WO1998058069A1 - Expression of fructose 1,6 bisphosphate aldolase in transgenic plants - Google Patents

Abstract

Fructose-1,6-bisphosphate aldolase (FDA) is an enzyme reversibly catalyzing the reaction converting triosephosphate into fructose-1,6-bisphosphate. In the leaf, this enzyme is located in the chloroplast (starch synthesis) and the cytosol (sucrose biosynthesis). Transgenic plants were generated that expess the E. coli fda gene in the chloroplast to improve plant yield by increasing leaf starch biosynthetic ability in particular and sucrose production in general. Leaves from plants expressing the fda transgene showed a significantly higher starch accumulation, as compared to control plants expressing the null vector, particularly early in the photoperiod, but had lower leaf sucrose. Transgenic plants also had a significantly higher root mass. Furthermore, transgenic potatoes expressing fda exhibited improved uniformity of solids.

Description

EXPRESSION OF FRUCTOSE 1.6 BISPHOSPHATE

ALDOLASE IN TRANSGENIC PLANTS

This invention relates to the expression of fructose 1,6 bisphosphate aldolase (FDA) in transgenic plants to increase or improve plant growth and development, yield, vigor, stress tolerance, carbon allocation and storage into various storage pools, and distribution of starch. Transgenic plants expressing FDA have increased carbon assimilation, export and storage in plant source and sink organs, which results in growth, yield and quality improvements in crop plants.

Recent advances in genetic engineering have provided the prerequisite tools to transform plants to contain alien (often referred to as "heterologous") or improved endogenous genes. These genes can lead either to an improvement of an already existing pathway in plant tissues or to an introduction of a novel pathway to modify product levels, increase metabolic efficiency, and or save on energy cost to the cell. It is presently possible to produce plants with unique physiological and biochemical traits and characteristics of high agronomic and crop processing importance. Traits that play an essential role in plant growth and development, crop yield potential and stability, and crop quality and composition include enhanced carbon assimilation, efficient carbon storage, and increased carbon export and partitioning.

Atmospheric carbon fixation (photosynthesis) by plants represents the major source of energy to support processes in all living organisms. The primary sites of photosynthetic activity, generally referred to as "source organs", are mature leaves and, to a lesser extent, green stems. The major carbon products of source leaves are starch, which represents the transitory storage form of carbohydrate in the chloroplast, and sucrose, which represents the predominant form of carbon transport in higher plants. Other plant parts named "sink organs" (e.g., roots, fruit, flowers, seeds, tubers, and bulbs) are generally not autotrophic and depend on import of sucrose or other major translocatable carbohydrates for their growth and development. The storage sinks deposit the imported metabolites as sucrose and other oligosaccharides, starch and other polysaccharides, proteins, and triglycerides. In leaves, the primary products of the Calvin Cycle (the biochemical pathway leading to carbon assimilation) are glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), also known as triose phosphates (triose-P). The condensation of G3P and DHAP into fructose 1,6 bisphosphate (FBP) is catalyzed reversibly by the enzyme fructose 1 ,6 bisphosphate aldolase (FDA), and various isozymes are known. The acidic isoenzyme appears to be chloroplastic and comprises about 85% of the total leaf aldolase activity. The basic isoenzyme is cytosolic. Both isoenzymes appear to be encoded by the nuclear genome and are encoded by different genes (Lebherz et al., 1984).

In the leaf, the chloroplast FDA is an essential enzyme in the Calvin Cycle, where its activity generates metabolites for starch biosynthesis. Removal of more than 40% of the plastidic aldolase enzymatic activity by antisense technology reduced leaf starch accumulation as well as soluble proteins and chlorophyll levels but also reduced plant growth and root formation (Sonnewald et al., 1994). In contrast, the cytosolic FDA is part of the sucrose biosynthetic pathway where it catalyzes the reaction of FBP production. Moreover, cytosolic FDA is also a key enzyme in the glycolytic and gluconeogenesis pathways in both source and sink plant tissues. In the potato industry, production of higher starch and uniform solids tubers is highly desirable and valuable. The current potato varieties that are used for french fry production, such as Russet Burbank and Shepody, suffer from a non-uniform deposition of solids between the tuber pith (inner core) and the cortex (outer core). French fry strips that are taken from pith tissue are higher in water content when compared to outer cortex french fry strips; cortex tissue typically displays a solids level of twenty-four percent whereas pith tissue typically displays a solids level of seventeen percent. Consequently, in the french fry production process, the pith strips need to be blanched, dried, and par-fried for longer times to eliminate the excess water. Adequate processing of the pith fries results in the over-cooking of fries from the high solids cortex. The blanching, drying, and par frying times of the french fry processor need to be adjusted accordingly to accommodate the low solids pith strips and the high solids cortex strips. A higher solids potato with a more uniform distribution of starch from pith to cortex would allow for a more uniform finished fty product, with higher plant throughput and cost savings due to reduced blanch, dry and par-fry times. Although various fructose 1,6 bisphosphate aldolases have been previously characterized, it has been discovered that overexpression of the enzyme in a transgenic plant provides advantageous results in the plant such as increasing the assimilation, export and storage of carbon, increasing the production of oils and/or proteins in the plant and improving tuber solids uniformity.

The present invention provides structural DNA constructs that encode a fructose 1 ,6 bisphosphate aldolase (FDA) enzyme and that are useful in increasing carbon assimilation, export, and storage in plants.

In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of producing genetically transformed plants that have elevated carbon assimilation, storage, export, and improved solids uniformity comprising the steps of: (a) Inserting into the genome of a plant a recombinant, double-stranded DNA molecule comprising

(i) a promoter that functions in the cells of a target plant tissue, (ii) a structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1 ,6 bisphosphate aldolase enzyme, (iii) a 3' non-translated DNA sequence that functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence;

(b) obtaining transformed plant cells; and

(c) regenerating from transformed plant cells genetically transformed plants that have elevated FDA activity.

In another aspect of the present invention there is provided a recombinant, double- stranded DNA molecule comprising in sequence

(i) a promoter that functions in the cells of a target plant tissue,

(ii) a structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1,6 bisphosphate aldolase enzyme,

(iii) a 3' non-translated DNA sequence that functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence.

In a further aspect of the present invention, the structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1,6 bisphosphate aldolase enzyme is coupled with a chloroplast transit peptide to facilitate transport of the enzyme to the plastid. In accordance with the present invention, an improved means for increasing carbon assimilation, storage and export in the source tissues of various plants is provided. Further means of improved carbon accumulation in sinks (such as roots, tubers, seeds, stems, and bulbs) are provided, thus increasing the size of various sinks (larger roots, tubers, etc.) and subsequently increasing yield and crop productivity. The increased carbon availability to these sinks would also improve composition and use efficiency in the sink (oil, protein, starch and/or sucrose production, and/or solids uniformity).

Various advantages may be achieved by the aims of the present invention, including: First, increasing the expression of the FDA enzyme in the chloroplast would increase the flow of carbon through the Calvin Cycle and increase atmospheric carbon assimilation during early photoperiod. This would result in an increase in photosynthetic efficiency and an increase in chloroplast starch production (a leaf carbon storage form degraded during periods when photosynthesis is low or absent). Both of these responses would lead to an increase in sucrose production by the leaf and a net increase in carbon export during a given photoperiod. This increase in source capacity is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance.

Second, increasing FDA expression in the cytosol of photosynthetic cells would lead to an increase in sucrose production and export out of source leaves. This increase in source capacity is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance.

Third, expression of FDA in sink tissues can show several desirable traits, such as increased amino acid and/or fatty acid pools via increases in carbon flux through giycolysis (and thus pyruvate levels) in seeds or other sinks and increased starch levels as result of increased production of glucose 6-phosphate in seeds, roots, stems, and tubers . where starch is a major storage nonstructural carbohydrate (reverse giycolysis). This increase in sink strength is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance. Fourth, the invention is particularly desirable for use in the commercial production of foods derived from potatoes. Potatoes used for the production of french fries and other products suffer from a non-uniform distribution of solids between the tuber pith (inner core) and the cortex (outer core). Thus, french fry strips from the pith regions of such tubers have a low solids content and a high water content in comparison to cortex strips from the same tubers. Therefore, the french fry processor attempts to adjust the processing parameters so that the final inner strips are sufficiently cooked while the outer cortex strips are not overcooked. The results of such adjustments, however, are highly variable and may lead to poor quality product. Transgenic potatoes expressing fda will provide to the french-fry and potato chip processor a raw product that consistently displays a higher tuber solids uniformity with acceptable agronomic traits. In the french fry plant production process, inner pith fry strips from higher solids uniformity tubers will require less time to blanch, less time to dry to a specific solids content, and less time to par-fry before freezing and shipping to retail and institutional end-users.

Therefore, with respect to potatoes, the present invention provides 1) a higher quality, more uniform finish fry product in which french fries from all tuber regions, when processed, are nearly the same, 2) a higher through-put in the french fry processing plant due to lower processing times, and 3) processor cost savings due to lower energy input required for lower blanch, dry, and par-fry times. A raw tuber product that displays a higher solids uniformity will also produce a potato chip that has a reduced saddle curl, and a reduced tendency for center bubble, which are undesirable qualities in the potato chip industry. Reduced fat content would also result; this would contribute to improved consumer appeal and lower oil use (and costs) for the processor. The increase in solids uniformity will also translate to an increase in overall tuber solids. For both the french fry and chipping industries, this overall tuber solids increase will also result in higher throughput in the processing plant due to lower processing times, and cost savings due to lower energy input for blanching, drying, par-frying, and finish frying.

Figure 1 shows the nucleotide sequence and deduced amino acid sequence of a fructose 1,6 bisphosphate aldolase gene from E. coli (SΕQ ID No:l).

Figure 2 shows a plasmid map for plant transformation vector pMON 17524.

Figure 3 shows a plasmid map for plant transformation vector pMON 17542. Figure 4 shows the change in diurnal fluctuations of sucrose, glucose, and starch levels in tobacco leaves expressing the fda transgene (pMON17524) and control (pMON17227). The light period is from 7:00 to 19:00 hours. Only fully expanded and non-senescing leaves were sampled.

Figure 5 shows a plasmid map for plant transformation vector pMON 13925.

Figure 6 shows a plasmid map for plant transformation vector pMON17590.

Figure 7 shows a plasmid map for plant transformation vector pMON13936.

Figure 8 shows a plasmid map for plant transformation vector pMON 17581.

Figure 9 shows potato tuber cross-sections of improved solids uniformity Segal Russet Burbank lines (top row) versus unimproved nontransgenic Russet Burbank (bottom row).

This invention is directed to a method for producing plant cells and plants demonstrating an increased or improved growth and development, yield, quality, starch storage uniformity, vigor, and/or stress tolerance. The method utilizes a DNA sequence encoding an fda (fructose 1.6 bisphosphate aldolase) gene integrated in the cellular genome of a plant as the result of genetic engineering and causes expression of the FDA enzyme in the transgenic plant so produced. Plants that overexpress the FDA enzyme exhibit increased carbon flow through the Calvin Cycle and increased atmospheric carbon assimilation during early photoperiod resulting in an increase in photosynthetic efficiency and an increase in starch production. Thus, such plants exhibit higher levels of sucrose production by the leaf and the ability to achieve a net increase in carbon export during a given photoperiod. This increase in source capacity leads to increased plant growth that in turn generates greater biomass and/or increases the size of the sink and ultimately providing greater yields of the transgenic plant. This greater biomass or increased sink size may be evidenced in different ways or plant parts depending on the particular plant species or growing conditions of the plant overexpressing the FDA enzyine. Thus, increased size resulting from overexpression of FDA may be seen in the seed, fruit, stem, leaf, tuber, bulb or other plant part depending upon the plant species and its dominant sink during a particular growth phase and upon the environmental effects caused by certain growing conditions, e.g. drought, temperature or other stresses. Transgenic plants overexpressing FDA may therefore have increased carbon assimilation, export and storage in plant source and sink organs, which results in growth, yield, and uniformity and quality improvements.

Plants overexpressing FDA may also exhibit desirable quality traits such as increased production of starch, oils and/or proteins depending upon the plant species overexpressing the FDA. Thus, overexpression of FDA in a particular plant species may affect or alter the direction of the carbon flux thereby directing metabolite utilization and storage either to starch production, protein production or oil production via the role of FDA in the giycolysis and gluconeogenesis metabolic pathways.

The mechanism whereby the expression of exogenous FDA modifies carbon relationships is believed to derive from source-sink relationships. The leaf tissue is a sucrose source, and if more sucrose resulting from the activity of increased FDA expression is transported to a sink, it results in increased storage carbon (sugars, starch, oil, protein, etc.) or nitrogen (protein, etc.) per given weight of the sink tissue.

The expression in a plant of a gene that exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region, which adds polyadenylate nucleotides to the 3' end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of RNA. This RNA is then used as a template for the production of the protein encoded therein by the cells protein biosynthetic machinery.

A number of promoters that are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and the figwort mosaic virus (FMV) 35S-promoters, the light-inducible promoter from the small subunit of ribulose-l,5-bisphosphate carboxylase (ssRUBISCO), a very abundant plant polypeptide, and the chlorophyll a/b binding protein gene promoters, etc. All of these promoters have been used to create various types of DNA constructs that have been expressed in plants; see, e.g., PCT publication WO 84/02913. Promoters that are known to or are found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant viruses and include, but are not limited to. the enhanced CaMV35S promoter and promoters isolated from plant genes such as ssRUBISCO genes. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of fructose 1 ,6 bisphosphate aldolase enzyme to cause the desired increase in carbon assimilation, export or storage. Expression of the double-stranded DNA molecules of the present invention can be driven by a constitutive promoter, expressing the DNA molecule in all or most of the tissues of the plant. Alternatively, it may be preferred to cause expression of the fda gene in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc. The promoter chosen will have the desired tissue and developmental specificity. Those skilled in the art will recognize that the amount of fructose 1,6 bisphosphate aldolase needed to induce the desired increase in carbon assimilation, export, or storage may vary with the type of plant. Therefore, promoter function should be optimized by selecting a promoter with the desired tissue expression capabilities and approximate promoter strength and selecting a transformant that produces the desired fructose 1,6 bisphosphate aldolase activity or the desired change in metabolism of carbohydrates in the target tissues. This selection approach from the pool of transformants is routinely employed in expression of heterologous structural genes in plants because there is variation between transformants containing the same heterologous gene due to the site of gene insertion within the plant genome (commonly referred to as "position effect"). In addition to promoters that are known to cause transcription (constitutively or tissue- specific) of DNA in plant cells, other promoters may be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed in the target tissues of interest and then isolating the promoter regions by methods known in the art. In particular, it may be desirable to use a bundle sheath cell specific (or cell enhanced expression) promoter for use with C4 plants such as corn, sorghum, and sugarcane to obtain the yield benefits of overexpression of FDA and not use a constitutive promoter or a promoter with mesophyll cell enhanced expression properties.

For the purpose of expressing the^ gene in source tissues of the plant, such as the leaf or stem, it is preferred that the promoters utilized in the double-stranded DNA molecules of the present invention have relatively high expression in these specific tissues. For this purpose, one may also choose from a number of promoters for genes with leaf- specific or leaf-enhanced expression. Examples of such genes known from the literature are the chloroplast glutamine synthetase GS2 from pea (Edwards et al., 1990), the chloroplast fructose- 1 ,6-bisphosphatase (FBPase) from wheat (Lloyd et al., 1991), the nuclear photosynthetic ST-LSl from potato (Stockhaus et al., 1989), and the phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes from Arabidopsis thaliana (Leyva et al., 1995). Also shown to be active in photosynthetically active tissues are the ribulose-l,5-bisphosphate carboxylase (RUBISCO), isolated from eastern larch (Larix la icinά) (Campbell et al., 1994); the cab gene, encoding the chlorophyll a/b-binding protein of PSII, isolated from pine (cab6; Yamamoto et al., 1994), wheat (Cab-1; Fejes et al., 1990), spinach (CAB-1; Luebberstedt et al., 1994), and rice (cablR: Luan et al., 1992); the pyruvate orthophosphate dikinase (PPDK) from maize (Matsuoka et al, 1993); the tobacco Lhcbl*2 gene (Cerdan et al., 1997); the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene (Truernit et al., 1995); and the thylacoid membrane proteins, isolated from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS; Oelmueller et al., 1992). Other chlorophyll a/b-binding proteins have been studied and described in the literature, such as LhcB and PsbP from white mustard (Sinapis alba; Kretsch et al., 1995). Homologous promoters to those described here may also be isolated from and tested in the target or related crop plant by standard molecular biology procedures. For the purpose of expressing the fda in sink tissues of the plant, for example the tuber of the potato plant; the fruit of tomato; or seed of maize, wheat, rice, or barley, it is preferred that the promoters utilized in the double- stranded DNA molecules of the present invention have relatively high expression in these specific tissues. A number of genes with tuber-specific or tuber-enhanced expression are known, including the class I patatin promoter (Bevan et al., 1986; Jefferson et al., 1990); the potato tuber ADPGPP genes, both the large and small subunits (Muller et al., 1990); sucrose synthase (Salanoubat and Belliard, 1987. 1989); the major tuber proteins including the 22 kDa protein complexes and proteinase inhibitors (Hannapel, 1990); the granule bound starch synthase gene (GBSS) (Rohde et al., 1990); and the other class I and II patatins (Rocha-Sosa et al., 1989; Mignery et al., 1988). Other promoters can also be used to express a fructose 1,6 bisphosphate aldolase gene in specific tissues, such as seeds or fruits. The promoter for β- conglycinin (Tierney, 1987) or other seed-specific promoters, such as the napin and phaseolin promoters, can be used to over-express an fda gene specifically in seeds. The zeins are a group of storage proteins found in maize endosperm. Genomic clones for zein genes have been isolated (Pedersen et al., 1982), and the promoters from these clones, including the 15 kDa, 16 kDa, 19 kDa, 22 kDa, 27 kDa, and gamma genes, could also be used to express an fda gene in the seeds of maize and other plants. Other promoters known to function in maize, wheat, or rice include the promoters for the following genes: waxy, Brittle, Shrunken 2, branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins, and sucrose synthases. Particularly preferred promoters for maize endosperm expression, as well as in wheat and rice, of an fda gene is the promoter for a glutelin gene from rice, more particularly the Osgt-1 promoter (Zheng et al., 1993); the maize granule-bound starch synthase (waxy) gene (zmGBS); the rice small subunit ADPGPP promoter (osAGP) ;and the zein promoters, particularly the maize 27 kDa zein gene promoter (zm27) (see, generally, Russell et al., 1997). Examples of promoters suitable for expression of an fda gene in wheat include those for the genes for the ADPglucose pyrophosphorylase (ADPGPP) subunits, for the granule bound and other starch synthases, for the branching and debranching enzymes, for the embryogenesis- abundant proteins, for the gliadins, and for the glutenins. Examples of such promoters in rice include those for the genes for the ADPGPP subunits, for the granule bound and other starch synthases, for the branching enzymes, for the debranching enzymes, for sucrose synthases, and for the glutelins. A particularly preferred promoter is the promoter for rice glutelin, Osgt-1. Examples of such promoters for barley include those for the genes for the ADPGPP subunits, for the granule bound and other starch synthases, for the branching enzymes, for the debranching enzymes, for sucrose synthases, for the hordeins, for the embryo globulins, and for the aleurone-specific proteins. The solids content of root tissue may be increased by expressing an fda gene behind a root-specific promoter. An example of such a promoter is the promoter from the acid chitinase gene (Samac et al., 1990). Expression in root tissue could also be accomplished by utilizing the root-specific subdomains of the CaMV35S promoter that have been identified (Benfey et al., 1989).

The RNA produced by a DNA construct of the present invention may also contain a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs, as presented in the following examples, wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence.

In monocots. an intron is preferably included in the gene construct to facilitate or enhance expression of the coding sequence. Examples of suitable introns include the HSP70 intron and the rice actin intron, both of which are known in the art. Another suitable intron is the castor bean catalase intron (Suzuki et al., 1994) Polyadenylation signal

The 3' non-translated region of the chimeric plant gene contains a polyadenylation signal that functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the RNA. Examples of suitable 3' regions are (1) the 3' transcribed, non- translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein genes and the small subunit of the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene.

Plastid-directed expression of fructose- 1,6-bisphosphate aldolase activity In one embodiment of the invention, the fda gene may be fused to a chloroplast transit peptide, in order to target the FDA protein to the plastid. As used hereinafter, chloroplast and plastid are intended to include the various forms of plastids including amyloplasts. Many plastid-localized proteins are expressed from nuclear genes as precursors and are targeted to the plastid by a chloroplast transit peptide (CTP), which is removed during the import steps. Examples of such chloroplast proteins include the small subunit of ribulose-l,5-biphosphate carboxylase (ssRUBISCO, SSU), 5- enolpyruvateshikimate-3-phosphate synthase (EPSPS), ferredoxin, ferredoxin oxidoreductase, the light-harvesting-complex protein I and protein II, and thioredoxin F. It has been demonstrated that non-plastid proteins may be targeted to the chloroplast by use of protein fusions with a CTP and that a CTP sequence is sufficient to target a protein to the plastid. Those skilled in the art will also recognize that various other chimeric constructs can be made that utilize the functionality of a particular plastid transit peptide to import the fructose- 1 ,6-diphosphate aldolase enzyme into the plant cell plastid. The fda gene could also be targeted to the plastid by transformation of the gene into the chloroplast genome (Daniell et al., 1998). Fructose 1 ,6 bisphosphate aldolases As used herein, the term "fructose 1, 6-bisphophate aldolase" means an enzyme

(E.C. 4.1.2.13) that catalyzes the reversible cleavage of fructose 1,6-bisphosphate to form glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). Aldolase enzymes are divided into two classes, designated class I and class II (Witke and Gotz, 1993). Various fda genes encoding the enzyme have been sequenced, as have numerous proteins, such as the cytosolic enzyme from maize (GenBank Accession S07789;S10638), cytosolic enzyme from rice (GenBank Accession JQ0543), cytosolic enzyme from spinach (GenBank Accession S31091 ;S22093), from Arabidopsis thaliana (GenBank Accession S11958), from spinach chloroplast (GenBank Accession S31090;A21815;S22092), from yeast (S. cerevisiae) (GenBank Accession S07855; S37882; S12945; S39178; S44523 ;X75781 ), from Rhodobacter sphaeroides (GenBank Accession B40767;D41080), from B. subtilis (GenBank Accession S55426; D32354: E32354; D41835), from garden pea (GenBank Accession S29048; S34411), from garden pea chloroplast (GenBank Accession S29047; S34410), from maize (GenBank Accession S05019), from Chlamydomonas reinhardtii (GenBank Accession S48639; S58485; S58486; S34367), from Corynebacterium glutamicum (GenBank Accession S09283; X17313), from

Campylobacter jejuni (GenBank Accession S52413), from Haemophilus influenzae (strain Rd KW20) (GenBank Accession C64074), from Streptococcus pneumonia (GenBank Accession AJ005697), from rice (GenBank Accession X53130), and from the maize anaerobically regulated gene (GenBank Accession XI 2872). The class I enzymes may be isolated from higher eukaryotes, such as animals and plants, and in some prokaryotes, including Peptococcus aerogens, (Lebherz and Rutter, 1973), Lactobacillus casei (London and Kline, 1973), Escherichia coli (Stribling and Perham, 1973), Mycobacterium smegmatis (Bai et al., 1975), and most staphylococcal species (Gotz et al., 1979). The gene for the FDA enzyme may be obtained by known methods and has already been done so for several organisms, such as rabbit (Lai et al., 1974), human (Besmond et al, 1983), rat (Tsutsumi et al., 1984), Trypanosoma brucei (Clayton, 1985), and Arabidopsis thaliana (Chopra et al., 1990). These class I enzymes are invariably tetrameric proteins with a total molecular weight of about 160 kDa and function by imine formation between the substrate and a lysine residue in the active site (Alfounder et al., 1989).

In animal, three class I isozymes, classified as A, B, and C, are expressed in the cytosol of muscle, liver, and brain tissue respectively, and they differ from plant aldolases in their expression and compartmentation patterns (Joh et al., 1986). In the leaves of higher plants, FDA is a class I enzyme, and two different isoenzymes within the class have been documented. One is contained in the chloroplast and the other in the cytosol (Lebherz et al., 1984). The acidic plant isozyme appear to be chloroplastic and comprises about 85% of the total leaf aldolase activity. The basic plant isozyme is cytosolic, and both isozymes appear to be encoded by the nuclear genome and are encoded by different genes (Lebherz et al., 1984).

The class II type aldolases are normally dimeric with molecular mass of approximately 80 kDa, and their activity depends on divalent metal ions. The class II enzymes may be isolated from prokaryotes, such as blue-green algae and bacteria, and eukaryotic green algae and fungi (Baldwin et al., 1978). The gene for the FDA class II enzyme may be obtained by known methods and has already been done so from several organisms including Saccharomyces cerevisiae (Jack and Harris, 1971), Bacillus stear other mophilus (Jack, 1973), and Escherichia coli (Baldwin et al., 1978). It is believed that highly homologous class II fructose 1 , 6-bisphophate aldolases with similar catalyzing activity will also be found in other species of microorganism, such as Saccharomyces {Saccharomyces cerevisiae); Bacillus {Bacillus subtilis); Rhodobacter {Rhodobacter sphaeroides); Plasmodium {Plasmodium falciparium, Plasmodium berghei); Trypanosoma {Trypanosoma brucei); Chlamydomonas {Chlamydomas reinhardtii); Candida {Candida albicans); Corynebacterium {Coryne bacterium glutamicum);

Campylobacter {Campylobacter jejuni); and Haemophilus {Haemophilus. influenza). Such sequences can be readily isolated by methods well known in the art, for example by nucleic acid hybridization. The hybridization properties of a given pair of nucleic acids are an indication of their similarity or identity. Nucleic acid sequences can be selected on the basis of their ability to hybridize with known fda sequences. Low stringency conditions may be used to select sequences with less homology or identity.

One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20°C to about 55°C. High stringency conditions may be used to select for nucleic acid sequences with higher degrees of identity to the disclosed sequences. Conditions typically employed may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N- laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50°C and about 70°C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50°C. The skilled individual will recognize that numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate fda sequences having similarity to fda sequences known in the art and are not limited to those explicitly disclosed herein. Preferably, such an approach is used to isolate fda sequences having greater than about 60% identity with the disclosed E.colifda sequence, more preferably greater than about 70% identity, most preferably greater than about 80% identity.

Depending on growth conditions Euglena gracilis, Chlamydomonas mundana, and Chlamydomomas rheinhardi produce either a class I or a class II aldolase (Cremona, 1968; Russell and Gibbs, 1967; Guerrini et al., 1971).

The isolation of a class llfda gene from E. coli is described in the following examples. Its DNA sequence is given as SΕQ ID NO:l and shown in Figure 1. The amino acid sequence is shown in SΕQ ID NO:2 and shown in Figure 1. This gene can be . used as isolated by inserting it into plant expression vectors suitable for the transformation method of choice as described. The E. coli FDA enzyme has an apparent pH optimum range near pH 7-9 and retains activity in the lower pH range of 5-7 (Baldwin et al., 1978; Alfounder et al, 1989).

Thus, many different genes that encode a fructose 1 ,6 bisphosphate aldolase activity may be isolated and used in the present invention. Synthetic gene construction

A carbohydrate metabolizing enzyme considered in this invention includes any sequence of amino acids, such as protein, polypeptide. or peptide fragment, that demonstrates the ability to catalyze a reaction involved in the synthesis or degradation of starch or sucrose. These can be sequences obtained from a heterologous source, such as algae, bacteria, fungi, and protozoa, or endogenous plant sequences, by which is meant any sequence that can be naturally found in a plant cell, including native (indigenous) plant sequences as well as sequences from plant viruses or plant pathogenic bacteria. It will be recognized by one of ordinary skill in the art that carbohydrate metabolizing enzyme gene sequences may also be modified using standard techniques such as site-specific mutation or PCR, or modification of the sequence may be accomplished by producing a synthetic nucleic acid sequence and will still be considered a carbohydrate biosynthesis enzyme nucleic acid sequence of this invention. For example, "wobble" positions in codons may be changed such that the nucleic acid sequence encodes the same amino acid sequence, or alternatively, codons can be altered such that conservative amino acid substitutions result. In either case, the peptide or protein maintains the desired enzymatic activity and is thus considered part of this invention.

A nucleic acid sequence to a carbohydrate metabolizing enzyme may be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized in whole or in part. The structural gene sequences may be cloned, for example, by isolating genomic DNA from an appropriate source and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR). Alternatively, the gene sequences may be synthesized, either completely or in part, especially where it is desirable to provide plant-preferred sequences. Thus, all or a portion of the desired structural gene may be synthesized using codons preferred by a selected plant host. Plant-preferred codons may be determined, for example, from the codons used most frequently in the proteins expressed in a particular plant host species. Other modifications of the gene sequences may result in mutants having slightly altered activity.

If desired, the gene sequence of the fda gene can be changed without changing the protein sequence in such a manner as may increase expression and thus even more positively affect carbohydrate content in transformed plants. A preferred manner for making the changes in the gene sequence is set out in PCT Publication WO 90/10076. A gene synthesized by following the methodology set out therein may be introduced into plants as described below and result in higher levels of expression of the FDA enzyme. This may be particularly useful in monocots such as maize, rice, wheat, sugarcane, and barley. Combinations with other transgenes

The effect of fda in transgenic plants may be enhanced by combining it with other genes that positively affect carbohydrate assimilation or content, such as a gene encoding for a sucrose phosphorylase as described in PCT Publication WO 96/24679, or ADPGPP genes such as the E. coli glgC gene and its mutant glgClβ. PCT Publication WO 91/19806 discloses how to incorporate the latter gene into many plant species in order to increase starch or solids. Another gene that can be combined with fda to increase carbon assimilation, export or storage is a gene encoding for sucrose phosphate synthase (SPS). PCT Publication WO 92/16631 discloses one such gene and its use in transgenic plants. Plant transformation/regeneration In developing the nucleic acid constructs of this invention, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature, many of which are commercially available. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell. A recombinant DNA molecule of the invention typically includes a selectable marker so that transformed cells can be easily identified and selected from non- transformed cells. Examples of such include, but are not limited to, a neomycin phosphotransferase (nptll) gene (Potrykus et al., 1985), which confers kanamycin resistance. Cells expressing the nptll gene can be selected using an appropriate antibiotic such as kanamycin or G418. Other commonly used selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (tlinchee et al., 1988), which confers glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985); and a methotrexate resistant DHFR gene (Thillet et al., 1988).

Plants that can be made to have enhanced carbon assimilation, increased carbon export and partitioning by practice of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, oil seed rape, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, a vine, watermelon, wheat, yams, and zucchini.

A double-stranded DNA molecule of the present invention containing an fda gene can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al. (1983), Bevan (1984), Klee et al. (1985) and EPO publication 120.516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation. chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen. DNA may also be inserted into the chloroplast genome (Darnell et al, 1998).

A plasmid expression vector suitable for the introduction of an fda gene in monocots using microprojectile bombardment is composed of the following: a promoter that is specific or enhanced for expression in the starch storage tissues in monocots, generally the endosperm, such as promoters for the zein genes found in the maize endosperm (Pedersen et al., 1982); an intron that provides a splice site to facilitate expression of the gene, such as the Hsp70 intron (PCT Publication W093/19189); and a 3' polyadenylation sequence such as the nopaline synthase 3' sequence (NOS 3'; Fraley et al., 1983). This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA. A particularly useful Agrobacterium-hased plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMON530 (Rogers et al., 1987). Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb Stul- Hindlll fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the Smal site is removed by digestion with Xmal, treatment with Klenow polymerase and ligation. Plasmid pMON530 retains all the properties of pMON505 and the CaMV35S-NOS expression cassette and now contains a unique cleavage site for Smal between the promoter and polyadenylation signal.

Binary vector pMON505 is a derivative of pMON200 (Rogers et al, 1987) in which the Ti plasmid homology region, LIH, has been replaced with a 3.8 kb Hindlll to Smal segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski, 1985). This segment contains the RK2 origin of replication, oriV, and the origin of transfer, oriT, for conjugation into Agrobacterium using the tri-parental mating procedure (Horsch and Klee, 1986). Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spectinomycin/streptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of transformants and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts of the vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end of the pTiT37 nopaline-type T-DNA. Southern blot analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the . plant genome, that is, the entire plasmid is the T-DNA that is inserted into the plant genome. One end of the integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences.

Another particularly useful Ti plasmid cassette vector is pMON 17227. This vector is described in PCT Publication WO 92/04449 and contains a gene encoding an enzyme conferring glyphosate resistance (denominated CP4), which is an excellent selection marker gene for many plants, including potato and tomato. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the FMV promoter as described therein. When adequate numbers of cells (or protoplasts) containing the fda gene or cDNA are obtained, the cells (or protoplasts) are regenerated into whole plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), various floral crops, such as sunflower, and nut-bearing trees, such as almonds, cashews, walnuts, and pecans. See, e.g., Ammirato et al. (1984); Shimamoto et al. (1989); Fromm (1990); Vasil et al. (1990); Vasil et al. (1992); Hayashimoto (1990); and Datta et al. (1990). The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.

The term "promoter" or "promoter region" refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase or other factors necessary for start of transcription at the correct site. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its .ability to produce mRNA.

"Regeneration" refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant). "Transformation" refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication.

A "transformed cell" is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell. The term "gene" refers to chromosomal DNA, plasmid DNA, cDNA, synthetic

DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.

"Identity" refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW vl.6 (Thompson et al., 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 50 bases or amino acids in length, the number of matches are divided by 50 and multiplied by 100 to obtain a percent identity. "C-terminal region" refers to the region of a peptide, polypeptide, or protein chain from the middle thereof to the end that carries the amino acid having a free carboxyl group.

The phrase "DNA segment heterologous to the promoter region" means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached.

The term "encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA that encodes any of the enzymes discussed herein.

The term "genome" as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs of the present invention introduced into bacterial host cells can therefore be either chromosomally integrated or plasmid- localized. The term "genome" as it applies to plant cells encompasses noj only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components of the cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized.

The terms "microbe" or "microorganism" refer to algae, bacteria, fungi, and protozoa. The term "mutein" refers to a mutant form of a peptide, polypeptide, or protein.

"N-terminal region" refers to the region of a peptide, polypeptide, or protein chain from the amino acid having a free amino group to the middle of the chain.

"Overexpression" refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.

The term "plastid" refers to the class of plant cell organelles that includes amyloplasts, chloroplasts. chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating and contain what is commonly referred to as the "chloroplast genome," a circular DNA molecule that ranges in size from about 120 kb to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region.

The phrase "simple carbohydrate substrate" means a monosaccharide or an oligosaccharide but not a polysaccharide; simple carbohydrate substrate includes glucose, fructose, sucrose, lactose. More complex carbohydrate substrates commonly used in media such as corn syrup, starch, and molasses can be broken down to simple carbohydrate substrates.

The term "solids" refers to the nonaqueous component of a tuber (such as in potato) or a fruit (such as in tomato) comprised mostly of starch and other polysaccharides, simple carbohydrates, nonstructural carbohydrated, amino acids, and other organic molecules.

The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, truncations, etc., can be made to the methods and genes described herein while not departing from the spirit and scope of the present invention. EXAMPLES EXAMPLE 1 cDNA cloning and overexpression

Unless otherwise stated, basic DNA manipulations and genetic techniques, such as PCR, agarose electrophoresis, restriction digests, ligations, E. coli transformations, colony screens, and Western blots were performed essentially by the protocols described in Sambrook et al. (1989) or Maniatis et al. (1982).

The E. coli fda gene sequence (SEQ ID NO: 1) was obtained from Genbank (Accession Number XI 4682) and nucleotide primers with homology to the 5' and 3' end were designed for PCR amplification. E. coli chromosomal DNA was extracted and the E. coli fda gene was amplified by PCR using the 5' oligonucleotide 5'GGGGCCATGGCTAAGATTTTTGATTTCGTA3' (SEQ ID NO:3) and the 3' oligonucleotide 5'CCCCGAGCTCTTACAGAACGTCGATCGCGTTCAG3' (SEQ ID

NO:4). The PCR cycling conditions were as follows: 94 C, 5 min (1 cycle); addition of polymerase; 94^°C, 1 min.. 60^°C, 1 min., 72^°C, 2 min.30 sec. (35 cycles). The 1.08 kb PCR product was gel purified and ligated into an E.coli expression vector, pMON5723, to form a vector construct that was used for transformation of frozen competent E.coli JM101 cells. The pMON5723 vector contains the E.coli recA promoter and the T7 gene 10 leader (G10L) sequences, which enable high level expression in E.coli (Wong et al., 1988). After induction of the transformed cells, a distinct protein band of about 40 kDa was apparent on an SDS PAGE gel, which correlates with the size of the subunit polypeptide chain of the dimeric aldolase II. It was shown that most of the induced protein was present in the soluble phase. A gel slice containing the highly induced protein was isolated and antibodies were produced in a goat, which was injected with the homogenized gel slice (emulsified in Freund's complete adjuvant).

The fda gene sequence was subsequently cloned into another E.coli expression vector, under the control of the taq promoter. Induction with IPTG of JM101 cells transformed with this vector showed the same 40 kDa overexpressed protein band. This new clone was used in an enzyme assay for FDA activity. Cells transformed with this vector construct were grown in a liquid culture, induced with IPTG, and grown for another 3 hours. Subsequently, a 3 mL cell culture was spun down, dissolved in 1.00mM Tris and sonicated. The cell pellet was spun down, and the crude cell extract supernatant was assayed for FDA activity, using a coupled enzymatic assay as described by Baldwin et al. (1978). This assay was routinely performed at 30 C.

The reaction was performed in a 1 mL final volume in excess presence of the enzymes triosephosphate isomerase (TIM) and alpha-glycerophosphate dehydrogenase (GDH) in a reaction mixture containing final concentrations of lOOmM Tris pH 8.0, 4.75 mM fructose 1,6 bisphosphate, 0.15 mM NADH, 500 U/mL TIM, and 30 U/mL GDH.

The decrease in absorbance at 340nm, after addition of the cell extract supernatant, was recorded using an HP diode array spectrophotometer. One international unit (LU.) of aldolase activity is that causing the oxidation of 2 μmol of NADH/min in this assay system.

Cell extracts containing the vector with the fda sequence showed a substantial increase in aldolase activity (13.1 I.U./mg protein) as compared to cells transformed with the control vector (0.15 I.U./mg protein). The activity was shown to be inhibited by EDTA, known to specifically inhibit class II aldolases. EXAMPLE 2

Plant transformation and fda expression in tobacco Targeting of FDA protein

E.coli fructose 1 ,6 bisphosphate aldolase was targeted to the plastid in plants in order to assess its influence on carbohydrate metabolism and starch biosynthesis in these plant organelles. To accomplish the import of the E.coli aldolase into the plastids, a vector was constructed in which the aldolase was fused to the Arabidopsis small subunit transit peptide (CTP1) (Stark et al, 1992) or the maize small subunit CTP (Russell et al., 1993), creating constructs in which the CTV-fda fusion gene was located between the 35S promoter from the figwort mosaic virus (P-FMV35S; Gowda et al., 1989) and the 3'- nontranslated region of the nopaline synthase gene (NOS 3'; Fraley et al., 1983) sequences. The vector construct containing the expression cassette [P- FMV/CTPl/ ώ/NOS3'] was subsequently used for tobacco protoplast transformation, which was performed as described in Fromm et al. (1987), with the following modifications. Tobacco cultivar Xanthi line D (Txd) cell suspensions were grown in 250- mL flasks, at 25°C and 138 rpm in the dark. For maintenance, a sub-culture volume of 9 mL was removed and added to 40 mL of fresh Txd media containing MS salts, 3% sucrose, 0.2 g/L inositol, 0.13 g/L asparagine, 80 μL of a 50 mg/mL stock of PCPA, 5 μL of a 1 mg/mL stock of kinetin, and 1 mL of lOOOx vitamins (1.3 g/L nicotinic acid, 0.25 g/L thiamine, 0.25 g/L pyridoxine HCL, and 0.25 g/L calcium pantothenate) every 3 to 4 days. Protoplasts were isolated from 1 -day-old suspension cells that came from a 2-day- old culture. Sixteen milliliters of cells were added to 40 mL of fresh Txd media and allowed to grow 24 hours prior to digestion and isolation of the protoplasts. The centrifugation stage for the enzyme mix has been eliminated. The electroporation buffer and protoplast isolation media were filter sterilized rather than autoclaved. The electroporation buffer did not have 4 mM CaCl2 added. The suspension cells were digested in enzyme for 1 hour. Protoplasts were counted on a hemacytometer, counting only the protoplasts that look intact and circular. Bio-rad Gene Pulser cuvettes (catalog # 165-2088) with a 0.4-cm gap and a maximum volume of 0.8 mL were used for the electroporations. Fifty to 100 μg of DNA containing the gene of interest along with 5 μg of internal control DNA containing the luciferase gene were added per cuvette. The final protoplast density at electroporation was 2xlθ6/mL and electroporater settings were a 500 μFarad capacitance and 140 volts on the Bio-rad Gene Pulser. Protoplasts were put on ice after resuspension in electroporation buffer and remained on ice in cuvettes until 10 minutes after electroporation. Protoplasts were added to 7 mL of Txd media + 0.4 M mannitol and conditioning media after electroporation. At this stage coconut water was no longer used. The protoplasts were grown in 1- hour day/night photoperiod regime at 26°C and were spun down and assayed or frozen 20-24 hours after electroporation.

Western blot analysis performed on the protoplast extracts, obtained after transformation, showed processing into the mature FDA in the tobacco protoplasts. Expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the aldolase subunit and the size of the protein also observed after overexpression of the aldolase in E. coli.

The expression cassette [P-FMV/CTPl//tfα/NOS3'] was subsequently cloned into the Notl site of pMON 17227 (described in PCT Publication WO 92/04449), in the same orientation as the selectable marker expression cassette, to form the plant transformation vector pMON17524, as shown in Figure 2 (SEQ ID NO: 5). An additional construct was made and used for tobacco protoplast transformation, fusing the fda gene to the Arabidopsis EPSPS transit peptide (CTP2), which is described in US patent 5,463,175. The transit peptide was cloned (through the Sphl site) into the Sphl site located immediately upstream from the N-terminus of the fda gene sequence in the CTP\-fda fusion (described above). This new CTP2-fda fusion gene was then cloned into a vector between the FMV promoter and the NOS 3' sequences. When this construct containing the CT?2/fda gene sequences was used for tobacco protoplast transformation, expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the aldolase subunit and the size of the protein also observed after overexpression of the aldolase in E. coli.

The Notl cassette [P-FMV/CTP2//dα/NOS3'] from this construct was then cloned into the Notl site of pMON 17227, in the same orientation as the selectable marker expression cassette, to form the plant transformation vector pMON 17542, which is shown in Figure 3 (SΕQ ID NO:6).

For cytoplasmic expression of the FDA in tobacco protoplasts, a construct was made in which the fda gene sequence (without being coupled to a transit peptide) was cloned into a vector backbone, between the FMV promoter and the NOS 3' sequences. Using this construct for tobacco protoplast transformation also showed expression of a protein of the same size, migrating at approximately 40 kDa. fda expression in tobacco plants

Two constructs, containing the fda gene, fused to the Arabidopsis small subunit CTPl (pMON17524) (SΕQ ID NO:5, Figure 2) and the Arabidopsis ΕPSPS (CTP2) transit peptide (pMONl 7542) (SΕQ ID NO:6, Figure 3), were used for tobacco plant transformation, as described in US patent 5,463,175. A vector without the CTV-fda sequences, pMON 17227 (described in PCT Publication WO 92/04449), was used as a negative control. The plant transformation vectors were mobilized into the ABI Agrobacterium strain. Mating of the plant vector into the ABI strain was done by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980). Growth chamber-grown tobacco transformant lines were generated and first screened by Western blot analysis to identify expressors using goat antibody raised against E.coli-expressedfda. Subsequently, for pMON17524-expressing tobacco lines, leaf nonstructural carbohydrates were analyzed (sucrose, glucose, and hydrolyzed starch into glucose) by means of a YSI Instrument, Model 2700 Select Biochemistry Analyzer.

Starting at flowering stage, leaf samples were also taken from these plants and analyzed for diurnal changes in leaf nonstructural carbohydrates. Five hundred milligrams to 1 g fresh tobacco leaf tissue samples were harvested and extracted in 5 mL of hot Na-phosphate buffer (40 g/L NaH₂PO₄ and 10 g/L Na₂H₂PO₄ in double de-ionized water) by homogenization with a Polytron. Test tubes were then placed in an 85°C water bath for 15 minutes. Tubes were centrifuged for 12 minutes at 3000 rpm and the supernatants saved for soluble sugar analysis. The pellet was resuspended in 5 mL of hot Na-phosphate buffer mixed with a Vortex and centrifuged as described above. The supernatant was carefully removed and added to the previous supernatant fraction for soluble sugar (sucrose and glucose) analysis by YSI using appropriate membranes. The starch was extracted from the pellet using the Megazyme Kit (Megazyme,

Australia). To the pellet, 200 μL of 50% ethanol and 3 mL of thermostable alpha-amylase (300U) were added and the mixture vortexed. Samples were then incubated in boiling water for 6 minutes and stirred after 2 and 4 minutes. Tubes were placed in 50°C water bath and 4 mL of 200 mM acetate buffer (pH 4.5) were added followed by 0.1 mL amyloglucosidase (20 U). Incubation occurred for 1 hour. Test tubes were then centrifuged for 15 minutes at 3000 rpm. Aliquots were taken from the supernatant and analyzed for glucose by YSI. The free glucose was adjusted to anhydrous glucose (as it occurs in starch by multiplying by the ratio 162/182). The total volume per tube was 7.1 mL.

As seen in Table 1, expression of the fda gene in tobacco correlated with a significant increase in leaf starch levels. However, referring to Figure 4, when a diurnal profile of starch levels was established in the7#α-expressing leaves, this increase was apparent mainly early in the photoperiod, which is a phase when leaves are known to have peak photosynthetic activity. This increase in starch has no apparent negative effect on the plant because the increased starch is turned over during the dark period. There was no apparent increase in steady state levels of sucrose or glucose in tobacco leaves expressing E.coli fda as compared to the control. Table 1

Leaf Carbohydrate Levels of Plants Expressing the fda Transgene¹ (pMON17524)

High Expressors Low Expressors Negative

(>0.01% total protein) (< 0.01%) Control

(mg/g fresh weight)

STARCH 35.08 ± 2.84 23.25 ± 3.20 16.69 ± 2.92

SUCROSE 0.97 + 0.17 0.86 ± 0.25 0.66 ± 0.19

GLUCOSE 1.88 + 0.17 1.58 ± 0.20 1.68 ± 0.26

Leaf samples were harvested at midday.

A second set of transgenic tobacco plants transformed with the construct pMON 17542 were grown in the greenhouse. Tobacco plants containing a vector without the CTV-fda sequences, pMON 17227, were used as negative control. Of all the pMON17542-lines screened for expression by Western blot analysis, 18 were high expressors (>0.01% of the total cellular protein) and 15 lines were low expressors (<0.01%). Fifteen plants containing the null vector, pMON17227, were used as control. Fully expanded leaves from plants expressing the fda transgene and negative controls were tested for sucrose export by collecting phloem exudate from excised leaf systems. The phloem exudation technique is described in Groussol et al. (1986). Leaves were harvested at 11:30 AM and placed in an exudation medium, containing 5 mM EDTA at pH 6.0, and allowed to exude for a period of 4 hours under full light and high humidity. The exudation solution was immediately analyzed for sucrose level, as described above in the carbohydrate analysis method. As seen in Table 2, a significant increase in sucrose export out of source leaves was observed in plants expressing the fda transgene.

This increase in sucrose export by^α-expressing leaves is an illustration of an increase in source capacity, very likely due to an increased carbon flow through the Calvin Cycle (in response to increased triose-P utilization) and thus an increase in net carbon utilization by the leaf. As seen in Table 2, the increase in sucrose loading in the phloem correlates with the level of fda expression. Table 2 Levels of Sucrose in Phloem Exudate from Excised Leaves of fda Transgenic Tobacco Plants (pMON17542) Water uptake sucrose in phloem exudate

(μl/g F.Wt./h) (ng/leaf) (ng/g F.Wt.)

fda high expressors 320 ± 20 330 ± 60 108 ± 22

fda low expressors 340 ± 10 210 + 10 77 ± 3

Control 390 + 30 160 + 10 56 + 3

Referring to Table 3, preliminary analysis of plant growth and development revealed no significant differences in number of leaves or pods per plant, plant height, stem diameter, or apparent seed weight per plant, between plants expressing the fda gene and the vector control under the specific growing and analysis conditions. However, as seen in Table 4,

plants had a significantly higher root mass. This may be an indication that, under these conditions, roots represented a more dominant sink that attracted excess carbohydrate produced by the source leaves. Furthermore, the present illustration shows that the increase in root mass in the presence of the E.coli fda gene was accomplished with no apparent negative effect on shoot growth, inflorescence, or seed set. Therefore, this increase in root growth and final root dry weight is a desirable plant trait because it would lead to a rapid seedling establishment following germination and greater plant ability to tolerate drought, cold stress, other environmental challenges, and transplanting. In different plants and under different growing conditions, other plant parts (such as seed, fruit, stem, leaf, tuber, bulb, etc.) are expected to show the weight increase ^' observed in tobacco roots overexpressing the fda transgene. Table 3 Assessment of Certain Plant Growth and Development Parameters in Tobacco Expressing the fda Transgene¹ (pMON 17542) #pods/plant #leaves/plant Plant height Seed weight (cm) (g/plant)

high expressors 162 ± 40 25.4 ± 0.8 65.3 ± 3.1 18.8 ± 2.4

Control 156 ± 28 24.4 ± 0.5 65.8 ± 5.1 17.3 ± 2.6

To achieve this analysis, 14 high-expressor lines were compared to 15 control plants. Measurements were made prior to seed harvest (most pods have reached maturity). The number of leaves was confirmed by counting the number of nodes to account for leaf drop.

Table 4 Tobacco Root Dry Weight of Plants Expressing the E.coli fda Transgene¹ (pMON 17542) Root Dry Weight (g/plant)

fda high expressors 64.0 ± 3.9 fda low expressors 62.7 ± 5.4

Control 31.7 ± 1.6

Roots from 5 high and 7 low expressing lines and 6 control plants were excised and washed carefully then placed in a 65°C drying oven for at least 48 hours. Roots were removed from the oven and allowed to equilibrate in the laboratory for 2 hours before dry weight determination. EXAMPLE 3

Plant transformation and fda expression in corn plants

Targeting of FDA protein

Vectors containing the fda gene with and without the plastid targeting peptide were made for transformation in corn and are also suitable for other monocots, including rice, wheat, barley, sugarcane, triticale, etc. For the cytosolic expression of the fda gene in corn plants, a construct was made in which the fda gene sequence was fused to the backbone of a vector containing the enhanced CaMV 35S promoter (e35S; Kay et al., 1987), the HSP70 intron (US patent 5,593,874), and the NOS3' polyadenylation sequence (Fraley et al., 1983). This created a Notl cassette [P-e35S/HSP70 intron//c.α/NOS3'] that was cloned into the Notl site of pMON30460, a monocot transformation vector, to form the plant transformation vector pMON13925, as shown in Figure 5. pMON30460 contains an expression cassette for the selectable marker neomycin phosphotransferase typell gene (nptll) [P-35S/NPTII /NOS3'] and a unique Notl site for cloning the gene of interest. The final vector (pMON13925) was constructed so that the gene of interest and the selectable marker gene were cloned in the same orientation. A vector fragment containing the expression cassettes for these gene sequences could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

For the chloroplast-targeted expression of the fda gene in corn plants, a construct was made in which the fda gene sequence, coupled to the maize RUBISCO small subunit CTP (Russell et al., 1993), was fused to the backbone of a vector containing the enhanced (CaMV) 35S promoter, the HSP70 intron, and the NOS3' polyadenylation sequences. This created a Notl cassette [P-e35S/HSP70 intron/mzSSuCTP//<ώ/NOS3'] that was cloned into the Notl site (in the same orientation as the selectable marker cassette [P-35S/NPTII /NOS3']) of the monocot transformation vector pMON30460, to form the vector pMON 17590, as shown in Figure 6. From this vector a fragment containing the fda gene expression cassette and the selectable marker cassette could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

For the cytosolic endosperm-specific expression of the aldolase gene in corn, the fda gene sequence was cloned into a vector (in the same orientation as the selectable marker cassette^"[P-35S/NPTII /NOS3']) containing the glutelin gene promoter P-osgtl (Zheng et al., 1993), the HSP70 intron, and the NOS3' polyadenylation sequences to form the vector pMON 13936, as shown in Figure 7. From this vector a fragment containing the fda gene expression cassette [P-osgtl/ΗSP70intron/ fi.α/NOS3'] and the selectable marker cassette could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation. Maize plant transformation

Transgenic maize plants transformed with the vectors pMON13925 (described above) or pMON17590 (described above) were produced using microprojectile bombardment, a procedure well-known to the art (Fromm, 1990; Gordon-Kamm et al., 1990; Walters et al., 1992). Embryogenic callus initiated from immature maize embryos was used as a target tissue. Plasmid DNA at lmg/mL in TE buffer was precipitated onto M10 tungsten particles using a calcium chloride / spermidine procedure, essentially as described by Klein et al. (1988). In addition to the gene of interest, the plasmids also contained the neomycin phosphotransferase II gene (nptll) driven by the 35S promoter from Cauliflower Mosaic Virus. The embryogenic callus target tissue was pretreated on culture medium osmotically buffered with 0.2M mannitol plus 0.2M sorbitol for approximately four hours prior to bombardment (Vain et al., 1993). Tissue was bombarded two times with the DNA-coated tungsten particles using the gunpowder version of the BioRad Particle Delivery System (PDS) 1000 device. Approximately 16 hours following bombardment, the tissue was subcultured onto a medium of the same composition except that it contained no mannitol or sorbitol, and it contained an appropriate aminoglycoside antibiotic, such as G418", to select for those cells that contained and expressed the 35S/nptII gene. Actively growing tissue sectors were transferred to fresh selective medium approximately every 3 weeks. About 3 months after bombardment, plants were regenerated from surviving embryogenic callus essentially as described by Duncan and Widholm (1988). Aldolase activity from transgenic maize

In order to measure leaf aldolase activity, corn leaf samples were taken and immediately frozen on dry ice. Aldolase enzyme was extracted from the leaf tissue by grinding the leaf tissue at 4°C in 1.2 mL of the extraction buffer (100 mM Hepes, pH 8.0, 5 mM MgCl₂, 5^"mM MnCl₂, 100 mM KC1, 10 mM DTT, 1% BSA, 1 mM PMSF, 10 . μg/mL leupeptin, 10 μg/mL aprotinin). The extract was centrifuged at 15,000 x g, at 4 C for 3 minutes, and the non-desalted supernatant was assayed for enzyme activity. This extraction method gave about 60% recovery of E. coli FDA activity. Total aldolase activity was determined in 0.98 mL of reaction mixture that consisted of 100 mM EPPS-NaOH, pH 8.5, 1 mM fructose-bisphosphate 0.1 mM NADH, 5 mM MgCl₂, 4 units of alpha-glycerophosphate dehydrogenase, and 15 units of triosephosphate isomerase. The reaction was initiated by addition of 20 μL of leaf extract. The resulting data, generated from a single experiment, are presented in Table 5.

Table 5 Aldolase Activity from Transgenic Maize Leaves

Lines A340/min/20μL Activity %

H99 (control) 0.113 100 pMON 17590 0.233 206 pMON13925 0.251 222

A phenotype was visible in the primary transformants (RO plants) expressing the E. coli FDA when the protein was targeted to the chloroplast. The leaves were chlorotic but seed set was normal. Rl plants were grown in both field and in greenhouse experiments. Starch was not detectable in the leaves using an iodine staining and pollination was delayed. It is believed that the phenotype in these com plants may be the result of the promoter (e35S) used in both the pMON 17590 and pMON13925 vectors not being preferred for causing FDA expression in com. Because e35S is believed to cause mesophyll enhanced expression and the Calvin Cycle in a C4 plant such as com occurs predominantly in the bundle sheath cells, the use of a promoter directing enhanced expression in the bundle sheath cells (such as the ssRUBISCO promoter) may be preferred. Vectors containing such a promoter and driving expression of FDA have been prepared and are being tested in maize.

In particular, the maize RuBISCO small subunit (PmzSSU, a bundle sheath cell- specific promoter) has been used to construct vectors for cell-specific fda expression in maize. A class I aldolase (fdal), an fda without an iron sulfur cluster and with different properties fxomjdall, was utilized to improve carbon metabolism in C4 crops (e.g. maize) . The gene for the class I aldolase was amplified from the genome of Staphylococcus aureus and activity was comfirmed. Transformation vectors were then constructed to express both classes of aldolase (fdal and fdαll) in a cell-specific manner in maize. The following cassettes have been made: pMON13899: PmzSSU/hsp70/mzSSU CTP/fdαl pMON13990PmzSSU/hsp70/mzSSU CTP/t /α/7 pMON13988:P35S/hsp70//rf /.

These vectors were used for com transformation as described generally above. The biochemical and physiological analysis of the primary transformants should allow for the identification of aldolase gene overexpression that will lead to increase growth and development and yield in maize.

Also, two vectors were used for transformation of com which would target the expression of the E. coli fda II gene in the maize endosperm. The vector pMON 13936 uses the rice gtl promoter to drive expression of aldolase in the cytoplasm of the endosperm cells. Another vector (pMON 36416) uses the same promoter with the maize RuBISCO small subunit transit peptide to localize the protein in the amyloplasts. Homozygous lines of the cytosolic aldolase transformants have been identified (Homozygosity of 37 plants was confirmed using western blot analysis) and seed from these plants were collected for grain composition analysis (moisture, protein, starch, and oil). Of the 53 pMON 36416 primary transformants screened for amylopast-targeted aldolase expression, 1 1 were positive. These plants will be tested for homozygosity selection/propagation and kernels from the homozygotes will be used for composition analysis. EXAMPLE 4 Plant transformation and fda expression in potato plants Targeting of fda expression

The plant expression vector, pMON17542 (described earlier), in which the fda gene is expressed behind the FMV promoter and the aldolase enzyme is fused to the chloroplast transit peptide CTP2, was used for Agrobacterium-mediated potato transformation. A second potato transformation vector was constructed by cloning the Notl cassette [P-FMV/CTP2//dα/NOS3'] (described earlier) into the unique Notl site of . pMON23616. pMON23616 is a potato transformation vector containing the nopaline-type T-DNA right border region (Fraley et al., 1985), an expression cassette for the neomycin phosphotransferase typell gene [P-35S/NPTII /NOS3'] (selectable marker), a unique Notl site for cloning the gene expression cassette of interest, and the T-DNA left border region (Barker et al., 1983). Cloning of the Notl cassette [P-FMV/CTP2//tfα/NOS3'j (described earlier) into the Notl site of pMON23616 results in the potato transformation vector pMON 17581, as shown in Figure 8. The vector pMON17581 was constructed such that the gene of interest and the selectable marker gene were transcribed in the same direction. Potato plant transformation

The plant transformation vectors were mobilized into the ABI Agrobacterium strain. Mating of the plant vector into the ABI strain was done by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980). The vector pMON 17542 was used for potato transformation via Agrobacterium transformation of Russet Burbank potato callus, following the method described in PCT Publication WO 96/03513 for glyphosate selection of transformed lines. After transformation with the vector pMONl 7542, transgenic potato plantlets that came through selection on glyphosate were screened for expression of E. coli aldolase by leaf Western blot analysis. Out of 1 12 independent lines assayed. 50yύ.α-expressing lines (45%o) were identified, with fda expression levels ranging between 0.12% and 1.2 % of total extractable protein. The plant transformation vector PMON 17581 was used for Agrobacterium- mediated transformation of HS31-638 potato callus. HS31-638 is a Russet Burbank potato line previously transformed with the mutant ADPglucose pyrophosphorylase (glgCIό) gene from E.coli (U.S. Patent 5,498,830). The potato callus was transformed following the method described in PCT Publication WO 96/03513, substituting kanamycin (administered at a concentration of 150-200 mg/L) for glyphosate as a selective agent. The transgenic potato plants were screened for expression of the fda gene by assaying leaf punches from tissue culture plantlets. Western blot analysis (using antibodies raised against the E. coli aldolase) of leaf tissue from the pMON 17581 -transformed lines identified 12 expressing lines out of 56 lines screened. Expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the E. coli (classll) aldolase subunit and the size of the protein observed after overexpression of the aldolase in E. coli. Specific gravity measurements of transgenic potato plants

From the 50^ -expressing potato lines obtained after transformation with pMON 17542, 7 of the highest expressing lines were micropropagated in tissue culture, and 8 copies of each line were planted in pots 14 inches in diameter and 12 inches deep, containing a mixture of: Vi Metro 350 potting media, VΛ fine sand, ! Ready Earth potting media. Wild-type Russet Burbank plantlets from tissue culture were planted as controls. All plants were cultivated for approximately 5 months in the greenhouse in which daytime temperature was approximately 21-23°C while nighttime temperature was approximately 13 C. Plants were watered every other day throughout their active growing period and fertilized with Peter's 20-20-20 commercial fertilizer once a week, at levels similar to commercial applications. Fertilization was carried out only for the first 2 Vi months, at which point fertilization was stopped completely. Plants were allowed to naturally senesce, and at approximately 50% senescence, tubers were harvested.

For each line at harvest, all tubers from all 8 pots were pooled and a total weight was obtained. Then for each line, tubers 30 g or greater were pooled and specific gravity was determined on this group of tubers. Specific gravity is the weight of the tubers in air divided by the weight in air minus the weight in water. Results of these weight measurements are presented in Table 6.

Table 6 Specific gravity measurements from transgenic potato plants

Line # Total Overall Combined % Increase in Combined Weight of Specific

Weight % Yield Weight Total Weight Tubers over 30g Gravity

Increas of Tubers (Tubers over (% of Total Weight) e over 30g 30g)

RB 6609 4477 67.10% 1.087

40652 5138 neg 1307 neg 25.40% 1.08

4061 1 7170 8.5% 4533 1.3% 63.20% 1.083

40608 7470 13.0% 1070 neg 14.30% 1.081

40632 7776 21.8% 5453 21.8% 70.10% 1.088

40614 8688 31.5% 5468 22.2% 62.90% 1.083

40631 8800 33.2% 6188 38.2% 70.30% 1.084

40610 9746 47.0% 7777 73.0% 80% 1.087

This table summarizes the tuber yield and specific gravity for all seven lines grown in the greenhouse. The results indicate that, in comparison to the control, all but one of the fda lines show an increase in overall tuber yield, and that in four lines, there is a corresponding increase in percentage of tubers that weigh more than 30 g. For combined tubers over 30 g, the percent of total weight is near that of the control, and for two lines is greater than the control. This indicates that five out of the six of the lines show higher overall yield and are not making smaller tubers. In other words, with the increase in overall yield, there is a corresponding increase in percentage of bigger tubers (over 30 g). However, there is no increase in specific gravity of the tubers. In conclusion, it appears that expression of fda in potato produces greater numbers of tubers per plant without a sacrifice in tuber size. This represents a yield benefit in that the farmer could potentially be able to produce the same amount of tubers using less acreage. Similar experiments will also be performed by co-expression of fda with other carbohydrate metabolizing genes, such as glgC16, in order to determine how such combinations will affect tuber yield, tuber solids deposition and overall tuber specific gravity. Aldolase activity from transgenic potato

After being cultivated for 3 months (post planting) in the greenhouse, leaf samples were taken from 6 of the highest / /α-expressing potato lines, obtained after transformation with pMON17542, and assayed for aldolase activity.

In order to measure potato leaf aldolase activity, duplicate leaf samples from each line were taken and immediately frozen on dry ice. Aldolase was extracted from 0.2 g of leaf tissue by grinding at 4°C in 1.2 mL of the extraction buffer: 100 mM Hepes, pH 8.0, 5 mM MgCl₂, 5 mM MnCl₂, 100 mM KCl, 10 mM DTT, 1% BSA, ImM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin. The extract was assayed for aldolase activity as described earlier.

Six independent transgenic potato lines expressing fda were tested for aldolase activity. The expression of fda in leaves is an indicator of the expression in the whole plant because the FMV promoter used to drive expression of the respective encoding DNAs directs gene expression constitutively in most, if not all, tissues of potato plants. Table 7 summarizes the quantitative protein expression data for each of the lines, and the percent activity for each individual line.

Table 7

Aldolase Activity from

Transgenic Russet Burbank Potato Leaves

Exp. #1 Exp. #2 Average

Lines Act (U/gFW) %Act Act (U/gFW) %Act % Activity

Control 4.461 100 4.732 100 100

40608 6.969 156 8.055 170 163

40610 8.489 190 7.326 155 173

40652 5.812 130 6.367 135 132

40632 5.257 118 4.244 90 104

40631 5.764 129 4.968 105 117

40611 5.715 128 5.836 123 126

Solids uniformity in transgenic potato

Twenty-five Russet Burbank lines expressing fda (potato lines designated "Maestro"), obtained after transformation with pMON 17542, and fifteen Russet Burbank Simple Solid lines, also containing g/gC16 (PCT Publication WO 91/19806 and US Patent 5,498,830), expressing fda (potato lines designated "Segal"), obtained after transformation with pMON 17581, were field tested at two different sites. For each field site, 36 plants per line (three repetitions of 12 plants per line) were evaluated for tuber solids distribution. At harvest, tubers were pre-sorted at each field site into a ten to twelve ounce category, and nine tubers from each replicated plot were analyzed in groups of three.

For a typical 10-12 ounce tuber having a diameter of 7-8 cm, starch distribution was evaluated by removing the center longitudinal slice (13 mm) from each tuber. Slices were then peeled and laid flat on a cutting board where the inner tuber region (pith region) was removed by a 14-mm cork punch. The tissue from pith to cortex (perimedullary region) was removed by an up-to-a 2-inch cork punch. The remaining cortex tissue was approximately an 8-mm wide ring from the outermost region of the slice.

Specific gravity was then determined by weighing both the pooled pith punches and pooled cortex punches in air and then in water:

Specific gravity = Air Wt./(Air Wt.-Water Wt.) After calculating specific gravity, solids levels were determined by the following equation:

-214.9206 + (218.1852*Sp. Gravity) The degree of solids uniformity (Solids Uniformity Index) is determined by calculating the pith to cortex solids ratio (pith solids divided by cortex solids). The three groups of three tubers per plot were averaged, at which point the average of three plot replications was calculated per field site.

Analyses of several previous solids uniformity field trials (data not shown) have demonstrated nontransgenic, wild-type Russet Burbank potato to have a typical pith to cortex tuber solids ratio within the range of 68% to 72%, depending on growing region and agricultural practices. Tables 8-1 1 provide the pith to cortex solids ratios by plant line number, with a higher pith to cortex solids ratio indicating a greater degree of solids uniformity.

Tables 8 and 9 represent the data from one field site (site 1) for Segal and Maestro, respectively, and illustrate that the majority of Segal and Maestro lines have higher pith to cortex solids ratios than that of 68.4% for the Russet Burbank control, with some lines approaching an 82% pith to cortex solids ratio.

Tables 10 and 1 1 represent the data from another field site (site 2) for Segal and Maestro, respectively, and also illustrate that the majority of Maestro and Segal lines have higher pith to cortex solids ratios than that of the Russet Burbank control, with some lines approaching an 88% pith to cortex solids ratio. In the site 2 field trial, the Russet Burbank control had an atypical, abnormally high pith-to-cortex solids uniformity ratio of 79.3%, which was most likely due to environmental growing conditions. The site 2 results demonstrate that expression in Russet Burbank potato of E. coli fda, alone or with co- expression of g/gC16, increases tuber solids uniformity even in a growing season when tuber solids uniformity is already extremely high in nontransgenic Russet Burbank. That is, the fda gene continues to perform when agricultural conditions are already conducive to an abnormally high solids uniformity level. Table 8. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio. Segal Russet Burbank Lines. Site 1

Line Ratio

S-29 79.1

S-9 75.8

S-20 71.3

S-15 71.3

S-21 70.5

S-5 70.2

S-18 70.0

RB control 68.4

S-32 68.3

S-16 65.6

Table 9. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio. Maestro Russet Burbank Lines. Site 1

Line Ratio M-13 74.0

M-12 73.6

M-l 73.4

M-3 73.0

M-6 72.4 M-9 71.2

M-l l 70.6

M-l 8 70.5

M-17 69.9

M-l 9 69.4 M-5 69.3

M-20 68.9

RB control 68.4

M-8 68.3

M-43 67.7 M-23 67.3

M-7 67.0

M-39 . 66.6

M-22 66.0

M-10 65.4 M-27 61.4 Table 10. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio Segal Russet Burbank Lines. Site 2

Line Ratio

S-33 87.4

S-54 87.1

S-05 86.8

S-29 85.1

S-21 84.3

S-16 83.2

S-20 81.5

S-18 80.7

S-32 80.6

RB control 79.3

S-09 79.0

Table 11. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio Maestro Russet Burbank Lines. Site 2 Line Ratio

M-04 87.7

M-18 83.9

M-17 83.8

M-03 83.7

M-09 83.4

M-15 83.2

M-29 82.9

M-44 82.3

M-08 82.2

M-43 81.6

M-22 81.1

M-05 80.8

M-01 80.5

M-20 80.2

M-45 79.6

M-39 79.5

M-27 . 79.5

RB control 79.3

M-13 78.9

M-22 78.8

M-l 9 78.7

M-07 78.2

M-12 77.9

M-23 77.3

M-06 76.5

M-10 75.0

M-l l 74.1 The effect of aldolase on pith to cortex solids ratios in the Segal lines is slightly more dramatic than in Maestro lines. We believe this phenotype is due to expression of fda in a background in which the Russet Burbank host expresses glg \6 at a relatively low to moderate level, and that the combination of fda plus glgCλβ provides improved benefits. Cross sectional tuber slices (Figure 9) of three Segal lines with improved solids uniformity illustrate a greater deposition of starch within the inner regions of the tuber. Specifically, an increase in cortex volume accompanied by relocation of the xylem ring towards the center of the tuber, plus a more opaque pith tissue due to an increase in starch density, are evident in the transgenic lines. This physiological alteration may be due to an increase in sucrose translocation from source to sink, which may influence phloem element distribution during tuber development or sucrose availability for starch biosynthesis across the tuber. Example 5 Plant transformation and FDA expression in cotton plants The E. coli fda vectors pMON17524 [FMV/CTPl//dα] (Figure 2) and pMON17542 [FMV/CT?2/fda] (Figure 3) were transformed into cotton using Agrobacterium as described by Umbeck et al. (1987) and in US Patent 5004863. The protein was targeted to the chloroplast using either the Arabidopsis SSU CTP 1 (pMON17524) or the Arabidopsis EPSPS (pMON17542) chloroplast transit peptide. Aldolase expression in cotton

Five-week-old calli transformed with both vectors were analyzed by Western blot analyses and by aldolase assays. Western blot analysis indicated a large amount of protein at the position of the full-length FDA standard and a lesser amount at the same position in the control callus extracts. It appeared that the protein was fully processed. To verify that FDA was expressed in the tissue and for comparison of activity, calli transformed with the two vectors were extracted in a buffer that would prevent loss of activity of the transgene . product. BSA was added to final concentration of 1 mg/mL, which limited the analysis of processing on import by Western blot. Aldolase assays were performed plus or minus 25 mM EDTA, which inhibits the E. coli enzyme but not the plant enzyme. The results of the assays are shown in Table 12. Table 12 Aldolase Activity in Cotton Calli and Cotton Leaf

Δ A340 e"3/mg protein/5 min

Colony# -EDTA +EDTA Fold Increase

Controls

Cotton Leaf (Coker) 4.0 4.2 -

Uninoculated Calli 7.7 5.6 1.3X

Inoculated Calli (E35S/GUS) #1 6.8 6.1 -

#2 3.5 4.0 -

FDA calli pMON 17542 #1 3.5 2.3 1.5X

#3 5.5 2.6 2.1X

#5 9.2 3.8 2.4X

#4 19.8 3.6 5.5X pMON 17524 #2 15.2 5.8 2.6X

#3 12.5 4.0 3. IX

#5 14.4 2.9 4.9X

#6 4.1 1.2 3.5X

The results indicate that there is good expression of the fda gene in cotton callus. Almost all calli had at least twofold higher aldolase activity, and the increase was sensitive to inhibition by EDTA. Processing appeared complete by Western blot analysis using these samples.

REFERENCES CITED

Alefounder et al. (1989) Biochem. J. 257:529-534

Ammirato et al. (1984) Handbook of Plant Cell Culture - Crop Species. Macmillan Publ. Co..

Bai et al. (1975) Arch. Biochem. Biophys. 168: 230-234.

Baldwin et al. (1978) Biochem. J. 169: 633-641

Barker et al. (1983) Plant Mol Biol 2 (6): 335-350.

Benfey et al. (1989) EMBO J. 5: 2195-2202. Besmond et al. (1983) Biochem. Biophys. Res. Commun. 117, 601-609.

Bevan (1984) Nucleic Acids Res. 12 (22): 8711-8721.

Bevan et al. (1986) Nucleic Acids Res. 14 (11):4625-4638.

Campbell et al. (1994) Canadian Journal of Forest Research 24 (8):1689-1693.

Cerdan et al. (1997) Plant Molecular Biology 33 (2): p245-255. Chopra et al. (1990) Plant Molecular Biology 15:517-520.

Clayton (1985) EMBO J. 4, 2997-3003.

Cremona (1968) G. Bot. Ital. 102, 253-259.

Daniell et al. (1998) Nature Biotechnology 16:345-348.

Datta et al. (1990) Bio-technology 8:736-740. Ditta et al. (1980) Proc Natl Acad Sci USA 77(12): 7347-7351.

Duncan and Widholm (1988) Plant Cell Reports 7: 452-455.

Edwards et al. (19901 Proc Natl Acad Sci USA 87 (9): p3459-3463.

Fejes et al. (19901. Plant Mol Biol 15 (6): p921-932.

Fraley et al. (1983) Proc Natl Acad Sci USA 80: 4803-4807. Fraley et al. (1985) Bio/Technology 3 (7): 629-635.

Fromm, M., (1990) UCLA Symposium on Molecular Strategies for Crop Improvement, April 16-22, 1990. Keystone, CO.

Fromm et al. (1987) Methods in Enzymology. 153:351-366.

Gordon-Kamm et al. (1990) Plant Cell 2: 603-618. Gotz et al. (1979) FEMS Microbiol. Lett. 5:253-257.

Gowda et al. (1989). Journal of Cellular Biochemistry supplement 13D, 301 (Abstract).

Groussol et al. (1986) Phvsiologie Vegetale 24(1):123-134. Guerrini et al. (1971) Arch. Biochem. Biophys. 146, 249-255.

Hannapel (1990) Plant Phvsiol. 94: 919-925.

Hayashimoto et al. (1990) Plant Phvsiol. 93:857-863.

Herrera-Estrella et al. (1983) Nature 303:209. Hinchee et al.. Bio/Technology 6:915-922 (1988V

Horsch and Klee. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:4428-4432.

Jack (1973) Ph.D. Dissertation, University of Cambridge.

Jack and Harris (1971) Biochem. J. 124, 680-690.

Jefferson et al. (1990) Plant Mol. Biol. 14: 995-1006. Joh et al. (1986) J. Mol. Biol. 190:401-410.

Kay et al. (1987) Science 236: 1299-1302.

Klee et al. (1985) Bio-Technology 3(7): 637-642.

Klein et al. (1988) Bio/Technology 6: 559-563.

Kretsch et al. (1995) Plant Journal 7 (5): p715-729. Lai et al., (1974) Science 183, 1204-1206.

Lebherz and Rutter (1973) J. Biol. Chem. 248:1650-1659.

Lebherz et al (1984) J. Biol. Chem. 259 (2):1011-1017.

Leyva et al. (1995) Plant Physiology 108(11:39-46.

London and Kline (1973) Bacteriol. Rev. 37:453-478. Lloyd et al. (1991V Mol. Gen. Genet. 225 (2):209-216.

Luan et al. (1992). Plant Cell 4 (8):971-981.

Luebberstedt et al. (1994) Plant Physiology 104 (3):997-1006.

Maniatis et al. (1982) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Matsuoka et al. (1993V Proc. Natl. Acad. Sci. U. S. A. 90(20):9586-9590.

Mignery et al (1988) Gene 62:27-44.

Muller et al (1990) Mol. Gen. Genet. 224:136-146.

Oelmueller et al. (1992). Res. Photosynth.. Proc. Int. Congr. Photosvnth.. 9th,

Volume 3: p219-24. Editor(s): Murata, Norio. Publisher: Kluwer, Dordrecht, Neth.

Pedersen et al. (1982) Cell 29:1015-1026.

Potrvkus et al. (1985V Mol. Gen. Genet. 799:183-188. Rocha-Sosa et al. (1989) EMBO J. 8 (l):23-29.

Rogers et al. (1987) Improved vectors for plant transformation: expression cassette vectors and new selectable markers. In Methods in Enzymology. Edited by R. Wu and L. Grossman. p253-277. San Diego: Academic Press. Rohde et al. (19901 J. Genet. & Breed. 44:311-315. Russell et al. (1993) Plant Cell Reports 13:24-27. Russell and Fromm (1997) Transgenic Research 6 (2):157-168. Russel and Gibbs (1967) Biochim. Biophys. Acta 132, 145-154 Salanoubat and Belliard (1987) Gene 60:47-56. Salanoubat and Belliard (1989) Gene 84:181-185. Samac et al. (19901 Plant Phvsiol. 93:907-914. Sambrook et al. (1989) Molecular cloning: A laboratory manual, 2nd ed., Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, N. Y. Schmidhauser and Helinski. (1985) J. Bacteriol. 164-155. Sonnewald et al. (1994) Plant Cell and Environment 17:649-658. Stalker et al. (19881 J. Biol. Chem. 263:6310-6314. Stark et al. (1992) Science 258: 287-292. Stockhaus et al. (1989). EMBO Journal 8(9):2445-2451. Stribling and Perham (1973) Biochem. J. 131 :833-841. Suzuki et al. (1994) Plant Mol. Biol. 25(31:507-516. Thillet et al. (1988) J. Biol. Chem. 263:12500-12508. Thompson et al. (1994) Nucl. Acids Res. 22:4673-4680. Tiemey et al. (1987) Planta 172:356-363. Truemit et al. (1995) Planta 196 (3):564-570. Tsutsumi et al. (1984) J. Biol. Chem. 259, 14572-14575. Umbeck et al. (1987) Biotechnology. 5, 263-266. . Vain et al. (1993) Plant Cell Reports 12: 84-88. Vasil et al. (1990) Bio/Technology 8:429-434. Vasil et al. (1992) Bio/Technology 10:667-674. Walters et al. (1992) Plant Molecular Biology 18: 189-200.

Witke and Goetz (1993) Journal of Bacteriology 175(22): 7495-7499 .. Wong et al. (1988) Gene 68: 193-203. Yamamoto et al. (1994) Plant and Cell Physiology 35(5):773-778. Zheng et al. (1993) Plant J. 4: 3357-3366.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Gerard Barry

NordineCheikh

Ganesh Kishore

(ii) TITLE OF INVENTION: Expression of Fructose 1,6 Bisphosphate

Aldolase in Transgenic Plants

(iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Arnold, White & Durkee

(B) STREET: P.O. Box 4433

(C) CITY: Houston

(D) STATE: Texas (E) COUNTRY: United States of America

(F) ZIP: 77210-4433

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US Unknown

(B) FILING DATE: Concurrently Herewith

(C) CLASSIFICATION: Unknown

(vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US Prov . App. Serial No. 60/049,995

(B) FILING DATE: June 17, 1997

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Patricia A. Kammerer

(B) REGISTRATION NUMBER: 29,775

(C) REFERENCE/DOCKET NUMBER: MOBT086

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (713) 787-1400

(B) TELEFAX: (713) 787-1440

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1080 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

ATGTCTAAGA TTTTTGATTT CGTAAAACCT GGCGTAATCA CTGGTGATGA CGTACAGAAA 60

GTTTTCCAGG TAGCAAAAGA AAACAACTTC GCACTGCCAG CAGTAAACTG CGTCGGTACT 120 GACTCCATCA ACGCCGTACT GGAAACCGCT GCTAAAGTTA AAGCGCCGGT TATCGTTCAG 180

TTCTCCAACG GTGGTGCTTC CTTTATCGCT GGTAAAGGCG TGAAATCTGA CGTTCCGCAG 240

GGTGCTGCTA TCCTGGGCGC GATCTCTGGT GCGCATCACG TTCACCAGAT GGCTGAACAT 300

TATGGTGTTC CGGTTATCCT GCACACTGAC CACTGCGCGA AGAAACTGCT GCCGTGGATC 360

GACGGTCTGT TGGACGCGGG TGAAAAACAC TTCGCAGCTA CCGGTAAGCC GCTGTTCTCT 420 TCTCACATGA TCGACCTGTC TGAAGAATCT CTGCAAGAGA ACATCGAAAT CTGCTCTAAA 480

TACCTGGAGC GCATGTCCAA AATCGGCATG ACTCTGGAAA TCGAACTGGG TTGCACCGGT 540

GGTGAAGAAG ACGGCGTGGA CAACAGCCAC ATGGACGCTT CTGCACTGTA CACCCAGCCG 600

GAAGACGTTG ATTACGCATA CACCGAACTG AGCAAAATCA GCCCGCGTTT CACCATCGCA 660

GCGTCCTTCG GTAACGTACA CGGTGTTTAC AAGCCGGGTA ACGTGGTTCT GACTCCGACC 720 ATCCTGCGTG ATTCTCAGGA ATATGTTTCC AAGAAACACA ACCTGCCGCA CAACAGCCTG 780 AACTTCGTAT TCCACGGTGG TTCCGGTTCT ACTGCTCAGG AAATCAAAGA CTCCGTAAGC 840

TACGGCGTAG TAAAAATGAA CATCGATACC GATACCCAAT GGGCAACCTG GGAAGGCGTT 900

CTGAACTACT ACAAAGCGAA CGAAGCTTAT CTGCAGGGTC AGCTGGGTAA CCCGAAAGGC 960

GAAGATCAGC CGAACAAGAA ATACTACGAT CCGCGCGTAT GGCTGCGTGC CGGTCAGACT 1020 TCGATGATCG CTCGTCTGGA GAAAGCATTC CAGGAACTGA ACGCGATCGA CGTTCTGTAA 1080

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 359amino acids (B) TYPE: amino

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2

Met Ser Lys lie Phe Asp Phe Val Lys Pro Gly Val lie Thr Gly

5 10 15 Asp Asp Val Gin Lys Val Phe Gin Val Ala Lys Glu Asn Asn Phe

20 25 30

Ala Leu Pro Ala Val Asn Cys Val Gly Thr Asp Ser lie Asn Ala

35 40 45

Val Leu Glu Thr Ala Ala Lys Val Lys Ala Pro Val lie Val Gin 50 55 60

Phe Ser Asn Gly Gly Ala Ser Phe lie Ala Gly Lys Gly Val Lys 65 70 75

Ser Asp Val Pro Gin Gly Ala Ala lie Leu Gly Ala lie Ser Gly 80 85 90 Ala His His Val His Gin Met Ala Glu His Tyr Gly Val Pro Val

95 100 105 lie Leu His Thr Asp His Cys Ala Lys Lys Leu Leu Pro Trp He 110 115 120

Asp Gly Leu Leu Asp Ala Gly Glu Lys His Phe Ala Ala Thr Gly 125 120 135

Lys Pro Leu Phe Ser Ser His Met He Asp Leu Ser Glu Glu Ser 140 145 150

Leu Gin Glu Asn He Glu He Cys Ser Lys Tyr Leu Glu Arg Met 155 160 165

Ser Lys He Gly Met Thr Leu Glu He Glu Leu Gly Cys Thr Gly 170 175 180

Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp Ala Ser Ala 185 190 195

Leu Tyr Thr Gin Pro Glu Asp Val Asp Tyr Ala Tyr Thr Glu Leu 200 205 210 Ser Lys He Ser Pro Arg Phe Thr He Ala Ala Ser Phe Gly Asn 215 220 225

Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr 230 235 240

He Leu Arg Asp Ser Gin Glu Tyr Val Ser Lys Lys His Asn Leu 245 250 255

Pro His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser 260 265 270

Thr Ala Gin Glu He Lys Asp Ser Val Ser Tyr Gly Val Val Lys 275 280 285

Met Asn He Asp Thr Asp Thr Gin Trp Ala Thr Trp Glu Gly Val 290 295 300

Leu Asn Tyr Tyr Lys Ala Asn Glu Ala Tyr Leu Gin Gly Gin Leu 305 310 315

Gly Asn Pro Lys Gly Glu Asp Gin Pro Asn Lys Lys Tyr Tyr Asp 320 325 330 Pro Arg Val Trp Leu Arg Ala Gly Gin Thr Ser Met He Ala Arg

335 340 345

Leu Glu Lys Ala Phe Gin Glu Leu Asn Ala He Asp Val Leu 350 355

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GGGGCCATGG CTAAGATTTT TGATTTCGTA

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCCCGAGCTC TTACAGAACG TCGATCGCGT TCAG (2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10847 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

1 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC

51 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT 101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT

151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG

201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT

251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG

301 GCGTGCATGC TTCACGGTGC AAGCAGCCGT CCAGCAACTG CTCGTAAGTC 351 CTCTGGTCTT TCTGGAACCG TCCGTATTCC AGGTGACAAG TCTATCTCCC

401 ACAGGTCCTT CATGTTTGGA GGTCTCGCTA GCGGTGAAAC TCGTATCACC 451 GGTCTTTTGG AAGGTGAAGA TGTTATCAAC ACTGGTAAGG CTATGCAAGC 501 TATGGGTGCC AGAATCCGTA AGGAAGGTGA TACTTGGATC ATTGATGGTG 551 TTGGTAACGG TGGACTCCTT GCTCCTGAGG CTCCTCTCGA TTTCGGTAAC 601 GCTGCAACTG GTTGCCGTTT GACTATGGGT CTTGTTGGTG TTTACGATTT

651 CGATAGCACT TTCATTGGTG ACGCTTCTCT CACTAAGCGT CCAATGGGTC

701 GTGTGTTGAA CCCACTTCGC GAAATGGGTG TGCAGGTGAA GTCTGAAGAC

751 GGTGATCGTC TTCCAGTTAC CTTGCGTGGA CCAAAGACTC CAACGCCAAT

801 CACCTACAGG GTACCTATGG CTTCCGCTCA AGTGAAGTCC GCTGTTCTGC 851 TTGCTGGTCT CAACACCCCA GGTATCACCA CTGTTATCGA GCCAATCATG

901 ACTCGTGACC ACACTGAAAA GATGCTTCAA GGTTTTGGTG CTAACCTTAC

951 CGTTGAGACT GATGCTGACG GTGTGCGTAC CATCCGTCTT GAAGGTCGTG

1001 GTAAGCTCAC CGGTCAAGTG ATTGATGTTC CAGGTGATCC ATCCTCTACT

1051 GCTTTCCCAT TGGTTGCTGC CTTGCTTGTT CCAGGTTCCG ACGTCACCAT 1101 CCTTAACGTT TTGATGAACC CAACCCGTAC TGGTCTCATC TTGACTCTGC

1151 AGGAAATGGG TGCCGACATC GAAGTGATCA ACCCACGTCT TGCTGGTGGA

1201 GAAGACGTGG CTGACTTGCG TGTTCGTTCT TCTACTTTGA AGGGTGTTAC

1251 TGTTCCAGAA GACCGTGCTC CTTCTATGAT CGACGAGTAT CCAATTCTCG

1301 CTGTTGCAGC TGCATTCGCT GAAGGTGCTA CCGTTATGAA CGGTTTGGAA 1351 GAACTCCGTG TTAAGGAAAG CGACCGTCTT TCTGCTGTCG CAAACGGTCT

1401 CAAGCTCAAC GGTGTTGATT GCGATGAAGG TGAGACTTCT CTCGTCGTGC

1451 GTGGTCGTCC TGACGGTAAG GGTCTCGGTA ACGCTTCTGG AGCAGCTGTC

1501 GCTACCCACC TCGATCACCG TATCGCTATG AGCTTCCTCG TTATGGGTCT

1551 CGTTTCTGAA AACCCTGTTA CTGTTGATGA TGCTACTATG ATCGCTACTA 1601 GCTTCCCAGA GTTCATGGAT TTGATGGCTG GTCTTGGAGC TAAGATCGAA

1651 CTCTCCGACA CTAAGGCTGC TTGATGAGCT CAAGAATTCG AGCTCGGTAC

1701 CGGATCCAGC TTTCGTTCGT ATCATCGGTT TCGACAACGT TCGTCAAGTT

1751 CAATGCATCA GTTTCATTGC GCACACACCA GAATCCTACT GAGTTCGAGT

1801 ATTATGGCAT TGGGAAAACT GTTTTTCTTG TACCATTTGT TGTGCTTGTA 1851 ATTTACTGTG TTTTTTATTC GGTTTTCGCT ATCGAACTGT GAAATGGAAA

1901 TGGATGGAGA AGAGTTAATG AATGATATGG TCCTTTTGTT CATTCTCAAA

1951 TTAATATTAT TTGTTTTTTC TCTTATTTGT TGTGTGTTGA ATTTGAAATT 2001 ATAAGAGATA TGCAAACATT TTGTTTTGAG TAAAAATGTG TCAAATCGTG

2051. GCCTCTAATG ACCGAAGTTA ATATGAGGAG TAAAACACTT GTAGTTGTAC 2101 CATTATGCTT ATTCACTAGG CAACAAATAT ATTTTCAGAC CTAGAAAAGC

2151 TGCAAATGTT ACTGAATACA AGTATGTCCT CTTGTGTTTT AGACATTTAT

2201 GAACTTTCCT TTATGTAATT TTCCAGAATC CTTGTCAGAT TCTAATCATT

2251 GCTTTATAAT TATAGTTATA CTCATGGATT TGTAGTTGAG TATGAAAATA

2301 TTTTTTAATG CATTTTATGA CTTGCCAATT GATTGACAAC ATGCATCAAT 2351 CGACCTGCAG CCACTCGAAG CGGCCGCGTT CAAGCTTGAG CTCAGGATTT

2401 AGCAGCATTC CAGATTGGGT TCAATCAACA AGGTACGAGC CATATCACTT

2451 TATTCAAATT GGTATCGCCA AAACCAAGAA GGAACTCCCA TCCTCAAAGG

2501 TTTGTAAGGA AGAATTCTCA GTCCAAAGCC TCAACAAGGT CAGGGTACAG 2551 AGTCTCCAAA CCATTAGCCA AAAGCTACAG GAGATCAATG AAGAATCTTC

2601 AATCAAAGTA AACTACTGTT CCAGCACATG CATCATGGTC AGTAAGTTTC

2651 AGAAAAAGAC ATCCACCGAA GACTTAAAGT TAGTGGGCAT CTTTGAAAGT

2701 AATCTTGTCA ACATCGAGCA GCTGGCTTGT GGGGACCAGA CAAAAAAGGA 2751 ATGGTGCAGA ATTGTTAGGC GCACCTACCA AAAGCATCTT TGCCTTTATT

2801 GCAAAGATAA AGCAGATTCC TCTAGTACAA GTGGGGAACA AAATAACGTG

2851 GAAAAGAGCT GTCCTGACAG CCCACTCACT AATGCGTATG ACGAACGCAG

2901 TGACGACCAC AAAAGAATTC CCTCTATATA AGAAGGCATT CATTCCCATT 2951 TGAAGGATCA TCAGATACTG AACCAATCCT TCTAGAAGAT CTCCACAATG 3001 GCTTCCTCTA TGCTCTCTTC CGCTACTATG GTTGCCTCTC CGGCTCAGGC

3051 CACTATGGTC GCTCCTTTCA ACGGACTTAA GTCCTCCGCT GCCTTCCCAG

3101 CCACCCGCAA GGCTAACAAC GACATTACTT CCATCACAAG CAACGGCGGA

3151 AGAGTTAACT GCATGCAGGT GTGGCCTCCG ATTGGAAAGA AGAAGTTTGA

3201 GACTCTCTCT TACCTTCCTG ACCTTACCGA TTCCGGTGGT CGCGTCAACT 3251 GCATGCAGGC CATGGCTAAG ATTTTTGATT TCGTAAAACC TGGCGTAATC

3301 ACTGGTGATG ACGTACAGAA AGTTTTCCAG GTAGCAAAAG AAAACAACTT 3351 CGCACTGCCA GCAGTAAACT GCGTCGGTAC TGACTCCATC AACGCCGTAC 3401 TGGAAACCGC TGCTAAAGTT AAAGCGCCGG TTATCGTTCA GTTCTCCAAC 3451 GGTGGTGCTT CCTTTATCGC TGGTAAAGGC GTGAAATCTG ACGTTCCGCA 3501 GGGTGCTGCT ATCCTGGGCG CGATCTCTGG TGCGCATCAC GTTCACCAGA

3551 TGGCTGAACA TTATGGTGTT CCGGTTATCC TGCACACTGA CCACTGCGCG 3601 AAGAAACTGC TGCCGTGGAT CGACGGTCTG TTGGACGCGG GTGAAAAACA 3651 CTTCGCAGCT ACCGGTAAGC CGCTGTTCTC TTCTCACATG ATCGACCTGT 3701 CTGAAGAATC TCTGCAAGAG AACATCGAAA TCTGCTCTAA ATACCTGGAG 3751 CGCATGTCCA AAATCGGCAT GACTCTGGAA ATCGAACTGG GTTGCACCGG

3801 TGGTGAAGAA GACGGCGTGG ACAACAGCCA CATGGACGCT TCTGCACTGT

3851 ACACCCAGCC GGAAGACGTT GATTACGCAT ACACCGAACT GAGCAAAATC

3901 AGCCCGCGTT TCACCATCGC AGCGTCCTTC GGTAACGTAC ACGGTGTTTA

3951 CAAGCCGGGT AACGTGGTTC TGACTCCGAC CATCCTGCGT GATTCTCAGG 4001 AATATGTTTC CAAGAAACAC AACCTGCCGC ACAACAGCCT GAACTTCGTA

4051 TTCCACGGTG GTTCCGGTTC TACTGCTCAG GAAATCAAAG ACTCCGTAAG

4101 CTACGGCGTA GTAAAAATGA ACATCGATAC CGATACCCAA TGGGCAACCT

4151 GGGAAGGCGT TCTGAACTAC TACAAAGCGA ACGAAGCTTA TCTGCAGGGT

4201 CAGCTGGGTA ACCCGAAAGG CGAAGATCAG CCGAACAAGA AATACTACGA 4251 TCCGCGCGTA TGGCTGCGTG CCGGTCAGAC TTCGATGATC GCTCGTCTGG

4301 AGAAAGCATT CCAGGAACTG AACGCGATCG ACGTTCTGTA AGAGCTCGGT

4351 ACCGGATCCA ATTCCCGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA

4401 GATTGAATCC TGTTGCCGGT CTTGCGATGA TTATCATATA ATTTCTGTTG

4451 AATTACGTTA AGCATGTAAT AATTAACATG TAATGCATGA CGTTATTTAT 4501 GAGATGGGTT TTTATGATTA GAGTCCCGCA ATTATACATT TAATACGCGA

4551 TAGAAAACAA AATATAGCGC GCAAACTAGG ATAAATTATC GCGCGCGGTG

4601 TCATCTATGT TACTAGATCG GGGATCGATC CCCGGGCGGC CGCCACTCGA

4651 GTGGTGGCCG CATCGATCGT GAAGTTTCTC ATCTAAGCCC CCATTTGGAC

4701 GTGAATGTAG ACACGTCGAA ATAAAGATTT CCGAATTAGA ATAATTTGTT 4751 TATTGCTTTC GCCTATAAAT ACGACGGATC GTAATTTGTC GTTTTATCAA

4801 AATGTACTTT CATTTTATAA TAACGCTGCG GACATCTACA TTTTTGAATT

4851 GAAAAAAAAT TGGTAATTAC TCTTTCTTTT TCTCCATATT GACCATCATA

4901 CTCATTGCTG ATCCATGTAG ATTTCCCGGA CATGAAGCCA TTTACAATTG

4951 AATATATCCT GCCGCCGCTG CCGCTTTGCA CCCGGTGGAG CTTGCATGTT 5001 GGTTTCTACG CAGAACTGAG CCGGTTAGGC AGATAATTTC CATTGAGAAC

5051 TGAGCCATGT GCACCTTCCC CCCAACACGG TGAGCGACGG GGCAACGGAG

5101 TGATCCACAT GGGACTTTTc CTAGCTTGGC TGCCATTTTT GGGGTGAGGC

5151 CGTTCGCGCG GGGCGCCAGC TGGGGGGATG GGAGGCCCGC GTTACCGGGA

5201 GGGTTCGAGA AGGGGGGGCA CCCCCCTTCG GCGTGCGCGG TCACGCGCCA 5251 GGGCGCAGCC CTGGTTAAAA ACAAGGTTTA TAAATATTGG TTTAAAAGCA

5301 GGTTAAAAGA CAGGTTAGCG GTGGCCGAAA AACGGGCGGA AACCCTTGCA

5351 AATGCTGGAT TTTCTGCCTG TGGACAGCCC CTCAAATGTC AATAGGTGCG

5401 CCCCTCATCT GTCATCACTC TGCCCCTCAA GTGTCAAGGA TCGCGCCCCT 5451 CATCTGTCAG TAGTCGCGCC CCTCAAGTGT CAATACCGCA GGGCACTTAT

5501 CCCCAGGCTT GTCCACATCA TCTGTGGGAA ACTCGCGTAA AATCAGGCGT

5551 TTTCGCCGAT TTGCGAGGCT GGCCAGCTCC ACGTCGCCGG CCGAAATCGA

5601 GCCTGCCCCT CATCTGTCAA CGCCGCGCCG GGTGAGTCGG CCCCTCAAGT 5651 GTCAACGTCC GCCCCTCATC TGTCAGTGAG GGCCAAGTTT TCCGCGTGGT

5701 ATCCACAACG CCGGCGGCCG GCCGCGGTGT CTCGCACACG GCTTCGACGG

5751 CGTTTCTGGC GCGTTTGCAG GGCCATAGAC GGCCGCCAGC CCAGCGGCGA

5801 GGGCAACCAG CCCGGTGAGC GTCGGAAAGG GTCGATCGAC CGATGCCCTT

5851 GAGAGCCTTC AACCCAGTCA GCTCCTTCCG GTGGGCGCGG GGCATGACTA 5901 TCGTCGCCGC ACTTATGACT GTCTTCTTTA TCATGCAACT CGTAGGACAG

5951 GTGCCGGCAG CGCTCTGGGT CATTTTCGGC GAGGACCGCT TTCGCTGGAG

6001 CGCGACGATG ATCGGCCTGT CGCTTGCGGT ATTCGGAATC TTGCACGCCC

6051 TCGCTCAAGC CTTCGTCACT GGTCCCGCCA CCAAACGTTT CGGCGAGAAG

6101 CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT 6151 GGCGTTCGCG ACGCGAGGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG

6201 CTTCCGGCGG CATCGGGATG CCCGCGTTGC AGGCCATGCT GTCCAGGCAG

6251 GTAGATGACG ACCATCAGGG ACAGCTTCAA GGATCGCTCG CGGCTCTTAC

6301 CAGCCTAACT TCGATCACTG GACCGCTGAT CGTCACGGCG ATTTATGCCG

6351 CCTCGGCGAG CACATGGAAC GGGTTGGCAT GGATTGTAGG CGCCGCCCTA 6401 TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA GCCGGGCCAC

6451 CTCGACCTGA ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCACTCC

6501 AAGAATTGGA GCCAATCAAT TCTTGCGGAG AACTGTGAAT GCGCAAACCA

6551 ACCCTTGGCA GAACATATCC ATCGCGTCCG CCATCTCCAG CAGCCGCACG

6601 CGGCGCATCT CGGGCAGCGT TGGGTCCTGG CCACGGGTGC GCATGATCGT 6651 GCTCCTGTCG TTGAGGACCC GGCTAGGCTG GCGGGGTTGC CTTACTGGTT

6701 AGCAGAATGA ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC

6751 AAAACGTCTG CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT

6801 TTCGTAAAGT CTGGAAACGC GGAAGTCAGC GCCCTGCACC ATTATGTTCC

6851 GGATCTGCAT CGCAGGATGC TGCTGGCTAC CCTGTGGAAC ACCTACATCT 6901 GTATTAACGA AGCGCTGGCA TTGACCCTGA GTGATTTTTC TCTGGTCCCG

6951 CCGCATCCAT ACCGCCAGTT GTTTACCCTC ACAACGTTCC AGTAACCGGG

7001 CATGTTCATC ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC

7051 GGTATCATTA CCCCCATGAA CAGAAATTCC CCCTTACACG GAGGCATCAA

7101 GTGACCAAAC AGGAAAAAAC CGCCCTTAAC ATGGCCCGCT TTATCAGAAG 7151 CCAGACATTA ACGCTTCTGG AGAAACTCAA CGAGCTGGAC GCGGATGAAC

7201 AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA GCTTTACCGC

7251 AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA

7301 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC 7351 AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC 7401 ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT TAACTATGCG

7451 GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC

7501 GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT

7551 CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA

7601 GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 7651 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA

7701 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT

7751 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA

7801 AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC

7851 CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 7901 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT

7951 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC

8001 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC

8051 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG

8101 GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT 8151 ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

8201 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG

8251 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

8301 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 8351 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT

8401 CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA

8451 GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

8501 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT 8551 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

8601 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA

8651 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC

8701 TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA

8751 GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGT 8801 CGGGAGCACA GGATGACGCC TAACAATTCA TTCAAGCCGA CACCGCTTCG

8851 CGGCGCGGCT TAATTCAGGA GTTAAACATC ATGAGGGAAG CGGTGATCGC

8901 CGAAGTATCG ACTCAACTAT CAGAGGTAGT TGGCGTCATC GAGCGCCATC

8951 TCGAACCGAC GTTGCTGGCC GTACATTTGT ACGGCTCCGC AGTGGATGGC

9001 GGCCTGAAGC CACACAGTGA TATTGATTTG CTGGTTACGG TGACCGTAAG 9051 GCTTGATGAA ACAACGCGGC GAGCTTTGAT CAACGACCTT TTGGAAACTT

9101 CGGCTTCCCC TGGAGAGAGC GAGATTCTCC GCGCTGTAGA AGTCACCATT

9151 GTTGTGCACG ACGACATCAT TCCGTGGCGT TATCCAGCTA AGCGCGAACT

9201 GCAATTTGGA GAATGGCAGC GCAATGACAT TCTTGCAGGT ATCTTCGAGC

9251 CAGCCACGAT CGACATTGAT CTGGCTATCT TGCTGACAAA AGCAAGAGAA 9301 CATAGCGTTG CCTTGGTAGG TCCAGCGGCG GAGGAACTCT TTGATCCGGT

9351 TCCTGAACAG GATCTATTTG AGGCGCTAAA TGAAACCTTA ACGCTATGGA

9401 ACTCGCCGCC CGACTGGGCT GGCGATGAGC GAAATGTAGT GCTTACGTTG

9451 TCCCGCATTT GGTACAGCGC AGTAACCGGC AAAATCGCGC CGAAGGATGT

9501 CGCTGCCGAC TGGGCAATGG AGCGCCTGCC GGCCCAGTAT CAGCCCGTCA 9551 TACTTGAAGC TAGGCAGGCT TATCTTGGAC AAGAAGATCG CTTGGCCTCG

9601 CGCGCAGATC AGTTGGAAGA ATTTGTTCAC TACGTGAAAG GCGAGATCAC

9651 CAAGGTAGTC GGCAAATAAT GTCTAACAAT TCGTTCAAGC CGACGCCGCT

9701 TCGCGGCGCG GCTTAACTCA AGCGTTAGAT GCTGCAGGCA TCGTGGTGTC

9751 ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 9801 GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC

9851 GGTCCTCCGA TCGAGGATTT TTCGGCGCTG CGCTACGTCC GCKACCGCGT

9901 TGAGGGATCA AGCCACAGCA GCCCACTCGA CCTCTAGCCG ACCCAGACGA

9951 GCCAAGGGAT CTTTTTGGAA TGCTGCTCCG TCGTCAGGCT TTCCGACGTT

10001 TGGGTGGTTG AACAGAAGTC ATTATCGTAC GGAATGCCAA GCACTCCCGA 10051 GGGGAACCCT GTGGTTGGCA TGCACATACA AATGGACGAA CGGATAAACC

10101 TTTTCACGCC CTTTTAAATA TCCGTTATTC TAATAAACGC TCTTTTCTCT

10151 TAGGTTTACC CGCCAATATA TCCTGTCAAA CACTGATAGT TTAAACTGAA

10201 GGCGGGAAAC GACAATCTGA TCCCCATCAA GCTTGAGCTC AGGATTTAGC

10251 AGCATTCCAG ATTGGGTTCA ATCAACAAGG TACGAGCCAT ATCACTTTAT 10301 TCAAATTGGT ATCGCCAAAA CCAAGAAGGA ACTCCCATCC TCAAAGGTTT

10351 GTAAGGAAGA ATTCTCAGTC CAAAGCCTCA ACAAGGTCAG GGTACAGAGT

10401 CTCCAAACCA TTAGCCAAAA GCTACAGGAG ATCAATGAAG AATCTTCAAT

10451 CAAAGTAAAC TACTGTTCCA GCACATGCAT CATGGTCAGT AAGTTTCAGA

10501 AAAAGACATC CACCGAAGAC TTAAAGTTAG TGGGCATCTT TGAAAGTAAT 10551 CTTGTCAACA TCGAGCAGCT GGCTTGTGGG GACCAGACAA AAAAGGAATG

10601 GTGCAG^'AATT GTTAGGCGCA CCTACCAAAA GCATCTTTGC CTTTATTGCA

10651 AAGATAAAGC AGATTCCTCT AGTACAAGTG GGGAACAAAA TAACGTGGAA 10701 AAGAGCTGTC CTGACAGCCC ACTCACTAAT GCGTATGACG AACGCAGTGA

10751. CGACCACAAA AGAATTCCCT CTATATAAGA AGGCATTCAT TCCCATTTGA 10801 AGGATCATCA GATACTGAAC CAATCCTTCT AGAAGATCTA AGCTTAT

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10901 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

1 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC 51 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT

101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT

151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG

201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT

251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG 301 GCGTGCATGC TTCACGGTGC AAGCAGCCGT CCAGCAACTG CTCGTAAGTC

351 CTCTGGTCTT TCTGGAACCG TCCGTATTCC AGGTGACAAG TCTATCTCCC

401 ACAGGTCCTT CATGTTTGGA GGTCTCGCTA GCGGTGAAAC TCGTATCACC

451 GGTCTTTTGG AAGGTGAAGA TGTTATCAAC ACTGGTAAGG CTATGCAAGC

501 TATGGGTGCC AGAATCCGTA AGGAAGGTGA TACTTGGATC ATTGATGGTG 551 TTGGTAACGG TGGACTCCTT GCTCCTGAGG CTCCTCTCGA TTTCGGTAAC

601 GCTGCAACTG GTTGCCGTTT GACTATGGGT CTTGTTGGTG TTTACGATTT

651 CGATAGCACT TTCATTGGTG ACGCTTCTCT CACTAAGCGT CCAATGGGTC

701 GTGTGTTGAA CCCACTTCGC GAAATGGGTG TGCAGGTGAA GTCTGAAGAC

751 GGTGATCGTC TTCCAGTTAC CTTGCGTGGA CCAAAGACTC CAACGCCAAT 801 CACCTACAGG GTACCTATGG CTTCCGCTCA AGTGAAGTCC GCTGTTCTGC

851 TTGCTGGTCT CAACACCCCA GGTATCACCA CTGTTATCGA GCCAATCATG

901 ACTCGTGACC ACACTGAAAA GATGCTTCAA GGTTTTGGTG CTAACCTTAC

951 CGTTGAGACT GATGCTGACG GTGTGCGTAC CATCCGTCTT GAAGGTCGTG

1001 GTAAGCTCAC CGGTCAAGTG ATTGATGTTC CAGGTGATCC ATCCTCTACT 1051 GCTTTCCCAT TGGTTGCTGC CTTGCTTGTT CCAGGTTCCG ACGTCACCAT

1101 CCTTAACGTT TTGATGAACC CAACCCGTAC TGGTCTCATC TTGACTCTGC

1151 AGGAAATGGG TGCCGACATC GAAGTGATCA ACCCACGTCT TGCTGGTGGA

1201 GAAGACGTGG CTGACTTGCG TGTTCGTTCT TCTACTTTGA AGGGTGTTAC

1251 TGTTCCAGAA GACCGTGCTC CTTCTATGAT CGACGAGTAT CCAATTCTCG 1301 CTGTTGCAGC TGCATTCGCT GAAGGTGCTA CCGTTATGAA CGGTTTGGAA

1351 GAACTCCGTG TTAAGGAAAG CGACCGTCTT TCTGCTGTCG CAAACGGTCT

1401 CAAGCTCAAC GGTGTTGATT GCGATGAAGG TGAGACTTCT CTCGTCGTGC

1451 GTGGTCGTCC TGACGGTAAG GGTCTCGGTA ACGCTTCTGG AGCAGCTGTC

1501 GCTACCCACC TCGATCACCG TATCGCTATG AGCTTCCTCG TTATGGGTCT 1551 CGTTTCTGAA AACCCTGTTA CTGTTGATGA TGCTACTATG ATCGCTACTA

1601 GCTTCCCAGA GTTCATGGAT TTGATGGCTG GTCTTGGAGC TAAGATCGAA

1651 CTCTCCGACA CTAAGGCTGC TTGATGAGCT CAAGAATTCG AGCTCGGTAC

1701 CGGATCCAGC TTTCGTTCGT ATCATCGGTT TCGACAACGT TCGTCAAGTT

1751 CAATGCATCA GTTTCATTGC GCACACACCA GAATCCTACT GAGTTCGAGT 1801 ATTATGGCAT TGGGAAAACT GTTTTTCTTG TACCATTTGT TGTGCTTGTA

1851 ATTTACTGTG TTTTTTATTC GGTTTTCGCT ATCGAACTGT GAAATGGAAA

1901 TGGATGGAGA AGAGTTAATG AATGATATGG TCCTTTTGTT CATTCTCAAA

1951 TTAATATTAT TTGTTTTTTC TCTTATTTGT TGTGTGTTGA ATTTGAAATT

2001 ATAAGAGATA TGCAAACATT TTGTTTTGAG TAAAAATGTG TCAAATCGTG 2051 GCCTCTAATG ACCGAAGTTA ATATGAGGAG TAAAACACTT GTAGTTGTAC

2101 CATTATGCTT ATTCACTAGG CAACAAATAT ATTTTCAGAC CTAGAAAAGC

2151 TGCAAATGTT ACTGAATACA AGTATGTCCT CTTGTGTTTT AGACATTTAT

2201 GAACTTTCCT TTATGTAATT TTCCAGAATC CTTGTCAGAT TCTAATCATT

2251 GCTTTATAAT TATAGTTATA CTCATGGATT TGTAGTTGAG TATGAAAATA 2301 TTTTTTAATG CATTTTATGA CTTGCCAATT GATTGACAAC ATGCATCAAT

2351 CGACCTGCAG CCACTCGAAG CGGCCGCGTT CAAGCTTGAG CTCAGGATTT

2401 AGCAGCATTC CAGATTGGGT TCAATCAACA AGGTACGAGC CATATCACTT

2451 TATTCAAATT GGTATCGCCA AAACCAAGAA GGAACTCCCA TCCTCAAAGG

2501 TTTGTAAGGA AGAATTCTCA GTCCAAAGCC TCAACAAGGT CAGGGTACAG 2551 AGTCTCCAAA CCATTAGCCA AAAGCTACAG GAGATCAATG AAGAATCTTC

2601 AATCAAAGTA AACTACTGTT CCAGCACATG CATCATGGTC AGTAAGTTTC

2651 AGAAAAAGAC ATCCACCGAA GACTTAAAGT TAGTGGGCAT CTTTGAAAGT

2701 AATCTTGTCA ACATCGAGCA GCTGGCTTGT GGGGACCAGA CAAAAAAGGA 2751 ATGGTGCAGA ATTGTTAGGC GCACCTACCA AAAGCATCTT TGCCTTTATT

2801 GCAAAGATAA AGCAGATTCC TCTAGTACAA GTGGGGAACA AAATAACGTG

2851 GAAAAGAGCT GTCCTGACAG CCCACTCACT AATGCGTATG ACGAACGCAG

2901 TGACGACCAC AAAAGAATTC CCTCTATATA AGAAGGCATT CATTCCCATT 2951 TGAAGGATCA TCAGATACTG AACCAATCCT TCTAGAAGAT CTAAGCTTAT

3001 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC

3051 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT

3101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT

3151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG 3201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT

3251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG

3301 GCGTGCATGC AGGCcatggC TAAGATTTTT GATTTCGTAA AACCTGGCGT

3351 AATCACTGGT GATGACGTAC AGAAAGTTTT CCAGGTAGCA AAAGAAAACA

3401 ACTTCGCACT GCCAGCAGTA AACTGCGTCG GTACTGACTC CATCAACGCC 3451 GTACTGGAAA CCGCTGCTAA AGTTAAAGCG CCGGTTATCG TTCAGTTCTC

3501 CAACGGTGGT GCTTCCTTTA TCGCTGGTAA AGGCGTGAAA TCTGACGTTC

3551 CGCAGGGTGC TGCTATCCTG GGCGCGATCT CTGGTGCGCA TCACGTTCAC

3601 CAGATGGCTG AACATTATGG TGTTCCGGTT ATCCTGCACA CTGACCACTG

3651 CGCGAAGAAA CTGCTGCCGT GGATCGACGG TCTGTTGGAC GCGGGTGAAA 3701 AACACTTCGC AGCTACCGGT AAGCCGCTGT TCTCTTCTCA CATGATCGAC

3751 CTGTCTGAAG AATCTCTGCA AGAGAACATC GAAATCTGCT CTAAATACCT

3801 GGAGCGCATG TCCAAAATCG GCATGACTCT GGAAATCGAA CTGGGTTGCA

3851 CCGGTGGTGA AGAAGACGGC GTGGACAACA GCCACATGGA CGCTTCTGCA

3901 CTGTACACCC AGCCGGAAGA CGTTGATTAC GCATACACCG AACTGAGCAA 3951 AATCAGCCCG CGTTTCACCA TCGCAGCGTC CTTCGGTAAC GTACACGGTG

4001 TTTACAAGCC GGGTAACGTG GTTCTGACTC CGACCATCCT GCGTGATTCT

4051 CAGGAATATG TTTCCAAGAA ACACAACCTG CCGCACAACA GCCTGAACTT

4101 CGTATTCCAC GGTGGTTCCG GTTCTACTGC TCAGGAAATC AAAGACTCCG

4151 TAAGCTACGG CGTAGTAAAA ATGAACATCG ATACCGATAC CCAATGGGCA 4201 ACCTGGGAAG GCGTTCTGAA CTACTACAAA GCGAACGAAG CTTATCTGCA

4251 GGGTCAGCTG GGTAACCCGA AAGGCGAAGA TCAGCCGAAC AAGAAATACT

4301 ACGATCCGCG CGTATGGCTG CGTGCCGGTC AGACTTCGAT GATCGCTCGT

4351 CTGGAGAAAG CATTCCAGGA ACTGAACGCG ATCGACGTTC TGTAAGAGCT

4401 CGGTACCGGA TCCAATTccc GATCGTTCAA ACATTTGGCA ATAAAGTTTC 4451 TTAAGATTGA ATCCTGTTGC CGGTCTTGCG ATGATTATCA TATAATTTCT

4501 GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC ATGACGTTAT

4551 TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA CATTTAATAC

4601 GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC

4651 GGTGTCATCT ATGTTACTAG ATCGGGGATC GATCCCCGGG CGGCCGCCAC 4701 TCGAGTGGTG GCCGCATCGA TCGTGAAGTT TCTCATCTAA GCCCCCATTT

4751 GGACGTGAAT GTAGACACGT CGAAATAAAG ATTTCCGAAT TAGAATAATT

4801 TGTTTATTGC TTTCGCCTAT AAATACGACG GATCGTAATT TGTCGTTTTA

4851 TCAAAATGTA CTTTCATTTT ATAATAACGC TGCGGACATC TACATTTTTG

4901 AATTGAAAAA AAATTGGTAA TTACTCTTTC TTTTTCTCCA TATTGACCAT 4951 CATACTCATT GCTGATCCAT GTAGATTTCC CGGACATGAA GCCATTTACA

5001 ATTGAATATA TCCTGCCGCC GCTGCCGCTT TGCACCCGGT GGAGCTTGCA

5051 TGTTGGTTTC TACGCAGAAC TGAGCCGGTT AGGCAGATAA TTTCCATTGA

5101 GAACTGAGCC ATGTGCACCT TCCCCCCAAC ACGGTGAGCG ACGGGGCAAC

5151 GGAGTGATCC ACATGGGACT TTTCCTAGCT TGGCTGCCAT TTTTGGGGTG 5201 AGGCCGTTCG CGCGGGGCGC CAGCTGGGGG GATGGGAGGC CCGCGTTACC

5251 GGGAGGGTTC GAGAAGGGGG GGCACCCCCC TTCGGCGTGC GCGGTCACGC

5301 GCCAGGGCGC AGCCCTGGTT AAAAACAAGG TTTATAAATA TTGGTTTAAA

5351 AGCAGGTTAA AAGACAGGTT AGCGGTGGCC GAAAAACGGG CGGAAACCCT

5401 TGCAAATGCT GGATTTTCTG CCTGTGGACA GCCCCTCAAA TGTCAATAGG 5451 TGCGCCCCTC ATCTGTCATC ACTCTGCCCC TCAAGTGTCA AGGATCGCGC

5501 CCCTCATCTG TCAGTAGTCG CGCCCCTCAA GTGTCAATAC CGCAGGGCAC

5551 TTATCCCCAG GCTTGTCCAC ATCATCTGTG GGAAACTCGC GTAAAATCAG

5601 GCGTTTTCGC CGATTTGCGA GGCTGGCCAG CTCCACGTCG CCGGCCGAAA 5651 TCGAGCCTGC CCCTCATCTG TCAACGCCGC GCCGGGTGAG TCGGCCCCTC

5701 AAGTGTCAAC GTCCGCCCCT CATCTGTCAG TGAGGGCCAA GTTTTCCGCG

5751 TGGTATCCAC AACGCCGGCG GCCGGCCGCG GTGTCTCGCA CACGGCTTCG

5801 ACGGCGTTTC TGGCGCGTTT GCAGGGCCAT AGACGGCCGC CAGCCCAGCG 5851 GCGAGGGCAA CCAGCCCGGT GAGCGTCGGA AAGGGTCGAT CGACCGATGC

5901 CCTTGAGAGC CTTCAACCCA GTCAGCTCCT TCCGGTGGGC GCGGGGCATG

5951 ACTATCGTCG CCGCACTTAT GACTGTCTTC TTTATCATGC AACTCGTAGG

6001 ACAGGTGCCG GCAGCGCTCT GGGTCATTTT CGGCGAGGAC CGCTTTCGCT

6051 GGAGCGCGAC GATGATCGGC CTGTCGCTTG CGGTATTCGG AATCTTGCAC 6101 GCCCTCGCTC AAGCCTTCGT CACTGGTCCC GCCACCAAAC GTTTCGGCGA

6151 GAAGCAGGCC ATTATCGCCG GCATGGCGGC CGACGCGCTG GGCTACGTCT

6201 TGCTGGCGTT CGCGACGCGA GGCTGGATGG CCTTCCCCAT TATGATTCTT

6251 CTCGCTTCCG GCGGCATCGG GATGCCCGCG TTGCAGGCCA TGCTGTCCAG

6301 GCAGGTAGAT GACGACCATC AGGGACAGCT TCAAGGATCG CTCGCGGCTC 6351 TTACCAGCCT AACTTCGATC ACTGGACCGC TGATCGTCAC GGCGATTTAT

6401 GCCGCCTCGG CGAGCACATG GAACGGGTTG GCATGGATTG TAGGCGCCGC

6451 CCTATACCTT GTCTGCCTCC CCGCGTTGCG TCGCGGTGCA TGGAGCCGGG

6501 CCACCTCGAC CTGAATGGAA GCCGGCGGCA CCTCGCTAAC GGATTCACCA

6551 CTCCAAGAAT TGGAGCCAAT CAATTCTTGC GGAGAACTGT GAATGCGCAA 6601 ACCAACCCTT GGCAGAACAT ATCCATCGCG TCCGCCATCT CCAGCAGCCG

6651 CACGCGGCGC ATCTCGGGCA GCGTTGGGTC CTGGCCACGG GTGCGCATGA

6701 TCGTGCTCCT GTCGTTGAGG ACCCGGCTAG GCTGGCGGGG TTGCCTTACT

6751 GGTTAGCAGA ATGAATCACC GATACGCGAG CGAACGTGAA GCGACTGCTG

6801 CTGCAAAACG TCTGCGACCT GAGCAACAAC ATGAATGGTC TTCGGTTTCC 6851 GTGTTTCGTA AAGTCTGGAA ACGCGGAAGT CAGCGCCCTG CACCATTATG

6901 TTCCGGATCT GCATCGCAGG ATGCTGCTGG CTACCCTGTG GAACACCTAC

6951 ATCTGTATTA ACGAAGCGCT GGCATTGACC CTGAGTGATT TTTCTCTGGT

7001 CCCGCCGCAT CCATACCGCC AGTTGTTTAC CCTCACAACG TTCCAGTAAC

7051 CGGGCATGTT CATCATCAGT AACCCGTATC GTGAGCATCC TCTCTCGTTT 7101 CATCGGTATC ATTACCCCCA TGAACAGAAA TTCCCCCTTA CACGGAGGCA

7151 TCAAGTGACC AAACAGGAAA AAACCGCCCT TAACATGGCC CGCTTTATCA

7201 GAAGCCAGAC ATTAACGCTT CTGGAGAAAC TCAACGAGCT GGACGCGGAT

7251 GAACAGGCAG ACATCTGTGA ATCGCTTCAC GACCACGCTG ATGAGCTTTA

7301 CCGCAGCTGC CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA 7351 TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC

7401 AGACAAGCCC GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCGC 7451 AGCCATGACC CAGTCACGTA GCGATAGCGG AGTGTATACT GGCTTAACTA 7501 TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATATG CGGTGTGAAA 7551 TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT 7601 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT

7651 ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA

7701 ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT

7751 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA

7801 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 7851 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT

7901 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG

7951 AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT

8001 AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC

8051 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 8101 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG

8151 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC

8201 GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT

8251 TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG

8301 CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 8351 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA

8401 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA

8451 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC

8501 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 8551 TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT

8601 GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC

8651 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT

8701 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 8751 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA

8801 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC

8851 AGGTCGGGAG CACAGGATGA CGCCTAACAA TTCATTCAAG CCGACACCGC

8901 TTCGCGGCGC GGCTTAATTC AGGAGTTAAA CATCATGAGG GAAGCGGTGA

8951 TCGCCGAAGT ATCGACTCAA CTATCAGAGG TAGTTGGCGT CATCGAGCGC 9001 CATCTCGAAC CGACGTTGCT GGCCGTACAT TTGTACGGCT CCGCAGTGGA

9051 TGGCGGCCTG AAGCCACACA GTGATATTGA TTTGCTGGTT ACGGTGACCG

9101 TAAGGCTTGA TGAAACAACG CGGCGAGCTT TGATCAACGA CCTTTTGGAA

9151 ACTTCGGCTT CCCCTGGAGA GAGCGAGATT CTCCGCGCTG TAGAAGTCAC

9201 CATTGTTGTG CACGACGACA TCATTCCGTG GCGTTATCCA GCTAAGCGCG 9251 AACTGCAATT TGGAGAATGG CAGCGCAATG ACATTCTTGC AGGTATCTTC

9301 GAGCCAGCCA CGATCGACAT TGATCTGGCT ATCTTGCTGA CAAAAGCAAG

9351 AGAACATAGC GTTGCCTTGG TAGGTCCAGC GGCGGAGGAA CTCTTTGATC

9401 CGGTTCCTGA ACAGGATCTA TTTGAGGCGC TAAATGAAAC CTTAACGCTA

9451 TGGAACTCGC CGCCCGACTG GGCTGGCGAT GAGCGAAATG TAGTGCTTAC 9501 GTTGTCCCGC ATTTGGTACA GCGCAGTAAC CGGCAAAATC GCGCCGAAGG

9551 ATGTCGCTGC CGACTGGGCA ATGGAGCGCC TGCCGGCCCA GTATCAGCCC

9601 GTCATACTTG AAGCTAGGCA GGCTTATCTT GGACAAGAAG ATCGCTTGGC

9651 CTCGCGCGCA GATCAGTTGG AAGAATTTGT TCACTACGTG AAAGGCGAGA

9701 TCACCAAGGT AGTCGGCAAA TAATGTCTAA CAATTCGTTC AAGCCGACGC 9751 CGCTTCGCGG CGCGGCTTAA CTCAAGCGTT AGATGCTGCA GGCATCGTGG

9801 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA

9851 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC

9901 CTTCGGTCCT CCGATCGAGG ATTTTTCGGC GCTGCGCTAC GTCCGCKACC

9951 GCGTTGAGGG ATCAAGCCAC AGCAGCCCAC TCGACCTCTA GCCGACCCAG 10001 ACGAGCCAAG GGATCTTTTT GGAATGCTGC TCCGTCGTCA GGCTTTCCGA

10051 CGTTTGGGTG GTTGAACAGA AGTCATTATC GTACGGAATG CCAAGCACTC

10101 CCGAGGGGAA CCCTGTGGTT GGCATGCACA TACAAATGGA CGAACGGATA

10151 AACCTTTTCA CGCCCTTTTA AATATCCGTT ATTCTAATAA ACGCTCTTTT

10201 CTCTTAGGTT TACCCGCCAA TATATCCTGT CAAACACTGA TAGTTTAAAC 10251 TGAAGGCGGG AAACGACAAT CTGATCCCCA TCAAGCTTGA GCTCAGGATT

10301 TAGCAGCATT CCAGATTGGG TTCAATCAAC AAGGTACGAG CCATATCACT

10351 TTATTCAAAT TGGTATCGCC AAAACCAAGA AGGAACTCCC ATCCTCAAAG

10401 GTTTGTAAGG AAGAATTCTC AGTCCAAAGC CTCAACAAGG TCAGGGTACA

10451 GAGTCTCCAA ACCATTAGCC AAAAGCTACA GGAGATCAAT GAAGAATCTT 10501 CAATCAAAGT AAACTACTGT TCCAGCACAT GCATCATGGT CAGTAAGTTT

10551 CAGAAAAAGA CATCCACCGA AGACTTAAAG TTAGTGGGCA TCTTTGAAAG

10601 TAATCTTGTC AACATCGAGC AGCTGGCTTG TGGGGACCAG ACAAAAAAGG

10651 AATGGTGCAG AATTGTTAGG CGCACCTACC AAAAGCATCT TTGCCTTTAT

10701 TGCAAAGATA AAGCAGATTC CTCTAGTACA AGTGGGGAAC AAAATAACGT 10751 GGAAAAGAGC TGTCCTGACA GCCCACTCAC TAATGCGTAT GACGAACGCA

10801 GTGACGACCA CAAAAGAATT CCCTCTATAT AAGAAGGCAT TCATTCCCAT

10851 TTGAAGGATC ATCAGATACT GAACCAATCC TTCTAGAAGA TCTAAGCTTA

10901 T

Claims

1. A recombinant, double-stranded DNA molecule containing a) a promoter functional in plant cells, and b) a DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1,6-bisphosphate aldolase and operatively linked to the promoter in sense orientation.

2. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1,6- bisphosphate aldolase is derived from a prokaryotic organism.

3. The DNA molecule according to claim 2, wherein the prokaryotic organism is Escherichia coli.

4. The DNA molecule according to claim 1 , wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1,6-bisphosphate aldolase has at least about 60% identity with a prokaryotic DNA sequence coding for fructose- 1,6-bisphosphate aldolase class II.

5. The DNA molecule according to claim 1, wherein the DNA sequence coding for the polypeptide having the enzymatic activity of a fructose- 1,6-bisphosphate aldolase is a sequence capable of hybridizing with the coding region depicted as SEQ ID NO. 1.

6. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1,6-bisphosphate aldolase has at least about 60% identity with the coding region depicted as SEQ ID NO. 1.

7. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1 ,6-bisphosphate aldolase has at least about 70%) identity with the coding region depicted as SEQ ID NO. 1.

8. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1,6-bisphosphate aldolase has at least about 80%) identity with the coding region depicted as SEQ ID NO. 1.

9. The DNA molecule according to claim 1 , wherein the DNA sequence coding for the polypeptide having the enzymatic activity of a fructose- 1 ,6-bisphosphate aldolase has the coding region depicted as SEQ ID NO. 1, or encodes the same peptide as SEQ ID NO. 1 in accordance with the degeneracy of the genetic code.

10. A transgenic plant cell containing in its genome a recombinant DNA molecule according to any of claims 1-9.

11. A transgenic plant containing plant cells according to claim 10.

12. The transgenic plant of claim 11 , wherein the plant exhibits a property selected from the group consisting of increased photosynthesis rates, increased yields, increased growth rates and improved solids uniformity compared with plants that do not contain the recombinant DNA molecule.

13. The transgenic plant according to claim 11 , which is a crop plant.

14. The transgenic plant according to claim 11, selected from the group consisting of com, wheat, rice, tomato, potato, carrots, sweet potato, yams, artichoke, alfalfa, peanut, barley, cotton, soybean, canola, sunflower, sugarbeet, apple, pear, orange, peach, sugarcane, strawberry, raspberry, banana, grape, plantain, tobacco, lettuce, cassava, cruciferous vegetables, forestry species and horticultural species.

15. The transgenic plant of claim 11 , wherein the plant is a potato.

16. A food product derived from the potato of claim 15.

17. The food product of claim 16, which is a french fry or a potato chip.

18. Propagation material derived from the transgenic plant of claim 11.

19. A process for increasing the photosynthesis rate in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and regenerating the transformed cells to produce a transgenic plant.

20. A process for increasing the yield in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and regenerating the transformed cells to produce a transgenic plant.

21. A process for increasing the growth rate in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and regenerating the transformed cells to produce a transgenic plant.

22. A process for improving the solids uniformity in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and regenerating the transformed cells to produce a transgenic plant.

23. In a method for the processing of potatoes into fries or chips, the improvement comprising, utilizing a potato that overexpresses the fda transgene providing a higher solids uniformity in such potato.