVQIPLFWNIQVSTWLRHYVYERIVKPGKKAGFFQLLATQTVS AVWHGLYPGYIIFFVQSALMIDGSKAIYRWQQAIPPKMAMLRNVLVLINFLYTVVVLNY SSVGFMVLSLHETLVAFKSVYYIGTVIPIAVLLLSYLVPVKPVRPKTRKEE The amino acid sequences of LCPAT from fruit fly, human, mouse, S. pombe, and Aspergillus oryzae.

(1) The amino acid sequences of LCPAT from fruit fly Sequence 1 (accession number AAR99097 (SEQ ED NO:38)) MLEPPKFEENDCYNGSRTFTWLADMVGLSVDLVNFLICQISALFLASLFRSMLHPS

KVSSKLRHTFALSIGLAFGYFCFGQQ AIHIAGLPAICYIVIRTQDPRIVQRA VLLV AMSYLL CVHLMRQLYDYGSYALDITGPLMIITQKVTSLAFSIHDGFVRGDEELTKAQQYHAMKM PSALE YFSYVWHFQSELAGPLVFYKDYEEFVEGYNLLSTPPGNGNLDSSKREVVLEPSPTK AVIRKVVGSLVCAFEFMKFVKI YPVKDMKEDDFMNNTSMVYKYWYAMMATTCIRFKY YHAWLLADAICNNSGLGFTGYDKDGNSKWDLISNINVLSFEFSTNMRDAINNWNCGTN RWLRTLVYERVPQQYGTLLTFALSAVWHGFYPGYYLTFATGAVVVTAARTGRRLFRHR FQSTQ VTRMF YDELTCLITRVVLGYATFPFVLLEFMGSIKLYLRFYLCLHIISLVTEFILPKFI RGERRLRTSNGNGNVRLSGSGNTKDAVTTSVESTAALTAGNDLNEDKEEDKHAQCKVH TPTQQQPAAGPHKTTVEQPTEQPNNVNLRSRPQQQQPHLEKKAMPPTCARDAVSVPHD QCEMDQLSSKLKEKIEAETKNΓEEFIDKTVTETVSGIVEFKNDLMRDIEFPKLKLPGSNGA ISLDSSNGGGLRKRNISSVHDNGTDPGHATADLHPPLEENGAAFLKKEIEVINAVVQQAV PAVLSNGHAK

Sequence 2 (accession number AAO41223 (SEQ ED NO: 39)) MAEFEEDLPHNGLMDGIASGVGVPVEALRLLLTELAGYPVAALYQKFISVIADKT

VHHMFFAGCGAGLCYFNYGLDTYHSLIAELTTYFLVLLLRKKTQEFLAENFVFHMSYLLL GYFYTSSND YDILWTMPHCILVLRMIGYGFDITDGLKEESELSKDQKETALKKPPSLLEL LAFSYFPSGFLVGPQFPFRRYKAFVDGEFRQHEGNVEAGVRRFGAGAFYLIVCQVGLRY LPDSYFLTPEFAQVSFVKRIYLLGFW AKFSL YKYISCWLLTEGALICIGLTYKGEDKNGQP DWSGCSNVKLKLLETGNTMEHYVQSFNVNTNQWVGQYIYKRLKFLNNRTISYGAALGF LAVWHGYHSGYYMTFLMEYMVVSTEKQITRFYTKVVLPQWGHELNNSDIYKLLYFITL KSYNVVYMGWCLTAFVFLKYERWIVVYGAVSYYGFTFLVLWAAFYHTFNHFFRSSSRK LAGEDQKLQDSNTDKLVEEKKPEDKKSE

(2) The amino acid sequences of LCPAT from human

Sequence 1 (accession number EAXO 1013 (SEQ ED NO:40))

MKCCFHHIIPRVNFVVCQLFALLAAIWFRTYLHSSKTSSFIRHVVATLLGL YLALF CFGWYALHFLVQSGΪSYCEMIIIGVENMHNYCFVFALGYLTVCQVTR VYEFDYGQYSADF SGPMMΠTQKITSLACEEHDGMFRKDEELTSSQRDLA VRRMPSLLE YLS YNCNFMGILAG PLCSYKD YITFIEGRSYHITQSGENGKEETQYERTEPSPNTA VVQKLLVCGLSLLFHLTICT TLPVEYNIDEHFQATASWPTKIIYLYISLLAARPKYYFAWTLADAINNAAGFGFRGYDEN GAARWDLISNLRIQQIEMSTSFKMFLDNWNIQTALWLKRVCYERTSFSPTIQTFILSAIWH GVYPGYYLTFLTGVLMTLAARAMRNNFRHYFIEPSQLKLFYDVITWIVTQVAISYTVVPF VLLSIKPSLTFYSSWYYCLHILGILVLLLLPVKKTQRRKNTHENIQLSQSKKFDEGENSLG QNSFSTTNNVCNQNQEIASRHSSLKQ

Sequence 2 (accession number Q6ZWT7 (SEQ ID NO:41)) MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAIWFRTYLHSSKTSSFIR HVVATLLGLYLALFCFGWYALHFLVQSGISYCIMIIIGVENMHNYCFVFALGYLTVCQVT RVYIFD YGQYSADFSGPMMnTQKITSLACEIHDGMFRKDEELTSSQRDLA VRRMPSLLE YLS YNCNFMGILAGPLCSYKD YITFIEGRSYHΓΓQSGENGKEETQYERTEPSPNTA VVQKL LVCGLSLLFHLTICTTLPVE YNIDEHFQATASWPTKn YLYISLLAARPKYYFA WTLAD AI NNAAGFGFRGYDENGAARWDLISNLRIQQIEMSTSFKMFLDNWNIQTALWLKR VCYER TSFSPTIQTFILSAIWHGVYPGYYLTFLTGVLMTLAARAMRNNFRHYFIEPSQLKLFYDVI TWIVTQVAISYTVVPFVLLSIKPSLTFYSSWYYCLHILGILVLLLLP VKKTQRRKNTHENI QLSQSRKFDEGENSLGQNSFSTTNNVCNQNQEIASRHSSLKQ

Sequence 3 (accession number Q6P1A2 (SEQ ID NO:85)) MASSAEGDEGTVVALAGVLQSGFQELSLNKLATSLGASEQALRLIISIFLGYPFAL

FYRHYLFYKETYLIHLFHTFTGLSIA YFNFGNQLYHSLLCIVLQFLILRLMGRTITA VLTTF CFQMA YLLAGYYYTATGNYDIKWTMPHCVLTLKLIGLA VD YFDGGKDQNSLSSEQQK YAIRGVPSLLEVAGFSYFYGAFLVGPQFSMNHYMKLVQGELIDIPGKIPNSIIPALKRLSL GLFYLVGYTLLSPHITEDYLLTEDYDNHPFWFRCMYMLIWGKFVLYKYVTCWLVTEGV CILTGLGFNGFEEKGKAKWDACANMKVWLFETNPRFTGTIASFNINTNAWVARYIFKRL KFLGNKELSQGLSLLFLALWHGLHSGYLVCFQMEFLIVIVERQAARLIQESPTLSKLAAIT VLQPFYYLVQQTIHWLFMGYSMT AFCLFTWDKWLKVYKSIYFLGHIFFLSLLFILP YIHK AMVPRKEKLKKME

Sequence 4 (accession number Q6ZNC8 (SEQ ID NO: 86))

MAAEPQPSSLSYRTTGSTYLHPLSELLGIPLDQVNFVVCQLVALFAAFWFRIYLRP GTTSSDVRHA VATIFGIYFVIFCFGWYSVHLFVLVLMCYAIMVTASVSNIHRYSFFV AMG YLTICHISRIYIFHYGILTTDFSGPLMIVTQKITTLAFQVHDGLGRRAEDLSAEQHRLAIKV KPSFLEYLSYLLNFMSVIAGPCNNFKDYIAFIEGKHIHMKLLEVNWKRKGFHSLPEPSPTG AVIHKLGITLVSLLLFLTLTKTFPVTCLVDDWFVHKASFPARLC YL YVVMQASKPKYYF AWTLADA VNNAAGFGFSGVDKNGNFCWDLLSNLNIWKIETATSFKM YLENWNIQTAT WLKCVCYQRVPWYPTVLTFILS ALWHGVYPGYYFTFLTGILVTLAARA VRNNYRHYFL SSRALKAVYDAGTWAVTQLAVSYTVAPFVMLAVEPTISLYKSMYFYLHIISLLIILFLPM KPQAHTQRRPQTLNSINKRKTD

Sequence 5 (accession number XPJ)Ol 129292 (SEQ ID NO:87))

MVMMMMMKVLLLLMKQRGAGLPAPAGVEPRPSSHHPKARVRLQGDESVRPRG CSQLWAFTRHSPRQRGFSARSLFWFVVLPAPTFVPNFPWRWLGGVPHIVPPAATPGPFV VCRLSQRGVGGRDIPGRRNRGVRGKDALPCSHPRSAPHDAGQPFSGDARHPRAEREVG

RALLPATAPGEGGRMGVRVCMRSLPFAAAALGSGGRVPEQPPVRMDRVVERVRKAAL WGAWRGAACPARASERPPERLMHGSGDGLLGFSFVRASLTVFGEEAGPSFLLAVLCAV

VWGGRGED VVSDVQACPAEQGFLLAEPSVFGVNFVVCQLFALLAAIWFRTYLHSSKTSS

FIRHVVATLLGL YLALFCFGWYALHFLVQSGISYCIMiπGVENMHNYCFVFALGYLTVC QVTRVYIFD YGQ YS ADFSGPMMIITQKITSLACEfflDGMFRKDEELTSSQRDLA VRRMPS LLEYLS YNCNFMGILAGPLCSYKD YITFIEGRSYHITQSGENGKEETQYERTEPSPNTA VV QKLLVCGLSLLFHLTICTTLP VE YNIDEHFQATASWPTKII YLYISLLAARPKYYFA WTLA DAINNAAGFGFRGYDENGAARWDLISNLRIQQIEMSTSFKMFLDNWNIQTALWLKRVC YERTSFSPTIQTFILSAIWHGVYPGYYLTFLTGVLMTLAARAMRNNFRHYFIEPSQLKLFY DVITWIVTQV AISYTVVPFVLLSIKPSLTFYSSWYYCLHILGILVLLLLPVKKTQRRKNTH ENIQLSQSKKFDEGENSLGQNSFSTTNNVCNQNQEIASRHSSLKQ

Sequence 6 (accession number XPJ)Ol 131044 (SEQ ID NO:88))

MVNFVVCQLV ALFAAFWFRIYLRPGTTSSDVRHA V ATIFGIYFVIFCFGWYSVHL FVLVLMCYAIMVTASVSNIHRYSFFVAMGYLTICHISRIYIFHYGILTTDFSGPLMIVTQKI TTLAFQVHDGLGRRAEDLS AEQHRLAIKVKPSFLE YLS YLLNFMSVIAGPCNNFKD YIAF IEGKHIHMKLLEVNWKRKGFHSLPEPSPTGAVIHKLGITLVSLLLFLTLTKTFPVTCLVDD WFVHKASFPARLC YL YVVMQASKPKYYFA WTLADA VNNAAGFGFSGVDKNGNFCWD LLSNLNIWKIETATSFKM YLENWNIQTATWLKCVCYQR VPWYPTVLTFILS ALWHGVYP GYYFTFLTGILVTLAARAVRNNYRHYFLSSRALKAVYDAGTWAVTQLAVSYTVAPFVM LAVEPTISLYKSMYFYLHIISLLIILFLPMKPQAHTQRRPQTLNSINKRKTD (3) The amino acid sequences of LCPAT from mouse Sequence 1 (accession number AAH24653 (SEQ ID NO:42))

MAARPPASLS YRTTGSTCLHPLSQLLGIPLDQVNFVACQLFALS AAFWFRIYLHPG KASPEVRHTLATILGIYFVVFCFGWYAVHLFVLVLMCYGVMVSASVSNIHRYSFFVAMG YLTICHISRIYIFHYGILTTDFSGPLMIVTQKITTLAFQVHDGLGRKAEDLSAEQHRLAVK AKPSLLE YLS YHLNFMSVIAGPCNNFKD YV AFIEGRHIHMKLLEVNWTQRGFQSLPEPSP TGAVIQKLCVTLMSLLLFLTLSKSFPVTFLIDDWFVHKANFLSRLWYLYVVMQAAKPKY YFAWTLADAVHNAAGFGFNGMDTDGKSRWDLLSNLNIWKIETATSFKMYLENWNIQT STWLKCVCYERVSWYPTVLTFLLSALWHGVYPGYYFTFLTGVPVTLAARAVRNNYRH HFLSSKARKIA YDVVTWA VTQLA VSYTAAPFVMLA VEPTISLYKSVFFFLHIICLLIILFLP

IKPHQPQRQSRSPNSVKKKAD

Sequence 2 (accession number AAH25429 (SEQ ED NO:43))

MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAVWFRTYLHSSKTSSFIR HVVATLLGLYLAFFCFGWYALHFLVQSGISYCIMIIAGVESMQQCCFVFALGYLSVCQIT RVYIFD YGQYSADFSGPMMnTQKITSLA YEIHDGMFRKDEELTPSQRGLA VRRMPSLLE YVSYTCNFMGILAGPLCSYKD YIAFIEGRASHVAQPSENGKDEQHGKADPSPNAA VTEK LLVCGLSLLFHLTISNMLPVEYNIDEHFQATASWPTKATYLYVSLLAARPKYYFAWTLA DAINNAAGFGFRGYDKNGVARWDLISNLRIQQIEMSTSFKMFLDNWNIQTALWLKRVC YERATFSPTIQTFFLSAIWHGVYPGYYLTFLTGVLMTLAARAVRNNFRHYFLEPPQLKLF YDLITWVATQITIS YTVVPFVLLSIKPSFTFYSSWYYCLHVCSILVLLLLPVKKSQRRTSTQ ENVHLSQAKKFDERDNPLGQNSFSTMNNVCNQNRDTGSRHSSLTQ

(4) The amino acid sequences of LCPAT from S. pombe Sequence (accession number CAA16861 (SEQ ID NO:44))

MA YLIDIPFE YFSSFLGVHPDQLKLLFCFLSAYPFAGILKRLPS APWIRNLFSISIGLF YLIGVHHL YDGVLVLLFD ALFT YFV AAFYRSSRMPWΠFIVILGHTFSSHVIR YIYPSENTD ITASQMVLCMKLTAFAWSVYDGRLPSSELSSYQKDRALRKIPNILYFLGYVFFFPSLLVG PAFD YVD YERFITLSMFKPLADPYEKQITPHSLEPALGRCWRGLLWLILFITGSSIYPLKFL LTPKFASSPILLKYGYVCITAFV ARMKYYGA WELSDGACILSGIGYNGLDSSKHPRWDR VKNIDPIKFEFADNIKCALEAWNMNTNKWLRNYVYLRVAKKGKRPGFKSTLSTFTVSA MWHGVSAGYYLTFVSAAFIQTVAKYTRRHVRPFFLKPDMETPGPFKRVYDVIGMVATN LSLS YLIISFLLLNLKESMVWKEL YFIVHIYILIALA VFNSPIRSKLDNKIRSRVNSYKLKSY EQSMKSTSDTDMLNMSVPKREDFENDE (5) The amino acid sequences of LCPAT from Aspergillus oryzae Sequence (accession number BAE61812 (SEQ ID NO:45))

MLPYVDLLKLIASFLLSYPLAALLKRIPDAQPWKKNAFIIAVSLFYLVGLFDLWDG LRTLA YSAAGIYAIA YYIDGSLMPWIGFEFLMGHMSISfflYRQIIDDAHVTDITGAQMVLV

MKLSSFCWNVHDGRLSQEQLSDPQKYAAIKDFPGILD YLG YVLFFPSLFAGPSFEYVD YR

RWIDTTLFDVPPGTDPSKVPPTRKKRKIPRSGTPAAKKALAGLGWILAFLQLGSLYNQEL VLDETFMQYSFVQRVWILHMLGFTARLKYYGVWYLTEGACVLSGMGYNGFDPKSGKV FWNRLENVDPWSLETAQNSHGYLGSWNKNTNHWLRNYVYLRVTPKGKKPGFRASLAT FVTSAFWHGFYPGYYLTFVLGSFIQTVAKNFRRHVRPFFLTPDGSRPTAYKKYYDIASYV VTQLTLSFAVMPFIFLSFGDSIKVWHSVYFYGIVGNIVSLAFFVSPARGLLLKKLKARNKP HVPRAVSSENIRQPTLGLPNDAIQEFDDAVQEIRAEIESRQRRGSLAHMPIGDELKAAVED

KIGRGH

Alignment of the LPCAT sequences from different species that reveals four conserved motifs unique for this novel type of LPCAT enzymes (FIG. 2). They are not present in the previously identified glycerol-3-phosphate acyltransferases, lyso-phosphatidic acid acyltransferases, and known LPCAT enzymes. The sequences of these motifs are as follows. The letter "φ" represents a certain amino acid. Motif 1: M V(I) L(I) φ φ K L(V,I) φ φ φ φ φ φ D G (or Met Xaa Xaa Xaa Xaa Lys Xaa

Xaa Xaa Xaa Xaa Xaa Xaa Asp GIy (SEQ ID NO:46), wherein the Xaa at position 2 can be VaI or He, the Xaa at position 3 can be Leu or He, the Xaa at position 7 can be Leu, VaI, or He, while the other Xaa's in the motif may be any amino acid.

Motif 2: R φ K Y Y φ φ W φ φ φ E(D) A(G) φ φ φ φ φ G φ G F(Y) φ G (or Arg Xaa Lys Tyr Tyr Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa GIy Xaa GIy Xaa Xaa GIy (SEQ ID NO:47), wherein the Xaa at position 12 is GIu or Asp, wherein the Xaa at position 13 is Ala or GIy, wherein the Xaa at position 22 is Phe or Tyr, while the other Xaa's in the motif may be any amino acid.

Motif 3: E φ φ φ φ φ φ φ φ φ φ φ W N φ φ T(V) φ φ W (or GIu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Asn Xaa Xaa Xaa Xaa Xaa Trp (SEQ ID NO:48) wherein the Xaa at position 17 is Thr or VaI, while the other Xaa's in the motif may be any amino acid.

Motif 4: S A φ W H G φ φ P G Y φ φ T(F) F (or Ser Ala Xaa Trp His GIy Xaa Xaa Pro GIy Tyr Xaa Xaa Xaa Phe (SEQ ID NO:49) wherein Xaa at position 14 is Thr or Phe, while the other Xaa's in the motif may be any amino acid. FIG. 3 depicts another alignment of LPCAT sequences from different plant species that revealed the following motifs:

Motif 5 (SEQ ID NO:81): E A φ φ I I(L) S G φ G F S(T) G W; Motif 6 (SEQ ID NO:82): W D R A φ N V D; Motif 7 (SEQ ID NO:83): W N I Q V S T W L φ φ Y V Y; and

Motif 8 (SEQ ID NO:84): G F φ Q L L φ T Q T φ S A φ W H G L Y P G Y.

Example 7: Analysis of LPCAT from the alga Thalassiosira pseudonana Materials and Methods Isolation of the LPCAT cDNA from T. pseudonana: PCR primers were designed for nucleotide sequence of the putative TpLPCAT obtained by a BLAST search of the sequenced T. pseudonana genome using the yeast LPCAT sequence. Plasmid from a cDNA library of T. pseudonana was used as template. A 50 μl PCR reaction contained 50 ng of plasmid DNA, 20 pM of each primer: 5'- GGTATGCTCATCTGCTACCCCCTC -3' (SEQ ID NO:89) and 5'- TTAAGTCTCCTTCGTCTTTGGTGTAG -3' (SEQ ID NO:90) and 1 μl of BD Advantage™ 2 Polymerase Mix (Clontech Laboratories, Inc.), and was amplified in a thermocycler during 30 cycles of the following program: 94°C for 30 seconds, 58⁰C for 30 seconds, and 72°C for one minute 30 seconds. The PCR product was purified, and subsequently cloned into the pYES2.1/V5-His-TOPO expression vector (In vitrogen). Expression of TpLPCAT in yeast: The TpLPCAT in pYES2.1/V5-His-TOPO plasmid was transformed into yeast lpcat mutant ByO2431 using the method provided by the producer's manual (Invitrogen). Yeast cells transformed with pYES2.1/V5-His-TOPO plasmid only were used as a control. Transformants were selected by growth on synthetic complete medium lacking uracil (SC-ura), supplemented with 2% (w/v) glucose. The colonies were transferred into liquid SC-ura with 2% (w/v) glucose and grown at 28°C overnight. The overnight cultures were diluted to an OD 0.4 in induction medium (SC-ura + 2% Galactose + 1% Raffinose), and were induced by incubating at 28°C for 24 hours. The yeast cells were collected and broken using glass beads. The protein concentrations in the lysates were normalized using the Biorad assay (Bradford 1976) and then assayed for LPCAT activity.

Identification of LPCAT from the algae Thalassiosira pseudonana

Isolation of the LPCAT cDNA from T. pseudonana A full-length T. pseudonana LPCAT cDNA clone was amplified by PCR from an algae cDNA library. The nucleotide sequence had an open reading frame of 1,323 bp encoding a polypeptide of 440 amino acids with a calculated molecular mass of 49.75 kD.

Expression of TpLPCAT in Yeast: To confirm the function of the protein encoded by the TpLPCAT, the full-length coding region of TpLPCAT was cloned into a yeast expression vector pYES2.1/V5-His-TOPO under the control of the galactose-inducible GALl promoter, and the construct was used to transform a LPCAT-deficient yeast strain ByO2431(a yeast lpcat strain). Yeast cells harboring an empty pYES2.1 vector plasmid were used as a control. We also discovered that the yeast lpcat strain is hypersensitive to lyso-PAF (lyso-Platelet-activating factor, l-O-alkyl-sn-glycero-3-phosphocholine). Expression of the TpLPCAT in yeast lpcat mutant was able to overcome lyso-PAF the sensitivity of the lpcat mutant (FIG. 4).

The microsomal membrane fractions prepared from lysates of the induced yeast cells were assayed for LPCAT activity using 14C-labelled Lyso-PC as acceptor, and different unlabeled acyl-CoAs as acyl donors. Under our assay conditions, expression of the TpLPCAT in yeast lpcat mutant resulted in a restoration of LPCAT function and produced a recombinant LPCAT protein capable of incorporating a range of different acyl-CoAs into PC including 14:0-, 16:0-, 16:1-, 18:0-, 18:1-, 18:2-, and 22:6(DHA)-, with the most preference of 18: 1-CoA, and efficiently utilization of the very long chain polyunsaturated fatty acid~22:6-CoA(DHA) (FIGs. 5 and 6).

Example 8: Arabidopsis Gene Assays Experimental procedure:

TA-cloning and yeast complementation: Total RNA was prepared from Arabidopsis seedlings using RNeasy Plant Mini Kit (Qiagen). RT-PCR of the ORFs of Arabidopsis Atlgl2460, Atlg63050 was performed with primer pairs designed based on sequences of gene annotation available at TAIR (The Arabidopsis Information Resources). The cDNA was cloned into vector pYES2.1 using pYES2.1 TOPO TA Cloning Kit according to the manufacturer's protocol (Invitrogen). Correctly-oriented positive colonies were identified through double digestion with restriction enzyme, followed by verification through DNA sequencing. The construct was introduced into yeast strain YOR175c, BY02431. Yeast extract, Yeast Nitrogen Base, Bacto-peptone, and Bacto-agar were purchased from DIFCO™, D-glucose, D-galactose and D-raffinose were from Sigma. SC minimal medium and plates was prepared according to Invitrogen's recipe described for the pYES2.1 TOPO TA Cloning Kit.

Lyso-PAF sensitivity: Yeast strains BY02431 carrying pYES 2.1-AtLPCATs or the empty vector were first grown in 15 ml of SC-Leu-His-ura medium containing 2% glucose. Yeast transformant strains of AtLPCATs were first grown in YPD overnight. Protein expression induction were carried out by protocol described in Invitrogen manufacturer manual for yeast expression vector pYES2.1. After 12 hours induction, 5 μl cultures were inoculated onto YPD plate with 10 μg/ml lysoPAF. The plates were incubated at 28°C for two days. The final lysoPAF is 10 μg/ml.

In vitro assay: Yeast strains BY02431 carrying pYES 2.1-AtLPCATs (or the empty vector) were first grown in 15 ml of SC-Leu-His-ura medium containing 2% glucose. Yeast transformant strains of AtLPCATs were first grown in YPD overnight. Protein expression induction was carried out by protocol described in Invitrogen manufacturer manual for yeast expression vector pYES2.1. After 24 hours of growth in the galactose induction conditions, the cells were washed first with distilled water and then with wall-breaking buffer (50 mM sodium phosphate, pH7.4; 1 mM EDTA; 1 mM PMSF; 5% glycerol) and spun down at 4,000 rpm (Eppendorf Centrifuge 5145C) to re-pellet cells. The cells, resuspended in 1 ml cell wall-breaking buffer, were shaken vigorously in the presence of acid-washed glass beads (diameter 0.5 mm) in a mini-bead beater at 5,000 rpm for three 1-minute intervals. The resultant homogenate was centrifuged at 1,500 g for five minutes at 4⁰C. The supernatant was decanted for in vitro assay. Protein concentration was measured using Bio-Rad Protein Assay Kit for final AtSATl activity calculation.

AtLPCAT substrate specificity was determined by counting incorporation of 14C-labeled lysophosphatidylcholine or 14C-labled palmitoyl-CoA into phosphatidylcholine. All assays were performed at least twice. 200 ml reaction mixture contained 50mg microsomal protein, 50 mM acyl-CoA and 45 mM palmitoyl-PC, pH7.4. 14C-lysophosphatidylcholine (1.4 nCi/nmol) or 14C-palmityl-CoA (5.5 nCi/nmol) was used to assess fatty-CoA or lyso-lipid substrate specificity. Reaction was allowed for ten minutes at 30°C. All radiolabel chemicals for these assays were purchased from ARC, Inc.

Lyso-PAF sensitivity test (FIG. 7): The yeast lpcat strain is deficient in its endogenous LPCAT and hypersensitive to lyso-PAF (lyso-Platelet-activating factor, l-O-alkyl-sn-glycero-3-phosphocholine). The lpcat yeast mutant is incapable of growth in the presence of 10 ug/ml lyso-PAF (lyso-Platelet-activating factor, l-O-alkyl-sn-glycero-3-phosphocholine). However, when the Arabidopsis LPCAT genes, Atlgl2640 and Atlg63050, were introduced into the yeast mutant, the transformants could survive on lyso-PAF-containing YPD plate. These results indicated that the Arabidopsis genes encode for LPCAT. In vitro enzyme characterization with the yeast cell free lysate expressing the Arabidopsis LPCATs was further conducted.

Lyso-lipid substrate specificity (FIG. 8): LPA (lysophosphatidic acid), LPC

(lysophosphatidic choline), LPE (lysophosphatidylethanolamine), LPG (lysophosphatidylglycerol), LPI (lysophosphatidyl inositol) and LPS (lysophosphatidyl serine) were first tested as substrates to compare their acyltransferase activity. The results clearly showed that Atlgl2640 and Atlg63050 both exhibited high activity towards LPC (FIG. 8).

Example 9: By NCBI-BLASTp search with default Algorithm parameters (expect threshold=10; word size=3; matrix=BLOSUM62; gap costs=existence: 11 Extension: 1; compositional adjustments=compositi on-based statistics) following sequences (E value<5el8) from various organisms including human and mouse were identified as YOR175cp homologs.

SEQ ID NO:87: Human_XP_001129292 protein sequence, PREDICTED: similar to

O-acyl transferase (membrane bound) domain containing 2 [Homo sapiens].

SEQ ID NO:93: Human_XP_001129292 CDS sequence ATGGTGATGATGATGATGATGAAGGTGCTGCTGCTGCTGATGAAGCAAAGGG GAGCCGGTCTCCCTGCGCCCGCGGGCGTCGAACCCAGGCCCAGCTCTCACCACCCAA AGGCCCGGGTGCGGCTGCAGGGTGACGAAAGCGTCAGACCCCGGGGCTGCTCTCAG CTTTGGGCTTTCACCCGGCACTCTCCCAGACAAAGGGGCTTCTCAGCCAGGTCGCTG TTTTGGTTTGTCGTCCTCCCAGCCCCCACCTTCGTCCCCAACTTCCCCTGGCGCTGGC TCGGCGGCGTCCCTCACATCGTCCCTCCGGCCGCCACCCCGGGCCCCTTTGTTGTCTG CCGTCTCTCCCAGAGAGGGGTGGGGGGCCGCGACATTCCAGGGAGGAGAAACCGAG GAGTGAGGGGCAAAGACGCTCTTCCATGCTCTCACCCGAGGAGCGCGCCCCACGAC GCTGGCCAGCCGTTCTCCGGCGACGCCCGCCATCCCCGGGCCGAGCGGGAGGTGGG CCGGGCGTTGTTGCCGGCGACAGCCCCCGGGGAGGGTGGTCGTATGGGCGTGCGGG TGTGCATGCGGTCCCTGCCCTTCGCGGCAGCGGCGCTCGGATCCGGTGGTCGGGTCC CGGAGCAGCCCCCGGTGCGCATGGACCGGGTTGTGGAAAGGGTGCGGAAGGCTGCG CTTTGGGGAGCCTGGCGTGGTGCTGCCTGCCCCGCGCGCGCCTCTGAGCGACCCCCG GAGAGGCTGATGCATGGGTCTGGGGATGGGCTGCTTGGCTTCTCATTTGTCAGAGCA AGCTTGACAGTGTTTGGAGAGGAAGCAGGCCCATCCTTTCTATTGGCAGTTCTCTGT GCTGTTGTCTGGGGAGGAAGAGGAGAGGATGTTGTGTCTGATGTACAGGCTTGTCCT GCAGAACAGGGCTTCTTGCTGGCTGAACCCAGTGTATTTGGTGTCAACTTTGTAGTG TGCCAACTCTTTGCCTTGCTAGCAGCCATTTGGTTTCGAACTTATCTACATTCAAGCA AAACTAGCTCTTTTATAAGACATGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCACT TTTTTGCTTTGGATGGTATGCCTTACACTTTCTTGTACAAAGTGGAATTTCCTACTGT ATCATGATCATCATAGGAGTGGAGAACATGCACAATTACTGCTTTGTGTTTGCTCTG GGATACCTCACAGTGTGCCAAGTTACTCGAGTCTATATCTTTGACTATGGACAATATT CTGCTGATTTTTCAGGCCCAATGATGATCATTACTCAGAAGATCACTAGTTTGGCTTG CGAAATTCATGATGGGATGTTTCGGAAGGATGAAGAACTGACTTCCTCACAGAGGG ATTTAGCTGTAAGGCGCATGCCAAGCTTACTGGAGTATTTGAGTTACAACTGTAACT TCATGGGGATCCTGGCAGGCCCACTTTGCTCTTACAAAGACTACATTACTTTCATTGA AGGCAGATCATACCATATCACACAATCTGGTGAAAATGGAAAAGAAGAGACACAGT ATGAAAGAACAGAGCCATCTCCAAATACTGCGGTTGTTCAGAAGCTCTTAGTTTGTG GGCTGTCCTTGTTATTTCACTTGACCATCTGTACAACATTACCTGTGGAGTACAACAT TGATGAGCATTTTCAAGCTACAGCTTCGTGGCCAACAAAGATTATCTATCTGTATATC TCTCTTTTGGCTGCCAGACCCAAATACTATTTTGCATGGACGCTAGCTGATGCCATTA ATAATGCTGCAGGCTTTGGTTTCAGAGGGTATGACGAAAATGGAGCAGCTCGCTGGG ACTTAATTTCCAATTTGAGAATTCAACAAATAGAGATGTCAACAAGTTTCAAGATGT TTCTTGATAATTGGAATATTCAGACAGCTCTTTGGCTCAAAAGGGTGTGTTATGAAC GAACCTCCTTCAGTCCAACTATCCAGACGTTCATTCTCTCTGCCATTTGGCACGGGGT ATACCCAGGATATTATCTAACGTTTCTAACAGGGGTGTTAATGACATTAGCAGCAAG AGCTATGAGAAATAACTTTAGACATTATTTCATTGAACCTTCCCAACTGAAATTATTT TATGATGTTATAACATGGATAGTAACTCAAGTAGCAATAAGTTACACAGTTGTGCCA TTTGTGCTTCTTTCTATAAAACCATCACTCACGTTTTACAGCTCCTGGTATTATTGCCT GCACATTCTTGGTATCTTAGTATTATTGTTGTTGCCAGTGAAAAAAACTCAAAGAAG AAAGAATACACATGAAAACATTCAGCTCTCACAATCCAAAAAGTTTGATGAAGGAG AAAATTCTTTGGGACAGAACAGTTTTTCTACAACAAACAATGTTTGCAATCAGAATC AAGAAATAGCCTCGAGACATTCATCACTAAAGCAGTGA

SEQ ID NO:85: Human_NP_005759 protein sequence, O-acyltransferase (membrane bound) domain containing 5 [Homo sapiens]:

MASSAEGDEGTVVALAGVLQSGFQELSLNKLATSLGASEQALRLIISIFLGYPFAL FYRHYLFYKETYLIHLFHTFTGLSIAYFNFGNQLYHSLLCIVLQFLILRLMGRTITA VLTTF CFQMA YLLAGYYYTATGNYDIKWTMPHCVLTLKLIGLA VD YFDGGKDQNSLSSEQQK YAIRGVPSLLEVAGFSYFYGAFLVGPQFSMNHYMKLVQGELIDIPGKIPNSIIPALKRLSL GLFYLVGYTLLSPHITEDYLLTEDYDNHPFWFRCMYMLIWGKFVLYKYVTCWLVTEGV CILTGLGFNGFEEKGKAKWDACANMKVWLFETNPRFTGTIASFNINTNAWVARYIFKRL KFLGNKELSQGLSLLFLALWHGLHSGYLVCFQMEFLIVIVERQAARLIQESPTLSKLAAIT VLQPFYYLVQQTIHWLFMGYSMTAFCLFTWDKWLKVYKSIYFLGHIFFLSLLFILPYIHK AMVPRKEKLKKME

SEQ ID NO:94: Human_NP_005759 cDNA sequence

ATGGCGTCCTCAGCGGAGGGGGACGAGGGGACTGTGGTGGCGCTGGCGGGG GTTCTGCAGTCGGGTTTCCAGGAGCTGAGCCTTAACAAGTTGGCGACGTCCCTGGGC GCGTCAGAACAGGCGCTGCGGCTGATCATCTCCATCTTCCTGGGTTACCCCTTTGCTT TGTTTTATCGGCATTACCTTTTCTACAAGGAGACCTACCTCATCCACCTCTTCCATAC CTTTACAGGCCTCTCAATTGCTTATTTTAACTTTGGAAACCAGCTCTACCACTCCCTG CTGTGTATTGTGCTTCAGTTCCTCATCCTTCGACTAATGGGCCGCACCATCACTGCCG TCCTCACTACCTTTTGCTTCCAGATGGCCTACCTTCTGGCTGGATACTATTACACTGC CACCGGCAACTACGATATCAAGTGGACAATGCCACATTGTGTTCTGACTTTGAAGCT GATTGGTTTGGCTGTTGACTACTTTGACGGAGGGAAAGATCAGAATTCCTTGTCCTCT GAGCAACAGAAATATGCCATACGTGGTGTTCCTTCCCTGCTGGAAGTTGCTGGTTTC TCCTACTTCTATGGGGCCTTCTTGGTAGGGCCCCAGTTCTCAATGAATCACTACATGA AGCTGGTGCAGGGAGAGCTGATTGACATACCAGGAAAGATACCAAACAGCATCATT CCTGCTCTCAAGCGCCTGAGTCTGGGCCTTTTCTACCTAGTGGGCTACACACTGCTCA GCCCCCACATCACAGAAGACTATCTCCTCACTGAAGACTATGACAACCACCCCTTCT GGTTCCGCTGCATGTACATGCTGATCTGGGGCAAGTTTGTGCTGTACAAATATGTCA CCTGTTGGCTGGTCACAGAAGGAGTATGCATTTTGACGGGCCTGGGCTTCAATGGCT TTGAAGAAAAGGGCAAGGCAAAGTGGGATGCCTGTGCCAACATGAAGGTGTGGCTC TTTGAAACAAACCCCCGCTTCACTGGCACCATTGCCTCATTCAACATCAACACCAAC GCCTGGGTGGCCCGCTACATCTTCAAACGACTCAAGTTCCTTGGAAATAAAGAACTC TCTCAGGGTCTCTCGTTGCTATTCCTGGCCCTCTGGCACGGCCTGCACTCAGGATACC TGGTCTGCTTCCAGATGGAATTCCTCATTGTTATTGTGGAAAGACAGGCTGCCAGGC TCATTCAAGAGAGCCCCACCCTGAGCAAGCTGGCCGCCATTACTGTCCTCCAGCCCT TCTACTATTTGGTGCAACAGACCATCCACTGGCTCTTCATGGGTTACTCCATGACTGC CTTCTGCCTCTTCACGTGGGACAAATGGCTTAAGGTGTATAAATCCATCTATTTCCTT GGCCACATCTTCTTCCTGAGCCTACTATTCATATTGCCTTATATTCACAAAGCAATGG TGCCAAGGAAAGAGAAGTTAAAGAAGATGGAATAA SEQ ID NO:95: Human_NP_077274 protein sequence, leukocyte receptor cluster (LRC) member 4 protein [Homo sapiens]:

MSPEEWTYLVVLLISIPIGFLFKKAGPGLKRWGAAAVGLGLTLFTCGPHTLHSLV TILGTWALIQAQPCPCHALALAWTFS YLLFFRALSLLGLPTPTPFTNAVQLLLTLKLVSLA SEVQDLHLAQRKEMASGFSKGPTLGLLPDVPSLMETLSYSYCYVGIMTGPFFRYRTYLD WLEQPFPGAVPSLRPLLRRAWPAPLFGLLFLLSSHLFPLEAVREDAFYARPLPARLFYMIP VFFAFRMRFYVA WIAAECGCIAAGFGA YPV AAKARAGGGPTLQCPPPSSPEKAASLE YD YETIRNIDCYSTDFCVR VRDGMR YWNMTVQWWLAQYIYKS APARSYVLRSAWTMLLS AYWHGLHPGYYLSFLTIPLCLAAEGRLESALRGRLSPGGQKAWDWVHWFLKMRAYDY MCMGFVLLSLADTLR YW ASIYFCIHFLALAALGLGLALGGGSPSRRKAASQPTSLAPEK LREE

SEQ ID NO:96: Human_NP_077274 cDNA sequence ATGTCGCCTGAAGAATGGACGTATCTAGTGGTTCTTCTTATCTCCATCCCCATC GGCTTCCTCTTTAAGAAAGCCGGTCCTGGGCTGAAGAGATGGGGAGCAGCCGCTGTG GGCCTGGGGCTCACCCTGTTCACCTGTGGCCCCCACACTTTGCATTCTCTGGTCACCA TCCTCGGGACCTGGGCCCTCATTCAGGCCCAGCCCTGCCCCTGCCACGCCCTGGCTCT GGCCTGGACTTTCTCCTATCTCCTGTTCTTCCGAGCCCTCAGCCTCCTGGGCCTGCCC ACTCCCACGCCCTTCACCAATGCCGTCCAGCTGCTGCTGACGCTGAAGCTGGTGAGC CTGGCCAGTGAAGTCCAGGACCTGCATCTGGCCCAGAGGAAGGAAATGGCCTCAGG CTTCAGCAAGGGGCCCACCCTGGGGCTGCTGCCCGACGTGCCCTCCCTGATGGAGAC ACTCAGCTACAGCTACTGCTACGTGGGAATCATGACAGGCCCGTTCTTCCGCTACCG CACCTACCTGGACTGGCTGGAGCAGCCCTTCCCCGGGGCAGTGCCCAGCCTGCGGCC CCTGCTGCGCCGCGCCTGGCCGGCCCCGCTCTTCGGCCTGCTGTTCCTGCTCTCCTCT CACCTCTTCCCGCTGGAGGCCGTGCGCGAGGACGCCTTCTACGCCCGCCCGCTGCCC GCCCGCCTCTTCTACATGATCCCCGTCTTCTTCGCCTTCCGCATGCGCTTCTACGTGG CCTGGATTGCCGCCGAGTGCGGCTGCATTGCCGCCGGCTTTGGGGCCTACCCCGTGG CCGCCAAAGCCCGGGCCGGAGGCGGCCCCACCCTCCAATGCCCACCCCCCAGCAGT CCGGAGAAGGCGGCTTCCTTGGAGTATGACTATGAGACCATCCGCAACATCGACTGC TACAGCACAGATTTCTGCGTGCGGGTGCGCGATGGCATGCGGTACTGGAACATGACG GTGCAGTGGTGGCTGGCGCAGTATATCTACAAGAGCGCACCTGCCCGTTCCTATGTC CTGCGGAGCGCCTGGACCATGCTGCTGAGCGCCTACTGGCACGGCCTCCACCCGGGC TACTACCTGAGCTTCCTGACCATCCCGCTGTGCCTGGCTGCCGAGGGCCGGCTGGAG TCAGCCCTGCGGGGGCGGCTGAGCCCAGGGGGCCAGAAGGCCTGGGACTGGGTGCA CTGGTTCCTGAAGATGCGCGCCTATGACTACATGTGCATGGGCTTCGTGCTGCTCTCC TTGGCCGACACCCTTCGGTACTGGGCCTCCATCTACTTCTGTATCCACTTCCTGGCCC TGGCAGCCCTGGGGCTGGGGCTGGCTTTAGGTGGGGGCAGCCCCAGCCGGCGGAAG GCAGCATCCCAGCCCACCAGCCTTGCCCCGGAGAAGCTCCGGGAGGAGTAA

SEQ ID NO:97: Human_NP_620154 protein sequence, O-acyltransferase (membrane bound) domain containing 2 [Homo sapiens]:

MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAIWFRTYLHSSKTSSFIR HVVATLLGL YLALFCFGWYALHFLVQSGISYCMIIIGVENMHNYCFVFALGYLTVCQVT RVYIFD YGQYSADFSGPMMΠTQKITSLACEIHDGMFRKDEELTSSQRDLA VRRMPSLLE YLS YNCNFMGILAGPLCSYKD YITFIEGRSYHITQSGENGKEETQ YERTEPSPNTA VVQKL LVCGLSLLFHLTICTTLPVEYNIDEHFQATASWPTKΠYLYISLLAARPKYYFA WTLADAI NNAAGFGFRGYDENGAARWDLISNLRIQQIEMSTSFKMFLDNWNIQT ALWLKRVCYER TSFSPTIQTFILSAIWHGVYPGYYLTFLTGVLMTLAARAMRNNFRHYFIEPSQLKLFYDVI TWIVTQVAIS YTVVPFVLLSIKPSLTFYSS W YYCLHILGILVLLLLPVKKTQRRKNTHENI QLSQSKKFDEGENSLGQNSFSTTNNVCNQNQEIASRHSSLKQ

SEQ ID NO:98: Human_NP_620154 cDNA sequence ATGGCCACCACCAGCACCACGGGCTCCACCCTGCTGCAGCCCCTCAGCAACG CCGTGCAGCTGCCCATCGACCAGGTCAACTTTGTAGTGTGCCAACTCTTTGCCTTGCT AGCAGCCATTTGGTTTCGAACTTATCTACATTCAAGCAAAACTAGCTCTTTTATAAGA CATGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCACTTTTTTGCTTTGGATGGTATGC CTTACACTTTCTTGTACAAAGTGGAATTTCCTACTGTATCATGATCATCATAGGAGTG GAGAACATGCACAATTACTGCTTTGTGTTTGCTCTGGGATACCTCACAGTGTGCCAA GTTACTCGAGTCTATATCTTTGACTATGGACAATATTCTGCTGATTTTTCAGGCCCAA TGATGATCATTACTCAGAAGATCACTAGTTTGGCTTGCGAAATTCATGATGGGATGT TTCGGAAGGATGAAGAACTGACTTCCTCACAGAGGGATTTAGCTGTAAGGCGCATGC CAAGCTTACTGGAGTATTTGAGTTACAACTGTAACTTCATGGGGATCCTGGCAGGCC CACTTTGCTCTTACAAAGACTACATTACTTTCATTGAAGGCAGATCATACCATATCAC ACAATCTGGTGAAAATGGAAAAGAAGAGACACAGTATGAAAGAACAGAGCCATCTC CAAATACTGCGGTTGTTCAGAAGCTCTTAGTTTGTGGGCTGTCCTTGTTATTTCACTT GACCATCTGTACAACATTACCTGTGGAGTACAACATTGATGAGCATTTTCAAGCTAC AGCTTCGTGGCCAACAAAGATTATCTATCTGTATATCTCTCTTTTGGCTGCCAGACCC AAATACTATTTTGCATGGACGCTAGCTGATGCCATTAATAATGCTGCAGGCTTTGGTT TCAGAGGGTATGACGAAAATGGAGCAGCTCGCTGGGACTTAATTTCCAATTTGAGAA TTCAACAAATAGAGATGTCAACAAGTTTCAAGATGTTTCTTGATAATTGGAATATTC AGACAGCTCTTTGGCTCAAAAGGGTGTGTTATGAACGAACCTCCTTCAGTCCAACTA TCCAGACGTTCATTCTCTCTGCCATTTGGCACGGGGTATACCCAGGATATTATCTAAC GTTTCTAACAGGGGTGTTAATGACATTAGCAGCAAGAGCTATGAGAAATAACTTTAG ACATTATTTCATTGAACCTTCCCAACTGAAATTATTTTATGATGTTATAACATGGATA GTAACTCAAGTAGCAATAAGTTACACAGTTGTGCCATTTGTGCTTCTTTCTATAAAAC CATCACTCACGTTTTACAGCTCCTGGTATTATTGCCTGCACATTCTTGGTATCTTAGT ATTATTGTTGTTGCCAGTGAAAAAAACTCAAAGAAGAAAGAATACACATGAAAACA TTCAGCTCTCACAATCCAAAAAGTTTGATGAAGGAGAAAATTCTTTGGGACAGAACA GTTTTTCTACAACAAACAATGTTTGCAATCAGAATCAAGAAATAGCCTCGAGACATT CATCACTAAAGCAGTGA

SEQ ID NO:88: Human_XP_001131044 protein sequence, PREDICTED: similar to O-acyl transferase (membrane bound) domain containing 1 isoform 1

SEQ ID NO:99: Human_XP_001131044 cDNA sequence

ATGGTGAATTTTGTGGTATGCCAGCTTGTTGCTCTGTTTGCTGCTTTCTGGTTT CGCATCTACTTACGTCCTGGTACAACCAGCTCTGATGTCCGGCATGCGGTTGCCACC ATTTTTGGCATCTATTTTGTCATCTITTGTTTCGGCTGGTACTCTGTGCATCTTTTTGT GCTGGTGTTAATGTGCTATGCAATCATGGTCACTGCTAGTGTATCCAATATTCACAG ATATTCCTTTTTTGTAGCAATGGGATATCTTACAATATGCCACATCAGCCGAATATAC ATCTTCCACTATGGAATTCTCACTACGGATTTTTCTGGGCCTCTGATGATTGTCACTC AGAAGATCACAACCTTGGCATTCCAGGTTCATGATGGATTAGGTCGAAGAGCTGAA GACCTTTCTGCTGAACAACATCGACTTGCTATCAAAGTGAAACCCTCTTTTTTGGAAT ACTTAAGTTACCTTCTCAATTTCATGAGTGTCATAGCTGGTCCTTGTAACAATTTCAA GGACTACATAGCCTTCATTGAGGGGAAGCATATACACATGAAGTTGCTGGAGGTGA ACTGGAAGCGAAAAGGTTTCCACAGCTTGCCAGAACCTTCTCCCACAGGAGCTGTGA TACACAAGTTGGGCATCACCTTGGTGTCTCTCCTTTTGTTTTTGACGCTAACGAAGAC CTTTCCTGTCACCTGCCTTGTGGATGACTGGTTTGTCCATAAAGCAAGCTTTCCGGCT CGACTCTGCTACTTATATGTTGTCATGCAAGCCTCAAAGCCCAAGTATTACTTTGCAT GGACATTAGCTGATGCAGTGAATAACGCAGCTGGCTTTGGGTTCAGCGGAGTGGATA AGAATGGGAATTTCTGTTGGGATCTGCTTTCGAACCTAAACATCTGGAAAATTGAGA CTGCCACAAGTTTCAAAATGTACTTGGAAAACTGGAATATTCAGACAGCTACTTGGC TAAAGTGTGTGTGCTATCAGCGGGTTCCATGGTACCCCACGGTGCTAACCTTCATCCT GTCTGCTTTGTGGCATGGTGTCTACCCTGGATACTATTTTACCTTCTTAACTGGAATT CTTGTCACATTAGCAGCTAGAGCGGTCAGGAACAACTACAGACATTACTTCCTTTCT TCAAGAGCTCTCAAGGCTGTGTATGATGCAGGCACCTGGGCCGTCACTCAGCTGGCT GTCTCTTACACGGTAGCACCCTTTGTGATGTTGGCAGTTGAACCGACCATCAGCTTAT ACAAGTCCATGTACTTTTATTTGCACATCATAAGTCTCCTGATAATACTATTTCTGCC AATGAAACCACAAGCTCATACGCAAAGGCGGCCTCAGACTCTGAACTCTATTAATAA GAGAAAAACAGATTGA

SEQ ID NO:88: Human_XP_001125855 protein sequence, PREDICTED: similar to

O-acyltransferase (membrane bound) domain containing 2 [Homo sapiens]

MVNFVVCQLV ALFAAFWFRIYLRPGTTSSDVRHA VATIFGIYFVIFCFGWYSVHL FVLVLMCYAIMVTASVSNIHR YSFFVAMGYLTICHISRIYIFHYGILTTDFSGPLMIVTQKI TTLAFQVHDGLGRRAEDLS AEQHRLAIKVKPSFLE YLS YLLNFMSVIAGPCNNFKD YIAF IEGKHIHMKLLEVNWKRKGFHSLPEPSPTGAVIHKLGITLVSLLLFLTLTKTFPVTCLVDD WFVHKASFPARLCYL YVVMQASKPKYYFA WTLADA VNNAAGFGFSGVDKNGNFCWD LLSNLNIWKIETATSFKMYLENWNIQTATWLKCVCYQRVPWYPTVLTFILS ALWHGVYP GYYFTFLTGILVTLAARAVRNNYRHYFLSSRALKAVYDAGTWAVTQLAVSYTVAPFVM LAVEPTISLYKSMYFYLHIISLLIILFLPMKPQAHTQRRPQTLNSINKRKTD

SEQ ID NO: 100: Human_XP_001125855 cDN A sequence

ATGTTCTTTAAAAAATTATCTTGCAGGTTCTGCATCACTCTTTCTTCTCTCATG CTCTTGACCCAGAGGGTCACGTCCCTCTCTCTGGACATTTGTGAGGGGAAAGTGAAG GCAGCATCTGGAGGCTTCAGGAGCAGGAGCTCTTTGTCTGAGCATGTGTGTAAGGCA CTGCCCTATTTCAGCTACTTGCTCTTTTTCCCTGCTCTCCTGGGAGGCTCTCTGTGCTC CTTCCAGCGATTTCAGGCTCGTGTTCAAGGGTCCAGTGCTTTGCATCCCAGACACTCT TTCTGGGCTCTGAGCTGGAGGGGTCTGCAGATTCTTGGACTAGAATGCCTAAACGTG GCAGTGAGCAGGGTGGTGGATGCAGGAGCGGGACTGACTGATTGCCAGCAATTCGA GTGCATCTATGTCGTGTGGACCACAGCTGGGCTTTTCAAGCTCACCTACTACTCCCAC TGGATCCTGGACGACTCCCTCCTCCACGCAGCGGGCTTTGGGCCTGAGCTTGGTCAG AGCCCTGGAGAGGAGGGATATGTCCCCGATGCAGACATCTGGACCCTGGAAAGAAC CCACAGGATATCTGTGTTCTCAAGAAAGTGGAACCAAAGCACAGCTCGATGGCTCCG ACGGCTTGTATTCCAGCACAGCAGGGCTTGGCCGTTGTTGCAGACATTTGCCTTCTCT GCCTGGTGGCATGGACTCCATCCAGGACAGGTGTTTGGTTTCGTTTGCTGGGCCGTG ATGGTGGAAGCTGACTACCTGATTCACTCCTTTGCCAATGAGTTTATCAGATCCTGGC CGATGAGGCTGTTCTATAGAACCCTCACCTGGGCCCACACCCAGTTGATCATTGCCT ACATCATGCTGGCTGTGGAGGTCAGGAGTCTCTCCTCTCTCTGGTTGCTCTGTAATTC GTACAACAGTGTCTTTCCCATGGTGTACTGTATTCTGCTTTTGCTATTGGCGAAGAGA AAGCACAAATGTAACTGA

SEQ ID NO:43: Mouse_NP_080313 deduced protein sequence, 0-acyltransferase (membrane bound) domain containing 2 isoform a [Mus musculus]

MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAVWFRTYLHSSKTSSFIR HVVATLLGLYLAFFCFGWYALHFLVQSGISYCIMIIAGVESMQQCCFVFALGYLSVCQIT RVYLRD YGQYSADFSGPMMΠTQKITSLA YEIHDGMFRKDEELTPSQRGLA VRRMPSLLE YVSYTCNFMGILAGPLCS YKDYIAFIEGRASHVAQPSENGKDEQHGKADPSPNAAVTEK LLVCGLSLLFHLTISNMLPVEYNIDEHFQATASWPTKATYLYVSLLAARPKYYFAWTLA DAINNAAGFGFRGYDKNGVAR WDLISNLRIQQIEMSTSFKMFLDNWNIQTALWLKRVC YERATFSPTIQTFFLSAIWHGVYPGYYLTFLTGVLMTLAARAVRNNFRHYFLEPPQLKLF YDLITWVATQITIS YTVVPFVLLSIKPSFTFYSSWYYCLHVCSILVLLLLPVKKSQRRTSTQ ENVHLSQAKKFDERDNPLGQNSFSTMNNVCNQNRDTGSRHSSLTQ

SEQ ID NO: 111: Mouse_NP_080313 cDNA sequence ATGGCCACCACCAGCACCACGGGCTCCACCCTGCTGCAGCCCCTCAGCAACG

CCGTGCAACTGCCCATCGATCAGGTCAACTTTGTAGTGTGCCAGCTCTTTGCCTTGTT AGCAGCCGTTTGGTTTCGAACTTATCTACACTCAAGCAAAACTAGCTCTTTTATCAGA CACGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCATTTTTTTGCTTTGGATGGTATGC CTTACACTTTCTTGTACAAAGTGGGATTTCCTACTGCATCATGATCATAGCAGGAGTG GAGAGCATGCAGCAATGTTGCTTTGTGTTTGCTTTGGGATACCTCTCAGTGTGTCAGA TTACTAGAGTCTATATCTTTGATTATGGACAATATTCTGCTGATTTTTCAGGCCCAAT GATGATCATTACGCAGAAGATCACTAGTTTGGCTTACGAAATTCACGACGGGATGTT TCGGAAGGATGAAGAACTGACTCCGTCGCAGAGGGGATTAGCTGTGAGGCGCATGC CAAGTCTCCTGGAGTATGTAAGTTATACCTGCAACTTCATGGGCATCCTGGCAGGCC CACTGTGCTCCTACAAAGACTACATTGCCTTCATTGAAGGCAGAGCATCCCACGTGG CACAGCCCAGTGAAAATGGAAAAGACGAGCAGCATGGGAAAGCAGATCCATCTCCA AATGCAGCAGTTACGGAGAAGCTCCTGGTCTGTGGACTCTCCTTATTATTCCACCTG ACCATCTCCAACATGCTACCCGTGGAGTACAACATCGATGAGCATTTCCAAGCCACT GCGTCGTGGCCGACTAAAGCCACCTATCTGTACGTCTCTCTCTTGGCTGCCAGACCTA AGTACTATTTTGCATGGACCTTAGCTGACGCCATTAACAATGCTGCGGGCTTCGGTTT CAGAGGATACGACAAGAATGGAGTGGCTCGCTGGGACTTAATTTCCAACTTGAGAA TTCAGCAAATAGAGATGTCAACAAGTTTTAAGATGTTTCTTGATAACTGGAATATCC AGACAGCTCTTTGGCTCAAAAGGGTGTGCTATGAACGAGCAACCTTCAGTCCGACAA TCCAGACATTCTTTCTCTCTGCCATTTGGCATGGGGTCTACCCAGGATACTATCTGAC ATTCCTAACGGGAGTGCTAATGACGTTAGCAGCTCGGGCTGTGAGAAATAACTTTAG GCACTATTTCCTGGAGCCCCCTCAACTTAAGTTATTTTATGACCTCATAACCTGGGTG GCCACCCAGATAACAATAAGTTACACAGTTGTTCCGTTTGTGCTCCTCTCCATAAAA CCGTCGTTCACGTTTTACAGCTCCTGGTATTACTGCCTTCACGTCTGTAGTATCTTGG TGTTGCTGTTGCTGCCTGTGAAAAAGTCTCAAAGAAGAACGAGCACACAGGAAAAT GTTCATCTCTCACAGGCCAAAAAGTTTGACGAAAGGGACAATCCTCTGGGACAGAA CAGTTTTTCCACGATGAATAACGTTTGCAATCAGAACCGAGACACTGGCTCCAGACA CTCGTCACTAACACAGTGA

SEQ ID NO: 101: Mouse_NP_084210 deduced protein sequence, leukocyte receptor cluster (LRC) member 4 [Mus musculus]

MTPEEWTYLMVLLISIPVGFLFKKAGPGLKRWGAAAVGLGLTLFTCGPHSLHSLI TILGTWALIQAQPCSCHALALAWTFS YLLFFRALSLLGLPTPTPFTNAVQLLLTLKLVSLA SEVQDLHLAQRKEIASGFHKEPTLGLLPEVPSLMETLSYSYCYVGIMTGPFFR YRTYLDW LEQPFPEAVPSLRPLLRRAWPAPLFGLLFLLSSHLFPLEAVREDAFYARPLPTRLFYMIPVF FAFRMRFYV A WIAAECGCIAAGFGA YPVAAKARAGGGPTLQCPPPSSPEIAASLE YD YE TIRNIDCYGTDFCVR VRDGMR YWNMTVQWWLAQYIYKSAPFRSYVLRSAWTMLLSAY WHGLHPGYYLSFMTIPLCLAAEGYLESALRRHLSPGGQKAWDWVHWFLKMRAYDYM CMGFVLLSMADTLR YWASIYFWVHFLALACLGLGLVLGGGSPSKRKTPSQATSSQAKE KLREE

SEQ ID NO: 102: Mouse_NP_084210 deduced cDNA sequence

ATGACACCCGAAGAATGGACATATCTAATGGTCCTTCTTATCTCCATCCCTGT TGGCTTCCTCTTTAAGAAAGCTGGACCTGGGCTGAAGAGATGGGGGGCAGCAGCTGT GGGCCTGGGGCTCACCTTATTCACCTGTGGCCCCCACAGTTTGCATTCTCTGATCACC ATCTTGGGAACCTGGGCCCTCATTCAGGCCCAGCCCTGCTCCTGCCATGCCCTGGCTC TTGCCTGGACCTTCTCCTATCTCCTCTTCTTCCGAGCCCTCAGCCTGCTGGGCCTGCC CACTCCCACGCCCTTCACCAATGCTGTCCAGCTGCTGTTGACACTGAAGTTGGTGAG TCTAGCTAGTGAAGTCCAGGATCTGCATCTGGCTCAGAGAAAGGAAATAGCCTCCGG CTTCCACAAGGAGCCTACGCTGGGCCTCCTGCCTGAGGTCCCCTCTTTGATGGAGAC ACTCAGCTATAGCTACTGTTACGTGGGAATCATGACAGGCCCATTCTTCCGCTACCG CACCTACCTGGATTGGCTGGAACAGCCCTTCCCGGAAGCCGTGCCCAGCCTGAGGCC CCTGCTGCGCCGCGCCTGGCCAGCCCCGCTCTTTGGCCTGCTCTTCCTGCTGTCCTCC CATCTCTTCCCACTGGAAGCTGTGCGTGAGGACGCCTTCTACGCCCGCCCGCTGCCC ACCCGCCTCTTCTACATGATCCCGGTCTTCTTCGCCTTCCGCATGCGCTTCTACGTTG CCTGGATTGCGGCCGAGTGCGGTTGCATTGCCGCGGGCTTCGGGGCCTACCCTGTGG CTGCCAAAGCCCGGGCCGGGGGCGGCCCCACCCTCCAATGCCCACCCCCTAGCAGTC CGGAGATTGCAGCTTCCCTGGAGTATGACTATGAGACCATCCGTAACATCGACTGCT ATGGCACAGACTTCTGCGTGCGTGTGCGGGATGGCATGCGATACTGGAACATGACCG TGCAGTGGTGGCTGGCACAGTACATCTACAAGAGCGCACCTTTCCGCTCCTACGTTT TGAGGAGTGCCTGGACCATGCTGTTGAGTGCCTACTGGCATGGCCTCCACCCTGGTT ACTACCTAAGCTTCATGACCATCCCGCTGTGCCTGGCTGCTGAGGGCTATTTGGAGT CAGCCTTGCGGAGACACCTGAGCCCCGGGGGCCAGAAAGCCTGGGACTGGGTCCAC TGGTTCCTGAAGATGCGTGCCTACGACTACATGTGCATGGGCTTTGTGCTCCTTTCCA TGGCTGACACACTCCGGTACTGGGCCTCCATCTACTTCTGGGTCCACTTTCTAGCCCT GGCTTGCTTGGGGCTGGGGCTGGTTTTGGGTGGGGGCAGCCCCAGC AAGAGGAAGA CACCATCCCAGGCCACCAGCAGCCAAGCGAAGGAAAAGCTCCGGGAAGAGTGA

SEQ ID NO: 103: Mouse_NP_660112 deduced protein sequence, membrane bound

O-acyltransferase domain containing 5 [Mus musculus]

MASTADGDMGETLEQMRGLWPGVEDLSLNKLATSLGASEQALRLIFSIFLGYPLA LFYRHYLFYKDSYLIHLFHTFTGLSIAYFNFGHQFYHSLLCVVLQFLILRLMGRTVTAVIT TLCFQMA YLLAGYYYTATGD YDIKWTMPHCVLTLKLIGLCID YYDGGKDGNSLTSEQQ KYAIRGVPSLLEVAGFSYFYGAFLVGPQFSMNHYMKLVRGQLTDIPGKMPNSTIPALKR LSLGLVYLVGYTLLSPHITDD YLLTED YDNRPFWFRCMYMLIWGKFVL YKYVTCWLVT EGVCILSGLGFNGFDENGTVRWDACANMKVWLFETTPRFNGTIASFNINTNAWVARYIF KRLKFLGNKELSQGLSLLFLALWHGLHSGYLICFQMEFLIVIVEKQVSSLIRDSPALSSLA SITALQPFYYLVQQTIHWLFMGYSMTAFCLFTWDKWLKVYRSIYFLGHVFFLSLLFILPYI HKAMVPRKEKLKKRE

SEQ ID NO: 104: Mouse_NP_660112 deduced cDNA sequence

ATGGCGTCTACAGCGGACGGGGACATGGGAGAGACGCTGGAGCAGATGCGG GGGCTGTGGCCGGGTGTCGAGGATCTGAGCCTTAACAAGTTGGCGACGTCTCTGGGC GCGTCGGAACAGGCGCTGCGGCTCATCTTCTCCATCTTCCTGGGCTACCCGTTGGCTC TGTTTTACCGGCATTACCTTTTCTACAAGGACAGCTACCTCATCCATCTCTTCCACAC CTTCACGGGCCTCTCAATTGCTTATTTCAACTTTGGCCACCAGTTCTACCACTCCTTG CTATGTGTCGTGCTTCAGTTCCTCATCCTGCGACTCATGGGCCGCACCGTCACTGCCG TTATTACTACCCTTTGCTTCCAGATGGCCTACCTTCTTGCCGGATATTACTACACAGC CACCGGTGACTACGATATCAAGTGGACAATGCCACATTGTGTCTTGACACTGAAGCT AATTGGGCTGTGTATTGACTACTACGATGGAGGCAAAGACGGGAATTCCTTGACCTC TGAGCAACAGAAATATGCCATACGGGGTGTCCCTTCATTGCTGGAAGTTGCTGGCTT CTCCTACTTCTATGGAGCCTTCTTGGTAGGGCCCCAATTTTCAATGAACCACTACATG AAGCTGGTGCGGGGACAGCTGACTGACATACCAGGGAAGATGCCAAACAGCACCAT ACCTGCTCTCAAGCGCCTGAGTCTGGGCCTTGTCTACCTGGTGGGCTACACCCTGCTG AGCCCCCACATCACAGACGACTATCTCCTCACAGAAGACTATGATAACCGCCCTTTC TGGTTCCGCTGCATGTACATGCTGATCTGGGGCAAATTTGTGCTGTACAAATACGTC ACCTGCTGGCTGGTCACAGAAGGAGTGTGCATTCTGTCGGGCCTGGGCTTTAATGGC TTCGATGAAAATGGGACCGTGAGATGGGATGCCTGTGCCAACATGAAAGTGTGGCT CTTTGAAACCACCCCTCGCTTCAATGGCACCATCGCCTCTTTCAACATCAATACCAAT GCCTGGGTAGCCCGTTACATCTTCAAACGCCTCAAGTTCCTTGGAAATAAAGAGCTC TCACAAGGTCTCTCCTTGCTGTTCTTGGCCCTCTGGCATGGCCTACACTCAGGATACC TGATTTGCTTCCAGATGGAATTCCTCATTGTTATCGTGGAAAAGCAGGTCAGCAGTC TAATTCGGGACAGCCCTGCCCTGAGCAGCCTGGCCTCCATCACTGCCCTACAGCCCT TCTACTACTTGGTGCAACAGACCATCCACTGGCTGTTCATGGGTTACTCTATGACTGC CTTCTGCCTCTTCACATGGGACAAATGGCTTAAGGTGTACAGATCCATCTATTTCCTT GGACATGTCTTCTTCTTGAGCCTACTATTCATATTGCCTTATATCCACAAAGCAATGG TGCCAAGAAAAGAAAAGTTAAAAAAGAGGGAATGA

SEQ DD NO: 105: Mouse_NP_705774 deduce protein sequence, membrane bound O-acyltransferase domain containing 1 [M. musculus]

MAARPPASLS YRTTGSTCLHPLSQLLGIPLDQVNFVACQLFALS AAFWFRIYLHPG KASPEVRHTLATILGIYFVVFCFGWYAVHLFVLVLMCYGVMVTASVSNIHRYSFFVAMG YLTICHISRIYIFHYGILTTDFSGPLMIVTQKITTLAFQVHDGLGRKAEDLSAEQHRLAVK AKPSLLEYLSYHLNFMSVIAGPCNNFKDYVAFIEGRHIHMKLLEVNWTQRGFQSLPEPSP MGAVIQKLCVTLMSLLLFLTLSKSFPVTFLIDDWFVHKANFLSRLWYLYVVMQAAKPK YYFAWTLADAVHNAAGFGFNGMDTDGKSRWDLLSNLNIWKIETATSFKMYLENWNIQ TSTWLKCVCYERVPWYPTVLTFLLSALWHGVYPGYYFTFLTGVPVTLAARAVRNNYRH HFLSSKARKIAYDVVTWAVTQLAVSYTAAPFVMLAVEPTISLYKSVFFFLHIICLLIILFLP IKPHQPQRQSRSPNSVKKKAD

SEQ ID NO: 106: Mouse_NP_705774 cDNA sequence ATGGCAGCACGGCCGCCCGCCAGCCTCTCTTACCGTACCACCGGCTCCACCTG

CCTGCACCCGCTCAGCCAGCTCCTGGGCATCCCGCTGGATCAGGTTAACTTTGTGGC TTGCCAGCTCTTTGCCTTGTCTGCTGCTTTCTGGTTCAGAATCTACTTACATCCTGGTA AAGCCAGCCCTGAGGTCCGGCACACCTTGGCCACCATTTTGGGCATCTATTTTGTTGT GTTTTGTTTTGGTTGGTATGCTGTACATCTCTTTGTGCTGGTGTTGATGTGTTATGGGG TCATGGTCACTGCAAGTGTATCCAATATTCACAGGTATTCCTTTTTTGTAGCCATGGG CTACCTTACGATATGCCACATCAGCCGCATTTACATCTTCCACTATGGAATTCTCACT ACAGATTTTTCTGGGCCCCTGATGATTGTCACTCAGAAGATCACGACGTTGGCTTTCC AAGTTCATGATGGATTGGGTCGAAAAGCTGAAGACCTTTCTGCTGAGCAACACCGAC TTGCTGTGAAAGCGAAGCCCTCGCTTCTGGAATACTTAAGCTACCATCTCAACTTTAT GAGTGTCATAGCCGGCCCTTGCAACAATTTCAAGGACTACGTAGCCTTCATCGAAGG GAGACATATACACATGAAGTTGCTGGAAGTGAACTGGACGCAAAGGGGTTTCCAGA GTTTGCCAGAGCCTTCTCCCATGGGAGCTGTGATACAGAAGTTGTGTGTGACCTTGA TGTCTCTCCTGTTGTTTTTGACGCTCTCCAAGTCCTTTCCCGTCACCTTCCTTATTGAT GACTGGTTTGTACATAAGGCCAACTTTCTGAGTCGTCTCTGGTACTTATATGTCGTCA TGCAAGCCGCAAAGCCCAAGTATTACTTTGCGTGGACATTAGCAGATGCGGTGCACA ATGCAGCTGGATTCGGGTTCAATGGCATGGACACGGATGGGAAGTCTCGCTGGGATT TACTATCTAACCTGAACATCTGGAAGATTGAGACTGCCACGAGTTTCAAAATGTACT TGGAAAACTGGAATATTCAGACATCTACGTGGCTGAAATGTGTGTGCTATGAGCGGG TTCCCTGGTACCCCACAGTGCTCACCTTCCTCCTGTCTGCTCTGTGGCACGGCGTCTA CCCTGGATACTACTTCACATTCCTAACTGGAGTCCCTGTCACATTGGCAGCCAGAGC GGTGAGGAACAACTACAGACACCACTTCCTCTCTTCCAAAGCTCGAAAGATTGCCTA TGACGTGGTGACCTGGGCTGTCACTCAGTTGGCTGTCTCTTACACGGCAGCGCCTTTC GTCATGTTGGCAGTCGAGCCAACCATCAGTTTATACAAGTCCGTGTTCTTTTTTTTAC ACATCATATGTCTGCTGATAATCCTCTTTCTGCCAATCAAACCACACCAGCCTCAAA GGCAGTCTCGGAGTCCAAATTCTGTAAAGAAGAAGGCAGACTGA SEQ ID NO: 107: Mouse_NP_001076810 deduced protein sequence, O-acyltransferase (membrane bound) domain containing 2 isoform b [M. musculus]

MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAVWFRTYLHSSKTSSFIR HVVATLLGLYLAFFCFGWY ALHFLVQSGISYCIMIIAGVESMQHPMMnTQKITSLA YEIH DGMFRKDEELTPSQRGLA VRRMPSLLEYVSYTCNFMGILAGPLCSYKDYIAFIEGRASHV AQPSENGKDEQHGKADPSPNAA VTEKLLVCGLSLLFHLTISNMLP VE YNIDEHFQATAS WPTKATYLYVSLLAARPKYYFAWTLADAINNAAGFGFRGYDKNGVARWDLISNLRIQQ IEMSTSFKMFLDNWNIQTALWLKRVCYERATFSPTIQTFFLSAIWHGVYPGYYLTFLTGV LMTLAARAVRNNFRHYFLEPPQLKLFYDLITWVATQITISYTVVPFVLLSIKPSFTFYSSW YYCLHVCSILVLLLLPVKKSQRRTSTQENVHLSQAKKFDERDNPLGQNSFSTMNNVCNQ NRDTGSRHSSLTQ

SEQ ID NO: 108: Mouse_NP_001076810 cDNA sequence ATGGCCACCACCAGCACCACGGGCTCCACCCTGCTGCAGCCCCTCAGCAACG CCGTGCAACTGCCCATCGATCAGGTCAACTTTGTAGTGTGCCAGCTCTTTGCCTTGTT AGCAGCCGTTTGGTTTCGAACTTATCTACACTCAAGCAAAACTAGCTCTTTTATCAGA CACGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCATTTTTTTGCTTTGGATGGTATGC CTTACACTTTCTTGTACAAAGTGGGATTTCCTACTGCATCATGATCATAGCAGGAGTG GAGAGCATGCAGCACCCAATGATGATCATTACGCAGAAGATCACTAGTTTGGCTTAC GAAATTCACGACGGGATGTTTCGGAAGGATGAAGAACTGACTCCGTCGCAGAGGGG ATTAGCTGTGAGGCGCATGCCAAGTCTCCTGGAGTATGTAAGTTATACCTGCAACTT CATGGGCATCCTGGCAGGCCCACTGTGCTCCTACAAAGACTACATTGCCTTCATTGA AGGCAGAGCATCCCACGTGGCACAGCCCAGTGAAAATGGAAAAGACGAGCAGCATG GGAAAGCAGATCCATCTCCAAATGCAGCAGTTACGGAGAAGCTCCTGGTCTGTGGA CTCTCCTTATTATTCCACCTGACCATCTCCAACATGCTACCCGTGGAGTACAACATCG • ATGAGCATTTCCAAGCCACTGCGTCGTGGCCGACTAAAGCCACCTATCTGTACGTCT CTCTCTTGGCTGCCAGACCTAAGTACTATTTTGCATGGACCTTAGCTGACGCCATTAA CAATGCTGCGGGCTTCGGTTTCAGAGGATACGACAAGAATGGAGTGGCTCGCTGGG ACTTAATTTCCAACTTGAGAATTCAGCAAATAGAGATGTCAACAAGTTTTAAGATGT TTCTTGATAACTGGAATATCCAGACAGCTCTTTGGCTCAAAAGGGTGTGCTATGAAC GAGCAACCTTCAGTCCGACAATCCAGACATTCTTTCTCTCTGCCATTTGGCATGGGGT CTACCCAGGATACTATCTGACATTCCTAACGGGAGTGCTAATGACGTTAGCAGCTCG GGCTGTGAGAAATAACTTTAGGCACTATTTCCTGGAGCCCCCTCAACTTAAGTTATTT TATGACCTCATAACCTGGGTGGCCACCCAGATAACAATAAGTTACACAGTTGTTCCG TTTGTGCTCCTCTCCATAAAACCGTCGTTCACGTTTTACAGCTCCTGGTATTACTGCC TTCACGTCTGTAGTATCTTGGTGTTGCTGTTGCTGCCTGTGAAAAAGTCTCAAAGAAG AACGAGCACACAGGAAAATGTTCATCTCTCACAGGCCAAAAAGTTTGACGAAAGGG ACAATCCTCTGGGACAGAACAGTTTTTCCACGATGAATAACGTTTGCAATCAGAACC GAGACACTGGCTCCAGACACTCGTCACTAACACAGTGA

SEQ ID NO: 109: Mouse_XP_134120 deduced protein sequence, PREDICTED: similar to O-acyltransferase (membrane bound) domain containing 1 [M musculus].

MPHCLQGTASESDFSVNTARGENACILWFPWLRPSVGKPTFTLLISSASISFCPAGL STSYKKATESPVVTSLLQGHRLGTLGRTVGLTFRMDWLQLFFLHPLSFYQGAAFPFALLF NYLCILDTFSTRAR YLFLLAGGGVLAFAAMGPYSLLIFIPALCA V ALVSFLSPQEVHRLTF FFQMGWQTLCHLGLHYTEYYLGEPPPVRFYITLSSLMLLTQRVTSLSLDICEGKVEAPRR GIRSKSSFSEHLWDALPHFS YLLFFPALLGGSLCSFRRFQACVQRSSSLYPSISFRALTWR GLQILGLECLKV ALRSAVSAGAGLDDCQRLECIYLMWSTA WLFKLTYYSHWILDDSLLH AAGFGAEAGQGPGEEGYVPDVDIWTLETTHRISLFARQWNRSTALWLRRLVFRKSRRW PLLQTFAFSAWWHGLHPGQ VFGFLCWSVMVKAD YLIHTFANVCIRSWPLRLLYRALTW AHTQLIIA YIMLA VEGRSLSSLCQLCCSYNSLFP VMYGLLLFLLAERKDKRNSAFSF

SEQ ID NO: 110: Mouse_XP_134120 deduced cDNA sequence ATGCCACACTGCCTGCAAGGTACAGCCTCTGAGAGTGACTTTTCAGTAAACAC

TGCGAGGGGAGAGAATGCCTGCATACTTTGGTTTCCATGGCTCCGCCCCTCTGTTGG GAAGCCAACCTTTACATTGCTTATCTCCAGTGCTTCCATTTCATTTTGTCCGGCAGGC CTTTCTACATCCTATAAAAAGGCTACGGAGAGCCCAGTTGTGACTTCCCTTTTACAA GGGCACCGCTTAGGGACTCTAGGAAGGACAGTGGGCCTCACATTCAGGATGGATTG GCTCCAGCTCTTTTTTCTGCATCCTTTATCATTTTATCAAGGGGCTGCATTCCCCTTTG CGCTTCTGTTTAATTATCTCTGCATCTTGGACACCTTTTCCACCCGGGCCAGGTACCT CTTTCTCCTGGCTGGAGGAGGTGTCCTGGCTTTTGCTGCCATGGGTCCCTACTCTCTG CTCATCTTCATCCCTGCGCTCTGCGCTGTGGCTCTGGTCTCCTTCCTCAGTCCACAGG AAGTCCATAGGCTGACCTTCTTCTTTCAGATGGGCTGGCAGACCCTGTGCCATCTGG GTCTTCACTACACCGAATACTACCTGGGTGAGCCTCCACCCGTGAGGTTCTACATCA CTCTTTCTTCCCTCATGCTCTTGACGCAGAGAGTCACATCCCTCTCACTGGACATTTG TGAAGGGAAGGTGGAGGCCCCGAGGCGGGGCATCAGGAGCAAGAGTTCTTTCTCTG AGCACCTGTGGGATGCTCTACCTCATTTCAGCTACTTGCTCTTTTTCCCTGCTCTCCTG GGAGGCTCCCTGTGTTCCTTCCGGAGGTTTCAGGCTTGCGTTCAAAGATCAAGCTCTT TGTATCCGAGTATCTCTTTTCGGGCTCTGACCTGGAGGGGTCTGCAGATTCTCGGGCT GGAGTGCCTCAAGGTGGCGCTGAGGAGCGCGGTGAGTGCTGGAGCTGGACTGGATG ACTGCCAGCGGCTGGAGTGCATCTACCTCATGTGGTCCACAGCCTGGCTCTTTAAAC TCACCTATTACTCCCATTGGATCCTGGACGACTCTCTCCTCCACGCGGCGGGCTTTGG CGCTGAGGCTGGCCAGGGGCCTGGAGAGGAGGGATACGTCCCCGACGTGGACATTT GGACCCTGGAAACTACCCACAGGATCTCCCTGTTCGCCAGGCAGTGGAACCGAAGC ACAGCTCTGTGGCTCAGGAGGCTCGTCTTCCGGAAGAGCCGGCGCTGGCCCCTGCTG CAGACATTTGCCTTCTCTGCCTGGTGGCACGGGCTCCACCCAGGTCAGGTGTTCGGC TTCCTGTGCTGGTCTGTAATGGTGAAAGCCGATTATCTGATTCACACTTTTGCCAACG TATGTATCAGATCCTGGCCCCTGCGGCTGCTTTATAGAGCCCTCACTTGGGCTCATAC CCAACTCATCATTGCCTACATCATGCTGGCGGTGGAGGGCCGGAGCCTTTCCTCTCTC TGCCAACTGTGCTGTTCTTACAACAGTCTCTTCCCTGTGATGTACGGTCTTTTGCTTTT TCTGTTAGCGGAGAGAAAAGACAAACGTAACTGA

All of the above human and mouse YOR175cp homologs were aligned with YOR175cp sequence with MegAlign program of Lasergene7.0 software package (FIG. 20). Mouse proteins NP_660112 and NP_084210, human proteins NP_005759 and NP_077274 were characterized.

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Claims (18)

CLAIMSWhat is claimed is:

1. An isolated, purified or recombinant nucleic acid molecule comprising the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ED NO:7, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO.24, SEQ ID NO:26, SEQ ID NO:28, SEQ DD NO:30, SEQ DD NO:32, or SEQ DD NO:34.

2. An isolated peptide encoded by the nucleic acid molecule of claim 1.

3. An isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ DD NO:4, SEQ DD NO:6, SEQ DD NO:8, SEQ DD NO: 10, SEQ DD NO:11; SEQ DD NO:13, SEQ DD NO:15, SEQ DD NO:17; SEQ DD NO:19, SEQ DD NO:21, SEQ DD NO:25, SEQ DD NO:27, SEQ DD NO:29, SEQ DD NO:31, SEQ DD NO:33, SEQ DD NO:35, and an amino acid sequence having at least 60% homology to any thereof.

4. The isolated peptide of claim 2, wherein the homology is at least 70% to the amino acid sequence.

5. The isolated peptide of claim 2 wherein the peptide consists of the amino acid sequence.

6. A method for identifying a /ysσ-phosphatidylcholine acyltransferase comprising: screening a peptide or a nucleic acid sequence encoding the peptide for at least one motif selected from the group consisting of SEQ DD NO:46, SEQ DD NO:47, SEQ DD NO:48, SEQ DD NO:49, SEQ DD NO:81, SEQ DD NO:82, SEQ DD NO:83, SEQ DD NO:84, or the corresponding nucleic acid sequence or sequences.

7. A method of identifying a peptide for (yso-phosphatidylcholine acyltransferase activity, the method comprising: screening the peptide for the presence of one or more of the following motifs of SEQ DD NO:46, SEQ DD NO:47, SEQ DD NO:48, SEQ DD NO:49, SEQ DD NO:81, SEQ DD NO:82, SEQ ID NO:83, and SEQ DD NO:84.

8. A method for increasing fatty acid production in a cell, the method comprising: transforming a cell with a nucleic acid molecule encoding a lyso-phosphatidylcholine acyltransferase; and growing the cell under conditions wherein the lyso-phosphatidylcholine acyltransferase is expressed.

9. The method according to claim 8, further comprising isolating the fatty acid.

10. The method according to claim 8, wherein the lyso-phosphatidylcholine acyltransferase comprises at least one motif selected from the group consisting of SEQ ID NO:46, SEQ ED NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ DD NO:81, SEQ ID NO: 82, SEQ DD NO:83, SEQ DD NO:84, and any combination thereof.

11. A method of altering oil content in a plant, the method comprising: screening for a peptide encoded by a nucleotide sequence for at least one motif selected from the group consisting of SEQ DD NO:46, SEQ DD NO:47, SEQ DD NO:48, SEQ DD NO:49, SEQ DD NO:81, SEQ DD NO:82, SEQ DD NO:83, SEQ DD NO:84, and any combination thereof; selecting the peptide based upon the presence of at least one of the four motifs; and expressing the nucleotide sequence encoding the peptide in the plant to alter the oil content of the plant.

12. A process of obtaining seeds, the process comprising: (a) transforming a plant by: i. transforming a plant cell with a recombinant DNA construct comprising a nucleic acid segment encoding a lyso-phosphatidylcholine acyltransferase comprising at least one motif selected from the group consisting of SEQ DD NO:46, SEQ DD NO:47, SEQ DD NO:48, SEQ DD NO:49, SEQ DD NO:81, SEQ DD NO:82, SEQ DD NO:83, SEQ DD NO: 84, and any combination thereof and a promoter for driving the expression of the nucleic acid segment in the plant cell to form a transformed plant cell, ii. regenerating the transformed plant cell into a transgenic plant, and iii. selecting transgenic plants that have enhanced levels of fatty acids in the seeds compared wild type strains of the same plant; (b) cultivating the transformed plant for one or more generations; and

(c) harvesting seeds from plants cultivated per (b).

13. A seed produced by a plant having increased lyso-phosphatidylcholine acyltransferase activity.

14. A process for obtaining oil comprising enhanced levels of fatty acids, the process comprising: extracting oil from the seed of claim 13.

15. Oil produced by the process of claim 14.

16. A composition comprising the oil of claim 15, wherein the composition is selected from the group of a food product, a pharmaceutical composition, and a nutraceutical composition.

17. A method of screening for a LPC acyltransferases (LPCAT), wherein the method comprises: expressing a candidate gene in a yeast LPCAT mutant, plating the yeast LPCAT mutant into a lyso-PAF (platelet-activating factor) environment, and detecting yeast colonies showing higher tolerance to lyso-PAF, wherein colonies exhibiting higher tolerance to lyso-PAF indicate that the candidate gene is a LPCAT gene.

18. The method according to claim 17, wherein the candidate gene is identified by screening a gene to determine the presence of one of more of nucleic acid sequences encoding at least one motif selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO.84, and any combination thereof.