WO2013024121A2 - Augmentation de l'activité du transporteur de sucrose dans les graines de plantes - Google Patents

Augmentation de l'activité du transporteur de sucrose dans les graines de plantes Download PDF

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WO2013024121A2
WO2013024121A2 PCT/EP2012/065958 EP2012065958W WO2013024121A2 WO 2013024121 A2 WO2013024121 A2 WO 2013024121A2 EP 2012065958 W EP2012065958 W EP 2012065958W WO 2013024121 A2 WO2013024121 A2 WO 2013024121A2
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plant
protein
sequence
seed
suc
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WO2013024121A3 (fr
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Norbert Sauer
Benjamin Peter Ulrich POMMERRENIG
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Basf Plant Science Company Gmbh
Friedrich-Alexander-Universität Erlangen-Nürnberg
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • Described herein are inventions in the field of genetic engineering of plants, including isolated nucleic acid molecules encoding a sucrose transporter protein to improve agronomic, horticultural and quality traits. These inventions relate generally to nucleic acid sequences encoding proteins that are related to the presence of seed storage compounds in plants. More specifically, the present invention relates to a sucrose transporter 5 nucleic acid sequence SEQ ID NO: 1 and the use of this sequence in transgenic plants. In particular, the invention is directed to methods for manipulating the content of sugar-related compounds, for increasing the oil level and/or altering the fatty acid composition in plants and seeds.
  • Plant seed oils comprise both neutral and polar lipids (see Table 1).
  • the neutral lipids consist primarily of triacylglycerol, which is the main storage lipid that accumulates in oil bodies in seeds.
  • the polar lipids are mainly found in the various membranes of the seed cells, e.g. microsomal membranes, the cell membrane and the mitochondrial and plastidial membranes.
  • the neutral and polar lipids contain several common fatty acids (see Table 2) and a range of less common fatty acids.
  • the fatty acid composition of membrane lipids is highly regulated and only a select number of fatty acids are found in membrane lipids.
  • Lipids are synthesized from fatty acids and their synthesis may be divided into two parts: the prokaryotic pathway and the eukaryotic pathway (Browse et al. 1986, Biochemical J. 235:25-31 ; Ohlrogge & Browse 1995, Plant Cell 7:957-970).
  • the prokaryotic pathway is located in plastids that are the primary site of fatty acid biosynthesis.
  • Fatty acid synthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (AC- Case).
  • Malonyl-CoA is converted to malonyl-acyl carrier protein (ACP) by the malonyl- CoA:ACP transacylase.
  • ACP malonyl-acyl carrier protein
  • the enzyme beta-keto-acyl-ACP-synthase III catalyzes a condensation reaction, in which the acyl group from acetyl-CoA is transferred to malonyl- ACP to form 3-ketobutyryl-ACP.
  • the nascent fatty acid chain on the ACP cofactor is elongated by the step-by-step addition (condensation) of two carbon atoms donated by malonyl-ACP until a 16- or 18-carbon saturated fatty acid chain is formed.
  • the plastidial delta-9 acyl-ACP de- saturase introduces the first unsaturated double bond into the fatty acid.
  • Thioesterases cleave the fatty acids from the ACP cofactor and free fatty acids are exported to the cytoplasm where they participate as fatty acyl-CoA esters in the eukaryotic pathway. In this pathway the fatty acids are esterified by glycerol-3-phosphate acyltransferase and lyso- phosphatidic acid acyl-transferase to the sn-1 and sn-2 positions of glycerol-3-phosphate, respectively, to yield phosphatidic acid (PA).
  • PA phosphatidic acid
  • the PA is the precursor for other polar and neutral lipids, the latter being formed in the Kennedy pathway (Voelker 1996, Genetic Engineering ed.: Setlow 18:11 1 -1 13; Shanklin & Cahoon 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:61 1 -641 ; Frentzen 1998, Lipids 100:161 -166; Millar et al. 2000, Trends Plant Sci. 5:95-101 ).
  • lipids in seeds are synthesized from carbohydrate-derived precursors. Plants have a complete glycolytic pathway in the cytosol (Plaxton 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185-214) and it has been shown that a complete pathway also exists in the plastids of rapeseeds (Kang & Rawsthorne 1994, Plant J. 6:795-805). Sucrose is the pri- mary source of carbon and energy, transported from the leaves into the developing seeds. During the storage phase of seeds, sucrose is converted in the cytosol to provide the metabolic precursors glucose-6-phosphate and pyruvate.
  • Acetyl-CoA in the plastids is the central precursor for lipid biosynthesis.
  • Acetyl-CoA can be formed in the plastids by different reactions and the exact contribution of each reaction is still being debated (Ohlrogge & Browse 1995, Plant Cell 7:957-970). It is however accepted that a large part of the acetyl-CoA is derived from glucose-6-phospate, phos- phoenolpyruvate and pyruvate that are imported from the cytoplasm into the plastids.
  • sucrose is produced in the source organs (leaves, or anywhere that photosynthesis occurs) and is transported to the developing seeds that are also termed sink organs.
  • sucrose is the precursor for all the storage compounds, i.e. starch or lipids. Therefore, it is clear that carbohydrate metabolism, in which sucrose plays a central role is very important to the accumulation of seed storage compounds.
  • lipid and fatty acid content and/or composition of seed oil can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the seed oil content of a plant, and in some cases, has allowed for the alteration of seed oils in ways that could not be accomplished by breeding alone (see, e.g., Topfer et al., 1995, Science 268:681 -686).
  • introduction of a A 12 -hydroxylase nucleic acid sequence into transgenic tobacco resulted in the introduction of a novel fatty acid, ricinoleic acid, into the tobacco seed oil (Van de Loo et al. 1995, Proc. Natl. Acad. Sci USA 92:6743-6747).
  • Tobacco plants have also been engineered to produce low levels of petroselinic acid by the introduction and expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992, Proc. Natl. Acad. Sci USA 89:1 1 184-1 1188).
  • the modification of seed oil content in plants has significant medical, nutritional and economic ramifications.
  • the long chain fatty acids (C18 and longer) found in many seed oils have been linked to reductions in hypercholesterolemia and other clinical disorders related to coronary heart disease (Brenner 1976, Adv. Exp. Med. Biol. 83:85-101 ). Therefore, consumption of a plant having increased levels of these types of fatty acids may reduce the risk of heart disease.
  • Enhanced levels of seed oil content also increase large-scale production of seed oils and thereby reduce the cost of these oils.
  • nucleic acid sequences and proteins regulating lipid and fatty acid metabolism must be identified.
  • desaturase nucleic acids such as the A 6 -desaturase nucleic acid, A 12 -desaturase nucleic acid and acyl-ACP desaturase nucleic acid have been cloned and demonstrated to encode enzymes required for fatty acid synthesis in various plant species.
  • Oleosin nucleic acid sequences from such different species as canola, soybean, car- rot, pine and Arabidopsis thaliana have also been cloned and determined to encode proteins associated with the phospholipid monolayer membrane of oil bodies in those plants.
  • the plant hormones eth- ylene e.g. Zhou et al., 1998, Proc. Natl. Acad. Sci. USA 95:10294-10299; Beaudoin et al., 2000, Plant Cell 2000:1 103-1 115
  • auxin e.g. Colon-Carmona et al., 2000, Plant Physiol. 124:1728-1738
  • nucleic acid sequences can be used to alter or increase the level of oil in plants, including transgenic plants, such as canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor, peanut, cranmbe and Jatropha, which are oilseed plants containing high amounts of lipid compounds.
  • transgenic plants such as canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor, peanut, cranmbe and Jatropha, which are oilseed plants containing high amounts of lipid compounds.
  • the Arabidopsis sucrose transporter 5 (SUC5 protein - SEQ ID NO: 2) represents a su- crose/H + symporter. Its gene was previously shown to be expressed in the endosperm.
  • SUC5 protein is also essential for the delivery of biotin to the endosperm and the embryo in developing seeds of biotin biosynthesis-defective Arabidopsis mutants (biol and bio2). Embryo development, seed germination, seedling development, triacylglycerol (TAG) accumulation, and fatty acid composition were compared in single mutant (suc5, biol or bio2), double mutant (suc5/bio1 and suc5/bio2) and wild type seeds.
  • TAG triacylglycerol
  • suc5 mutants were like wild type plants, biol and bio2 mutants showed multiple developmental defects and had reduced TAG contents and altered fatty acid compositions in their dry seeds. These phenotypes were severely enhanced in suc5/bio1 and suc5/bio2 double mutants. Externally supplied biotin suppressed the phenotypes of biol and bio2 single and suc5/bio1 and suc5/bio2 double mutants, but higher biotin concentration were needed for double than for single mutants. Results of genetic and metabolic anal- yses demonstrate that the SUC5 protein acts as biotin transporter in plants.
  • Example 15 and 16 it was shown that the over-expression of the SUC 5 gene in plants alone or in combination with the GPT1 (SEQ ID NO: 83) nucleic acid sequence and the NTT1 (SEQ ID NO: 84) nucleic acid sequence results in an increased fatty acid content in plant seeds.
  • Biotin vitamin B 7 or vitamin H
  • Bacteria, plants, some fungi and few animals are capable of synthesizing the biotin needed for these reactions.
  • VHT1 gene (Avht1) was complemented with an Arabidopsis cDNA library and screened for growth on low extracellular biotin concentrations (Ludwig et al., 2000). Surprisingly, this screening identified a sequence with high similarity to sucrose transporter cDNAs from different plant species [e.g. AtSUCI and AtSUC2 from Arabidopsis or SoSUTI from spinach (Spinacea oleracea; Sauer, 2007)]. Functional analyses of the encoded protein demonstrated that it was, in fact, a Arabidopsis sucrose transporter (named SUC5; At1 g71890) with transport characteristics similar to those of previously published sucrose transporters (Ludwig et ai, 2000).
  • bio1. 1 Arabidopsis plants with defects in biotin biosynthesis were first identified in analyses of em- bryo-lethal mutants.
  • One mutant, bio1. 1 (At5g57590), was shown to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid (Schneider et ai, 1989; Muralla et ai, 2008), the other, bio2. 1 (At2g43360), in the conversion of dethiobiotin to biotin (Baldet and Ruffet, 1996; Patton et ai, 1996; Weaver et ai, 1996; Patton et ai, 1998). The developmental arrest observed in homozygous (bio1.
  • Two different pSL/C5/reporter lines under the control of a 2030-bp SUC5 promoter were generated. These lines expressed the open reading frames (ORFs) of the soluble and freely mobile green fluorescent protein (sGFP) or of a non-mobile version of GFP (tmGFP9) that is membrane-attached by N-terminal transmembrane helices (Stadler et al., 2005a). After BASTA-selection of T1 seedlings, we obtained numerous transformed T1 plants for both constructs.
  • ORFs open reading frames
  • sGFP soluble and freely mobile green fluorescent protein
  • tmGFP9 non-mobile version of GFP
  • bio2.1 seeds showed a biotin-dependent phenotype.
  • the bio2.1 seeds were yellowish and pale.
  • increasing concentrations (0.1 mM or 1 mM biotin) of supplemented biotin this phenotype disappeared gradually.
  • a significantly stronger phenotype was observed in developing seeds of bio2.1/suc5.5 double mutants that were not supplemented with biotin (0 mM biotin in Figure 3a). Seeds from these plants were white and smaller than seeds of bio2. 1 single mutants, which indicated a stronger biotin-deficiency.
  • increasing concentrations of supplemented biotin gradually reduced this phenotype.
  • biotin limitation should not only affect seed and seedling morphology, but also reduce the capacity to synthesize TAG. If SUC5 acts as biotin trans- porter, an additional suc5 mutation should further reduce these TAG levels. In fact, when the total TAG content in seeds of wt plants, single and double mutants were analysed, we observed an 80% reduction of the TAG content in seeds from bio2.1 single mutants that were not supplemented with biotin (0 mM biotin), and this reduction was even more pronounced (almost 95%) in seeds from non-supplemented bio2.1/suc5.5 double mutants (Figure 7a).
  • the examples presented address the question whether or not the Arabidopsis SUC5 protein does act as biotin transporter in planta.
  • SUC5 is expressed in the epidermis of torpedo-stage or older embryos ( Figure 2e to 2i) demonstrating that SUC5 is not only involved in the transport of its substrate(s) from the maternal tissue into the endosperm but also in the transport from the endosperm into the embryo.
  • the results demonstrate that SUC5 is important for the transport of biotin across these boundaries and provide the first direct evidence that biotin transport by SUC proteins is physiologically relevant in planta.
  • Example 1 comparative analyses of two newly characterized suc5 mutants (suc5.4 and suc5.5), of two previously characterized biotin biosynthetic mutants (bio1.1 and bio2.1; Schneider et al., 1989; Patton et al., 1998), and of double mutants (bio1/suc5 or bio2/suc5) resulting from crosses of transport-defective and biosynthesis-defective lines are presented.
  • suc5 sin- gle mutants showed no phenotypic alterations under the conditions analysed, bio1.1 and bio2.
  • SUC5 is responsible for biotin transport in planta
  • Acetyl-CoA carboxylase ACCase
  • ACCase a biotin enzyme that catalyses the first and rate-limiting step in fatty acid biosynthesis
  • Example 1 underlines the importance of sufficiently high biotin concentrations for optimal TAG formation in Arabidopsis embryos.
  • biotin-depleted seeds of bio2.1/suc5.5 ( Figure 4) look empty, have a wrinkled appearance, and resemble seeds of the low-seed- oil mutant wrinkledl (writ, Focks and Benning, 1998).
  • Example 1 suggest that fatty acid biosynthesis and ACCase activity are strongly affected by changes in the availability of biotin. Moreover, the data suggest that the concentration of biotin is adjusted to the specific needs of an organ under different developmental conditions. Besides biotin biosynthesis, biotin supply from adjacent tissues is an alternative mechanism to adjust cellular biotin concentrations. The results presented in Example 1 demonstrate that SUC5 is responsible for the supply of biotin to the endosperm and the embryo under conditions of biotin limitation.
  • the present invention relates to a polynucleotide comprising a nucleic acid sequences selected from the group consisting of:
  • nucleic acid sequence which is at least 70% identical to the nucleic acid sequence of (a) or (b), wherein said nucleic acid sequence encodes a polypeptide or biologically active portion thereof having sucrose transporter 5 activity ;
  • nucleic acid sequence being a fragment of any one of (a) to (c), wherein said frag- ment encodes a polypeptide or biologically active portion thereof having sucrose transporter 5 activity.
  • polynucleotide as used in accordance with the present invention relates to a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide being a biotin and sucrose transporter, i.e. a polypeptide capable of transporting biotin and sucrose through a membrane.
  • sucrose transporter 5 polypeptides encoded by the polynucleo- tides of the present invention shall be capable of increasing the amount of seed storage compounds, preferably, fatty acids or lipids, when present in plant seeds.
  • the polypeptides encoded by the polynucleotide of the present invention are also referred to as SUC 5 protein herein below. Suitable assays for measuring the activities mentioned before are described in the accompanying Examples.
  • the polynucleotide of the present invention upon expression in the seed of a transgenic plant is capable of significantly increasing the amount by weight of at least one seed storage compound. More preferably, such an increase as referred to in accordance with the present invention is an increase of the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control. Whether an increase is significant can be determined by statistical tests well known in the art including, e.g., Student ' s t-test. The percent increase rates of a seed storage compound are, preferably, determined compared to an empty vector control.
  • An empty vector control is a transgenic plant, which has been transformed with the same vector or construct as a transgenic plant according to the present invention except for such a vector or construct is lacking the polynucleotide of the present invention.
  • an untreated plant i.e. a plant which has not been genetically manipulated
  • a wildtype regenerate from the in vitro culture may be used as a control.
  • a polynucleotide encoding a polypeptide having a biological activity as specified above has been obtained in accordance with the present invention from Arabidopsis thaliana.
  • the corresponding polynucleotides preferably, comprises the nucleic acid sequence shown in SEQ ID NO: 1 encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. It is to be understood that a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 may be also encoded due to the degenerated genetic code by other polynucleotides as well.
  • polynucleotide as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide of the present invention.
  • polynucleotide variants preferably, also comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid se- quences shown in SEQ ID NO: 1 by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a biological activity as specified above.
  • Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecu- lar Biology, John Wiley & Sons, N. Y.
  • SSC sodium chloride/sodium citrate
  • 0.1 % SDS 0.1 % SDS at 50 to 65°C.
  • the skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 ⁇ SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C.
  • DNA:DNA hybrids are, preferably, 0.1 ⁇ SSC and 20°C to 45°C, preferably between 30°C and 45°C.
  • the hybridization conditions for DNA:RNA hybrids are, preferably, 0.1 ⁇ SSC and 30°C to 55°C, preferably between 45°C and 55°C.
  • conserved domains of the polypeptide of the present invention may be identified by a sequence comparison of the nucleic acid sequences of the polynucleotides or the ami- no acid sequences of the polypeptides of the present invention. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used.
  • variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequences shown in SEQ ID NO: 1 retaining a biological activity as specified above.
  • the percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region.
  • a series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results.
  • the program PileUp J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153
  • Gap and BestFit Needle- man and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math.
  • the percent sequence identity between two nucleic acid or polypeptide sequences can be also determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814).
  • PC Vector NTI 7.0
  • a gap- opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids.
  • a gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings.
  • the gap-opening penalty is 10
  • the gap extension penalty is 0.05 with blosum62 matrix.
  • a thymidine nucleotide sequence is equivalent to an uracil nucleotide.
  • a polynucleotide comprising a fragment of any of the aforementioned nucleic acid sequences is also encompassed as a polynucleotide of the present invention.
  • the fragment shall encode a polypeptide which still has a biological activity as specified above. Accordingly, the polypeptide may comprise or consist of the domains of the polypeptide of the present invention conferring the said biological activity.
  • a fragment as meant herein preferably, comprises at least 20, at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of any one of the aforementioned nucleic acid sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the aforementioned amino acid sequences.
  • the variant polynucleotides or fragments referred to above preferably, encode polypeptides retaining at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the sucrose transporter 5 activity exhibited by the polypeptide shown in SEQ ID NO: 2.
  • the activity may be tested as described in the accompanying Examples.
  • the polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well.
  • the polynucleotide of the present invention may comprise in addition to an open reading frame further un- translated sequence at the 3' and at the 5' terminus of the coding gene region: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5' terminus of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' terminus of the coding gene region.
  • the polynucleotides of the present invention may encode fusion proteins wherein one partner of the fusion protein is a polypeptide being encoded by a nucleic acid sequence recited above.
  • Such fusion proteins may comprise as additional part other enzymes of the fatty acid or lipid biosynthesis pathways, polypeptides for monitoring expression (e.g., green, yellow, blue or red fluorescent proteins, alkaline phosphatase and the like) or so called "tags" which may serve as a detectable marker or as an auxiliary measure for purification purposes.
  • tags for the different purposes are well known in the art and comprise FLAG-tags, 6-histidine-tags, MYC-tags and the like.
  • Variant polynucleotides as referred to in accordance with the present invention may be ob-tained by various natural as well as artificial sources.
  • polynucleotides may be obtained by in vitro and in vivo mutagenesis approaches using the above mentioned mentioned specific polynucleotides as a basis.
  • polynucleotids being homologs or orthologs may be obtained from various animal, plant, bacteria or fungus species. Paralogs may be identified from Arabidopsis thaliana.
  • the polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context such as a gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form.
  • An isolated polynucleotide can, for example, comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.
  • the polynucleotide preferably, is double or single stranded DNA including cDNA or RNA.
  • the term encompasses single- as well as double-stranded polynucleotides. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides.
  • the polynucleotide encoding a polypeptide having a biological activity as specified encompassed by the present invention is also, preferably, a polynucleotide having a nucleic acid sequence which has been adapted to the specific codon- usage of the organism, e.g., the plant species, in which the polynucleotide shall be expressed (i.e. the target organism).
  • a polynucleotide having a nucleic acid sequence which has been adapted to the specific codon- usage of the organism, e.g., the plant species, in which the polynucleotide shall be expressed (i.e. the target organism).
  • This is, in general, achieved by changing the codons of a nucleic acid sequence obtained from a first organism (i.e. the donor organism) encoding a given amino acid sequence into the codons normally used by the target organism whereby the amino acid sequence is retained. It is in principle acknowleged that the genetic code is redundant (i.e
  • 61 codons are used to encode only 20 amino acids. Thus, a majority of the 20 amino acids will be encoded by more than one codon.
  • the codons for the amino acids are well known in the art and are universal to all organisms. However, among the different codons which may be used to encode a given amino acid, each organism may preferably use certain codons. The presence of rarely used codons in a nucleic acid sequence will result a depletion of the respective tRNA pools and, thereby, lower the translation efficiency.
  • a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as referred to above wherein said nucleic acid sequence is optimized for expression in the target organism with respect to the codon usage.
  • a plurality of known genes from the said organism may be investigated for the most commonly used codons encoding the amino acids.
  • the codons of a nuclei acid sequence from the donor organism will be optimized by replacing the codons in the donor sequence by the codons most commonly used by the target organism for encoding the same amino acids. It is to be understood that if the same codon is used preferably by both organisms, no replacement will be necessary.
  • the polynucleotide of the present invention has an optimized nucleic acid for codon usage in the envisaged target organism wherein at least 20%, at least 40%, at least 60%, at least 80% or all of the relevant codons are adapted.
  • the polypeptides being encoded by the polynucleotides of the present invention is a sucrose transporter 5 polypeptide involved in the regulation of seed storage compounds.
  • the polypeptides encoded by the polynucleotides of the present invention are, advantageously, capable of in- creasing the amount of seed storage compounds in plants significantly.
  • the polynucleotides of the present invention are, in principle, useful for the enrichment and synthesis of seed storage compounds such as fatty acids or lipids.
  • they may be used to generate transgenic plants or seeds thereof having a modified, preferably increased, amount of seed storage compounds. Such transgenic plants or seeds may be used for the manufacture of seed oil or other lipid and/or fatty acid containing compositions.
  • the present invention relates to vector comprising the polynucleotide of the present invention.
  • the vector is an expression vector.
  • vector preferably, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous recombination or heterologous insertion as described in detail below.
  • the vector encompassing the polynucleotides of the pre- sent invention preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art.
  • the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion, see below.
  • Vectors can be introduced into prokaryotic or eukary- otic cells via conventional transformation or transfection techniques.
  • An "expression vector" according to the present invention is characterized in that it comprises an expression control sequence such as promoter and/or enhancer sequence operatively linked to the polynucleotide of the present invention. Preferred vectors, expression vectors and transfor- mation or transfection techniques are specified elsewhere in this specification in detail.
  • the present invention encompasses a host cell comprising the polynucleotide or vector of the present invention.
  • Host cells are primary cells or cell lines derived from multicellular organisms such as plants or animals.
  • host cells encompass prokaryotic or eukaryotic single cell organisms (also referred to as microorganisms), e.g. bacteria or fungi including yeast or bacteria.
  • Primary cells or cell lines to be used as host cells in accordance with the present invention may be derived from the multicellular organisms, preferably from plants. Specifically pre- ferred host cells, microorganisms or multicellular organism from which host cells may be obtained are disclosed below.
  • the polynucleotides or vectors of the present invention may be incorporated into a host cell or a cell of a transgenic non-human organism by heterologous insertion or homologous re- combination.
  • Heterologous refers to a polynucleotide which is inserted (e.g., by ligation) or is manipulated to become inserted to a nucleic acid sequence context which does not naturally encompass the said polynucleotide, e.g., an artificial nucleic acid sequence in a genome of an organism.
  • a heterologous polynucleotide is not endogenous to the cell into which it is introduced, but has been obtained from another cell.
  • heterologous polynucle- otides encode proteins that are normally not produced by the cell expressing the said heterologous polynucleotide.
  • An expression control sequence as used in a targeting construct or expression vector is considered to be "heterologous” in relation to another sequence (e.g., encoding a marker sequence or an agronomically relevant trait) if said two sequences are either not combined or operatively linked in a different way in their natural environment.
  • said sequences are not operatively linked in their natural environment (i.e. originate from different genes).
  • said regulatory sequence is covalently joined (i.e. ligated) and adjacent to a nucleic acid to which it is not adjacent in its natural environment.
  • “Homologous” as used in accordance with the present invention relates to the insertion of a polynucleotide in the sequence context in which the said polynucleotide naturally occurs.
  • a heterologous polynucleotide is also incorporated into a cell by homologous recombination.
  • the heterologous polynucleotide is flanked by nucleic acid sequences being homologous to a target sequence in the genome of a host cell or a non- human organism. Homologous recombination now occurs between the homologous sequences.
  • Also provided in accordance with the present invention is a method for the manufacture of a polypeptide having sucrose transporter 5 activity comprising:
  • the polypeptide may be obtained, for example, by all conventional purification techniques including affinity chromatography, size exclusion chromatography, high pressure liquid chromatography (HPLC) and precipitation techniques including antibody precipitation. It is to be understood that the method may - although preferred -not necessarily yield an essentially pure preparation of the polypeptide. It is to be understood that depending on the host cell which is used for the aforementioned method, the polypeptides produced thereby may become posttranslationally modified or processed otherwise.
  • HPLC high pressure liquid chromatography
  • the present invention pertains to a polypeptide encoded by the polynucleotide of the present invention or which is obtainable by the aforementioned method of the present invention.
  • polypeptide as used herein encompasses essentially purified polypeptides or polypeptide preparations comprising other proteins in addition. Further, the term also relates to the fusion proteins or polypeptide fragments being at least partially encoded by the polynucleotide of the present invention referred to above. Moreover, it includes chemically modified polypeptides. Such modifications may be artificial modifications or naturally occurring modifications such as phosphorylation, glycosylation, myristylation and the like.
  • polypeptide amino acid peptide
  • peptide or “protein” are used interchangeable throughout this specification.
  • the polypeptide of the present invention shall exhibit the biological activities referred to above, i.e. it should be a sucrose transporter 5 and, more preferably, it shall be capable of increasing the amount of seed storage compounds, preferably, fatty acids or lipids, when present in plant seeds as referred to above. Most preferably, if present in plant seeds, the polypeptide shall be capable of significantly increasing the seed storage of lipids.
  • Encompassed by the present invention is, furthermore, an antibody which specifically rec- ognizes the polypeptide of the invention.
  • Antibodies against the polypeptides of the invention can be prepared by well known methods using a purified polypeptide according to the invention or a suitable fragment derived therefrom as an antigen.
  • a fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either from the polypeptide of the invention by proteolytic digestion or may be a synthetic peptide.
  • the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody or primatized, chimerized or fragment thereof.
  • antibodies by the present invention are: a bispecific antibody, a synthetic antibody, an antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these.
  • the antibody of the present invention shall specifically bind (i.e. does significantly not cross react with other polypeptides or peptides) to the polypeptide of the invention. Specific binding can be tested by various well known techniques. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques originally described in Kohler and Milstein, Nature 256 (1975) 495, and Galfre, Meth.
  • Enzymol. 73 (1981 ) 3 which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • the antibodies can be used, for example, for the im- munoprecipitation, immunolocalization or purification (e.g., by affinity chromatography) of the polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention.
  • the present invention also relates to a transgenic non-human organism comprising the polynucleotide, the vector or the host cell of the present invention.
  • said non-human transgenic organism is a plant.
  • non-human transgenic organism preferably, relates to a plant, an animal or a multicellular microorganism.
  • the polynucleotide or vector may be present in the cytoplasm of the organism or may be incorporated into the genome either heterologous or by homologous recombination.
  • Host cells in particular those obtained from plants or animals, may be introduced into a developing embryo in order to obtain mosaic or chimeric organisms, i.e. non-human transgenic organisms comprising the host cells of the present invention.
  • the non-human transgenic organism expresses the polynucleotide of the present invention in order to produce the polypeptide in an amount resulting in a detectable sucrose transporter 5 activity due to the presence of the said polypeptide.
  • Suitable transgenic organisms are, preferably, all those organisms which are capable of synthesizing fatty acids or lipids. Preferred organisms and methods for transgenesis are disclosed in detail below.
  • a transgenic organism or tissue may comprise one or more transgenic cells.
  • the organism or tissue is substantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99% of the cells in said organism or tissue are transgenic).
  • transgene refers to any nucleic acid se- quence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations including techniques such as chimera- or genoplasty.
  • said sequence is resulting in a genome which is significantly different from the overall genome of an organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or de- creased).
  • a transgene may comprise an endogenous polynucleotide (i.e.
  • a polynucleotide having a nucleic acid sequence obtained from the same organism or host cell may be obtained from a different organism or hast cell, wherein said different organism is, preferably an organism of another species and the said different host cell is, preferably, a different microorganism, a host cell of a different origin or derived from a an organism of a different species.
  • a plant to be used in accordance with the present invention are oil producing plant species.
  • the said plant is selected from the group consisting of canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, ta- getes, cotton, oil palm, coconut palm, flax, castor and peanut,
  • the present invention relates to a method for the manufacture of a lipid and/or a fatty acid comprising the steps of:
  • lipid and "fatty acid” as used herein refer, preferably, to those recited in Table 1 (for lipids) and Table 2 (for fatty acids), below. However, the terms, in principle, also encompass other lipids or fatty acids which can be obtained by the lipid metabolism in a host cell or an organism referred to in accordance with the present invention.
  • the said lipid and/or fatty acids constitute seed oil.
  • the present invention pertains to a method for the manufacture of a plant having a modified amount of a seed storage compound, preferably a lipid or a fatty acid, comprising the steps of:
  • seed storage compound refers to compounds being a sugar or, more preferably, a lipid or a fatty acid.
  • the amount of said seed storage compound is significantly increased compared to a control, preferably an empty vector control as specified above.
  • the increase is, more preferably, an increase in the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control.
  • polynucleotides or the vector referred to in accordance with the above method of the present invention may be introduced into the plant cell by any of the aforementioned insertion or recombination techniques.
  • the aforementioned method of the present invention may be also used to manufacture a plant having altered total oil content in its seeds or a plant having altered total seed oil content and/or altered levels of seed storage compounds in its seeds.
  • Such plants are suitable sources for seed oil and may be used for the large scale manufacture thereof.
  • the present invention provides novel isolated nucleic acid and amino acid sequences, i.e., the polynucleotides and polypeptides of the present invention, associated with the metabolism of seed storage compounds in plants.
  • a polynucleotide comprising a nucleic acid from Arabidopsis thaliana encoding the sucrose transporter s polypeptide of the present invention, i.e. a Lipid Metabolism Protein (SUC 5 protein), or a portion thereof.
  • SUC 5 protein Lipid Metabolism Protein
  • nucleic acid molecules originating from a plant like Brassica napus or related organisms including Arabidopsis are especially suited to modify the lipid and fatty acid metabolism in a host such as the host cells or transgenic non-human organisms of the present invention, especially in microorganisms and plants.
  • nucleic acids from the plant Arabidopsis thaliana or related organisms can be used to identify those DNA sequences and enzymes in other species, which are useful to modify the biosynthesis of precursor molecules of fatty acids in the respective organisms.
  • the present invention further provides an isolated nucleic acid comprising a fragment of at least 15 nucleotides of a polynucleotide of the present invention, preferably, a polynucleotide comprising a nucleic acid from a plant encoding the polypeptides of the present invention.
  • the present invention also encompasses an oligonucleotide which specifically binds to the polynucleotides of the present invention. Binding as meant in this context refers to hybridization by Watson-Crick base pairing discussed elsewhere in the specification in detail.
  • An oligonucleotide as used herein has a length of at most 100, at most 50, at most 40, at most 30 or at most 20 nucleotides in length which are complementary to the nucleic acid sequence of the polynucleotides of the present invention.
  • the sequence of the oligonucleotide is, preferably, selected so that a perfect match by Watson-Crick base pairing will be obtained.
  • the oligonucleotides of the present invention may be suitable as primers for PCR- based amplification techniques.
  • polypeptides encoded by the nucleic acids and heterologous polypeptides comprising polypeptides encoded by the nucleic acids, and antibodies to those polypeptides.
  • the present invention relates to and provides the use of the polynucleotides of the present invention in the production of transgenic plants having a modified level or com- position of a seed storage compound.
  • the present invention can be used to, for example, increase the percentage of oleic acid relative to other plant oils.
  • a method of producing a transgenic plant with a modified level or composition of a seed storage compound includes the steps of transforming a plant cell with an expression vector comprising a polynucleotide of the present invention, and generating a plant with a modified level or composition of the seed storage compound from the plant cell.
  • the plant is an oil producing species selected from the group consisting of canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut, for example.
  • compositions and methods described herein can be used to alter the composition of a SUC 5 protein in a transgenic plant and to increase or decrease the level of a SUC 5 protein in a transgenic plant comprising increasing or decreasing the expression of a SUC protein nucleic acid in the plant.
  • Increased or decreased expression of the SUC 5 protein nucleic acid can be achieved through transgenic overex- pression, co-suppression approaches, antisense approaches, and in vivo mutagenesis of the SUC 5 protein nucleic acid or micro-RNA based techniques.
  • the present invention can also be used to increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in seed oil.
  • the present invention includes and provides a method for altering (increasing or decreasing or changing the specific profile) of the total oil content in a seeds comprising: Transforming a plant with a nucleic acid construct that comprises as operably linked components, a promoter and nucleic acid sequences capable of modulating the level of the polynucleotides or polypeptides of the present invention, and growing the plant.
  • the present invention includes and provides a method for altering (increasing or decreasing) the level of eicosenoic acid in a seed comprising: transforming a plant with a nucleic acid construct that comprises as operably linked components, a promoter, a struc- tual nucleic acid sequence capable of altering (increasing or decreasing) the level of ei- cosenoic acid, and growing the plant
  • a seed produced by a transgenic plant transformed by a polynucleotide of the present invention wherein the seed contains the said polynucleotide and wherein the plant is true breeding for a modified level of a seed storage compound.
  • the present invention additionally includes seed oil produced by the aforementioned seed.
  • vectors comprising a polynucleotide of the present invention, host cells containing the vectors, and descendent plant materials produced by transforming a plant cell with the nucleic acids and/or vectors.
  • the compounds, compositions, and methods described herein can be used to increase or decrease the relative percentages of a lipid in a seed oil, increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch or other carbohy- drate in a seed or plant.
  • the manipulations described herein can also be used to improve seed germination and growth of the young seedlings and plants and to enhance plant yield of seed storage compounds.
  • transgenic plant expressing a polynucleotide of the present invention from Arabisopsis thalaiana in the transgenic plant
  • the transgenic plant is Ara- bidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris or a species different from Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris.
  • compositions and methods of the modification of the efficiency of production of a seed storage compound are also included herein.
  • Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris this also means Arabidopsis thaliana and/or Brassica napus and/or Glycine max and/or Oryza sativa and/or Triticum aestivum and/or Zea mays and/or Helianthus anuus and/or Beta vulgaris.
  • polynucleotides encoding a SUC 5 protein as well as the corresponding polypeptide from Arabidopsis thaliana as well as active fragments, analogs, and orthologs thereof.
  • active fragments, analogs, and orthologs can also be from different plant species as one skilled in the art will appreciate that other plant species will also contain those or related nucleic acids.
  • polynucleotides and polypeptides of the present invention have also uses that include modulating plant growth, and potentially plant yield, preferably increasing plant growth under adverse conditions (drought, cold, light, UV).
  • antagonists of the present invention may have uses that include modulating plant growth and/or yield, through preferably increasing plant growth and yield.
  • over-expression polypeptides of the present invention using a constitutive promoter may be useful for increasing plant yield under stress conditions (drought, light, cold, UV) by modulating light utilization efficiency.
  • polynucleotides and pol- ypeptides of the present invention will improve seed germination and seed dormancy and, hence, will improve plant growth and/or yield of seed storage compounds.
  • the polynucleotides of the present invention may further comprise an operably linked promoter or partial promoter region.
  • the promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
  • the constitutive promoter can be, for example, the superpromoter (Ni et al., Plant J. 7:661 -676, 1995; US5955646) or the PtxA promoter (WO 05/085450, Song H. et al. ).
  • the tissue-specific promoter can be active in vegetative tissue or reproductive tissue.
  • the tissue-specific promoter active in reproductive tissue can be a seed-specific promoter.
  • the tissue-specific promoter active in vegetative tissue can be a root-specific, shoot-specific, meristem-specific, or leaf-specific promoter.
  • the polynucleotides of the present invention can still further comprise a 5' non-translated sequence, 3' non-translated sequence, introns, or the combination thereof.
  • the present invention also provides a method for altering (increasing or decreasing) the number and/or size of one or more plant organs of a plant expressing a polynucleotide of the present invention, preferably, from Arabidopsis thaliana encoding a polypeptide of the present invention. More specifically, seed size and/or seed number and/or weight might be manipulated. Moreover, root length can be increased. Longer roots can alleviate not only the effects of water depletion from soil but also improve plant anchorage/standability, thus reducing lodging. Also, longer roots have the ability to cover a larger volume of soil and improve nutrient uptake. All of these advantages of altered root architecture have the po- tential to increase crop yield.
  • the number and size of leaves might be increased by the nucleic acid sequences provided in this application. This will have the advantage of improving photosynthetic light utilization efficiency by increasing photosynthetic light-capture capacity and photosynthetic efficiency. It is a further object of the present invention to provide methods for producing such aforementioned transgenic plants.
  • the present invention is based, in part, on the isolation and characterization of nucleic acid molecules a sucrose 5 transporter from plants including Arabidopsis, canola (Brassica napus or Brassica oleracea) and other related crop species like maize, barley, linseed, sugar beet, or sunflower.
  • this invention in one aspect, provides an isolated nucleic acid from a plant (Arabidopsis thaliana) encoding a SUC 5 protein, or a portion thereof.
  • a plant Arabidopsis thaliana
  • One aspect of the invention pertains to an isolated nucleic acid molecule that encodes a SUC 5 polypeptide or a biologically active portion thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of a SUC 5 protein-encoding nucleic acid (e.g., SUC 5 protein DNA).
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of a gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded
  • an "isolated" nucleic acid molecule is one which is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is substantially free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism, from which the nucleic acid is derived.
  • the isolated SUC 5 protein nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a Brassica napus cell).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having a nucleotide sequence of the polynucleotide of the present invention, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a Arabidopsis thaliana or Brassica napus SUC 5 protein cDNA can be isolated from an a Arabidopsis thaliana or Brassica napus library using all or portion of one of the sequences of the polynucleotide of the present invention as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual.
  • nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence of SEQ ID NO: 1).
  • mRNA can be isolated from plant cells (e.g., by the guanidinium- thiocyanate extraction procedure of Chirgwin et al. 1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO: 1.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a SUC 5 protein nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid of the invention comprises one of the nucleotide sequences shown of the polynucleotide of the present invention.
  • the sequence of SEQ ID NO: 1 corresponds to the Arabidopsis thaliana SUC 5 protein cDNA of the invention.
  • These cDNAs comprise sequences encoding SUC 5 proteins (i.e., the "coding region", indicated in SEQ ID NO: 1 ), as well as 5' untranslated sequences and 3' untranslated sequences.
  • the nucleic acid molecules can comprise only the coding region of any of the sequences in SEQ ID NO: 1 or can contain whole genomic fragments isolated from genomic DNA.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule, which is a complement of one of the nucleotide sequences shown in SEQ ID NO: 1 , or a portion thereof.
  • a nucleic acid molecule which is complemen- tary to one of the nucleotide sequences shown in SEQ ID NO: 1 is one which is sufficiently complementary to one of the nucleotide sequences shown in SEQ ID NO: 1 such that it can hybridize to one of the nucleotide sequences shown in SEQ ID NO: 1 , thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleotide sequence shown in SEQ ID NO: 1 , or a portion thereof. Specific algorithms for the determination of the degree of identity are found elsewhere in this specification.
  • an isolated nucleic acid molecule of the invention com- prises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences shown in SEQ ID NO: 1 , or a portion thereof.
  • hybridization conditions include washing with a solution having a salt concentration of about 0.02 molar at pH 7 at about 60°C. Specific hybridization conditions are to be found elsewhere in this specification.
  • the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the sequences in SEQ ID NO: 1 , for ex- ample a fragment, which can be used as a probe or primer or a fragment encoding a biologically active portion of a SUC 5 protein.
  • the nucleotide sequences determined from the cloning of the SUC 5 protein gene from Arabidopsis thaliana allows for the generation of probes and primers designed for use in identifying and/or cloning SUC 5 protein homo- logues in other cell types and organisms, as well as SUC 5 protein homologues from other plants or related species. Therefore this invention also provides compounds comprising the nucleic acids disclosed herein, or fragments thereof.
  • the probe/primer typically comprises substantially purified oligo- nucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO: 1 , an anti-sense sequence of one of the sequences set forth in SEQ ID NO: 1 , or naturally occurring mutants thereof.
  • Primers based on a nucleotide se- quence of SEQ ID NO: 1 can be used in PCR reactions to clone SUC 5 protein homologues.
  • Probes based on the SUC 5 protein nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a genomic marker test kit for identifying cells which express a SUC 5 protein, such as by measuring a level of a SUC 5 protein-encoding nucleic acid in a sample of cells, e.g., detecting SUC 5 protein mRNA levels or determining whether a genomic SUC 5 protein gene has been mutated or deleted.
  • the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid encoded by a sequence of SEQ ID NO: 2 such that the protein or portion thereof maintains the same or a similar function as the wild-type protein.
  • the language "sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue, which has a simi- lar side chain as an amino acid residue in one of the ORFs of a sequence of SEQ ID NO: 2) amino acid residues to an amino acid sequence such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the production of seed storage compounds in plants, construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes. How to determine the de- gree of identical or equivalent amino acids between two sequences is set forth elsewhere in this specification in detail.
  • Transport proteins such as the sucrose transporter 5 play a role in the biosynthesis of seed storage compounds. Examples of such activities are described herein.
  • SUC 5 protein-encoding nucleic acid sequences are set forth in SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 and 79.
  • sugar and/or fatty acid production is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, canola, manihot, pepper, sunflower, sugar beet and tagetes, solana- ceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut) and perennial grasses and forage crops, these crop plants are also preferred target plants for genetic engineering as one further embodiment of the present invention.
  • Portions of proteins encoded by the SUC 5 protein nucleic acid molecules of the invention are preferably biologically active portions of one of the SUC 5 proteins.
  • biologically active portion of a SUC 5 protein is intended to include a portion, e.g., a domain/ motif, of a SUC 5 protein that has an activity as set forth above.
  • an assay of enzymatic activity may be performed. Such assay methods are well known to those skilled in the art, and as described in Example 14 of the Exemplification.
  • Biologically active portions of a SUC 5 protein include peptides comprising amino acid sequences derived from the amino acid sequence of a SUC 5 protein (e.g., an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 or the amino acid sequence of a protein homologous to a SUC 5 protein, which include fewer amino acids than a full length SUC 5 protein or the full length protein which is homologous to a SUC 5 protein) and exhibit at least one activity of a SUC 5 protein.
  • amino acid sequences derived from the amino acid sequence of a SUC 5 protein e.g., an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 or the amino acid sequence of a protein homologous to a SUC 5 protein, which include fewer amino acids than a full length SUC 5 protein or the full length protein which is homologous to a SUC 5 protein
  • biologically active portions comprise a domain or motif with at least one activity of a SUC 5 protein and in accordance with the present invention, preferably, the sucrose transporter 5 activity.
  • other biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.
  • the biologically active portions of a SUC 5 pro- tein include one or more selected domains/motifs or portions thereof having biological activity.
  • Additional nucleic acid fragments encoding biologically active portions of a SUC 5 protein can be prepared by isolating a portion of one of the sequences, expressing the encoded portion of the SUC 5 protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the SUC 5 protein or peptide.
  • the invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO: 1 (and portions thereof) due to degeneracy of the genetic code and thus encode the same SUC 5 protein as that encoded by the nucleotide sequences shown in SEQ ID NO: 1.
  • the nucleic acid molecule of the invention encodes a full length protein which is substantially homologous to an amino acid sequence of a polypeptide encoded by an open reading frame shown in SEQ ID NO: 1.
  • the full-length nucleic acid or protein or fragment of the nucleic acid or protein is from Arabidopsis thaliana.
  • SUC 5 protein nucleotide sequences shown in SEQ ID NO:1 it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of SUC 5 proteins may exist within a population (e.g., the Arabidopsis thaliana population).
  • Such genetic polymorphism in the SUC 5 protein gene may exist among individuals within a population due to natural variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a SUC 5 protein, preferably, an Arabidopsis thaliana SUC 5 protein.
  • Such natural variations can typically result in 1 -40% variance in the nucleotide sequence of the SUC 5 protein gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SUC 5 protein that are the result of natural variation and that do not alter the functional activity of SUC 5 proteins are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural variants and non- Arabidopsis thaliana orthologs of the SUC 5 protein cDNA of the invention can be isolated based on their homology SUC 5 protein nucleic acid disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989: 6.3.1 -6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C.
  • SSC 6X sodium chloride/sodium citrate
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the nucleic acid encodes a natural Arabidopsis thaliana SUC 5 protein.
  • the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO: 1 , thereby leading to changes in the amino acid sequence of the encoded SUC 5 protein, without altering the functional ability of the SUC 5 protein.
  • nucleotide substitutions leading to amino acid substitutions at "nonessential" amino acid residues can be made in a sequence of SEQ ID NO: 2.
  • non- essential amino acid residue is a residue that can be altered from the wild-type sequence of one of the SUC 5 proteins (SEQ ID NO: 2) without altering the activity of said SUC 5 protein, whereas an "essential" amino acid residue is required for SUC 5 protein activity.
  • Other amino acid residues e.g., those that are not conserved or only semi-conserved in the domain having SUC 5 protein activity) may not be essential for activity and thus are likely to be amenable to alteration without altering SUC 5 protein activity.
  • nucleic acid molecules encoding SUC 5 proteins that contain changes in amino acid residues that are not essential for SUC 5 protein activity.
  • SUC 5 proteins differ in amino acid sequence from a sequence yet retain at least one of the SUC 5 protein activities described herein.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and has one or more activities set forth above.
  • the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79, more preferably at least about 60-70% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 even more
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid).
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in one sequence e.g., one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79
  • the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"
  • An isolated nucleic acid molecule encoding a SUC 5 protein homologous to a protein sequence encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47
  • Mutations can be introduced into one of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 by stand- ard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted nonessential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted non-essential amino acid residue in a SUC 5 protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a SUC 5 protein coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a SUC 5 protein activity de- scribed herein to identify mutants that retain SUC 5 protein activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Examples 1 1 -13 of the Exemplification).
  • SUC 5 proteins are preferably produced by recombinant DNA techniques.
  • a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described herein), and the SUC 5 protein is expressed in the host cell.
  • the SUC 5 protein can then be isolat- ed from the cells by an appropriate purification scheme using standard protein purification techniques.
  • a SUC 5 protein or peptide thereof can be synthesized chemically using standard peptide synthesis techniques.
  • native SUC 5 protein can be isolated from cells, for example using an anti-SUC 5 protein antibody, which can be produced by standard techniques utilizing a SUC 5 protein or fragment there- of of this invention.
  • the invention also provides SUC 5 protein chimeric or fusion proteins.
  • a SUC 5 protein "chimeric protein” or “fusion protein” comprises a SUC 5 protein polypeptide operatively linked to a non-SUC 5 protein polypeptide.
  • An "SUC 5 protein polypeptide” re- fers to a polypeptide having an amino acid sequence corresponding to a SUC 5 protein
  • a “non-SUC 5 protein polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SUC 5 protein, e.g., a protein which is different from the SUC 5 protein, and which is derived from the same or a different organism.
  • the term "operatively linked" is intended to indicate that the SUC 5 protein polypeptide and the non-SUC 5 protein polypeptide are fused to each other so that both sequences fulfil the proposed function attributed to the sequence used.
  • the non-SUC 5 protein polypeptide can be fused to the N-terminus or C-terminus of the SUC 5 protein polypeptide.
  • the fusion protein is a GST-SUC 5 protein (glutathione S-transferase) fusion protein in which the SUC 5 protein sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant SUC 5 protein s.
  • the fusion protein is a SUC 5 protein containing a heterologous signal sequence at its N- terminus.
  • expression and/or secretion of a SUC 5 protein can be increased through use of a heterologous signal sequence.
  • a SUC 5 protein chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An SUC 5 protein-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SUC 5 protein.
  • an antisense nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can be hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire SUC 5 protein coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a SUC 5 protein.
  • coding region refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding SUC 5 protein.
  • noncoding region refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of SUC 5 protein mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of SUC 5 protein mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SUC 5 protein mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
  • An antisense or sense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylamino-methyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydro-uracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine, 1 -methyl-guanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine, 7- methylguanine, 5-methyl-aminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • a double-strand interfering RNA construct can be used to cause a down-regulation of the SUC 5 protein mRNA level and SUC 5 pro- tein activity in transgenic plants. This requires transforming the plants with a chimeric construct containing a portion of the SUC 5 protein sequence in the sense orientation fused to the antisense sequence of the same portion of the SUC 5 protein sequence.
  • a DNA linker region of variable length can be used to separate the sense and antisense fragments of SUC 5 protein sequences in the construct.
  • the antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a SUC 5 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complement to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • the antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic including plant promoters are preferred.
  • the antisense nucleic acid molecule of the invention is anomehc nucleic acid molecule.
  • anomehc nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual units, the strands run parallel to each other (Gaultier et al. 1987, Nucleic Acids Res. 15:6625-6641 ).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methyl-ribonucleotide (Inoue et al. 1987, Nucleic Acids Res. 15:6131 -6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987, FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribo- zymes are catalytic RNA molecules with ribonuclease activity, which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff & Gerlach 1988, Nature 334:585-591 )
  • a ribozyme having specificity for a SUC 5 protein -encoding nucleic acid can be designed based upon the nucleotide sequence of a SUC 5 protein cDNA disclosed herein or on the basis of a heterologous sequence to be isolated according to methods taught in this invention.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SUC 5 protein-encoding mRNA (see, e.g., Cech et al., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,1 16,742).
  • SUC 5 protein mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D. & Szostak J.W. 1993, Science 261 :1411 -1418).
  • SUC 5 protein gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a SUC 5 protein nucleotide sequence (e.g., a SUC 5 protein promoter and/or enhancers) to form triple helical structures that prevent transcription of a SUC 5 protein gene in target cells (See generally, Helene C. 1991 , Anticancer Drug Des. 6:569-84; Helene C. et al. 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. 1992, Bioassays 14:807-15).
  • vectors preferably expression vectors, containing a nucleic acid encoding a SUC 5 protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicat- ed along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used inter-changeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence ⁇ ) in a manner which allows for expression of the nucleotide sequence and both sequences are fused to each other so that each fulfils its proposed function (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., SUC 5 proteins, mutant forms of SUC 5 proteins, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of SUC 5 proteins in prokaryotic or eukaryotic cells.
  • SUC 5 protein genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos M.A. et al. 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al. 1991 , Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, Bennet & Lasure, eds., p.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins.
  • Such fusion vectors typically serve one or more of the following purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson 1988, Gene 67:31 -40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • the coding sequence of the SUC 5 protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant SUC 5 protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. 1988, Gene 69:301 -315) and pET 1 1d (Studier et al. 1990, Gene Expression Technolo- gy: Methods in Enzymology 185, Academic Press, San Diego, California 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 11 d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA pol- ymerase (T7 gn1).
  • This viral polymerase is supplied by host strains BL21 (DE3) or
  • HMS174 (DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein
  • nucleic acid sequence of the nucleic acid is altered so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression (Wada et al. 1992, Nucleic Acids Res. 20:211 1 -2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the SUC 5 protein expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSed (Baldari et al. 1987, Embo J. 6:229-234), pMFa (Kurjan & Herskowitz 1982, Cell 30:933-943), pJRY88 (Schultz et al. 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi include those detailed in: van den Hondel & Punt 1991 , "Gene transfer systems and vector development for filamentous fungi," in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1 -28, Cambridge University Press: Cambridge.
  • the SUC 5 proteins of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow & Summers 1989, Virology 170:31 -39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed 1987, Nature 329:840) and pMT2PC (Kaufman et al. 1987, EMBO J. 6:187- 195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the SUC 5 proteins of the invention may be expressed in unicellular plant cells (such as algae, see Falciatore et al. (1999, Marine Biotechnology 1 :239- 251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants).
  • plant expression vectors include those detailed in:
  • a plant expression cassette preferably contains regulatory sequences capable to drive gene expression in plant cells, and which are operably linked so that each sequence can fulfil its function such as termination of transcription, including polyadenylation signals.
  • Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.
  • a plant expres- sion cassette preferably contains other operably linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al. 1987, Nucleic Acids Res. 15:8693-871 1 ).
  • Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner.
  • promoters driving constitutive expression (Benfey et al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al.
  • seed-specific plant promoters are known to those of ordinary skill in the art and are identified and characterized using seed-specific mRNA libraries and expression profiling techniques. Seed-specific promoters include the napin-gene promoter from rapeseed (US 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al. 1991 , Mol. Gen.
  • Suitable promoters to note are the Ipt2 or Ipt1 -gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (pro- moters from the barley hordein-gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, and the rye secalin gene). Plant gene expression can also be facilitated via an inducible promoter (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol.
  • Chemically inducible promoters are especially suitable if gene expression is desired in a time specific manner. Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404), and an ethanol inducible promoter (WO 93/21334).
  • Promoters responding to biotic or abiotic stress conditions are also suitable promoters such as the pathogen inducible PRP1-gene promoter (Ward et al., 1993, Plant. Mol. Biol. 22:361 - 366), the heat inducible hsp80-promoter from tomato (US 5,187,267), cold inducible alpha- amylase promoter from potato (WO 96/12814) or the wound-inducible pinll-promoter (EP 375091).
  • Suitable sequences for use in plant gene expression cassettes are targeting- sequences necessary to direct the gene-product in its appropriate cell compartment (for review see Kermode 1996, Crit. Rev. Plant Sci. 15:285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chro- moplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells.
  • promoters that confer plastid-specific gene expression as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNA-polymerase promoter are described in WO 95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsis described in WO 99/46394.
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to SUC 5 protein mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the anti- sense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokar- yotic or eukaryotic cell.
  • a SUC 5 protein can be expressed in bacterial cells, insect cells, fungal cells, mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, or plant cells.
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells
  • algae such as Chinese hamster ovary cells (CHO) or COS cells
  • ciliates or plant cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection,” “conjugation” and “transduction” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation.
  • Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin, kanamycin, and methotrexate or in plants that confer resistance towards an herbicide such as glyphosate or glufosinate.
  • a nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a SUC 5 protein or can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • drug selection e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • a vector is prepared which contains at least a portion of a SUC 5 protein gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SUC 5 protein gene.
  • this SUC 5 protein gene is an Arabidopsis thaliana or Brassica napus SUC 5 protein gene, but it can be a homologue from a related plant or even from a mammalian, yeast, or insect source.
  • the vector is designed such that, upon homologous recombination, the endogenous SUC 5 protein gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a knock-out vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous SUC 5 protein gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous SUC 5 protein).
  • DNA- RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al. 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, American Scientist 87:240-247).
  • Ho- mologous recombination procedures in Arabidopsis thaliana or other crops are also well known in the art and are contemplated for use herein.
  • the altered portion of the SUC 5 protein gene is flanked at its 5' and 3' ends by additional nucleic acid of the SUC 5 protein gene to allow for homologous recombination to occur between the exogenous SUC 5 protein gene carried by the vector and an endogenous SUC 5 protein gene in a microorganism or plant.
  • the additional flanking SUC 5 protein nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into a microorganism or plant cell (e.g., via polyethylenegly- col mediated DNA).
  • Cells in which the introduced SUC 5 protein gene has homologously recombined with the endogenous SUC 5 protein gene are selected using art-known techniques.
  • recombinant microorganisms can be produced which contain selected systems, which allow for regulated expression of the introduced gene.
  • selected systems which allow for regulated expression of the introduced gene.
  • inclusion of a SUC 5 protein gene on a vector placing it under control of the lac operon permits expression of the SUC 5 protein gene only in the presence of IPTG.
  • Such regulatory systems are well known in the art.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) a SUC 5 protein.
  • the invention further provides methods for producing SUC 5 proteins using the host cells of the invention.
  • the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding a SUC 5 protein has been introduced, or which contains a wild- type or altered SUC 5 protein gene in its genome) in a suitable medium until SUC 5 protein is produced.
  • the method further comprises isolating SUC 5 proteins from the medium or the host cell.
  • Another aspect of the invention pertains to isolated SUC 5 proteins SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 ,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80 and biologically active portions thereof.
  • An "isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of SUC 5 protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of SUC 5 protein having less than about 30% (by dry weight) of non-SUC 5 protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-SUC 5 protein, still more preferably less than about 10% of non-SUC 5 protein, and most preferably less than about 5% non-SUC 5 protein.
  • the SUC 5 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • substantially free of chemical precursors or other chemicals includes preparations of SUC 5 protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language "substantially free of chemical precursors or other chemicals” includes prepara- tions of SUC 5 protein having less than about 30% (by dry weight) of chemical precursors or non-SUC 5 protein chemicals, more preferably less than about 20% chemical precursors or non-SUC 5 protein chemicals, still more preferably less than about 10% chemical precursors or non-SUC 5 protein chemicals, and most preferably less than about 5% chemical precursors or non-SUC 5 protein chemicals.
  • isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SUC 5 protein is derived.
  • such proteins are produced by recombinant expression of, for example, an Arabidopsis thaliana or Brassica napus SUC 5 protein in other plants than Arabidopsis thaliana or Brassica napus or microorganisms, algae or fungi.
  • an isolated SUC 5 protein or a portion thereof of the invention can participate in the metabolism of compounds necessary for the production of seed storage compounds in Brassica napus or of cellular membranes, or has one or more of the activities set forth above.
  • the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 such that the protein or portion thereof maintains its sucrose transporter 5 activity.
  • the portion of the protein is preferably a biologically active portion as described herein.
  • a SUC 5 protein of the invention has an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1.
  • the SUC 5 protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1.
  • the SUC 5 protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50- 60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90- 95%, and even more preferably at least about 96%, 97%, 98%, 99% or more homologous to one of the amino acid sequences encoded by a nucleic acid of SEQ ID NO: 1.
  • a preferred SUC 5 protein of the present invention also preferably possess at least one of the SUC 5 protein activities described herein.
  • a preferred SUC 5 protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1 , and which has one or more of the activities set forth above.
  • the SUC 5 protein is substantially homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and retains the functional activity of the protein of one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail above.
  • the SUC 5 protein is a protein which comprises an amino acid sequence which is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence and which has at least one of the SUC 5 protein activities SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 ,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80 described herein.
  • the invention pertains to a full Arabidopsis thali- ana protein which is substantially homologous to an entire amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1.
  • Dominant negative mutations or trans-dominant suppression can be used to reduce the activity of a SUC 5 protein in transgenic seeds in order to change the levels of seed storage compounds.
  • a mutation that abolishes the activity of the SUC 5 protein is created and the inactive non-functional SUC 5 protein gene is overexpressed in the transgenic plant.
  • the inactive trans-dominant SUC 5 protein competes with the active endogenous SUC 5 protein for substrate or interactions with other proteins and dilutes out the activity of the active SUC 5 protein. In this way the biological activity of the SUC 5 protein is reduced without actually modifying the expression of the endogenous SUC 5 protein gene.
  • Homologues of the SUC 5 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the SUC 5 protein.
  • the term "homologue” refers to a variant form of the SUC 5 protein that acts as an agonist or antagonist of the activity of the SUC 5 protein.
  • An agonist of the SUC 5 protein can retain substantially the same, or a subset, of the biological activities of the SUC 5 protein.
  • An antagonist of the SUC 5 protein can inhibit one or more of the activities of the naturally occurring form of the SUC 5 protein, by, for example, competitively binding to a downstream or upstream member of the cell membrane component metabolic cascade which includes the SUC 5 protein, or by binding to a SUC 5 protein which mediates transport of compounds across such membranes, thereby preventing translocation from taking place.
  • homologues of the SUC 5 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the SUC 5 protein for SUC 5 protein agonist or antagonist activity.
  • a variegated library of SUC 5 protein variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of SUC 5 protein variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SUC 5 protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SUC 5 protein sequences therein.
  • libraries of fragments of the SUC 5 protein coding sequences can be used to generate a variegated population of SUC 5 protein fragments for screening and subsequent selection of homologues of a SUC 5 protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a SUC 5 protein coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nu- clease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SUC 5 protein.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SUC 5 protein homologues.
  • the most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis REM
  • REM Recursive ensemble mutagenesis
  • cell based assays can be exploited to analyze a variegated SUC 5 protein library, using methods well known in the art.
  • nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Arabidopsis thaliana and related organisms; mapping of genomes of organisms related to Arabidopsis thaliana; identification and localization of Arabidopsis thaliana sequences of interest; evolutionary studies; determination of SUC 5 protein regions required for function; modulation of a SUC 5 protein activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; and modulation of seed storage compound accumulation.
  • the nucleic acid and protein molecules of the invention may serve as markers for specific re- gions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of Arabidopsis thaliana or Brassica napus proteins. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related plants.
  • the SUC 5 protein nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies.
  • the metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.
  • Manipulation of the SUC 5 protein nucleic acid molecules of the invention may result in the production of SUC 5 proteins having functional differences from the wild-type SUC 5 pro- teins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
  • SUC 5 protein of the invention may directly affect the accumulation and/or composition of seed storage compounds.
  • increased transport can lead to altered accumulation of compounds and/or solute partitioning within the plant tissue and organs which ultimately could be used to affect the accumulation of one or more seed storage compounds during seed development.
  • An example is provided by Mitsukawa et al. (1997, Proc. Natl. Acad. Sci. USA 94:7098-7102), where overexpression of an Arabidopsis high- affinity phosphate transporter gene in tobacco cultured cells enhanced cell growth under phosphate-limited conditions. Phosphate availability also affects significantly the production of sugars and metabolic intermediates (Hurry et al.
  • a method of producing a transgenic plant having an increased level of fatty acids in the seed comprising, transforming a plant cell with an expression vector comprising a nucle- ic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the plant cell the transgenic plant, analyzing the production of fatty acids in the seed of the transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variant of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
  • polynucleotide sequence having at least 70% sequence identity with the polynucleotide sequence of a), b) or c);
  • polynucleotide sequence is operatively linked to a seed-specific promoter.
  • B The method of producing a transgenic plant according to A, wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence comprises a polynucleotide having at least 90% sequence identity with the polynucleotide sequence of a), b) or c).
  • C The method of producing a transgenic plant according to A and B, wherein the seed- specific promoter is the USP promoter or the SUC5 promoter.
  • a method of increasing the level of the total fatty acids in the seed of a plant comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the cell a transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variety of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide sequence as defined in SEQ ID NO:1 ;
  • polynucleotide sequence is operatively linked to a seed-specific promoter.
  • a transgenic plant with increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polynucleotide sequence selected from the group consisting of:
  • polynucleotide sequence is operatively linked to a seed-specific promoter.
  • a transgenic plant with an increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polypeptide encoding the polypeptide as described by SEQ ID NO: 2 or a polypeptide having at least 70% sequence identity with the polypeptide as defined by SEQ ID NO: 2.
  • a method of producing a transgenic plant having an increased level of 20:1 (1 1 ) fatty acid in the seed comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5, a plastidic translocator GPT1 and a plastidic translocator NTT1 polypeptide sequence, generating from the plant cell a transgenic plant, analyzing the production of fatty acid in the seed of the transgenic plant, and selecting a transgenic plant having an increased level of 20:1 fatty acid as compared to a corresponding untransformed wild type variant of the plant, wherein the expression vector comprises polynucleotide sequences selected from the group consisting of:
  • polynucleotide sequences are operatively linked to a seed-specific promoter.
  • K The method of producing a transgenic plant according to J, wherein the nucleic acid sequences encoding a sucrose transporter 5, a plastidic translocator GPT1 and a plastidic translocator NTT1 polypeptide sequence comprise polynucleotides having at least 90% sequence identity with the polynucleotide sequences of a), b) or c).
  • a method of increasing the level of 20:1 (11) fatty acid in the seed of a plant comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, a plastidic translocator GPT1 and a plastidic translocator NTT1 , generating from the cell a transgenic plant, and selecting a transgenic plant having an increased level of 20:1 fatty acid as compared to a corresponding untransformed wild type variety of the plant, wherein the nucleic acid comprises polynucleotide sequences selected from the group consisting of:
  • polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences of a) or b), wherein the polynucleotide sequences are operatively linked to a seed-specific promoter.
  • a method of increasing the level of 20: 1 (11 ) fatty acid in the seed of a plant according to N wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide, a plastidic translocator GPT1 and a plastidic translocator NTT1 sequence comprises polynucleotide sequences having at least 90% sequence identity with the polynucleotide sequences of a), b) or e).
  • R. A transgenic plant with increased 20:1 (11 ) fatty acid content in the seed of the plant as compared to a wild type variety of the plant comprising polynucleotide sequences selected from the group consisting of:
  • polynucleotide sequences are operatively linked to a seed-specific pro- moter.
  • FIG. 1 Analysis of pSUC5/sGFP and pSUC5/tmGFP9 plants. Confocal images of developing seeds [(a) to (e)] and isolated embryos [(f) to (i)] from pSUC5/sGFP plants [(a), (d) and (e)] or pSUC5/tmGFP9 [(b), (c), and (f) to (i)] plants are presented.
  • Figure 3 Development of seeds and embryos in siliques of wt plants and of different single and double mutants. After an initial growth (12 d) on agar medium with 1 mM biotin, plants were transferred to soil and watered with the indicated supplements of biotin.
  • Space bars are 20 ⁇ in (a) and (b).
  • Seeds of wt and suc5.5 plants are shaped nor- mally under all growth conditions. Seeds of bio2. 1 plants are wrinkled and seeds of bio2. 1/suc5.5 double mutants have the appearance of "empty bags", when their parent plants were not supplemented with biotin. A supplement of 0.1 mM biotin complemented this defect partly (bio2.1 seeds are almost normal looking; bio2.1/suc5.5 plants are still wrinkled), and the seeds looked normal, when the parent plants were watered with 1 mM biotin. Bars are 50 ⁇ .
  • FIG. 5 Comparative analysis of seedlings from wt plants and from homozygous single or double mutants on MS medium or MS medium supplemented with biotin. Seedlings were photographed after 10 days at 21 °C under long-day conditions (16 h light / 8 h dark). Par- ent plants of all seeds with a bio mutation had been watered with 1 mM biotin, parent plants of wt and suc5 mutant seeds only with water. Typical phenotypes are shown for pairs of seedlings grown either on biotin-free MS medium (left) or on MS medium supplemented with biotin (right; final biotin concentration: 1 mM). Names of different double mutant lines are given in brackets. All pictures were taken at the same magnification. Bar is 2 mm.
  • FIG. 8 Schematic map of the expression vector pDEST-USP:SUC5, which was used for transformation of Arabidopsis thaliana plants with the SUC5 gene (SEQ ID NO: 1 ) alone under the transcriptional control of the USP promoter.
  • Figure 9. Schematic map of the expression vector pDEST- USP:GPT/USP:NTT/USP:SUC5, which was used for transformation of Arabidopsis thaliana plants. Additionally to the SUC5 gene (SEQ ID NO: 1 ) the plasmid contains genes for the plastidic translocators GPT1 (SEQ ID NO: 83) and NTT1 (SEQ ID NO: 84). All three genes are cloned behind the transcriptional control of the USP-promoter. Transformation of Arabidopsis wt plants with this construct resulted in the generation of independent transgenic lines, which were consecutively named BioOI3-1 to BioOI3-20.
  • FIG. 12 Fatty acid composition of TAG from dry seeds obtained from wt and the 3 BioOI3-lines with the strongest increase in total TAG (lines 3-1 , 3-4 and 3-15). Increase in total TAG is mostly due to an increase in fatty acids from C20 to C22. The increase in the level of C20:1 (1 1 ) fatty acid averages 20% in these 3 lines. Error bars represent meas- urements of seeds from 10 different plants per line.
  • FIG. 13 Histogram showing the total TAG content of different plants from wt, BioOI3-1 , 3-4 and 3-15. Individual TAG content from 10 plants of each line is plotted in 20 g TAG/mg seeds-intervals (x-axis) against the number of individual plants with this TAG content. TAG content in wt and in the 3 plotted transgenic lines shows a Gaussian distribution in which the peak of the distribution curve is shifted towards higher TAG levels in the transgenic lines compared with wt.
  • Figure 14 In the alignment shown in Figure 14 the nine disaccharide transporters from Arabidopsis thaliana share 25.3% identical positions (black/ white) and 77.0% consensus positions (grey/ white).
  • FIG. 15 Total TAG content of dry seeds obtained from wt (white bar) and transgenic BioOil4 plants (black bars) is shown in Figure 15. Total TAG content is shown in g/mg dry seeds for 1 1 independent transgenic BioOil4 lines. Error bars represent measurements of seeds from 10 different plants per line. An average increase of 4.7% TAG was observed throughout all BioOil4 plants.
  • Figure 16 shows an elevation of total biotin content in the seeds of SUC5- overexpressing plants.
  • Figure 17 shows an elevation of total biotin content in the seeds of SUC5- overexpressing plants.
  • Figure 18 shows the SUC5 promoter sequence as used in constructs used for construction of above BioOil4 plants. Sequences highlighted in grey or dark grey show primers used for amplification of the promoter sequence from genomic Arabidopsis DNA.
  • SL/C5-mRNA levels were measured by qPCR in developing seeds harvested from siliques of wt or SL/C5-overexpressing lines (BioOil3, BioOil4) at the indicated days after flowering (DAF).
  • ACTIN2 ACT2 was used as internal reference gene for the determination of relative expression.
  • FIG. 20 Sucrose and raffinose content in ripe seeds of wt, BioOil3 and BioOM. Bars and errors represent mean values and standard deviations from 3 independent measurements.
  • Figure 21 Uptake of sucrose and biotin by wt, BioOil3 and BioOM embryos. Bars and errors in (a) and (b) represent mean values and standard deviations from 3 independent measurements.
  • the position of the T-DNA insertion in the suc5.4 mutant was determined by sequencing PCR fragments that were with the primers LB2 (5'- GCTTCCTATTATATCTTCCCAAATTACCAATACA-3') and AtSUC5g540f (5'- CGCAAACGCGTGTTTCTCCT-3').
  • the double insertion in suc5.5 was determined with the primers LBa1 (5'-TGGTTCACGTAGTGGGCCATCG-3') and AtSUC5g540f (5'-end of insertion) or with LBa1 and AtSUC5g2136r ( 5 ' -TG C ACAACAATACTGTATT AG ATG G -3 ' ; 3'-end of the insertion).
  • the seeds from heterozygous, biotin- watered BI01/bio1.
  • pSUC5/sGFP For construction of pSUC5/sGFP, 2030 bp of pSUC5 were amplified using the primers At- SUC5-2030f (5'-AAGCTTAACAATTTATGTAGTTTAGAACG-3') and AtSUC5-1 r (5'- CCATGGTGAAAAGAAAAACGAGCAGACAA-3') that introduced Hindlll and Ncol cloning sites to the 5' and 3'-ends, respectively. The resulting fragment was used to replace pSUC2 in pEP/pUC19, a plasmid containing a pSUC2/GFP cassette (Imlau et ai, 1999).
  • pEP-S5-GFP the pSUC5/sGFP fragment was excised with Hindlll and Sacl and cloned into the respective sites of pAF16 (Stadler et ai, 2005b).
  • the resulting plasmid was used for Arabidopsis transformation.
  • pSUC5/tmGFP9 For construction of pSUC5/tmGFP9, a genomic 1 152-bp fragment encoding the 232 N- terminal amino acids of STP9 (Schneidereit et ai, 2003) was excised from plasmid pMH4 (Stadler et ai, 2005b) with Ncol and inserted into the unique Ncol site separating pSUC5 and the GFP ORF in pEP-S5-GFP.
  • the 3916-bp pSUC5/tmGFP9 cassette was excised with Hindll l/Sacl and cloned into the respective sites of pAF16 yielding pMH21 that was used for Arabidopsis transformation.
  • Fatty acid methyl esters (FAMEs) of pooled Arabidopsis seeds were obtained by methyla- tion with 0.5 M sulphuric acid in methanol containing 2% (v/v) dimethoxypropane at 80°C for 1 h.
  • FAMEs were extracted in 2 ml of n-hexane, dried under N 2 and analysed by gas- chromatography (GC).
  • GC analysis was performed with an Agilent GC 6890 system coupled with a flame ionization detector equipped with a capillary 122-2332 DB-23 column (30 m x 0.32 mm; 0.5 ⁇ coating thickness; Agilent). Helium was used as carrier gas (1 ml min- 1 ).
  • Cloning processes such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli and yeast cells, growth of bacteria and sequence analysis of recombinant DNA were carried out as de- scribed in Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309- 6) or Kaiser, Michaelis and Mitchell (1994, "Methods in Yeast Genetics", Cold Spring Harbor Laboratory Press: ISBN 0-87969-451 -3).
  • the details for the isolation of total DNA relate to the working up of 1 gram fresh weight of plant material.
  • CTAB buffer 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCI pH 8.0; 1.4 M NaCI; 20 mM EDTA.
  • N-Laurylsarcosine buffer 10% (w/v) N- laurylsarcosine; 100 mM Tris HCI pH 8.0; 20 mM EDTA.
  • the plant material was triturated under liquid nitrogen in a mortar to give a fine powder and transferred to 2 ml Eppendorf vessels.
  • the frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 ⁇ of N-laurylsarcosine buffer, 20 ⁇ of ⁇ -mercaptoethanol and 10 ⁇ of proteinase K solution, 10 mg/ml) and incubated at 60°C for one hour with continuous shaking.
  • the homogenate obtained was distributed into two Eppendorf vessels (2 ml) and extracted twice by shaking with the same volume of chlo- roform/isoamyl alcohol (24:1 ). For phase separation, centrifugation was carried out at 8000g and RT for 15 min in each case.
  • the DNA was then precipitated at -70°C for 30 min using ice-cold isopropanol.
  • the precipitated DNA was sedimented at 4°C and 10,000 g for 30 min and resuspended in 180 ⁇ of TE buffer (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
  • the DNA was treated with NaCI (1.2 M final concentration) and precipitated again at -70°C for 30 min using twice the volume of absolute ethanol.
  • the DNA was dried and subsequently taken up in 50 ⁇ of H 2 0 + RNAse (50 mg/ml final concentration).
  • the DNA was dissolved overnight at 4°C and the RNAse digestion was subsequently carried out at 37°C for 1 h. Storage of the DNA took place at 4°C.
  • RNA is isolated from siliques of Arabidopsis plants according to the following procedure: RNA preparation from Arabidopsis seeds - "hot” extraction:
  • Resuspension buffer 0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC- treated water as this solution can not be DEPC-treated
  • RNA from wild-type of Arabidopsis is isolated as described (Hosein, 2001 , Plant Mol. Biol. Rep., 19, 65a-65e; Ruuska,S.A., Girke,T., Benning.C, & Ohlrogge,J.B., 2002, Plant Cell, 14, 1 191 -1206).
  • the mRNA is prepared from total RNA, using the Amersham Pharmacia Biotech mRNA purification kit, which utilizes oligo(dT)-cellulose columns.
  • RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70°C.
  • Hyseq Pharmaceuticals Incorporated (Sunnyville, CA) for further processing of mRNA from each tissue type into cDNA libraries and for use in their proprietary processes in which similar inserts in plasmids are clustered based on hybridization patterns.
  • first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12°C (2 h), 16°C (1 h) and 22°C (1 h). The reaction was stopped by incubation at 65°C (10 min) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37°C (30 min). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns.
  • EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12°C, overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37°C, 30 min). This mixture was subjected to separation on a low melting agarose gel.
  • DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.
  • Brassica napus cDNA libraries were generated at Hyseq Pharmaceuticals Incorporated (Sunnyville, CA) No amplification steps were used in the library production to retain expression information.
  • Hyseq's genomic approach involves grouping the genes into clusters and then sequencing representative members from each cluster.
  • cDNA libraries were generat- ed from oligo dT column purified mRNA. Colonies from transformation of the cDNA library into E.coli were randomly picked and the cDNA insert were amplified by PCR and spotted on nylon membranes. A set of 33_ P radiolabeled oligonucleotides were hybridized to the clones and the resulting hybridization pattern determined to which cluster a particular clone belonged.
  • cDNA clones and their DNA sequences were obtained for use in overexpression in transgenic plants and in other molecular biology processes described herein.
  • Hyseq clones corresponding to full-length sequences and partial cDNAs from Arabidopsis thaliana had been identified in the in-house proprietary Hyseq databases.
  • the Hyseq clones of Ar- abidopsis thaliana were sequenced at DNA Landmarks using a ABI 377 slab gel sequencer and BigDye Terminator Ready Reaction kits (PE Biosystems, Foster City, CA). Sequence alignments were done to determine whether the Hyseq clones were full-length or partial clones. In cases where the Hyseq clones were determined to be partial cDNAs the following procedure was used to isolate the full-length sequences.
  • Full-length cDNAs were iso- lated by RACE PCR using the SMART RACE cDNA amplification kit from Clontech allowing both 5'- and 3' rapid amplification of cDNA ends (RACE).
  • the RACE PCR primers were designed based on the Hyseq clone sequences.
  • the isolation of full-length cDNAs and the RACE PCR protocol used were based on the manufacturer's conditions.
  • the RACE product fragments were extracted from agarose gels with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions.
  • Recombinant vectors were transformed into TOP10 cells (Invitrogen) using standard conditions (Sambrook et al. 1989). Transformed cells were grown overnight at 37°C on LB agar containing 50 ⁇ g/ml kanamycin and spread with 40 ⁇ of a 40 mg/ml stock solution of X-gal in dimethylformamide for blue-white selection. Single white colonies were selected and used to inoculate 3 ml of liquid LB containing 50 ⁇ g/ml kanamycin and grown overnight at 37°C. Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Subsequent analyses of clones, and restriction mapping, was performed according to standard molecular biology techniques (Sambrook et al. 1989).
  • Full-length cDNAs were isolated and cloned into binary vectors by using the following pro- cedure: Gene specific primers were designed using the full-length sequences obtained from Hyseq clones or subsequent RACE amplification products. Full-length sequences and genes were amplified utilizing Hyseq clones or cDNA libraries as DNA template using touch-down PCR. In some cases, primers were designed to add an "AACA" Kozak-like sequence just upstream of the gene start codon and two bases downstream were, in some cases, changed to GC to facilitate increased gene expression levels (Chandrashekhar et al. 1997, Plant Molecular Biology 35:993-1001 ).
  • PCR reaction cycles were: 94°C, 5 min; 9 cycles of 94°C, 1 min, 6°C, 1 min, 72°C, 4 min and in which the anneal temperature was lowered by 1 °C each cycle; 20 cycles of 94°C, 1 min, 55°C, 1 min, 72°C, 4 min; and the PCR cycle was ended with 72°C, 10 min.
  • Amplified PCR products were gel purified from 1 % agarose gels using GenElute -EtBr spin columns (Sigma) and after standard enzymatic digestion, were ligated into the plant binary vector pBPS-GB1 for transformation of Ara- bidopsis. The binary vector was amplified by overnight growth in E.
  • coli DH5 in LB media and appropriate antibiotic and plasmid was prepared for downstream steps using Qiagen MiniPrep DNA preparation kit.
  • the insert was verified throughout the various cloning steps by determining its size through restriction digest and inserts were sequenced to ensure the expected gene was used in Arabidopsis transformation.
  • Gene sequences can be used to identify homologous or heterologous genes (orthologs, the same SUC 5 protein gene from another plant) from cDNA or genomic libraries. This can be done by designing PCR primers to conserved sequences identified by multiple sequence alignments. Orthologs are often identified by designing degenerate primers to full-length or partial sequences of genes of interest.
  • Gene sequences can be used to identify homologues or orthologs from cDNA or genomic libraries.
  • Homologous genes e. g. full-length cDNA clones
  • 32P radioactive
  • Partially homologous or heterologous genes that are related but not identical can be identi- fied in a procedure analogous to the above-described procedure using low stringency hybridization and washing conditions.
  • the ionic strength is normally kept at 1 M NaCI while the temperature is progressively lowered from 68 to 42°C.
  • Radio labeled oligonucleotides are prepared by phosphorylation of the 5' end of two complementary oligonucleotides with T4 polynucleotide kinase.
  • the complementary oligonucleotides are annealed and ligated to form concatemers.
  • the double stranded concatemers are then radiolabeled by for example nick transcription.
  • Hy- bridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
  • c-DNA clones can be used to produce recombinant protein for example in E. coil (e. g. Qi- agen QIAexpress pQE system). Recombinant proteins are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant proteins can be used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al. (1994, BioTechniques 17:257-262). The antibody can then be used to screen expression cDNA libraries to identify homologous or heterologous genes via an immunological screening (Sambrook et al. 1989, "Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press or Ausubel et al. 1994, “Current Protocols in Molecular Biology,” John Wiley & Sons).
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of poly-(A)+ RNA is separated by gel electrophoresis in 1.25% agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem. 152:304), transferred by capillary attraction using 10 x SSC to positively charged nylon membranes (Hybond N+, Amersham, Braunschweig), immobilized by UV light and pre-hybridized for 3 hours at 68°C using hybridization buffer (10% dextran sulfate w/v, 1 M NaCI, 1 % SDS, 100 ⁇ g/ml of herring sperm DNA).
  • the labelling of the DNA probe with the Highprime DNA labelling kit is carried out during the pre-hybridization using alpha-32P dCTP (Amersham, Braunschweig, Germany).
  • Hybridization is carried out after addition of the labelled DNA probe in the same buffer at 68°C overnight.
  • the washing steps are carried out twice for 15 min using 2 x SSC and twice for 30 min using 1 x SSC, 1 % SDS at 68°C.
  • the exposure of the sealed filters is carried out at - 70°C for a period of 1 day to 14 days.
  • cDNA libraries can be used for DNA sequencing according to standard methods, in particu- lar by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Rothstadt, Germany). Random sequencing can be carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands). Plasmid DNA can be prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing am- picillin (see Sambrook et al.
  • FASTA Very sensitive protein sequence database searches with estimates of statistical significance (Pearson W.R. 1990, Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzy- mol. 183:63-98).
  • BLAST Very sensitive protein sequence database searches with esti- mates of statistical significance (Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 215:403-410).
  • PREDATOR High- accuracy secondary structure prediction from single and multiple sequences. (Frishman & Argos 1997, 75% accuracy in protein secondary structure prediction. Proteins 27:329-335).
  • CLUSTALW Multiple sequence alignment (Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22:4673-4680).
  • TMAP Transmembrane region prediction from multiply aligned sequences (Persson B. & Argos P. 1994, Prediction of transmembrane segments in proteins utilizing multiple sequence alignments, J. Mol. Biol. 237: 182-192).
  • ALOM2 Transmembrane region prediction from single sequences (Klein P., Kanehisa M., and DeLisi C. 1984, Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221 -226. Version 2 by Dr. K. Nakai).
  • PROSEARCH Detection of PROSITE protein sequence patterns. Kolakowski L.F. Jr., Leunissen J.A.M. and Smith J.E. 1992, ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13:919- 921 ).
  • binary vectors such as pBinAR can be used (Hofgen & Willmitzer 1990, Plant Sci. 66:221 -230). Construction of the binary vectors can be performed by ligation of the cDNA in sense or antisense orientation into the T-DNA. 5' to the cDNA a plant promoter activates transcription of the cDNA. A polyadenylation sequence is located 3' to the cDNA. Tissue-specific expression can be achieved by using a tissue specific promoter. For example, seed-specific expression can be achieved by cloning the napin or LeB4 or USP promoter 5' to the cDNA. Also any other seed specific promoter element can be used. For constitutive expression within the whole plant the CaMV 35S promoter can be used.
  • the expressed protein can be targeted to a cellular compartment using a signal peptide, for example for plastids, mitochondria, or endoplasmic reticulum (Kermode 1996, Crit. Rev. Plant Sci. 15:285-423).
  • the signal peptide is cloned 5-prime in frame to the cDNA to achieve subcellular localization of the fusion protein.
  • plant binary vectors are the pBPS-GB1 , pSUN2-GW or pBPS-GB047 vectors into which the SUC 5 protein gene candidates are cloned.
  • These binary vectors contain an antibiotic resistance gene driven under the control of the AtAct2-l promoter and a USP seed-specific promoter or the PtxA promoter in front of the candidate gene with the NOSpA terminator or the OCS terminator.
  • Partial or full-length SUC 5 protein cDNA are cloned into the multiple cloning site of the plant binary vector in sense orientation behind the USP seed-specific or PtxA promoters.
  • the recombinant vector containing the gene of in- terest is transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells are selected for on LB agar containing 50 ⁇ g/ml kanamycin grown overnight at 37°C.
  • Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analysis of subsequent clones and restriction mapping is performed according to standard molecular biology techniques (Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual. 2 nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY).
  • Agrobacterium mediated plant transformation with the SUC 5 protein nucleic acids described herein can be performed using standard transformation and regeneration techniques (Gelvin, Stanton B. & Schilperoort R.A, Plant Molecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Gottatur:BT1 1 -P; Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and Biotechnology, S. 360, CRC Press, Boca Raton 1993).
  • Agrobacterium mediated transformation can be performed using the GV3 (pMP90) (Koncz & Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.
  • Arabidopsis thaliana can be grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1 194-1 199; Bent et al. 1994, Science 265:1856-
  • rapeseed can be transformed with the LMR nucleic acids of the present invention via cotyledon or hypocotyl transformation (Moloney et al. 1989, Plant Cell Report 8:238-242; De Block et al. 1989, Plant Physiol. 91 :694-701 ).
  • Use of antibiotic for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using a selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al. (1994, Plant Cell Report 13:282-285).
  • the Arabidopsis thaliana sucrose transporter 5 gene was cloned into a binary vector and expressed either under the USP promoter or the PtxA promoter (the promoter of the Pisum sativum PtxA gene), which is a promoter active in virtually all plant tissues.
  • the PtxA promoter the promoter of the Pisum sativum PtxA gene
  • there is no expression activity detectable by GUS staining and low expression activity detectable with the more sensitive method of RT-PCR (Song, H-S. et al., WO 05/085450). Only in plant lines comprising multiple copies of a transgenic ptxA- promoter/GUS expression construct some expression could be detected in part of the flowers and the siliques (for more details see Song, H-S.
  • the superpromoter which is a constitutive promoter (Stanton B. Gelvin, US 5,428,147 and US 5,217,903) or seed-specific promoters like USP (unknown seed protein) from Vicia faba (Baeumlein et al. 1991 , Mol. Gen. Genetics 225:459-67), or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233-239) as well as promoters conferring seed- specific expression in monocot plants like maize, barley, wheat, rye, rice etc. were used.
  • Transformation of soybean can be performed using for example a technique described in EP 0424 047, U.S. Patent No. 5,322,783 (Pioneer Hi-Bred International) or in EP 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No. 5,169,770 (University Toledo), or by any of a number of other transformation procedures known in the art.
  • Soybean seeds are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) tween for 20 minutes with con- tinuous shaking.
  • the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours.
  • the seed coats are peeled off, and cotyledons are detached from the embryo axis.
  • the embryo axis is examined to make sure that the meristematic region is not damaged.
  • the excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.
  • the method of plant transformation is also applicable to other crops.
  • seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05 % (v/v) Tween for 20 minutes, at room temperature with continuous shaking.
  • the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours.
  • the seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of their water content.
  • the seeds are then stored at room temperature in a sealed Petri dish until further use.
  • Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8.
  • appropriate antibiotics e.g. 100 mg/l streptomycin, 50 mg/l kanamycin
  • the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and re-suspended in MS (Murashige & Skoog 1962, Physiol. Plant. 15:473-497) medium supplemented with 100 mM acetosyringone. Bacteria cultures are incubated in this pre- induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 44% moisture content are imbibed for 2 h at room tempera- ture with the pre-induced Agrobacterium suspension culture. (The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes).
  • the embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supple- merited with 500 mg/l carbenicillin or 300 mg/l cefotaxime to kill the agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25°C, under 440 ⁇ nr ⁇ s ' l and 12 hours photoperiod.
  • the seedlings Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favour the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25°C, under 440 ⁇ nr ⁇ s ' l light intensity and 12 h photoperiod for about 80 days.
  • Samples of the primary transgenic plants are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization wherein DNA is electro- phoresed on a 1 % agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics).
  • the PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labeled probe by PCR as recommended by the manufacturer.
  • a rice (or other monocot) sucrose transporter 5 gene under a plant promoter like PtxA could be transformed into corn, or another crop plant, to generate effects of monocot sucrose transporter 5 genes in other monocots, or dicot sucrose transporter 5 genes in other dicots, or monocot genes in dicots, or vice versa.
  • the plasmids containing these coding sequences, 5' of a promoter and 3' of a terminator would be constructed in a manner similar to those described for construction of other plasmids herein.
  • In vivo mutagenesis of microorganisms can be performed by incorporation and passage of the plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) that are impaired in their capabilities to maintain the integrity of their genetic information.
  • E. coli or other microorganisms e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae
  • Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp W.D. 1996, DNA repair mechanisms, in: Escherichia col ⁇ and Salmonella, p. 2277- 2294, ASM: Washington.) Such strains are well known to those skilled in the art.
  • the activity of a recombinant gene product in the transformed host organism can be measured on the transcriptional or/and on the translational level.
  • a useful method to ascertain the level of transcription of the gene is to perform a Northern blot (for reference see, for exam- pie, Ausubel et al.
  • RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene.
  • detectable tag usually radioactive or chemiluminescent
  • SUC 5 proteins that bind to DNA can be measured by several well- established methods, such as DNA band-shift assays (also called gel retardation assays).
  • DNA band-shift assays also called gel retardation assays.
  • the effect of such SUC 5 protein on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar H. et al. 1995, EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both prokaryotic and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
  • lipid metabolism membrane-transport proteins can be performed according to techniques such as those described in Gennis R.B. (1989 Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137, 199-234 and 270-322).
  • An SUC 5 protein can be recovered from plant material by various methods well known in the art. Organs of plants can be separated mechanically from other tissue or organs prior to isolation of the seed storage compound from the plant organ. Following homogenization of the tissue, cellular debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from cells grown in culture, then the cells are removed from the cul- ture by low-speed centrifugation and the supernatant fraction is retained for further purification.
  • the supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin, while the sample is not.
  • chromatography steps may be repeated as necessary, using the same or different chromatography resins.
  • One skilled in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified.
  • the purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
  • the identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, analytical chromatography such as high performance liquid chromatography, NIRS, enzymatic assay, or microbiologically.
  • HPLC high-performance liquid chromatography
  • spectroscopic methods spectroscopic methods
  • staining methods staining methods
  • thin layer chromatography thin layer chromatography
  • analytical chromatography such as high performance liquid chromatography, NIRS, enzymatic assay, or microbiologically.
  • the effect of the genetic modification in plants on a desired seed storage compound can be assessed by growing the modified plant under suitable conditions and analyzing the seeds or any other plant organ for increased production of the desired product (i.e., a lipid or a fatty acid).
  • a desired seed storage compound such as a sugar, lipid or fatty acid
  • Such analysis techniques are well known to one skilled in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatog- raphy such as high performance liquid chromatography (see, for example, Ullman 1985, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and 443-613, VCH: Weinheim; Fallon, A. et al.
  • plant lipids are extracted from plant material as described by Cahoon et al. (1999, Proc. Natl. Acad. Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry 442:141-145).
  • Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology. Ayr/Scotland :Oily Press. - (Oily Press Lipid Library; Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland:Oily Press, 1989 Repr. 1992. - IX,307 S.
  • TAG Triacylglycerol
  • DAG Diacylglycerol
  • MGDG Monogalactosyldiacylglycerol
  • DGDG Digalactosyldiacylglycerol
  • PC Phosphatidylcholine
  • PE Phosphatidylethanolamine
  • the marked up (*) fatty acids do not normally occur in plant seed oils, but their production in transgenic plant seed oil is of importance in plant biotechnology.
  • Positional analysis of the fatty acid composition at the sn-1 , sn-2 or sn-3 positions of the glycerol backbone is determined by lipase digestion (see, e.g., Siebertz & Heinz 1977, Z. Naturforsch. 32c: 193-205, and Christie 1987, Lipid Analysis 2 nd Edition, Pergamon Press, Wales, ISBN 0-08-023791 -6).
  • Total seed oil levels can be measured by any appropriate method. Quantification of seed oil contents is often performed with conventional methods, such as near infrared analysis (NIR) or nuclear magnetic resonance imaging (NMR). NIR spectroscopy has become a standard method for screening seed samples whenever the samples of interest have been amenable to this technique. Samples studied include canola, soybean, maize, wheat, rice, and others. NIR analysis of single seeds can be used (see e.g. Velasco et al., "Estimation of seed weight, oil content and fatty acid composition in intact single seeds of rapeseed” (Brassica napus L.) by near-infrared reflectance spectroscopy, "Euphytica,” Vol. 106, 1999, pp. 79-85).
  • NIR near infrared analysis
  • NMR nuclear magnetic resonance imaging
  • NMR has also been used to analyze oil content in seeds (see e.g. Robertson & Morrison, "Analysis of oil content of sunflower seed by wide-line NMR," Journal of the American Oil Chemists Society, 1979, Vol. 56, 1979, pp. 961 -964, which is herein incorporated by reference in its entirety).
  • a typical way to gather information regarding the influence of increased or decreased protein activities on lipid and sugar biosynthetic pathways is for example via analyzing the carbon fluxes by labeling studies with leaves or seeds using ⁇ c-acetate or 14 C -pyruvate
  • Material to be analyzed can be disintegrated via sonification, glass milling, liquid nitrogen and grinding or via other applicable methods.
  • the material has to be centrifuged after disintegration.
  • the sediment is re-suspended in distilled water, heated for 10 minutes at 100°C, cooled on ice and centrifuged again followed by extraction in 0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at 90°C leading to hydrolyzed oil and lipid compounds resulting in transmethylated lipids.
  • fatty acid methyl esters are extracted in petrolether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperature gradient between 170°C and 240°C for 20 minutes and 5 min. at 240°C.
  • Chropack Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm
  • the identity of resulting fatty acid methylesters is defined by the use of standards available form commercial sources (i.e., Sigma).
  • molecule identity is shown via deri- vatization and subsequent GC-MS analysis.
  • the localization of triple bond fatty acids is shown via GC-MS after derivatization via 4,4-Dimethoxy-oxazolin-Derivaten (Christie, Oily Press, Dundee, 1998).
  • soluble sugars and starch For the extraction of soluble sugars and starch, 50 seeds are homogenized in 500 ⁇ of 80% (v/v) ethanol in a 1.5-ml polypropylene test tube and incubated at 70°C for 90 min. Following- ing centrifugation at 16,000 g for 5 min, the supernatant is transferred to a new test tube. The pellet is extracted twice with 500 ⁇ of 80% ethanol. The solvent of the combined su- pernatants is evaporated at room temperature under a vacuum. The residue is dissolved in 50 ⁇ of water, representing the soluble carbohydrate fraction.
  • the pellet left from the etha- nol extraction which contains the insoluble carbohydrates including starch, is homogenized in 200 ⁇ of 0.2 N KOH, and the suspension is incubated at 95°C for 1 h to dissolve the starch. Following the addition of 35 ⁇ of 1 N acetic acid and centrifugation for 5 min at 16,000 g, the supernatant is used for starch quantification.
  • Enzymatic assays of hexokinase and fructokinase are performed spectrophotometrically according to Renz et al. (1993, Planta 190:156-165), of phosphogluco-isomerase, ATP- dependent 6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase, Fructose-1 ,6-bisphosphate aldolase, those phosphate isomerase, glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase are performed according to Burrell et al. (1994, Planta 194:95-101 ) and of UDP-Glucose- pyrophosphorylase according to Zrenner et al. (1995, Plant J. 7:97-107).
  • yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.
  • plant expression vectors comprising the nucleic acids disclosed herein, or frag- ments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soybean, rapeseed, rice, maize, wheat, Medicago truncatula, etc., using standard protocols.
  • the resulting transgenic cells and/or plants derived there from can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.
  • sequences disclosed herein, or fragments thereof can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke at al. 1998, Plant J. 15:39-48).
  • the resultant knockout cells can then be evaluated for their composition and content in seed storage compounds, and the effect on the phenotype and/or genotype of the mutation.
  • methods of gene inactivation include US 6004804 "Non-Chimeric Mutational Vectors" and Puttaraju et al. (1999, "Spliceosome-mediated RNA frans-splicing as a tool for gene therapy” Nature Biotech. 17:246-252).
  • the genomic SUC5 sequence (SEQ ID NO: 1 ) was amplified from genomic Arabidopsis thaliana DNA (ecotype Wassilewskija, Ws) with Phusion High Fidelity Polymerase (Finnzymes, Espoo, Fl) using the oligonucleotides AtSUC5+1-Ascl (5'- GAG AGA GAG AGA GGC GCG CCA TGG GAG CCT TGG AAG CAG AAA G -3') and AtSUC5+2082r-Notl (5'-GAG AGA GAG AGA GCG GCC GCC TAA TGG AAT CCC ATA GCC CCT GAC -3').
  • PCR fragments were then cloned into the TOPO PCR Blunt II vector (Life Technologies, Carlsbad, CA, USA) and sequenced using the insert flanking vectors M13-f and M13-r.
  • Error-free clones were directionally cloned into the Gateway entry vector VC- LJB1006-1 (Entry_C vector) behind the USP promoter sequence using the restriction endo- nucleases Ascl and Notl (New England Biolabs, Frankfurt, Germany).
  • the resulting entry vector pEntryC-USPp::SUC5 was then subjected into a Gateway reaction using the Gateway LR Clonase Plus II Enzyme Mix (Life Technologies, Carlsbad, CA, USA) together with the entry vectors VC-LJB2174-3 (Entry_A vector, carrying a Napin promoter sequence) and VC-LLL895-1 (Entry_B vector, carrying a USP promoter sequence) and the destination vector VC-LLL1 164-1 , containing a streptomycin/ spectinomycin resistance gene for bacterial selection and a Imazamox resistance gene (AtAHAS) for transgenic plant selection.
  • Gateway LR Clonase Plus II Enzyme Mix (Life Technologies, Carlsbad, CA, USA) together with the entry vectors VC-LJB2174-3 (Entry_A vector, carrying a Napin promoter sequence) and VC-LLL895-1 (Entry_B vector, carrying a USP promoter sequence)
  • the Entry_A and Entry_B vectors did not contain any ORF sequences within the cassettes integrated in the final expression clone and were solely used for providing the attachment sides needed for the Gateway reaction.
  • the resulting expression clone pDEST-USP:SUC5 ( Figure 8) was then transformed into the Agrobacterium tumefaciens strain C58 (Deblaere et al. 1985. Nucl. Acids Res 13:4777-88).
  • Arabidopsis thaliana plants (Ws) were transformed using the floral dip method described by Clough and Bent (1998. Plant J. 16, 735-743). Transgenic plants were identified by selecting the germinating seeds of the dipped plants with the herbicide Imazamox.
  • Resistant plants were further tested with PCR using a USP promoter specific forward primer ⁇ USP-fwd, 5'- CTG CAG CAA ATT TAC ACA TTG CCA CTA-3') and a SUC5 specific reverse primer ⁇ SUC5-rev, 5'-TAC ACT TCA CGA CCC ATC CA-3') for correct insert integration.
  • Seeds from transformed Arabidopsis thaliana T2-plants are analyzed by gas chromatography (GC) for total oil content and fatty acid profile.
  • GC gas chromatography
  • Arabidopsis ecotype Wassilewskija, WS
  • SEQ ID NO 1 sucrose transporter 5 gene
  • Table 3 A table of the function of the SUC 5 protein
  • Total fatty acid content of seeds of control and transgenic plants are measured with bulked seeds (usually 5 mg seed weight) of a single plant.
  • Two different types of controls are used: Untransformed Ws wt plants, that are grown under exactly the same conditions as the transformed plants and BPS empty (without SUC 5 protein gene of interest) binary vector construct (GB1 ). Seeds from transgenic and control plants are sown on potting soil (65% peat, 25% washed sand, 10% clay granulate), stratified in the dark for 3 days at 4°C and then transferred to the growth chamber.
  • sucrose transporter 5 gene expression is driven by a seed specific USP promoter.
  • the p values reveal significant increases in at least 2 independent transgenic events in the T3 seed gen- eration of at least 5% respectively. The results suggest that sucrose transporter 5 over- expression with a seed specific promoter allows the manipulation of total seed oil content.
  • the genomic SUC5 sequence (SEQ ID NO: 1) was amplified from genomic Arabidopsis thaliana DNA (ecotype Wassilewskija, Ws) with Phusion High Fidelity Polymerase (Finnzymes, Espoo, Fl) using the oligonucleotides AtSUC5+1-Ascl (5'- GAG AGA GAG AGA GGC GCG CCA TGG GAG CCT TGG AAG CAG AAA G -3') and AtSUC5+2082r-Notl (5'-GAG AGA GAG AGA GCG GCC GCC TAA TGG AAT CCC ATA GCC CCT GAC -3').
  • PCR fragments were then cloned into the TOPO PCR Blunt II vector (Life Technologies, Carlsbad, CA, USA) and sequenced using the insert flanking vectors M13-f and M13-r.
  • Error-free clones were directionally cloned into the Gateway entry vector VC- LJB1006-1 (Entry _C vector) behind the USP promoter sequence using the restriction endo- nucleases Ascl and Notl (New England Biolabs, Frankfurt, Germany).
  • the resulting entry vector pEntryC-USPp::SUC5 was then subjected into a Gateway reaction using the Gate- way LR Clonase Plus II Enzyme Mix (Life Technologies, Carlsbad, CA, USA) together with the entry vectors GPT1 [EntrA-pUSP] (Entry_A vector, carrying the GPT1 (SEQ ID NO: 83) cDNA sequence under control of the USP promoter) and NTT1 [EntrB-pUSP] (Entry_B vector, carrying the NTT1 (SEQ ID NO: 84) cDNA sequence under control of the USP promoter sequence) and the destination vector VC-LLL1 164-1 , containing a streptomycin/ spectino- mycin resistance gene for bacterial selection and a Imazamox-resistance gene (AtAHAS) for transgenic plant selection.
  • GPT1 [EntrA-pUSP] Entry_A vector, carrying the GPT1 (SEQ ID NO:
  • Resistant plants were further tested with PCR using a USP promoter specific forward primer (USP-fwd, 5'- CTG CAG CAA ATT TAC ACA TTG CCA CTA -3') and a SUC5 specific reverse primer (SUC5- rev, 5'- TAC ACT TCA CGA CCC ATC CA -3') for correct insert integration. Plants success- fully transformed with the pDEST-USP:GPT/USP:NTT/USP:SUC5 are designated below as BioOI3-plants.
  • Seeds from transgenic BioOI3 plants of the T2 generation and control plants were sown on potting soil (65% peat, 25% washed sand, 10% clay granulate), stratified in the dark for 3 days at 4°C and then transferred to the growth chamber. Plants were grown under short day conditions (8 h light/16 h dark cycles) for 4 weeks and then long day conditions (16 h light/8 h dark cycles at 180-200 pinoles nr 2 sec- 1 ) at 22°C with 60% relative humidity. After induction of flowering, inflorescences were framed with Plexiglas tubes. Plants were watered during seed development and ripe seeds were collected not before 10 weeks after sowing. Seeds were filtered and stored at room temperature in the dark at low humidity until fatty acid analysis.
  • FIG 12 shows the FA composition of the 3 BioOI3 lines with the highest overall TAG increase. Interestingly, such significant increases could only be measured for C20 or longer fatty acids. These FAs are being elongated in the plant cytosol, whereas elongation of fatty acids until C18 takes place in the plastids. This specific increase in FAs elongated in the cytosol becomes also apparent, when the weight percentages (weight %) of the individual FAs are calculated (Table 4). In wt plants the weight % of TAG FAs elongated in the plastids (up to C18) is 76.6% against 23.4% for the TAG FAs elongated in the cytosol.
  • Total TAG content of dry seeds obtained from wt (white bar) and transgenic BioOil4 plants (black bars) is shown in Figure 15.
  • Total TAG content is shown in g/mg dry seeds for 1 1 independent transgenic BioOil4 lines. Error bars represent measurements of seeds from 10 different plants per line. An average increase of +4.7% TAG was observed throughout all BioON4 plants. The BioON4 line 4-16 showed an increase of +9.5 % TAG.
  • the SUC5 gene is under the control of its endogenous promoter.
  • the endogenous SUC5 promoter is disclosed in Figure 18.
  • Figure 18 shows the SUC5 promoter sequence as used in constructs used for construction of above BioOil4 plants. Sequences highlighted in grey or dark grey show primers used for amplification of the promoter sequence from genomic Arabidopsis DNA.
  • Biotin levels were measured using the FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins from Molecular Probes (Life Technologies). Levels of free biotin were obtained by homogenizing seed tissue in aqueous solution, total biotin levels (i.e. free biotin plus protein bound biotin) were obtained by hydrolyzing homogenized seed tissue in 2N sulphuric acid. Results show an elevation of total biotin content in the seeds of SUC5-overexpressing plants as shown in Figure 17.
  • SL/C5-mRNA levels were measured by qPCR in developing seeds harvested from siliques of wt or SL/C5-overexpressing lines (BioOil3, BioOil4) at the indicated days after flowering (DAF).
  • ACTIN2 ACT2
  • SUC5 mRNA levels are strongly elevated in the SL/C5-overexpressing lines BIOOH3 and BIOOH4 in contrast to the wt ( Figure 19).
  • SUC5 expression in BioOil3 exceeds wt peak expression of SUC5 already at 4-DAF.
  • SUC5 expression is also increased already at 4-DAF and stays high until 8-DAF, a time when SUC5 expression in the wt has declined to almost zero.
  • sucrose and raffinose levels are unaltered between seeds (>21 -DAF) of wt and the SL/C5-overexpressing lines, indicating that elevated sucrose import during seed development in BioOil3 and BioOM plants rather leads to higher TAG synthesis than to greater carbohydrate storage - see Figure 20.
  • Siliques (8-DAF) from wt and SUC5 overexpressing BioOil3 and BioOM plants were collected and dissected under the binocular with fine forceps. Developing seeds were selected and zygotic embryos in the upturned U stage were transferred into 25 mM sodium phosphate buffer pH 7.0. For every uptake experiment 50 embryos were incubated at 22 °C in 200 ⁇ solution containing 25 mM NaHP0 (pH 5.5) and 2 mM CaCI 2 added with the radiolabeled substrate at the indicated concentration. Incubation time was 6 h for biotin and 90 min for sucrose. After incubation samples were filtered on glass microfibres filters 696 (VWR, Darmstadt, Germany) and washed with an excess of distilled H 2 0. Incorporation of radioactivity was determined by scintillation counting.
  • WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis. Plant J. 50, 825-838.
  • Floral dip a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
  • Pantothenate synthetase is essential but not limiting for pantothenate biosynthesis in Arabidopsis. Plant Mol. Biol. 66, 1 -14.
  • AtSUC3 a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in carpel cell layer. Plant J. 24, 869-882.
  • SUC1 and SUC2 two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein. Plant J. 6, 67-77.
  • AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related At- SUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis eco- types. Plant J. 40, 120-130.
  • Brassicaceae express multiple isoforms of biotin carboxyl carrier protein in a tissue-specific manner. Plant Physiol. 125, 2016-2028.

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Abstract

La présente invention concerne l'utilisation de séquences d'acides nucléiques codant pour un polypeptide du transporteur de sucrose dans des plantes transgéniques. L'invention concerne notamment des procédés d'augmentation de composés apparentés à des acides gras et d'augmentation du niveau d'huile et de modification de la composition en acides gras dans les graines de plantes.
PCT/EP2012/065958 2011-08-18 2012-08-15 Augmentation de l'activité du transporteur de sucrose dans les graines de plantes WO2013024121A2 (fr)

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