MXPA99006823A - Transgenic potatoes having reduced levels of alpha glucan l- or h-type tuber phosphorylase activity with reduced cold-sweetening - Google Patents

Abstract

Potato plants which exhibit reduced levels of&agr;glucan L-type tuber phosphorylase (GLTP) or&agr;glucan H-type tuber phosphorylase (GHTP) enzyme activity within the potato tuber are provided. The conversion of starches to sugars in potato tubers, particularly when stored at temperatures below 7°C, is reduced in tubers exhibiting reduced GLTP or GHTP enzyme activity. Reducing cold-sweetening in potatoes allows for potato storage at cooler temperatures, resulting in prolonged dormancy, reduced incidence of disease, and increased storage life. Methods for producing potato plants which produce tubers exhibiting reduced GLTP or GHTP enzyme activity are also provided. Reduction of GLTP or GHTP activity within the potato tuber may be accomplished by such techniques as suppression of gene expression using homologous antisense RNA, the use of co-suppression, regulatory silencing sequences, chemical and protein inhibitors, or the use of site-directed mutagenesis or the isolation of alternative alleles to obtain GLTP or GHTP variants with reduced starch affinity or activity.

Description

. TRANSGENIC POTATOES THAT HAVE REDUCED LEVELS OF ACTIVITY ALPHA GLUCAN PHOSPHORYLASE OF TUBERCLE OF TYPE L OR H WITH ENDULZING IN REDUCED FRIÓ FIELD OF INVENTION The invention relates to the inhibition of the accumulation of sugars in potatoes by reducing the level of enzymatic activity of a glucan-glucan of tubercle type L or glucan phosphorylase of tubercle of type H in the potato plant.

BACKGROUND OF THE INVENTION The effects experienced by plants caused by a wide variety of factors including disease, environment and storage of potato tubers (Solanum tuberosum) represent determinants REF .: 30732 main tuber quality. The periods of dormant life between harvest and shoot are essential to maintain quality potatoes. Processing potatoes are generally stored between 7 and 12 JC. Cold storage at 2 to 6 ° C, versus storage at 7 to 12 ° C, provides the greatest longevity by reducing respiration, moisture loss, microbial infection, heating costs and the need for chemical outbreak inhibitors (Burton 1989 ). However, low temperatures lead to cold induced sweetening, and the resulting high sugar levels contribute to an unacceptable brown or black color in the fried product (Coffin et al., 1987 Weaver et al., 1978). The sugars that accumulate are predominantly glucose, fructose and sucrose. It is mainly glucose and fructose (reducing sugars) that react with free amino groups when they are heated during the various cooking processes such as frying by means of the Maillard reaction, resulting in the formation of brown pigments (Burton, 1989). , Shallenberger et al., 1959). Sucrose produces a black coloration when fried due to caramelization and carbonization. The ideal content of reducing sugars is generally accepted as 0.1% fresh tuber weight with 0.33% as the upper limit and higher levels of reducing sugars are sufficient to cause the formation of brown and black pigments that result in a fried product unacceptable (Davies and Viola 1992). Although the accumulation of reducing sugars can be delayed in storage at higher temperatures (7 to 12 ° C), this increases the microbial infection and the need to use outbreak inhibitors. Given the negative environmental and health risks associated with chemical use, the development of pesticide-resistant pathogens, and the fact that the use of current outbreak inhibitors may be banned soon, there is a need to obtain potato varieties that can support the long-term stress and cold storage without the use of chemical products, without the accumulation of reducing sugars and with greater retention of starch. The metabolism of carbohydrates is a complex process in plant cells. The manipulation of a number of different enzymatic processes can potentially affect the accumulation of reducing sugars during cold storage. For example, the inhibition of starch decomposition would reduce the accumulation of free sugar. Other methods can also serve to increase the cold storage properties of potatoes by reducing the sugar content, including starch resynthesis using reducing sugars, elimination of sugars through glycolysis and respiration, or conversion of sugars into other sugars. forms that would not participate in the Maillard reaction. However, many of the enzymatic processes are reversible, and the role of most of the enzymes involved in the metabolism of carbohydrates is poorly understood. The challenge remains to identify an enzyme that will provide the desired result, reach the function at low temperatures, and still retain the qualities of the product desired by producers, processors and consumers. It has been suggested that phosphofructate inase (PFK) has an important role in the process of cold-induced sweetening (Kruger and Hammond, 1988, Ap Rees et al., 1988, Dixon et al., 1981, Claassen et al., 1991). Reese et al. (1988) suggested that cold treatment had a disproportionate effect on different routes in carbohydrate metabolism in the sense that glycolysis was more severely reduced due to the cold sensitivity of PFK. The reduction in the activity of PFK would then lead to an increased availability of hexose phosphates for the production of sucrose. In European Patent 0438904 (Burrell et al., July 31, 1991) it is described that the increasing PFK activity reduces the accumulation of sugars during storage by eliminating hexoses through glycolysis and additional metabolism. An enzyme PFK of E. coli was expressed in potato tubers and the report claimed the increase in the activity of PFK and the reduction of sucrose content in tubers tested at harvest. However, it is shown that pyrophosphate: fructose 6-phosphate phosphotransterase (PFP) remains active at low temperatures (Claasen et al., 1991). The activity of PFP can supply 6-fructose phosphate for glycolysis just as PFK can do, since the two enzymes catalyze the same reaction. Therefore, the effectiveness of this strategy to improve the quality of cold storage of potato tubers remains a question. Addition- ally, the elimination of sugars through glycolysis and additional metabolism would not be a preferred method to increase the storage properties of potato tubers due to the resulting loss of valuable dry matter through respiration. It has also been suggested that ADPglucose pyrophosphorylase (ADPGPP) has an important role in the process of cold induced sweetening. It was described in International Application WO 94/28149 (Barry et al., Filed May 18, 1994) that increasing the activity of ADPGPP reduces the accumulation of sugars during storage by re-synthesizing the starch using reducing sugars. An enzyme ADPGPP of E. coli was expressed in potato tubers under the control of a cold-induced promoter and the report said to increase ADPGPP activity and lower the content of reducing sugars in tubers tested at harvest and after storage at temperatures low. However, this strategy does not eliminate the catabolism of starch but increases the rate of starch resynthesis. Therefore, the catabolism of sugars occurs through glycolysis and respiration and the reincorporation in starch is limited. The up-regulation of ADPGPP would not be a preferred method to increase the storage properties of potato tubers due to the resulting loss of valuable dry matter by respiration. Again, a method that involves reducing the catabolism of starch would be preferable since it would retain dry matter. The degradation of starch is thought to involve various enzymes including α-amylase (endoamylase), β-amylase (exoamylase), amyloglucosidase and α-glucan phosphorylase (starch phosphorylase). By retarding the catabolism of starch, the accumulation of reducing sugars should be avoided and the elimination of sugars by glycolysis and additional metabolism would be minimized.

Three different a-glucan phosphorylase enzymes have bdescribed. The tuber-L-type 1,4-glucan phosphorylase isozyme (EC 2.4.1.1) (GLTP) (Nakano and Fukui, 1986) has low affinity for highly branched glycans, such as glycogen, and is located in amioplast. The monomer consists of 916 amino acids and comparisons of sequences with rabbit muscle phosphorylases and Escherichia coli revealed a high level of homology, 51% and 40% amino acids, respectively. The nucleotide sequence of the GLTP gene and the amino acid sequence of the GLTP enzyme are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The isozyme H-glucan phosphorylase of tubercle type H (GHTP) (Mori et al., 1991) has high affinity for glycogen and is located in the cytoplasm. The gene encodes 838 amino acids and shows 63% sequence homology with the L-type phosphorylase although it lacks the 78-residue insertion and the amino-terminal extension of 50 residues found in the L-type polypeptide. The nucleotide sequence of the GHTP gene and the amino acid sequence of the GHTP gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. A third isozyme has bdescribed (Sonnewald et al., 1995) consisting of 974 amino acids and is highly homologous to tuber type L phosphorylase with 81% identity with respect to most of the polypeptide. However, the regions containing the transit peptide and the insertion sequence are highly different. It refers to the isozyme as the leaf type L phosphorylase since the mRNA accumulates in the same way in the leaf and the tuber, while the L-type phosphorylase mRNA of the tuber accumulates strongly in potato tubers and only weakly in leaf tissues. L-type phosphorylase of the tubercle is present mainly in the tubers and leaf type L phosphorylase is more abundant in the leaves (Sonnewald et al., 1995). The nucleotide sequence of the leaf L-type phosphorylase gene and the amino acid sequence of the leaf L-type phosphorylase enzyme are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively. However, the role of the various starch degrading enzymes is not clear and there has bconsiderable debate about the opposite results. For example, reduced expression of sheet L-type phosphorylase (Sonnewald et al., 1995) had no significant influence on the accumulation of starch. Sonnewald et al. (1995) reported that the constitutive expression of an antisense RNA specific for the leaf type L gene resulted in a strong reduction of the L-type activity of a glucan phosphorylase in leaf tissue, although it had no effect on the tissue of potato tuber. Since the antisense repression of the a-glucan phosphorylase activity had no significant influence on the accumulation of starch in the leaves of transgenic potato plants, the authors concluded that the decomposition of the starch was not catalyzed by phosphorylases. Considering the high level of sequence homology betwidentified isozymes to glucan phosphorylase, a similar negative response would be expected with the isozymes of tuber type H (GHTP) and type L (GLTP). In view of the foregoing, there remains a need to obtain potato plants that produce tubers that exhibit reduced conversion of starches to sugars during propagation and during storage at ambient and reduced temperatures, particularly at temperatures below 7 ° C.

SYNTHESIS OF THE INVENTION The inventors have found that surprisingly, the reduction of the level of enzymatic activity of L-type tuberculo-glucan-phosphorylase (GLTP) or glucan-phosphorylase of H-type tubercle (GHTP) in the potato tuber results in a substantial reduction of accumulation. of sugars in the tuber during propagation and storage, in relation to potatoes of the wild type, especially at storage temperatures below 10: 'C, and specifically at 4 ° C. It is notable that, given the complexity of the carbohydrate metabolism in the tuber, the reduction in the activity of a single enzyme is effective in the accumulation of reducing sugars in the tuber. The discovery of the inventors is even more surprising in view of the previously discussed work of Sonnewald et al. (1995) in which it was reported that the reduced expression of leaf type L phosphorylase had no significant influence on the accumulation of starch in leaves. of potato plants. The present invention presents tremendous commercial advantages. Tubers in which cold-induced sweetening is inhibited or reduced can be stored at lower temperatures without producing high levels of reducing sugars in the tuber which causes unacceptable browning of potato chips products. The cold storage of tubers results in a longer shelf life, prolonged latent life limiting respiration and delaying the outbreak and lower incidence of disease.

The reduction of the activity of GLTP or GHTP in potato and tuber plants can be achieved by any of a number of known methods including, without limitation, antisense inhibition of GLTP or GHTP mRNA co-suppression, site-directed mutagenesis of gene sites. OHIR or wild type GLTP, chemical or protein inhibition, or plant cultivation programs. Therefore, in broad terms, the invention features modified potato plants that have a reduced level of activity of either L-type tuberculin phosphorylase (GLTP) or glucan-type phosphorylase of tuber type H (GHTP > in tubers produced by The plants, in relation to that of tubers produced by an unmodified potato plant In a preferred embodiment, the invention provides a potato plant transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence. when transcribed in the plant, it inhibits the expression of an endogenous GLTP gene or GHTP gene As will be discussed in detail below, the aforementioned DNA sequence can be inserted into the expression cassette in sense or antisense orientation. The potato plants of the present invention could have reduced activity levels of one of GLTP or GHTP independently, or they could have r reduced activity levels of both GLTP and GHTP. As discussed above, the inventors have found that reducing the activity levels of the GLTP or GHTP enzymes in potato plants results in potato tubers in which the accumulation of sugars, especially in prolonged storage periods at temperatures above below 10 ° C it is reduced. Therefore the invention extends further to the methods for the production of reducing sugars in tubers produced by a potato plant which comprises reducing the activity level of GLTP or GHTP in the potato plant. In a preferred embodiment such methods involve the introduction into the potato plant of an expression cassette having a plant promoter sequence operably linked to a DNA sequence that when transcribed in the plant, inhibits the expression of a GLTP gene or of an endogenous GHTP gene. As described above, the DNA sequence can be inserted into the expression cassette in a sense or antisense orientation. As described in detail in the examples herein, improvements in cold storage characteristics have been observed in the Desiree potato variety transformed by the methods of the present invention. A direct measure of improved cold storage characteristics is a reduction in the activity level of the GLTP or GHTP enzyme detected in potatoes after harvesting and cold storage. Transformed potato varieties have been developed where the total activity of a glucan phosphorylase measured as μmol of NADPH produced mg "1 protein-1 h_1 in tubers of plants stored at 4 ° C for 189 days is as much as 70% less than total a-glucan phosphorylase activity in tubers of non-transformed plants stored under the same conditions Another relatively direct measure of improved cold storage characteristics is a reduction in sweetness in potatoes observed after a period of cold storage. Transformed plants have been developed where the sum of the glucose and fructose concentrations in the tubers stored at 4 ° C for 91 days is 39% lower than the sum of the glucose and fructose concentrations in tubers of a non-transformed plant stored under Same conditions Another measure of cold storage characteristics improves One that demonstrates a practical advantage of the present invention is a reduction of the darkening of a fried potato during the -processing (cooking). As discussed above, the accumulation of sugars in the potatoes during cold storage contributes to the unacceptable browning of the fried product. The darkening reduced to frying can be quantified as a measure of the reflectance or fried potato stripe, fried potato. The techniques for measuring the results of French fries are discussed in the present. The transformed potato varieties of the present invention have been developed where the result of the fried potato for the tubers of the plants stored at 4 ° C for 124 days was as much as 89% higher than the results of the French fries for the tubers of non-transformed plants stored under the same conditions. By reducing the activity of GLTP and / or GHTP in tubers of potato plants thereby inhibiting the accumulation of sugars during storage at low temperatures, the present invention makes it possible to store potatoes at lower temperatures than would be possible with the potatoes of the wild type of the same crop. As previously discussed, storage of potatoes at lower temperatures than those traditionally used could result in increased storage life, increased latent life due to reduced respiration, and reduced disease incidence and outbreak. It will be apparent to the person skilled in the art that such additional benefits also constitute improved cold storage characteristics and can be measured and quantified by known techniques.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings illustrating the embodiments of the invention: Figure 1 is a schematic diagram of the antisense sequence of T-glucan phosphorylase type L inserted into the transformation vector pB1121; Figure 2 is a schematic diagram of the antisense sequence of tuberculin H type glucan phosphorylase inserted into the transformation vector pB1121; Figure 3 shows the basic structure of the three isolated isoforms of glucan phosphorylase. The transit peptide (TS) and the insertion sequence (IS) are characteristic of L-type phosphorylases and are not found in type H phosphorylase. The percentages indicate the nucleic acid sequence homologies between the isoforms; Figure 4 is a schematic diagram of carbohydrate interconversions in potatoes (Sowokinos 1990); Figure 5 is a comparison of the amino acid sequences of the three phosphorylase isoforms found in the potato for the region sought by the antisense GLTP construct used in the Examples herein. The highlighted amino acids are identical. The amino acid sequence of a leaf L-type glucan phosphorylase is in the upper part (amino acids 21-238 of SEQ ID NO: 6), the amino acid sequence of a L-type glucan phosphorylase of tubercle is in the medium (amino acids 49 - 266 of SEQ ID NO: 2), and the amino acid sequence of tuberculin H-type glucan phosphorylase is in the lower part (amino acids 46-264 of SEQ ID NO: 4); Figures 6A and 6B are a comparison of the nucleotide sequences of the three phosphorylase isoforms found in the potato for the region sought by the antisense GLTP construct used in the Examples herein. The highlighted nucleotides are identical. The nucleotide sequence of α-glucan phosphorylase type L is at the top (nucleotides 389-1045 of SEQ ID NO: 5), the nucleotide sequence of tuberculin L-type glucan phosphorylase is in the medium (nucleotides 338-993 of SEQ ID NO: 1), and the nucleotide sequence of a glucan Phosphorylase type H of tubercle is in the lower part (nucleotides 147-805 of SEQ ID NO: 3); Figure 7 is a Northern blot of polyadenylated RNA isolated from wild-type potato tubers and lines 3, 4, 5 and 9 transformed with the tuber L-type glucan phosphorylase. The dyeing was probed with a radiorotulated probe specific for the tuberculin L-type glucan phosphorylase. Figure 8 is a Northern blot of total RNA isolated from wild-type potato tubers and lines 1 and 2 transformed with α-glycan phosphorylase type H. Dyeing was probed with a radio-labeled probe specific for α-glucan phosphorylase type H. Figure 9 shows the fried product obtained from (A) wild type and transformants of tuberculin L-type glucan phosphorylase (b) ATL1 (C) ATL3 (D) ATL4 (E) ATL5 (F) ATL9 tubers grown in the field after 86 days of storage at 4 ° C ("ATL" = L-type transformant of antisense tuber); Figure 10 shows the activity and Western spotting gel of isozyme type H and type L of a 1,4 glucan phosphorylase extracted from wild-type tubers and tubers transformed with the antisense construct for the L-type isoform and Figure 11 shows the activity gel and Western spotting of isozymes type H and type L of a 1,4 glucan phosphorylase extracted from wild-type tubers and tubers transformed with the antisense construct for the isoform type H.

DESCRIPTION OF THE PREFERRED MODALITY Potato plants are provided that have a reduced level of activity of a glucan-type glucanase of tuber type L (GLTP) or of a glucan phosphorylase of tubercle type H (GHTP) in tubers produced by plants in relation to the activity of tubers produced by potato plants not modified. In the exemplified case, the reduction of a glucan phosphorylase activity is achieved by transforming a potato plant with an expression cassette having a plant promoter sequence operably linked to a DNA sequence that, when transcribed in the plant, it inhibits the expression of a GLTP gene or an endogenous GHTP gene. Although in the exemplified case the DNA sequence is inserted into the expression cassette in the antisense orientation, a reduction in the activity of a glucan phosphorylase with the DNA sequence inserted into the expression cassette in a sense or antisense orientation can be achieved. . 1. Homology Dependent Silencing The control of gene expression using antisense and sense gene fragments is a conventional laboratory practice and is well documented in the literature. Antisense and sense suppression are both phenomena dependent on sequence homology of genes that can be described as phenomena of "homology-dependent silencing". A review of scientific research articles published during 1996 reveals several hundred reports of homology-dependent silencing in transgenic plants. The mechanisms underlying the homology-dependent silencing are not completely understood although the characteristics of the phenomena have been studied in many plant genes and this body of work has been extensively reviewed (Meyer and Saedler 1996, Matzke and Matzke 1995, Jorgensen 1995, Weintraub 1990, Van der Krol and others 1988). Mutation dependent on homology seems to be a general phenomenon that can be used to control the activity of many endogenous genes. Examples of genes that exhibit reduced expression after introduction of homologous sequences include dihydroflavanol reductase (Van der Krol 1990), polygalacturonidase (Smith and others 1990), phytoene synthase (Fray and Grierson 1993), pectinesterase (Seymour et al. 1993) phenylalanine ammonia-lyase (De Carvalho et al. 1992), β-1,3-glucanase (Hart et al. 1992), chitinase (Dorlhac et al. 1994), nitrate reductase (Napoli et al. 1990) and chalcone synthase (14). Transformation of Russet Burbank potato plants with sense or antisense constructions of the coat protein virus of the potato leaf-binding virus has been reported to confer resistance to potato leaf-binding virus infection (Kawchuk et al. others, 1991). The transfer of a sense sequence or homologous antisense generally generates transformants with reduced endogenous gene expression. As discussed in detail in the examples herein, transformed potato plants that exhibit phenotypes that indicate reduced GLTP or GHTP expression can be easily identified. In the antisense suppression technique, a genetic construct or expression cassette is assembled which, when inserted into a plant cell, results in the expression of an RNA that is of sequence complementary to the mRNA produced by the target gene. It is theorized that the complementary RNA sequences form a duple thereby inhibiting translation to protein. The theory that underlies both sense and antisense inhibition has been discussed in the literature, including in Antisense Research and Applications (CRC Press, 1993). 125-148. The complementary sequence may be equivalent in length to the entire sequence of the target gene although a fragment is generally sufficient and is more convenient to work with. For example, Cannon et al. (1990) discloses that an antisense sequence as short as 41 base pairs is sufficient to achieve genetic inhibition. U.S. Patent No. 5,585,545 (Bennett et al., December 17, 1996) discloses genetic inhibition by an antisense sequence of only 20 base pairs. There are many examples in the patent, patent literature that include descriptions and claims of methods for suppressing gene expression through the introduction of antisense sequences to an organism, including for example, U.S. Patent No. 5,545,815 (Fischer et al., August 13, 1996) and U.S. Patent No. 5,387,757 (Bridges et al., February 7, 1995). Sense sequence homology-dependent silencing is conducted in a manner similar to antisense suppression except that the nucleotide sequence is inserted into the expression cassette in the normal sense orientation. A number of patents, including U.S. Patents 5,034,323, 5,231,020 and 5,283,184, describe the introduction of sense sequences leading to the suppression of gene expression. Both forms of homology-dependent silencing, sense and antisense suppression, are useful to achieve deregulation of GLTP or GHTP of the present invention. It is recognized in the art that both techniques are equally useful strategies for genetic suppression. For example, U.S. Patent No. 5,585,545 (Bennett et al., December 17, 1996) and U.S. Patent No. 5,451,514 (Boudet et al., September 15, 1995) claim methods of inhibiting gene expression or recombinant DNA sequences useful in methods for suppressing gene expression for sense and antisense suppression techniques. 2. Alternative Techniques to Reduce the GHTP and / or GLTP Activity in Tubers While Homology Dependent Silencing is a preferred technique for the deregulation of GLTP or GHTP in potato plants of the present invention, there are several commonly used alternative strategies available to reduce the activity of a product. specific genetics that will be understood by those skilled in the art having application in the present invention. The insertion of a related gene or promoter in a plant can induce a rapid turnover of endogenous homologous transcripts, a process that is called co-suppression and is believed to have many similarities with the mechanism responsible for the inhibition of antisense RNA (Jorgensen 1995, Brusslan and Tobin, 1995). Several regulatory DNA sequences can be altered (promoters, polyadenylation signals, post-transcriptional processing sites) or can be used to alter the expression levels (enhancers and silencers) of a specific mRNA. Another strategy to reduce the expression of a gene and its encoded protein is the use of ribozymes designed to specifically dissociate the target mRNA rendering it incapable of producing a fully functional protein (Hasseloff and Gerlach, 1988). The identification of alleles that occur naturally or the development of genetically constructed alleles of an enzyme that have been identified as important in the determination of a particular trait can alter activity levels and can be exploited by classical cropping programs (Oritz and Hua an, 1994). Site-directed mutagenesis is often used to modify the activity of an identified genetic product. The structural coding sequence for a phosphorylase enzyme can be mutagenized in E. coli or another suitable host and classified to determine phosphorolysis of reduced starch. Alternatively, naturally occurring alleles of phosphorylase with reduced affinity and / or specific activity can be identified. Additionally the activity of a particular enzyme can be altered using several inhibitors. These procedures are routinely used and can be found in textbooks such as Sambrook and others (1989). 3. Variants of GLTP and GHTP Enzymes and Sequences Used for Homology-Dependent Silencing As described in the background of the invention, and in more detail by Nakano et al. (1986) Mori et al. (1991) and Sonnewald et al. (1990) , there are three known isozymes of a glucan phosphorylase that are produced in potato plants. The present invention relates to the deregulation of the GLTP and / or GHTP isozymes. While it is believed that the GLTP and GHTP genes of all commercially known potato varieties are substantially identical, it is expected that the principles and techniques of the present invention would be effective in potato plants that have full length polynucleotide variants or subsequences that encode polypeptides having the enzymatic starch catabolizing activity of the GHTP and GLTP enzymes described. The terms "GLTP" and "GHTP", as used herein and in the claims, are intended to encompass the variants described above.The above variants may include variants of GLTP and GHTP nucleotide sequences which differ from those employed and yet still encode to the same polypeptide due to the degeneracy of codon, as well as variants encoding proteins capable of recognition by antibodies cultured against the amino acid sequences of GLTP and GHTP set forth in SEQ ID Nos. 2 and 4. Similarly, the experts in the will recognize that GLTP and / or GHTP homology-dependent silencing in potato plants can be achieved with sense or antisense sequences other than those exemplified, first the region of the GLTP cDNA sequence or GHTP from which the antisense sequence is derived is not essential. Second as described hereinabove, the length of the antisense sequence used can vary considerably. Additionally the sense or antisense sequence need not be identical to that of the GLTP or target GHTP gene to be deleted. As described in the Examples herein the inventors have observed that the transformation of potato plants with antisense DNA sequences derived from the GHTP gene not only substantially suppresses the activity of GHTP genes., but causes some degree of suppression of GLTP gene activity. The antisense sequences of GHTP and GLTP genes have 56.8% sequence identity. The sequence identity between the GLTP antisense sequence and the corresponding sheet-like glucan phosphorylase sequence described by Sonnewald et al. (1990) is 71.3%. In the inventors' research to date, the same phenomenon of cross-deregulation has not been observed when potato plants are transformed with antisense DNA sequences derived from the GLTP gene. However, these results clearly indicate that the absolute sequence identity between the target endogenous a-glucan-phosphorylase gene and the recombinant DNA is not essential since the GLTP activity was suppressed by an antisense sequence having approximately 57% sequence identity with the target GLTP sequence. Accordingly, those skilled in the art will understand that sense or antisense sequences other than those exemplified herein and different from those having absolute sequence identity with the target endogenous GLTP or GHTP gene will be effective in causing deletion of the GLTP or GHTP gene. endogenous when it is introduced into the cells of potato plants. Useful sense or antisense sequences may differ from the exemplified antisense sequences or from other sequences derived from the endogenous GLTP or GLTP gene sequences by means of conservative amino acid substitutions or differences in the percentage of paired nucleotides or amino acids in portions of the sequences that They are aligned for comparison purposes. U.S. Patent 5,585,545 (Bennett et al., December 17, 1996) provides a useful description with respect to techniques for comparing sequence identity for polynucleotide and polypeptide conservative amino acid substitutions and hybridization conditions indicative of degrees of sequence identity. The relevant parts of that description are summarized here. The percentage of sequence identity for polynucleotides and polypeptides can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can include additions or deletions (ie, gaps). compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences .. The percentage is calculated: (a) by determining the number of positions at which the nucleic acid base or identical amino acid residue occurs in both sequences to produce the number of paired positions; (b) dividing the number of paired positions by the total number of positions in the comparison window, (c) multiplying the result by 100 to give the percentage of sequence identity. Optimal alignment of sequences for comparison can be driven by computerized implementations of known algorithms (eg, GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison WI or BlastX and BlastN available from the National Center for Biotechnology Information, or by inspection Polypeptides that are substantially similar share sequences as indicated above except that those residual positions that are not identical may differ by amino acid changes Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine, a group of amino acids that has chains lateral aliphatic hydrochloride as is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids with basic side chains is lysine, arginine and histidine; and a group of amino acids having side chains with sulfur content is cysteine and methionine. Another indication that the nucleotide sequences are substantially identical is if two molecules specifically hybridize to each other under severe conditions. Severe conditions depends on the sequence and will be different in different circumstances. In general, severe conditions are selected from approximately 10 ° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (low pH and defined ionic strength) at which 50% of the target sequence is hybridized to a perfectly matched probe. The Tm of a hybrid, which is a function of the length and base composition of the probe, can be calculated as described in Sambrook et al. (1989). Typically, stringency conditions for a Southern blot protocol involve washing at 65 ° C with 0.2XSSC. For the preferred oligonucleotide probes, wash conditions are typically at about 42 ° C in 6XSSC. 4. General Methods Several methods are available to introduce and express foreign DNA sequences in plant cells. Briefly, the steps involved in preparing antisense glucan phosphorylase cDNAs and introducing them into plant cells include: (1) isolating mRNA from potato plants and preparing cDNA from the mRNA; (2) classify the cDNA to determine the desired sequences; (3) ligating a promoter to the desired cDNA in the opposite orientation for the expression of the phosphorylase genes; (4) transforming suitable host plant cells; and (5) selecting and regenerating cells that transcribe the inverted sequences. In the exemplified case, the DNA derived from potato GLTP and GHTP genes is used to create expression cassettes having a plant promoter sequence operatively linked to an antisense DNA sequence which, when transcribed in the plant, inhibits the expression of a GLTP gene or an endogenous GHTP gene. Agrobacterium tumefaciens is used as a vehicle for the transmission of DNA to stem explants from shoots of potato plants. A regenerated plant of the transformed explants transcribes the antisense DNA that inhibits the activity of the enzyme. The recombinant DNA technology described herein involves conventional laboratory techniques that are well known in the art and are described in standard references such as Sambrook et al. (1989). In general, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed in accordance with the manufacturer's specifications.

. Preparation of GLTP and GHTP cDNA The cDNA is prepared from potato tuber mRNA isolated by reverse transcription. A primer anneals the mRNA, providing a 3 'end which can be used for extension by the enzyme reverse transcriptase. The enzyme binds in the usual 5 '-3' elongation, as directed by base pairing complementary to mRNA tuning to form a hybrid molecule consisting of an RNA strand of annealing with base pairing with the strand of cDNA complementary After degradation of the original mRNA, a DNA polymerase is used to synthesize the complementary DNA strand to convert the single-stranded cDNA into double DNA. After DNA amplification, the double-stranded cDNA is inserted into a vector for propagation in E. coli. Typically, the identification of clones harboring the desired cDNA would be carried out by nucleic acid hybridization or immunological detection of the encoded protein if an expression vector is used. In the exemplified case, the question is simplified in the sense that the DNA sequences of the GHTP and CLIP genes are known, as the sequences of suitable primers are known (Brisson et al., 1990 Fukui et al., 1991). The primers used hybridize within the GHTP and GLTP genes. Therefore, it is expected that the prepared amplified cDNAs represent portions of the GLTP and GHTP genes without further analysis. E. coli transformed with pUC19 plasmids carrying the phosphorylase DNA insert were detected by color selection. Appropriate E. coli strains transformed with plasmids that do not carry inserts grow as blue colonies. Strains transformed with pBluescript plasmids carrying inserts grow as white colonies. Plasmids isolated from transformed E. coli were sequenced to confirm the sequence of the phosphorylase inserts. 6. Construction of vectors The prepared cDNAs can be inserted in the sense orientation or antisense in expression cassette into expression vectors for the transformation of potato plants to inhibit the expression of the GLTP and / or GHTP genes in potato tubers. As in the exemplified case, which involves antisense suppression, the desired recombinant vector will comprise an expression cassette designed to initiate transcription of antisense cDNAs in plants. Additional sequences are included to allow the vector to be cloned into a phage or bacterial host. The vector will preferably contain a prokaryotic origin of replication having a wide range of host a selectable marker should also be included to allow the selection of bacterial cells carrying the desired construct. Suitable prokaryotic selectable markers include resistance to antibiotics such as ampicillin. Other DNA sequences encoding additional functions may also be present in the vector as is known in the art. For example, in the case of Agrobacterium transformations, the T-DNA sequences will also be included for subsequent transfer to plant chromosomes. For expression in plants, the recombinant expression cassette will contain, in addition to the desired sequence, a plant promoter region, a transcription initiation site (if the sequence to be transcribed lacks one) and a sequence of transcript termination. the transcript Typically, unique restriction enzyme sites are included at the 5 'and 3' ends to allow easy insertion into a preexisting vector. The sequences that control the expression of eukaryotic genes are well known in the art. The transcription of DNA into mRNA is regulated by a region of DNA that is referred to as the promoter. The promoter region contains a sequence of bases that tells the RNA polymerase to associate with the DNA, and to initiate the transcription of the mRNA using one of the strands of DNA as a template to be a complementary strand of RNA corresponding elements of promoter sequence include the consensus sequence in TATA box (TATAAT), which is generally 20 to 30 base pairs (bp) upstream (by convention -30 to -20 bp in relation to the transcription start site) of the transcription start site. In most cases the TATA box is required for the initiation of accurate transcription. The TATA box is the only upstream promoter element that has a relatively fixed location with respect to the starting point. The consensus sequence in the CAAT box is centered at -75 although it can operate at distances that vary considerably from the starting point and in either of the two orientations. Another common promoter element is the GC box at -90 which contains the consensus sequence GGGCGG. It can occur in multiple copies and in either orientation. Other sequences that confer tissue specificity, response to environmental signals or maximum transcription efficiency can also be found in the promoter region. Such sequences are often found within 400 bp of transcription initiation site, although they may extend as far as 2000 bp or more. In heterologous promoter / structural gene combinations, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural location. However, some variation in this distance can be adapted without loss of the promoter function. The particular promoter used in the expression cassette is not essential to the invention. Any of a number of promoters that direct transcription in plant cells is suitable. The promoter can be constitutive or inducible. 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 in plasmids that induce tumors of Agrobacterium tumefaciens), the colimovirus promoters such as cauliflower mosaic virus (CaMV) 19S and 35S and the 35S promoters. of the scrofularia mosaic virus, the light-inducible promoter of the small subunit of ribulose-1, 5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the chlorophyll a / b binding protein gene promoter etc. All of these promoters have been used to create several types of DNA constructs that have been expressed in plants; see, for example, PCT WO 8402913. The CaMV 35S promoter used in the examples herein has been shown to be highly active and constitutively expressed in most tissues (Bevan et al., 1986). A number of other genes with increased or specific tuber expression are known, including potato tuber ADPGPP genes, large and small subunits (Muller et al., 1990). Other promoters that are contemplated as useful in this invention include those that show increased or specific expression in potato tubers, which are promoters normally associated with the expression of enzymatic modification genes or starch biosynthetics, or that exhibit different expression patterns, for example, they are expressed or expressed at different times during the development of the tubers. Examples of these promoters include those for the genes for granule-bound starch synthases and others, branching enzymes (Blennow et al., 1991; WO 9214827; WO 9211375), disproportionate enzyme (Takaha et al., 1993) debranching enzymes, amylases, phosphorylases (Nakano et al 1989; Mori et al., 1991), pectin esterases (Ebbelaar et al., 1993), the 40 kD glycoprotein; ubiquitin, aspartic proteinase inhibitor (Stukerlj et al., 1990); the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shahar et al, 1992; GenBank accession numbers M95196 and M95197), putative trypsin inhibitor and other tuber cDNAs (Stiekema et al., 1988) and for amylases and sporamines (Yoshida et al.; 1992; Ohta et al., 1991). In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes. In the exemplified case the 3 'terminator sequence of nopaline synthase NOS (Bevan et al. 1983) was used. The polyadenylation sequences without also commonly added to the construction of the vector if the mRNA encoded by the structural gene must be efficiently transferred (Alber and Kawasaki, 1982). It is believed that polyadenylation has an effect on the stabilization of mRNAs. Polyadenylation sequences include, but are not limited to, the Agrobacterium octopine synthase signal (Gielen et al., 1984) or the nopaline synthase signal (Depicker et al. 1982). Typically, the vector will also contain a selectable marker gene by which transformed plant cells can be identified in culture. Typically, the marker gene encodes antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin and gentamicin. In the case exemplified, the marker gene confers resistance to kanamycin. After transforming plant cells, those cells that contain the vector will be identified by their ability to grow in a medium containing the particular antibiotic. 7. Transformation of plant cells Although in the case exemplified, shoot stem explants from potato plants were transformed by means of inoculation with Agrobacterium tumefaciens carrying the antisense sequence bound to a binary vector, the direct transformation techniques known in the art. The technique can also be used to transfer the recombinant DNA. The vector can be microinjected directly into plant cells. Alternatively, the nucleic acids can be introduced into the plant cell by high speed ballistic penetration by small particles having the nucleic acid of interest embedded within the matrix of the particles or on the surface. The fusion of protoplasts with bodies with lipid surfaces can be used, such as mini cells, cells or lysosomes that carry the DNA of interest. DNA can also be introduced into plant cells by electroporation, where the plant protoplasts are electroporated in the presence of plasmids carrying the expression cassette. In contrast to direct transformation methods, the exemplified case uses vector transformation using Agrobacterium tumefaciens. Agrobacterium tumefaciens is a Gram-negative soil bacterium that causes a neoplastic disease known as crown gill in dicotyledonous plants. The induction of tumors is caused by tumor induction plasmids known as Ti plasmids. The Ti plasmids direct the synthesis of "opines" in the infected plant. The "opines" are used as a source of carbon and / or nitrogen by the Agrobacteria.

The bacterium does not enter the plant cell, but transfers only part of the Ti plasmid, a portion called T DNA, which is stably integrated into the plant's genome, where it expresses the functions necessary to synthesize "opines" and to transform plant cell. The Vir genes (virulence) in the Ti plasmid, outside the T DNA region, are necessary for the T-DNA transfer. However, the vir region is not transferred. Of course, the vir region, while required for T-DNA transfer, does not need to be physically bound to the T-DNA and can be provided in a separate plasmid. Tumor-inducing portions of T-DNA can be interrupted or deleted without loss of transfer and integration functions, so that healthy and normal transformed plant cells can be produced that have lost all the properties of tumor cells, but still harbor and express certain T-DNA loaves, particularly the T-DNA boundary regions. Therefore, the modified Ti plasmids in which the disease-causing genes have been deleted can be used as vectors for the transfer of the sense and antisense gene constructs of the present invention in potato plants (see generally Winnacker 1987).

The transformation of plant cells with Agrobacterium and the regeneration of whole plants typically involve co-cultivation of Agrobacterium with cultured protoptastos isolated or transformation of intact cells or tissues with Agrobacterium. In the exemplified case, stem explants of potato shoot cultures are transformed with Agrobacterium. Alternatively, the cauliflower mosaic virus (CaMV) can be used as a vector to introduce sense or antisense DNA in plants of the Solanaceae family.

For example, U.S. Patent No. 4,407,956 (Howell, October 4, 19B3) describes the use of cauliflower mosaic virus DNA as a vegetable carrier. 8. Selection and regeneration of transformed plant cells After transformation, the transformed plant cells or plants carrying the sense or antisense DNA must be identified. Typically a selectable marker is used, such as resistance to antibiotics. In the case exemplified, the transformed plant cells were selected by culturing the cells in culture medium containing kanamycin. Other selectable markers will be apparent to those skilled in the art. For example, the presence of "opines" can be used to identify transformants if the plants are transformed with Agrobacterium. Expression of foreign DNA can be confirmed by detection of RNA encoded by the inserted DNA using well-known methods such as staining the protein from an unincubated natural gel identical to nitrocellulose and probing with polyclonal antibodies specific for isoforms of glucan phosphorylase type H and type L of tuber. The levels of reducing sugars (glucose and fructose) in tuber tissues were quantified by HPLC (Tables 2, 3 and 4). The degree of Maillard reaction, which is proportional to the concentration of reducing sugars in tubers, was examined by determining the results of French fries after frying (Table 5 and Figure 6).

. Definitions As used herein and in the claims, the term "approximately three months", "approximately four months" and "approximately six months" refer, respectively, to periods of time of three months plus or minus two weeks, four months plus or minus two weeks and six months plus or minus two weeks; "antisense orientation" refers to the orientation of the nucleic acid sequence of a structural gene that is inserted into an expression cassette in an inverted manner with respect to its naturally occurring orientation. When the sequence is double-stranded, the strand that is the tempered strand in the orientation that occurs naturally becomes the coding strand, and vice versa; "result of fried potato" from a tuber means the measurement of the reflectance recorded by a Direct Reading Concise Spectrophotometer model E-15-FP (Agtron Inc. 1095 Spice Island Drive No. 100, Sparks Nevada 89431) of a potato chip cut in the center fried at 205 ° F (96.1 ° C) in soybean oil for approximately 3 minutes until the bubbling is stopped; "cold storage" or "storage at reduced temperature" or its variants, means keeping at temperatures below 10 ° C, which can be achieved by cooling or ambient temperatures; "endogenous", as used with reference to genes to glucan phosphorylase of a potato plant, will mean a naturally occurring gene that was present in the genome of the potato plant prior to the introduction of an expression cassette carrying a DNA sequence derived from an a-glucan phosphorylase gene; "expression" refers to the transcription and translation of a structural gene so that a protein is synthesized; "heterologous sequence" or "heterologous expression cassette" is one that originates from a foreign species, or if it is of the same species, is substantially modified from its original form; "Improved cold storage characteristics include, without limitation, improvements in the result of the fried potato and reduction in the accumulation of sugars in tubers measured at harvest or after a period of storage below 10 ° C, and includes also improvements, advantages and benefits that can result from the storage of potatoes at lower temperatures than those traditionally used, such as being, without limitation, increased storage life of potatoes, increased latent life due to reduced respiration and potato sprouting, and reduced incidence of Disease Unless further qualified by a specific measurement or specific assay, an improvement in a cold storage characteristic refers to a difference in the characteristic described in relation to that of a control, wild type or unmodified potato plant; "modified" or variant thereof, when used to describe plants or tubers Potato circles, is used to distinguish a potato plant or tuber that has been altered from its natural state through: the introduction of a nucleotide sequence of the same species or of a different one, either in sense or antisense orientation , either by recombinant DNA technology or by traditional cross-culture methods including the introduction of modified regulatory or structural sequences; modification of a natural nucleotide sequence by site-directed mutagenesis or otherwise; or the treatment of the potato plant with chemical or protein inhibitors. An "unmodified" potato plant or tuber means a natural, wild type or control potato plant or tuber that has not been modified as described above; "nucleic acid sequence" or "nucleic acid segment" refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to the 3' end. Includes plasmids for self-replication of infectious polymers of DNA or RNA and DNA p non-functional RNA; "operably linked" refers to a functional linkage between a promoter and a second sequence wherein the promoter sequence initiates RNA transcription corresponding to the second sequence; "planting" includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds and plant cells; "promoter" refers to a region of DNA upstream of the structural gene and involved in the recognition and ligation of RNA polymerase and other proteins that initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. "reduced activity" or its variants, when used with reference to the level of GLTP or GHTP enzyme activity in a potato tuber includes the reduction of GLTP or GHTP enzyme activity that results from the reduced expression of the GLTP or GHTP gene product , reduced substrate affinity of the GLTP or GHTP enzyme and reduced catalytic activities of the GLTP or GHTP enzyme; "Reduced" or its variants may be used herein with reference to, without limitation, the activity levels of the GLTP or GHTP enzyme in potato tuber, accumulation of sugars in potato tubers and darkening of potato chips when frying. Unless further qualified by a specific measure or assay, reduced levels or reduced activity refers to a statistically demonstrable significant difference in the characteristic described in relation to the characteristic in an unmodified or wild type or control potato plant; "Stress" or its variants, when used in relation to the stress experienced by potato plants and tubers, includes the effects of environment, fertility, humidity, temperature, handling, disease, atmosphere and aging that impact the quality of the tuber or plant that can be experienced by potato plants through all stages of their life cycle and by tubers at all stages of growth and development cycle and during subsequent harvest, transport, storage and processing; "resistance to stress" or its variants, will mean reduced effects of temperature, aging, disease, atmosphere, physical manipulation, humidity, environmental chemical residues, pests and other stresses; "suitable host" refers to a microorganism or cell that is compatible with a recombinant plasmid, DNA sequence or recombinant expression cassette and will allow the plasmid to replicate, be incorporated into its genome or be expressed; and "uninterrupted" refers to a sequence of DNA (for example cDNA) containing an open reading frame that lacks intervening untranslated sequences.

EXAMPLE 1 This example describes the reduction of GHTP and / or GLTP activity in tubers of potato plants by transforming potato plants with expression cassettes containing DNA sequences derived from GLTP and GHTP genetic sequences linked to the promoter in the antisense orientation.

A. Isolation of potato tuber mRNA The total RNA of potato was purified at 4 ° C using autoclaved reagents of 1 g of ground tuber tissue to obtain a fine powder under liquid nitrogen with a mortar and pestle. The powder was transferred to a 30 ml corex tube and 3 volumes of 100 mM Tris-Cl, pH 8.0 100 mM NaCl, and 10 mM EDTA (10 x TNE) containing 0.2% (w / v) SDS were added. and 0.5% (v / v) 2-mercaptoethanol. An equal volume of phenol-chloroform (1: 1) was added and the sample was subjected to a gentle vortex before centrifugation at 4 ° C in an SS 34 rotor at 8000 rpm. for 5 minutes. The organic phase was reextracted with 0.5 volume of 10 x TNE containing 0.2% (w / v) of SDS and 0.5% (v / v) of 2-mercaptoethanol and the combined aqueous phases were extracted with chloroform. The nucleic acids were precipitated from the aqueous phase with sodium acetate and absolute ethanol, a pellet was obtained by centrifugation and resuspended in 3 ml of 1 x TNE. An equal volume of 5 M LiCl was added and the sample was stored at -20 ° C for 4 hours before centrifuging at 8,000 r.p.m. in a SS34 rotor at 4 ° C for 10 minutes. The RNA pellet was washed with 70% ethanol, dried and resuspended in water treated with DEPC. Poly (A +) RNA was isolated using oligo (dT) cellulose column chromatography (Boehringer Mannheim). Poly (A +) RNA was isolated from the total RNA resuspended in RNase-free water. The columns were prepared using a 10 ml Bio-Rad Poly-Prep column in an autoclave to which 50 mg of aligo (dT) cellulose suspended in 1 ml of loading buffer B containing 20 mM Tris-Cl, pH was added. 7.4, 0.1 M NaCl, 1 mM EDTA and 0.1% (w / v) SDS. The column was washed with 3 volumes of 0.1 M NaOH with 5 mM EDTA and then water treated with DEPC until the pH of the effluent was less than 8, as determined with pH paper. Then, the column was washed with 5 volumes of loading buffer A containing 40 mM Tris-Cl, pH 7.4, 1 M NaCl, 1 mM EDTA and 0.1% (w / v) SDS. The RNA samples were heated at 65 ° C for 5 minutes at which time 400 μl of loading buffer A, preheated to 65 ° C, was added. The sample was mixed and allowed to cool to room temperature for 2 min. before the application to the column. The eluate was collected, heated at 65 ° C for 5 mm. cooled to room temperature for 2 min. and reapplied to the column. This was followed by a 5 volume wash with loading buffer A followed by a 4 volume wash with load buffer B. Poly (A +) RNA was eluted with 3 volumes of 10 mM Tris-Cl, pH 7.4, 1 mM EDTA and 0.05% (w / v) SDS. The fractions were collected and those containing RNA were identified in a plaque assay with ethidium bromide, a petri dish with 1% agarose made with TAE containing EtBr. The RNA was precipitated, resuspended in 10 μl, and a 1 μl aliquot quantified with a spectrophotometer.

B. Isolation of GLTP DNA Sequences and GHTP The nucleotide sequences used in the development of the antisense construct were selected at random from the 5 'sequence of GLTP (SEQ ID NO 1) and GHTP (SEQ ID NO: 3). The DNA sequences used to develop the antisense constructs were obtained using polymerase chain reaction-reverse transcription. Specific primers of GHTP (SPH1 and SPH2) and GLTP (SPL1 and SPL2) were designed according to the published sequences (Brisson et al., 1990, Fukui et al., 1991) with minor modifications to facilitate restriction with enzymes: Primer SPL1: 5 'ATTCGAAAAGCTCGAGATTTGCATAGA3' (SEQ ID NO: 7) (additional CG creates Xho 1 site); SPL2 primer: 5 'GTGTGCTCTCGAGCATTGAAAGC3' (SEQ ID NO: 8) (changed C to G to create the Xho 1 site); SPH1 primer: 5 'GTTTATTTTCCATCGATGGAAGGTGGTG3' (SEQ ID NO: 9) (added CGAT to create the Cia 1 site); Primer SPH2: 5? TAATATCCTGAATCGATGCACTGC3 '(SEQ ID NO: 10) (changed G to T to create the Cia 1 site); Reverse transcription was carried out in a volume of 15 μl containing 1 x CPR buffer (10 mM Tris-Cl, pH 8.2, 50 mM KCl, 0.001% gelatin, 1.5 mM MgCl2), 670 μM of each dNTP, 6 μg of tuber of total potato cv. Russet Burbank RNA, 1 mM each primer (SPH1 and SRL2, or SPH1 and SPH2) and 200 U reverse transcriptase of the Maloney murine leukemia virus (BRL). The reaction was fixed at 37 ° C during minutes, then it was stopped with heat at 94 ° C for 5 minutes and it was. chilled on ice. To the reverse transcription reaction was added 2.5 U of DNA polymerase Taq (BRL) in 35 μl of 1. x CPR buffer. DNA amplification was performed on a Perkin Elmer 480 programmed during cycles with a denaturation step from 1 min to 94 ° C, an annealing step at 58 ° C (SPHl and SPH2) or 56 ° C (SPL1 and SPL2) for 1 min, and an extension step at 72 ° C for 2 min. The CPR was completed with an extension of 10 min. final to 72 UC.

C. Construction of SP Vectors for Phosphorylase Inhibition In order to express the antisense constructs in plant cells, it was necessary to fuse the genes to the regulatory regions of appropriate plants. This was achieved by cloning the antisense DNA into a plasmid vector containing the necessary sequences. The blunt end of the amplified DNA was made and was cloned into a pUC19 vector at the Smal site. The recombinant plasmid was transformed into E. coli DH5a cells of subcloning efficiency (BRL). Transformed cells were plated on LB plates (15 g / 1 Bactotyptone 5 g / 1 yeast extract, 10 g / 1 NaCl, pH 7.3 and solidified with 1.5% agar) containing ampicillin at 100 μg / ml. The selection of plasmids containing bacteria with inserted plant phosphorylase sequence was achieved using color selection. The polylinker and polymerase promoter sequences of T7 and T3 RNA are present in the N-terminal portion of the lacZ gene fragment. Plasmids pUC19 without inserts in the polylinker grow as blue colonies in appropriate bacterial strains such as DH5a. The color selection was made by spreading 100 μl of 2% X-gal (prepared in dimethylformamide) on LB plates containing 50 μg / ml of ampicillin 30 minutes before plating the transformants. Colonies containing plasmids without inserts will be blue after incubation for 12 to 18 hours at 37 ° C and colonies with plasmids containing inserts will remain white. An isolated plasmid was sequenced to confirm the sequence of the phosphorylase inserts. The sequences were determined using the ABI Prism Dye Terminator Cycle Sequencing Core Kit (Applied Biosystems, Foster City CA), reverse primers and M13 universal primers, and an automatic ABI DNA sequencer. The constructed plasmid was purified by the rapid alkaline extraction procedure of a 5 ml overnight culture (Bir boim and Doly, 1979). The orientation of the SPH and SRL fragments in pUC19 was determined by digestion of restriction enzymes. The recombinant pUC19 vectors and the binary vector pB1121 (Clonetech) were restricted, operated on an agarose gel and the fragments purified by gel separation as described by Thuring et al. (1975). The ligation fused the antisense sequence to the binary vector pB1121. The ligation contained the vector pB1121 which had been digested with BamHl and Sacl, together with the phosphorylase DNA product of SPH or SRL, which had been cut from the pUC19 subclone with BamHl and Sacl. The ligated DNA was transformed into E. coli DH5a cells from SCE, and the transformed cells were plated on LB plates containing ampicillin. The nucleotide sequences of the SPL and SPH antisense DNA are nucleotides 338 to 993 of SEQ ID NO: 1 and nucleotides 147 to 799 of SEQ ID NO: 3, respectively. The selection of pB1121 with phosphorylase inserts was performed with specific CAMV and NOS primers. Samples 1 and 2 representing the fragments of tuber type T-type phosphorylase DNA and tuber type L were harvested from a plate after culturing overnight. These samples were inoculated in 5 ml of LB media and cultured overnight at 37 ° C. Plasmids were isolated by the rapid alkaline extraction procedure, and 7DNA was electroporated into Agrobacterium tumefaciens. The constructs were constructed in vector pB1121 containing the 35S promoter of CaMV (Kay et al., 1987) and the terminator sequence NOS 3 '(Bevan et al., 1983). Plasmid pB1121 is prepared from the following well-characterized DNA segments. A fragment of 0.93 kb isolated from transposon Tn7 that encodes resistance to bacterial spectinomycin / streptomycin (Spc / Str) and is a determinant for selection in E. coli and Agrobacterium tumefaciens (Fling et al., 1985). This binds to a chimeric kanamycin resistance gene designed for expression in plants to allow selection of the transformed tissue. The chimeric gene consists of the promoter 355 of the cauliflower mosaic virus of 0.35 kb (P-35S) (Odell et al., 1985), the type II gene of neomycin phosphotransferase of 0.83 kb (NPTII), and the region not 3 'translated from 026 kb of the nopaline synthase gene (NOS 3 r) (Fraley et al., 1983). The next segment is a 0.75 kb replication origin of plasmid RK2 (ori-V) (Stalker et al., 1981). This binds to a Sa / 1 to Pvul segment of 3.1 kb of pBR322 that provides the origin of replication for maintenance in E. coli (ori-322) and the bom site for conjugational transfer in Agrobacterium tumefaciens cells. Then there is a 0.36 kb Pvul fragment of plasmid pTiT37 which contains the right boundary region of nopaline type T DNA (Fraley et al., 1985). The antisense sequence was constructed for expression in the tuber by placing the gene under the control of a non-specific constitutive tissue promoter.

D. Transformation / regeneration of plants The SPL and SPH vectors were transformed into Desiree potato culture according to De Block (1988). To transform "Desiree" potatoes, the cultures of sterile "Desiree" shoots were maintained in test tubes containing 8 ml of SI (Murashige and Skoog medium (MS) supplemented with 2% sucrose and 0.5 g / 1 of MES pH 5.7, solidified with 6 g / 1 of Phytagar). When the seedlings reached approximately 5 cm in length, pieces of leaves were cut with a single cut along the base and inoculated with a 1: 10 dilution of an Agrobacterium tumefaciens overnight culture. Stem explants were co-cultured for two days at 20 ° C on SI medium (De Bock 1988). After co-culture, the explants were transferred to S4 medium (MS medium with sucrose supplemented with 0.5 g / 1 of MES pH 5.7, 200 mg / 1 of glutamine, 0.5 g / 1 of PVP, 20 g / 1 of mannitol, 20 g / 1 of glucose, 40 mg / 1 of adenine, 1 mg / 1 of trans zeatin, 0.1 mg / 1 of NAA, 1 g / 1 of carbenicillin, 50 mg / 1 of kanamycin, solidified with 6 g / 1 of fitagar) for 1 week and then two weeks to induce callus formation. After 3 weeks, the explants were transferred to S6 medium (S4 without NAA and with half the concentration (500 mg / 1) of carbenicillin). After another 2 weeks, the explants were transferred to S8 medium (S6 with only 250 mg / 1 carbenicillin and 0.01 mg / 1 gibberellic acid, GA3) to promote shoot formation. Outbreaks began to develop approximately 2 weeks after transfer to S8 shoot induction medium. These shoots were cut and transferred to jars of SI medium for implantation. After approximately 6 weeks of multiplication in the implant medium, the plants were transferred to the soil and gradually solidified. Desiree plants regenerated in culture were transplanted in 1 gallon (3,785 1) pots and were grown to maturity under greenhouse conditions. The tubers were harvested and allowed to become super at room temperature for 2 days. All tubers larger than 2 cm in length were collected and stored at 4 ° C under high humidity.

E. Field tests Unprocessed controls, plants expressing the SPL construct and plants expressing the SPH construct were propagated in field trials in a randomized design in unique replicas. All the plants were grown side by side in the same field and were exposed to similar pesticide, fertilizer and irrigation regimes. The tubers were harvested and stored at 10 ° C for 2 weeks before randomly selecting a fraction of the tubers from each line to place in storage at 4 ° C, F. Analysis of sugars The tubers were stored at 4 ° C and the reconditioning was not allowed at room temperature before the analysis of sugars. An intact longitudinal slice (1 cm thick, variable width and equal to the outer dimensions of the tuber) of the central portion of each tubercle was cut, thus representing all tuber tissues. At each harvest, the central slices of four tubers for each clone (three replicates) were collectively cut into 1 cm cubes, 15 g of the pooled tissue was randomly selected for analysis. Glucan phosphorylase (see below) and sugars were extracted with 15 ml of Tris buffer (50 mM, pH 7.0) containing 2 mM sodium bisulfite, 2 mM EDTA, 0.5 mM PMSF and 10% (w / w) of glycerol with a polytron homogenizer at 4 ° C. The extracts were centrifuged at 4 ° C (30,000 g, 30 minutes) and the reducing sugars (glucose and fructose) were measured at a 10-fold dilution of the supernatant using a Spectra Physics high-performance liquid chromatograph in interface with a detector. refractive index. The separation was carried out at 80 ° C on an Aminex HPX 87C 30 x 0.78 cm column (Biorad) using 0.6 ml / min. of water as the mobile phase. The calibration of the instrument was by means of authentic patterns of d-glucose and d-fructose.

D. Analysis of a-glucan phosphorylase activity Tubers stored at 40 ° C were not allowed to warm up before extraction and analysis of a-glucan phosphorylase and isozyme activity. The in vitro activity of glucan phosphorylase in the phosphorolytic direction was assayed as described by Steup (1990). Briefly, samples of extracts obtained for analysis of sugars (see above) were added to a reaction medium that coupled phospholysis of starch to the reduction of NADP through the sequential actions of phosphoglucomutase and glucose-6-phosphate dehydrogenase. The rate of NADP reduction during the reaction is stoichiometric with the glucose-1-phosphate production index of the starch substrate. The reduction of NADP was continued at 340 nm on a Varigan Cary double beam spectrophotometer. The protein levels in the extracts were determined according to Bradford (1976). Glucan phosphorylase activity gels were processed essentially according to Steup (1990). The proteins were separated on natural polyacrylamide gels (8.5%) containing 1.5% glycogen. After electrophoresis at 80 V for 15 hours (4 ° C), the gels were incubated (1-2 hours) at 37 ° C in 0.1 M citrate-NaOH buffer (pH 6.0) containing 20 mM glucose-1 P and 0.05% (w / v) of hydrolyzed potato starch. Then the gels were sprayed and stained with an iodine solution. For Western blot analysis, the proteins were subjected to electrophoresis on polyacrylamide gels containing glycogen as described above. The proteins were electrotrown to nitrocellulose and the dyeings were probed with polyclonal antibodies grown against GHTP and GLTP. The immunostains were developed with anti-rabbit secondary antibodies conjugated with alkaline phosphatase (Sigma).

H. Determination of fried potato color Five transgenic potato lines expressing the GLTP antisense sequence, two transgenic lines expressing the GHTP antisense sequence, non-transgenic Desiree control lines and two control lines transformed with vector T-DNA pB1121, were grown under field conditions in Alberta, Canada. The tubers were harvested and stored at 10 ° C and 4 ° C. The color of the fried potato was determined for all the potato lines by taking centric cuts of representative samples of each line and fried at 205 ° F (96.1 ° C) in soybean oil for approximately 3 minutes until the bubbling stopped.

I. Results All the tubers were harvested from plants of the same crop (Desiree), the same age and were grown side by side under identical growth conditions. Northern analysis of tubers showed a considerable reduction of endogenous GLTP transcription in transgenic plants expressing the homologous antisense transcript (Figure 7). The glucan phosphorylase assays showed that the activities (μmol NADPH mg "1 protein h" 1) were reduced (Table 1) at harvest and for at least six months after harvest in transgenic plants expressing the GLTP antisense DNA. The results tabulated in Table 1 show that the activity of a glucan phosphorylase in tubers stored at 4 ° C for 189 days was reduced from approximately 16% to 70% in various varieties of potatoes transformed in relation to the wild-type control strain. Activity and Western spotted gel analyzes showed reduced expression specific for homologous enzymes and lower expression reduction for heterologous enzymes (Figures 10 and 11). This specificity for homologous products can result from differences between phosphorylases (Figures 6A and 6B). Analysis of tubers at harvest (0 days) shows that those expressing the transcription of antisense GLTP have up to 5 times less reducing sugars than control tubers (Table 2). Likewise, after 91 days of storage at 4 ° C the transformed tubers contained 28 to 39% less concentrations of reducing sugars than the wild-type control strain. Glucose and fructose concentrations were significantly reduced in tubers expressing antisense GLTP transcription (Tables 3 and 4). These results suggest that the reduced GLTP activity delays the catabolism of starch in reducing sugars in tubers, while in the control tubers the sugars continue to accumulate. The correlation between the total phosphorylase activity and the concentration of reducing sugars is not direct, suggesting that certain phosphorylase isozymes may play a more important role in starch catabolism, that specific levels of reduced expression of particular phosphorylase isozymes may be more optimal than others, and / or there may be unidentified interactions involved in lower reducing sugar levels.

Transgenic potato plants that express transcription of GLTP or antisense GHTP have been cultured under field conditions and their tubers stored at 4 ° C. The color of the fried potato, which correlated with the sugar content, was determined before cold storage and after 86 and 124 days of cold storage. The potato chip color of all transgenic plants expressing the antisense GLTP transcript was significantly (lighter) improved relative to that of the control (darker) tubers stored under identical conditions (Table 5 and Figure 9) . The results of potato tuber fries from "Desiree" potato plants expressing GLTP transcription were improved by at least 4.3 points and 8.9 points as determined with a Condensed Direct Reading Spectrophotometer Model E-15-FP Agtron (Agtron Inc. 1095 Spice Island Drive Number 100, Sparks Nevada 89431) after storage at 10 ° C and 4 ° C, respectively, for 86 days. The results of the potato chips of GLTP transformants measured after 124 days of storage at 4 ° C were improved by 44% to 89% in relation to the wild type (Table 5). The Desiree crop is not a commercially desirable potato to cut into slices due to its high content of natural sugars and propensity to rapidly sweeten in cold storage. However, the significant improvements in the color of the fried potato were noticed with the "Desiree" potatoes transformed. It is expected that superior color clarification will be achieved if the methods of the invention were applied to varieties of commercially processed potatoes. Analysis of tubers stored at 10 ° C and 4 ° C shows that those expressing the transcript of antisense GHTP sometimes provided fries that were fried with a lighter color than the control tubers, indicating a lower accumulation of reducing sugars (Table 5). The results showing heterologous and homologous reduction in phosphorylase activity (Figures 10 and 11) indicate that the improvement may be the result of the reduction of one or both phosphorylases of tubers. However, these results suggest that L-type phosphorylase plays a more important role in the catabolism of starch in reducing sugars. Additionally, the results show that the difference in the levels of reducing sugars (Table 2) and the results of the fried potato (Table 5) between plants of wild type of tubers and those that express RNA antisense of phosphorylase of tubercle is maintained during storage long-term. As shown in Table 5, the results of the fried potato are approximately equal to 86 days and to 124 days. No other increase in reducing sugar concentrations was evident after storage of 49 and 91 days at 4 ° C (Table 2). This balance in the concentration of sugars was probably associated with the kinetics of the tuber phosphorylases. The ability to maintain levels of sugars. lower has the potential to extend the storage period in at least several months. At present, processing potatoes are generally stored for a maximum of 3 to 6 months at 10 ° C to 120 ° C before the accumulation of sugars reaches levels that reduce the quality. The fresh product must be imported until the seasonal potatoes are available. The extension of the period of storage of potatoes in many months can reduce import costs. Table 6 presents a summary of the percentage of improvement in several improved cold storage characteristics of tubers, tubers of potato plants transformed with antisense DNA derived from the sequence of the GLTP gene (ATL3-ATL9) and the genetic sequence of GHTP (ATH1 and ATH2) in relation to the control plants did not transformed. It is evident from the results summarized in Table 6 that substantial improvements in cold storage characteristics of tubers can be consistently obtained through the methods of the present invention. Especially noteworthy are the improvements in the percentages of the results of the fried potato with respect to the wild type observed after storage at 4 ° C for approximately four months (124 days). Improvements were observed in the relative fried potato results of up to 89% in relation to the wild type. The improved results of the fried potato reflect the commercial utility of the invention. That is to say, by reducing cold induced sweetening, the tubers can be stored at lower temperatures, without causing unacceptable browning of the potato chips products. The reduction in sugar accumulation of transformed potato lines relative to the wild type, both at harvest and after 91 days of storage, also demonstrates significant advantages of the invention. The accumulation of reduced sugars is related to the improvements observed in the results of the fried potato and also reflects improved specific heaviness of the tubers, another important commercial measure of the quality of the tubers. Even in the harvest, substantial improvements in the result of the fried potato and the accumulation of reduced sugars were noted for the transformed lines in relation to the wild type. Therefore, the benefits of the invention are not limited to the improvements that arise only after prolonged periods of cold storage, but are present at the time of harvest. In this sense, the invention is not limited only to the improvements in the characteristics of cold storage but also encompasses the improvements in the quality characteristics of the tubers, such as the result of fried potatoes or the accumulation of sugars that are present. present in e! moment of harvest, resulting in an earlier maturity. With reference to the specific improvements summarized in Table 6, it can be seen that the GLTP-type transformants (ATL3-ATL9) exhibited up to 66%, 70% and 69% reduction in the activity of a glucan phosphorylase relative to the wild type. , in the harvest and after storage for 91 and 189 days, respectively. Most also showed improvements in excess of 10% and 30% in relation to the wild type at harvest and after storage for 91 and 189 days. After storage for 91 and 189 days, the GHTP-type transformants (ATH1 and ATH2) exhibited, respectively, up to 28% and 39% relative improvement over the wild type and showed in general at least 10% improvement. The GLTP-type transformants exhibited up to 80% and 39% reduction of sugar accumulation relative to the wild type at harvest and at 91 days, respectively. At harvest, all GLTP-type transformants exhibited at least 10% and at least 30% relative improvement. At 91 days all GLTP-type transformants exhibited at least 10% and most exhibited at least 30% relative improvement. The GLTP-type transformants exhibited up to 46%, 89% and 89% improvement of potato chips results relative to the wild type at harvest, and after storage for 86 days and 124 days, respectively. Almost all exhibited at least 10% and 30% relative improvement in the harvest and after storage for 86 and 124 days. After 124 days of storage, at least 1 of the GHTP-type transformants exhibited up to 25% relative improvement in the result of the potato chips. The results clearly show that substantial improvements in the cold storage characteristics of tubers can easily be obtained by the methods of the invention. The results vary due to, among other things, the location within the genome of the plant where the sense or recombinant antisense DNA is inserted, and the number of insertion events that occur. It is important to note that despite the variability in the results between the various transformed lines, there was little variation in the results between the samples within a single transformed potato line (see footnotes in Tables 1 to 5). The results are presented in Table 6 for all lines of potato plants that were successfully transformed with antisense DNA of GHTP or GLTP. Therefore, all transformants show at least some improvement in one or more cold storage characteristics such as increased result of fried potato (lighter color when cooking) and reduced sugar accumulation and most show very substantial improvements. Given the large proportion of positive transformants observed in the examples herein, it is expected that, using the cold storage characteristics testing procedures described in the examples, potato plants transformed through the methods of the invention can be easily classified to identify transformed lines that exhibit significantly cold storage characteristics. improved. By applying the techniques described herein to the commercially important potato varieties, it will be possible to easily create and select transformants that have significantly improved cold storage characteristics. Those transformants that show the relative greater improvements with respect to wild-type controls can be used in the development of new varieties of commercial potatoes.

Table 1 Effects of an antisense transcript on glucan phosphorylase activity measured in enzymatic extracts of "Desiree" tubers grown in the field.

Glucan phosphorylase activity Storage Period at 4 ° C (days) Clone 0 49 91 140 189 μmol NADPH mg "1 protein rT1 Wf 10.50 11, 83 9.94 11, 90 13.04 ATL3 4.90 4.86 4.49 4.73 4:88 ATL4 11.45 7.17 8.09 11.32 10.99 ATL5 3.58 3.56 2.97 4.59 4.79 ATL9 3.59 '3.88 3.84 4.72 3.98 LSDa_-b 1, 97 2.94 1, 59 2.34 2.58 SD0.01 2.87 4.28 2.31 3.41 3.75 Ctopc / _: 01d WT vs. 0.01 ATL's Days NS Clone x Days 0.05 WT 1 1, 49 8.90 12:66 13.66 ATH-1 10.40 9.69 10.79 10.10 ATH-2 6.46 6.40 6.56 8.38 LSDar 2.02 0.41 3.00 NS Clonc 0.01 Tvs. 0.01 ATH's Days 0.05 Clopx Days NS aWT, untransformed wild type tubers, bLSD, less significant difference at 0.05 or 0.01 level for each storage period. c Variable sources in factor analysis. d Significance levels for indicated sources of variation.

Table 2 Effects of antisense GLTP transcription on the low temperature induced sweetening of "Desiree" tubers grown in the field.

Reducing sugars (glucose + fructose) Storage Period at 4 ° C (days) Clone 49 91 gg wta fresh weight 5.63 31.8 28.0 ATL3 1, 88 17.3 17.3 ATL4 1, 11 14.3 20.1 ATL5 1.51 18.3 17.0 17 ATL9 1, 36 17.3 18.5 WT vs. ATL's 0.01 0.01 0.05 Clonc 0.0 Days 0.01 Clone x Days NS aWT, untransformed wild type tubers. b Orthogonal comparisons for ANOVA 's in each storage period. c Variable sources in factor analysis. d Significance levels for indicated sources of variation.

Table 3 Effects of antisense GLTP transcription on the fructose accumulation induced by low temperature of "Desiree" tubers grown in the field.

Fructose Storage Period at 4 ° C (days) Clone 49 91 mg g wta fresh weight 3.53 15.10 12.20 ATL3 1.21 8.40 8.79 ATL4 0.79 7.22 8.56 ATL5 0.61 10.00 8.09 ATL9 0.54 8, 38 8.72 WT vs. ATL'sb 0.01 0.01 NS Clop 0.01 d Days 0.01 Clone x Days NS aWT, untransformed wild type tubers.

Orthogonal comparisons for ANOVA 's in each storage period. c Variable sources in factor analysis. d Significance levels for indicated sources of variation. Table 4 Effects of antisense GLTP transcription on glucose accumulation induced by low temperature of "Desiree" tubers grown in the field.

Glucose Storage Period at 4 ° C (days) Clone 49 91 mg g "1 wta fresh weight 2,10 16,60 15,90 AT 3 0,68 8,94 8,49 ATL 4 0,32 7,07 11, 06 ATL5 1.05 8.33 8.91 ATL9 0.83 8.87 9.78 WT vs. ATL's "0.01 0.01 0.05 Cionc 0.01 d Days 0.01 Clone x Days NS 3WT, untransformed wild type tubers, ^ Orthogonal comparisons for ANOVA 's in each storage period. c Variable sources in factor analysis. d Significance levels for indicated sources of variation. Table 5 Color of the average fried potato of "Desiree" tubers grown in the field. The evaluation of the color of the fried potato was assigned using an Agtron meter similar to that used by the industry to measure the color of potato chips. In this index, the higher the lighter number is the product of fried potato although the color does not represent a linear relationship with the index.

Storage Temperature, Period and Reading Agtron8 Harvest 86 days 86 days 124 days wt 26 25.3 15.4 17.1 ATL3e 25 37.4 26.7 30.8 ATL4 35 43.2 29.1 32.3 ATL5 36 29.6 24.7 24.6 ATL9 38 38.7 24.3 26.6 ATH1d 26 49.7 17.5 17.4 ATH2 29 31, 2 15.6 15.9 Glv.P1 '31 15.7 15.7 GMP2 35 16.7 16.6 3Agtron Inc. 1095 Spice Island Drive No. 100, Sparks Nevada 89431. Agtron model E-15-FP (Condensed Direct Reading Spectrophotometer). The ratio of reflectance measurements in two spectral modes, almost infrared and green. The results represent the measurement of 6 to 8 potato chips of 3 tubers randomly selected approximately 3 to 4 cm in diameter. ^ T, negative control, untransformed wild-type tubers. CATL, tubers transformed with the a-glucan phosphorylase type L tuber. dATH, tubers transformed with the a-glucan phosphorylase type H tuber. eGMP, negative control tubers transformed with T DNA pB1121.

Table 6 Summary of Results REFERENCES Alber and Kawasaki (1982) Mol. And Appl Genet 1: 419-434.

Rees et al. (19) Symp. Soc. Exp. Biol. 42: 377-393.

Bevan et al. (1983) Nature [Nature] (London) 304: 184-187.

Bevan et al. (1986) Nucleic Acids Res. [Imv. of Nucleic acids] 14 (11): 4625-4638.

Birnboim et al. (1979) Nucleic Acids Res. [Inv. of Nucleic acids] 7: 1513-1523. Blennow et al. (1991) Phytochemistry [Phytochemistry] 30: 437-444.

Bradford, M.M. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. [A rapid and sensitive method for the quantification of amounts in micrograms of protein using the principle of fixation with protein staining. Bioquim. Anal.] 72: 243-254. Brisson et al. (1989) The Plant Cell [The Plant Cell] 1: 559-566. Brusslan and Tobin (1995) Plant Molecular Biology 27: 809-813. Burton, W.G. (1989) The Potato [The Pope] Longman Scientific and Technical. Cannon et al. (1990) Plant Molecular Biology 15: 39-47. Claassen et al. (1991) Plant Physiol. [Fisiol. of Plants] 95: 1243-1249. Coffin et al. (1987) J. Food Sci. 52: 639-645. Davies and Viola (1992) Postharvest News and Information [Information and Post-harvest News] 3: 97-100. De Block, M. (1988) Theoretical and Applied Genetics [76] 767-774. De Carvalho et al. (1992) EMBO J. 11: 2595-2602. Depicker and others (1982) Mol. And Appl. Genet 1: 561-573. Dixon et al. (1981) Phytochemistry [Phytochemistry] 20: 969-972. Dorlhac and others 1994 Mol. Gen. Genetic. 243: 613-621. Ebbelaar et al. (1993) Int. Symp. on Gen. Manip. of Plant Metabolism and Growth, 29-31 March, Norwich UK Abstract No. 9. Ecker and Davies (1986) Proc. Nati Acad. Sci. 83: 5373-5376. Fling et al. (1985) Nucleic Acids Research 13 no. 19, 7095-7106. Fraley et al. (1983) Proc Nati Acad Sci USA 80, 4803-4807. Fraley and others (1985) Bio / Technology [Bio / Technology] 3, 629-635. Fray and Grierson 1993 Plant Mol. Biol. 22: 589-602. Gielen et al. (1984) EMBO J. 3: 835-846. Hasseloff, J. And W. L. Gerlach (1988) Nature [Nature] 334: 585-591. Hart and others (1992) Mol. Gen. Genetic. 235: 179-188. Jorgensen, R. A. (1995) Science [Science] 268: 686-691. Kawchuk and others (1990) Molecular Plant-Microbe Interactions [Plant-Microbe Molecular Interactions] 3: 301-307. Kawchuk et al. (1991) Molecular Plant-Microbe inter. [Plant-Microbe Molecular Interactions] 4: 247-253.

Kay et al. (1987) Science [Science] 236: 1299-1302. Kruger, N. J. and Hammond, J. B. W. (1988) Plant Physiol. [Fisiol. of Plants] 86: 645-648. Laemmli, U.K. (1970) Nature [Nature] (London) 227: 680-685. Lin et al. (1988) Plant Physiol. [Fisiol. of Plants] 86: 1131-1135. Loiselle et al. (1990) American Potato Journal [American Potato Bulletin] 67: 633-646. Lynch et al., (1992) Can. J. Plant Sci. 72: 535-543. Matzke and Matzke (1995) Plant Pnysiol. [Fisiol. of Plants] 107: 679. 'Meyer and Saedler (1996) Annu. Rev. Plant Physiol. 47: 23-48. Mori et al. (1991) J. Biol. Chem. 286: 18446-18453. Muller et al. (1990) Mol. Gen. Genet. 224: 136-146. Nakano et al. (1989) J. Biochem. 106: 691-695. Nakano, K. and Fukui, T. (1986) J. Biol. Chem. 266: 8230-8256. Napoli et al. (1990) Plant Cell 2: 279-289. Odell et al. (1985) Ñature [Nature] 313, 810-812 Ohta et al. (1991) Mol. Gen. Genet. 225: 369- 378. Ortíz, R. and Hua an, Z. (1994) In: Potato Genetics. [Genetics of the potato] Bradshaw, J. E. and Mackay G.R. (eds.). Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual [Molecular Cloning, a Laboratory Manual]. 2nd Ed. Cold Spring Habor Laboratory Press, N.Y. Seymour and others 1993 Plant Mol. Biol. 23: 1-9. Shahar et al. (1992) Plant Cell 4: 135-147. Shallenberger et al. (1959) Agrie, and Food Chem. 7: 274-277. Smith and others (1988) Nature [Nature] 334: 724-726. Smith et al. (1990) Mol. Gen. Genet. 244: 447-481. Sonnewald et al. (1995) Plant Molecular Biology 27: 567-576. Sowokinos, J. (1990) In: The Molecular and Cellular Biology of the Potato. M. A. Mayo and W. D. Parks (eds.). Stalker et al. (1981) Mol. Gen Genet 181, 8-12.

Steup, M. (1990) Starch Degrading Enzymes in "Methods in Plant Biochemistry" [Starch Degradation Enzymes in "Methods in Plant Biochemistry" Vol. 3. P.M. Dey and J.B. Harborne. eds. Academic Press, London. Stiekema et al. (1988) Plant Mol. Biol. 11: 255-269. Stukerlj and others (1990) Nucí. Acids Res. 18: 46050. Takaha et al., (1993) J. Biol. Chem. 268: 1391-1396. Thuring et al. (1975) Analytical Biochemistry 66: 213-220. Van der Krol et al. (1988) Gene [Gen] 72: 45-50. Van der Krol (1990) Plant Cell 2: 291-299. Weaver and others (1978) Am. Pot. J. 55: 83-93. Weintraub (1990) Scientific American [American Scientist] 1: 34-40. Winnacker, Ernst L. (1987) From Genes to Clones.

[From Genes to Clones]. VCH Verlagsgessellschaft mbH, Federal Republic of Germany. Yoshida et al. (1992) Geneg 10: 255-259.

All publications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains. All publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated as incorporated by reference.

While the foregoing invention has been described with some details by way of illustration and exemplified for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

LIST OF SEQUENCES GENERAL INFORMATION: (i) APPLICANT: Her Majesty the Queen in Right Cares Represented by the Department Agriculture and Agri-Food Canada (ii) TITLE OF THE INVENTION: TRANSGENIC POTS Q HAVE REDUCED LEVELS OF ACTIVITY ALPHA GLUCA PHOSPHORYLASE OF TUBERCULOSIS OF THE TYPE L OR H C ENDULZING IN REDUCED FRIÓ (iii) SEQUENCE NUMBER: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: McKay-Carey & Company (B) STREET: 2125 Commerce Place, 10155-102nd Street (C) CITY: Edmonton (D) STATE: Alberta (E) COUNTRY: Canada (F) POSTAL CODE: T5J 4G8 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Hard disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: WO (B) SUBMISSION DATE: 10-FEB-1998 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60 / 036,946 (B) DATE OF SUBMISSION: 10-FEB-1997 (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 868,786 (B) DATE OF SUBMISSION: 04-JUN-1997 (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: McKay-Carey, Mary Jane (B) REGISTRATION NUMBER: 3790 (C) REFERENCE NUMBER / DOCUMENT: 24002WO0 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (403) 424-0222 (B) TELEFAX: (403) 421-0834 (2) INFORMATION FOR SECTION ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3101 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosu (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 44..2944 (D) OTHER INFORMATION: / product- "alpha glyca phosphorylase of tuber type L potato" (ix) CHARACTERISTICS: (A) ) NAME / KEY: mat_peptido (B) LOCATION 194..2941 (ix) CHARACTERISTICS: (A) NAME / KEY: sig_peptido (B) LOCATION: 44..193 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l ATCACTCTCA TTCGAAAAGC TAGATTTGCA TAGAGAGCAC AAA ATG GCG ACT GCA 5 Met Ala Thr Ala -50 AAT GGA GCA CAC TTG TTC AAC CAT TAC AGC TCC AAT TCC AGA TTC ATC 10 Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn Ser Arg Phe lie -45 -40 -35 CAT TTC ACT TCT AGA AAC ACA AGC TCC AAA TTG TTC CTT ACC AAA ACC 15 His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe Leu Thr Lys Thr -30 -25 -20 -15 TCC CAT TTT CGG AGA CCC AAA CGC TGT TTC CAT GTC AAC AAT ACC TTG 19 Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val Asn Asn Thr Leu -10 -5 1 AGT GAG AAA ATT CAC CAT CCC ATT ACT GAA CAA GGT GGT GAG AGC GAC 24 Ser Glu Lys lie His Pro Pro lie Thr Glu Gln Gly Gly Glu Ser Asp 5 10 15 CTG AGT TCT TTT GCT CCT GAT GCC GCA TCT ATT ACC TCA AGT ATC AAA 29 Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser lie Thr Ser Ser lie Lys 20 25 30 TAC CAT GCA GAA TTC ACÁ CCT GTA TTC TCT CCT GAA AGG TTT GAG CTC 34 Tyr His Wing Glu Phe Thr Pro Val Phe Ser Pro Glu Arg Phe Glu Leu 35 40 45 50 CCT AAG GTC TTC TTT GCA ACA GCT CAUT AGT GTT CGT GAT TCG CTC CTT 39 Pro Lys Wing Phe Phe Wing Thr Wing Gln Ser Val Arg Asp Ser Leu Leu 55 60 65 ATT AAT TGG AAT GCT ACG TAT GAT ATT TAT GAA AAG CTG AAC ATG AAG 43 lie Asn Trp Asn Wing Thr Tyr Asp lie Tyr Glu Lys Leu Asn Met Lys 70 75 80 CAA GCG TAC TAT CTA TCC ATG GAA TTT CTG CAG GGT AGA GCA TTG TTA 48 Gln Wing Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly Arg Wing Leu Leu 85 90 95 AAT GCA ATT GGT AAT CTG GAG CTT ACT GGT GCA TTT GCG GAA GCT TTG 53 Asn Ala lie Gly Asn Leu Glu Leu Thr Gly Ala Phe Ala Glu Ala Leu 100 105 110 AAA AAC CTT GGC CAC AAT CTA GAA AAT GTG GCT TCT CAG GAA CCA GAT 583 Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser Gln Glu Pro Asp 115 120 125 130 GCT GCT CTT GGA AAT GGG GGT TTG GGA CGG CTT GCT TCC TGT TTT CTG 631 Wing Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Wing Ser Cys Phe Leu 135 140 145 GAC TCT TTG GCA ACA CTA AAC TAC CCA GCA TGG GGC TAT GGA CTT AGG 67 Asp Ser Leu Wing Thr Leu Asn Tyr Pro Wing Trp Gly Tyr Gly Leu Arg 150 155 160 TAC AAG TAT GGT TTA TTT AAG CAG CGG ATT AA AAA GAT GGT CAG GAG 727 Tyr Lys Tyr Gly Leu Phe Lys Gln Arg lie Thr Lys Asp Gly Gln Glu 165 170 175 GAG GTG GCT GAA GAT TGG CTT GAA ATT GGC AGT CCA TGG GAA GTT GTG 775 Glu Val Wing Glu Asp Trp Leu Glu He Gly Ser Pro Trp Glu Val Val 180 185 190 AGG AAT GAT GTT TCA TAT CCT ATC AAA TTC TAT GGA AAA GTC TCT ACA 823 Arg Asn Asp Val Ser Tyr Pro He Lys Phe Tyr Gly Lys Val Ser Thr 195 200 205 210 GGA TGA GAT GGA AAG AGG TGA TGG ATG ATG GGT AAG GAG AAG GAG 871 Gly As Asp Gly Lys Arg Tyr Trp He Gly Gly Glu Asp He Lys Wing 215 220 225 GTT GCG TAT GAT GTT CCC ATA CCA GGG TAT AAG ACC AGA ACC ACÁ ATC 91 Val Ala Tyr Asp Val Pro Pro Gly Tyr Lys Thr Arg Thr Thr He 230 235 240 AGC CTT CGA CTG TGG TCT ACA CAG GTT CCA TCA GCG GAT TTT GAT TTA 96 Ser Leu Arg Leu Trp Ser Thr Gln Val Pro Ser Wing Asp Phe Asp Leu 245 250 255 TCT GCT TTC AAT GCT GGA GAG CAC ACC AAA GCA TGT GAA GCC CA GCA 101 Be Wing Phe Asn Wing Gly Glu His Thr Lys Wing Cys Glu Wing Gln Wing 260 265 270 AAC GCT GAG AAG ATA TGT TAC ATA CTC TAC CCT GGG GAT GAA TCA GAG 106 Asn Wing Glu Lys He Cys Tyr He Leu Tyr Pro Gly Asp Glu Ser Glu 275 280 285 290 GAG GGA AAG ATC CTT CGG TTG AAG CAA CA TAT ACC TTA TGC TCG GCT 111 Glu Gly Lys He Leu Arg Leu Lys Gln Gln Tyr Thr Leu Cys Ser Wing 295 300 305 TCT CTC CAA GAT ATT ATT TCT CGA TTT GAG AGG AGA TCA GGT GAT CGT 115 Ser Leu Gln Asp He He Ser Arg Phe Glu Arg Arg Ser Gly Asp Arg 310 315 320 ATT AAG TGG GAA GAG TTT CCT GAA AAA GTT GCT GTG CAG ATG AAT GAC 120 lie Lys Trp Glu Glu Phe Pro Glu Lys Val Wing Val Gln Met Asn Asp 325 330 335 ACT CAC CCT ACÁ CTT TGT ATC CCT GAG CTG ATG AGA ATA TTG ATA GAT 125 Thr His Pro Thr Leu Cys He Pro Glu Leu Met Arg He Leu He Asp 340 345 350 CTG AAG GGC TTG AAT TGG AAT GAA GCT TGG AAT ATT ACT CAA AGA ACT 130 Leu Lys Gly Leu Asn Trp Asn Glu Wing Trp Asn He Thr Gln Arg Thr 355 360 365 370 GTG GCC TAC ACA AAC CAT ACT GTT TTG CCT GAG GCA CTG GAG AAA TGG 1351 Val Wing Tyr Thr Asn His Thr Val Leu Pro Glu Wing Leu Glu Lys Trp 375 380 385 AGT TAT GAA TTG ATG CAG AAA CTC CTT CCC AGA CAT GTC GAA ATC ATT 139 Ser Tyr Glu Leu Met Gln Lys Leu Pro Arg His Val Glu He He 390 395 400 GAG GCG ATT GAC GAG GAG CTG GTA CAT GAA ATT GTA TTA AAA TAT GGT 144 Glu Ala He Asp Glu Glu Leu Val His Glu He Val Leu Lys Tyr Gly 405 410 415 TCA ATG GAT CTG AAC AAA TTG GAG GAA AAG TTG ACT ATG AGA ATC 149 Ser Met Asp Leu Asn Lys Leu Glu Glu Lys Leu Thr Thr Met Arg He 420 425 430 TTA GAA AAT TTT GAT CTT CCC AGT TCT GTT GCT GAA TTA TTT ATT AAG 154 Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu Leu Phe He Lys 435 440 445 450 CCT GAA ATC TCA GTT GAT GAT GAT ACT GAA ACA GTA GAA GTC CAT GAC 159 Pro Glu He Ser Val Asp Asp Asp Thr Glu Thr Val Glu Val His Asp 455 460 465 AAA GTT GAA GCT TCC GAT AAA GTT GTG ACT AAT GAT GAA GAT GAC ACT 163 Lys Val Glu Ala Ser Asp Lys Val Val Thr Asn Asp Glu Asp Asp Thr 470 475 480 GGT AAG AAA ACT AGT GTG AAG ATA GAA GCA GCT GCA GAA AAA GAC ATT 168 Gly Lys Lys Thr Ser Val Lys He Glu Ala Ala Wing Glu Lys Asp He 485 490 495 GAC AAG AAA ACT CCC GTG AGT CCG GAA CCA GCT GTT ATA CCA CCT AAG 173 Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val He Pro Pro Lys 500 505 510 AAG GTA CGC ATG GCC AAC TTG TGT GTT GTG GGC GGC CAT GCT GTT AAT 178 Lys Val Arg Met Wing Asn Leu Cys Val Val Gly Gly His Wing Val Asn 515 520 525 530 GGA GTT GCT GAG ATC CAT AGT GAA ATT GTG AAG GAG GAG GTT TTC AAT 183 Gly Val Ala Glu He His Ser Glu He Val Lys Glu Glu Val Phe Asn 535 540 545 GAC TTC TAT GAG CTC TGG CCG GAA AAG TTC CAA AAC AAA ACÁ AAT GGA 187 Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gln Asn Lys Thr Asn Gly 550 555 560 GTG ACT CCA AGA AGA TGG ATT CGT TTC TGC AAT CCT CCT CTT AGT GCC 192 Val Thr Pro Arg Arg Trp He Arg Phe Cys Asn Pro Pro Leu Ser Wing 565 570 575 ATC ATA ACT AAG TGG ACT GGT ACÁ GAG GAT TGG GTC CTG AAA ACT GAA 197 He He Thr Lys Trp Thr Gly Thr Glu Asp Trp Val Leu Lys Thr Glu 580 585 590 AAG TTG GCA GAA TTG CAG AAG TTT GCT GAT AAT GAA GAT CTT CAA AAT 202 Lys Leu Wing Glu Leu Gln Lys Phe Wing Asp Asn Glu Asp Leu Gln Asn 595 600 605 610 GAG TGG AGG GAA GCA AAA AGG AGC AAC AAG ATT AAA GTT GTC TCC TTT 207 Glu Trp Arg Glu Wing Lys Arg Ser Asn Lys He Lys Val Val Ser Phe 615 620 625 CTC AAA GAA AAG ACÁ GGG TAT TCT GTT GTC CCA GAT GCA ATG TTT GAT 211 Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp Wing Met Phe Asp 630 635 640 ATT CAG GTA AAA CGC ATT CAT GAG TAC AAG CGA CA CTG TTA AAT ATC 216 He Gln Val Lys Arg He His Glu Tyr Lys Arg Gln Leu Leu Asn He 645 650 655 TTC GGC ATC GTT TAT CGG TAT AAG AAG ATG AAA GAA ATG ACÁ GCT GCA 221 Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu Met Thr Ala Wing 660 665 670 GAA AGA AAG ACT AAC TTC GTT CCT CGA GTA TGC ATA TTT GGG GGA AAA 226 Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys He Phe Gly Gly Lys 675 680 685 690 GCT TTT GCC ACÁ TAT GTG CAÁ GCC AAG AGG ATT GTA AAA TTT ATC ACÁ 2311 Wing Phe Wing Thr Tyr Val Gln Wing Lys Arg He Val Lys Phe He Thr 695 700 705 GAT GTT GGT GCT ACT ATA AAT CAT GAT CCA GAA ATC GGT GAT CTG TTG 235 Asp Val Gly Ala Thr He Asn His Asp Pro Glu He Gly Asp Leu Leu 710 715 720 AAG GTA GTC TTT GTG CCA GAT TAC AAT GTC AGT GTT GCT GAA TTG CTA 240 Lys Val Val Phe Val Pro Asp Tyr Asn Val Val Ser Wing Glu Leu Leu 725 730 735 ATT CCT GCT AGC GAT CTA TCA GAA CAT ATC AGT ACG GCT GGA ATG GAG 245 He Pro Wing Ser Asp Leu Ser Glu His He Ser Thr Wing Gly Met Glu 740 745 750 GCC AGT GGA ACC AGT AAT ATG AAG TTT GCA ATG AAT GGT TGT ATC CAA 250 Wing Ser Gly Thr Ser Asn Met Lys Phe Wing Met Asn Gly Cys He Gln 755 760 765 770 ATT GGT ACA TTG GAT GGC GCT AAT GTT GAA ATA AGG GAA GAG GTT GGA 255 He Gly Thr Leu Asp Gly Ala Asn Val Glu He Arg Glu Glu Val Gly 775 780 785 GAA GAA AAC TTC TTT CTC TTT GGT GCT CA GCT CAT GAA ATT GCA GGG 259 Glu Glu Asn Phe Phe Leu Phe Gly Wing Gln Wing His Glu He Wing Gly 790 795 800 CTT AGA AAA GAA AGA GCT GAC GGA AAG TTT GTA CCT GAT GAA CGT TTT 264 Leu Arg Lys Glu Arg Wing Asp Gly Lys Phe Val Pro Asp Glu Arg Phe 805 810 815 GAA GAG GTG AAG GAA TTT GTT AGA AGC GGT GCT TTT GGC TCT TAT AAC 2695 Glu Glu Val Lys Glu Phe Val Arg Ser Gly Wing Phe Gly Ser Tyr Asn 820 825 830 TAT GAT GAC CTA ATT GGA TCG TTG GAA GGA AAT GAA GGT TTT GGC CGT 2743 Tyr Asp Asp Leu He Gly Ser Leu Glu Gly Asn Glu Gly Phe Gly Arg 835 840 845 850 GCT GAC TAT TTC CTT GTG GGC AAG GAC TTC CCC AGT TAC ATA GAA TGC 2791 Wing Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser Tyr He Glu Cys 855 860 865 CAA GAG AAA GTT GAT GAG GCA TAT CGC GAC CAG AAA AGG TGG ACA ACG 283 Gln Glu Lys Val Asp Glu Wing Tyr Arg Asp Gln Lys Arg Trp Thr Thr 870 875 880 ATG TCA ATC TTG AAT ACA GCG GGA TCG TAC AAG TTC AGC AGT GAC AGA 288 Met Ser Asn Leu Thr Ala He Gly Ser Tyr Lys Asp Phe Ser Ser Arg 885 890 895 ACA ATC CAT GAA TAT GCC AAA GAC ATT TGG AAC ATT GAA GCT GTG GAA 293 Thr He His Glu Tyr Ala Lys Asp He Trp Asn He Glu Ala Val Glu 900 905 910 ATA GCA TAA GAGGGGGAAG TGAATGAAAA ATAACAAAGG CACAGTAAGT 298 He Ala * 915 AGTTTCTCTT TTTATCATGT GATGAAGGTA TATAATGTAT GTGTAAGAGG ATGATGTTAT 304 TACCACATAA TAAGAGATGA AGAGTCTCAT TTTGCTTCAA AAAAAAAAAA AAAAAAA 310 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 967 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (/ xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Met Ala Thr Ala Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn -50 -45 -40 -35 Be Arg Phe He His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe -30 -25 -20 Leu Thr Lys Thr Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val -15 -10 -5 Asn Asn Thr Leu Ser Glu Lys He His His Pro He Thr Glu Gln Gly 1 5 10 Gly Glu Ser Asp Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser He Thr 15 20 25 30 Being Ser He Lys Tyr His Wing Glu Phe Thr Pro Val Phe Ser Pro Glu 35 40 45 Arg Phe Glu Leu Pro Lys Wing Phe Phe Wing Thr Wing Gln Ser Val Arg 50 55 60 Asp Ser Leu Leu lie Asn Trp Asn Wing Thr Tyr Asp lie Tyr Glu Lys 65 70 75 Leu Asn Met Lys Gln Ala Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly 80 85 90 Leu Ala Arg Leu Ala Asn Asn Leu Glu Gly He Leu Thr Gly Ala Phe Ala 95100105110 Ala Glu Leu Lys Asn Leu Gly His Asn Leu Glu Asn Ala Val Ser 115 120 125 Gln Glu Pro Asp Wing Wing Leu Gly Asn Gly Gly Leu Gly Arg Leu Wing 130 135 140 Be Cys Phe Leu Asp Be Leu Wing Thr Leu Asn Tyr Pro Wing Trp Gly 145 150 155 Tyr Gly Leu Arg Tyr Lys Tyr Gly Leu Phe Lys Gln Arg He Thr Lys 160 165 170 Asp Gly Gln Glu Glu Val Ala Glu Asp Trp Leu Glu He Gly Ser Pro 175 180 185 190 Trp Glu Val Val Arg Asn Asp Val Ser Tyr Pro He Lys Phe Tyr Gly 195 200 205 Lys Val Ser Thr Gly Ser Asp Gly Lys Arg Tyr Trp He Gly Gly Glu 210 215 220 Asp He Lys Wing Val Wing Tyr Asp Val Pro He Pro Gly Tyr Lys Thr 225 230 235 Arg Thr Thr He Ser Leu Arg Leu Trp Ser Thr Gln Val Pro Ser Wing 240 245 250 Asp Phe Asp Leu Be Wing Phe Asn Wing Gly Glu His Thr Lys Wing Cys 255 260 265 270 Glu Ala Gln Ala Asn Ala Glu Lys He Cys Tyr He Leu Tyr Pro Gly 275 280 285 sp Glu Ser Glu Glu Gly Lys He Leu Arg Leu Lys Gln Gln Tyr Thr 290 295 300 Leu Cys Ser Ala Ser Leu Gln Asp He He Ser Arg Phe Glu Arg Arg 305 310 315 Ser Gly Asp Arg He Lys Trp Glu Glu Phe Pro Glu Lys Val Wing Val 320 325 330 Gln Met Asn Asp Thr His Pro Thr Leu Cys He Pro Glu Leu Met Arg 335 340 345 350 He Leu He Asp Leu Lys Gly Leu Asn Trp Asn Glu Wing Trp Asn He 355 360 365 Thr Gln Arg Thr Val Wing Tyr Thr Asn His Thr Val Leu Pro Glu Wing 370 375 380 Leu Glu Lys Trp Ser Tyr Glu Leu Met Gln Lys Leu Leu Pro Arg His 385 390 395 Val Ala Glu He He Glu Glu Asp Glu Leu He His Glu He Val Val Leu Lys 400 405 410 Tyr Gly Met Asp Leu Ser Asn Glu Lys Leu Leu Glu Lys Thr 415 420 425 430 Thr Met Arg He Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu 435 440 445 Leu Phe He Lys Pro Glu He Ser Val Asp Asp Asp Thr Glu Thr Val 450 455 460 Glu Val His Asp Lys Val Glu Wing Ser Asp Lys Val Val Thr Asn Asp 465 470 475 Glu Asp Asp Thr Gly Lys Lys Thr Ser Val Lys He Glu Ala Wing Wing 480 485 490 Glu Lys Asp He Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val 495 500 505 510 He Pro Pro Lys Lys Val Arg Met Wing Asn Leu Cys Val Val Gly Gly 515 520 525 His Wing Val Asn Gly Val Wing Glu He His Ser Glu He Val Lys Glu 530 535 540 Glu Val Phe Asn Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gln Asn 545 550 555 Lys Thr Asn Gly Val Thr Pro Arg Arg Trp He Arg Phe Cys Asn Pro 560 565 570 Pro Leu Ser Ala He He Thr Lys Trp Thr Gly Thr Glu Asp Trp Val 575 580 585 590 Leu Lys Thr Glu Lys Leu Wing Glu Leu Gln Lys Phe Wing Asp Asn Glu 595 600 605 sp Leu Gln Asn Glu Trp Arg Glu Wing Lys Arg Ser Asn Lys He Lys 610 615 620 Val Val Ser Phe Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp 625 630 635 Wing Met Phe Asp He Gln Val Lys Arg He His Glu Tyr Lys Arg Gln 640 645 650 Leu Leu Asn He Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu 655 660 665 670 Met Thr Ala Ala Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys He 675 680,685.

Phe Gly Gly Lys Wing Phe Wing Thr Tyr Val Gln Wing Lys Arg He Val 690 695 700 Lys Phe He Thr Asp Val Gly Wing Thr He Asn His Asp Pro Glu He 705 710 715 Gly Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val 720 725 730 Wing Glu Leu Leu He Pro Wing Being Asp Leu Ser Glu His He Being Thr 735 740 745 750 Wing Gly Met Glu Wing Being Gly Thr Being Asn Met Lys Phe Wing Met Asn 755 760 765 Gly Cys He Gln He Gly Thr Leu Asp Gly Wing Asn Val Glu He Arg 770 775 780 Glu Glu Val Gly Glu Glu Asn Phe Phe Leu Phe Gly Ala Gln Ala His 785 790 795 Glu He Wing Gly Leu Arg Lys Glu Arg Wing Asp Gly Lys Phe Val Pro 800 805 '810 sp Glu Arg Phe Glu Glu Val Lys Glu Phe Val Arg Ser Gly Wing Phe 815 820 825 830 Gly Ser Tyr Asn Tyr Asp Asp Leu He Gly Ser Leu Glu Gly Asn Glu 835 840 845 Gly Phe Gly Arg Wing Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser 850 855 860 Tyr He Glu Cys Gln Glu Lys Val Asp Glu Wing Tyr Arg Asp Gln Lys 865 870 875 Arg Trp Thr Thr Met Ser He Leu Asn Thr Wing Gly Ser Tyr Lys Phe 880 885 890 Being Ser Asp Arg Thr He His Glu Tyr Ala Lys Asp He Trp Asn He 895 900 905 910 Glu Ala Val Glu He Ala * 915 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2655 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 12.2528 (D) OTHER INFORMATION: / product = "phosphorylase alpha glucan of H type tuber" (ix) FEATURE: (A) NAME / KEY: mat_peptido (B) LOCATION: 12. . 2525 (xi) DESCRIPTION OF THE SEQUENCE: SEA ID NO: 3: GTTTATTTTC C ATG GAA GGT GGT GCA AAA TCG AAT GAT GTA TCA GCA GCA 5 Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala 1 5 10 CCT ATT GCT CAA CCA CTT TCT GAA GAC CCT ACT GAC ATT GCA TCT AAT 98 Pro He Wing Gln Pro Leu Ser Glu Asp Pro Thr Asp He Wing Ser Asn 15 20 25 ATC AAG TAT CAT GCT CAAT TAT ACT CCT CAT TTT TCT CCT TTC AAG TTT 146 He Lys Tyr His Wing Gln Tyr Thr Pro His Phe Ser Pro Phe Lys Phe 30 35 40 45 GAG CCA CTA CA GCA TAC TAT GCT GCT ACT GCT GAC AGT GTT CGT GAT 194 Glu Pro Leu Gln Wing Tyr Tyr Wing Wing Thr Wing Asp Ser Val Arg Asp 50 55 60 CGC TTG ATC AAA CAA TGG AAT GAC ACC TAT CTT CAT TAT GAC AAA GTT 242 Arg Leu He Lys Gln Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val 65 70 75 AAT CCA AAG CAÁ ACÁ TAC TAC TTA TCA ATG GAG TAT CTC CAG GGG CGA 290 Asn Pro Lys Gln Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gln Gly Arg 80 85 90 GCT TTG ACA AAT GCA GTT GGA AAC TTA GAC ATC CAC AAT GCA TAT GCT 338 Wing Leu Thr Asn Wing Val Gly Asn Leu Asp He His Asn Wing Tyr Wing 95 100 105 GAT GCT TTA AAC AAA CTG GGT CAG CAG CTT GAG GAG GTC GTT GAG CAG 386 Asp Ala Leu Asn Lys Leu Gly Gln Gln Leu Glu Glu Val Val Glu Gln 110 115 120 125 GAA AAA GAT GCA GCA TTA GGA AAT GGT GGT TTA GGA AGG CTC GCT TCA 434 Glu Lys Asp Wing Wing Leu Gly Asn Gly Gly Leu Gly Arg Leu Wing Ser 130 135 140 TGT TTT CTT GAT TCC ATG GCC ACA TTG AAC CTT CCA GCA TGG GGT TAT 482 Cys Phe Leu Asp Ser Met Wing Thr Leu Asn Leu Pro Wing Trp Gly Tyr 145 150 155 GGC TTG AGG TAC AGA TAT GGA CTT TTT AAG CAG CTT ATC ACA AAG GCT 530 Gly Leu Arg Tyr Arg Tyr Gly Leu Phe Lys Gln Leu He Thr Lys Wing 160 165 170 GGG CAA GAA GAA GTT CCT GAA GAT TGG TTG GAG AAA TTT AGT CCC TGG 578 Gly Gln Glu Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp 175 180 185 GAA ATT GTA AGG CAT GAT GTT GTC TTT CCT ATC AGG TTT TTT GGT CAT 626 Glu He Val Arg His Asp Val Val Phe Pro He Arg Phe Phe Gly His 190 195 200 205 GTT GAA GTC CTC CCT TCT GGC TCG CGA AAA TGG GTT GGT GGA GAG GTC 674 Val Glu Val Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Val 210 215 220 CTA CAG GCT CTT GCA TAT GAT GTG CCA ATT CCA GGA TAC AGA ACT AAA 722 Leu Gln Ala Leu Ala Tyr Asp Val Pro Pro Pro Gly Tyr Arg Thr Lys 225 230 235 AAC ACT AAT AGT CTT CGT CTC TGG GAA GCC AAA GCA AGC TCT GAG GAT 770 Asn Thr Asn Ser Leu Arg Leu Trp Glu Ala Lys Ala Ser Ser Glu Asp 240 245 250 TTC AAC TTG TTT CTG TTT AAT GAT GGA CAG TAT GAT GCT GCT GCA CAG 818 Phe Asn Leu Phe Leu Phe Asn Asp Gly Gln Tyr Asp Ala Wing Ala Gln 255 260 265 CTT CAT TCT AGG GCT CAG CAG ATT TGT GCT GTT CTC TAC CCT GGG GAT 866 Leu His Ser Arg Ala Gln Gln He Cys Ala Val Leu Tyr Pro Gly Asp 270 275 280 285 GCT ACA GAG AAT GGA AAA CTC TTA CGG CTA AAG CAA CAA TTT TTT CTG 914 Thr wing Glu Asn Gly Lys Leu Leu Arg Leu Lys Gln Gln Phe Phe Leu 290 295 300 TGC AGT GCA TCG CTT CAG GAT ATT ATT GCC AGA TTC AAA GAG AGA GAA 962 Cys Ser Ala Be Leu Gln Asp He He Ala Arg Phe Lys Glu Arg Glu 305 310 315 GAT GGA AAG GGT TCT CAC CAG TGG TCT GAA TTC CCC AAG AAG GTT GCG 1010 Asp Gly Lys Gly Ser His Gln Trp Ser Glu Phe Pro Lys Lys Val Wing 320 325 330 ATA CAA CTA AAT GAC ACA CAT CCA ACT CTT ACG ATT CCA GAG CTG ATG 1058 He Gln Leu Asn Asp Thr His Pro Thr Leu Thr He Pro Glu Leu Met 335 340 345 CGG TTG CTA ATG GAT GAT GAA GGA CTT GGG TGG GAT GAA TCT TGG AAT 1106 Arg Leu Leu Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn 350 355 360 365 ATC ACT ACT AGG ATT ATT GCC TAT ACG AAT CAT ACA GTC CTA CCT GAA 1154 He Thr Thr Arg Thr He Wing Tyr Thr Asn His Thr Val Leu Pro Glu 370 375 380 GCA CTT GAA AAA TGG TCT CAG GCA GTC ATG TGG AAG CTC CTT CCT AGA 1202 Wing Leu Glu Lys Trp Ser Gln Wing Val Met Trp Lys Leu Leu Pro Arg 385 390 395 CAT ATG GAA ATC ATT GAA GAA ATT GAC AAA CGG TTT GTT GCT ACA ATA 1250 His Met Glu He He Glu Glu He Asp Lys Arg Phe Val Wing Thr He 400 405 410 ATG TCA GAA AGA CCT GAT CTT GAG AAT AAG ATG CCT AGC ATG CGC ATT 1298 Met Ser Glu Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg He 415 420 425 TTG GAT CAC AAC GCC ACA AAA CCT GTT GTG CAT ATG GCT AAC TTG TGT 1346 Leu Asp His Asn Wing Thr Lys Pro Val Val His Met Wing Asn Leu Cys 430 435 440 445 GTT GTC TCT TCA CAT ACG GTA AAT GGT GTT GCC CAG CTG CAT AGT GAC 1394 Val Val Ser Ser His Thr Val Asn Gly Val Ala Gln Leu His Ser Asp 450 455 460 ATC CTG AAG GCT GAG TTA TTT GCT GAT TAT GTC TCT GTA TGG CCC ACC 1442 He Leu Lys Wing Glu Leu Phe Wing Asp Tyr Val Ser Val Trp Pro Thr 465 470 475 AAG TTC CAG AAT AAG ACC AAT GGT ATA ACT CCT CGT AGG TGG ATC CGA 1490 Lys Phe Gln Asn Lys Thr Asn Gly He Thr Pro Arg Arg Trp He Arg 480 485 490 TTT TGT AGT CCT GAG CTG AGT CAT ATA ATT ACC AAG TGG TTA AAA ACA 1538 Phe Cys Ser Pro Glu Leu Ser His He He Thr Lys Trp Leu Lys Thr 495 500 505 GAT CAA TGG GTG ACG AAC CTC GAA CTG CTT GCT AAT CTT CGG GAG TTT 1586 Asp Gln Trp Val Thr Asn Leu Glu Leu Leu Wing Asn Leu Arg Glu Phe 510 515 520 525 GCT GAT AAT TCG GAG CTC CAT GCT GAA TGG GAA TCA GCC AAG ATG GCC 1634 Wing Asp Asn Ser Glu Leu His Wing Glu Trp Glu Wing Ala Lys Met Wing 530 535 540 AAC AAG CAG CGT TTG GCA CAG TAT ATA CTG CAT GTG ACÁ GGT GTG AGC 168 Asn Lys Gln Arg Leu Wing Gln Tyr He Leu His Val Thr Gly Val Ser 545 550 555 ATC GAT CCA AAT TCC CTT TTT GAC ATA CAA GTC AAA CGT ATC CAT GAA 173 He Asp Pro Asn Ser Leu Phe Asp He Gln Val Lys Arg He His Glu 560 565 570 TAC AAA AGG CAG CTT CTA AAT ATT CTG GGC GTC ATC TAT AGA TAC AAG 177 Tyr Lys Arg Gln Leu Leu Asn He Leu Gly Val He Tyr Arg Tyr Lys 575 580 585 AAG CTT AAG GGA ATG AGC CCT GAA GAA AGG AAA AAT ACA ACT CCT CGC 182 Lys Leu Lys Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg 590 595 600 605 ACA GTC ATG ATT GGA GGA AAA GCA TTT GCA ACÁ TAC ACA AAT GCA AAA 1874 Thr Val Met He Gly Gly Lys Wing Phe Wing Thr Tyr Thr Asn Wing Lys 610 615 620 CGA ATT GTC AAG CTC GTG ACT GAT GTT GGC GAC GTT GTC AAT AGT GAC 192 Arg He Val Lys Leu Val Thr Asp Val Gly Asp Val Val Asn Ser Asp 625 630 635 CCT GAC GTC AAT GAC TAT TTG AAG GTG GTT TTT GTT CCC AAC TAC AAT 197 Pro Asp Val Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn 640 645 650 GTA TCT GTG GCA GAG ATG CTT ATT CCG GGA AGT GAG CTA TCA CAA CAC 2018 Val Ser Val Wing Glu Met Leu He Pro Gly Ser Glu Leu Ser Gln His 655 660 665 ATC AGT ACT GCA GGC ATG GAA GCA AGT GGA ACA AGC AAC ATG AAA TTT 2066 Be Ser Thr Wing Gly Met Glu Wing Ser Gly Thr Ser Asn Met Lys Phe 670 675 680 685 GCC CTT AAT GGA TGC CTT ATC ATT GGG ACÁ CTA GAT GGG GCC AAT GTG 2114 J Ala Leu Asn Gly Cys Leu He He Gly Thr Leu Asp Gly Wing Asn Val 690 695 700 GAA ATT AGG GAG GAA ATT GGA GAA GAT AAC TTC TTT CTT TTT GGT GCA 2162 Glu He Arg Glu Glu He Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala 705 710 715 ACÁ GCT GAT GAA GTT CCT CAA CTG CGC AAA GAT CGA GAG AAT GGA CTG 2210 Thr Wing Asp Glu Val Pro Gln Leu Arg Lys Asp Arg Glu Asn Gly Leu 720 725 730 TTC AAA CCT GAT CCT CGG TTT GAA GAG GCA AAA CAA TTT ATT AGG TCT 2258 Phe Lys Pro Asp Pro Arg Phe Glu Glu Ala Lys Gln Phe He Arg Ser 735 740 745 GGA GCA TTT GGG ACG TAT GAT TAT AAT CCC CTC CTT GAA TCA CTG GAA 2306 Gly Wing Phe Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu 750 755 760 765 GGG AAC TCG GGA TAT GGT CGT GGA GAT TAT TTT CTT GTT GGT CAT GAT 2354 Gly Asn Ser Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp 770 775 780 TTT CCG AGC TAC ATG GAT GCT CAG GCA AGG GTT GAT GAA GCT TAC AAG 2402 Phe Pro Ser Tyr Met Asp Ala Gln Ala Arg Val Asp Glu Ala Tyr Lys 785 790 795 GAC AGG AAA AGA TGG ATA AAG ATG TCT ATA CTG AGC ACT AGT GGG AGT 2450 Asp Arg Lys Arg Trp He Lys Met Ser He Leu Ser Thr Ser Gly Ser 800 805 810 GGC AAA TTT AGT AGT GAC CGT ACA ATT TCT CAA TAT GCA AAA GAG ATC 2498 Gly Lys Phe Ser Being Asp Arg Thr He Ser Gln Tyr Ala Lys Glu He 815 820 825 TGG AAC ATT GCC GAG TGT CGC GTG CCT TGA GCACACTTCT GAACCTGGTA 2548 Trp Asn He Wing Glu Cys Arg Val Pro * 830 835 TCTAATAAGG ATCTAATGTT CATTGTTTAC TAGCATATGA ATAATGTAAG TTCAAGCACA 2608 ACATGCTTTC TTATTTCCTA CTGCTCTCAA GAAGCAGTTA TTTGTTG 2655 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: "(A) LENGTH: 839 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala Pro He Ala 1 5 10 15 Gln Pro Leu Ser Glu Asp Pro Thr Asp He Wing Ser Asn He Lys Tyr 25 30 His Wing Gln Tyr Thr Pro His Phe Ser Pro Phe Lys Phe Glu Pro Leu 35 40 45 Gln Ala Tyr Tyr Ala Ala Thr Ala Asp Ser Val Arg Asp Arg Leu He 50 55 60 Lys Gln Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val Asn Pro Lys 65 70 75 '80 Gln Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gln Gly Arg Ala Leu Thr 85 90 95 Asn Ala Val Gly Asn Leu Asp He His Asn Ala Tyr Ala Asp Ala Leu 100 105 110 Asn Lys Leu Gly Gln Gln Leu Glu Glu Val Val Glu Gln Glu Lys Asp 115 120 125 Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu 130 135 140 Asp Ser Met Wing Thr Leu Asn Leu Pro Wing Trp Gly Tyr Gly Leu Arg 145 150 155 160 Tyr Arg Tyr Gly Leu Phe Lys Gln Leu He Thr Lys Wing Gly Gln Glu 165 170 175 Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp Glu He Val 180 185 190 Arg His Asp Val Val Phe Pro He Arg Phe Phe Gly His Val Glu Val 195 200 205 Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Glu Val Leu Gln Ala 210 215 220 Leu Ala Tyr Asp Val Pro He Pro Gly Tyr Arg Thr Lys Asn Thr Asn 225 230 235 240 Ser Leu Arg Leu Trp Glu Wing Lys Wing Being Ser Glu Asp Phe Asn Leu 245 250 255 Phe Leu Phe Asn Asp Gly Gln Tyr Asp Ala Ala Ala Gln Leu His Ser 260 265 270 Arg Wing Gln Gln He Cys Wing Val Leu Tyr Pro Gly Asp Wing Thr Glu 275 280 285 Asn Gly Lys Leu Leu Arg Leu Lys Gln Gln Phe Phe Leu Cys Ser Wing 290 295 300 Ser Leu Gln Asp He He Wing Arg Phe Lys Glu Arg Glu Asp Gly Lys 305 310 315 320 Gly Ser His Gln Trp Ser Glu Phe Pro Lys Lys Val Wing He Gln Leu 325 330 335 Asn Asp Thr His Pro Thr Leu Thr He Pro Glu Leu Met Arg Leu Leu 340 345 350 Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn He Thr Thr 355 360 365 Arg Thr He Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu 370 375 380 Lys Trp Ser Gln Ala Val Met Trp Lys Leu Leu Pro Arg His Met Glu 385 390 395 400 He He Glu Glu He Asp Lys Arg Phe Val Wing Thr He Met Ser Glu 405 410 415 Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg He Leu Asp His 420 425 430 Asn Ala Thr Lys Pro Val Val His Met Ala Asn Leu Cys Val Val Ser 435 440 445 Ser His Thr Val Asn Gly Val Wing Gln Leu His Ser Asp He Leu Lys 450 455 460 Wing Glu Leu Phe Wing Asp Tyr Val Ser Val Trp Pro Thr Lys Phe Gln 465 470 475 480 Asn Lys Thr Asn Gly He Thr Pro Arg Arg Trp He Arg Phe Cys Ser 485 490 495 Pro Glu Leu Ser His He He Thr Lys Trp Leu Lys Thr Asp Gln Trp 500 505 510 Val Thr Asn Leu Glu Leu Leu Wing Asn Leu Arg Glu Phe Wing Asp Asn 515 520 525 Ser Glu Leu His Wing Glu Trp Glu Be Wing Lys Met Wing Asn Lys Gln 530 535 540 Arg Leu Ala Gln Tyr He Leu His Val Thr Gly Val Ser He Asp Pro 545 550 555 560 Asn Ser Leu Phe Asp He Gln Val Lys Arg He His Glu Tyr Lys Arg 565 570 575 Gln Leu Leu Asn He Leu Gly Val He Tyr Arg Tyr Lys Lys Leu Lys 580 '585 590 Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg Thr Val Met 595 600 605 He Gly Gly Lys Wing Phe Wing Thr Tyr Thr Asn Wing Lys Arg He Val 610 615 620 Lys Leu Val Thr Asp Val Gly Asp Val Val Asn As Asp Pro Asp Val 625 630 635 640 Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn Val Ser Val 645 650 655 Ala Glu Met Leu He Pro Gly Ser Glu Leu Ser Gln His He Ser Thr. 660 665 670 Wing Gly Met Glu Wing Being Gly Thr Being Asn Met Lys Phe Wing Leu Asn 675 680 685 Gly Cys Leu He He Gly Thr Leu Asp Gly Wing Asn Val Glu He Arg 690 695 700 Glu Glu He Gly Glu Asp Asn Phe Phe Leu Phe Gly Wing Thr Wing Asp 705 710 715 720 Glu Val Pro Gln Leu Arg Lys Asp Arg Glu Asn Gly Leu Phe Lys Pro 725 730 735 Asp Pro Arg Phe Glu Glu Wing Lys Gln Phe He Arg Ser Gly Wing Phe 740 745 750 Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu Gly Asn Ser 755 760 765 Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp Phe Pro Ser 770 775 780 Tyr Met Asp Ala Gln Ala Arg Val Asp Glu Ala Tyr Lys Asp Arg Lys 785"790 795 800 Arg Trp He Lys Met Ser He Leu Ser Thr Ser Gly Ser Gly Lys Phe 805 810 815 Ser Ser Asp Arg Thr He Ser Gln Tyr Wing Lys Glu He Trp Asn He 820 825 830 Wing Glu Cys Arg Val Pro * 835 (2) INFORMATION FOR SEQ ID NO: 5; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3171 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSITION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 87..3011 (D) OTHER INFORMATION: / product = "phosphorylase alf glucan L-type potato leaf" (ix) CHARACTERISTICS: (A) ) NAME / KEY: mat_peptidO (B) LOCATION: 330..3008 (ix) FEATURE: (A) NAME / KEY: sig_peptido (B) LOCATION: 87..329 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: TTTTTTTTTTT CAACATGCAC AACAATTATT TTGATTAAAT TTTGTATCTA AAAATTTAGC 60 ATTTTGAAAT TCAGTTCAGA GACATC ATG GCA ACT TTT GCT GTC TCT GGA TTG 113 Met Ala Thr Phe Ala Val Ser Gly Leu -81 -80 -75 AAC TCA ATT TCA AGT ATT TCT AGT TTT AAT AAC AAT TTC AGA AGC AAA 161 Asn Be Ser Be Ser Be Ser Phe Asn Asn Asn Phe Arg Ser Lys -70 -65 -60 AAC TCA AAC ATT TTG TTG AGT AGA AGG AGG ATT TTA TTG TTC AGT TTT 209 Asn Ser Asn He Leu Leu Ser Arg Arg Arg Leu Leu Phe Ser Phe -55 -50 -45 AGA AGA AGA AGA AGA AGT TTC TCT GTT AGC AGT GTT GCT AGT GAT CAA 257 Arg Arg Arg Arg Arg Ser Phe Ser Val Ser Ser Val Ala Ser Asp Gln -40 -35 -30 -25 AAG CAG AAG ACA AAG GAT TCT TCC TCT GAT GAA GGA TTT ACATTA GAT 305 Lys Gln Lys Thr Lys Asp Ser Ser As As Glu Gly Phe Thr Leu Asp -20 -15 -10 GTT TTT CAG CCG GAC TCC ACG TCT GTT TTA TCA AGT ATA AAT TAT CAC 353 Val Phe Gln Pro Asp Ser Thr Ser Val Leu Ser Ser He Lys Tyr His -5 1 5 GCT GAG TTC ACA CCA TCA TTT TCT CCT GAG AAG TTT GAA CTT CCC AAG 401 Wing Glu Phe Thr Pro Ser Phe Ser Pro Glu Lys Phe Glu Leu Pro Lys 10 15 20 GCA TAC TAT GCA ACT GCA GAG AGT GTT CGA GAT ACG CTC ATT ATA AAT 449 Ala Tyr Tyr Ala Thr Ala Glu Ser Val Arg Asp Thr Leu He He Asn 25 30 35 40 TGG AAT GCC ACÁ TAC GAA TTC TAT GAA AAG ATG AAT GTA AAG CAG GCA 49 Trp Asn Ala Thr Tyr Glu Phe Tyr Glu Lys Met Asn Val Lys Gln Ala 45 50 55 TAT TAC TTG TCT ATG GAA TTT CTT CAG GGA AGA GCT TTA CTC AAT GCT 54 Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly Arg Ala Leu Leu Asn Wing 60 65 70 ATT GGT AAC TTG GGG CTA ACC GGA CCT TAT GCA GAT GCT TTA ACT AAG 59 He Gly Asn Leu Gly Leu Thr Gly Pro Tyr Wing Asp Wing Leu Thr Lys 75 80 85 CTC GGA TAC AGT TTA GAG GAT GTA GCC AGG CAG GAA CCG GAT GCA GCT 64 Leu Gly Tyr Ser Leu Glu Asp Val Wing Arg Gln Glu Pro Asp Wing Wing 90 95 100 TTA GGT AAT GGA GGT TTA GGA AGA CTT GCT TCT TGC TTT CTG GAC TCA 689 Leu Gly Asn Gly Gly Leu Gly Arg Leu Wing Ser Cys Phe Leu Asp Ser 105 110 115 120 ATG GCG ACÁ CTA AAC TAC CCT GCA TGG GGC TAT GGA CTT AGA TAC CAA 73 Met Wing Thr Leu Asn Tyr Pro Wing Trp Gly Tyr Gly Leu Arg Tyr Gln 125 130 135 TAT GGC CTT TTC AAA CAG CTT ATT AA AAA GAT GGA CAG GAG GAA GTT 78 Tyr Gly Leu Phe Lys Gln Leu He Thr Lys Asp Gly Gln Glu Glu Val 140 145 150 GCT GAA AAT TGG CTC GAG ATG GGA AAT CCA TGG GAA ATT GTG AGG AAT 83 Wing Glu Asn Trp Leu Glu Met Gly Asn Pro Trp Glu He Val Arg Asn 155 160 165 GAT ATT TCG TAT CCC GTA AAA TTC TAT GGG AAG GTC ATT GAA GGA GCT t Asp He Ser Tyr Pro Val Lys Phe Tyr Gly Lys Val He Glu Gly Wing 170 175 180 GAT GGG AGG AAG GAA TGG GCT GGC GGA GAA GAT ATA ACT GCT GTT GCC 92 Asp Gly Arg Lys Glu Trp Wing Gly Gly Glu Asp He Thr Wing Ala Wing 185 190 195 200 TAT GAT GTC CCA ATA CCA GGA TAT AAA AA AAA ACA ACG ATT AAC CTT 97 Tyr Asp Val Pro He Pro Gly Tyr Lys Thr Lys Thr Thr He Asn Leu 205 210 215 CGA TTG TGG ACÁ ACÁ AAG CTA GCT GCA GAA GCT TTT GAT TTA TAT GCT 102 Arg Leu Trp Thr Thr Lys Leu Ala Wing Glu Wing Phe Asp Leu Tyr Wing 220 225 230 TTT AAC AAT GGA GAC CAT GCC AAA GCA TAT GAG GCC CAG AAA AAG GCT 107 Phe Asn Asn Gly Asp His Wing Lys Wing Tyr Glu Wing Gln Lys Lys Wing 235 240 245 GAA AAG ATT TGC TAT GTC TTA TAT CCA GGT GAC GAA TCG CTT GAA GGA 112 Glu Lys He Cys Tyr Val Leu Tyr Pro Gly Asp Glu Ser Leu Glu Gly 250 255 260 AAG ACG CTT AGG TTA AAG CAG CAA TAC ACA CTA TGT TCT GCT TCT CTT 116 Lys Thr Leu Arg Leu Lys Gln Gln Tyr Thr Leu Cys Ser Ala Ser Leu 265 270 275 280 CAG GAC ATT ATC GCA CGG TTC GAG AAG AGA TCA GGG AAT GCA GTA AAC 121 Gln Asp He He Wing Arg Phe Glu Lys Arg Ser Gly Asn Wing Val Asn 285 290 295 TGG GAT CAG TTC CCC GAA AAG GTT GCA GTA CAG ATG AAT GAC ACT CAT 126 Trp Asp Gln Phe Pro Glu Lys Val Wing Val Gln Met Asn Asp Thr His 300 305 310 CCA ACTA CTT TGT ATA CCA GAA CTT TTA AGG ATA TTG ATG GAT GTT AAA 131 Pro Thr Leu Cys He Pro Glu Leu Leu Arg He Leu Met Asp Val Lys 315 320 325 GGT TTG AGC TGG AAG CAG GCA TGG GAA ATT ACT CAA AGA ACG GTC GCA 136 Gly Leu Ser Trp Lys Gln Wing Trp Glu He Thr Gln Arg Thr Val Wing 330 335 340 TAC ACT AAC CAC ACT GTT CTA CCT GAG GCT CTT GAG AAA TGG AGC TTC 140 Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu Lys Trp Ser Phe 345 350 355 360 ACÁ CTT CTG CTG CTG CTT CTT CTG CTG CAC GTG GAG ATC ATA GCA ATG 145 Thr Leu Leu Gly Glu Leu Leu Pro Arg His Val Glu He He Wing Met 365 370 375 ATA GAT GAG GAG CTC TTG CAT ACT ATA CTT GCT GAA TAT GGT ACT GAA 150 He Asp Glu Glu Leu Leu His Thr He Leu Wing Glu Tyr Gly Thr Glu 380 385 390 GAT CTT GAC TTG TTG CAA GAA AAG CTA AAC CAA ATG AGG ATT CTG GAT 155 Asp Leu Asp Leu Leu Gln Glu Lys Leu Asn Gln Met Arg He Leu Asp 395 400 405 AAT GTT GAA ATA CCA AGT TCT GTT TTG GAG TTG CTT ATA AAA GCC GAA 160 Asn Val Glu He Pro Ser Ser Val Leu Glu Leu Leu He Lys Ala Glu 410 415 420 GAA AGT GCT GCT GAT GAT GAA AAG GCA GCA GAT GAA GAA GAA CAA GAA GAA 164 Glu Ser Ala Ala Asp Val Glu Lys Ala Ala Asp Glu Glu Gln Glu Glu 425 430 435 440 GAA GGT AAG GAT GAC AGT AAA GAT GAG GAA ACT GAG GCT GTA AAG GCA 169 Glu Gly Lys Asp Asp Ser Lys Asp Glu Glu Thr Glu Ala Val Lys Wing 445 450 455 GAA ACT ACG AAC GAA GAG GA'G GAA ACT GAG GTT AAG AAG GTT GAG GTG 174 Glu 'Thr Thr Asn Glu Glu Glu Glu Thr Glu Val Lys Lys Val Glu Val 460 465 470 GAG GAT AGT CAA GCA AAA ATA AAA CGT ATA TTC GGG CCA CAT CCA AAT 179 Glu Asp Ser Gln Wing Lys He Lys Arg He Phe Gly Pro His Pro Asn 475 480 485 AAA CCA CAG GTG GTT CAC ATG GCA AAT CTA TGT GTA GTT AGC GGG CAT 184 Lys Pro Gln Val Val His Met Wing Asn Leu Cys Val Val Ser Gly His 490 495 500 GCA GTT AAC GGT GTT GCT GAG ATT CAT AGT GAA ATA GTT AAG GAT GAA 1889 Wing Val Asn Gly Val Wing Glu He His Ser Glu He Val Lys Asp Glu 505 510 '515 520 GTT TTC AAT GAA TTT TAC AAG TTA TGG CCA GAG AAA TTC CAA AAC AAG 193 Val Phe Asn Glu Phe Tyr Lys Leu Trp Pro Glu Lys Phe Gln Asn Lys 525 530 535 ACÁ AAT GGT GTG ACÁ CCA AGA AGA TGG CTA AGT TTC TGT AAT CCA GAG 198 Thr Asn Gly Val Thr Pro Arg Arg Trp Leu Ser Phe Cys Asn Pro Glu 540 545 550 TTG AGT GAA ATT ATA ACC AAG TGG ACA GGA TCT GAT TGAT TTA GTA 203 Leu Ser Glu He He Thr Lys Trp Thr Gly Ser Asp Asp Trp Leu Val 555 560 565 AAC ACT GAA AAA TTG GCA GAG CTT CGA AAG TTT GCT GAT AAC GAA GAA 208 Asn Thr Glu Lys Leu Wing Glu Leu Arg Lys Phe Wing Asp Asn Glu Glu 570 575 580 CTC CAG TCT GAG TGG AGG AAG GCA AAA GGA AAT AAC AAA ATG AAG ATT 212 Leu Gln Ser Glu Trp Arg Lys "Ala Lys Gly Asn Asn Lys Met Lys He 585 590 595 600 GTC TCT CTC ATT AAA GAA AAA ACA GGA TAC GTG GTC AGT CCC GAT GCA 217 Val Ser Leu He Lys Glu Lys Thr Gly Tyr Val Val Ser Pro Asp Wing 605 610 615 ATG TTT GAT GTT CAG ATC AAG CGC ATC CAT GAG TAT AAA AGG CAG CTA 222 Met Phe Asp Val Gln He Lys Arg He His Glu Tyr Lys Arg Gln Leu 620 625 630 TTA AAT ATA TTT GGA ATC GTT TAT CGC TAT AAG AAG ATG AAA GAA ATG 227 Leu Asn He Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu Met 635 640 645 AGC CCT GAA GAA CGA AAA GAA AAG TTT GTC CCT CGA GTT TGC ATA TTT 232 Ser Pro Glu Glu Arg Lys Glu Lys Phe Val Pro Arg Val Cys He Phe 650 655 660 GGA GGA AAA GCA TTT GCT ACTA TAT GTT CAG GCC AAG AGA ATT GTA AAA 236 Gly Gly Lys Ala Phe Ala Thr Tyr Val Gln Ala Lys Arg He Val Lys 665 670 675 680 TTT ATC ACT GAT GTA GGG GAA ACÁ GTC AAC CAT GAT CCC GAG ATT GGT 241 Phe He Thr Asp Val Gly Glu Thr Val Asn His Asp Pro Glu He Gly 685 690 695 GAT CTT TTG AAG GTT GTA TTT GTT CCT GAT TAC AAT GTC AGT GTA GCA 246 Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val Ala 700 705 710 GAA GTG CTA ATT CCT GGT AGT GAG TTG TCC CAG CAT ATT AGT ACT GCT 251 Glu Val Leu He Pro Gly Ser Glu Leu Ser Gln His He Ser Thr Wing 715 720 725 GGT ATG GAG GCT AGT GGA ACC AGC AAC ATG AAA TTT TCA ATG AAT GGC 256 Gly Met Glu Wing Ser Gly Thr Ser Asn Met Lys Phe Ser Met Asn Gly 730 735 740 TGC CTC CTC ATC GGG ACTA TTA GAT GGT GCC AAT GTT GAG ATA AGA GAG 260 Cys Leu Leu He Gly Thr Leu Asp Gly Wing Asn Val Glu He Arg Glu 745 750 755 760 GAA GTT GGA GAG GAC AAT TTC TTT CTT TTC GGA GCT CAG GCT CAT GAA 26 Glu Val Gly Glu Asp Asn Phe Phe Leu Phe Gly Wing Gln Ala His Glu 765 770 775 ATT GCT GGC CTA CGA AAG GAA AGA GCC GAG GGA AAG TTT GTC CCG GAC 27 He Wing Gly Leu Arg Lys Glu Arg Wing Glu Gly Lys Phe Val Pro Asp 780 785 790 CCA AGA TTT GAA GAA GTA AAG GCG TTC ATT AGG ACA GGC GTC TTT GGC 27 Pro Arg Phe Glu Glu Val Lys Wing Phe He Arg Thr Gly Val Phe Gly 795 800 805 ACC TAC AAC TAT GAA GAA CTC ATG GGA TCC TTG GAA GGA AAC GAA GGC 28 Thr Tyr Asn Tyr Glu Glu Leu Met Gly Ser Leu Glu Gly Asn Glu Gly 810 815 820 TAT GGT CGT GCT GAC TAT TTT CTT GTA GGA AAG GAT TTC CCC GAT TAT 28 Tyr Gly Arg Wing Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Asp Tyr 825 830 835 840 ATA GAG TGC CAA GAT AAA GTT GAT GAA GCA TAT CGA GAC CAG AAG AAA 28 He Glu Cys Gln Asp Lys Val Asp Glu Ala Tyr Arg Asp Gln Lys Lys 845 850 855 TGG ACC AAA ATG TCG ATC TTA AAC ACÁ GCT GGA TCG TTC AAA TTT AGC 29 Trp Thr Lys Met Ser He Leu Asn Thr Wing Gly Ser Phe Lys Phe Ser 860 865 870 AGT GAT CGA ATÁ CAT CAA TAT GCA AGA GAT ATA TGG AGA ATT GAA 29 Be Asp Arg Thr He His Gln Tyr Ala Arg Asp He Trp Arg He Glu 875 880 885 CCT GTT GAA TTA CCT TAA AAGTTAGCCA GTTAAAGGAT GAAAGCCAAT 30 Pro Val Glu Leu Pro * 890 TTTTTTCCCCC TGAGGTTCTC CCATACTGTT TATTAGTACA TATATTGTCA ATTGTTGCTA 31 CTGAAATGAT AGAAGTTTTG AATATTTACT GTCAATAAAA TACAGTTGAT TCCATTTGAA 31 AAAAAAAAAA 317 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 975 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Wing Thr Phe Wing Val Being Gly Leu Asn Being I Be Ser Being Be -81 -80 -75 -70 Ser Phe Asn Asn Asn Phe Arg Ser Lys Asn Ser Asn He Leu Leu Ser -65 -60 -55 -50 Arg Arg Arg He Leu Leu Phe Ser Phe Arg Arg Arg Arg Arg Ser Phe -45 -40 -35 Ser Val Ser Ser Val Ala Ser Asp Gln Lys Gln Lys Thr Lys Asp Ser -30 -25 -20 Ser Ser Asp Glu Gly Phe Thr Leu Asp Val Phe Gln Pro Asp Ser Thr -15 -10 -5 Ser Val Leu Ser Ser He Lys Tyr His Wing Glu Phe Thr Pro Ser Phe 1 5 10 15 Ser Pro Glu Lys Phe Glu Leu Pro Lys Wing Tyr Tyr Wing Thr Wing Glu 20 25 30 Ser Val Arg Asp Thr Leu He He Asn Trp Asn Wing Thr Tyr Glu Phe 40 45 Tyr Glu Lys Met Asn Val Lys Gln Wing Tyr Tyr Leu Ser Met Glu Phe 50 55 60 Leu Gln Gly Arg Ala Leu Leu Asn Ala He Gly Asn Leu Gly Leu Thr 65 70 75 Gly Pro Tyr Ala Asp Ala Leu Thr Lys Leu Gly Tyr Ser Leu Glu Asp 80 85 90 95 Val Ala Arg Gln Glu Pro Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly 100 105 110 Arg Leu Ala Ser Cys Phe Leu Asp Ser Met Ala Thr Leu Asn Tyr Pro 115 120 125 Wing Trp Gly Tyr Gly Leu Arg Tyr Gln Tyr Gly Leu Phe Lys Gln Leu 130 135 140 He Thr Lys Asp Gly Gln Glu Glu Val Wing Glu Asn Trp Leu Glu Met 145 150 155 Gly Asn Pro Trp Glu Lie Val Arg Asn Asp He Ser Tyr Pro Val Lys 160 165 170 175 Phe Tyr Gly Lys Val He Glu Gly Wing Asp Gly Arg Lys Glu Trp Wing 180 185 190 Gly Gly Glu Asp He Thr Wing Val Wing Tyr Asp Val Pro Pro Gly 195 200 205 Tyr Lys Thr Lys Thr Thr He Asn Leu Arg Leu Trp Thr Thr Lys Leu 210 215 220 Ala Ala Glu Ala Phe Asp Leu Tyr Ala Phe Asn Asn Gly Asp His Ala 225 230 235 Lys Ala Tyr Glu Ala Gln Lys Lys Ala Glu Lys He Cys Tyr Val Leu 240 245 250 255 Tyr Pro Gly Asp Glu Ser Leu Glu Gly Lys Thr Leu Arg Leu Lys Gln 260 265 270 Gln Tyr Thr Leu Cys Ser Ala Ser Leu Gln Asp He He Wing Arg Phe 275 280 285 Glu Lys Arg Ser Gly Asn Wing Val Asn Trp Asp Gln Phe Pro Glu Lys 290 295 300 Val Ala Val Gln Met Asn Asp Thr His Pro Thr Leu Cys He Pro Glu 305 310 315 Leu Leu Arg He Leu Met Asp Val Lys Gly Leu Ser Trp Lys Gln Ala 320 325 330 335 Trp Glu He Thr Gln Arg Thr Val Wing Tyr Thr Asn His Thr Val Leu 340 345 350 Pro Glu Ala Leu Glu Lys Trp Ser Phe Thr Leu Leu Gly Glu Leu Leu 355 360 365 Pro Arg His Val Glu He He Ala Met He Asp Glu Glu Leu Leu His 370 375 380 Thr He Leu Wing Glu Tyr Gly Thr Glu Asp Leu Asp Leu Leu Gln Glu 385 390 395 Lys Leu Asn Gln Met Arg He Leu Asp Asn Val Glu He Pro Ser Ser 400 405 410 415 Val Leu Glu Leu Leu He Lys Wing Glu Glu Be Wing Wing Asp Val Glu 420 425 430 Lys Wing Wing Asp Glu Glu Gln Glu Glu Glu Gly Lys Asp Asp Ser Lys 435 440 445 Asp Glu Glu Thr Glu Ala Val Lys Ala Glu Thr Thr Asn Glu Glu Glu 450 455 460 Glu Thr Glu Val Lys Lys Val Glu Val Glu Asp Ser Gln Ala Lys He 465 470 475 Lys Arg He Phe Gly Pro His Pro Asn Lys Pro Gln Val Val His Met 480 485 490 495 Wing Asn Leu Cys Val Val Ser Gly His Wing Val Asn Gly Val Wing Ala 500 505 510 He His Ser Glu He Val Lys Asp Glu Val Phe Asn Glu Phe Tyr Lys 515 520 525 Leu Trp Pro Glu Lys Phe Gln Asn Lys Thr Asn Gly Val Thr Pro Arg 530.535 540 Arg Trp Leu Ser Phe Cys Asn Pro Glu Leu Ser Glu He He Thr Lys 545 550 555 Trp Thr Gly Ser Asp Asp Trp Leu Val Asn Thr Glu Lys Leu Wing Glu 560 565 570 575 Leu Arg Lys Phe Wing Asp Asn Glu Glu Leu Gln Ser Glu Trp Arg Lys 580 585 590 Ala Lys Gly Asn Asn Lys Met Lys He Val Ser Leu He Lys Glu Lys 595 600 605 Thr Gly Tyr Val Val Ser Pro Asp Wing Met Phe Asp Val Gln He Lys 610 615 620 Arg He His Glu Tyr Lys Arg Gln Leu Leu Asn He Phe Gly He Val 625 630 635 Tyr Arg Tyr Lys Lys Met Lys Glu Met Ser Pro Glu Glu Arg Lys Glu 640 645 650 655 15: Lys Phe Val Pro Arg Val Cys He Phe Gly Gly Lys Ala Phe Ala Thr 660 665 670 Tyr Val Gln Ala Lys Arg He Val Lys Phe He Thr Asp Val Gly Glu 675 680 685 Thr Val Asn His Asp Pro Glu He Gly Asp Leu Leu Lys Val Val Phe 690 695 700 Val Pro Asp Tyr Asn Val Ser Val Val Glu Val Leu He Pro Gly Ser 705 710 715 Glu Leu Ser Gln His He Ser Thr Wing Gly Met Glu Wing Ser Gly Thr 720 725 730 735 Be Asn Met Lys Phe Ser Met Asn Gly Cys Leu Leu He Gly Thr Leu 740 745 750 sp Gly Wing Asn Val Glu He Arg Glu Glu Val Gly Glu Asp Asn Phe 755 760 765 Phe Leu Phe Gly Wing Gln Wing His Glu He Wing Gly Leu Arg Lys Glu 770 775 780 Arg Ala Glu Gly Lys Phe Val Pro Asp Pro Arg Phe Glu Glu Val Lys 785 790 795 Wing Phe He Arg Thr Gly Val Phe Gly Thr Tyr Asn Tyr Glu Glu Leu 800 805 810 815 Met Gly Ser Leu Glu Gly Asn Glu Gly Tyr Gly Arg Wing Asp Tyr Phe 820 825 830 Leu Val Gly Lys Asp Phe Pro Asp Tyr He Glu Cys Gln Asp Lys Val 835 840 845 sp Glu Wing Tyr Arg Asp Gln Lys Lys Trp Thr Lys Met Ser He Leu 850 855 860 sn Thr Wing Gly Ser Phe Lys Phe Ser Ser Asp Arg Thr He His Gln 865 870 875 Tyr Wing Arg Asp He Trp Arg He Glu Pro Val Glu Leu Pro 880 885 890 (2) INFORMATION FOR SEQ ID NO: 7: (í ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) CHARACTERISTICS: (A) NAME / KEY: misc_característica (B) LOCATION: 1..27 (D) OTHER INFORMATION: / function = "starter" / label = SPL1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 ATTCGAAAAG CTCGAGATTT GCATAGA 2 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) CHARACTERISTIC: (A) NAME / KEY: misc_característica (B) LOCATION: 1..27 (D) OTHER INFORMATION: / function = "primer" / label = SPL2 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8 GTTTATTTTC CATCGATGGA AGGTGGT 2 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME / KEY: misc_característica (B) LOCATION: 1..23 (D) OTHER INFORMATION: / function = "primer" / label = SPHl (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GTGTGCTCTC GAGCATTGAA AGC 2 (2) INFORMATION FOR SEQ ID NO: 10; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME / KEY: misc_characteristic (B) LOCATION: 1.25 (D) OTHER INFORMATION: / function = "primer" / label = SPH2 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 ATAATATCCT GAATCGATGC ACTGC 2 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from a description of the present invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (50)

1. A potato plant having improved tuber cold storage characteristics, characterized in that it comprises a modified potato plant having a reduced level of activity from an enzyme to glucan phosphorylase selected from the group consisting of phosphorylase of the L-type tubercle of a glucan (GLTP) and glucan phosphorylase type H (GHTP) in tubers produced by the plant in relation to that of tubers produced by an unmodified potato plant.

2. The potato plant according to claim 1, characterized in that it is transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits the expression of a to endogenous phosphorylase glucan selected from the group consisting of a GLTP gene and a GHTP gene.

3. A potato plant having improved cold storage characteristics, characterized in that it comprises a potato plant transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence comprising at least 20 nucleotides of a gene which encodes a glucan phosphorylase selected from the group consisting of L-type glucanilase of glucan (GLTP) and glucan phosphorylase type H (GHTP).

4. The potato plant according to claim 3, characterized in that the encoded a glucan phosphorylase is GLTP.

5. The potato plant according to claim 3, characterized in that the encoded a glucan phosphorylase is GHTP.

6. The potato plant according to claim 3, characterized in that the DNA sequence comprises nucleotides 338 to 993 of SEQ ID NO. 1.

7. The potato plant according to claim 3, characterized in that the DNA sequence comprises nucleotides 147 to 799 of SEQ ID NO. 3.

8. The potato plant according to any of claims 2, 3, 4, 5, 6 or 7, characterized in that the DNA sequence is linked to the promoter sequence in an antisense orientation.

9. The potato plant according to claim 4, characterized in that the sum of the glucose and fructose concentration in the tubers of the plant measured at harvest is at least 10% lower than the sum of the concentration of glucose and fructose in the tubers of an untransformed plant measured in the harvest.

10. The potato plant according to claim 4, characterized in that the sum of the concentration of glucose and fructose in the tubers of the plant measured at harvest is at least 30% lower than the sum of the concentration of glucose and fructose in the tubers of an untransformed plant measured in the harvest.

11. The potato plant according to claim 4, characterized in that the sum of the concentration of glucose and fructose in the tubers of the plant measured at harvest is at least 80% lower than the sum of the concentration of glucose and fructose in the tubers of an untransformed plant measured in the harvest.

12. The potato plant according to claim 4, in which the sum of the concentration of glucose and fructose in the tubers of the plant stored at 4 ° C for about three months is at least 10% less than the sum of the concentration of glucose and fructose in the tubers of an untransformed plant stored under the same conditions.

13. The potato plant according to claim 4, characterized in that the sum of the concentration of glucose and fructose in the tubers of the plant stored at 4 ° C for about three months is at least 30% internal to the sum of the concentration of glucose and fructose in the tubers of an untransformed plant stored under the same conditions.

14. The potato plant according to claim 4, characterized in that the sum of the concentration of glucose and fructose in the tubers of the plant stored at 4 ° C for approximately three months is at least 39% internal to the sum of the concentration of glucose and fructose in the tubers of an untransformed plant stored under the same conditions.

15. The potato plant according to claim 4, characterized in that the activity of a total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant measured at harvest is at least 10% lower than the activity of a total glucan phosphate in tubers of an untransformed plant measured at harvest.

16. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" "1" h-1 in tubers of the plant measured at harvest is at least 30% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant measured at harvest.

17. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein-1 h" 1 in tubers of the plant measured at harvest is at least 66% lower than the activity of a total glucan phosphate in tubers of an untransformed plant measured at harvest.

18. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately three months is at least 10% lower than the activity of total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions

19. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately three months is at least 30% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

20. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately three months is at least 70% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

The potato plant according to claim 5, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately three months is of at least 10% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

22. The potato plant according to claim 5, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately three months is at least 28% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

23. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately six months is at least 10% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

24. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h-1 in tubers of the plant stored at 4 ° C for approximately six months is at least 30% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

25. The potato plant according to claim 4, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately six months is at least 69% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

26. The potato plant according to claim 5, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein-1 h" 1 in tubers of the plant stored at 4 ° C for approximately six months is at least 10% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

27. The potato plant according to claim 5, characterized in that the activity of total glucan phosphorylase measured as μmol NADPH produced mg "1 protein" 1 h "1 in tubers of the plant stored at 4 ° C for approximately six months is at least 39% lower than the activity of a total glucan phosphorylase in tubers of an untransformed plant stored under the same conditions.

28. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant measured in the harvest is at least 5% higher than the ratings of the fried potato for tubers of an untransformed plant measurements in the harvest.

29. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant measured at harvest is at least 30% higher than the ratings of the fried potato for tubers of an untransformed plant measurements in the harvest.

30. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant measured in the harvest is at least 46% higher than the ratings of the fried potato for tubers of a non-transfonmada plant measurements in the harvest.

31. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant measured in the harvest is at least 5% higher than the ratings of the fried potato for tubers of an untransformed plant measurements in the harvest.

32. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant measured at harvest is at least 10% higher than the ratings of the fried potato for tubers of an untransformed plant measurements in the harvest.

33. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about three months is at least 5% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

34. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about three months is at least 30% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

35. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about three months is at least 89% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

36. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about three months is at least 5% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

37. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about three months is at least 10% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

38. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about four months is at least 5% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

39. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about four months is at least 30% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

40. The potato plant according to claim 4, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for approximately four months is at least 89% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

41. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for about four months is at least 5% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

42. The potato plant according to claim 5, characterized in that the rating of the fried potato for tubers of the plant stored at 4 ° C for approximately four months is at least 25% higher than the ratings of the fried potato for tubers of a plant not transformed measures stored under the same conditions.

43. A method for improving the cold storage characteristics of a potato tuber, characterized in that it comprises providing a potato plant that has been modified to reduce the level of activity in the tubers of an enzyme to glucan phosphorylase selected from the group consisting of L-type glucanilase phosphorylase of glucan (GLTP) and glucan phosphorylase type H (GH? P)

44. The method according to claim 43, characterized in that it comprises: introducing into the potato plant an expression cassette having a plant promoter sequence operatively linked to a DNA sequence which, when transcribed in the plant, inhibits the expression of a endogenous a-glucan a-glucan gene selected from the group consisting of a GLTP gene and a GHTP gene.

45. A method to improve the cold storage characteristics of a potato tuber, characterized in that it comprises: introducing into an potato plant an expression cassette having a plant promoter sequence operatively linked to a DNA sequence comprising at least 20 nucleotides of a gene encoding a glucan phosphorylase selected from the group consisting of GLTP and GHTP.

46. The method according to claim 45, characterized in that the encoded a glucan phosphorylase is GLTP.

47. The method according to claim 45, characterized in that the encoded a glucan phosphorylase is GHTP.

48. The method according to claim 45, characterized in that the DNA sequence comprises nucleotides 338 to 993 of SEQ ID NO: 1.

49. The method according to claim 45, characterized in that the DNA sequence comprises nucleotides 147 to 799 of SEQ ID NO: 3.

50. The method according to any of claims 44, 45, 46, 47, 48 or 49 wherein the DNA sequence is linked to the promoter sequence in an antisense orientation.