Methods for Elevating Fat/Oil Content in Plants

Abstract

In some embodiments, the present invention provides a method of elevating lipid content in vegetative (non-seed) plant or algal cells, plant tissues, or whole plants by genetically modifying the plant or algae to express a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of animal origin or plant origin. Also provided are genetically-modified plant or algal cells, plant tissues, or whole plants with elevated cellular lipid content, expressing a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of animal (e.g. human) origin or plant origin.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. Provisional Application Ser. No. 61/739,499, filed Dec. 19, 2012, and U.S. Non-provisional application Ser. No. 13/830,012, filed Mar. 14, 2013, both of which are hereby incorporated by reference in their entirety, including any figures, tables, or drawings.

BACKGROUND OF THE INVENTION

[0002] Plants are a primary source of human and/or animal food, excellent feedstock for fuels, and useful for production of desirable chemicals. Plants synthesize and store lipids, primarily, in cytosolic lipid droplets. In plants, seeds are the primary site of oil synthesis and storage; vegetable oils (such as triacylglycerol) are used as a form of energy during seed germination. Vegetable oils can be synthesized in non-seed (such as leaf) tissues; however, their abundance is low and the stored lipids are presumed to be metabolized rapidly, perhaps for the recycling of fatty acids for energy or the synthesis of membrane lipids.

[0003] Plants that can accumulate oils in non-seed tissues are commercially attractive. The biomass of non-seed parts (such as leaves, stems) of plants is generally far greater than the amount accounted for by seeds. Thus, the transformation of non-seed tissues into oil-producing machinery can significantly increase the energy-production capacity. Currently, the regulation and transient accumulation of stored oils in non-seed tissues are not well understood, and the production of oils in non-seed plant tissues for industrial applications remains challenging. Cellular lipid droplets are dynamic organelles that regulate triglyceride storage in mammalian cells. Lipid droplets are composed of a core of neutral lipids surrounded by a phospholipid monolayer and associated proteins. Various proteins associated with lipid metabolism, including fat specific protein 27 (FSP27), perilipins, (Bernardinelli-Seip congenital lipodystrophy type 2 protein), FIT1 (fat storage-inducing transmembrane protein 1), and FIT2 (fat storage-inducing transmembrane protein 2) have been well characterized for their ability to regulate fat metabolism in mammalian species.

BRIEF SUMMARY OF THE INVENTION

[0004] In some embodiments, the present invention provides a method of elevating oil content in algae, plants, or plant parts by genetically modifying the plant to express a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of animal or plant origin. In one specific embodiment, the present invention provides a method of elevating oil content in vegetative (non-seed) plant tissues or algae.

[0005] In some embodiments, the present invention also provides genetically-modified algal cells, plant cells, tissues, or whole plants with elevated cellular oil content, wherein the algal cell, plant cell, tissue, or whole plant expresses a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of exogenous origin, for example, of exogenous animal origin or exogenous plant origin. In certain embodiments, the proteins or polypeptides associated with lipid metabolism useful according to the present invention are of mammalian origin. In some embodiments, the present invention provides a method for obtaining a plant cell or algal cell with elevated lipid content, wherein the method comprises:

[0006] genetically modifying the plant cell or algal cell to express an exogenous protein or polypeptide associated with lipid metabolism, thereby obtaining a genetically-modified plant cell or algal cell with elevated lipid content;

[0007] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity; and

[0008] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the genetically-modified plant cell or algal cell as compared to a wild-type (native) plant cell or algal cell of the same type.

[0009] In some embodiments, the present invention provides a method for obtaining a plant cell or algal cell with elevated lipid content, wherein the method comprises:

[0010] transforming the plant cell or algal cell with a vector comprising a nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism, wherein the nucleic acid is operably linked to a promoter and/or a regulatory sequence;

[0011] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity;

[0012] wherein the transformed plant cell or algal cell expresses the protein or polypeptide associated with lipid metabolism; and

[0013] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the transformed plant cell or algal cell as compared to a wild-type (native) plant cell or algal cell of the same type.

[0014] In certain embodiments, the genetically-modified plant cell is contained in a plant tissue, plant part, or whole plant.

[0015] In some embodiments, the genetically-modified plant cell or algal cell comprises, in its genome or in its plastome, a nucleic acid molecule encoding a protein or polypeptide associated with lipid metabolism.

[0016] In some embodiments, the protein or polypeptide associated with lipid metabolism is not of plant origin. In certain embodiments, the protein or polypeptide associated with lipid metabolism is of animal origin, such as of insect, vertebrate, fish, bird, amphibian, or mammalian (e.g., mouse, human) origin. In some embodiments, the protein or polypeptide associated with lipid metabolism is of plant origin.

[0017] In some embodiments, a T-DNA binary vector system is used for plant transformation. In one embodiment, plant transformation is performed using the floral dip method.

[0018] In certain embodiments, to elevate cellular lipid content and/or to induce lipid droplet production, the plant cell or the algal cell can be genetically engineered to expresses one or more proteins or polypeptides associated with lipid metabolism including, but not limited to, fat specific protein 27 (FSP27); perilipins including PLIN1 (perilipin 1) and PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)); SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein); FIT1 (fat storage-inducing transmembrane protein 1), and FIT2 (fat storage-inducing transmembrane protein 2); acyl-CoA:diacylglycerol acyltransferase 1 (DGAT-1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT-1); cell death activator (Cidea); leafy cotyledon 2 (LEC2); and WRINKLED1 (WRIT).

[0019] In certain embodiments, to elevate cellular lipid content and/or to induce lipid droplet production, the plant cell or the algal cell can be genetically engineered to expresses one or more proteins or polypeptides associated with lipid metabolism including, but not limited to FSP27, PLIN1, PLIN2, SEIPIN, FIT1, FIT2, and LEC2.

[0020] In certain specific embodiments, the transgenic plants or algae express a combination of proteins or polypeptides associated with lipid metabolism, wherein the protein or polypeptide associated with lipid metabolism is selected from: DGAT-1 and FSP27; DGAT-1, cgi58 (mutation), and FSP27; DGAT-1, PDAT-1, and FSP27; DGAT-1, PDAT-1, cgi58 (mutation), FSP27; FSP27, PLIN2, and cgi58 (mutation); DGAT-1, FSP27, PLIN2, and cgi58 (mutation); and DGAT-1, PDAT-1, FSP27, PLIN2, and cgi58 (mutation). In a further embodiment of the invention, the transgenic plants or algae express any combination of proteins or polypeptides associated with lipid metabolism selected from: DGAT-1, FSP27, cgi58 (mutation), PDAT-1, PLIN2, FIT1, FIT2, SEIPIN, LEC2, and WRIT. In certain other embodiments, various proteins or polypeptides associated with lipid metabolism expressed in a transgenic plant or algae are of different origin. For example, in an embodiment of the invention, a plant or algal cell expresses human FSP27 and SEIPIN.

[0021] In another embodiment, the present invention provides a method for obtaining an algae or bacterial cell with elevated lipid content, wherein the method comprises:

[0022] transforming an algae or bacterial cell with a vector comprising a nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism, wherein the nucleic acid is operably linked to a promoter and/or a regulatory sequence;

[0023] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity;

[0024] wherein the transformed algae or bacterial cell expresses the protein or polypeptide associated with lipid metabolism; and

[0025] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the transformed algae or bacterial cell as compared to a wild-type (native) algae or bacterial cell of the same type.

[0026] In certain embodiments, the algal cell can be genetically engineered to expresses any combinations of proteins associated with lipid metabolism and peptides including, but not limited to, FSP27; perilipins including PLIN1 and PLIN2; SEIPIN; FIT1 and FIT2; DGAT-1; PDAT-1; Cidea; LEC2; and WRIT.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1A is a diagram that illustrates embodiments of the transfer DNA (T-DNA) region of the binary vector for transformation of A. thaliana with the mouse fat specific protein 27 (FSP27) cDNA. The FSP27 open reading frame was inserted downstream from the 2.times. 35S promoter, either in-frame with green fluorescent protein (GFP) (pMDC43) or without (pMDC32). Binary vectors are known in the art, as described in Curtis and Grossniklaus (Plant Physiology, October 2003, Vol. 133, pp. 462-469), which is herein incorporated by reference in its entirety. Plasmid vectors were transformed into Agrobacterium tumefaciens LBA4404 and clones were selected and verified by PCR. Arabidopsis plants were transformed by the floral dip method of Bent and Clough (Plant J. 1998 December; 16(6):735-43.). Both wild-type plants (A. thaliana, ecotype Columbia), and plants with a T-DNA insertional mutation in the At4g24160 locus were used for transformations. The T-DNA knockout is in an exon of the Arabidopsis homolog of the human CGI-58 gene, and in Arabidopsis plants with this mutation there is an increase in cytosolic lipid droplets in leaves (James et al., Proc. Natl. Acad. Sci. USA. 2010 Oct. 12; 107(41):17833-8).

[0028] FIG. 1B are Confocal Laser Scanning Microscopy images of leaves of approximately 30-d-old Arabidopsis seedlings stained with the neutral lipid-specific stain, Nile blue. Red autofluorescence is from chlorophyll and shows the location of chloroplasts distributed around the perimeter of leaf mesophyll cells. Lipid droplets (blue) are distributed throughout the cytosol of the cells and are more abundant in transgenic seedlings expressing mouse FSP27 than in non-transformed cells (WT). Bar is 20 microns.

[0029] FIG. 2 shows representative Confocal Laser Scanning Microscopy images of leaves of approximately 30-day-old A. thaliana seedlings stained with Nile blue--a neutral lipid-specific stain. Red autofluorescence emitted from chlorophylls shows the location of chloroplasts distributed around the perimeter of leaf mesophyll cells. Lipid droplets (blue) are distributed throughout the cytosol of the cells and are more abundant in transgenic seedlings expressing mouse FSP27 than in non-transformed cells (WT). Bar is 20 microns.

[0030] FIG. 3 shows representative Confocal Laser Scanning Microscopy images of leaves of approximately 30-day-old A. thaliana seedlings stained with BODIPY 493/503--a neutral lipid-specific stain. Red autofluorescence emitted from chlorophylls shows the location of chloroplasts distributed around the perimeter of leaf mesophyll cells. Lipid droplets (yellow-green with BODIPY staining) are distributed throughout the cytosol of the cells and are more abundant in transgenic seedlings expressing mouse FSP27 than in non-transformed cells (cgi58). Bar is 20 microns.

[0031] FIG. 4 shows representative Confocal Laser Scanning Microscopy images of leaves of approximately 30-day-old A. thaliana seedlings stained with Nile blue--a neutral lipid-specific stain. Red autofluorescence emitted from chlorophylls shows the location of chloroplasts distributed around the perimeter of leaf mesophyll cells. GFP fluorescence (green) marks the location of the mouse FSP27-GFP fusion protein. Lipid droplets (blue) are distributed throughout the cytosol of the cells and are more abundant in the cgi58 mutant background than in the wild-type background. More lipid droplets are formed in leaves of transformed plants than in untransformed leaves (see also FIG. 2). Scale bars represent 20 microns.

[0032] FIG. 5 shows the content of total fatty acids extracted from 15-day-old A. thaliana seedlings sown on solidified nutrient medium. The total fatty acid content is shown on a fresh weight basis. Transgenic plants (mouse FSP27-GFP in the cgi58 mutant background) in the T1 generation are selected using hygromycin medium. Despite the inclusion of heterozygotes in the analysis, the FSP27-transformed plants exhibit a measureable increase in total lipid content. Also, it is postulated that the transfer of FSP27 stabilizes the variable cgi58 phenotype (reduced standard deviation in the FSP27 expressing plants). Values are the means and standard deviation of three replicates.

[0033] FIG. 6 shows the content of total fatty acids extracted from 15-day-old A. thaliana seedlings sown on solidified nutrient medium. The total fatty acid content is shown on a dry weight basis. Transgenic plants (expressing mouse FSP27-GFP or mouse autosomal dominant retinitis pigmentosa (ADRP)) in the T1 generation are selected on hygromycin medium. All FSP27-GFP or ADSP transgenic plants have a higher average lipid content in the T1 generation than that of the non-transformed plants, and one line (cgi58-43fsp27line1) has a statistically higher lipid content (P<0.05) than that of non-transformed plants. Values are the means and standard deviations of five replicates.

[0034] FIG. 7A-C show confocal fluorescence micrographs of leaves in Arabidopsis plants expressing ADRP (lower left; A-C) or FSP27 (lower right; A-C) in the cgi58 knockout background. Red autofluorescence is marking chloroplasts; green fluorescence is from the neutral-lipid-specific stain-BODIPY 493/503, showing the accumulation of lipid droplets in leaves. The upper left is wild-type; upper left is the cgi58 knockout background alone.

[0035] FIG. 8 shows that amino acids 120-220 of FSP27 are associated with lipid accumulation. Amino acids 120-220 of FSP27 and the full length FSP27 are expressed in human adipocytes using lentivirus. X-axis shows total triglycerides in adipocytes. Note that the human adipocytes already have huge amount of triglycerides, and the expression of FSP27 (full length) and FSP27 (120-220) significant increase triglyceride contents in adipocytes by almost 40%. *, p<0.05, t-test.

[0036] FIG. 9 shows sequence similarity between mouse and zebra fish FSP27 protein. NP.sub.--848460.1: CIDE-3 Mus musculus (mouse); NP.sub.--001038512.1: CIDE-3 Danio rerio (zebra fish).

[0037] FIG. 10 shows motif locations of various SEIPIN homologs from H. sapiens, S. cereviciae, and A. thaliana.

[0038] FIG. 11 shows sequence alignment of various SEIPIN homologs from H. sapiens, S. cereviciae, and A. thaliana.

[0039] FIG. 12 shows developmental and tissue-specific expression profiles of Arabidopsis SEIPIN genes identified by semi-quantitative reverse transcriptase (RT)-PCR analysis of Arabidopsis SEIPIN isoforms. Constitutively-expressed elongation factor (EF)1-alpha is included for comparison. SEIPIN2 and SEIPIN3 appear to be more constitutively expressed and may function in a partially redundant manner. Whereas, SEIPIN1 seems only to be expressed in seeds and seedlings.

[0040] FIG. 13 shows lipid droplet staining in wild type and genetically modified yeast. Green fluorescence is from the neutral-lipid-specific stain-BODIPY 493/503, showing the accumulation of lipid droplets. The top left panel shows lipid droplets in wild type yeast, top middle panel shows lipid droplets in ylr404w.DELTA., which is a yeast having a deletion of yeast SEIPIN protein. The top right panel shows lipid droplets in ylr404w.DELTA., expressing yeast SEIPIN. The bottom left panel shows lipid droplets in ylr404w.DELTA., expressing yeast A. thaliana SEIPIN1, the bottom middle panel shows lipid droplets in ylr404w.DELTA., expressing A. thaliana SEIPIN2, and the bottom right panel shows lipid droplets in ylr404w.DELTA., expressing A. thaliana SEIPIN3. Expression of A. thaliana SEIPIN1, 2, or 3 restores lipid droplet accumulation in ylr404w.DELTA..

[0041] FIG. 14 shows quantification of lipid droplets in terms of the number of lipid droplets per cell in wild type and genetically modified yeast. Number of lipid droplets is significantly reduced in ylr404w.DELTA. compared to wild type yeast. Expression of A. thaliana SEIPIN1, 2, or 3 restores lipid droplet accumulation in ylr404w.DELTA. to certain extent with A. thaliana SEIPIN3 having the maximum effect in terms of the number of lipid droplets per yeast cell.

[0042] FIG. 15 shows lipid droplet staining in wild type and genetically modified yeast. Green fluorescence is from the neutral-lipid-specific stain-BODIPY 493/503, showing the accumulation of lipid droplets. The size of lipid droplets is significant increased in ylr404w.DELTA. compared to wild type yeast. Expression of A. thaliana SEIPIN1, 2, or 3 did not restore the number of lipid droplets in ylr404w.DELTA. to those observed in wild type yeast. Expression of A. thaliana SEIPINs also increased the size of lipid droplets in ylr404w.DELTA. compared to wild type yeast, with A. thaliana SEIPIN1 producing the biggest lipid droplets amongst the mutants tested.

[0043] FIG. 16 shows quantification of lipid droplets in terms of the size of lipid droplets in wild type and genetically modified yeast. The size of lipid droplets is significant increased in ylr404w.DELTA. compared to wild type yeast. Expression of A. thaliana SEIPIN1, 2, or 3 did not restore the size of lipid droplets in ylr404w.DELTA. to those observed in wild type yeast. Expression of A. thaliana SEIPINs also increased the size of lipid droplets in ylr404w.DELTA. compared to wild type yeast with A. thaliana SEIPIN1 producing the biggest lipid droplets amongst the mutants tested.

[0044] FIG. 17 further illustrates changes in the size of the lipid droplets in wild type and genetically modified yeast.

[0045] FIG. 18 shows localization of A. thaliana SEIPIN1 to lipid droplets when expressed in yeast. The top left panel indicates Nile Red staining of lipid droplets and the top right column shows green fluorescence indicating localization of A. thaliana SEIPIN1-GFP. The bottom left panel shows endoplasmic reticulum with blue fluorescence coming from cyano fluorescence protein (CFP) fused to HDEL, which is a C-terminal tetrapeptide found in yeast and plants allowing the sorting of the proteins in the lumen of the endoplasmic reticulum. The bottom right panel shows the merged figure of the other three panels indicating that A. thaliana SEIPIN1-GFP colocalises with lipid droplets in yeast.

[0046] FIG. 19 shows localization of A. thaliana SEIPIN2 to lipid droplets when expressed in yeast. The top left panel indicates Nile Red staining of lipid droplets and the top right column shows green fluorescence indicating localization of A. thaliana SEIPIN2-GFP. The bottom left panel shows endoplasmic reticulum with blue fluorescence coming from CFP fused to HDEL. The bottom right panel shows the merged figure of the other three panels indicating that A. thaliana SEIPIN2-GFP colocalises with lipid droplets yeast.

[0047] FIG. 20 shows localization of A. thaliana SEIPIN3 to lipid droplets when expressed in yeast. The top left panel indicates Nile Red staining of lipid droplets and the top right column shows green fluorescence indicating localization of A. thaliana SEIPIN3-GFP. The bottom left panel shows endoplasmic reticulum with blue fluorescence coming from CFP fused to HDEL. The bottom right panel shows the merged figure of the other three panels indicating that A. thaliana SEIPIN3-GFP colocalises with lipid droplets yeast.

[0048] FIG. 21 shows quantification of lipid droplets in terms of the amount of triacylglyceride (TAG) amount in lipid droplets in the wild type and genetically modified yeast. The amount of TAG in lipid droplets is significant decreased in ylr404w.DELTA. compared to wild type yeast. Expression of yeast SEIPIN and A. thaliana SEIPIN1, 2, or 3 restored the amount of TAG in the lipid droplets in ylr404w.DELTA. to those observed in wild type yeast. (* represents p=0.02.)

[0049] FIGS. 22 and 23 show quantification of different types of TAG in lipid droplets in the wild type and genetically modified yeast. (* represents p=0.05.)

[0050] FIG. 24 provides a summary of the morphologies of lipid droplets in in the wild type and genetically modified yeast. The phrase "Not numbers" indicates that A. thaliana SEIPIN does not restore the number of lipid droplets in ylr404w.DELTA. to those found in the wild type yeast. The phrase "Not size" indicates that A. thaliana SEIPIN does not restore the size of lipid droplets in ylr404w.DELTA. to those found in the wild type yeast. The phrase ".uparw. numbers" indicates that A. thaliana SEIPIN increases the number of lipid droplets in ylr404w.DELTA. when expressed therein; and the phrase ".uparw. size" indicates that A. thaliana SEIPIN increases the size of lipid droplets in ylr404w.DELTA. when expressed therein.

[0051] FIG. 25 shows schematic representation of transient expression of exogenous genes in N. benthamiana.

[0052] FIG. 26 shows RT-PCR confirming the expression of exogenous genes in N. benthamiana.

[0053] FIG. 27 shows lipid droplet and chloroplast staining of various N. benthamiana lines expressing exogenous genes. Red autofluorescence is marking chloroplasts; green fluorescence is from the neutral-lipid-specific stain-BODIPY 493/503, showing the accumulation of lipid droplets in leaves.

[0054] FIG. 28 shows average number of lipid droplets in various N. benthamiana lines expressing exogenous genes.

[0055] I: Mock.

[0056] II: 35S:P19.

[0057] III: 35S:P19+35S:AtSEIPIN1.

[0058] IV: 35S:P19+35 S:AtSEIPIN2.

[0059] V: 35S:P19+35S:AtSEIPIN3.

[0060] VI: 35S:P19+35S:AtSEIPIN1+35S:AtSEIPIN2.

[0061] VII: 35S:P19+35 S:AtSEIPIN1+35S:AtSEIPIN3.

[0062] VIII: 35 S:P19+35 S:AtSEIPIN2+35 S:AtSEIPIN3.

[0063] IX: 35S:P19+35 S:AtSEIPIN1+35S:AtSEIPIN2+35S:AtSEIPIN3.

[0064] X: 35S:P19+35S:AtLEC2, XI: 35S:P19+35S:AtLEC2+35S:AtSEIPIN1.

[0065] XII: 35S:P19+35S:AtLEC2+35S:AtSEIPIN2.

[0066] IX: 35S:P19+35S:AtLEC2+35S:AtSEIPIN3.

[0067] XIV: 35S:P19+35S:AtLEC2+35S:AtSEIPIN1+35S:AtSEIPIN2+35S:AtSEIPIN3.

[0068] (#0.005<p<0.05, * p<0.005.)

[0069] FIG. 29 shows average number of lipid droplets of various sizes in various N. benthamiana lines expressing exogenous genes.

[0070] I: Mock.

[0071] II: 35S:P19.

[0072] III: 35S:P19+35S:AtSEIPIN1.

[0073] IV: 35S:P19+35S:AtSEIPIN2.

[0074] V: 35S:P19+35S:AtSEIPIN3.

[0075] VI: 35S:P19+35S:AtSEIPIN1+35S:AtSEIPIN2.

[0076] VII: 35S:P19+35S:AtSEIPIN1+35S:AtSEIPIN3.

[0077] VIII: 35S:P19+35S:AtSEIPIN2+35S:AtSEIPIN3.

[0078] IX: 35S:P19+35S:AtSEIPIN1+35S:AtSEIPIN2+35S:AtSEIPIN3.

[0079] X: 35S:P19+35S:AtLEC2, XI: 35S:P19+35S:AtLEC2+35S:AtSEIPIN1.

[0080] XII: 35S:P19+35S:AtLEC2+35S:AtSEIPIN2.

[0081] IX: 35S:P19+35S:AtLEC2+35S:AtSEIPIN3.

[0082] XIV: 35S:P19+35S:AtLEC2+35S:AtSEIPIN1+35S:AtSEIPIN2+35S:AtSEIPIN3.

[0083] (#0.005<p<0.05, * p<0.005.)

[0084] FIG. 30 shows lipid droplet and chloroplast staining of various N. benthamiana lines expressing exogenous genes.

[0085] FIG. 31 shows transient expression of mouse FIT2 in N. benthamiana leaf tissue. Top left panel shows leaves transfected with empty vector, bottom left panel shows leaves transfected with 35S-P19, and large panel on the right shows leaves transfected with P19 and mouse FIT2. The presence of green fluorescence in P19 and mouse FIT2 transfected leaves indicates accumulation of lipid droplets in these leaves.

[0086] FIG. 32 shows transient expression of A. thaliana LEC2 in N. benthamiana leaf tissue. Red autofluorescence is marking chloroplasts; green fluorescence is from the neutral-lipid-specific stain-BODIPY 493/503, showing the accumulation of lipid droplets in leaves. Top left panel shows leaves transfected with empty vector, bottom left panel shows leaves transfected with 35S-P19, and large panel on the right shows leaves transfected with P19 and A. thaliana LEC2. The presence of green fluorescence in P19 and A. thaliana LEC2 transfected leaves indicates accumulation of lipid droplets in these leaves.

[0087] FIG. 33 shows transient expression of GFP-mouse FIT2 in N. benthamiana leaf tissue. Top left panel shows green fluorescence originating from GFP-mouse FIT2 marking the ER. Top middle panel shows lipid droplets stained in yellow with Nile Red stain. Top right panel shows overlap of green endoplasmic reticulum fluorescence and yellow lipid droplet staining Bottom left panel shows overlap of green endoplasmic reticulum fluorescence and yellow lipid droplet staining, further showing red autofluorescence marking chloroplasts. Bottom right panel shows a portion of the bottom left panel magnified to more clearly indicate the colocalization of endoplasmic reticulum and lipid droplets. These figures suggest that GFP-mouse FIT2 colocalize with lipid droplets in N. benthamiana leaves.

[0088] FIG. 34 shows that stable expression of FIT2 increased lipid droplets accumulation in A. thaliana leaves. The top left panel shows Nile Red staining of wild type A. thaliana leaves and the top right panel shows a portion of the top left panel magnified to more clearly display Nile Red staining. The bottom left panel shows Nile Red staining of A. thaliana leaves in which GFP-FIT2 is overexpressed and the bottom right panel shows a portion of the bottom left panel magnified to more clearly display Nile Red staining Increased Nile Red staining of A. thaliana leaves in which GFP-FIT2 is overexpressed indicates that FIT2 causes the accumulation of lipid droplets.

[0089] FIG. 35 shows expression of GFP-mouse FIT2 in A. thaliana. Top left panel shows green fluorescence originating from GFP-mouse FIT2 indicating the ER. Top middle panel shows lipid droplets stained in yellow with Nile Red stain. Top right panel shows overlap of green endoplasmic reticulum fluorescence and yellow lipid droplet staining Bottom left panel shows overlap of green endoplasmic reticulum fluorescence and yellow lipid droplet staining further showing red autofluorescence marking chloroplasts. These figures suggest that GFP-mouse FIT2 colocalizes with lipid droplets in A. thaliana leaves.

[0090] FIG. 36 shows the oil contents of A. thaliana seeds sown on solidified nutrient medium. The total fatty acid content is shown on percent basis. Transgenic plants expressing mouse FSP27 or mouse autosomal dominant ADRP in the T2 or T3 generation are grown. Cgi-58 32 FSP 27, T2 lines 1-4 and cgi-58 32 FSP27, T3 lines 1-4 transgenic plants have a significantly higher average lipid content than that of the non-transformed plants. Values are the means and standard deviations.

BRIEF DESCRIPTION OF THE SEQUENCES

[0091] SEQ ID NO:1 is the amino acid sequence of a human fat specific protein 27 (FSP27) (GenBank Accession Q96AQ7).

[0092] SEQ ID NO:2 is the amino acid sequence of a mouse fat specific protein 27 (FSP27) (GenBank Accession NP 848460).

[0093] SEQ ID NO:3 is the amino acid sequence of a human PLN1 (perilipin 1) (GenBank Accession NP 002657).

[0094] SEQ ID NO:4 is the amino acid sequence of a mouse PLN1 (perilipin 1) (GenBank Accession Q96AQ7).

[0095] SEQ ID NO:5 is the amino acid sequence of a human PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)) (GenBank Accession NP.sub.--001106942).

[0096] SEQ ID NO:6 is the amino acid sequence of a mouse PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)) (GenBank Accession NP.sub.--031434).

[0097] SEQ ID NO:7 is the amino acid sequence of a human SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein) (GenBank Accession Q96G97).

[0098] SEQ ID NO:8 is the amino acid sequence of a mouse SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein) (GenBank Accession AAH43023).

[0099] SEQ ID NO:9 is the amino acid sequence of a human FIT1 (fat storage-inducing transmembrane protein 1) (GenBank Accession A5D6W6).

[0100] SEQ ID NO:10 is the amino acid sequence of a mouse FIT1 (fat storage-inducing transmembrane protein 1) (GenBank Accession NP.sub.--081084).

[0101] SEQ ID NO:11 is the amino acid sequence of a human FIT2 (fat storage-inducing transmembrane protein 2) (GenBank Accession Q8N6M3).

[0102] SEQ ID NO:12 is the amino acid sequence of a mouse FIT2 (fat storage-inducing transmembrane protein 2) (GenBank Accession NP.sub.--775573).

[0103] SEQ ID NO:13 is the mRNA sequence of the At4g24160 gene (GenBank Accession BT029749).

[0104] SEQ ID NO:14 is the amino acid sequence of the full length polypeptide encoded at the At4g24160 locus (GenBank Accession ABM06019).

[0105] SEQ ID NO:15 is the amino acid sequence of a diacylglycerol acyltransferase 1 [Jatropha curcas] (GenBank Accession ACA49853). SEQ ID NO:16 is the amino acid sequence of a phospholipid: diacylglycerol acyltransferase 1 [Jatropha curcas] (GenBank Accession AED91921).

[0106] SEQ ID NO:17 is the amino acid sequence of a phospholipid:diacylglycerol acyltransferase 1 [Laccaria bicolor] (GenBank Accession EDR11533).

[0107] SEQ ID NO:18 is the amino acid sequence of a phospholipid:diacylglycerol acyltransferase 1 [Scheffersomvces stipitis] (GenBank Accession ABN67418).

[0108] SEQ ID NO:19 is the amino acid sequence of an adipose triglyceride lipase [Homo sapiens] (GenBank Accession AAW81962).

[0109] SEQ ID NO:20 is the amino acid sequence of an adipose triglyceride lipase [Mus musculus] (GenBank Accession AAW81963).

[0110] SEQ ID NO:21 is the amino acid sequence of a cell death activator [Homo sapiens] (GenBank Accession AAQ65241).

[0111] SEQ ID NO:22 is the amino acid sequence of a cell death activator [Mus musculus] (GenBank Accession NP.sub.--031728).

[0112] SEQ ID NO:23 is the amino acid sequence of a WRINKLED1 [A. thaliana] (GenBank Accession AAP80382).

[0113] SEQ ID NO:24 is the amino acid sequence of a cell death activator CIDE-3 [Danio rerio] (GenBank Accession NP.sub.--001038512).

[0114] SEQ ID NO:25 is the amino acid sequence of human lysophosphatidic acid acyltransferase alpha (LPAAT) (GenBank Accession NP.sub.--116130).

[0115] SEQ ID NO:26 is the amino acid sequence of mouse lysophosphatidic acid acyltransferase alpha isoform 1 (GenBank Accession NP.sub.--001156851).

[0116] SEQ ID NO:27 is the amino acid sequence of mouse Glycerol-3-phosphate acyltransferase 1, mitochondrial (GenBank Accession NP.sub.--032175).

[0117] SEQ ID NO:28 is the amino acid sequence of wild boar (Sus scrofa) Glycerol-3-phosphate acyltransferase 1, partial (GenBank Accession AAP74372).

[0118] SEQ ID NO:29 is the amino acid sequence of mouse Complement factor D (adipsin) (GenBank Accession AAI38780).

[0119] SEQ ID NO:30 is the amino acid sequence of wild boar (Sus scrofa) Complement factor D (adipsin), partial (GenBank Accession AAQ63882).

[0120] SEQ ID NO:31 is the amino acid sequence of mouse phosphatidate phosphatase PLIN1 isoform a (GenBank Accession NP.sub.--001123884).

[0121] SEQ ID NO:32 is the amino acid sequence of mouse phosphatidate phosphatase PLIN2 isoform 1 (GenBank Accession NP.sub.--001158357).

[0122] SEQ ID NO:33 is the amino acid sequence of A. thaliana SEIPIN1 (GenBank Accession AED92296).

[0123] SEQ ID NO:34 is the amino acid sequence of A. thaliana SEIPIN2 (GenBank Accession AEE31126).

[0124] SEQ ID NO:35 is the amino acid sequence of A. thaliana SEIPIN3 (GenBank Accession AEC08966).

[0125] SEQ ID NO:36 is the amino acid sequence of A. thaliana LEC2 (GenBank Accession ABE65660).

[0126] SEQ ID NO:37 is the amino acid sequence of tomato bushy stunt virus P19 protein (GenBank Accession AEC08966).

DETAILED DISCLOSURE OF THE INVENTION

[0127] In some embodiments, the present invention relates the use of proteins associated with lipid metabolism originated from animals or plants to elevate the lipid content in vegetative tissues (such as leaves) of plants. In certain embodiments, the proteins or polypeptides associated with lipid metabolism useful according to the present invention are of mammalian origin.

[0128] As lipid has more than twice the energy content of carbohydrate or protein, the present invention can be used to increase energy content in crop biomass, useful for production of biofuel, renewable chemical feedstocks, animal feed, and nutritional products. The term "lipid," as used throughout, encompasses oils (such as triglyceride), and in some embodiments "lipid" is oil.

[0129] For the purpose of this invention, the term "protein or polypeptide associated with lipid metabolism" refers to a protein or polypeptide which is a "lipid droplet-associated protein or polypeptide," "endoplasmic reticulum (ER) associated protein or polypeptide that localizes to domains of ER that form lipid droplets," "lipid droplet forming protein or polypeptide," or "lipid forming protein or polypeptide." In some embodiments, a protein associated with lipid metabolism, designated as fat storage protein 27 (FSP27), is expressed in leaves of transgenic Arabidoposis thaliana plants.

[0130] Neutral lipid-specific fluorescent staining of cystolic lipid droplets reveals a marked increase in the number and size of lipid droplets in the mesophyll cells of the levels of transgenic plants, when compared with non-transformed plants of the same type. The expression of a fluorescent-tagged mouse FSP27 protein in transgenic plants shows the FSP27 protein associated with the lipid droplets in plant cells, similar to that of mouse adipocytes. When the FSP27 protein is expressed in the Arabidopsis cgi58 mutant background, lipid droplet formation and lipid content in leaves are further augmented, when compared to transgenic Arabidopsis plants that only express FSP27 or Arabidopsis cgi58 mutant.

[0131] In some embodiments, the present invention provides a method of elevating lipid content in a plant or plant part by genetically modifying the plant to express a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of animal origin in the plant or plant part. In one specific embodiment, the present invention provides a method of elevating lipid content in vegetative (non-seed) plant tissues.

[0132] In some embodiments, the present invention also provides genetically-modified algal cells, plant cells, tissues, or whole plants with elevated cellular lipid content, wherein the algal cells, plant cells, tissues or whole plants express a protein or polypeptide associated with lipid metabolism (such as fat-specific protein 27) of animal origin or plant origin.

Genetically-Modified Plants with Elevated Lipid Content and/or Lipid Droplet Production

[0133] In some embodiments, the present invention provides a method for obtaining a plant cell or an algal cell with elevated lipid content, wherein the method comprises:

genetically modifying the plant cell or the algal cell to express an exogenous protein or polypeptide associated with lipid metabolism, thereby obtaining a genetically-modified plant cell with elevated lipid content;

[0134] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity; and

[0135] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the genetically-modified plant cell or algal cell, when compared to a wild-type (native) plant cell or algal cell of the same type.

[0136] In some embodiments, the present invention provides a method for obtaining a plant cell or an algal cell with elevated lipid content, wherein the method comprises:

[0137] transforming the plant cell or the algal cell with a vector comprising a nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism, yielding a transformed cell wherein the nucleic acid is operably linked to a promoter and/or a regulatory sequence;

[0138] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity;

[0139] wherein the transformed plant cell or algal cell expresses the protein or polypeptide associated with lipid metabolism; and

[0140] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the transformed plant cell or algal cell as compared to a wild-type (native) plant cell or algal cell of the same type.

[0141] In certain embodiments, the genetically-modified plant cell is contained in an algal cell, a plant tissue, plant part, or whole plant.

[0142] In some embodiments, the genetically-modified plant cell comprises, in its genome, a nucleic acid molecule encoding a protein or polypeptide associated with lipid metabolism.

[0143] In some embodiments, the protein or polypeptide associated with lipid metabolism is not of plant origin. In certain embodiments, the protein or polypeptide associated with lipid metabolism is of animal origin, such as of insect, vertebrate, amphibian, or mammalian (e.g., mouse, human) origin. In another embodiment, the protein or polypeptide associated with lipid metabolism is of plant origin.

[0144] In some embodiments, a T-DNA binary vector system is used for plant transformation. A T-DNA binary vector system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. In one embodiment, the T-DNA region located on the binary vector comprises a vector nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism.

[0145] T-DNA binary vector systems are routinely used in plant transformation. A variety of vectors and expression cassettes useful for performing plant transformation are described in Curtis and Grossniklaus (2003), which is herein incorporated by reference in its entirety. Non-limiting examples of vectors and expression cassettes useful in accordance with the present invention include pMDC32, pMDC7, pMDC30, pMDC45, pMDC44, pMDC43, pMDC83, pMDC84, pMDC85, pMDC139, pMDC140, pMDC141, pMDC107, pMDC111, pMDC110, pMDC162, pMDC163, pMDC164, pMDC99, pMDC100, and pMDC123.

[0146] In some embodiments, plant transformation is performed using the floral dip method, as describe in Bent and Clough (1998), which is herein incorporated by reference in its entirety.

[0147] In certain embodiments, to elevate cellular lipid content and/or to induce lipid droplet production, the plant cell can be genetically engineered to expresses one or more proteins or polypeptides associated with lipid metabolism including, but not limited to, fat specific protein 27 (FSP27); perilipins including PLIN1 (perilipin 1) and PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)); SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein); FIT1 (fat storage-inducing transmembrane protein 1), and FIT2 (fat storage-inducing transmembrane protein 2); acyl-CoA:diacylglycerol acyltransferase 1 (DGAT-1); phospholipid:diacylglycerol acyltransferase 1 (PDAT-1); cell death activator (Cidea); and WRINKLED1 (WRI1).

[0148] In certain specific embodiments, the plant cell or the algal cell can be genetically engineered to express one or more functional domains of the proteins associated with lipid metabolism, wherein the functional domain is involved lipid metabolism, including, but not limited to, the synthesis, protection, accumulation, storage, or breakdown of lipids.

[0149] In another embodiment, to elevate cellular lipid content and/or to induce lipid droplet production, the plant cell or the algal cell can be genetically engineered to over-express one or more proteins or polypeptides associated with lipid metabolism of plant origin.

[0150] A variety of proteins associated with lipid metabolism are known in the art; amino acid sequences of proteins associated with lipid metabolism, as well as cDNA sequences encoding proteins associated with lipid metabolism, are publically available, such as via the GenBank database.

[0151] Fat Specific Protein 27 (FSP27), a lipid droplet (LD) associated protein in adipocytes, regulates triglyceride (TG) storage. FSP27 plays a key role in LD morphology to accumulate TGs. FSP27 facilitates LD clustering and promotes their fusion to form enlarged droplets, resulting in triglyceride accumulation. Functional domains of FSP27 responsible for LD formation have been characterized (see Jambunathan et al., 2011, which is hereby incorporated by reference in its entirety). Specifically, amino acids 173-220 of human FSP27 are necessary and sufficient for both the targeting of FSP27 to LDs and the initial clustering of the droplets. Amino acids 120-140 of human FSP27 are essential but not sufficient for LD enlargement, whereas amino acids 120-210 of human FSP27 are necessary and sufficient for both clustering and fusion of LDs to form enlarged droplets. In addition, FSP27-mediated enlargement of LDs, but not their clustering, is associated with triglyceride accumulation. CIDEC (human ortholog of FSP27) results in the accumulation of multiple, small LD's in white adipocytes in vivo.

[0152] In certain embodiments, the plant cell or the algal cell can be genetically engineered to express one or more functional domains of FSP27, including, but not limited to, amino acids 173-220 of human FSP27, amino acids 120-140 of human FSP27, amino acids 120-210 of human FSP27, or any fragment having no fewer than 10 consecutive amino acids (such as, more than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 consecutive amino acids) of the aforementioned functional domains.

[0153] In certain embodiments, the plant cell or the algal cell can be genetically engineered to express a FSP protein or peptide that corresponds to amino acids 120-220 of mouse FSP27 of SEQ ID NO:2 (GenBank Accession No. NP.sub.--848460), or any fragment thereof having no fewer than 10 consecutive amino acids (such as, more than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 consecutive amino acids).

[0154] Members of the PAT family (also called the perilipin (PLIN) family), which regulate lipolysis, are a family of proteins associated with lipid metabolism that have been well characterized in the art. Perilipins function as a protective coating from the body's natural lipases, such as hormone-sensitive lipase, which break triglycerides into glycerol and free fatty acids for use in metabolism--a process called lipolysis.

[0155] Acyl-CoA: diacylglycerol acyltransferase 1 (DGAT-1) and phospholipid: diacylglycerol acyltransferase 1 (PDAT-1) proteins are essential for triacylglyceride (Oil) biosynthesis in plants and seeds. DGAT-1 is also responsible for triglyceride biosynthesis in mammals. See Zhang et al. (2009) Plant Cell 21, 3885-901, PMID: 20040537, which is hereby incorporated as reference in its entirety.

[0156] Mutations in cgi58 (plant ortholog is also called cgi58) can be used to increase in plant oil contents. See James et al. (2010) PNAS 107, 17833-1838, PMID: 20876112, which is hereby incorporated as reference in its entirety.

[0157] Yeast gene SEIPIN (human ortholog is also called SEIPIN) can be used to increase the size of oil droplets in mammalian cells. See Szymanski et al. (2007) PNAS 104, 20890-5, PMID: 18093937, which is hereby incorporated as reference in its entirety.

[0158] FIT1 and FIT2 proteins, which belong to the FIT family (also have orthologues in yeast), play an important role in lipid droplet formation. Gross et al. (2011) PNAS 108, 19581-19586; PMID: 22106267, which is hereby incorporated as reference in its entirety.

[0159] Mammalian genes PLIN1 and PLIN2 play a role in protecting against breakdown of fat (called hydrolysis or lipolysis).

[0160] Cgi58 activate lipases (e.g., adipose triglyceride lipase (ATGL)), which catalyze the breakdown of lipids.

[0161] Cell death activator (Cidea), a novel gene identified by the inventors, plays a role in triglyceride accumulation in humans.

[0162] In certain embodiments, the plant cell or the algal cell can be genetically engineered to expresses any combinations of proteins associated with lipid metabolism and peptides including, but not limited to, fat specific protein 27 (FSP27); perilipins including PLIN1 (perilipin 1) and PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)); SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein); FIT1 (fat storage-inducing transmembrane protein 1), and FIT2 (fat storage-inducing transmembrane protein 2); acyl-CoA:diacylglycerol acyltransferase 1 (DGAT-1); phospholipid:diacylglycerol acyltransferase 1 (PDAT-1); cell death activator (Cidea); and WRINKLED1 (WRIT).

[0163] In one embodiment, the plant cell can be genetically engineered to expresses one or more proteins associated with lipid metabolism in a cgi58 (mutation) background, wherein the cgi58 (mutation) background results in enhanced lipid/oil content in plants.

[0164] In certain specific embodiments, the transgenic plants or algae express a combination of nucleic acids expressing proteins associated with lipid metabolism selected from: DGAT-1 and FSP27; DGAT-1, cgi58 (mutation), and FSP27; DGAT-1, PDAT-1, and FSP27; DGAT-1, PDAT-1, cgi58 (mutation), FSP27; FSP27, PLIN2, and cgi58 (mutation); DGAT-1, FSP27, PLIN2, and cgi58 (mutation); and DGAT-1, PDAT-1, FSP27, PLIN2, and cgi58 (mutation). In some embodiments, any protein or polypeptide associated with lipid metabolism of animal origin can be used in accordance with the present invention. In certain embodiments, suitable proteins or polypeptides associated with lipid metabolism can be originated from insects, fish, birds, vertebrates, amphibians, and mammalian species including, but not limited to apes, chimpanzees, orangutans, humans, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs, and hamsters.

[0165] In certain embodiments, the plant cell or the algal cell can be genetically engineered to expresses a protein or polypeptide associated with lipid metabolism comprising any of SEQ ID NOs: 1-12 and 14-36, a homolog or variant thereof, or a functional fragment of a protein or polypeptide associated with lipid metabolism comprising any of SEQ ID NOs: 1-12, 14-36 or a homolog or variant thereof, wherein the functional variant and the functional fragment induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity.

[0166] In certain embodiments, a variant of a protein or polypeptide associated with lipid metabolism comprising a sequence of SEQ ID NOs:1-12, 14-36 comprises an amino acid sequence that may share about at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater sequence similarity at the respective amino acid sequence of SEQ ID NOs:1-12, 14-36.

[0167] The term "homolog," as used herein, refers to genes or proteins related to each other by descent from a common ancestral DNA (such as genes) or protein sequence. In certain embodiments, a homolog of a protein or polypeptide associated with lipid metabolism comprising a sequence of SEQ ID NOs:1-12, 14-36 comprises an amino acid sequence that may share about at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater sequence similarity at the respective amino acid sequence of SEQ ID NOs:1-12, 14-36.

[0168] The sequence identity will typically be greater than 75%, preferably greater than 80%, more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.

[0169] Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, word length=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/NIH website.

[0170] Furthermore, as various proteins associated with lipid metabolism have been well characterized in the art, a skilled artisan can readily make modifications to native or naturally-occurring sequences without substantially affecting their function of regulating lipid metabolism. In certain embodiments, the present invention relates to use of proteins or polypeptides associated with lipid metabolism comprising no more than 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative modification(s) (e.g., conservative substitutions, additions, deletions) to any of naturally-occurring sequences, such as SEQ ID NOs:1-12, 14-36.

[0171] In addition, the present invention relates to the use of functional fragments of naturally-occurring proteins or polypeptides associated with lipid metabolism. In certain embodiments, the functional fragments comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 280, 300, 330, or 350 consecutive amino acids of any of SEQ ID NOs:1-12, 14-36.

[0172] In certain embodiments, plant species that can be genetically-modified in accordance with the current invention include, but are not limited to, monocots, dicots, crop plants (i.e., any plant species grown for purposes of agriculture, food production for animals including humans), trees (i.e., fruit trees, trees grown for wood production, trees grown for decoration, etc.), flowers of any kind (i.e., plants grown for purposes of decoration, for example, following their harvest), and cacti. More specific examples of plants that can be genetically-modified to express one or more proteins or polypeptides associated with lipid metabolism include, but are not limited to, Viridiplantae, Streptophyta, Embryophyta, Tracheophyta, Euphyllophytes, Spermatophyta, Magnoliophyta, Liliopsida, Commelinidae, Poales, Poaceae, Oryza, Oryza sativa, Zea, Zea mays, Hordeum, Hordeum vulgare, Triticum, Triticum aestivum, Eudicotyledons, Core eudicots, Asteridae, Euasterids, Rosidae, Eurosids II, Brassicales, Brassicaceae, Arabidopsis, Magnoliopsida, Solananae, Solanales, Solanaceae, Solanum, and Nicotiana. Thus, the embodiments of the invention have uses over a broad range of plants including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Panneserum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Titicum, Vicia, Vitis, Vigna, and Zea.

[0173] In certain embodiments, plant species that can be genetically-modified in accordance with the current invention include, but are not limited to, corn, sugarcane, sorghum, millet, rice, wheat, barley, soybean, olive, peanut, castor, oleaginous fruits such as palm and avocado, Glycine sp., grape, canola, Arabidopsis, Brassica sp., cotton, tobacco, bamboo, sugar beet, sunflower, willow, switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus crossed with giganteus (Miscanthus X giganteus), Miscanthus sp., Sericea lespedeza (Lespedeza cuneata), ryegrass (Lolium multiflorum, lolium sp.), timothy, kochia (Kochia scoparia), forage soybeans, alfalfa, clover, turf grass, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.) including tall fescue, Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, kentucky bluegrass, yellow nutsedge, pine, poplar (Populus sp.), and eucalyptus, among others.

[0174] In certain specific embodiments, plant species that can be genetically-modified in accordance with the current invention include, but are not limited to, sorghum; switchgrass (panicum); wheat (triticum); sugarcane (for expression in leaves and stems); camelina, canola (for expression in oil seeds); soybean; safflower; and jatropha (e.g., for expression in seeds).

[0175] In certain embodiments, plant species that can be genetically-modified in accordance with the current invention include grasses such as the Poaceae (or Gramineae) family, the sedges (Cyperaceae), and the rushes (Juncaceae).

[0176] While A. thaliana is used in the present invention as an example of plant species to demonstration that plants transformed with proteins associated with lipid metabolism have elevated cellular lipid content and/or increased lipid droplet formation, those skilled in the art would readily obtain transgenic plants of other species with elevated cellular lipid content and/or increased lipid droplet formation, wherein transgenic plants express proteins associated with lipid metabolism.

[0177] Triacylglycerols (TG) can be synthesized in non-seed tissues; however, their abundance is low and these storage lipids are presumed to be metabolized rapidly, perhaps for the recycling of fatty acids for energy or the synthesis of membrane lipids.

[0178] In certain embodiments, the algal cells that can be genetically modified in accordance with the current invention include, but are not limited to, algae selected from Amphora, Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Euglena, Hematococcus, Isochrysis, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Parachlorella, Pavlova, Phaeodactylum, Pinguiococcus, Playtomonas, Pleurochrysis, Porphyra, Pseudoanabaena, Pyramimonas, Rhodomonas, Selenastrum, Scenedesmus, Sticococcus, Synechococcus, Tetraselmis, Thalassiosira, and Trichodesmium. In certain embodiments, the algal cells are selected from Botryococcus braunii, Chlorella spp., Dunaliella tertiolecta, Gracilaria spp., Pleurochrysis camerae (also called CCMP647), Sargassum spp., Ankistrodesmus spp., Botryococcus braunii, Chlorella protothecoides, Cyclotella DI-35, Dunaliella tertiolecta, Hantzschia DI-160, Nannochloris spp., Nannochloropsis spp., Nitzschia TR-114, Phaeodactylum tricornutum, Scenedesmus TR-84, Stichococcus spp., Tetraselmis suecica, Thalassiosira pseudonana, Crypthecodinium cohnii, Neochloris oleoabundans, and Schiochytrium spp.

[0179] In certain embodiments, the present invention provides a method of elevating lipid content and/or inducing lipid droplet accumulation in vegetative plant (non-seed) tissues or plant parts including, but not limited to, leaves, roots, stems, shoots, buds, tubers, fruits, and flowers. In another embodiment, the present invention provides elevated lipid content and/or induces lipid droplet accumulation in seeds.

[0180] In some embodiments, the present invention can be used to increase total fatty acid content of the plant cell or the algal cell. In certain embodiments, the present invention can be used to increase the level of fatty acids including leaf-specific fatty acids, including but not limited to, triacylglycerol, hydroxyl, epoxy, cyclic, acetylenic, saturated, polyunsaturated (such as omega-3, omega-6 fatty acids), and short-chain or long-chain fatty acids, which can be incorporated into neutral lipids that can be compartmentalized in lipid droplets, including TAGs, wax-esters, and steryl-esters.

[0181] In some embodiments, the method for obtaining a plant cell or an algal cell with elevated lipid content further comprises: downregulating, in the plant cell or the algal cell, the function of an At4924160 gene product.

[0182] Chanarin-Dorfman Syndrome is a neutral-lipid storage disorder (Lefevre et al., 2001; Bruno et al., 2008). CGI58, also known as ABHD5, associates with lipid droplets in human cells and participates in storage lipid hydrolysis. A mutation in this protein results in hyperaccumulation of lipid droplets in cells and the pathology associated with this syndrome. The CGI58 protein sequence includes a so-called "alpha/beta hydrolase fold" that is shared by members of the esterase/lipase/thioesterase family, suggesting that it might be a TAG lipase. Recent analyses of its functional properties have indicated that the mammalian polypeptide stimulates the activity of a lipase called ATGL (Adipose Triglyceride Lipase), which is the major lipase responsible for catalyzing the initial step of TAG breakdown in both adipose and non-lipid storing cell types (e.g. Lass et al., 2006; Yen & Farese, 2006; Schweiger et al., 2006; Yamaguchi et al., 2007). Interestingly, CGI58 also possesses lysophosphatidic acid acyltransferase (LPAAT) activity in vitro, suggesting that, in addition to its role in stimulating lipase activity, it may play a role in recycling of fatty acids into membrane phospholipids (Ghosh et al., 2008).

[0183] At4g24160 has been identified as a putative homolog of human CGI58, in A. thaliana. The gene in Arabidopsis is apparently expressed as two alternative transcripts (two distinct cDNAs corresponding to the same gene have been identified) and the predicted protein products share domain architecture with other lipases/esterases and acyltransferases. Arabidopsis mutant lines lacking the function of the CGI58 homolog (i.e., At4g24160) contained vegetative (i.e. non-seed) tissues with metabolic machinery capable of synthesizing and storing oil as TAG, demonstrating that there are mechanisms in place to regulate this process in non-seed tissues.

[0184] The term "down-regulating," as used herein, refers to reducing the expression or function of a gene of interest. In certain embodiments, the reduction in expression or function of a gene of interest may be least a 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, when compared to wild-type. The down-regulation of function may also be measured by assaying the enzymatic activity of a polypeptide that is regulated by a polypeptide encoded by the gene of interest.

[0185] In certain embodiments of the invention, down-regulation of the activity of a polypeptide encoded by a gene may be accomplished using antisense-mediated-, or dsRNA-mediated-, or other forms of RNA-mediated-interference (RNAi), as is well known in the art. Methods for identification of candidate nucleotide sequences for RNA-mediated gene suppression, and design of oligonucleotides and constructs to achieve RNA-mediated gene suppression, are well known (e.g. Reynolds et al., 2004; Lu and Mathews, 2008).

[0186] In one embodiment, the plant cell can be genetically engineered to expresses one or more proteins associated with lipid metabolism in a cgi58 (mutation) background, wherein the CGI58 (mutation) background results in enhanced lipid content in plants. In one embodiment, the plant cell of the present invention has a cgi58 (mutation) background described in US2010/0221400.

[0187] Methods for the genetic control of lipid accumulation in vegetative (non-seed) portions of plants by down-regulation of activity of At4g24160 or a homolog thereof are described in US2010/0221400, which is herein incorporated by reference in its entirety.

[0188] In certain embodiments, the present invention provides a transgenic plant cell or an algal cell with elevated lipid content, wherein the transgenic plant or algal cell expresses an exogenous protein or polypeptide associated with lipid metabolism, wherein the protein associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity; and wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the genetically-modified plant or algal cell, when compared to a wild-type plant cell. In certain embodiments, the genetically-modified plant cell is contained in a plant tissue, plant part, or whole plant. In one embodiment, the genetically-modified plant or algal cell comprises, in its genome, a transgene encoding a protein or polypeptide associated with lipid metabolism that induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity.

[0189] As used herein, the term "genetically modified plant or plant parts" refers to a plant or a plant part, whether it is attached or detached from the whole plant. It also includes progeny of the genetically modified plant or plant parts that are produced through sexual or asexual reproduction. Similarly, "transformed plant cell" refers to the initial transformant as well as progeny cells of the initial transformant in which the heterologous genetic sequence is found.

[0190] "Progeny" includes the immediate and all subsequent generations of offspring traceable to a parent.

[0191] In some embodiments, the present invention provides a method for obtaining an algal or bacterial cell with elevated lipid content, wherein the method comprises:

[0192] genetically modifying an algal or bacterial cell to express an exogenous protein or polypeptide associated with lipid metabolism, thereby obtaining a genetically-modified algae or bacterial cell with elevated lipid content;

[0193] wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity; and

[0194] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the genetically-modified algal or bacterial cell, when compared to a wild-type (native) algal or bacterial cell of the same type.

[0195] In another embodiment, the present invention provides a method for obtaining an algal or bacterial cell with elevated lipid content, wherein the method comprises:

[0196] transforming an algal or bacterial cell with a vector comprising a nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism, wherein nucleic acid is operably linked to a promoter and/or a regulatory sequence;

[0197] wherein the protein associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity;

[0198] wherein the transformed algal or bacterial cell expresses the protein or polypeptide associated with lipid metabolism; and

[0199] wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the transformed algal or bacterial cell as compared to a wild-type (native) algal or bacterial cell of the same type.

[0200] In certain embodiments, the algal cell can be genetically engineered to expresses any combinations of proteins associated with lipid metabolism and peptides including, but not limited to, fat specific protein 27 (FSP27); perilipins including PLIN1 (perilipin 1) and PLIN2 (also called autosomal dominant retinitis pigmentosa (ADRP)); SEIPIN (Bernardinelli-Seip congenital lipodystrophy type 2 protein); FIT1 (fat storage-inducing transmembrane protein 1), and FIT2 (fat storage-inducing transmembrane protein 2); acyl-CoA: diacylglycerol acyltransferase 1 (DGAT-1); phospholipid:diacylglycerol acyltransferase 1 (PDAT-1); cell death activator (Cidea); and WRINKLED1 (WRIT). In some embodiments, algae can be selected from the group consisting of Amphora, Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Euglena, Hematococcus, Isochrysis, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Parachlorella, Pavlova, Phaeodactylum, Pinguiococcus, Playtomonas, Pleurochrysis, Porphyra, Pseudoanabaena, Pyramimonas, Rhodomonas, Selenastrum, Scenedesmus, Sticococcus, Synechococcus, Tetraselmis, Thalassiosira, and Trichodesmium.

Nucleic Acid Constructs, Expression Cassettes, and Host Cells

[0201] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0202] As used herein, the terms "operon" and "single transcription unit" are used interchangeably to refer to two or more contiguous coding regions (nucleotide sequences that encode a gene product such as an RNA or a protein) that are coordinately regulated by one or more controlling elements (e.g., a promoter).

[0203] As used herein, the term "gene product" refers to RNA encoded by DNA (or vice versa) or protein that is encoded by an RNA or DNA, where a gene will typically comprise one or more nucleotide sequences that encode a protein, and may also include introns and other non-coding nucleotide sequences.

[0204] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0205] The term "naturally-occurring" or "native" as used herein as applied to a nucleic acid, a cell, or an organism, refers to a nucleic acid, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring, and includes "wild-type" plants.

[0206] The term "heterologous nucleic acid," as used herein, refers to a nucleic acid wherein at least one of the following is true: (a) the nucleic acid is foreign ("exogenous") to (i.e., not naturally found in) a given host microorganism or host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (e.g., is "endogenous to") a given host microorganism or host cell (e.g., the nucleic acid comprises a nucleotide sequence endogenous to the host microorganism or host cell); however, in the context of a heterologous nucleic acid, the same nucleotide sequence as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell, or a nucleic acid comprising a nucleotide sequence that differs in sequence from the endogenous nucleotide sequence but encodes the same protein (having the same or substantially the same amino acid sequence) as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell; (c) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to each other in nature, e.g., the nucleic acid is recombinant. An example of a heterologous nucleic acid is a nucleotide sequence encoding a protein or polypeptide associated with lipid metabolism operably linked to a transcriptional control element (for example, a promoter) to which an endogenous (naturally-occurring) sequence coding for a protein or polypeptide associated with lipid metabolism is not normally operably linked. Another example of a heterologous nucleic acid is a high copy number plasmid comprising a nucleotide sequence encoding a protein or polypeptide associated with lipid metabolism. Another example of a heterologous nucleic acid is a nucleic acid encoding a protein or polypeptide associated with lipid metabolism, where a host cell that does not normally produce a protein or polypeptide associated with lipid metabolism is genetically modified with the nucleic acid encoding a protein or polypeptide associated with lipid metabolism; because protein associated with lipid metabolism-encoding nucleic acids are not naturally found in the host cell, the nucleic acid is heterologous to the genetically modified host cell.

[0207] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", below).

[0208] Thus, for example, the term "recombinant" polynucleotide or nucleic acid refers to one which is not naturally occurring, for example, is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0209] By "construct" is meant a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences.

[0210] As used herein, the term "exogenous nucleic acid" refers to a nucleic acid that is not normally or naturally found in and/or produced by a given bacterium, organism, or cell in nature. As used herein, the term "endogenous nucleic acid" refers to a nucleic acid that is normally found in and/or produced by a given bacterium, organism, or cell in nature. An "endogenous nucleic acid" is also referred to as a "native nucleic acid" or a nucleic acid that is "native" to a given bacterium, organism, or cell.

[0211] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.

[0212] The terms "transformation" or "transformed" are used interchangeably herein with "genetic modification" or "genetically modified" and refer to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic change ("modification") can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell or into a plastome of the cell. In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids, plastids, and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.

[0213] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms "heterologous promoter" and "heterologous control regions" refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a "transcriptional control region heterologous to a coding region" is a transcriptional control region that is not normally associated with the coding region in nature.

[0214] A "host cell," as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (for example, a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (for example, an expression vector that comprises a nucleotide sequence encoding one or more gene products such as proteins or polypeptides associated with lipid metabolism), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a subject prokaryotic host cell is a genetically modified prokaryotic host cell (for example, a bacterium), by virtue of introduction into a suitable prokaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell; and a subject eukaryotic host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, for example, an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0215] As used herein the term "isolated" is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0216] Expression cassettes may be prepared comprising a transcription initiation or transcriptional control region(s) (for example, a promoter), the coding region for the protein of interest, and a transcriptional termination region. Transcriptional control regions include those that provide for over-expression of the protein of interest in the genetically modified host cell; those that provide for inducible expression, such that when an inducing agent is added to the culture medium, transcription of the coding region of the protein of interest is induced or increased to a higher level than prior to induction.

[0217] An expression cassette may contain at least one polynucleotide of interest to be co-transformed into the organism. Such an expression cassette is preferably provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0218] The cassette may include 5' and 3' regulatory sequences operably linked to a polynucleotide of interest. By "operably linked" is intended, for example, a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. When a polynucleotide comprises a plurality of coding regions that are operably linked such that they are under the control of a single promoter, the polynucleotide may be referred to as an "operon".

[0219] The expression cassette will optionally include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a polynucleotide sequence of interest and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, is optional, but may be native or analogous, or foreign or heterologous, to the intended host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By "foreign" is intended that the transcriptional initiation region is not found in the native organism into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.

[0220] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0221] Where appropriate, the proteins or polynucleotides of interest may be optimized for expression in the transformed organism. That is, the genes can be synthesized using plant or algae genomic preferred codons (for genomic transformation) or plastid-preferred codons corresponding to the plastids of the plant or algae of interest (for plastidic transformation). Methods are available in the art for synthesizing such codon optimized polynucleotides. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Of course, the skilled artisan will appreciate that for the transplastomic purposes described herein, sequence optimization should be conducted with plastid codon usage frequency in mind, rather than the plant or algae genome codon usage exemplified in these references.

[0222] It is now well known in the art that when synthesizing a protein or polynucleotide of interest for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of codon usage of the host cell. It is also well known that plastome codon usage may vary from that of the host plant genome. For purposes of the subject invention, "frequency of preferred codon usage" is viewed in the context of whether the transformation is to be genomic or plastidic. For example, in the case of the latter, the phrase refers to the preference exhibited by a specific host cell plastid in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a plastid can be calculated by averaging frequency of preferred codon usage in a number of genes expressed by the plastid. It usually is preferable that this analysis be limited to genes that are among those more highly expressed by the plastid or in the host cell's genome, as appropriate. Alternatively, the polynucleotide of interest may be synthesized to have a greater number of the host plastid's most preferred codon for each amino acid, or to reduce the number of codons that are rarely used by the host.

[0223] The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region), Elroy-Stein et al. (1989) PNAS USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMV Leader (Maize Dwarf Mosaic Virus) Virology 154:9-20; and human immunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature 325:622-625; tobacco mosaic virus leader (TMV), Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256; and maize chlorotic mottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

[0224] In preparing an expression cassette, the various proteins or polynucleotide may be manipulated, so as to provide for the polynucleotide sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the polynucleotide fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleotides, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0225] Tissue-specific promoters are well known in the art and can be used to localize expression of the heterologous coding sequence in desired plant parts.

[0226] In addition, expressed gene products may be localized to specific organelles in the target cell by ligating DNA or RNA coded for peptide leader sequences to the polynucleotide of interest. Such leader sequences can be obtained from several genes of either plant or other sources. These genes encode cytoplasmically-synthesized proteins directed to, for example, mitochondria (the F1-ATPase beta subunit from yeast or tobacco, cytochrome c1 from yeast), chloroplasts (cytochrome oxidase subunit Va from yeast, small subunit of rubisco from pea), endoplasmic reticulum lumen (protein disulfide isomerase), vacuole (carboxypeptidase Y and proteinase A from yeast, phytohemagglutinin from French bean), peroxisomes (D-aminoacid oxidase, uricase) and lysosomes (hydrolases).

[0227] A nucleic acid is "hybridizable" to another nucleic acid, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid can anneal to the other nucleic acid under the appropriate conditions of temperature and solution ionic strength.

[0228] Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001).

[0229] As used herein, "stringent" conditions for hybridization refers to conditions wherein hybridization is carried out overnight at 20-25.degree. C. below the melting temperature (Tm) of the DNA hybrid in 6.times.SSPE, 5.times.Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, is described by the following formula (Beltz et al., 1983):

Tm=81.5 C+16.6 Log [Na+]+0.41(% G+C)-0.61(% formamide)-600/length of duplex in base pairs.

[0230] Washes are typically carried out as follows:

[0231] (1) Twice at room temperature for 15 minutes in 1.times.SSPE, 0.1% SDS (low stringency wash).

[0232] (2) Once at Tm-20.degree. C. for 15 minutes in 0.2.times.SSPE, 0.1% SDS.

[0233] Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50 9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7 11.8). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Illustrative minimum lengths for a hybridizable nucleic acid are: at least about 15 nucleotides; at least about 20 nucleotides; and at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

[0234] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. A protein or polypeptide associated with lipid metabolism containing conserved amino acid substitutions as compared to a protein or polypeptide associated with lipid metabolism exemplified herein would fall within the scope of "variants" of proteins or polypeptides associated with lipid metabolism.

[0235] "Synthetic nucleic acids" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized," as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. The nucleotide sequence of the nucleic acids can be modified for optimal expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available. Fragments of full-length proteins can be produced by techniques well known in the art, such as by creating synthetic nucleic acids encoding the desired portions; or by use of Bal 31 exonuclease to generate fragments of a longer nucleic acid.

[0236] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-410. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0237] As used herein, the term "variant" refers either to a naturally occurring genetic mutant of protein associated with lipid metabolism or a recombinantly prepared variation of protein associated with lipid metabolism, each of which contains one or more mutations in its DNA.

[0238] The term "variant" may also refer to either a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion. In certain embodiments, the variants include less than 75, less than 70, less than 60, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, less than 5, less than 4, less than 3, or less than 2 amino acid substitutions, rearrangements, insertions, and/or deletions relative to a naturally-occurring or native protein or polypeptide associated with lipid metabolism.

[0239] In some embodiments, the transformation vector further comprises a nucleic acid that confers resistance to a selection agent selected from bar, pat, ALS, HPH, HYG, EPSP, and Hm1.

[0240] Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) as well as genes conferring resist insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. (See DeBlock et al. (1987) EMBO J, 6:2513-2518; DeBlock et al. (1989) Plant Physiol., 91:691-704; Fromm et al. (1990) 8:833-839. For example, resistance to glyphosate or sulfonylurea herbicides has been obtained by using genes coding for the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS). Resistance to glufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respective herbicides.

[0241] For purposes of the present invention, selectable marker genes include, but are not limited to genes encoding: neomycin phosphotransferase II (Fraley et a. (1986) CRC Critical Reviews in Plant Science, 4:1-25); cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA, 88:4250-4264); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) Bio/Technology, 11:715-718); tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Bio., 22:907-912); dihydrodipicolinate synthase and desensitized aspartade kinase (Perl et al. (1993) Bio/Technology, 11:715-718); bar gene (Toki et al. (1992) Plant Physiol., 100:1503-1507 and Meagher et al. (1996) and Crop Sci, 36:1367); tryptophane decarboxylase (Goddijn et al. (1993) Plant Mol. Biol., 22:907-912); neomycin phosphotransferase (NEO) (Southern et al. (1982) J. Mol. Appl. Gen., 1:327; hygromycin phosphotransferase (HPT or HYG) (Shimizu et al. (1986) Mol. Cell Biol., 6:1074); dihydrofolate reductase (DHFR) (Kwok et al. (1986) PNAS USA 4552); phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J., 6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (Anderson et al U.S. Pat. No. 4,761,373; Haughn et al. (1988) Mol. Gen. Genet. 221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA) (Comai et al. (1985) Nature 317:741); haloarylnitrilase (Stalker et al., published PCT applon WO87/04181); acetyl-coenzyme A carboxylase (Parker et al. (1990) Plant Physiol. 92:1220); dihydropteroate synthase (sul I) (Guerineau et al. (1990) Plant Mol. Biol. 15:127); 32 kD photosystem II polypeptide (psbA) (Hirschberg et al. (1983) Science, 222:1346); etc.

[0242] Also included are genes encoding resistance to: chloramphenicol (Herrera-Estrella et al. (1983) EMBO J., 2:987-992); methotrexate (Herrera-Estrella et al. (1983) Nature, 303:209-213; Meijer et al. (1991) Plant Mol Bio., 16:807-820 (1991); hygromycin (Waldron et al. (1985) Plant Mol. Biol., 5:103-108; Zhijian et al. (1995) Plant Science, 108:219-227 and Meijer et al. (1991) Plant Mol. Bio. 16:807-820); streptomycin (Jones et al. (1987) Mol. Gen. Genet., 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res., 5:131-137); bleomycin (Hille et al. (1986) Plant Mol. Biol., 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio., 15:127-136); bromoxynil (Stalker et al. (1988) Science, 242:419-423); 2,4-D (Streber et al. (1989) Bio/Technology, 7:811-816); glyphosate (Shaw et al. (1986) Science, 233:478-481); phosphinothricin (DeBlock et al. (1987) EMBO J., 6:2513-2518); spectinomycin (Bretagne-Sagnard and Chupeau (1996) Transgenic Research 5:131-137).

[0243] The bar gene confers herbicide resistance to glufosinate-type herbicides, such as phosphinothricin (PPT) or bialaphos, and the like. As noted above, other selectable markers that could be used in the vector constructs include, but are not limited to, the pat gene, also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, the HPH or HYG gene for hygromycin resistance, the EPSP synthase gene for glyphosate resistance, the Hm1 gene for resistance to the Hc-toxin, and other selective agents used routinely and known to one of ordinary skill in the art.

Screening Methods for Obtaining Plants with Elevated Lipid Content

[0244] In some embodiments, the invention provides methods for screening for a functional protein or polypeptide associated with lipid metabolism for elevating lipid content and/or inducing lipid droplet accumulation in a plant, bacterial, or algal cell, wherein the method comprises:

[0245] obtaining a test plant, bacterial, or algal cell genetically-modified to express a candidate exogenous protein or polypeptide associated with lipid metabolism; and

[0246] growing the genetically-modified test cell and selecting the genetically-modified test cell having elevated lipid content and/or increased lipid droplet level when compared to a native (wild-type) cell of the same type.

[0247] Embodiments of this invention also pertain to methods for screening for a functional protein or polypeptide associated with lipid metabolism for elevating lipid content and/or inducing lipid droplet accumulation in a plant, bacterial, or algal cell, wherein the method comprises:

[0248] transforming a test plant, bacterial, or algal cell with a vector nucleic acid sequence encoding a candidate exogenous protein or polypeptide associated with lipid metabolism, wherein the nucleic acid is operably linked to a promoter and/or a regulatory sequence; and

[0249] growing the genetically-modified test cell and selecting the genetically-modified test cell having elevated lipid content and/or increased lipid droplet level when compared to a native (wild-type) cell of the same type.

[0250] In certain embodiments of the screening method, the transformed or genetically-modified test cell is a plant cell. In certain embodiments, the plant test cell is in a plant tissue, plant part, or whole plant.

[0251] In certain embodiments of the screening method, vegetative plant (non-seed) cells, tissues or plant parts including, but not limited to, leaves, roots, stems, shoots, buds, tubers, fruits, and flowers, are genetically-modified or transformed. In another embodiment of the screening method, a plant seed cell or tissue is genetically-modified or transformed.

[0252] In some embodiments, a method may employ marker-assisted breeding to identify plants, including cultivars or breeding lines, displaying a trait of interest, such as elevated levels of neutral lipids in vegetative portions of plant biomass.

[0253] When an exogenous nucleic acid comprising a nucleotide sequence that encodes a protein or polypeptide associated with lipid metabolism is introduced into the host cell, lipid content of the test cell is elevated. In certain embodiments, a candidate protein or polypeptide associated with lipid metabolism is selected if there is an elevation of the lipid content of the cell of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, as compared to a non-genetically-modified host.

[0254] In some embodiments, for example, where the exogenous nucleic acid is a plurality of exogenous nucleic acids (such as, for example, a cDNA library, a genomic library, or a population of nucleic acids, each encoding a protein or polypeptide associated with lipid metabolism with a different amino acid sequence, etc.), the exogenous nucleic acids are introduced into a plurality of host cells, forming a plurality of test cells. In certain embodiments, the test cells are in some embodiments grown in normal culture conditions.

[0255] Methods of isolating the exogenous nucleic acid from a test cell are well known in the art. Suitable methods include, but are not limited to, any of a number of alkaline lysis methods that are standard in the art.

[0256] In some embodiments, a subject screening method will further comprise further characterizing a candidate gene product. In these embodiments, the exogenous nucleic acid comprising nucleotide sequence(s) encoding protein or polypeptide associated with lipid metabolism are isolated from a test cell; the gene product(s) are expressed in a cell and/or in an in vitro cell-free transcription/translation system. In some embodiments, the exogenous nucleic acid is subjected to nucleotide sequence analysis, and the amino acid sequence of the gene product deduced from the nucleotide sequence. In some embodiments, the amino acid sequence of the gene product is compared with other amino acid sequences in a public database of amino acid sequences, to determine whether any significant amino acid sequence identity to an amino acid sequence of a known protein exists. In addition, the gene product(s) are expressed in a cell and/or in an in vitro cell-free transcription/translation system; and the effect of the gene product(s) on a metabolic pathway intermediate or other metabolite is analyzed.

[0257] Exogenous nucleic acids that are suitable for introducing into a host cell, to produce a test cell, include, but are not limited to, naturally-occurring nucleic acids isolated from a cell; naturally-occurring nucleic acids that have been modified (for example, by mutation) before or subsequent to isolation from a cell; synthetic nucleic acids, e.g., nucleic acids synthesized in a laboratory using standard methods of chemical synthesis of nucleic acids, or generated by recombinant methods; synthetic or naturally-occurring nucleic acids that have been amplified in vitro, either within a cell or in a cell-free system; and the like.

[0258] Exogenous nucleic acids that are suitable for introducing into a host cell include, but are not limited to, genomic DNA; RNA; a complementary DNA (cDNA) copy of mRNA isolated from a cell; recombinant DNA; and DNA synthesized in vitro, e.g., using standard cell-free in vitro methods for DNA synthesis. In some embodiments, exogenous nucleic acids are a cDNA library made from cells, either prokaryotic cells or eukaryotic cells. In some embodiments, exogenous nucleic acids are a genomic DNA library made from cells, either prokaryotic cells or eukaryotic cells.

[0259] Nucleic acids will in some embodiments be mutated before being introduced into a host cell. Methods of mutating a nucleic acid are well known in the art and include well-established chemical mutation methods, radiation-induced mutagenesis, and methods of mutating a nucleic acid during synthesis. Chemical methods of mutating DNA include exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline N-oxide, diethylsulfate, benzopyrene, cyclophosphamide, bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (for example, diepoxybutane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide, bisulfan, and the like. Radiation mutation-inducing agents include ultraviolet radiation, .gamma.-irradiation, X-rays, and fast neutron bombardment. Mutations can also be introduced into a nucleic acid using, e.g., trimethylpsoralen with ultraviolet light. Random or targeted insertion of a mobile DNA element, e.g., a transposable element, is another suitable method for generating mutations. Mutations can be introduced into a nucleic acid during amplification in a cell-free in vitro system, e.g., using a polymerase chain reaction (PCR) technique such as error-prone PCR. Mutations can be introduced into a nucleic acid in vitro using DNA shuffling techniques (e.g., exon shuffling, domain swapping, and the like). Mutations can also be introduced into a nucleic acid as a result of a deficiency in a DNA repair enzyme in a cell, e.g., the presence in a cell of a mutant gene encoding a mutant DNA repair enzyme is expected to generate a high frequency of mutations (i.e., about 1 mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell. Examples of genes encoding DNA repair enzymes include but are not limited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof in other species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like). Methods of mutating nucleic acids are well known in the art, and any known method is suitable for use. See, e.g., Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and 6,773,900.

Isolation of Homologs

[0260] Isolation of additional homologs from other plant species may be accomplished by laboratory procedures well known and commonly used in the art. Standard techniques are used for identification, cloning, isolation, amplification, and purification of nucleic acid sequences and polypeptides. These techniques and various others are generally performed as described for instance in Sambrook et al., 1989. Genome walking techniques may be performed according to manufacturer's specifications (CLONTECH Laboratories, Inc., Palo Alto, Calif.).

[0261] One such technique for isolation of homologs is the use of oligonucleotide probes based on sequences disclosed in this specification to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by digestion with restriction endonucleases and then ligating the resultant segments with vector DNA to form concatemers that can be packaged into an appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as seed tissue, and a cDNA library is prepared from the mRNA.

[0262] A cDNA or genomic DNA library can be screened using a probe based upon the sequence of a cloned naturally-occurring protein or polypeptide sequence. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Usefully employed such probes include, without limitation, 5' UTRs which, may function as promoters. Alternatively, antibodies raised against a polypeptide, or homolog thereof, can be used to screen an mRNA expression library to isolate sequences of interest. Homologs may also be identified in silico, for instance by similarity-based database searches as described below.

[0263] Nucleic acid sequences can be screened for the presence of a protein encoding sequence that is homologous to genes of other organisms with known protein encoding sequence using any of a variety of search algorithms. Such search algorithms can be homology-based or predictive-based. Similarity-based searches (e.g., GAP2, BLASTX supplemented by NAP and TBLASTX) can detect conserved sequences during comparison of DNA sequences or hypothetically translated protein sequences to public and/or proprietary DNA and protein databases.

[0264] Existence of a gene is inferred if significant sequence similarity extends over the majority of the target gene. Since such methods may overlook genes unique to the source organism, for which homologous nucleic acid molecules have not yet been identified in databases, gene prediction programs may also be used. Gene prediction programs generally use "signals" in the sequences, such as splice sites or "content" statistics, such as codon bias, to predict gene structures (Stormo, 2000).

[0265] Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For example, polymerase chain reaction technology can be used to amplify the sequences of a gene of interest or the homolog gene directly from genomic DNA, from cDNA, from genomic libraries, and cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, in cloning nucleic acids sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of desired mRNA in samples, for nucleic acid sequencing, or for other purposes.

[0266] Appropriate primers and probes for identifying homolog sequences from plant tissues are generated from comparisons of the sequences provided herein. For a general overview of PCR, see, Innis, et al., eds., 1990.

[0267] PCR or other primers may be used under standard PCR conditions, preferably using nucleic acid sequences as identified in EST libraries or other GenBank accessions as a template. The PCR products generated by any of the reactions can then be used to identify nucleic acids useful in the context of the present invention by their ability to hybridize to known homolog genes found in GenBank and other databases.

Plant Transformation

[0268] To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al., 1988; and Sambrook et al., 1989. Methods of plant cell culture are well known in the art. A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences that will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

[0269] Vectors used for plant transformation may include, for example, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), plant artificial chromosomes (PACs), or any suitable cloning system. It is contemplated the utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. Introduction of such sequences may be facilitated by use of BACs or YACs, or even PACs. For example the use of BACs for Agrobacterium-mediated transformation was disclosed by Hamilton et al., 1999.

[0270] Particularly useful for transformation are expression cassettes that have been isolated from such vectors. DNA segments used for transforming plant cells will, of course, generally comprise the cDNA, gene or genes that one desires to introduce into and have expressed in the host cells. These DNA segments can further include structures such as promoter, enhancers, 3' untranslated regions (such as polyadenylation sites), polylinkers, or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction may encode a protein that will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and/or will impart an improved phenotype to the resulting transgenic plant. However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes.

[0271] A number of promoters that are active in plant cells have been described in the literature, and are preferred elements included in the context of the present invention. Such promoters would include but are not limited to those isolated from the following genes: nopaline synthase (NOS; Ebert et al., 1987) and octopine synthase (OCS): cauliflower mosaic virus (CaMV) 19S (Lawton et al. 1987) and 35S (Odell et al., 1985), as well as the enhanced CaMV 35S promoter (e35S; described by Kay et al., 1987); figwort mosaic virus (FMV) 35S; the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide); napin (Kridl et al., 1991); Adh (Walker et al., 1987); sucrose synthase (Yang et al., 1990); tubulin; actin (Wang et al., 1992); cab (Sullivan et al., 1989); PEPCase (Hudspeth et al., 1989); 7S-alpha'-conglycinin (Beachy et al., 1985); R gene complex promoters (Chandler et al. 1989); tomato E8; patatin; ubiquitin; mannopine synthase (mas); soybean seed protein glycinin (Gly); soybean vegetative storage protein (vsp); waxy; Brittle; Shrunken 2; Branching enzymes I and II; starch synthases; debranching enzymes; oleosins; glutelins; globulin 1; BETL1; and Arabidopsis banyuls promoter. The rice actin 1 promoter, the AGL11 promoter, the BETL1 promoter, and the e35S promoter may find use in the practice of the present invention. All of these promoters have been used to create various types of DNA constructs that have been expressed in plants (see, for example, Rogers et al., WO 84/02913).

[0272] Promoter hybrids can also be constructed to enhance transcriptional activity (Hoffman, U.S. Pat. No. 5,106,739, herein incorporated by reference), or to combine desired transcriptional activity, inducibility, and tissue or developmental specificity. Promoters that function in plants include but are not limited to promoters that are classified as, among others, inducible, viral, synthetic, constitutive, tissue-specific, developmentally-regulated, chemically or environmentally inducible, or senescence-related, for instance as described (Odell et al., 1985). Promoters that are tissue specific, tissue-enhanced, or developmentally regulated are also known in the art and envisioned to have utility in the practice of this present invention. For instance, a tissue specific promoter, such as the ST-LS1 promoter (e.g. Stockhaus et al., 1989), that is functional in plant vegetative tissues such as leaves, stems, and/or roots, may be of use. Such a promoter may also be expressed to at least some degree in seed or embryo tissues. In certain embodiments, the promoter to be utilized may be expressed preferentially in green parts of a plant such as leaves or stems. A senescence-related promoter (e.g. from SAG12) may also be utilized.

[0273] The promoters used in the present invention may be modified to affect their control characteristic. Promoters can be derived by means of ligation with operator regions, random or controlled mutagenesis, or other means well known in the art. Furthermore the promoter regions can be altered to contain multiple enhancer sequences to assist in elevating gene expression. Examples of such enhancer sequences have been reported (Kay et al., 1987).

[0274] Where an enhancer is used in conjunction with a promoter for the expression of a selected protein, it is believed that it will be preferred to place the enhancer between the promoter and the start codon of the selected coding region. However, one could also use a different arrangement of the enhancer relative to other sequences and still realize the beneficial properties conferred by the enhancer. For example, the enhancer could be placed 5' of the promoter region, within the promoter region, within the coding sequence, or 3' of the coding region. The placement and choice of sequences used as enhancers is known to those of skill in the art in light of the present disclosure. Transformation constructs prepared in accordance with the current invention will typically include a 3' untranslated region (3' UTR), and typically contains a polyadenylation sequence. One type of 3' UTR that may be used is a 3' UTR from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3'-end; Bevan et al., 1983). Other 3' UTR sequences can be used and are commonly known to those of skill in the art.

[0275] A number of selectable marker genes are known in the art and can be used in the present invention (Wilmink and Dons, 1993). By employing a selectable or screenable marker gene in addition to the gene of interest, one can provide or enhance the ability to identify transformants. Useful selectable marker genes for use in the present invention would include genes that confer resistance to compounds such as antibiotics like kanamycin and herbicides like glyphosate or dicamba. Other selectable markers known in the art may also be used and would fall within the scope of the present invention.

[0276] DNA constructs of the present invention may be introduced into the genome of the desired plant host by a variety of techniques that are well known in the art. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using DNA particle bombardment.

[0277] Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., 1984. Electroporation techniques are described in Fromm et al., 1985. Ballistic transformation techniques are described in Klein et al., 1987.

[0278] Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch, 1984; and Fraley, 1983.

[0279] After transformation by any of the above transformation techniques, the transformed plant cells or tissues may be grown in an appropriate medium to promote cell proliferation and regeneration. Plant regeneration from cultured protoplasts is described in Evans et al., 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21 73, CRC Press, Boca Raton, 1985. For gene gun transformation of wheat and maize, see, U.S. Pat. Nos. 6,153,812 and 6,160,208. See also, Christou, 1996. See, also, U.S. Pat. Nos. 5,416,011; 5,463,174; and 5,959,179 for Agrobacterium-mediated transformation of soy; U.S. Pat. Nos. 5,591,616 and 5,731,179 for Agrobacterium-mediated transformation of monocots such as maize; and U.S. Pat. No. 6,037,527 for Agrobacterium-mediated transformation of cotton. Other Rhizobiaceae may be used for plant cell transformation as well (e.g. Broothaerts et al., 2007).

[0280] To generate a subject genetically modified host cell according to the subject invention, one or more nucleic acids comprising nucleotide sequences encoding one or more proteins or polypeptides associated with lipid metabolism can be introduced stably or transiently into a parent host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, particle bombardment, Agrobacterium-mediated transformation, and the like. For stable transformation, a nucleic acid will generally further include a selectable marker, for example, any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.

[0281] Where a parent host cell has been genetically modified to produce two or more proteins or polypeptides associated with lipid metabolism, nucleotide sequences encoding the two or more proteins or polypeptides associated with lipid metabolism will in some embodiments each be contained on separate expression vectors. Where the host cell is genetically modified to express one or more proteins or polypeptides associated with lipid metabolism, nucleotide sequences encoding the one or more proteins or polypeptides associated with lipid metabolism will in some embodiments be contained in a single expression vector. Where nucleotide sequences encoding the one or more proteins or polypeptides associated with lipid metabolism are contained in a single expression vector, in some embodiments, the nucleotide sequences will be operably linked to a common control element (for example, a promoter), such that the common control element controls expression of all of the nucleotide sequences on the single expression vector.

[0282] Where nucleotide sequences encoding proteins or polypeptides associated with lipid metabolism are contained in a single expression vector, in some embodiments, the nucleotide sequences will be operably linked to different control elements (for example, a promoter), such that, the different control elements control expression of each of the nucleotide sequences separately on a single expression vector.

[0283] In many embodiments, the exogenous nucleic acid is inserted into an expression vector. Expression vectors that are suitable for use in prokaryotic and eukaryotic host cells are known in the art, and any suitable expression vector can be used. Suitable expression vectors are as described above.

[0284] As noted above, an exogenous nucleic acid will in some embodiments be isolated from a cell or an organism in its natural environment. In some embodiments, the nucleic acid of the cell or organism will be mutated before nucleic acid is isolated from the cell or organism. In other embodiments, the exogenous nucleic acid is synthesized in a cell-free system in vitro.

[0285] In some embodiments, the exogenous nucleic acid is a synthetic nucleic acid. In some embodiments, a synthetic nucleic acid comprises a nucleotide sequence encoding a variant protein or polypeptide associated with lipid metabolism, for example, a variant protein or polypeptide associated with lipid metabolism that differs in amino acid sequence by one or more amino acids from a naturally-occurring protein or polypeptide associated with lipid metabolism. In some embodiments, a variant protein or polypeptide associated with lipid metabolism differs in amino acid sequence by from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, or from about 50 amino acids to about 60 amino acids, compared to the amino acid sequence of a naturally-occurring parent protein or polypeptide associated with lipid metabolism.

[0286] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a naturally-occurring protein or polypeptide associated with lipid metabolism is mutated, using any of a variety of well-established methods, giving rise to a nucleic acid comprising a nucleotide sequence encoding a variant protein or polypeptide associated with lipid metabolism.

[0287] Suitable mutagenesis methods include, but are not limited to, chemical mutation methods, radiation-induced mutagenesis, and methods of mutating a nucleic acid during synthesis, as described above. Thus, for example, a nucleic acid comprising a nucleotide sequence encoding a naturally-occurring protein or polypeptide associated with lipid metabolism is exposed to a chemical mutagen, as described above, or subjected to radiation mutation, or subjected to an error-prone PCR, and the mutagenized nucleic acid introduced into a genetically modified host cell(s) as described above. Methods for random mutagenesis using a "mutator" strain of bacteria are also well known in the art and can be used to generate a variant. See, e.g., Greener et al., "An Efficient Random Mutagenesis Technique Using an E. coli Mutator Strain", Methods in Molecular Biology, 57:375-385 (1995). Saturation mutagenesis techniques employing a polymerase chain reaction (PCR) are also well known and can be used. See, e.g., U.S. Pat. No. 6,171,820.

[0288] An embodiment of the invention provides a host cell comprising a vector according to the invention. Other embodiments include plant plastid transformation vectors or nuclear transformation vectors containing nucleotide sequences encoding proteins or polypeptides associated with lipid metabolism, such as containing the full-length protein or polypeptide associated with lipid metabolism, or variants or fragments thereof, for the expression of the protein or polypeptide associated with lipid metabolism with elevated lipid content in the plant cell. These plant vectors may contain other sequences for the generation of chimeric proteins or polypeptides associated with lipid metabolism which may contain mutations, deletions, or insertions of nucleic acid sequences.

[0289] According to embodiments of the present invention, a wide variety of plants and plant cell systems can be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present invention by various transformation methods known in the art, including Agrobacterium-mediated transformation (Horsch et al., Science 227: 1227-1231, 1985) or plastid transformation (Staub and Maliga, Plant J. 6: 547-553, 1994; Hahn and Kuehnle, 2003, cited herein above).

[0290] In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops, including grain crops (for example, wheat, maize, rice, millet, barley), tobacco, fruit crops (for example, tomato, strawberry, orange, grapefruit, banana), forage crops (for example, alfalfa), root vegetable crops (for example, carrot, potato, sugar beets, yam), leafy vegetable crops (for example, lettuce, spinach); flowering plants (for example, petunia, rose, chrysanthemum), conifers and pine trees (for example, pine, fir, spruce); oil crops (for example, sunflower, rape seed); and plants used for experimental purposes (for example, Arabidopsis).

[0291] According to other embodiments of the present invention, desired plants may be obtained by engineering one or more of the vectors expressing proteins or polypeptides associated with lipid metabolism as described herein into a variety of plant cell types, including but not limited to, protoplasts, tissue culture cells, tissue and organ explants, pollens, embryos, as well as whole plants. In an embodiment of the present invention, the engineered plant material is selected or screened for transformants (those that have incorporated or integrated the introduced gene construct(s)) following the approaches and methods described below. An isolated transformant may then be regenerated into a plant and progeny thereof (including the immediate and subsequent generations) via sexual or asexual reproduction or growth. Alternatively, the engineered plant material may be regenerated into a plant before subjecting the derived plant to selection or screening for the marker gene traits. Procedures for regenerating plants from plant cells, tissues or organs, either before or after selecting or screening for marker gene(s), are well known to those skilled in the art.

[0292] According to another embodiment of the present invention, tissue-specific promoters may be used to target the expression of proteins or polypeptides associated with lipid metabolism in fruits, roots or leaves so that an edible plant part is provided low-temperature tolerance. Examples of tissue-specific promoters include those encoding rbsC (Coruzzi et al., EMBO J. 3:1671-1697, 1984) for leaf-specific expression and SAHH or SHMT (Sivanandan et al., Biochimica et Biophysica Acta 1731:202-208, 2005) for root-specific expression. Another exemplary root-specific promoter is taught by Ekramoddoullah et al., U.S. Pat. No. 7,285,656 B2. Also, the Cauliflower Mosaic Virus (CaMV) 35S promoter has been reported to have root-specific and leaf-specific modules in its promoter region (Benfey et al., EMBO J. 8:2195-2202, 1989). Other tissue-specific promoters are well known and widely available to those of ordinary skill in the art. Further, a wide variety of constitutive or inducible promoters are also well known and widely available to those of ordinary skill in the art.

[0293] Proplastid and chloroplast genetic engineering have been shown to varying degrees of homoplasmy for several major agronomic crops including potato, rice, maize, soybean, grape, sweet potato, and tobacco including starting from non-green tissues. Non-lethal selection on antibiotics is used to proliferate cells containing plastids with antibiotic resistance genes. Plastid transformation methods use two plastid-DNA flanking sequences that recombine with plastid sequences to insert chimeric DNA into the spacer regions between functional genes of the plastome, as is established in the field (see Bock and Hagemann, Prog. Bot. 61:76-90, 2000, and Guda et al., Plant Cell Reports 19:257-262, 2000, and references therein).

[0294] Antibiotics such as spectinomycin, streptomycin, and kanamycin can shut down gene expression in chloroplasts by ribosome inactivation. These antibiotics bleach leaves and form white callus when tissue is put onto regeneration medium in their presence. The bacterial genes aadA and neo encode the enzymes aminoglycoside-3N-adenyltransferase and neomycin phosphotransferase, which inactivate these antibiotics, and can be used for positive selection of plastids engineered to express these genes. Polynucleotides of interest can be linked to the selectable genes and thus can be enriched by selection during the sorting out of engineered and non-engineered plastids. Consequently, cells with plastids engineered to contain genes for these enzymes (and linkages thereto) can overcome the effects of inhibitors in the plant cell culture medium and can proliferate, while cells lacking engineered plastids cannot proliferate. Similarly, plastids engineered with polynucleotides encoding enzymes from the mevalonate pathway to produce IPP from acetyl CoA in the presence of inhibitors of the non-mevalonate pathway can overcome otherwise inhibitory culture conditions. By utilizing the polynucleotides disclosed herein in accord with this invention, an inhibitor targeting the non-mevalonate pathway and its components can be used for selection purposes of transplastomic plants produced through currently available methods, or any future methods which become known for production of transplastomic plants, to contain and express said polynucleotides and any linked coding sequences of interest.

[0295] This selection process of the subject invention is unique in that it is the first selectable trait that acts by pathway complementation to overcome inhibitors. This is distinguished from the state of the art of selection by other antibiotics to which resistance is conferred by inactivation of the antibiotic itself, e.g. compound inactivation as for the aminoglycoside 3'-adenyltransferase gene or neo gene. This method avoids the occurrence of resistant escapes due to random insertion of the resistance gene into the nuclear genome or by spontaneous mutation of the ribosomal target of the antibiotic, as is known to occur in the state of the art. Moreover, this method requires the presence of an entire functioning mevalonate pathway in plastids. For example, if one of the enzyme activities of the mevalonate pathway is not present in the plastid, resistance will not be conferred.

[0296] A transformed plant cell, callus, tissue, or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the .beta.-glucuronidase, luciferase, B or C1 genes) that may be present on the vector of the present invention. Such selection and screening methodologies are well known to those skilled in the art. Alternatively or in addition, screening may be for improved low-temperature tolerance as taught herein, for example, by observing a reduction in growth-inhibition.

[0297] Physical and biochemical methods may also be used to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, 51 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis (PAGE), Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art. In a specific embodiment, the selectable marker gene nptII, which specifies kanamycin-resistance, is used in nuclear transformation.

[0298] Following transformation, a plant may be regenerated, e.g., from single cells, callus tissue, or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant. Available techniques are reviewed in Vasil et al. (1984) in Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and III, Laboratory Procedures and Their Applications (Academic press); and Weissbach et al. (1989) Methods for Plant Mol. Biol.

[0299] The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

[0300] The particular choice of a transformation technology will be determined by its efficiency to transform certain target species, as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant plastids is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Applications

[0301] In certain embodiments, the present invention can be used to: [0302] a) provide higher efficiency and cost effective energy production; [0303] b) increase production of lipids which are beneficial for human health, e.g., omega-unsaturated fat in olives, canola, corns, peanuts, sunflower seeds, etc; [0304] c) generate plants for protein therapy. Some proteins play a positive regulatory role in improving the metabolic health in humans suffering from insulin resistance, type 2 diabetes, cardiovascular diseases etc.; [0305] d) produce genetically-modified plants with elevated lipid content for feeding animals including livestock such as cows to produce milk with high level of lipid droplets; [0306] e) produce genetically-modified algal cells with elevated lipid content for production of biofuels, and feed; and [0307] f) produce genetically-modified bacterial cells expressing proteins associated with lipid metabolism for cleaning oil spillage.

[0308] Increase the production of oils which are beneficial for human health. Our biochemical analysis shows that FSP27 expression in plants increase omega-6 and omega-3 unsaturated fatty acids.

[0309] Expressing fish homologs of FSP27 in combination with other nucleic acid molecules encoding proteins involved in the synthesis of long-chain polyunsaturated fatty acids in plants can be used to increase oil contents in plants, thereby producing plants with high omega-unsaturated fatty acid contents. In one embodiment, the transgenic plants of the present invention can serve as an inexpensive and safe source of dietary fatty acids.

[0310] Transgenic plants with high fat contents can be used to feed milk-producing cows, thereby increasing fat contents in dairy products.

[0311] The present invention can be used to increase oil contents in oil-producing plants including, but not limited to, olive, canola, sunflower, soybean, castor, and oleaginous fruits such as palm and avocado. The present invention can also be used to increase unsaturated oil contents in plants, to improve the quality and quantity of oil in plants, and to increase oil content in seeds.

The seeds of the transgenic plants with high lipid contents can be used to produce biodegradable plastic (also called as "bioplastic").

[0312] The proteins or polypeptides associated with lipid metabolism (such as FSP27) can be expressed in algae to increase biofuel production.

[0313] Common uses for oils comprising neutral lipids include the preparation of food for human consumption, feed for non-human animal consumption and industrial uses such as for preparation of biofuels.

[0314] As used herein, "industrial use" or "industrial usage" refers to non-food and non-feed uses for products prepared from plant parts prepared according to the present invention. As used herein, "biofuel" refers to a fuel combusted to provide power, heat, or energy, e.g. for an internal combustion engine, comprising at least 1%, 5%, 10%, 20% or more, by weight, of an oil, or product thereof, produced from a plant of the present invention, or by a method of the present invention.

[0315] Also included in this invention are plants, plant cell cultures, and plant parts thereof, oil obtained from the vegetative tissues of such plants and cells and progeny thereof, animal feed derived from the processing of such tissues, the use of the foregoing oil in food, animal feed, biofuels, cooking oil or industrial applications, and products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil.

Materials and Methods

Expression of A. thaliana SEIPIN Genes in Yeast Cells

[0316] The coding regions of three Arabidopsis SEIPIN genes, designated AtSEIPIN1, AtSEIPIN2, and AtSEIPIN3, were isolated from wild type Arabidopsis (Columbia-0 [Col-0]) by using reverse transcriptase (RT)-PCR. RNA was purified by RNeasy Plant Mini kit (Qiagen) and treated by DNase (Promega) to avoid any DNA contamination. About 100 ng total RNA from each sample was used for RT-PCR. The RT-PCR was performed by using SuperScript.RTM. One-Step RT-PCR System (Invitrogen). The RT-PCR program was set up as follows, reverse transcription at 42.degree. C. for 15 min, pre-denaturation at 95.degree. C. for 5 min, 35 amplification cycles (94.degree. C. for 30 sec, 50.degree. C. for 30 sec, 72.degree. C. 90 sec), and post-extension step at 72.degree. C. for 7 min. The Genbank accession numbers of AtSEIPIN1, AtSEIPIN2, and AtSEIPIN3 proteins are AED92296, AEE31126, and AEC08966, respectively. Wild type yeast strain (BY4742), SEIPIN-deletion yeast mutant (ylr404w.DELTA.), and yeast expression plasmids (pRS315-PGK, pRS315-ylr404w and pRS316-CFP-HDEL) were obtained. The coding regions of AtSEIPIN1, AtSEIPIN2 and AtSEIPIN3 genes were inserted into yeast expression vector pRS315-PGK using restriction enzymes BamHI and PstI (Promega). Then, the recombined yeast expression plasmids (pRS315-AtSEIPIN1, pRS315-AtSEIPIN2 and pRS315-AtSEIPIN3) containing Arabidopsis SEIPIN cDNAs were transformed into SEIPIN-deletion yeast mutant (ylr404w.DELTA.) with Frozen-EZ Yeast Transformation II Kit.TM. (Zymo Research). The transformed yeast cells were selected by synthetic complete (SC)-Leu medium and then further confirmed by colony PCR.

Transient Expression of A. thaliana Seipins and Mouse Fit2 in N. benthamiana by Infiltration

[0317] Arabidopsis SEIPIN coding regions were cloned (as described above) and inserted into plant expression vector pMDC32 respectively to construct plant expression plasmids (pMDC32-AtSEIPIN1, AtSEIPIN2 and AtSEIPIN3). The mouse FIT2 gene coding region was obtained and subcloned into pMDC32 vector to be expressed in plants. The recombined plant expression plasmids were transformed into Agrobacterium tumefaciens (GV3101) by electroporation. Agrobacteria containing appropriate cDNAs were mixed and diluted with infiltration buffer to make the final infiltration mixtures, which were used to infiltrate N. benthamiana leaf tissue. The recipe of infiltration buffer, N. benthamiana and Agrobacterium growth conditions, and infiltration procedures were described by Petrie et al., 2010. Tomato bushy stunt virus protein P19 (Genbank accession number: AAB02538) plant expression plasmid pORE04-P19 was obtained and was included in all infiltration mixtures to enhance the gene expression in N. benthamiana leaf tissue. A. thaliana LEAFY COTYLEDON2 (AtLEC2) in pORE04 was also included in appropriate infiltration mixtures to enhance the synthesis of triacylglycerol (TAG) and further to simulate "seed metabolism" in N. benthamiana leaf tissue. The expression of different genes in N. benthamiana leaf tissue was tested at the transcriptional level by using RT-PCR. RNA was purified from N. benthamiana leaf tissue by RNeasy Plant Mini kit (Qiagen), and treated by DNase (Promega) to avoid any DNA contamination. RT-PCR was performed by using One-Step Ex Tag RT-PCR kit (Takara). The reverse transcription step was incubation at 42.degree. C. for 15 min. The pre-denaturation step was at 95.degree. C. for 5 min. The post-extension step was at 72.degree. C. for 7 min. EF1.alpha. and P19 were amplified by 28 cycles with 94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 72.degree. C. for 1 min. AtLEC2 and AtSEIPIN1 were amplified by 35 cycles with 94.degree. C. for 30 sec, 50.degree. C. for 30 sec and 72.degree. C. for 1 min. AtSEIPIN2 and AtSEIPIN3 were amplified by 35 cycles with 94.degree. C. for 30 sec, 50.degree. C. for 30 sec and 72.degree. C. for 1.5 min. For samples infiltrated with less than two genes, infiltrated with three cDNAs, and infiltrated with more than three genes, 50 ng, 100 ng and 200 ng of total RNA were used for amplification, respectively.

[0318] Lipid Analysis and Colocalization

[0319] To visualize lipid droplets (LD) in yeast cells, yeast cells were grown in appropriate SC drop-out medium (with glucose or oleic acid) at 28.degree. C. to stationary phase (0D600.about.3.0), and lipid droplets were stained with 0.4 .mu.g/ml Bodipy FL (Invitrogen, from 4 mg/ml stock in DMSO) in 50 mM PIPES buffer (pH=7). To visualize lipid droplets in N. benthamiana leaf tissue, leaf discs were collected 5-7 days after infiltration, and lipid droplets were stained with 2 .mu.g/ml Bodipy FL (from 4 mg/ml stock in DMSO) in 50 mM PIPES buffer (pH=7). To colocalize Arabodopsis SEIPINs, ER and LDs in yeast cells, Arabidopsis SEIPINs were fused with GFP at both N and C terminus and inserted in yeast expression plasmid pRS315-PGK. Endoplasmic Reticulum (ER) was indicated by ER marker (pRS316-CFP-HDEL) co-expressed with GFP-fused Arabidopsis SEIPINs. LDs were stained with 0.4 .mu.g/ml Nile Red (Sigma Aldrich, from 1 mg/ml stock in DMSO) in 50 mM PIPES buffer (pH=7) to avoid overlapping of emission spectra with GFP and CFP. To colocalize mouse FIT2 and LDs in N. benthamiana leaf tissue, FIT2 was fused with GFP at N terminus and lipid droplets were stained with 2 .mu.g/ml Nile Red (from 1 mg/ml stock in DMSO) in 50 mM PIPES buffer (pH=7). Confocal images were acquired by Zeiss LSM10 confocal laser scanning microscope (funded by NSF-MRI grant #1126205). GFP and Bodipy FL was excited by 488 nm laser and the emission signal was collected in a spectra of 500-540 nm. CFP was excited by 405 nm laser and the fluorescent signal was collected from 450 nm to 500 nm. Nile Red was excited by 488 nm laser and the emission was acquired from 520 nm to 560 nm. Chloroplast autofluorescence was collected in spectra of 640-720 nm. Both 2-D images and single images in Z-stack series were saved as 512.times.512-pixel (for yeast) and 1024.times.1024-pixel (for N. benthamiana) images.

[0320] To profile the effects of AtSEIPINs on LD morphology in different organisms (yeast and tobacco), numbers and sizes of lipid droplets were quantified by using ImageJ. In yeast, 3 lines with more than 150 cells for each strain were used for number quantification, and 3 lines with 30 LDs for each strain were used for size quantification. For LD statistics in N. benthamiana, 9 confocal images from 3 individual infiltrations for each transient expression were used to quantify the number of LDs for different size categories.

[0321] Quantification of TAG Content and Composition in Different Yeast Strains

[0322] Yeast cells were grown in appropriate SC drop-out medium (with glucose) until stationary phase (OD.about.3.0) and about 50 OD600 units cells were used for lipid extraction. The cells were disrupted by glass beads and bead beater (BioSpec Mini-Beadbeater-16), and 5 .mu.g TAG (tri-15:0) standard was added into each sample. Total lipid was extracted by using hot (70.degree. C.) isopropanol and chloroform in a ratio of 450 mg sample:2 ml isopropanol:1 ml chloroform at 4.degree. C. overnight. Then the total lipid was further purified by adding 1 ml chloroform and 2 ml 1M KCl, followed by washing with 2 ml 1 M KCl twice. The purified lipid was dried under N.sub.2, and stored in 400 .mu.l 1:1 chloroform/methanol at -20.degree. C. The neutral lipid was separated from polar lipid by using solid phase extraction (SPE). The 6 ml silica column (Sigma Aldrich) was cleaned with 3 ml acetone, and then conditioned with 6 ml hexane. Each lipid sample was loaded onto one conditioned column. 5 mL of hexane/diethyl ether 4:1 and hexane/diethyl ether 1:1 were used to elute neutral lipids. Then, 3 mL methanol and 3 mL chloroform were loaded to the column to elute polar lipids. The neutral lipid and polar lipid samples were evaporated under nitrogen and re-dissolved in chloroform/methanol 1:1 for storage. To analyze TAG content and composition, 20 .mu.L of neutral lipid for each sample was mixed with 5 .mu.L 500 mM ammonium acetate and 230 .mu.L chloroform/methanol 1:1, and injected into triple quadrupole mass spectrometer. The spectra were acquired using Xcalibur (v.2.0.7), and processed by Metabolite Imager (v.1.0) to quantify the total amount and composition of TAG.

[0323] Staining of Lipid Droplets with Nile Red and BODIPY 493/503

[0324] Stock solutions contained BODIPY 493/503 dissolved in ethanol at a concentration of 1 mg/ml. This solution is stored in the dark at -20.degree. C.).

[0325] Nile Red is Dissolved in DMSO to Give a Stock Solution of 50 .mu.g/ml.

[0326] Paraformaldehyde is aspirated off after fixing the cells and the cells are rinsed with PBS. PBS+Nile red (at 1:2000 dilution) or PBS+BODIPY 493/503 (at 1:1000 dilution) is added to the cells and agitated for 15 minutes. The staining solution was aspired out and the cells were washed thrice with PBS. Cells were mounted to observe under the microscope.

EXAMPLES

[0327] Following are examples that illustrate procedures for practicing the invention. The examples should not be construed as limiting.

Example 1

Increase of Lipid Content and Induction of Lipid Droplet Formation in Plants Using Mammalian Proteins Associated with Lipid Metabolism

[0328] Plant transformation vectors are constructed and are propagated in Eschericia coli Top 10 cells. The vectors are sequenced for verification. Plasmid vectors are transformed into Agrobacterium, tunefaciens LBA4404, and the clones are selected and verified by PCR. Arabidopsis plants are transformed by the floral dip method as described in Bent and Clough, Plant J. 1998 December; 16(6):735-43, which is herein incorporated by reference in its entirety.

[0329] Both wild-type plants (A. thaliana, ecotype Columbia), and plants with a transfer DNA (T-DNA) insertion mutation in the At4g24160 locus are used for transformations. The T-DNA knockout is in an exon of the Arabidopsis homolog of the human CG1-58 gene. For Arabidopsis plants with CGI-58 mutation, there is an increase in cystosolic lipid droplets in leaves when compared to wild-type plants (James et al., Proc Natl Acad Sci USA. 2010 Oct. 12; 107(41):17833-8).

[0330] FIG. 1 is a diagram that illustrates the elements in the T-DNA regions of plant binary transformation vectors. Plants are allowed to set seed and the seed are screened on hygromycin medium for identification of transgenic plants.

[0331] Cystolic lipid droplets are normally low in abundance in leaves of wildtype plants and they can be visualized by neutral-lipid-specific fluorescent stains like Nile blue (FIG. 2) or Bodipy493/503 (FIG. 3). The loss of function mutant, cgi-58, results in more lipid droplets than in wildtype plants (James et al., Proc Natl Acad Sci USA. 2010 Oct. 12; 107(41):17833-17838; see also FIG. 3. vs. FIG. 2). Expression of mouse FSP27 in either the wild-type or the cgi-58 background accentuates lipid droplet accumulation (FIGS. 2-4).

[0332] Total fatty acid content is measured in seedlings as a crude estimate of changes in lipid content. Fatty acid methyl esters are quantified by gas chromatography-flame ionization detection (GC-FID) using heptadecanoic acid as an internal standard. Transgenic T1 seedlings are grown on hygromycin medium, and plants with five rosette leaves are combined for extraction. Total lipids are extracted and fatty acid methyl esters are prepared according to Chapman and Moore (Arch Biochcem Biophys. 1993 Feb. 15; 301(1):21-33), which is herein incorporated by reference in its entirety.

[0333] The results show that transformed lines expressing FSP27 in the T1 generation have higher total fatty acid content than that of corresponding non-transformed plants on a fresh weight (FIG. 5) and a dry weight (FIG. 6) basis. Transformed lines being homozygous for FSP27 will exhibit greater increase in total fat content. Also, there will be a greater increase in total fat content when neutral lipids are separated from polar membrane lipids, since changes in fat content will be in triacylglycerol levels only, but not to bulk changes in membrane lipids.

Example 2

Generation of FSP27 and PLIN2 Expressing Homozygous Transgenic Plants with High Lipid Content

[0334] Seven homozygous lines of FSP27-expressing plants in the cgi58 mutant background, as well as one homozygous line expressing PLIN2 (ADRP) are raised. The new plants are completely viable and healthy with higher lipid accumulation as shown by microscopic data (FIG. 7).

[0335] Seedlings are grown on solidified nutrient medium under selection. Seven Arabidopsis homozygous lines in T2 generation over-expressing the FSP27 in the cgi58 knockout background are identified. Also, one Arabidopsis homozygous line in T2 generation overexpressing the ADRP in the cgi58 knockout background is identified. Lines that are no longer segregating (homozygous) are selected for harvest and extraction. FIG. 7 shows representative confocal images of leaves having preponderance of lipid droplets in both lines as well as the cgi-58 knockout background.

Example 3

Identification of Triglyceride-Accumulatory Domain of FSP27

[0336] Using deletion-mutagenesis, the domain of amino acids 120-220 of the mouse FSP27 protein (SEQ ID NO: 2), which is associated with lipid accumulation in adipocytes, is dissected. The domain 120-220 of mouse FSP27 is a core-portion of FSP27 protein. As shown in FIG. 8, adipocytes expressing amino acids 120-220 of the mouse FSP27 protein accumulate lipids faster than adipocytes expressing the full length mouse FSP27 protein.

[0337] The present invention also provides genetically engineered plants expressing only the triglyceride-accumulating domain of FSP27 (such as amino acids 120-220 of mouse FSP27), in order to accumulate lipids/oils at a faster rate than the full length protein. For the plants that need to be harvested from time to time for biofuel production, expressing the triglyceride-accumulating domain can be useful for improving lipid/or production.

Example 4

Expression of Mammalian and Fish Analogs of FSP27/Cidec/Cide-3 in Plants to Increase Lipid Contents

[0338] Homologs of mammalian proteins associated with lipid metabolism can be used to increase lipid/oil contents in transgenic plants. FSP27 plays a key role in triglyceride accumulation in mammals such as mouse and humans. As shown in FIG. 9, mammalian FSP27 and the zebra fish homolog of FSP27 protein share higher than 85% sequence similarity. In one embodiment, mammalian FSP27 and/or fish homologs of FSP27 can be used for expression in plants to generate transgenic plants with high oil and/or lipid contents.

Example 5

Increase of Lipid Content in Plants by Expressing a Combination of Proteins Associated with Lipid Metabolism

[0339] In certain embodiments, to increase and maximize the efficiency of oil production in plants, transgenic plants are genetically modified to express a combination of proteins associated with lipid metabolism and peptides. Proteins or polypeptides associated lipid metabolism useful for improving plant lipid/oil content include, but are not limited to, proteins and peptides involved in lipid (such as triglyceride) metabolism, such as, for example, proteins involved in the synthesis, protection, accumulation, storage, and breakdown of lipid (such as triglyceride).

[0340] For instance, FSP27 expression in plants increase plant lipid/oil content, and FSP27 expressed in CGI58-mutants results in even greater increase in lipid/oil content. In certain embodiments, the present invention provides transgenic plants expressing a combination of proteins associated with lipid metabolism including, but not limited to, DGAT-1, PDAT-1, cgi58 mutation, SEIPIN, FIT1, FIT2, PLIN1, PLIN2, FSP27/Cidec/cide-3, and Cidea.

[0341] In certain embodiments, the transgenic plants express a combination of nucleic acids expressing proteins associated with lipid metabolism selected from: DGAT-1 and FSP27; DGAT-1, cgi58 (mutation), and FSP27; DGAT-1, PDAT-1, and FSP27; DGAT-1, PDAT-1, cgi58 (mutation), FSP27; FSP27, PLIN2, and cgi58 (mutation); DGAT-1, FSP27, PLIN2, and cgi58 (mutation); and DGAT-1, PDAT-1, FSP27, PLIN2, and cgi58 (mutation).

[0342] In one embodiment, a combination of "triglyceride accumulation" proteins is expressed in leaves of plants with globally up-regulated fatty acid biosynthesis. Plants with globally up-regulated fatty acid biosynthesis include, but are not limited to, plants with the WRINKLED1 transcription factor mis-expressed in leaves. The WRINKLED1 transcription is involved in the regulation of fatty acid biosynthesis. See Sanjaya et al., 2011, Plant Biotechnology Journal (2011) 9, pp. 874-883), which is hereby incorporated as reference in its entirety.

Example 6

Homologues of Human Lipodystrophy Genes in A. thaliana

[0343] Table 1 shows Homologues of Human Lipodystrophy genes in A. thaliana

TABLE-US-00001 Human gene Protein function Candidate Arabidopsis homolog(s).sup.a Agpat2 LPAT, synthesis of At1g80950; At1g51260; At3g57650; phosphatidic acid At3g18850; At1g75020; At4g30580 Bscl2 SEIPIN, role in LD At5g16460; At1g29760; At2g34380 morphology Akt2 Protein Kinase B At3g08730; At3g08720; At5g04510.sup.b; At310540.sup.b Zmpste24 Zinc metalloprotease; At4g01320 processing of lamin subunits Cgi-58 Co-activator of ATGL, At4g24160 also has LPAT activity Lipa Lysosomal acid lipase; At5g14180; At2g15230 hydrolyzes cholesteryl esters and TAGs .sup.aBest match by WU-BLAST against the Arabidopsis genome at TAIR [www.arabidopsis.org]. .sup.bContains pleckstrin homology domains and has phosphoinositide-3-dependent kinase activity.

Example 7

Increased Lipid Content in Plants by Expressing Proteins Associated with Lipid Metabolism or Combinations Thereof

[0344] Proteins associated with lipid metabolism of animal origin, for example, mouse and human, or of plant origin, for example, A. thaliana, were transiently expressed in vegetative tissues of plants, for example, N. benthamiana (a close relative of tobacco and species of Nicotiana indigenous to Australia) and A. thaliana. Increased lipid accumulation in lipid droplets of plants transiently expressing exogenous proteins or polypeptide associated with lipid metabolism was observed indicating that overexpression of exogenous proteins associated with lipid metabolism in vegetative tissue of plants can be used to increase lipid production in these plants and such plants provide a valuable means of producing higher yields of biofuel.

[0345] Further, plants permanently expressing exogenous proteins or polypeptide associated with lipid metabolism, for example, having the exogenous proteins associated with lipid metabolism incorporated in the genomes of the plants to produce transgenic plants, can also be used to produce higher amounts of lipids in such plants. These plants can also provide valuable means of producing higher yields of biofuel.

[0346] Examples of techniques of expressing endogenous lipid droplets in vegetative tissues of plants and increased lipid accumulation in plants expressing exogenous proteins associated with lipid metabolism are provided in FIGS. 25 to 36.

[0347] Over-expression of SEIPINs in leaves enhances the capacity for neutral lipid storage, and provides additional strategies to engineer increased neutral lipid accumulation in plant cells, including even subcellular "packages" of different sizes. Transient overexpression of SEIPINs in tobacco leaves increases lipid droplet numbers and influences the size of LDs (S1, large; S2, medium; S3 small). The current invention provides that permanent overexpression of proteins associated with lipid metabolism, such as SEIPINs, can be used to produce higher amounts of oil in plants as compared to wild type plants of the same type.

Example 8

Increase of Lipid Content in Yeast Cells by Expressing Proteins Associated with Lipid Metabolism or Combinations Thereof

[0348] Wild type cells of S. cerevisiae produce lipid droplets (see, FIG. 13, top left panel). A yeast SEIPIN (ScSEIPIN) plays an important role in the production of these lipid droplets in S. cerevisiae as shown by reduced accumulation of lipids in S. cerevisiae mutant (ylr404w.DELTA.) lacking ScSEIPIN activity (see, FIG. 13, top middle panel). The role of ScSEIPIN in lipid droplet production in yeast is further confirmed by restoration of lipid accumulation in ylr404w.DELTA. expressing ScSEIPIN. FIG. 13, bottom panels, further show that expression of exogenous SEIPINs, namely SEIPIN 1, 2, or 3 from A. thaliana also restores lipid accumulation in ylr404w.DELTA..

[0349] Further, expression of SEIPIN 1, 2, or 3 in ylr404w.DELTA. produces lipid droplets of varying morphologies (see FIGS. 13-16 and 24). For example, overexpression of AtSEIPIN1 produces lipid droplets of larger size than the wild type, whereas overexpression of AtSEIPIN2 or 3, without affecting the size of the lipid droplets, increases the number of lipid droplets per yeast cell compared to ylr404w.DELTA. mutant.

[0350] Furthermore, overexpression of AtSEIPIN 1, 2, or 3 in ylr404w.DELTA. restores the amount of TAG accumulation comparable to that found in the wild type yeast cells (see, FIGS. 21-23).

[0351] These data show that the three A. thaliana SEIPIN homologues provide different developmental expression profiles. All localize to discrete domains of ER in heterologous system (yeast). AtSEIPINs 2 and 3 partially complement yeast mutants, indicating they function generally in a similar manner to yeast and human SEIPIN in the regulation of LD number and shape. AtSEIPIN1 generates supersize LDs in yeast (and plants).

Example 9

Colocalization of Seipins and Lipid Droplets in Yeast

[0352] AtSEIPINs, when overexpressed in ylr404w.DELTA., localize to lipid droplets which further confirms the role of SEIPINs in lipid droplet accumulation in yeast (see, for example, FIGS. 17-20). AtSEIPIN-GFP and CFP-HDEL were overexpressed in a yeast cells. Conjugation with GFP allowed visualization of the location of AtSEIPINs in a cell by green fluorescence (see, FIGS. 18-20, top right panels), whereas expression of CFP-HDEL allowed visualization of endoplasmic reticulum as blue fluorescence in the yeast cell (see, FIGS. 18-20, bottom left panels). Lipid droplets in these yeast cells is visualized by Nile Red staining (see, FIGS. 18-20, top left panels).

[0353] Overlapping the top left, bottom left, and top right columns in FIGS. 18-20 indicates that green fluorescence coming from AtSEIPIN GFP fusion proteins largely co-localized with the yellow staining of lipid droplets. Blue fluorescence of CFR-HDEL did not colocalize with either the lipid droplets or the AtSEIPIN GFP fusion proteins.

Example 10

Expression of Lipid-Droplets Associated Proteins in Algae to Increase Algal Lipid Contents

[0354] Overexpression of various proteins associated with lipid metabolism from mammalian and plant origin, for example, FSP27, Cidea, PLIN1, PLIN2, SEIPIN, FIT1, and FIT2 in various cell types cause 3-10 fold increase in fat accumulation. Algae are widely used as an organism for production of biofuel. Accordingly, the current invention further provides algal cells expressing one or more of the proteins associated with lipid metabolism, either from animal or plant origin. These algal cells contain higher amounts of oil/fat.

[0355] Examples of various proteins or polypeptides associated with lipid metabolism that can be expressed in algae to produce increased oil in algae include, but are not limited to FSP27, Cidea, ADRP, PLIN1, FIT, /2, SEIPIN, SEIPIN 1, SEIPIN 2, SEIPIN 3, DGAT1, DGAT2, PDAT1, WRIT, and mutant CGI-58. Examples of algae that can be used according to the current invention to produce oil include, but are not limited to algae from Chlamydomonas spp., Botryococcus braunii, Chlorella spp., Dunaliella tertiolecta, Gracilaria spp., Pleurochrysis camerae (also called CCMP647), Sargassum spp., and Eudorina elegans.

[0356] Non-limiting examples of various fuel types that can be produced in algae expressing exogenous proteins associated with lipid metabolism include biodiesel, biobutanol, biogasoline, methane, ethanol, vegetable oil fuel, hydrocracking to traditional transport fuels, and jet fuel.

[0357] Thus, the algal cells of the current invention can be used to produce energy with higher efficiency and at a cost effective manner.

[0358] Algal cells of the current invention can also be used to increase production of oils which are beneficial for human health, e.g. omega-unsaturated fat in olives, canola oil, etc. For example, fatty acid analysis in FSP27 expressing plants show that besides increase in overall oil content the content of omega-3 fatty acids, particularly linoleic (18:2) and alpha-linolenic (18:3) fatty acid, is increased in these plants.

[0359] Certain proteins associated with lipid metabolism play a positive regulatory role in improving the metabolic health in humans suffering from insulin resistance, type 2 diabetes, cardiovascular disease, etc. Generating algae expressing such proteins associated with lipid metabolism can have therapeutic use based on the positive role played by these proteins.

[0360] Various techniques discussed in references 11-14 can be used to genetically manipulate algae according to the current invention and are expressly incorporated by reference herein. Methods of genetically manipulating algae, in addition to those described in references 11-14, are well known to a person of ordinary skill in the art and such methods are within the purview of the current invention.

[0361] Non-limiting examples of vectors used for transformation in algae include pPmr3 plasmid, pmfg-GLuc (mfg refers to "my favorite gene"), pALM32, and pALM33.

[0362] All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0363] The terms "a" and "an" and "the" and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0364] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

[0365] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0366] The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0367] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

REFERENCES

[0368] 1. Curtis and Grossniklaus, A gateway cloning vector set for high-throughput functinal analysis of genes in planta, Plant Physiology, Vol. 133, p 462-469 (2003). [0369] 2. Gross et al. (2011) PNAS 108, 19581-19586; PMID: 22106267. [0370] 3. Jambunathan et al., FSP27 promotes lipid droplet clustering and then fusion to regulate triglyceride accumulation (2011). [0371] 4. James et al. (2010) PNAS 107, 17833-1838, PMID: 20876112 [0372] 5. Sanjaya et al., 2011, Plant Biotechnology Journal (2011) 9, pp. 874-883. [0373] 6. Szymanski et al. (2007) PNAS 104, 20890-5, PMID: 18093937. [0374] 7. Zhang et al. (2009) Plant Cell 21, 3885-901, PMID: 20040537. [0375] 8. U.S. Application Publication No. 2010/0221400. [0376] 9. Petrie, J. R., Shrestha, P., Liu, Q., Mansour, M. P., Wood, C. C., Zhou, X. R., Nichols, P. D., Green, A. G., and Singh, S. P. (2010). Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production. Plant Methods 6, 8. [0377] 10. Szymanski, K. M., Binns, D., Bartz, R., Grishin, N. V., Li, W. P., Agarwal, A. K., Garg, A., Anderson, R. G., and Goodman, J. M. (2007). The lipodystrophy protein SEIPIN is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc Natl Acad Sci USA 104, 20890-20895. [0378] 11. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33, 949-956. [0379] 12. Neupert J, Shao N, Lu Y, Bock R. (2012), Genetic transformation of the model green alga Chlamydomonas reinhardtii. Methods Mol Biol.; 847:35-47. [0380] Lerche K, Hallmann A. (2013), Stable nuclear transformation of Eudorina elegans. BMC Biotechnol. 13:11. [0381] 13. Meslet-Cladiere L, Vallon O. (2011), Novel shuttle markers for nuclear transformation of the green alga Chlamydomonas reinhardtii. Eukaryot Cell; 10(12):1670-8. [0382] 14. U.S. Application Publication No. 2009/0176272.

Sequence CWU 1

1

371238PRTHomo sapiens 1Met Glu Tyr Ala Met Lys Ser Leu Ser Leu Leu Tyr Pro Lys Ser Leu 1 5 10 15 Ser Arg His Val Ser Val Arg Thr Ser Val Val Thr Gln Gln Leu Leu 20 25 30 Ser Glu Pro Ser Pro Lys Ala Pro Arg Ala Arg Pro Cys Arg Val Ser 35 40 45 Thr Ala Asp Arg Ser Val Arg Lys Gly Ile Met Ala Tyr Ser Leu Glu 50 55 60 Asp Leu Leu Leu Lys Val Arg Asp Thr Leu Met Leu Ala Asp Lys Pro 65 70 75 80 Phe Phe Leu Val Leu Glu Glu Asp Gly Thr Thr Val Glu Thr Glu Glu 85 90 95 Tyr Phe Gln Ala Leu Ala Gly Asp Thr Val Phe Met Val Leu Gln Lys 100 105 110 Gly Gln Lys Trp Gln Pro Pro Ser Glu Gln Gly Thr Arg His Pro Leu 115 120 125 Ser Leu Ser His Lys Pro Ala Lys Lys Ile Asp Val Ala Arg Val Thr 130 135 140 Phe Asp Leu Tyr Lys Leu Asn Pro Gln Asp Phe Ile Gly Cys Leu Asn 145 150 155 160 Val Lys Ala Thr Phe Tyr Asp Thr Tyr Ser Leu Ser Tyr Asp Leu His 165 170 175 Cys Cys Gly Ala Lys Arg Ile Met Lys Glu Ala Phe Arg Trp Ala Leu 180 185 190 Phe Ser Met Gln Ala Thr Gly His Val Leu Leu Gly Thr Ser Cys Tyr 195 200 205 Leu Gln Gln Leu Leu Asp Ala Thr Glu Glu Gly Gln Pro Pro Lys Gly 210 215 220 Lys Ala Ser Ser Leu Ile Pro Thr Cys Leu Lys Ile Leu Gln 225 230 235 2239PRTMus musculus 2Met Asp Tyr Ala Met Lys Ser Leu Ser Leu Leu Tyr Pro Arg Ser Leu 1 5 10 15 Ser Arg His Val Ala Val Ser Thr Ala Val Val Thr Gln Gln Leu Val 20 25 30 Ser Lys Pro Ser Arg Glu Thr Pro Arg Ala Arg Pro Cys Arg Val Ser 35 40 45 Thr Ala Asp Arg Lys Val Arg Lys Gly Ile Met Ala His Ser Leu Glu 50 55 60 Asp Leu Leu Asn Lys Val Gln Asp Ile Leu Lys Leu Lys Asp Lys Pro 65 70 75 80 Phe Ser Leu Val Leu Glu Glu Asp Gly Thr Ile Val Glu Thr Glu Glu 85 90 95 Tyr Phe Gln Ala Leu Ala Lys Asp Thr Met Phe Met Val Leu Leu Lys 100 105 110 Gly Gln Lys Trp Lys Pro Pro Ser Glu Gln Arg Lys Lys Arg Ala Gln 115 120 125 Leu Ala Leu Ser Gln Lys Pro Thr Lys Lys Ile Asp Val Ala Arg Val 130 135 140 Thr Phe Asp Leu Tyr Lys Leu Asn Pro Gln Asp Phe Ile Gly Cys Leu 145 150 155 160 Asn Val Lys Ala Thr Leu Tyr Asp Thr Tyr Ser Leu Ser Tyr Asp Leu 165 170 175 His Cys Tyr Lys Ala Lys Arg Ile Val Lys Glu Met Leu Arg Trp Thr 180 185 190 Leu Phe Ser Met Gln Ala Thr Gly His Met Leu Leu Gly Thr Ser Ser 195 200 205 Tyr Met Gln Gln Phe Leu Asp Ala Thr Glu Glu Glu Gln Pro Ala Lys 210 215 220 Ala Lys Pro Ser Ser Leu Leu Pro Ala Cys Leu Lys Met Leu Gln 225 230 235 3522PRTHomo sapiens 3Met Ala Val Asn Lys Gly Leu Thr Leu Leu Asp Gly Asp Leu Pro Glu 1 5 10 15 Gln Glu Asn Val Leu Gln Arg Val Leu Gln Leu Pro Val Val Ser Gly 20 25 30 Thr Cys Glu Cys Phe Gln Lys Thr Tyr Thr Ser Thr Lys Glu Ala His 35 40 45 Pro Leu Val Ala Ser Val Cys Asn Ala Tyr Glu Lys Gly Val Gln Ser 50 55 60 Ala Ser Ser Leu Ala Ala Trp Ser Met Glu Pro Val Val Arg Arg Leu 65 70 75 80 Ser Thr Gln Phe Thr Ala Ala Asn Glu Leu Ala Cys Arg Gly Leu Asp 85 90 95 His Leu Glu Glu Lys Ile Pro Ala Leu Gln Tyr Pro Pro Glu Lys Ile 100 105 110 Ala Ser Glu Leu Lys Asp Thr Ile Ser Thr Arg Leu Arg Ser Ala Arg 115 120 125 Asn Ser Ile Ser Val Pro Ile Ala Ser Thr Ser Asp Lys Val Leu Gly 130 135 140 Ala Ala Leu Ala Gly Cys Glu Leu Ala Trp Gly Val Ala Arg Asp Thr 145 150 155 160 Ala Glu Phe Ala Ala Asn Thr Arg Ala Gly Arg Leu Ala Ser Gly Gly 165 170 175 Ala Asp Leu Ala Leu Gly Ser Ile Glu Lys Val Val Glu Tyr Leu Leu 180 185 190 Pro Pro Asp Lys Glu Glu Ser Ala Pro Ala Pro Gly His Gln Gln Ala 195 200 205 Gln Lys Ser Pro Lys Ala Lys Pro Ser Leu Leu Ser Arg Val Gly Ala 210 215 220 Leu Thr Asn Thr Leu Ser Arg Tyr Thr Val Gln Thr Met Ala Arg Ala 225 230 235 240 Leu Glu Gln Gly His Thr Val Ala Met Trp Ile Pro Gly Val Val Pro 245 250 255 Leu Ser Ser Leu Ala Gln Trp Gly Ala Ser Val Ala Met Gln Ala Val 260 265 270 Ser Arg Arg Arg Ser Glu Val Arg Val Pro Trp Leu His Ser Leu Ala 275 280 285 Ala Ala Gln Glu Glu Asp His Glu Asp Gln Thr Asp Thr Glu Gly Glu 290 295 300 Asp Thr Glu Glu Glu Glu Glu Leu Glu Thr Glu Glu Asn Lys Phe Ser 305 310 315 320 Glu Val Ala Ala Leu Pro Gly Pro Arg Gly Leu Leu Gly Gly Val Ala 325 330 335 His Thr Leu Gln Lys Thr Leu Gln Thr Thr Ile Ser Ala Val Thr Trp 340 345 350 Ala Pro Ala Ala Val Leu Gly Met Ala Gly Arg Val Leu His Leu Thr 355 360 365 Pro Ala Pro Ala Val Ser Ser Thr Lys Gly Arg Ala Met Ser Leu Ser 370 375 380 Asp Ala Leu Lys Gly Val Thr Asp Asn Val Val Asp Thr Val Val His 385 390 395 400 Tyr Val Pro Leu Pro Arg Leu Ser Leu Met Glu Pro Glu Ser Glu Phe 405 410 415 Arg Asp Ile Asp Asn Pro Pro Ala Glu Val Glu Arg Arg Glu Ala Glu 420 425 430 Arg Arg Ala Ser Gly Ala Pro Ser Ala Gly Pro Glu Pro Ala Pro Arg 435 440 445 Leu Ala Gln Pro Arg Arg Ser Leu Arg Ser Ala Gln Ser Pro Gly Ala 450 455 460 Pro Pro Gly Pro Gly Leu Glu Asp Glu Val Ala Thr Pro Ala Ala Pro 465 470 475 480 Arg Pro Gly Phe Pro Ala Val Pro Arg Glu Lys Pro Lys Arg Arg Val 485 490 495 Ser Asp Ser Phe Phe Arg Pro Ser Val Met Glu Pro Ile Leu Gly Arg 500 505 510 Thr His Tyr Ser Gln Leu Arg Lys Lys Ser 515 520 4 517PRTMus musculus 4Met Ser Met Asn Lys Gly Pro Thr Leu Leu Asp Gly Asp Leu Pro Glu 1 5 10 15 Gln Glu Asn Val Leu Gln Arg Val Leu Gln Leu Pro Val Val Ser Gly 20 25 30 Thr Cys Glu Cys Phe Gln Lys Thr Tyr Asn Ser Thr Lys Glu Ala His 35 40 45 Pro Leu Val Ala Ser Val Cys Asn Ala Tyr Glu Lys Gly Val Gln Gly 50 55 60 Ala Ser Asn Leu Ala Ala Trp Ser Met Glu Pro Val Val Arg Arg Leu 65 70 75 80 Ser Thr Gln Phe Thr Ala Ala Asn Glu Leu Ala Cys Arg Gly Leu Asp 85 90 95 His Leu Glu Glu Lys Ile Pro Ala Leu Gln Tyr Pro Pro Glu Lys Ile 100 105 110 Ala Ser Glu Leu Lys Gly Thr Ile Ser Thr Arg Leu Arg Ser Ala Arg 115 120 125 Asn Ser Ile Ser Val Pro Ile Ala Ser Thr Ser Asp Lys Val Leu Gly 130 135 140 Ala Thr Leu Ala Gly Cys Glu Leu Ala Leu Gly Met Ala Lys Glu Thr 145 150 155 160 Ala Glu Tyr Ala Ala Asn Thr Arg Val Gly Arg Leu Ala Ser Gly Gly 165 170 175 Ala Asp Leu Ala Leu Gly Ser Ile Glu Lys Val Val Glu Phe Leu Leu 180 185 190 Pro Pro Asp Lys Glu Ser Ala Pro Ser Ser Gly Arg Gln Arg Thr Gln 195 200 205 Lys Ala Pro Lys Ala Lys Pro Ser Leu Val Arg Arg Val Ser Thr Leu 210 215 220 Ala Asn Thr Leu Ser Arg His Thr Met Gln Thr Thr Ala Trp Ala Leu 225 230 235 240 Lys Gln Gly His Ser Leu Ala Met Trp Ile Pro Gly Val Ala Pro Leu 245 250 255 Ser Ser Leu Ala Gln Trp Gly Ala Ser Ala Ala Met Gln Val Val Ser 260 265 270 Arg Arg Gln Ser Glu Val Arg Val Pro Trp Leu His Asn Leu Ala Ala 275 280 285 Ser Gln Asp Glu Ser His Asp Asp Gln Thr Asp Thr Glu Gly Glu Glu 290 295 300 Thr Asp Asp Glu Glu Glu Glu Glu Glu Ser Glu Ala Glu Glu Asn Val 305 310 315 320 Leu Arg Glu Val Thr Ala Leu Pro Asn Pro Arg Gly Leu Leu Gly Gly 325 330 335 Val Val His Thr Val Gln Asn Thr Leu Arg Asn Thr Ile Ser Ala Val 340 345 350 Thr Trp Ala Pro Ala Ala Val Leu Gly Thr Val Gly Arg Ile Leu His 355 360 365 Leu Thr Pro Ala Gln Ala Val Ser Ser Thr Lys Gly Arg Ala Met Ser 370 375 380 Leu Ser Asp Ala Leu Lys Gly Val Thr Asp Asn Val Val Asp Thr Val 385 390 395 400 Val His Tyr Val Pro Leu Pro Arg Leu Ser Leu Met Glu Pro Glu Ser 405 410 415 Glu Phe Arg Asp Ile Asp Asn Pro Ser Ala Glu Ala Glu Arg Lys Gly 420 425 430 Ser Gly Ala Arg Pro Ala Ser Pro Glu Ser Thr Pro Arg Pro Gly Gln 435 440 445 Pro Arg Gly Ser Leu Arg Ser Val Arg Gly Leu Ser Ala Pro Ser Cys 450 455 460 Pro Gly Leu Asp Asp Lys Thr Glu Ala Ser Ala Arg Pro Gly Phe Leu 465 470 475 480 Ala Met Pro Arg Glu Lys Pro Ala Arg Arg Val Ser Asp Ser Phe Phe 485 490 495 Arg Pro Ser Val Met Glu Pro Ile Leu Gly Arg Ala Gln Tyr Ser Gln 500 505 510 Leu Arg Lys Lys Ser 515 5 437PRTHomo sapiens 5Met Ala Ser Val Ala Val Asp Pro Gln Pro Ser Val Val Thr Arg Val 1 5 10 15 Val Asn Leu Pro Leu Val Ser Ser Thr Tyr Asp Leu Met Ser Ser Ala 20 25 30 Tyr Leu Ser Thr Lys Asp Gln Tyr Pro Tyr Leu Lys Ser Val Cys Glu 35 40 45 Met Ala Glu Asn Gly Val Lys Thr Ile Thr Ser Val Ala Met Thr Ser 50 55 60 Ala Leu Pro Ile Ile Gln Lys Leu Glu Pro Gln Ile Ala Val Ala Asn 65 70 75 80 Thr Tyr Ala Cys Lys Gly Leu Asp Arg Ile Glu Glu Arg Leu Pro Ile 85 90 95 Leu Asn Gln Pro Ser Thr Gln Ile Val Ala Asn Ala Lys Gly Ala Val 100 105 110 Thr Gly Ala Lys Asp Ala Val Thr Thr Thr Val Thr Gly Ala Lys Asp 115 120 125 Ser Val Ala Ser Thr Ile Thr Gly Val Met Asp Lys Thr Lys Gly Ala 130 135 140 Val Thr Gly Ser Val Glu Lys Thr Lys Ser Val Val Ser Gly Ser Ile 145 150 155 160 Asn Thr Val Leu Gly Ser Arg Met Met Gln Leu Val Ser Ser Gly Val 165 170 175 Glu Asn Ala Leu Thr Lys Ser Glu Leu Leu Val Glu Gln Tyr Leu Pro 180 185 190 Leu Thr Glu Glu Glu Leu Glu Lys Glu Ala Lys Lys Val Glu Gly Phe 195 200 205 Asp Leu Val Gln Lys Pro Ser Tyr Tyr Val Arg Leu Gly Ser Leu Ser 210 215 220 Thr Lys Leu His Ser Arg Ala Tyr Gln Gln Ala Leu Ser Arg Val Lys 225 230 235 240 Glu Ala Lys Gln Lys Ser Gln Gln Thr Ile Ser Gln Leu His Ser Thr 245 250 255 Val His Leu Ile Glu Phe Ala Arg Lys Asn Val Tyr Ser Ala Asn Gln 260 265 270 Lys Ile Gln Asp Ala Gln Asp Lys Leu Tyr Leu Ser Trp Val Glu Trp 275 280 285 Lys Arg Ser Ile Gly Tyr Asp Asp Thr Asp Glu Ser His Cys Ala Glu 290 295 300 His Ile Glu Ser Arg Thr Leu Ala Ile Ala Arg Asn Leu Thr Gln Gln 305 310 315 320 Leu Gln Thr Thr Cys His Thr Leu Leu Ser Asn Ile Gln Gly Val Pro 325 330 335 Gln Asn Ile Gln Asp Gln Ala Lys His Met Gly Val Met Ala Gly Asp 340 345 350 Ile Tyr Ser Val Phe Arg Asn Ala Ala Ser Phe Lys Glu Val Ser Asp 355 360 365 Ser Leu Leu Thr Ser Ser Lys Gly Gln Leu Gln Lys Met Lys Glu Ser 370 375 380 Leu Asp Asp Val Met Asp Tyr Leu Val Asn Asn Thr Pro Leu Asn Trp 385 390 395 400 Leu Val Gly Pro Phe Tyr Pro Gln Leu Thr Glu Ser Gln Asn Ala Gln 405 410 415 Asp Gln Gly Ala Glu Met Asp Lys Ser Ser Gln Glu Thr Gln Arg Ser 420 425 430 Glu His Lys Thr His 435 6 425PRTMus musculus 6Met Ala Ala Ala Val Val Asp Pro Gln Gln Ser Val Val Met Arg Val 1 5 10 15 Ala Asn Leu Pro Leu Val Ser Ser Thr Tyr Asp Leu Val Ser Ser Ala 20 25 30 Tyr Val Ser Thr Lys Asp Gln Tyr Pro Tyr Leu Arg Ser Val Cys Glu 35 40 45 Met Ala Glu Lys Gly Val Lys Thr Val Thr Ser Ala Ala Met Thr Ser 50 55 60 Ala Leu Pro Ile Ile Gln Lys Leu Glu Pro Gln Ile Ala Val Ala Asn 65 70 75 80 Thr Tyr Ala Cys Lys Gly Leu Asp Arg Met Glu Glu Arg Leu Pro Ile 85 90 95 Leu Asn Gln Pro Thr Ser Glu Ile Val Ala Ser Ala Arg Gly Ala Val 100 105 110 Thr Gly Ala Lys Asp Val Val Thr Thr Thr Met Ala Gly Ala Lys Asp 115 120 125 Ser Val Ala Ser Thr Val Ser Gly Val Val Asp Lys Thr Lys Gly Ala 130 135 140 Val Thr Gly Ser Val Glu Arg Thr Lys Ser Val Val Asn Gly Ser Ile 145 150 155 160 Asn Thr Val Leu Gly Met Val Gln Phe Met Asn Ser Gly Val Asp Asn 165 170 175 Ala Ile Thr Lys Ser Glu Leu Leu Val Asp Gln Tyr Phe Pro Leu Thr 180 185 190 Gln Glu Glu Leu Glu Met Glu Ala Lys Lys Val Glu Gly Phe Asp Met 195 200 205 Val Gln Lys Pro Ser Asn Tyr Glu Arg Leu Glu Ser Leu Ser Thr Lys 210 215 220 Leu Cys Ser Arg Ala Tyr His Gln Ala Leu Ser Arg Val Lys Glu Ala 225 230 235 240 Lys Gln Lys Ser Gln Glu Thr Ile Ser Gln Leu His Ser Thr Val His 245 250 255 Leu Ile Glu Phe Ala Arg Lys Asn Met His Ser Ala Asn Gln Lys Ile 260 265 270 Gln Gly Ala Gln Asp Lys Leu Tyr Val Ser Trp Val Glu Trp Lys Arg 275 280 285 Ser Ile Gly Tyr Asp Asp Thr Asp Glu Ser His Cys Val Glu His Ile 290 295 300 Glu Ser Arg Thr Leu Ala Ile Ala Arg Asn Leu Thr Gln Gln Leu Gln 305 310 315 320 Thr Thr Cys Gln Thr Val Leu Val Asn Ala Gln Gly Leu Pro Gln Asn 325 330

335 Ile Gln Asp Gln Ala Lys His Leu Gly Val Met Ala Gly Asp Ile Tyr 340 345 350 Ser Val Phe Arg Asn Ala Ala Ser Phe Lys Glu Val Ser Asp Gly Val 355 360 365 Leu Thr Ser Ser Lys Gly Gln Leu Gln Lys Met Lys Glu Ser Leu Asp 370 375 380 Glu Val Met Asp Tyr Phe Val Asn Asn Thr Pro Leu Asn Trp Leu Val 385 390 395 400 Gly Pro Phe Tyr Pro Gln Ser Thr Glu Val Asn Lys Ala Ser Leu Lys 405 410 415 Val Gln Gln Ser Glu Val Lys Ala Gln 420 425 7398PRTHomo sapiens 7Met Val Asn Asp Pro Pro Val Pro Ala Leu Leu Trp Ala Gln Glu Val 1 5 10 15 Gly Gln Val Leu Ala Gly Arg Ala Arg Arg Leu Leu Leu Gln Phe Gly 20 25 30 Val Leu Phe Cys Thr Ile Leu Leu Leu Leu Trp Val Ser Val Phe Leu 35 40 45 Tyr Gly Ser Phe Tyr Tyr Ser Tyr Met Pro Thr Val Ser His Leu Ser 50 55 60 Pro Val His Phe Tyr Tyr Arg Thr Asp Cys Asp Ser Ser Thr Thr Ser 65 70 75 80 Leu Cys Ser Phe Pro Val Ala Asn Val Ser Leu Thr Lys Gly Gly Arg 85 90 95 Asp Arg Val Leu Met Tyr Gly Gln Pro Tyr Arg Val Thr Leu Glu Leu 100 105 110 Glu Leu Pro Glu Ser Pro Val Asn Gln Asp Leu Gly Met Phe Leu Val 115 120 125 Thr Ile Ser Cys Tyr Thr Arg Gly Gly Arg Ile Ile Ser Thr Ser Ser 130 135 140 Arg Ser Val Met Leu His Tyr Arg Ser Asp Leu Leu Gln Met Leu Asp 145 150 155 160 Thr Leu Val Phe Ser Ser Leu Leu Leu Phe Gly Phe Ala Glu Gln Lys 165 170 175 Gln Leu Leu Glu Val Glu Leu Tyr Ala Asp Tyr Arg Glu Asn Ser Tyr 180 185 190 Val Pro Thr Thr Gly Ala Ile Ile Glu Ile His Ser Lys Arg Ile Gln 195 200 205 Leu Tyr Gly Ala Tyr Leu Arg Ile His Ala His Phe Thr Gly Leu Arg 210 215 220 Tyr Leu Leu Tyr Asn Phe Pro Met Thr Cys Ala Phe Ile Gly Val Ala 225 230 235 240 Ser Asn Phe Thr Phe Leu Ser Val Ile Val Leu Phe Ser Tyr Met Gln 245 250 255 Trp Val Trp Gly Gly Ile Trp Pro Arg His Arg Phe Ser Leu Gln Val 260 265 270 Asn Ile Arg Lys Arg Asp Asn Ser Arg Lys Glu Val Gln Arg Arg Ile 275 280 285 Ser Ala His Gln Pro Gly Pro Glu Gly Gln Glu Glu Ser Thr Pro Gln 290 295 300 Ser Asp Val Thr Glu Asp Gly Glu Ser Pro Glu Asp Pro Ser Gly Thr 305 310 315 320 Glu Gly Gln Leu Ser Glu Glu Glu Lys Pro Asp Gln Gln Pro Leu Ser 325 330 335 Gly Glu Glu Glu Leu Glu Pro Glu Ala Ser Asp Gly Ser Gly Ser Trp 340 345 350 Glu Asp Ala Ala Leu Leu Thr Glu Ala Asn Leu Pro Ala Pro Ala Pro 355 360 365 Ala Ser Ala Ser Ala Pro Val Leu Glu Thr Leu Gly Ser Ser Glu Pro 370 375 380 Ala Gly Gly Ala Leu Arg Gln Arg Pro Thr Cys Ser Ser Ser 385 390 395 8383PRTMus musculus 8Met Val Asn Asp Pro Pro Val Pro Ala Leu Leu Trp Ala Gln Glu Val 1 5 10 15 Gly His Val Leu Ala Gly Arg Ala Arg Arg Leu Met Leu Gln Phe Gly 20 25 30 Val Leu Phe Cys Thr Ile Leu Leu Leu Leu Trp Val Ser Val Phe Leu 35 40 45 Tyr Gly Ser Phe Tyr Tyr Ser Tyr Met Pro Thr Val Ser His Leu Ser 50 55 60 Pro Val His Phe His Tyr Arg Thr Asp Cys Asp Ser Ser Thr Ala Ser 65 70 75 80 Leu Cys Ser Phe Pro Val Ala Asn Val Ser Leu Ala Lys Ser Gly Arg 85 90 95 Asp Arg Val Leu Met Tyr Gly Gln Pro Tyr Arg Val Thr Leu Glu Leu 100 105 110 Glu Leu Pro Glu Ser Pro Val Asn Gln Asp Leu Gly Met Phe Leu Val 115 120 125 Thr Val Ser Cys Tyr Thr Arg Gly Gly Arg Ile Ile Ser Thr Ser Ser 130 135 140 Arg Ser Val Met Leu His Tyr Arg Ser Gln Leu Leu Gln Val Leu Asp 145 150 155 160 Thr Leu Leu Phe Ser Ser Leu Leu Leu Phe Gly Phe Ala Glu Gln Lys 165 170 175 Gln Leu Leu Glu Val Glu Leu Tyr Ser Asp Tyr Arg Glu Asn Ser Tyr 180 185 190 Val Pro Thr Thr Gly Ala Ile Ile Glu Ile His Ser Lys Arg Ile Gln 195 200 205 Met Tyr Gly Ala Tyr Leu Arg Ile His Ala His Phe Thr Gly Leu Arg 210 215 220 Tyr Leu Leu Tyr Asn Phe Pro Met Thr Cys Ala Phe Val Gly Val Ala 225 230 235 240 Ser Asn Phe Thr Phe Leu Ser Val Ile Val Leu Phe Ser Tyr Met Gln 245 250 255 Trp Val Trp Gly Ala Val Trp Pro Arg His Arg Phe Ser Leu Gln Val 260 265 270 Asn Ile Arg Gln Arg Asp Asn Ser His His Gly Ala Pro Arg Arg Ile 275 280 285 Ser Arg His Gln Pro Gly Gln Glu Ser Thr Gln Gln Ser Asp Val Thr 290 295 300 Glu Asp Gly Glu Ser Pro Glu Asp Pro Ser Gly Thr Glu Gly Gln Leu 305 310 315 320 Ser Glu Glu Glu Lys Pro Glu Lys Arg Pro Leu Asn Gly Glu Glu Glu 325 330 335 Gln Glu Pro Glu Ala Ser Asp Gly Ser Trp Glu Asp Ala Ala Leu Leu 340 345 350 Thr Glu Ala Asn Pro Pro Thr Ser Ala Ser Ala Ser Ala Leu Ala Pro 355 360 365 Glu Thr Leu Gly Ser Leu Arg Gln Arg Pro Thr Cys Ser Ser Ser 370 375 380 9292PRTHomo sapiens 9Met Glu Arg Gly Pro Val Val Gly Ala Gly Leu Gly Ala Gly Ala Arg 1 5 10 15 Ile Gln Ala Leu Leu Gly Cys Leu Leu Lys Val Leu Leu Trp Val Ala 20 25 30 Ser Ala Leu Leu Tyr Phe Gly Ser Glu Gln Ala Ala Arg Leu Leu Gly 35 40 45 Ser Pro Cys Leu Arg Arg Leu Tyr His Ala Trp Leu Ala Ala Val Val 50 55 60 Ile Phe Gly Pro Leu Leu Gln Phe His Val Asn Pro Arg Thr Ile Phe 65 70 75 80 Ala Ser His Gly Asn Phe Phe Asn Ile Lys Phe Val Asn Ser Ala Trp 85 90 95 Gly Trp Thr Cys Thr Phe Leu Gly Gly Phe Val Leu Leu Val Val Phe 100 105 110 Leu Ala Thr Arg Arg Val Ala Val Thr Ala Arg His Leu Ser Arg Leu 115 120 125 Val Val Gly Ala Ala Val Trp Arg Gly Ala Gly Arg Ala Phe Leu Leu 130 135 140 Ile Glu Asp Leu Thr Gly Ser Cys Phe Glu Pro Leu Pro Gln Gly Leu 145 150 155 160 Leu Leu His Glu Leu Pro Asp Arg Arg Ser Cys Leu Ala Ala Gly His 165 170 175 Gln Trp Arg Gly Tyr Thr Val Ser Ser His Thr Phe Leu Leu Thr Phe 180 185 190 Cys Cys Leu Leu Met Ala Glu Glu Ala Ala Val Phe Ala Lys Tyr Leu 195 200 205 Ala His Gly Leu Pro Ala Gly Ala Pro Leu Arg Leu Val Phe Leu Leu 210 215 220 Asn Val Leu Leu Leu Gly Leu Trp Asn Phe Leu Leu Leu Cys Thr Val 225 230 235 240 Ile Tyr Phe His Gln Tyr Thr His Lys Val Val Gly Ala Ala Val Gly 245 250 255 Thr Phe Ala Trp Tyr Leu Thr Tyr Gly Ser Trp Tyr His Gln Pro Trp 260 265 270 Ser Pro Gly Ser Pro Gly His Gly Leu Phe Pro Arg Pro His Ser Ser 275 280 285 Arg Lys His Asn 290 10292PRTMus musculus 10Met Glu Arg Gly Pro Thr Val Gly Ala Gly Leu Gly Ala Gly Thr Arg 1 5 10 15 Val Arg Ala Leu Leu Gly Cys Leu Val Lys Val Leu Leu Trp Val Ala 20 25 30 Ser Ala Leu Leu Tyr Phe Gly Ser Glu Gln Ala Ala Arg Leu Leu Gly 35 40 45 Ser Pro Cys Leu Arg Arg Leu Tyr His Ala Trp Leu Ala Ala Val Val 50 55 60 Ile Phe Gly Pro Leu Leu Gln Phe His Val Asn Ser Arg Thr Ile Phe 65 70 75 80 Ala Ser His Gly Asn Phe Phe Asn Ile Lys Phe Val Asn Ser Ala Trp 85 90 95 Gly Trp Thr Cys Thr Phe Leu Gly Gly Phe Val Leu Leu Val Val Phe 100 105 110 Leu Ala Thr Arg Arg Val Ala Val Thr Ala Arg His Leu Ser Arg Leu 115 120 125 Val Val Gly Ala Ala Val Trp Arg Gly Ala Gly Arg Ala Phe Leu Leu 130 135 140 Ile Glu Asp Leu Thr Gly Ser Cys Phe Glu Pro Leu Pro Gln Gly Leu 145 150 155 160 Leu Leu His Glu Leu Pro Asp Arg Lys Ser Cys Leu Ala Ala Gly His 165 170 175 Gln Trp Arg Gly Tyr Thr Val Ser Ser His Thr Phe Leu Leu Thr Phe 180 185 190 Cys Cys Leu Leu Met Ala Glu Glu Ala Ala Val Phe Ala Lys Tyr Leu 195 200 205 Ala His Gly Leu Pro Ala Gly Ala Pro Leu Arg Leu Val Phe Leu Leu 210 215 220 Asn Val Leu Leu Leu Gly Leu Trp Asn Phe Leu Leu Leu Cys Thr Val 225 230 235 240 Ile Tyr Phe His Gln Tyr Thr His Lys Val Val Gly Ala Ala Val Gly 245 250 255 Thr Phe Ala Trp Tyr Leu Thr Tyr Gly Ser Trp Tyr His Gln Pro Trp 260 265 270 Ser Pro Gly Ile Pro Gly His Gly Leu Phe Pro Arg Ser Arg Ser Met 275 280 285 Arg Lys His Asn 290 11262PRTHomo sapiens 11Met Glu His Leu Glu Arg Cys Glu Trp Leu Leu Arg Gly Thr Leu Val 1 5 10 15 Arg Ala Ala Val Arg Arg Tyr Leu Pro Trp Ala Leu Val Ala Ser Met 20 25 30 Leu Ala Gly Ser Leu Leu Lys Glu Leu Ser Pro Leu Pro Glu Ser Tyr 35 40 45 Leu Ser Asn Lys Arg Asn Val Leu Asn Val Tyr Phe Val Lys Val Ala 50 55 60 Trp Ala Trp Thr Phe Cys Leu Leu Leu Pro Phe Ile Ala Leu Thr Asn 65 70 75 80 Tyr His Leu Thr Gly Lys Ala Gly Leu Val Leu Arg Arg Leu Ser Thr 85 90 95 Leu Leu Val Gly Thr Ala Ile Trp Tyr Ile Cys Thr Ser Ile Phe Ser 100 105 110 Asn Ile Glu His Tyr Thr Gly Ser Cys Tyr Gln Ser Pro Ala Leu Glu 115 120 125 Gly Val Arg Lys Glu His Gln Ser Lys Gln Gln Cys His Gln Glu Gly 130 135 140 Gly Phe Trp His Gly Phe Asp Ile Ser Gly His Ser Phe Leu Leu Thr 145 150 155 160 Phe Cys Ala Leu Met Ile Val Glu Glu Met Ser Val Leu His Glu Val 165 170 175 Lys Thr Asp Arg Ser His Cys Leu His Thr Ala Ile Thr Thr Leu Val 180 185 190 Val Ala Leu Gly Ile Leu Thr Phe Ile Trp Val Leu Met Phe Leu Cys 195 200 205 Thr Ala Val Tyr Phe His Asn Leu Ser Gln Lys Val Phe Gly Thr Leu 210 215 220 Phe Gly Leu Leu Ser Trp Tyr Gly Thr Tyr Gly Phe Trp Tyr Pro Lys 225 230 235 240 Ala Phe Ser Pro Gly Leu Pro Pro Gln Ser Cys Ser Leu Asn Leu Lys 245 250 255 Gln Asp Ser Tyr Lys Lys 260 12262PRTMus musculus 12Met Glu His Leu Glu Arg Cys Ala Trp Phe Leu Arg Gly Thr Leu Val 1 5 10 15 Arg Ala Thr Val Arg Arg His Leu Pro Trp Ala Leu Val Ala Ala Met 20 25 30 Leu Ala Gly Ser Val Val Lys Glu Leu Ser Pro Leu Pro Glu Ser Tyr 35 40 45 Leu Ser Asn Lys Arg Asn Val Leu Asn Val Tyr Phe Val Lys Leu Ala 50 55 60 Trp Ala Trp Thr Val Cys Leu Leu Leu Pro Phe Ile Ala Leu Thr Asn 65 70 75 80 Tyr His Leu Thr Gly Lys Thr Ser Leu Val Leu Arg Arg Leu Ser Thr 85 90 95 Leu Leu Val Gly Thr Ala Ile Trp Tyr Ile Cys Thr Ala Leu Phe Ser 100 105 110 Asn Ile Glu His Tyr Thr Gly Ser Cys Tyr Gln Ser Pro Ala Leu Glu 115 120 125 Gly Ile Arg Gln Glu His Arg Ser Lys Gln Gln Cys His Arg Glu Gly 130 135 140 Gly Phe Trp His Gly Phe Asp Ile Ser Gly His Ser Phe Leu Leu Thr 145 150 155 160 Phe Cys Ala Leu Met Ile Val Glu Glu Met Ala Val Leu His Glu Val 165 170 175 Lys Thr Asp Arg Gly His His Leu His Ala Ala Ile Thr Thr Leu Val 180 185 190 Val Ala Leu Gly Phe Leu Thr Phe Ile Trp Val Trp Met Phe Leu Cys 195 200 205 Thr Ala Val Tyr Phe His Asp Leu Thr Gln Lys Val Phe Gly Thr Met 210 215 220 Phe Gly Leu Leu Gly Trp Tyr Gly Thr Tyr Gly Tyr Trp Tyr Leu Lys 225 230 235 240 Ser Phe Ser Pro Gly Leu Pro Pro Gln Ser Cys Ser Leu Thr Leu Lys 245 250 255 Arg Asp Thr Tyr Lys Lys 260 131702DNAArabidopsis thaliana 13agaaaattta gtagcaaact tctcgattcc ttgattcgtg ggaaaaagaa agtctagatt 60tttgtggatt ttgattttgt gattccgtga ttgtatgaac ttgagccgtt ttgcttcgag 120attaagaatg gcggaagaaa tctcaaagac gaaggtggga tcttcttcta ctgcttcggt 180ggctgattca tctgctgctg cgtcggctgc aacgaatgcg gccaaatcaa gatggaaaat 240tttgtggcct aattcgctcc ggtggattcc tacgtccacc gattacatca tcgccgccga 300gaaacgtctt ctctccatcc tcaagacgcc ttatgtacaa gagcaagtca gtattggttc 360aggaccacca ggttctaaaa tcaggtggtt taggtctacg agcaatgagt cacgttacat 420caacactgtt acatttgatg ccaaggaggg agctcctaca ctcgtcatgg ttcatggtta 480tggtgcttct caagggtttt tcttccgtaa ttttgatgct cttgccagtc gatttagagt 540gatcgctatt gatcaacttg ggtggggtgg ttcaagtagg cctgatttta catgtagaag 600cacagaagaa actgaggcat ggtttatcga ctcctttgag gaatggcgta aagcccagaa 660tctcagtaac tttattctat taggacattc ttttggaggc tatgttgctg ctaaatacgc 720gcttaagcat cctgaacatg ttcaacactt aattctggtg ggatctgctg ggttctcagc 780agaagcagat gccaaatcag aatggctcac taaatttaga gcaacatgga aaggtgcagt 840cctaaatcat ttatgggagt caaatttcac tcctcagaag ctggttagag gattaggtcc 900ttggggtcca ggtcttgtaa atcggtatac aactgcaaga tttggtgcac attcggaggg 960aactgggcta acagaagagg aagccaaatt gctaaccgat tatgtgtacc atactttggc 1020tgcaaaggct agtggagagt tatgcttgaa atacatcttc tcatttggag catttgctag 1080gaagcccctc ttacaaaggt atgtccacca aaaacattgc tgataaagtt tctgcatact 1140cacactcgat gactcctctt ttgtgtgcag tgcatcagag tggaaagtgc caacaacgtt 1200tatctatgga atgaatgatt ggatgaacta tcaaggtgcg gtggaagcga ggaaatccat 1260gaaggtccct tgcgaaatca ttcgggttcc acagggtggt cattttgtgt tcatagacaa 1320cccaattggt tttcattctg cagtgcttta tgcttgccgc aagtttatat ctcaagactc 1380ctctcatgat caacaactcc tagatggtct acgattggtt tagtcatagt atcttgttcc 1440ttttaccttc caaatttatt ctatatgtgt atacaagtat atatgaaaaa gaacataaaa 1500aagaattact ttctttattt gaatattcgg ttgtgtattg gagtttcaag tcctctttcc 1560atgtctaaaa gttctatttg taacgttctt gatttcactc taaaacctct taaagtgttt 1620caaatgtgat ctcattatcg acatccaagt tgtaatcttt cacaatccac aataatcttt 1680tatctcattt tttacatttt ac 170214418PRTArabidopsis thaliana 14Met Asn Leu Ser Arg Phe Ala Ser Arg Leu Arg Met Ala Glu Glu Ile 1 5 10 15 Ser Lys Thr Lys Val Gly Ser Ser Ser Thr Ala Ser Val Ala Asp Ser 20

25 30 Ser Ala Ala Ala Ser Ala Ala Thr Asn Ala Ala Lys Ser Arg Trp Lys 35 40 45 Ile Leu Trp Pro Asn Ser Leu Arg Trp Ile Pro Thr Ser Thr Asp Tyr 50 55 60 Ile Ile Ala Ala Glu Lys Arg Leu Leu Ser Ile Leu Lys Thr Pro Tyr 65 70 75 80 Val Gln Glu Gln Val Ser Ile Gly Ser Gly Pro Pro Gly Ser Lys Ile 85 90 95 Arg Trp Phe Arg Ser Thr Ser Asn Glu Ser Arg Tyr Ile Asn Thr Val 100 105 110 Thr Phe Asp Ala Lys Glu Gly Ala Pro Thr Leu Val Met Val His Gly 115 120 125 Tyr Gly Ala Ser Gln Gly Phe Phe Phe Arg Asn Phe Asp Ala Leu Ala 130 135 140 Ser Arg Phe Arg Val Ile Ala Ile Asp Gln Leu Gly Trp Gly Gly Ser 145 150 155 160 Ser Arg Pro Asp Phe Thr Cys Arg Ser Thr Glu Glu Thr Glu Ala Trp 165 170 175 Phe Ile Asp Ser Phe Glu Glu Trp Arg Lys Ala Gln Asn Leu Ser Asn 180 185 190 Phe Ile Leu Leu Gly His Ser Phe Gly Gly Tyr Val Ala Ala Lys Tyr 195 200 205 Ala Leu Lys His Pro Glu His Val Gln His Leu Ile Leu Val Gly Ser 210 215 220 Ala Gly Phe Ser Ala Glu Ala Asp Ala Lys Ser Glu Trp Leu Thr Lys 225 230 235 240 Phe Arg Ala Thr Trp Lys Gly Ala Val Leu Asn His Leu Trp Glu Ser 245 250 255 Asn Phe Thr Pro Gln Lys Leu Val Arg Gly Leu Gly Pro Trp Gly Pro 260 265 270 Gly Leu Val Asn Arg Tyr Thr Thr Ala Arg Phe Gly Ala His Ser Glu 275 280 285 Gly Thr Gly Leu Thr Glu Glu Glu Ala Lys Leu Leu Thr Asp Tyr Val 290 295 300 Tyr His Thr Leu Ala Ala Lys Ala Ser Gly Glu Leu Cys Leu Lys Tyr 305 310 315 320 Ile Phe Ser Phe Gly Ala Phe Ala Arg Lys Pro Leu Leu Gln Ser Ala 325 330 335 Ser Glu Trp Lys Val Pro Thr Thr Phe Ile Tyr Gly Met Asn Asp Trp 340 345 350 Met Asn Tyr Gln Gly Ala Val Glu Ala Arg Lys Ser Met Lys Val Pro 355 360 365 Cys Glu Ile Ile Arg Val Pro Gln Gly Gly His Phe Val Phe Ile Asp 370 375 380 Asn Pro Ile Gly Phe His Ser Ala Val Leu Tyr Ala Cys Arg Lys Phe 385 390 395 400 Ile Ser Gln Asp Ser Ser His Asp Gln Gln Leu Leu Asp Gly Leu Arg 405 410 415 Leu Val 15521PRTJatropha curcas 15Met Thr Ile Leu Glu Thr Thr Thr Ser Gly Gly Asp Gly Val Ala Glu 1 5 10 15 Ser Ser Ser Asp Leu Asn Val Ser Leu Arg Arg Arg Arg Lys Gly Thr 20 25 30 Ser Ser Asp Gly Ala Leu Pro Glu Leu Thr Ser Asn Ile Val Glu Leu 35 40 45 Glu Ser Glu Ser Gly Gly Gln Val Met Met Asp Pro Gly Met Val Thr 50 55 60 Glu Pro Glu Thr Glu Lys Ile Asn Gly Lys Asp Cys Gly Gly Asp Lys 65 70 75 80 Asp Lys Ile Asp Asn Arg Glu Asn Arg Gly Arg Ser Asp Ile Lys Phe 85 90 95 Thr Tyr Arg Pro Ser Val Pro Ala His Arg Ala Leu Arg Glu Ser Pro 100 105 110 Leu Ser Ser Asp Ala Ile Phe Lys Gln Ser His Ala Gly Leu Phe Asn 115 120 125 Leu Cys Ile Val Val Leu Val Ala Val Asn Ser Arg Leu Ile Ile Glu 130 135 140 Asn Leu Met Lys Tyr Gly Trp Leu Ile Lys Thr Gly Phe Trp Phe Ser 145 150 155 160 Ser Arg Ser Leu Arg Asp Trp Pro Leu Leu Met Cys Cys Leu Thr Leu 165 170 175 Pro Ile Phe Ser Leu Ala Ala Tyr Leu Val Glu Lys Leu Ala Tyr Arg 180 185 190 Lys Tyr Ile Ser Ala Pro Ile Val Ile Phe Phe His Met Leu Ile Thr 195 200 205 Thr Thr Ala Val Leu Tyr Pro Val Ser Val Ile Leu Ser Cys Gly Ser 210 215 220 Ala Val Leu Ser Gly Val Ala Leu Met Leu Phe Ala Cys Ile Val Trp 225 230 235 240 Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr Asp Met Arg Ala Ile 245 250 255 Ala Asn Ser Ala Asp Lys Gly Asp Ala Leu Ser Asp Thr Ser Gly Ala 260 265 270 Asp Ser Ser Arg Asp Val Ser Phe Lys Ser Leu Val Tyr Phe Met Val 275 280 285 Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Asp Ser Val 290 295 300 Arg Lys Gly Trp Val Val Arg Gln Phe Val Lys Leu Ile Ile Phe Thr 305 310 315 320 Gly Phe Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile Val Gln 325 330 335 Asn Ser Gln His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile Glu Arg 340 345 350 Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys Met Phe 355 360 365 Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu Leu Arg 370 375 380 Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Arg Thr Val 385 390 395 400 Glu Glu Tyr Trp Arg Met Trp Asn Met Pro Val His Lys Trp Met Val 405 410 415 Arg His Ile Tyr Phe Pro Cys Leu Arg His Lys Ile Pro Arg Gly Val 420 425 430 Ala Leu Leu Ile Ala Phe Phe Val Ser Ala Val Phe His Glu Leu Cys 435 440 445 Ile Ala Val Pro Cys His Met Phe Lys Leu Trp Ala Phe Ile Gly Ile 450 455 460 Met Phe Gln Ile Pro Leu Val Gly Ile Thr Asn Tyr Leu Gln Asn Lys 465 470 475 480 Phe Arg Ser Ser Met Val Gly Asn Met Ile Phe Trp Phe Ile Phe Cys 485 490 495 Ile Leu Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His Asp Leu Met 500 505 510 Asn Arg Lys Gly Asn Ala Glu Leu Arg 515 520 16671PRTArabidopsis thaliana 16Met Pro Leu Ile His Arg Lys Lys Pro Thr Glu Lys Pro Ser Thr Pro 1 5 10 15 Pro Ser Glu Glu Val Val His Asp Glu Asp Ser Gln Lys Lys Pro His 20 25 30 Glu Ser Ser Lys Ser His His Lys Lys Ser Asn Gly Gly Gly Lys Trp 35 40 45 Ser Cys Ile Asp Ser Cys Cys Trp Phe Ile Gly Cys Val Cys Val Thr 50 55 60 Trp Trp Phe Leu Leu Phe Leu Tyr Asn Ala Met Pro Ala Ser Phe Pro 65 70 75 80 Gln Tyr Val Thr Glu Arg Ile Thr Gly Pro Leu Pro Asp Pro Pro Gly 85 90 95 Val Lys Leu Lys Lys Glu Gly Leu Lys Ala Lys His Pro Val Val Phe 100 105 110 Ile Pro Gly Ile Val Thr Gly Gly Leu Glu Leu Trp Glu Gly Lys Gln 115 120 125 Cys Ala Asp Gly Leu Phe Arg Lys Arg Leu Trp Gly Gly Thr Phe Gly 130 135 140 Glu Val Tyr Lys Arg Pro Leu Cys Trp Val Glu His Met Ser Leu Asp 145 150 155 160 Asn Glu Thr Gly Leu Asp Pro Ala Gly Ile Arg Val Arg Ala Val Ser 165 170 175 Gly Leu Val Ala Ala Asp Tyr Phe Ala Pro Gly Tyr Phe Val Trp Ala 180 185 190 Val Leu Ile Ala Asn Leu Ala His Ile Gly Tyr Glu Glu Lys Asn Met 195 200 205 Tyr Met Ala Ala Tyr Asp Trp Arg Leu Ser Phe Gln Asn Thr Glu Val 210 215 220 Arg Asp Gln Thr Leu Ser Arg Met Lys Ser Asn Ile Glu Leu Met Val 225 230 235 240 Ser Thr Asn Gly Gly Lys Lys Ala Val Ile Val Pro His Ser Met Gly 245 250 255 Val Leu Tyr Phe Leu His Phe Met Lys Trp Val Glu Ala Pro Ala Pro 260 265 270 Leu Gly Gly Gly Gly Gly Pro Asp Trp Cys Ala Lys Tyr Ile Lys Ala 275 280 285 Val Met Asn Ile Gly Gly Pro Phe Leu Gly Val Pro Lys Ala Val Ala 290 295 300 Gly Leu Phe Ser Ala Glu Ala Lys Asp Val Ala Val Ala Arg Ala Ile 305 310 315 320 Ala Pro Gly Phe Leu Asp Thr Asp Ile Phe Arg Leu Gln Thr Leu Gln 325 330 335 His Val Met Arg Met Thr Arg Thr Trp Asp Ser Thr Met Ser Met Leu 340 345 350 Pro Lys Gly Gly Asp Thr Ile Trp Gly Gly Leu Asp Trp Ser Pro Glu 355 360 365 Lys Gly His Thr Cys Cys Gly Lys Lys Gln Lys Asn Asn Glu Thr Cys 370 375 380 Gly Glu Ala Gly Glu Asn Gly Val Ser Lys Lys Ser Pro Val Asn Tyr 385 390 395 400 Gly Arg Met Ile Ser Phe Gly Lys Glu Val Ala Glu Ala Ala Pro Ser 405 410 415 Glu Ile Asn Asn Ile Asp Phe Arg Gly Ala Val Lys Gly Gln Ser Ile 420 425 430 Pro Asn His Thr Cys Arg Asp Val Trp Thr Glu Tyr His Asp Met Gly 435 440 445 Ile Ala Gly Ile Lys Ala Ile Ala Glu Tyr Lys Val Tyr Thr Ala Gly 450 455 460 Glu Ala Ile Asp Leu Leu His Tyr Val Ala Pro Lys Met Met Ala Arg 465 470 475 480 Gly Ala Ala His Phe Ser Tyr Gly Ile Ala Asp Asp Leu Asp Asp Thr 485 490 495 Lys Tyr Gln Asp Pro Lys Tyr Trp Ser Asn Pro Leu Glu Thr Lys Leu 500 505 510 Pro Asn Ala Pro Glu Met Glu Ile Tyr Ser Leu Tyr Gly Val Gly Ile 515 520 525 Pro Thr Glu Arg Ala Tyr Val Tyr Lys Leu Asn Gln Ser Pro Asp Ser 530 535 540 Cys Ile Pro Phe Gln Ile Phe Thr Ser Ala His Glu Glu Asp Glu Asp 545 550 555 560 Ser Cys Leu Lys Ala Gly Val Tyr Asn Val Asp Gly Asp Glu Thr Val 565 570 575 Pro Val Leu Ser Ala Gly Tyr Met Cys Ala Lys Ala Trp Arg Gly Lys 580 585 590 Thr Arg Phe Asn Pro Ser Gly Ile Lys Thr Tyr Ile Arg Glu Tyr Asn 595 600 605 His Ser Pro Pro Ala Asn Leu Leu Glu Gly Arg Gly Thr Gln Ser Gly 610 615 620 Ala His Val Asp Ile Met Gly Asn Phe Ala Leu Ile Glu Asp Ile Met 625 630 635 640 Arg Val Ala Ala Gly Gly Asn Gly Ser Asp Ile Gly His Asp Gln Val 645 650 655 His Ser Gly Ile Phe Glu Trp Ser Glu Arg Ile Asp Leu Lys Leu 660 665 670 17546PRTLaccaria bicolor S238N-H82 17Asp Gly Arg Glu Phe Gln Val Gly Glu Ala Met Lys Ala Arg Gly Leu 1 5 10 15 Thr Ala Gln His Pro Val Val Ile Ile Pro Gly Ile Val Ser Thr Gly 20 25 30 Leu Glu Ser Trp Ser Thr Ser Pro Asp Tyr Arg Ala Phe Phe Arg Glu 35 40 45 Lys Leu Trp Gly Ala Phe Asn Met Leu Ser Gln Val Thr Phe Asn Lys 50 55 60 Glu Lys Trp Ile Ala Ala Met Met Leu Asp Pro Leu Thr Gly Leu Asp 65 70 75 80 Pro Pro Gly Ala Lys Val Arg Ala Ala Glu Gly Ile Asp Ala Ala Ser 85 90 95 Ser Phe Ile Gln Gly Phe Trp Ile Trp Ser Lys Val Val Glu Asn Leu 100 105 110 Ala Val Val Asn Tyr Asp Thr Asn Asn Leu Tyr Leu Ala Pro Tyr Asp 115 120 125 Trp Arg Leu Ser Tyr Tyr Asn Leu Glu Val Arg Asp Gly Tyr Phe Ser 130 135 140 Arg Leu Lys Ser Thr Ile Glu Gly Leu Lys Lys Arg Gln Asn Lys Lys 145 150 155 160 Val Val Ile Ala Ala His Ser Met Gly Ser Thr Val Arg His Arg His 165 170 175 Leu Tyr Thr Tyr Glu Thr Phe Lys Trp Val Glu Ser Pro Leu His Gly 180 185 190 Asn Gly Gly Ile Asp Trp Val Glu Asn His Ile Glu Ser Tyr Ile Ser 195 200 205 Ile Ala Gly Thr His Leu Ala Lys Ala Met Ser Ala Phe Leu Ser Gly 210 215 220 Glu Met Lys Asp Thr Val Gln Met Asn Pro Ala Gly Ala Tyr Val Leu 225 230 235 240 Glu Arg Phe Phe Ser Arg Lys Glu Arg Gln Arg Leu Phe Arg Ser Trp 245 250 255 Ala Gly Ser Ala Ser Met Trp Leu Lys Gly Gly Asn Ala Val Trp Gly 260 265 270 Ser Ala Leu His Ala Pro Asp Asp Ala Cys Asn Asn Thr His Thr His 275 280 285 Gly Glu Leu Ile Ala Phe Arg Ser Leu Ser Pro Gln Ser Asn Gly Asp 290 295 300 Thr Thr Arg Asn Met Thr Ala Glu Glu Ala Gly Leu Trp Ile Leu Gln 305 310 315 320 His Thr Pro Thr Ala Phe Gln Lys Met Leu Glu Thr Asn Tyr Ser Tyr 325 330 335 Gly Ile Glu Arg Asp Glu Glu Gln Leu Ser Arg Asn Asp Leu Asp His 340 345 350 Arg Lys Trp Thr Asn Pro Leu Glu Arg Phe Gln Leu Leu Pro Arg Ala 355 360 365 Pro Ser Met Lys Ile Tyr Cys Val Tyr Gly His Gly Lys Glu Thr Glu 370 375 380 Arg Ser Tyr Trp Tyr Val Gln Gly Lys Asp Ser Glu Ala Ala Asp Ala 385 390 395 400 Val Asp Thr Glu Cys Thr Asp Pro His Ser Ser Glu Cys Gly Val Leu 405 410 415 Ser Gln His Leu Gly Pro Pro Ser Leu Arg Glu Ser Trp Ile Asp Ser 420 425 430 Asp Tyr Thr Asn Asn Ser Ala Phe Pro Lys Leu Leu Asn Gly Val Lys 435 440 445 Met Gly Glu Gly Asp Gly Thr Val Ser Leu Val Ser Leu Gly Ala Met 450 455 460 Cys Val Glu Gly Trp Lys Arg Pro Arg Trp Asn Pro Ala Gly Ile Lys 465 470 475 480 Ile Thr Thr Val Glu Leu Pro His Arg Pro Thr Val Thr Met Pro Arg 485 490 495 Gly Gly Ala Asn Thr Ser Asp His Val Asp Ile Leu Gly Ser Thr Gly 500 505 510 Leu Asn Glu Val Ile Leu Lys Val Ala Thr Gly Val Gly His Glu Val 515 520 525 Thr Asp Asn Tyr Val Ser Asp Ile Gln Arg Tyr Ala Gln Arg Ile Gln 530 535 540 Trp Asp 545 18680PRTScheffersomyces stipitis CBS 6054 18Met Ser Asn Leu Ser Asn Arg Arg Arg Ser Lys Ser Glu Asp Ser Leu 1 5 10 15 Asp Val Ser Glu Gly Ala Ala Lys Ala Ser Gly Val Ala Tyr Leu Gly 20 25 30 Lys Val Phe Ser Ala His Thr Thr Gly Pro Asp Gly Gln Glu Gly His 35 40 45 His Ile His Gln His Ile Gly Lys Pro Ser Ser Ile Glu Glu Lys Asp 50 55 60 Thr Pro Arg Pro Pro Ile Ile Ser Thr Ser Ser Ser Ser Ser Thr Ser 65 70 75 80 Ser Lys Ser Lys Arg Lys Phe His Glu Lys Arg Arg Val Val Phe Ile 85 90 95 Phe Gly Ala Phe Leu Gly Leu Phe Leu Thr Ile Gly Tyr Ser Thr Tyr 100 105 110 Tyr Asn Pro Ser Ile Lys Asn Glu Ile Asp Lys Ile Val Arg Ile Asp 115 120 125 Arg Phe Asn Asp Phe Phe Glu Asp Trp Lys Asp Trp Lys Asp Ile Leu 130 135 140 Pro Val Gly Leu Gln Ser Ile Leu Ser Glu Gln Leu Gly Gln Lys Asp 145 150 155 160 Asp Ala Leu Gln Tyr

Ser Pro Asp Ser Phe Ser Val Gly Arg Arg Leu 165 170 175 Ala Ala Thr Met Asn Leu Thr Ser Glu Tyr Asn Val Leu Leu Val Pro 180 185 190 Gly Val Ile Ser Thr Gly Ile Glu Ser Trp Gly Val Ser Thr Glu Gly 195 200 205 Asp Cys Pro Ser Ile Ser His Phe Arg Lys Arg Leu Trp Gly Ser Phe 210 215 220 Tyr Met Leu Arg Thr Met Val Leu Asp Lys Lys Cys Trp Leu Lys His 225 230 235 240 Ile Met Leu Asp Pro Val Thr Gly Leu Asp Pro His Asn Ile Lys Met 245 250 255 Arg Ala Ala Gln Gly Phe Glu Ala Ala Asp Tyr Phe Met Val Gly Tyr 260 265 270 Trp Ile Trp Asn Lys Ile Leu Gln Asn Leu Ala Val Ile Gly Tyr Gly 275 280 285 Pro Asn Thr Met Gln Val Ala Ser Tyr Asp Trp Arg Leu Ala Phe Leu 290 295 300 Asp Leu Glu Lys Arg Asp Gly Tyr Phe Ser Lys Ile Lys Ser Gln Ile 305 310 315 320 Glu Val Thr Lys Asn Leu Asn Gly Lys Lys Ser Ile Ile Val Gly His 325 330 335 Ser Met Gly Ala Gln Ile Ser Tyr Tyr Phe Leu Lys Trp Val Glu Ala 340 345 350 Glu Asn Tyr Gly Gly Gly Gly Pro Asn Trp Val Asn Asp His Ile Glu 355 360 365 Ala Phe Val Asp Ile Ser Gly Ser Thr Leu Gly Thr Pro Lys Thr Ile 370 375 380 Pro Ala Leu Leu Ser Gly Glu Met Lys Asp Thr Val Gln Leu Asn Ala 385 390 395 400 Leu Ala Val Tyr Gly Leu Glu Gln Phe Phe Ser Arg Lys Glu Arg Val 405 410 415 Asp Leu Leu Arg Thr Phe Gly Gly Ile Ala Gly Met Leu Pro Lys Gly 420 425 430 Gly Ser Thr Ile Trp Gly Asp Leu Glu Arg Ala Pro Asp Asp Asp Ile 435 440 445 Ser Asp Tyr Ser Glu Asp Val Glu Gly Ala Ile Lys Lys Asn Asn Asp 450 455 460 Ser Phe Gly Asn Phe Ile Arg His Lys Lys Lys Asp Gly Thr Val Ser 465 470 475 480 Asn Phe Thr Ile Glu Gln Ser Ile Asp Met Leu Leu Asp Glu Ser Pro 485 490 495 Asn Trp Tyr Lys Glu Arg Val Glu His Gln Tyr Ser Tyr Gly Ile Ala 500 505 510 Lys Thr Lys Glu Glu Leu Glu Arg Asn Asn Lys Asp His Ser Lys Phe 515 520 525 Ser Asn Pro Leu Glu Ala Ala Leu Pro Asn Ala Pro Asp Met Lys Ile 530 535 540 Phe Cys Phe Tyr Gly Val Gly Lys Pro Thr Glu Arg Ala Tyr Asn Tyr 545 550 555 560 Val Asp Ala Asp Ser Gln Thr Gly Leu His Lys Val Ile Asp Pro Asp 565 570 575 Ala Glu Thr Pro Val Tyr Leu Gly Asp Gly Asp Gly Thr Val Ser Leu 580 585 590 Leu Ala His Thr Met Cys His Glu Trp Lys Lys Gly Ser Glu Ser Arg 595 600 605 Tyr Asn Pro Ser Gly Ile Pro Val Thr Ile Val Glu Ile Met Asn Glu 610 615 620 Pro Asp Arg Tyr Asp Ile Arg Gly Gly Ala Lys Thr Ala Asp His Val 625 630 635 640 Asp Ile Leu Gly Ser Ala Glu Leu Asn Glu Leu Val Leu Arg Val Ala 645 650 655 Ala Gly Val Gly Asp Gly Ile Glu Asp His Tyr Val Ser Asn Leu Arg 660 665 670 Tyr Ile Ala Glu Lys Met Ala Ile 675 680 19504PRTHomo sapiens 19Met Phe Pro Arg Glu Lys Thr Trp Asn Ile Ser Phe Ala Gly Cys Gly 1 5 10 15 Phe Leu Gly Val Tyr Tyr Val Gly Val Ala Ser Cys Leu Arg Glu His 20 25 30 Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly Ala Ser Ala 35 40 45 Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Val Cys Leu Gly Glu 50 55 60 Ala Gly Ala Lys Phe Ile Glu Val Ser Lys Glu Ala Arg Lys Arg Phe 65 70 75 80 Leu Gly Pro Leu His Pro Ser Phe Asn Leu Val Lys Ile Ile Arg Ser 85 90 95 Phe Leu Leu Lys Val Leu Pro Ala Asp Ser His Glu His Ala Ser Gly 100 105 110 Arg Leu Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Ile 115 120 125 Ile Ser His Phe Asn Ser Lys Asp Glu Leu Ile Gln Ala Asn Val Cys 130 135 140 Ser Gly Phe Ile Pro Val Tyr Cys Gly Leu Ile Pro Pro Ser Leu Gln 145 150 155 160 Gly Val Arg Tyr Val Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr 165 170 175 Glu Leu Lys Asn Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp 180 185 190 Ile Cys Pro Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr 195 200 205 Asn Thr Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys 210 215 220 Ala Leu Phe Pro Pro Glu Pro Leu Val Leu Arg Glu Met Cys Lys Gln 225 230 235 240 Gly Tyr Arg Asp Gly Leu Arg Phe Leu Gln Arg Asn Gly Leu Leu Asn 245 250 255 Arg Pro Asn Pro Leu Leu Ala Leu Pro Pro Ala Arg Pro His Gly Pro 260 265 270 Glu Asp Lys Asp Gln Ala Val Glu Ser Ala Gln Ala Glu Asp Tyr Ser 275 280 285 Gln Leu Pro Gly Glu Asp His Val Leu Glu His Leu Pro Ala Arg Leu 290 295 300 Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro Thr Asp Leu Leu Thr 305 310 315 320 Thr Leu Ser Asn Met Leu Pro Val Arg Leu Ala Thr Ala Met Met Val 325 330 335 Pro Tyr Thr Leu Pro Leu Glu Ser Ala Leu Ser Phe Thr Ile Cys Leu 340 345 350 Leu Glu Trp Leu Pro Asp Val Pro Glu Asp Ile Arg Trp Met Lys Glu 355 360 365 Gln Thr Gly Ser Ile Cys Gln Tyr Leu Val Met Arg Ala Lys Arg Lys 370 375 380 Leu Gly Arg His Leu Pro Ser Arg Leu Pro Glu Gln Val Glu Leu Arg 385 390 395 400 Arg Val Gln Ser Leu Pro Ser Val Pro Leu Ser Cys Ala Ala Tyr Arg 405 410 415 Glu Ala Pro Pro Gly Trp Met Arg Asn Asn Leu Ser Leu Gly Asp Ala 420 425 430 Leu Ala Lys Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly Leu Phe 435 440 445 Cys Thr Asn Val Ala Phe Pro Pro Glu Ala Leu Arg Met Arg Ala Pro 450 455 460 Ala Asp Pro Ala Pro Ala Pro Ala Asp Pro Ala Ser Pro Gln His Gln 465 470 475 480 Leu Ala Gly Pro Ala Pro Leu Leu Ser Thr Pro Ala Pro Glu Ala Arg 485 490 495 Pro Val Ile Gly Ala Leu Gly Leu 500 20486PRTMus musculus 20Met Phe Pro Arg Glu Thr Lys Trp Asn Ile Ser Phe Ala Gly Cys Gly 1 5 10 15 Phe Leu Gly Val Tyr His Ile Gly Val Ala Ser Cys Leu Arg Glu His 20 25 30 Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly Ala Ser Ala 35 40 45 Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Ala Cys Leu Gly Glu 50 55 60 Ala Gly Ala Asn Ile Ile Glu Val Ser Lys Glu Ala Arg Lys Arg Phe 65 70 75 80 Leu Gly Pro Leu His Pro Ser Phe Asn Leu Val Lys Thr Ile Arg Gly 85 90 95 Cys Leu Leu Lys Thr Leu Pro Ala Asp Cys His Glu Arg Ala Asn Gly 100 105 110 Arg Leu Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Ile 115 120 125 Ile Ser His Phe Ser Ser Lys Asp Glu Leu Ile Gln Ala Asn Val Cys 130 135 140 Ser Thr Phe Ile Pro Val Tyr Cys Gly Leu Ile Pro Pro Thr Leu Gln 145 150 155 160 Gly Val Arg Tyr Val Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr 165 170 175 Glu Leu Lys Asn Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp 180 185 190 Ile Cys Pro Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr 195 200 205 Asn Thr Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys 210 215 220 Ala Leu Phe Pro Pro Glu Pro Met Val Leu Arg Glu Met Cys Lys Gln 225 230 235 240 Gly Tyr Arg Asp Gly Leu Arg Phe Leu Arg Arg Asn Gly Leu Leu Asn 245 250 255 Gln Pro Asn Pro Leu Leu Ala Leu Pro Pro Val Val Pro Gln Glu Glu 260 265 270 Asp Ala Glu Glu Ala Ala Val Val Glu Glu Arg Ala Gly Glu Glu Asp 275 280 285 Gln Leu Gln Pro Tyr Arg Lys Asp Arg Ile Leu Glu His Leu Pro Ala 290 295 300 Arg Leu Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro Lys Asp Leu 305 310 315 320 Met Thr Thr Leu Ser Asn Met Leu Pro Val Arg Leu Ala Thr Ala Met 325 330 335 Met Val Pro Tyr Thr Leu Pro Leu Glu Ser Ala Val Ser Phe Thr Ile 340 345 350 Arg Leu Leu Glu Trp Leu Pro Asp Val Pro Glu Asp Ile Arg Trp Met 355 360 365 Lys Glu Gln Thr Gly Ser Ile Cys Gln Tyr Leu Val Met Arg Ala Lys 370 375 380 Arg Lys Leu Gly Asp His Leu Pro Ser Arg Leu Ser Glu Gln Val Glu 385 390 395 400 Leu Arg Arg Ala Gln Ser Leu Pro Ser Val Pro Leu Ser Cys Ala Thr 405 410 415 Tyr Ser Glu Ala Leu Pro Asn Trp Val Arg Asn Asn Leu Ser Leu Gly 420 425 430 Asp Ala Leu Ala Lys Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly 435 440 445 Leu Phe Cys Thr Asn Val Ala Phe Pro Pro Asp Ala Leu Arg Met Arg 450 455 460 Ala Pro Ala Ser Pro Thr Ala Ala Asp Pro Ala Thr Pro Gln Asp Pro 465 470 475 480 Pro Gly Leu Pro Pro Cys 485 21219PRTHomo sapiens 21Met Glu Ala Ala Arg Asp Tyr Ala Gly Ala Leu Ile Arg Pro Leu Thr 1 5 10 15 Phe Met Gly Ser Gln Thr Lys Arg Val Leu Phe Thr Pro Leu Met His 20 25 30 Pro Ala Arg Pro Phe Arg Val Ser Asn His Asp Arg Ser Ser Arg Arg 35 40 45 Gly Val Met Ala Ser Ser Leu Gln Glu Leu Ile Ser Lys Thr Leu Asp 50 55 60 Ala Leu Val Ile Ala Thr Gly Leu Val Thr Leu Val Leu Glu Glu Asp 65 70 75 80 Gly Thr Val Val Asp Thr Glu Glu Phe Phe Gln Thr Leu Gly Asp Asn 85 90 95 Thr His Phe Met Ile Leu Glu Lys Gly Gln Lys Trp Met Pro Gly Ser 100 105 110 Gln His Phe Pro Thr Cys Ser Pro Pro Lys Arg Ser Gly Ile Ala Arg 115 120 125 Val Thr Phe Asp Leu Tyr Arg Leu Asn Pro Lys Asp Phe Ile Gly Cys 130 135 140 Leu Asn Val Lys Ala Thr Met Tyr Glu Met Tyr Ser Val Ser Tyr Asp 145 150 155 160 Ile Arg Cys Thr Gly Leu Lys Gly Leu Leu Arg Ser Leu Leu Arg Phe 165 170 175 Leu Ser Tyr Ser Ala Gln Val Thr Gly Gln Phe Leu Ile Tyr Leu Gly 180 185 190 Thr Tyr Met Leu Arg Val Leu Asp Asp Lys Glu Glu Arg Pro Ser Leu 195 200 205 Arg Ser Gln Ala Lys Gly Arg Phe Thr Cys Gly 210 215 22217PRTMus musculus 22Met Glu Thr Ala Arg Asp Tyr Ala Gly Ala Leu Ile Arg Pro Leu Thr 1 5 10 15 Phe Met Gly Leu Gln Thr Lys Lys Val Leu Leu Thr Pro Leu Ile His 20 25 30 Pro Ala Arg Pro Phe Arg Val Ser Asn His Asp Arg Ser Ser Arg Arg 35 40 45 Gly Val Met Ala Ser Ser Leu Gln Glu Leu Ile Ser Lys Thr Leu Asp 50 55 60 Val Leu Val Ile Thr Thr Gly Leu Val Thr Leu Val Leu Glu Glu Asp 65 70 75 80 Gly Thr Val Val Asp Thr Glu Glu Phe Phe Gln Thr Leu Arg Asp Asn 85 90 95 Thr His Phe Met Ile Leu Glu Lys Gly Gln Lys Trp Thr Pro Gly Ser 100 105 110 Lys Tyr Val Pro Val Cys Lys Gln Pro Lys Lys Ser Gly Ile Ala Arg 115 120 125 Val Thr Phe Asp Leu Tyr Arg Leu Asn Pro Lys Asp Phe Leu Gly Cys 130 135 140 Leu Asn Val Lys Ala Thr Met Tyr Glu Met Tyr Ser Val Ser Tyr Asp 145 150 155 160 Ile Arg Cys Thr Ser Phe Lys Ala Val Leu Arg Asn Leu Leu Arg Phe 165 170 175 Met Ser Tyr Ala Ala Gln Met Thr Gly Gln Phe Leu Val Tyr Ala Gly 180 185 190 Thr Tyr Met Leu Arg Val Leu Gly Asp Thr Glu Glu Gln Pro Ser Pro 195 200 205 Lys Pro Ser Thr Lys Gly Trp Phe Met 210 215 23430PRTArabidopsis thaliana 23Met Lys Lys Arg Leu Thr Thr Ser Thr Cys Ser Ser Ser Pro Ser Ser 1 5 10 15 Ser Val Ser Ser Ser Thr Thr Thr Ser Ser Pro Ile Gln Ser Glu Ala 20 25 30 Pro Arg Pro Lys Arg Ala Lys Arg Ala Lys Lys Ser Ser Pro Ser Gly 35 40 45 Asp Lys Ser His Asn Pro Thr Ser Pro Ala Ser Thr Arg Arg Ser Ser 50 55 60 Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe Glu Ala 65 70 75 80 His Leu Trp Asp Lys Ser Ser Trp Asn Ser Ile Gln Asn Lys Lys Gly 85 90 95 Lys Gln Val Tyr Leu Gly Ala Tyr Asp Ser Glu Glu Ala Ala Ala His 100 105 110 Thr Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Asp Thr Ile Leu 115 120 125 Asn Phe Pro Ala Glu Thr Tyr Thr Lys Glu Leu Glu Glu Met Gln Arg 130 135 140 Val Thr Lys Glu Glu Tyr Leu Ala Ser Leu Arg Arg Gln Ser Ser Gly 145 150 155 160 Phe Ser Arg Gly Val Ser Lys Tyr Arg Gly Val Ala Arg His His His 165 170 175 Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Phe Gly Asn Lys Tyr 180 185 190 Leu Tyr Leu Gly Thr Tyr Asn Thr Gln Glu Glu Ala Ala Ala Ala Tyr 195 200 205 Asp Met Ala Ala Ile Glu Tyr Arg Gly Ala Asn Ala Val Thr Asn Phe 210 215 220 Asp Ile Ser Asn Tyr Ile Asp Arg Leu Lys Lys Lys Gly Val Phe Pro 225 230 235 240 Phe Pro Val Asn Gln Ala Asn His Gln Glu Gly Ile Leu Val Glu Ala 245 250 255 Lys Gln Glu Val Glu Thr Arg Glu Ala Lys Glu Glu Pro Arg Glu Glu 260 265 270 Val Lys Gln Gln Tyr Val Glu Glu Pro Pro Gln Glu Glu Glu Glu Lys 275 280 285 Glu Glu Glu Lys Ala Glu Gln Gln Glu Ala Glu Ile Val Gly Tyr Ser 290 295 300 Glu Glu Ala Ala Val Val Asn Cys Cys Ile Asp Ser Ser Thr Ile Met 305 310 315 320 Glu Met Asp Arg Cys Gly Asp Asn Asn Glu Leu Ala Trp Asn Phe Cys 325 330 335 Met Met Asp Thr Gly Phe Ser Pro Phe Leu Thr Asp Gln Asn Leu Ala

340 345 350 Asn Glu Asn Pro Ile Glu Tyr Pro Glu Leu Phe Asn Glu Leu Ala Phe 355 360 365 Glu Asp Asn Ile Asp Phe Met Phe Asp Asp Gly Lys His Glu Cys Leu 370 375 380 Asn Leu Glu Asn Leu Asp Cys Cys Val Val Gly Arg Glu Ser Pro Pro 385 390 395 400 Ser Ser Ser Ser Pro Leu Ser Cys Leu Ser Thr Asp Ser Ala Ser Ser 405 410 415 Thr Thr Thr Thr Thr Thr Ser Val Ser Cys Asn Tyr Leu Val 420 425 430 24239PRTDanio rerio 24Met Glu Asn Ala Lys Lys Ser Val Asp Val Leu Ser Thr Ser Leu Ser 1 5 10 15 Lys Cys Ile Ser Ala Cys Gly Ser Val Thr His Gln Ile Leu Pro Arg 20 25 30 Trp Thr Gln His Ser Arg Pro Phe Arg Val Ile Asn Ser Asp Arg Ser 35 40 45 Ile Lys Lys Gly Ile Met Ala Asp Asp Leu Glu Asp Leu His His Lys 50 55 60 Val Met Asp Val Phe His Ile His Cys Ile Ser Ala Leu Val Leu Asp 65 70 75 80 Glu Asp Gly Thr Gly Ile Asp Thr Gln Asp Phe Phe Gln Thr Leu Lys 85 90 95 Asp Asn Thr Val Leu Met Val Leu Gly Lys Gly Gln Lys Trp Ala Pro 100 105 110 Gln Thr Lys His Leu Pro Gly Gln Lys Lys Val Glu Arg Lys Arg Met 115 120 125 Thr Lys Lys Asp Pro Asp Cys Asn Trp Thr Gln Pro Arg Lys Asp Val 130 135 140 Ala Lys Leu Thr Phe Asp Leu Tyr Lys Lys His Pro Gln Asp Phe Ile 145 150 155 160 Gly Cys Leu Asn Val Gln Ala Thr Leu Tyr Gly Met Tyr Ser Val Ser 165 170 175 Tyr Val Leu His Cys Tyr Lys Ala Lys Arg Met Leu Arg Glu Ala Leu 180 185 190 Arg Trp Thr Leu Phe Thr Met Gln Thr Thr Gly His Val Leu Val Gly 195 200 205 Thr Ser Cys Tyr Ile Gln His Leu Ile Asp Glu Glu Glu Lys Thr Glu 210 215 220 Thr Glu Met Ile Thr Pro Ala Tyr Val Ile Lys Gln Leu Lys His 225 230 235 25283PRTHomo sapiens 25Met Asp Leu Trp Pro Gly Ala Trp Met Leu Leu Leu Leu Leu Phe Leu 1 5 10 15 Leu Leu Leu Phe Leu Leu Pro Thr Leu Trp Phe Cys Ser Pro Ser Ala 20 25 30 Lys Tyr Phe Phe Lys Met Ala Phe Tyr Asn Gly Trp Ile Leu Phe Leu 35 40 45 Ala Val Leu Ala Ile Pro Val Cys Ala Val Arg Gly Arg Asn Val Glu 50 55 60 Asn Met Lys Ile Leu Arg Leu Met Leu Leu His Ile Lys Tyr Leu Tyr 65 70 75 80 Gly Ile Arg Val Glu Val Arg Gly Ala His His Phe Pro Pro Ser Gln 85 90 95 Pro Tyr Val Val Val Ser Asn His Gln Ser Ser Leu Asp Leu Leu Gly 100 105 110 Met Met Glu Val Leu Pro Gly Arg Cys Val Pro Ile Ala Lys Arg Glu 115 120 125 Leu Leu Trp Ala Gly Ser Ala Gly Leu Ala Cys Trp Leu Ala Gly Val 130 135 140 Ile Phe Ile Asp Arg Lys Arg Thr Gly Asp Ala Ile Ser Val Met Ser 145 150 155 160 Glu Val Ala Gln Thr Leu Leu Thr Gln Asp Val Arg Val Trp Val Phe 165 170 175 Pro Glu Gly Thr Arg Asn His Asn Gly Ser Met Leu Pro Phe Lys Arg 180 185 190 Gly Ala Phe His Leu Ala Val Gln Ala Gln Val Pro Ile Val Pro Ile 195 200 205 Val Met Ser Ser Tyr Gln Asp Phe Tyr Cys Lys Lys Glu Arg Arg Phe 210 215 220 Thr Ser Gly Gln Cys Gln Val Arg Val Leu Pro Pro Val Pro Thr Glu 225 230 235 240 Gly Leu Thr Pro Asp Asp Val Pro Ala Leu Ala Asp Arg Val Arg His 245 250 255 Ser Met Leu Thr Val Phe Arg Glu Ile Ser Thr Asp Gly Arg Gly Gly 260 265 270 Gly Asp Tyr Leu Lys Lys Pro Gly Gly Gly Gly 275 280 26285PRTMus musculus 26Met Glu Leu Trp Pro Gly Ala Trp Thr Ala Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Ser Thr Leu Trp Phe Cys Ser Ser Ser Ala Lys Tyr Phe 20 25 30 Phe Lys Met Ala Phe Tyr Asn Gly Trp Ile Leu Phe Leu Ala Ile Leu 35 40 45 Ala Ile Pro Val Cys Ala Val Arg Gly Arg Asn Val Glu Asn Met Lys 50 55 60 Ile Leu Arg Leu Leu Leu Leu His Val Lys Tyr Leu Tyr Gly Ile Arg 65 70 75 80 Val Glu Val Arg Gly Ala His His Phe Pro Pro Thr Gln Pro Tyr Val 85 90 95 Val Val Ser Asn His Gln Ser Ser Leu Asp Leu Leu Gly Met Met Glu 100 105 110 Val Leu Pro Asp Arg Cys Val Pro Ile Ala Lys Arg Glu Leu Leu Trp 115 120 125 Ala Gly Ser Ala Gly Leu Ala Cys Trp Leu Ala Gly Ile Ile Phe Ile 130 135 140 Asp Arg Lys Arg Thr Gly Asp Ala Ile Ser Val Met Ser Glu Val Ala 145 150 155 160 Gln Thr Leu Leu Thr Gln Asp Val Arg Val Trp Val Phe Pro Glu Gly 165 170 175 Thr Arg Asn His Asn Gly Ser Met Leu Pro Phe Lys Arg Gly Ala Phe 180 185 190 His Leu Ala Val Gln Ala Gln Val Pro Ile Ile Pro Ile Val Met Ser 195 200 205 Ser Tyr Gln Asp Phe Tyr Ser Lys Lys Glu Arg Arg Phe Thr Ser Pro 210 215 220 Gly Arg Cys Gln Val Arg Val Leu Pro Pro Val Ser Thr Glu Gly Leu 225 230 235 240 Thr Pro Asp Asp Val Pro Ala Leu Ala Asp Ser Val Arg His Ser Met 245 250 255 Leu Thr Ile Phe Arg Glu Ile Ser Thr Asp Gly Leu Gly Gly Gly Asp 260 265 270 Cys Leu Lys Lys Pro Gly Gly Ala Gly Glu Ala Arg Leu 275 280 285 27827PRTMus musculus 27Met Glu Glu Ser Ser Val Thr Val Gly Thr Ile Asp Val Ser Tyr Leu 1 5 10 15 Pro Ser Ser Ser Glu Tyr Ser Leu Gly Arg Cys Lys His Thr Ser Glu 20 25 30 Asp Trp Val Asp Cys Gly Phe Lys Pro Thr Phe Phe Arg Ser Ala Thr 35 40 45 Leu Lys Trp Lys Glu Ser Leu Met Ser Arg Lys Arg Pro Phe Val Gly 50 55 60 Arg Cys Cys Tyr Ser Cys Thr Pro Gln Ser Trp Glu Arg Phe Phe Asn 65 70 75 80 Pro Ser Ile Pro Ser Leu Gly Leu Arg Asn Val Ile Tyr Ile Asn Glu 85 90 95 Thr His Thr Arg His Arg Gly Trp Leu Ala Arg Arg Leu Ser Tyr Ile 100 105 110 Leu Phe Val Gln Glu Arg Asp Val His Lys Gly Met Phe Ala Thr Ser 115 120 125 Val Thr Glu Asn Val Leu Ser Ser Ser Arg Val Gln Glu Ala Ile Ala 130 135 140 Glu Val Ala Ala Glu Leu Asn Pro Asp Gly Ser Ala Gln Gln Gln Ser 145 150 155 160 Lys Ala Ile Gln Lys Val Lys Arg Lys Ala Arg Lys Ile Leu Gln Glu 165 170 175 Met Val Ala Thr Val Ser Pro Gly Met Ile Arg Leu Thr Gly Trp Val 180 185 190 Leu Leu Lys Leu Phe Asn Ser Phe Phe Trp Asn Ile Gln Ile His Lys 195 200 205 Gly Gln Leu Glu Met Val Lys Ala Ala Thr Glu Thr Asn Leu Pro Leu 210 215 220 Leu Phe Leu Pro Val His Arg Ser His Ile Asp Tyr Leu Leu Leu Thr 225 230 235 240 Phe Ile Leu Phe Cys His Asn Ile Lys Ala Pro Tyr Ile Ala Ser Gly 245 250 255 Asn Asn Leu Asn Ile Pro Val Phe Ser Thr Leu Ile His Lys Leu Gly 260 265 270 Gly Phe Phe Ile Arg Arg Arg Leu Asp Glu Thr Pro Asp Gly Arg Lys 275 280 285 Asp Ile Leu Tyr Arg Ala Leu Leu His Gly His Val Val Glu Leu Leu 290 295 300 Arg Gln Gln Gln Phe Leu Glu Ile Phe Leu Glu Gly Thr Arg Ser Arg 305 310 315 320 Ser Gly Lys Thr Ser Cys Ala Arg Ala Gly Leu Leu Ser Val Val Val 325 330 335 Asp Thr Leu Ser Ser Asn Thr Ile Pro Asp Ile Leu Val Ile Pro Val 340 345 350 Gly Ile Ser Tyr Asp Arg Ile Ile Glu Gly His Tyr Asn Gly Glu Gln 355 360 365 Leu Gly Lys Pro Lys Lys Asn Glu Ser Leu Trp Ser Val Ala Arg Gly 370 375 380 Val Ile Arg Met Leu Arg Lys Asn Tyr Gly Tyr Val Arg Val Asp Phe 385 390 395 400 Ala Gln Pro Phe Ser Leu Lys Glu Tyr Leu Glu Gly Gln Ser Gln Lys 405 410 415 Pro Val Ser Ala Pro Leu Ser Leu Glu Gln Ala Leu Leu Pro Ala Ile 420 425 430 Leu Pro Ser Arg Pro Asn Asp Val Ala Asp Glu His Gln Asp Leu Ser 435 440 445 Ser Asn Glu Ser Arg Asn Pro Ala Asp Glu Ala Phe Arg Arg Arg Leu 450 455 460 Ile Ala Asn Leu Ala Glu His Ile Leu Phe Thr Ala Ser Lys Ser Cys 465 470 475 480 Ala Ile Met Ser Thr His Ile Val Ala Cys Leu Leu Leu Tyr Arg His 485 490 495 Arg Gln Gly Ile His Leu Ser Thr Leu Val Glu Asp Phe Phe Val Met 500 505 510 Lys Glu Glu Val Leu Ala Arg Asp Phe Asp Leu Gly Phe Ser Gly Asn 515 520 525 Ser Glu Asp Val Val Met His Ala Ile Gln Leu Leu Gly Asn Cys Val 530 535 540 Thr Ile Thr His Thr Ser Arg Lys Asp Glu Phe Phe Ile Thr Pro Ser 545 550 555 560 Thr Thr Val Pro Ser Val Phe Glu Leu Asn Phe Tyr Ser Asn Gly Val 565 570 575 Leu His Val Phe Ile Met Glu Ala Ile Ile Ala Cys Ser Ile Tyr Ala 580 585 590 Val Leu Asn Lys Arg Cys Ser Gly Gly Ser Ala Gly Gly Leu Gly Asn 595 600 605 Leu Ile Ser Gln Glu Gln Leu Val Arg Lys Ala Ala Ser Leu Cys Tyr 610 615 620 Leu Leu Ser Asn Glu Gly Thr Ile Ser Leu Pro Cys Gln Thr Phe Tyr 625 630 635 640 Gln Val Cys His Glu Thr Val Gly Lys Phe Ile Gln Tyr Gly Ile Leu 645 650 655 Thr Val Ala Glu Gln Asp Asp Gln Glu Asp Val Ser Pro Gly Leu Ala 660 665 670 Glu Gln Gln Trp Asp Lys Lys Leu Pro Glu Leu Asn Trp Arg Ser Asp 675 680 685 Glu Glu Asp Glu Asp Ser Asp Phe Gly Glu Glu Gln Arg Asp Cys Tyr 690 695 700 Leu Lys Val Ser Gln Ser Lys Glu His Gln Gln Phe Ile Thr Phe Leu 705 710 715 720 Gln Arg Leu Leu Gly Pro Leu Leu Glu Ala Tyr Ser Ser Ala Ala Ile 725 730 735 Phe Val His Asn Phe Ser Gly Pro Val Pro Glu Ser Glu Tyr Leu Gln 740 745 750 Lys Leu His Arg Tyr Leu Ile Thr Arg Thr Glu Arg Asn Val Ala Val 755 760 765 Tyr Ala Glu Ser Ala Thr Tyr Cys Leu Val Lys Asn Ala Val Lys Met 770 775 780 Phe Lys Asp Ile Gly Val Phe Lys Glu Thr Lys Gln Lys Arg Val Ser 785 790 795 800 Val Leu Glu Leu Ser Ser Thr Phe Leu Pro Gln Cys Asn Arg Gln Lys 805 810 815 Leu Leu Glu Tyr Ile Leu Ser Phe Val Val Leu 820 825 28201PRTSus scrofa 28Asn Ser Glu Asp Val Val Met His Ala Ile Gln Leu Leu Gly Asn Cys 1 5 10 15 Ile Thr Ile Thr His Thr Ser Arg Asn Asp Glu Phe Phe Ile Thr Pro 20 25 30 Ser Thr Thr Val Pro Ser Val Phe Glu Leu Asn Phe Tyr Ser Asn Gly 35 40 45 Val Leu His Val Phe Ile Met Glu Ala Ile Ile Ala Cys Ser Leu Tyr 50 55 60 Ala Val Leu Lys Lys Arg Gly Ser Gly Gly Pro Ala Ser Pro Ser Leu 65 70 75 80 Ile Ser Gln Glu Gln Leu Val Arg Lys Ala Ala Ser Leu Cys Tyr Leu 85 90 95 Leu Ser Asn Glu Gly Thr Ile Ser Leu Pro Cys Gln Thr Phe Tyr Gln 100 105 110 Ile Cys His Glu Thr Val Gly Arg Phe Ile Gln Tyr Gly Ile Leu Thr 115 120 125 Val Ala Glu Gln Asp Asp Gln Glu Asp Ile Ser Pro Ser Leu Ala Glu 130 135 140 Gln His Trp Asp Lys Lys Leu Pro Glu Pro Leu Ser Trp Arg Ser Asp 145 150 155 160 Glu Glu Asp Glu Asp Ser Asp Phe Gly Glu Glu Gln Arg Asp Cys Tyr 165 170 175 Leu Lys Val Ser Gln Ser Lys Glu His Gln Gln Phe Ile Thr Phe Leu 180 185 190 Gln Arg Leu Leu Gly Pro Leu Leu Glu 195 200 29259PRTMus musculus 29Met His Ser Ser Val Tyr Phe Val Ala Leu Val Ile Leu Gly Ala Ala 1 5 10 15 Val Cys Ala Ala Gln Pro Arg Gly Arg Ile Leu Gly Gly Gln Glu Ala 20 25 30 Ala Ala His Ala Arg Pro Tyr Met Ala Ser Val Gln Val Asn Gly Thr 35 40 45 His Val Cys Gly Gly Thr Leu Leu Asp Glu Gln Trp Val Leu Ser Ala 50 55 60 Ala His Cys Met Asp Gly Val Thr Asp Asp Asp Ser Val Gln Val Leu 65 70 75 80 Leu Gly Ala His Ser Leu Ser Ala Pro Glu Pro Tyr Lys Arg Trp Tyr 85 90 95 Asp Val Gln Ser Val Val Pro His Pro Gly Ser Arg Pro Asp Ser Leu 100 105 110 Glu Asp Asp Leu Ile Leu Phe Lys Leu Ser Gln Asn Ala Ser Leu Gly 115 120 125 Pro His Val Arg Pro Leu Pro Leu Gln Tyr Glu Asp Lys Glu Val Glu 130 135 140 Pro Gly Thr Leu Cys Asp Val Ala Gly Trp Gly Val Val Thr His Ala 145 150 155 160 Gly Arg Arg Pro Asp Val Leu His Gln Leu Arg Val Ser Ile Met Asn 165 170 175 Arg Thr Thr Cys Asn Leu Arg Thr Tyr His Asp Gly Val Val Thr Ile 180 185 190 Asn Met Met Cys Ala Glu Ser Asn Arg Arg Asp Thr Cys Arg Gly Asp 195 200 205 Ser Gly Ser Pro Leu Val Cys Gly Asp Ala Val Glu Gly Val Val Thr 210 215 220 Trp Gly Ser Arg Val Cys Gly Asn Gly Lys Lys Pro Gly Val Tyr Thr 225 230 235 240 Arg Val Ser Ser Tyr Arg Met Trp Ile Glu Asn Ile Thr Asn Gly Asn 245 250 255 Met Thr Ser 3083PRTSus scrofa 30Trp Gln Arg Glu Asp His Glu Val Pro Ala Gly Thr Leu Cys Asp Val 1 5 10 15 Ala Gly Trp Gly Val Val Ser His Thr Gly Arg Arg Pro Asp Arg Leu 20 25 30 Gln His Leu Leu Leu Pro Val Leu Asp Arg Thr Thr Cys Asn Leu Arg 35 40 45 Thr Tyr His Asp Gly Thr Ile Thr Glu Arg Met Met Cys Ala Glu Ser 50 55 60 Asn Arg Arg Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys 65 70 75 80 Gly Gly Val 31891PRTMus musculus 31Met Asn Tyr Val Gly Gln Leu Ala Gly Gln Val Phe Val Thr Val Lys 1 5 10 15 Glu Leu Tyr Lys Gly Leu Asn Pro

Ala Thr Leu Ser Gly Cys Ile Asp 20 25 30 Ile Ile Val Ile Arg Gln Pro Asn Gly Ser Leu Gln Cys Ser Pro Phe 35 40 45 His Val Arg Phe Gly Lys Met Gly Val Leu Arg Ser Arg Glu Lys Val 50 55 60 Val Asp Ile Glu Ile Asn Gly Glu Ser Val Asp Leu His Met Lys Leu 65 70 75 80 Gly Asp Asn Gly Glu Ala Phe Phe Val Gln Glu Thr Asp Asn Asp Gln 85 90 95 Glu Ile Ile Pro Met Tyr Leu Ala Thr Ser Pro Ile Leu Ser Glu Gly 100 105 110 Ala Ala Arg Met Glu Ser Gln Leu Lys Arg Asn Ser Val Asp Arg Ile 115 120 125 Arg Cys Leu Asp Pro Thr Thr Ala Ala Gln Gly Leu Pro Pro Ser Asp 130 135 140 Thr Pro Ser Thr Gly Ser Leu Gly Lys Lys Arg Arg Lys Arg Arg Arg 145 150 155 160 Lys Ala Gln Leu Asp Asn Leu Lys Arg Asp Asp Asn Val Asn Thr Ser 165 170 175 Glu Asp Glu Asp Met Phe Pro Ile Glu Met Ser Ser Asp Glu Asp Thr 180 185 190 Ala Pro Met Asp Gly Ser Arg Thr Leu Pro Asn Asp Val Pro Pro Phe 195 200 205 Gln Asp Asp Ile Pro Lys Glu Asn Phe Pro Ser Ile Ser Thr Tyr Pro 210 215 220 Gln Ser Ala Ser Tyr Pro Ser Ser Asp Arg Glu Trp Ser Pro Ser Pro 225 230 235 240 Ser Pro Ser Gly Ser Arg Pro Ser Thr Pro Lys Ser Asp Ser Glu Leu 245 250 255 Val Ser Lys Ser Ala Asp Arg Leu Thr Pro Lys Asn Asn Leu Glu Met 260 265 270 Leu Trp Leu Trp Gly Glu Leu Pro Gln Ala Ala Lys Ser Ser Ser Pro 275 280 285 His Lys Met Lys Glu Ser Ser Pro Leu Gly Ser Arg Lys Thr Pro Asp 290 295 300 Lys Met Asn Phe Gln Ala Ile His Ser Glu Ser Ser Asp Thr Phe Ser 305 310 315 320 Asp Gln Ser Pro Thr Met Ala Arg Gly Leu Leu Ile His Gln Ser Lys 325 330 335 Ala Gln Thr Glu Met Gln Phe Val Asn Glu Glu Asp Leu Glu Ser Leu 340 345 350 Gly Ala Ala Ala Pro Pro Ser Pro Val Ala Glu Glu Leu Lys Ala Pro 355 360 365 Tyr Pro Asn Thr Ala Gln Ser Ser Ser Lys Thr Asp Ser Pro Ser Arg 370 375 380 Lys Lys Asp Lys Arg Ser Arg His Leu Gly Ala Asp Gly Val Tyr Leu 385 390 395 400 Asp Asp Leu Thr Asp Met Asp Pro Glu Val Ala Ala Leu Tyr Phe Pro 405 410 415 Lys Asn Gly Asp Pro Gly Gly Leu Pro Lys Gln Ala Ser Asp Asn Gly 420 425 430 Ala Arg Ser Ala Asn Gln Ser Pro Gln Ser Val Gly Gly Ser Gly Ile 435 440 445 Asp Ser Gly Val Glu Ser Thr Ser Asp Ser Leu Arg Asp Leu Pro Ser 450 455 460 Ile Ala Ile Ser Leu Cys Gly Gly Leu Ser Asp His Arg Glu Ile Thr 465 470 475 480 Lys Asp Ala Phe Leu Glu Gln Ala Val Ser Tyr Gln Gln Phe Ala Asp 485 490 495 Asn Pro Ala Ile Ile Asp Asp Pro Asn Leu Val Val Lys Val Gly Asn 500 505 510 Lys Tyr Tyr Asn Trp Thr Thr Ala Ala Pro Leu Leu Leu Ala Met Gln 515 520 525 Ala Phe Gln Lys Pro Leu Pro Lys Ala Thr Val Glu Ser Ile Met Arg 530 535 540 Asp Lys Met Pro Lys Lys Gly Gly Arg Trp Trp Phe Ser Trp Arg Gly 545 550 555 560 Arg Asn Ala Thr Ile Lys Glu Glu Ser Lys Pro Glu Gln Cys Leu Thr 565 570 575 Gly Lys Gly His Asn Thr Gly Glu Gln Pro Ala Gln Leu Gly Leu Ala 580 585 590 Thr Arg Ile Lys His Glu Ser Ser Ser Ser Asp Glu Glu His Ala Ala 595 600 605 Ala Lys Pro Ser Gly Ser Ser His Leu Ser Leu Leu Ser Asn Val Ser 610 615 620 Tyr Lys Lys Thr Leu Arg Leu Thr Ser Glu Gln Leu Lys Ser Leu Lys 625 630 635 640 Leu Lys Asn Gly Pro Asn Asp Val Val Phe Ser Val Thr Thr Gln Tyr 645 650 655 Gln Gly Thr Cys Arg Cys Glu Gly Thr Ile Tyr Leu Trp Asn Trp Asp 660 665 670 Asp Lys Val Ile Ile Ser Asp Ile Asp Gly Thr Ile Thr Arg Ser Asp 675 680 685 Thr Leu Gly His Ile Leu Pro Thr Leu Gly Lys Asp Trp Thr His Gln 690 695 700 Gly Ile Ala Lys Leu Tyr His Lys Val Ser Gln Asn Gly Tyr Lys Phe 705 710 715 720 Leu Tyr Cys Ser Ala Arg Ala Ile Gly Met Ala Asp Met Thr Arg Gly 725 730 735 Tyr Leu His Trp Val Asn Glu Arg Gly Thr Val Leu Pro Gln Gly Pro 740 745 750 Leu Leu Leu Ser Pro Ser Ser Leu Phe Ser Ala Leu His Arg Glu Val 755 760 765 Ile Glu Lys Lys Pro Glu Lys Phe Lys Val Gln Cys Leu Thr Asp Ile 770 775 780 Lys Asn Leu Phe Phe Pro Asn Thr Glu Pro Phe Tyr Ala Ala Phe Gly 785 790 795 800 Asn Arg Pro Ala Asp Val Tyr Ser Tyr Lys Gln Val Gly Val Ser Leu 805 810 815 Asn Arg Ile Phe Thr Val Asn Pro Lys Gly Glu Leu Val Gln Glu His 820 825 830 Ala Lys Thr Asn Ile Ser Ser Tyr Val Arg Leu Cys Glu Val Val Asp 835 840 845 His Val Phe Pro Leu Leu Lys Arg Ser His Ser Cys Asp Phe Pro Cys 850 855 860 Ser Asp Thr Phe Ser Asn Phe Thr Phe Trp Arg Glu Pro Leu Pro Pro 865 870 875 880 Phe Glu Asn Gln Asp Met His Ser Ala Ser Ala 885 890 32931PRTMus musculus 32Met Leu Tyr Leu Glu Asp Asn Ser Glu Asp Glu Lys Thr Val Gln Glu 1 5 10 15 Ser Ser Leu Ser Lys Pro Ala Ser Val Tyr His Gly Lys Ala Pro Pro 20 25 30 Gly Ile Leu Ser Gln Thr Met Asn Tyr Val Gly Gln Leu Ala Gly Gln 35 40 45 Val Leu Val Thr Val Lys Glu Leu Tyr Lys Gly Ile Asn Gln Ala Thr 50 55 60 Leu Ser Gly Cys Ile Asp Val Val Val Val Arg Gln Gln Asp Gly Ser 65 70 75 80 Tyr Gln Cys Ser Pro Phe His Val Arg Phe Gly Lys Leu Gly Val Leu 85 90 95 Arg Ser Lys Glu Lys Val Ile Asp Ile Glu Ile Asn Gly Ser Ala Val 100 105 110 Asp Leu His Met Lys Leu Gly Asp Asn Gly Glu Ala Phe Phe Val Glu 115 120 125 Glu Thr Glu Glu Glu Tyr Glu Lys Leu Pro Ala Tyr Leu Ala Thr Ser 130 135 140 Pro Ile Pro Thr Glu Asp Gln Phe Phe Lys His Ile Glu Thr Pro Leu 145 150 155 160 Val Lys Ser Ser Gly Asn Glu Arg Pro Ala Gln Ser Ser Asp Val Ser 165 170 175 His Thr Leu Glu Ser Glu Ala Val Phe Thr Gln Ser Ser Val Lys Lys 180 185 190 Lys Lys Arg Arg Arg Lys Lys Cys Lys Gln Asp Asn Arg Lys Glu Glu 195 200 205 Gln Ala Ala Ser Pro Val Ala Glu Asp Val Gly Asp Val Gly Val Ser 210 215 220 Ser Asp Asp Glu Lys Arg Ala Gln Ala Ala Arg Gly Ser Ser Asn Ala 225 230 235 240 Ser Leu Lys Glu Glu Asp Tyr Lys Glu Pro Ser Leu Phe His Ser Gly 245 250 255 Asp Asn Tyr Pro Leu Ser Asp Gly Asp Trp Ser Pro Leu Glu Thr Thr 260 265 270 Tyr Pro Gln Ala Val Cys Pro Lys Ser Asp Ser Glu Leu Glu Val Lys 275 280 285 Pro Ser Glu Ser Leu Leu Arg Ser Glu Pro His Met Glu Trp Thr Trp 290 295 300 Gly Gly Phe Pro Glu Ser Thr Lys Val Thr Lys Arg Glu Arg Tyr Asp 305 310 315 320 Tyr His Pro Arg Thr Ala Thr Ile Thr Pro Ser Glu Asn Thr His Phe 325 330 335 Arg Val Ile Pro Ser Glu Asp Ser Leu Ile Arg Glu Val Glu Lys Asp 340 345 350 Ala Thr Val Glu Asp Thr Thr Cys Thr Ile Val Lys Pro Lys Pro Arg 355 360 365 Ala Leu Cys Lys Gln Leu Ser Asp Ala Ala Ser Thr Glu Leu Pro Glu 370 375 380 Ser Pro Leu Glu Ala Pro Gln Ile Ser Ser Leu Leu Asp Ala Asp Pro 385 390 395 400 Val Pro Ser Pro Ser Ala Glu Ala Pro Ser Glu Pro Lys Pro Ala Ala 405 410 415 Lys Asp Ser Pro Thr Lys Lys Lys Gly Val His Lys Arg Ser Gln His 420 425 430 Gln Gly Pro Asp Asp Ile Tyr Leu Asp Asp Leu Lys Ala Leu Glu Pro 435 440 445 Glu Val Ala Ala Leu Tyr Phe Pro Lys Ser Asp Thr Asp Pro Gly Ser 450 455 460 Arg Gln Trp Pro Glu Ser Asp Thr Phe Ser Gly Ser Gln Ser Pro Gln 465 470 475 480 Ser Val Gly Ser Ala Ala Ala Asp Ser Gly Thr Glu Cys Leu Ser Asp 485 490 495 Ser Ala Met Asp Leu Pro Asp Val Thr Leu Ser Leu Cys Gly Gly Leu 500 505 510 Ser Glu Asn Gly Glu Ile Ser Lys Glu Lys Phe Met Glu His Ile Ile 515 520 525 Thr Tyr His Glu Phe Ala Glu Asn Pro Gly Leu Ile Asp Asn Pro Asn 530 535 540 Leu Val Ile Arg Ile Tyr Asn Arg Tyr Tyr Asn Trp Ala Leu Ala Ala 545 550 555 560 Pro Met Ile Leu Ser Leu Gln Val Phe Gln Lys Ser Leu Pro Lys Ala 565 570 575 Thr Val Glu Ser Trp Val Lys Asp Lys Met Pro Lys Lys Ser Gly Arg 580 585 590 Trp Trp Phe Trp Arg Lys Lys Glu Ser Met Ile Lys Gln Leu Pro Glu 595 600 605 Thr Lys Glu Gly Lys Ser Glu Val Pro Pro Ala Asn Asp Leu Pro Ser 610 615 620 Asn Ala Glu Glu Pro Thr Ser Ala Arg Pro Ala Glu Asn Asp Thr Ser 625 630 635 640 Ser Asp Glu Gly Ser Gln Glu Leu Glu Glu Ser Ile Lys Val Asp Pro 645 650 655 Ile Thr Val Glu Thr Leu Ser His Cys Gly Thr Ala Ser Tyr Lys Lys 660 665 670 Ser Leu Arg Leu Ser Ser Asp Gln Ile Ala Lys Leu Lys Leu His Asp 675 680 685 Gly Pro Asn Asp Val Val Phe Ser Ile Thr Thr Gln Tyr Gln Gly Thr 690 695 700 Cys Arg Cys Ala Gly Thr Ile Tyr Leu Trp Asn Trp Asn Asp Lys Val 705 710 715 720 Ile Ile Ser Asp Ile Asp Gly Thr Ile Thr Lys Ser Asp Ala Leu Gly 725 730 735 Gln Ile Leu Pro Gln Leu Gly Lys Asp Trp Thr His Gln Gly Ile Ala 740 745 750 Arg Leu Tyr His Ser Ile Asn Glu Asn Gly Tyr Lys Phe Leu Tyr Cys 755 760 765 Ser Ala Arg Ala Ile Gly Met Ala Asp Met Thr Arg Gly Tyr Leu His 770 775 780 Trp Val Asn Asp Lys Gly Thr Ile Leu Pro Arg Gly Pro Leu Met Leu 785 790 795 800 Ser Pro Ser Ser Leu Phe Ser Ala Phe His Arg Glu Val Ile Glu Lys 805 810 815 Lys Pro Glu Lys Phe Lys Ile Glu Cys Leu Asn Asp Ile Lys Asn Leu 820 825 830 Phe Ala Pro Ser Arg Gln Pro Phe Tyr Ala Ala Phe Gly Asn Arg Pro 835 840 845 Asn Asp Val Tyr Ala Tyr Thr Gln Val Gly Val Pro Asp Cys Arg Ile 850 855 860 Phe Thr Val Asn Pro Lys Gly Glu Leu Ile Gln Glu Arg Thr Lys Gly 865 870 875 880 Asn Lys Ser Ser Tyr His Arg Leu Ser Glu Leu Val Glu His Val Phe 885 890 895 Pro Leu Leu Ser Lys Glu Gln Asn Ser Ala Phe Pro Cys Pro Glu Phe 900 905 910 Ser Ser Phe Cys Tyr Trp Arg Asp Pro Ile Pro Asp Leu Asp Leu Asp 915 920 925 Asp Leu Ala 930 33368PRTArabidopsis thaliana 33Met Arg Ile Leu Gln Asn Lys Thr Met Lys Glu Gln Asp Asn Gln Leu 1 5 10 15 Lys Ile Pro Glu Pro Leu Arg Ala Asp Trp Phe Met Val Leu Val Thr 20 25 30 Ile Gln Ala Asp Leu Ile Tyr Asn Ala Leu Val Val Leu Ser Ser Pro 35 40 45 Phe Phe Leu Leu Tyr Arg Ser Tyr Arg Arg Ala Val Val Thr Val Ser 50 55 60 Ala Ala Glu Lys Ala Val Lys Arg Ala Pro Ala Gln Ile Ala Gly Gly 65 70 75 80 Ala Gly Arg Val Val Arg Arg Thr Trp Phe Gly Ile Leu Gly Ala Cys 85 90 95 His Val Ser Met Val Met Val Leu Ala Leu Ile Leu Ala Val Val Ile 100 105 110 Gly Val Gly Ile Val Ser Leu Tyr Val Glu Lys Pro Val Val Val Arg 115 120 125 Asp Arg Leu Phe Phe Asp Tyr Thr Glu Glu Asn Pro Ser Ala Val Phe 130 135 140 Ser Phe Asp Lys Lys Lys Arg Ser Phe Ser Val Pro Val Gly His Ser 145 150 155 160 Val His Val Ser Leu Val Leu Trp Met Pro Glu Ser Glu Ile Asn Arg 165 170 175 Arg Ile Gly Val Phe Gln Leu Lys Val Glu Leu Leu Ser Leu Lys Gly 180 185 190 Glu Thr Ile Ala Arg Ser Ser Gln Pro Cys Met Leu Arg Phe Arg Ser 195 200 205 Lys Pro Ile Arg Leu Ala Arg Thr Phe Val Met Ser Val Pro Leu Ile 210 215 220 Ala Gly Ile Ala Asn Glu Ala Gln Thr Met Arg Ile Asp Ala Leu Lys 225 230 235 240 His Gln Glu Lys Met Pro Arg Thr Lys Ala Val Arg Ala Thr Leu Ile 245 250 255 Pro Arg Ala Gln Thr Arg Thr Leu Pro Gln Leu Tyr Glu Ala Glu Ile 260 265 270 Val Ile Asn Ser Lys Pro Pro Trp Ile Lys Arg Met Ala Tyr Asn Trp 275 280 285 Lys Trp Thr Leu Cys Val Trp Thr Ser Met Tyr Leu Tyr Val Ala Ile 290 295 300 Leu Thr Ala Leu Leu Trp Cys Phe Arg Pro Val Leu Phe Pro Tyr Thr 305 310 315 320 Ser Ser Arg Thr Ile Ser Glu Ser Glu Asn Leu Glu Ile Glu Val Val 325 330 335 Glu Glu Glu Gln Glu Gln Val Met Glu Arg Arg Arg Arg Glu Arg Arg 340 345 350 Asn Gln Pro Arg Arg Arg Asn Phe Ala Thr Thr Gln Lys Ser Tyr Thr 355 360 365 34526PRTArabidopsis thaliana 34Met Asp Ser Glu Ser Glu Ser Glu Ser Asn Pro Ser Thr Thr Asp Glu 1 5 10 15 Phe Asp Arg Phe Leu Asp Ala Pro Asp Glu Phe Tyr Tyr Asp Cys Leu 20 25 30 Pro Ile Arg Ser Asn Ser His Gln Pro Ser Ser Leu Leu Arg Arg Arg 35 40 45 Lys Ser Ala His Arg Arg Asp Leu Ile Ser Ser Asp Ile Glu Thr Glu 50 55 60 Pro Ser Ser Ser Ser Asp Gly Phe Asp Val Gly Glu Lys Ser Ser Tyr 65 70 75 80 Val Glu Lys Asn Ala Glu Leu Arg Gly Asp Ile Asp Thr Ser Asp Val 85 90 95 Ile Glu Ser Thr Lys Asp Ser Ile Asp Leu Ser Ser Glu Lys Glu Asn 100 105 110 Asp Leu Asp Val Ile Ser Ser Ser Gly Asn Asp Met Asp Val Ile Asp 115 120

125 Ser Gly Arg Asn Arg Val Asp Pro Phe Gln Glu Glu Ser Thr Val Thr 130 135 140 Thr Val Ser Ser Asp Asp Gln Gly Asp Asp Asp Tyr Ala Gly Ser Val 145 150 155 160 Pro Gln Phe Arg Glu Pro Pro Asn Ser Thr Glu Trp Ser Leu Leu Gly 165 170 175 Phe Leu Val Gly Leu Val Ile Lys Ala Ile Glu Phe Gln Val Ser Phe 180 185 190 Met Thr Ser Leu Leu Thr Phe Pro Pro Trp Leu Leu Arg Asn Cys Phe 195 200 205 Leu Phe Phe Phe Asp Pro Phe Ser Thr Ile Arg Phe Gly Arg Arg Phe 210 215 220 Leu Met Ala Arg Val Ala Gly Ile Ser Asp Met Ile Phe Gly Tyr Met 225 230 235 240 Asn Pro Phe Arg Leu Lys Asp Thr Lys Gln Met Leu Ser Ile Val Cys 245 250 255 Lys Phe Gly Trp Gly Met Phe Trp Ala Val Tyr Val Gly Ile Val Leu 260 265 270 Phe Gly Leu Leu Val Ser Ser Leu Met Ile Gly Gly Tyr Val Ile Asn 275 280 285 Arg Ile Ala Asp Lys Pro Phe Glu Val Lys Glu Thr Leu Asn Phe Asp 290 295 300 Tyr Thr Lys Asn Ser Pro Glu Ala Tyr Val Pro Ile Ser Ser Cys Ala 305 310 315 320 Gly Val Glu Cys Glu Gly Ser Cys Lys Glu Ser Asn Glu Met Ser Lys 325 330 335 Ile Arg Gly Leu Arg Val Ile Pro Arg Asp Gln Lys Leu Asp Ile Ile 340 345 350 Leu Ser Met Thr Leu Pro Glu Ser Ala Tyr Asn Lys Asn Leu Gly Met 355 360 365 Phe Gln Val Arg Val Asp Phe Leu Ser Val Asp Gly Gln Thr Ile Ala 370 375 380 Ser Ile Arg Arg Pro Cys Met Leu Arg Phe Arg Ser Glu Pro Ile Arg 385 390 395 400 Leu Val Gln Thr Phe Phe Lys Val Val Pro Leu Val Thr Gly Tyr Val 405 410 415 Ser Glu Ile Gln Thr Leu Ser Leu Lys Leu Lys Gly Phe Val Glu Lys 420 425 430 Asp Ile Pro Thr Ala Cys Leu Lys Ile Ile Ile Glu Gln Arg Ala Glu 435 440 445 Phe Arg Pro Gly Ala Gly Ile Pro Glu Leu Tyr Asp Ala Ser Leu Ser 450 455 460 Val Glu Ser Gly Leu Pro Phe Phe Arg Lys Ile Ile Trp Lys Trp Arg 465 470 475 480 Lys Thr Leu Phe Val Trp Ile Ser Met Ser Leu Phe Ile Thr Glu Leu 485 490 495 Leu Phe Thr Leu Val Cys Cys Arg Pro Leu Ile Ile Pro Arg Thr Gln 500 505 510 Pro Arg Asp Arg Ser Pro Ser Asn Pro Thr Gly Val Trp Arg 515 520 525 35509PRTArabidopsis thaliana 35Met Glu Ser Glu Ser Glu Ser Ser Ser Thr His Ser Ser Cys Asp Arg 1 5 10 15 Phe Leu Asp Ala Glu Asp Glu Phe Phe Tyr Asp Ser Phe Ser Asn His 20 25 30 Tyr Asp Cys Leu Asn Ser Ser Pro Pro Ala Asn Leu Arg Arg Arg Arg 35 40 45 Leu Pro Met Asp Thr Asp Ser Ser Ser Ser Ser Ser Thr Ser Ser Leu 50 55 60 Glu Ser Cys Glu Lys Arg Ser Thr Val Gly Glu Asn Asp Glu Leu Glu 65 70 75 80 Val Ser Leu Val Asp Phe Glu Thr Ile Glu Ile Asp Val Asp Val Thr 85 90 95 Asp Ser Ala Asn Ser Ser Ile Asp Ser Ile Ser Glu Lys Gly Glu Asp 100 105 110 Phe Glu Val Ile Asp Ser Cys Thr Asp Thr Glu Lys Asn Met Gly Glu 115 120 125 Asn Asp Ser Gly Arg Val Asp Pro Phe Thr Val Thr Thr Leu Asn Asp 130 135 140 Glu Arg Gly Glu Ile Tyr Thr Gly Pro Glu Tyr Thr Ser Thr Asp Trp 145 150 155 160 Ser Leu Thr Ser Leu Val Ile Arg Ser Ile Glu Phe Gln Val Ser Leu 165 170 175 Met Ile Thr Phe Ile Arg Phe Pro Pro Trp Leu Ile Ser Lys Cys Leu 180 185 190 Ser Phe Val Phe Asp Pro Tyr Arg Thr Met Arg Arg Gly Arg Arg Tyr 195 200 205 Leu Val Ser Trp Ile Val Gly Leu Cys Asp Ser Gly Leu Lys Asp Asp 210 215 220 Lys Pro Val Leu Glu Leu Val Arg Arg Val Thr Trp Gly Leu Phe Cys 225 230 235 240 Ala Val Tyr Val Gly Ile Met Leu Phe Ala Leu Leu Val Ser Ala Phe 245 250 255 Met Ile Ser Gly Phe Val Ile Thr Tyr Leu Ala His Glu Pro Leu Val 260 265 270 Ile Lys Glu Ser Leu Asn Phe Asp Tyr Thr Lys Ser Ser Pro Glu Ala 275 280 285 Tyr Val Pro Ile Ser Ser Cys Ala Gly Val Ala Phe Gly Leu Ser Gly 290 295 300 Lys Glu Ser Ile Glu Thr Gly Lys Val Lys Gly Leu Lys Asp Arg Thr 305 310 315 320 Glu Ile Thr Val Ser Met Thr Leu Pro Glu Ser Glu Tyr Asn Arg Asn 325 330 335 Leu Gly Met Phe Gln Val Arg Val Asp Phe Leu Ser Ala Ser Gly His 340 345 350 Val Leu Ala Ser Ser Arg Arg Pro Cys Met Val Lys Phe Ser Ser Glu 355 360 365 Pro Ile Arg Leu Val Gln Thr Leu Leu Lys Ile Ala Pro Leu Val Thr 370 375 380 Gly Tyr Val Ser Glu Ile Gln Thr Leu Asn Leu Lys Leu Lys Gly Leu 385 390 395 400 Val Glu Lys Asp Ile Ile Pro Thr Ala Cys Leu Lys Ile Met Ile Glu 405 410 415 Gln Arg Ala Glu Phe Arg Pro Gly Ala Gly Ile Pro Glu Ile Tyr Asp 420 425 430 Ala Ser Leu Phe Leu Glu Ser Lys Leu Pro Phe Leu Lys Arg Ile Ile 435 440 445 Trp Asn Trp Arg Lys Thr Leu Phe Val Trp Ile Ser Met Ser Leu Phe 450 455 460 Ile Met Glu Leu Leu Phe Ala Leu Val Phe Phe Arg Pro Leu Ile Ile 465 470 475 480 Pro Arg Thr Gly Gln Arg Thr Gln Gln Arg Asp Gly Thr His Ser Ile 485 490 495 Asn Asn Asn Leu Tyr Leu Asp Gly Gln Ala Gly Ser Arg 500 505 36363PRTArabidopsis thaliana 36Met Asp Asn Phe Leu Pro Phe Pro Ser Ser Asn Ala Asn Ser Val Gln 1 5 10 15 Glu Leu Ser Met Asp Pro Asn Asn Asn Arg Ser His Phe Thr Thr Val 20 25 30 Pro Thr Tyr Asp His His Gln Ala Gln Pro His His Phe Leu Pro Pro 35 40 45 Phe Ser Tyr Pro Val Glu Gln Met Ala Ala Val Met Asn Pro Gln Pro 50 55 60 Val Tyr Leu Ser Glu Cys Tyr Pro Gln Ile Pro Val Thr Gln Thr Gly 65 70 75 80 Ser Glu Phe Gly Ser Leu Val Gly Asn Pro Cys Leu Trp Gln Glu Arg 85 90 95 Gly Gly Phe Leu Asp Pro Arg Met Thr Lys Met Ala Arg Ile Asn Arg 100 105 110 Lys Asn Ala Met Met Arg Ser Arg Asn Asn Ser Ser Pro Asn Ser Ser 115 120 125 Pro Ser Glu Leu Val Asp Ser Lys Arg Gln Leu Met Met Leu Asn Leu 130 135 140 Lys Asn Asn Val Gln Ile Ser Asp Lys Lys Asp Ser Tyr Gln Gln Ser 145 150 155 160 Thr Phe Asp Asn Lys Lys Leu Arg Val Leu Cys Glu Lys Glu Leu Lys 165 170 175 Asn Ser Asp Val Gly Ser Leu Gly Arg Ile Val Leu Pro Lys Arg Asp 180 185 190 Ala Glu Ala Asn Leu Pro Lys Leu Ser Asp Lys Glu Gly Ile Val Val 195 200 205 Gln Met Arg Asp Val Phe Ser Met Gln Ser Trp Ser Phe Lys Tyr Lys 210 215 220 Phe Trp Ser Asn Asn Lys Ser Arg Met Tyr Val Leu Glu Asn Thr Gly 225 230 235 240 Glu Phe Val Lys Gln Asn Gly Ala Glu Ile Gly Asp Phe Leu Thr Ile 245 250 255 Tyr Glu Asp Glu Ser Lys Asn Leu Tyr Phe Ala Met Asn Gly Asn Ser 260 265 270 Gly Lys Gln Asn Glu Gly Arg Glu Asn Glu Ser Arg Glu Arg Asn His 275 280 285 Tyr Glu Glu Ala Met Leu Asp Tyr Ile Pro Arg Asp Glu Glu Glu Ala 290 295 300 Ser Ile Ala Met Leu Ile Gly Asn Leu Asn Asp His Tyr Pro Ile Pro 305 310 315 320 Asn Asp Leu Met Asp Leu Thr Thr Asp Leu Gln His His Gln Ala Thr 325 330 335 Ser Ser Ser Met Pro Pro Glu Asp His Ala Tyr Val Gly Ser Ser Asp 340 345 350 Asp Gln Val Ser Phe Asn Asp Phe Glu Trp Trp 355 360 37509PRTtomato bushy stunt virus 37Met Glu Ser Glu Ser Glu Ser Ser Ser Thr His Ser Ser Cys Asp Arg 1 5 10 15 Phe Leu Asp Ala Glu Asp Glu Phe Phe Tyr Asp Ser Phe Ser Asn His 20 25 30 Tyr Asp Cys Leu Asn Ser Ser Pro Pro Ala Asn Leu Arg Arg Arg Arg 35 40 45 Leu Pro Met Asp Thr Asp Ser Ser Ser Ser Ser Ser Thr Ser Ser Leu 50 55 60 Glu Ser Cys Glu Lys Arg Ser Thr Val Gly Glu Asn Asp Glu Leu Glu 65 70 75 80 Val Ser Leu Val Asp Phe Glu Thr Ile Glu Ile Asp Val Asp Val Thr 85 90 95 Asp Ser Ala Asn Ser Ser Ile Asp Ser Ile Ser Glu Lys Gly Glu Asp 100 105 110 Phe Glu Val Ile Asp Ser Cys Thr Asp Thr Glu Lys Asn Met Gly Glu 115 120 125 Asn Asp Ser Gly Arg Val Asp Pro Phe Thr Val Thr Thr Leu Asn Asp 130 135 140 Glu Arg Gly Glu Ile Tyr Thr Gly Pro Glu Tyr Thr Ser Thr Asp Trp 145 150 155 160 Ser Leu Thr Ser Leu Val Ile Arg Ser Ile Glu Phe Gln Val Ser Leu 165 170 175 Met Ile Thr Phe Ile Arg Phe Pro Pro Trp Leu Ile Ser Lys Cys Leu 180 185 190 Ser Phe Val Phe Asp Pro Tyr Arg Thr Met Arg Arg Gly Arg Arg Tyr 195 200 205 Leu Val Ser Trp Ile Val Gly Leu Cys Asp Ser Gly Leu Lys Asp Asp 210 215 220 Lys Pro Val Leu Glu Leu Val Arg Arg Val Thr Trp Gly Leu Phe Cys 225 230 235 240 Ala Val Tyr Val Gly Ile Met Leu Phe Ala Leu Leu Val Ser Ala Phe 245 250 255 Met Ile Ser Gly Phe Val Ile Thr Tyr Leu Ala His Glu Pro Leu Val 260 265 270 Ile Lys Glu Ser Leu Asn Phe Asp Tyr Thr Lys Ser Ser Pro Glu Ala 275 280 285 Tyr Val Pro Ile Ser Ser Cys Ala Gly Val Ala Phe Gly Leu Ser Gly 290 295 300 Lys Glu Ser Ile Glu Thr Gly Lys Val Lys Gly Leu Lys Asp Arg Thr 305 310 315 320 Glu Ile Thr Val Ser Met Thr Leu Pro Glu Ser Glu Tyr Asn Arg Asn 325 330 335 Leu Gly Met Phe Gln Val Arg Val Asp Phe Leu Ser Ala Ser Gly His 340 345 350 Val Leu Ala Ser Ser Arg Arg Pro Cys Met Val Lys Phe Ser Ser Glu 355 360 365 Pro Ile Arg Leu Val Gln Thr Leu Leu Lys Ile Ala Pro Leu Val Thr 370 375 380 Gly Tyr Val Ser Glu Ile Gln Thr Leu Asn Leu Lys Leu Lys Gly Leu 385 390 395 400 Val Glu Lys Asp Ile Ile Pro Thr Ala Cys Leu Lys Ile Met Ile Glu 405 410 415 Gln Arg Ala Glu Phe Arg Pro Gly Ala Gly Ile Pro Glu Ile Tyr Asp 420 425 430 Ala Ser Leu Phe Leu Glu Ser Lys Leu Pro Phe Leu Lys Arg Ile Ile 435 440 445 Trp Asn Trp Arg Lys Thr Leu Phe Val Trp Ile Ser Met Ser Leu Phe 450 455 460 Ile Met Glu Leu Leu Phe Ala Leu Val Phe Phe Arg Pro Leu Ile Ile 465 470 475 480 Pro Arg Thr Gly Gln Arg Thr Gln Gln Arg Asp Gly Thr His Ser Ile 485 490 495 Asn Asn Asn Leu Tyr Leu Asp Gly Gln Ala Gly Ser Arg 500 505

Claims

1. A method for obtaining a plant cell or algal cell with elevated lipid content, wherein the method comprises: genetically modifying a plant cell or algal cell to express an exogenous protein or polypeptide associated with lipid metabolism, thereby obtaining a genetically-modified plant cell or algal cell with elevated lipid content; wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity; and wherein the expression of the protein or polypeptide associated with lipid metabolism increases lipid content of the genetically-modified plant cell or algal cell as compared to a wild-type plant cell or algal cell of the same type.

2. A method according to claim 1, wherein the protein or polypeptide associated with lipid metabolism is selected from fat specific protein 27 (FSP27), PLIN1, PLIN2, SEIPIN, FIT1, FIT2, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT-1), phospholipid: diacylglycerol acyltransferase 1 (PDAT-1), cell death activator (Cidea), leafy cotyledon 2 (LEC2), and WRINKLED1 (WRIT) protein or polypeptide.

3. A method according to claim 1, wherein the protein or polypeptide associated with lipid metabolism is of animal origin.

4. A method according to claim 1, wherein the protein or polypeptide associated with lipid metabolism is a fat specific protein 27 (FSP27) protein or polypeptide.

5. A method according to claim 1, further comprising modifying the plant cell or algal cell to express a combination of exogenous proteins or polypeptides associated with lipid metabolism, wherein at least one exogenous protein or polypeptide associated with lipid metabolism is selected from fat specific protein 27 (FSP27), PLIN1, PLIN2, SEIPIN, FIT1, FIT2, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT-1), phospholipid: diacylglycerol acyltransferase 1 (PDAT-1), cell death activator (Cidea), LEC2, and WRINKLED1 (WRI1) protein or polypeptide.

6. A method according to claim 1, wherein the cell is a plant cell and the method further comprises regenerating the plant cell into a plant.

7. A method according to claim 1, wherein the genetic modification of the plant cell or algal cell comprises transforming the plant cell or algal cell with a vector comprising a nucleic acid sequence encoding an exogenous protein or polypeptide associated with lipid metabolism, wherein the nucleic acid is operably linked to a promoter and/or a regulatory sequence.

8. A method according to claim 1, wherein the exogenous protein or polypeptide associated with lipid metabolism is selected from Arabidopsis thaliana SEIPIN1, SEIPIN2, SEIPIN3, or leafy cotyledon 2 (LEC2).

9. A method according to claim 8, wherein lipid droplet size is enhanced as compared to lipid droplet size of a wild-type cell of the same type.

10. A transgenic plant cell or algal cell having elevated lipid content as compared to a wild-type plant cell or algal cell of the same type, wherein the transgenic plant cell or algal cell expresses an exogenous protein or polypeptide associated with lipid metabolism, wherein the protein or polypeptide associated with lipid metabolism induces adipogenesis, enhances the accumulation of cellular lipid droplets, and/or reduces lipase activity.

11. A transgenic plant cell or algal cell according to claim 10, wherein the protein or polypeptide associated with lipid metabolism is selected from fat specific protein 27 (FSP27), PLIN1, PLIN2, SEIPIN, FIT1, FIT2, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT-1), phospholipid:diacylglycerol acyltransferase 1 (PDAT-1), adipose triglyceride lipase (ATGL), cell death activator (Cidea), LEC2, and WRINKLED1 (WRI1) protein or polypeptide.

12. A transgenic plant cell or algal cell according to claim 11, wherein the protein associated with lipid metabolism is a fat specific protein 27 (FSP27) protein or polypeptide.

13. A transgenic plant cell according to claim 10, wherein the transcenic plant cell is in a plant or plant part.

14. A transgenic plant cell according to claim 13, which is a non-seed cell.

15. A transgenic plant cell according to claim 14, wherein the non-seed cell is a leaf, root, stem, shoot, bud, tuber, fruit, or flower cell.

16. A transgenic plant cell according to claim 13, wherein the cell is a seed cell of a plant.

17. A transgenic plant cell or algal cell according to claim 11, wherein the exogenous protein or polypeptide associated with lipid metabolism is selected from A. thaliana SEIPIN1, SEIPIN2, SEIPIN3, or LEC2.

18. A transgenic plant cell or algal cell according to claim 17, wherein lipid droplet size is enhanced as compared to lipid droplet size of a wild-type cell of the same type.

19. A method for screening for a functional protein or polypeptide associated with lipid metabolism for elevating lipid content and/or inducing lipid droplet accumulation in a plant or algal cell, wherein the method comprises: obtaining a test plant cell or algal cell genetically-modified to express a candidate exogenous protein or polypeptide associated with lipid metabolism; and growing the genetically-modified test cell and selecting the genetically-modified test cell having elevated lipid content and/or increased lipid droplet size or number, when compared to a wild-type cell of the same type.

20. A method according to claim 19, wherein the cell is a plant cell and the method further comprises regenerating the genetically-modified cell into a plant.

21. The method according to claim 20, further comprising obtaining progeny of said plant.