MXPA97002881A - Isolated promoter of the p - Google Patents

Abstract

A promoter that is capable of expressing a GDI in at least any of root, tuber, stem and tuber tissue of a dicotyledonous plant is described, in particular, the promoter is a promoter for alpha-amylase

Description

ISOLATED PROMOTER OF THE POPE The present invention relates to a promoter that includes a construct and an expression vector comprising it and a transformed cell comprising it. In addition, the present invention relates to a plant that comprises it. It is known that it is convenient to direct the expression of a gene of interest ("GDI") in certain tissues of an organism, such as a plant. For example, it may be convenient to produce crop protein products with an optimal amino acid composition and increase the nutritional value of the culture. It may also be convenient to use the crop? Ar-to express genes other than the plant, such as genes for mammalian products. Examples of the latter products include interferons, insulin, blood factors and plasminogen activators. However, yes. It may be convenient to achieve the expression of a GDI in certain tissues, sometimes it is important (s not necessary) to ensure that the GDI is not expressed in other tissues in a way that may occur-harmful effects. In addition, it is important not to alter the normal metabolism of the organism to such a degree that harmful defects occur. For example, an alteration in the nortal metabolism in a plant leaf or in the tip of the rair. could lead to insufficient development of the plant. CA-A-2006454 discloses a DNA sequence of an expression cell in which specific regulatory regions of the potato tuber are located. The expression cassette contains a patat gene with a paw a gene promoter. The DNA sequence is transferred to a plant genome using agrobacteria. According to CA-20G645, the DNA sequence allows heterologous products to be prepared in cultures. One of the key enzymes of the plant is the a-arrulasa. The of-a Ilase participates in the path responsible for the decomposition of starch to reducing sugars in potato tubers. The genes that encode the < * -amylase in potato plants have been isolated and characterized. See, for example, the teachings of EP- -0470145. Briefly, α-ainilase is coded by means of a family of genes consisting of at least five individual genes. Based on their homology, genes can be divided into two subfamilies, one of which is constituted by amylase or ama lasas type, and the other is constituted by arnilasa or amylase type 1. The two groups of cf-arn? lasas are expressed differently, not only at the molecular level, but also in different tissues of the potato plant. In this sense, the ef-arnilasas of type 3 are expressed in the root, in the tubers, in the buds and in the tissue of the stems. Honey s that the a-amylases of type 1 are expressed in buds and stem tissues, but not in tubers. The present invention seeks to provide a plant promoter that is capable of directing the expression of a gene of interest in specific tissues, or just in a specific tissue of an organism, typically a plant. In accordance with a first aspect of this invention, a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, or a variant, homologous or inquisitive fragment, is provided. A restriction apa of the 5.5 Kb EcoRI fragment isolated from Solanum tuberosum is shown in Figures 1, 2 and 8, which are discussed below. In accordance with a second aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, or a variant, homologous or fragment thereof but in which at least one part of the promoter is inactivated. According to a third aspect of the present invention there is provided a promoter comprising at least the nucleotide sequence shown as Seq.I.D. No. 1 or a variant, a homologous to a fragment of it. According to a fourth aspect of the present invention there is provided a promoter comprising the nucleotide sequence of any of the sequences shown as Seq.T.D. No. 4-17, preferably any of the sequences shown as Seq.I.D. No. 4-16, or a vanante, a homolog or a fragment thereof. According to a fifth aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, or a variant, a homologue or a fragment thereof, but at least the nucleotide sequence shown as Seq.ID No. I is inactive. According to a sixth aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, or a variant, a homologue or a fragment thereof, but wherein the minus any of the sequences shown as Seq.ID No. 2-6 is inactive. According to a seventh aspect of the present invention there is provided a construct comprising a promoter according to the present invention, fused to a GDI. According to an eighth aspect of the present invention there is provided an expression vector comprising a promoter according to the present invention. According to a ninth aspect of the present invention, a t ansformation vector comprising the promoter according to the present invention is provided. According to a tenth aspect of the present invention there is provided a transformed cell comprising the promoter according to the present invention. According to an eleventh aspect of the present invention there is provided a transgenic organism comprising the promoter according to the present invention. According to a twelfth aspect of the present invention there is provided the use of a promoter according to the present invention, co or a cold-inducible promoter. According to a thirteenth aspect of the present invention there is provided a construct comprising the promoter of the present invention and a sequence of nucleotides coding for α-amylase of the opposite direction. According to a fourteenth aspect of the present invention the use of a promoter according to the present invention is provided to express a GDI in the tuber and / or the shoot and / or the root and / or the stem of the na? plant, preferably in together or at least the tuber of a plant. Other aspects of the present invention include methods for expressing or transforming any of the expression vector, the transformation vector, the transformed cell, including n-situ expression within the transgenic organism, as well as the products of the ism. Additional aspects of the present invention include the uses of promoters to express GDI in vitro (e.g., in culture media such as a broth) e m v vo (e.g., in a transgenic organism). Preferably, in either the expression vector, the transformation vector, the transformed cell or the transgenic organism, the promoter is present in combination with at least one GDI. The vector transformation of Agrobactenum is preferably derived. Preferably the promoter is stably incorporated into the gonoma of the transgenic organism. Preferably the transgenic organism is a plan-t. Preferably the plant is a dicotiled nea plant. Better yet, the plant is a potato plant. An advantage of the present invention is that a promoter- corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, is capable of directing the expression of a GDI in any of the tissues of the root, tuber, sprout and stalk of a dichotilone, for example, a potato. The same applies to the vanante, the homologue or the fragment thereof. Surprisingly, however, is the fact that at least a part of the promoter sequence can be activated (for example, truncated) and yet a GDI is still expressed. More surprising is the fact that partially activated (eg, truncated) promoter sequences can direct the expression of a GDI in one or more specific tissues, such as tuber tissue, rather than in combination in root tissues, tuber, sprout and stem.

In this sense, the promoters were found to correspond to the 5.5 Kb EcoRI fragment, isolated from Solanum tuberosum, which contained the inactivated nucleotide sequences upstream (ie, towards the 5 'end) of the position - 691, with reference to figure 3, did not produce expression in any tissue of root, tubercle, stem or stem. Examples of such modified promoters include the modified promoters that only contain the n-cleotide sequences downstream of the -692 position, such as the promoter sequences SEQ.Δ.D. No. 2-3 However, it was found that the promoters corresponding to the EcoRl fragment of 5.5 Kt > , isolated from Sol num tuberosum containing inactivated nucleotide sequences upstream of the -1535 position (with reference to Figure 3) produced expression only in the tuber tissue. Examples of these types of promoters include those which contain only the nucl sequences located downstream of the position -1535, but in which at least the nucleotide sequences upstream of -691 (with reference to the figure 3), such as the promoter sequences SEQ.ID No. 4-17, in particular-SEQ.I.D. No. 6-17, more in particular, the SEO.T.D. No. 6.16. Additionally, it was found with the last type of promoters that if those promoters contained at least the Seq.l.D. No. 1, high expression yields were observed in tuber tissue. In such manner, preferred examples of promoter sequences for the tuber-specific expression of a GDI containing at least the sequence shown as Seq.l.D. No. 1, include those sequences shown as Seq.I.D. No. 4-17, better still, the sequences shown as Seq.l.D. No.1-17, even better still, the sequences shown as Seq.l.D. No. 6-16. It was further found that l- ^ promoters corresponding to the 5.5 KL EcpJRl fragment of Sol nurn tuberosum, which contained the nucleotide sequences inactivated downstream of the -1535 position (with reference to Figure 3) produced expression in the root tissue and / or bud and / or stem. Examples of those types of promoters include those that contain only the nucleotide sequences located upstream of the -1535 position (with reference to Figure 3). In addition, it was found that the promoters corresponding to the 5.5 Kb EcoRl fragment, isolated from Solanum tuberosum, which contained a Seq.l.D. No. 1 inactive, produced expression only in root tissue and / or bud and / or stem. Examples of those types of promoters include those that do not contain Seq.l.D. No. 1. Particularly preferred sequences with Seq.l.D. No. 4-16. The tissue-specific expression, such as the specific expression in the tuber, is par- ticularly advantageous for a number of reasons. First, a GDI can be expressed (as defined below) in a specific type of tissue. This is particularly advantageous if the GDI is an endogenous of the opposite direction for the organism in question, because the expression of the sequence of opposite direction in other tissues can be detrimental. Secondly, it is possible to express a GDI coding for an agent that of a resistant organism against a disease associated with one or more specific tissues. For example, GDI may be a toxin against common scab, which normally affects tuber tissue. Third, large quantities of the expression product of a GDI can be obtained, wherein the GDI, for example, is a compound beneficial to humans or animals (eg, a suitable food material or an enzyme having an effect pharmaceutical pharmacist). Additionally, that product is easily recoverable. Fourth, the use of the promoter according to the present invention allows a suitable nucleotide to be expressed, in order to change the metabolism of the organism in a specific site, in such a way that the levels of starch in the tuber are increased or even modified starch is produced in them. Another additional surprising advantage is that the promoter of the present invention, in particular the promoter of the first aspect of the present invention, is cold-chewable, that is to say, it leads to the expression under approximate conditions of 0 ° C to 12 ° C. , up to around 4 ° C. Thus, this promoter is very useful for expressing GDI under conditions that would be of some benefit in cold conditions, in particular-such as the expression of the a-amylase gene (or the active fragment thereof) of EP-B-04P0145 ( shown as SEQ.ID No. 18). It is more preferred that the GDI be a nucleotide sequence that is opposite in direction to the a-amylase gene (or its active fragment), such as that shown in SEQ.T.D. No. 19. The most preferred embodiments of each of the aspects of the present invention do not include the native promoter in its natural environment. The term "promoter" is used in the normal sense of the art, for example, an RNA polymerase binding site in the gene expression theory of 3acob-Monod. The promoters of the present invention are capable of expressing a GDI. In addition to the nucleotide sequences described above, the promoters of the present invention could include additional conserved regions, such as the Ppbnow cell or a TATA cell. The promoters may even contain other sequences that affect (eg, maintain, increase, decrease) GDI expression levels. For example, other suitable sequences include the intron Shl or an ADH intron. Other sequences include inducible elements, such as elements inducible by tempera! ura, chemical action, light or effort. In addition, the appropriate elements to increase transcription or translation may be present. An example of these latter elements is the TMV 5 'conductive sequence (see Sleat, Gene 217 C1987) 217-225; and Da are, Plant Mol. Biol. 23 [1993] 97). The promoter of the present invention can also be referred to as the Amy 3 promoter or the Amy 51 promoter or the promoter-ai farny 351 or the lfa-A promoter and 3. The present invention also contains combinations of promoters or elements. For example, a promoter of the present invention, such as a tuber-specific promoter (see above) can be used in combination with a promoter specific for the stem (see above). Other combinations are possible. For example, the promoter of the present invention, such as a specific promoter for stem or tuber, can be used in combination with a root promoter and / or a leaf promoter. The term "corresponding" in relation to the present invention means that the promoter sequence is not necessarily derived from Solanum tuberosurn. For example, the promoter could be prepared synthetically. It can even be derived from another source. The terms "vanant", "homologous" or "fragment" include any substitution, variation of, modification of, replacement of, omission of or addition of, one or more nucleic acids from or to or in the sequence that provides the sequence of resulting nucleotides with the ability to act-pair or a promoter in the expression system, such as the transformed cell or the transformed transgenic organism according to the present invention. In particular, the term "homologous" covers homology with respect to structure and / or function, providing a resulting nucleotide sequence that has the ability to act as a promoter. With respect to sequence homology, preferably there is at least 75%, better yet, at least 85%, still better still, at least 90% homology, most preferably at least 95%, and what is most preferred, at least 98% homology. The term "inactivated" means partial inactivation in the sense that the expression pattern of the complete promoter of Figure 8 is modified per where the partially-activated promoter still functions as a promoter. However, as mentioned above, the modified promoter is capable of expressing a GDI in at least one specific (but not all) tissue of the full-promoter of Figure 8. Therefore, with this particular aspect of In the invention, the promoter having a activated portion can still function as a promoter (hence, it is still referred to as a promoter), but where the promoter is able to express a GDI in one or more, but not all, the tissues in where a GDI is expressed by the full promoter shown in Figure 8. Examples of partial inactivation include altering the folding pattern of the promoter sequence or the binding species to parts of the nucleotide sequence, so that a part of the Nucleotide sequence is not recognized, for example, by RNA polymerase. Another, preferable, way to partially inactivate the promoter is to truncate it to form fragments thereof. Another way is to identify at least a part of the sequence of i so that the RNA-polyrnerase can not bind to that part or to another part. Accordingly, for a preferred embodiment of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanurn tuberosum, or a vanant, a homologue or a fragment thereof, but wherein the promoter is truncated. The term "truncated" includes shortened versions of the promoter shown in Figure 8. Accordingly, for a preferred embodiment of the present invention, a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum, is also provided. tuberosum, or a variant, a homologue or a fragment thereof, but wherein the promoter does not contain at least the nucleotide sequence of any of the sequences shown with or Seq.lD No. 4-16. Additionally, for a preferred embodiment of the present invention, there is also provided a promoter that provides a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment, isolated from Solanum tuberosum, or a variant, homologous or fragment thereof, but where the promoter does not contain at least the nucleotide sequence shown as Seq. T. D. Do not . 1. The term "construction", which is synonymous with terms such as "conjugate", "cassette" and "hybrid", includes a GDI directly or indirectly linked to the promoter. An example of an indirect link is the provision of a suitable spacer group, such as an intron sequence, for example, the mtron Shl or the ADH intron, between the promoter and the GDI. The same applies to the term "melt" in relation to the present invention, which includes direct or indirect bonding. In each case, it is much preferred that the terms do not cover the natural combination of the wild type alpha-alase gene, ordinarily associated with the wild-type gene promoter, or the wild-type promoter, and when both are in its natural environment The construct may also contain or express a marker that follows the selection of the genetic construct, for example, in a plant cell into which it has been transferred. There are several markers that can be used, for example, in plants, such as mañosa. Other examples of markers include those that provide resistance to antibiotics, such as resistance to G418, mead honey, bleo icine, kanamycin and gentamicin. The term "GDI" with reference to the present invention means any gene of interest. A GDI can be any- nucleotide that is foreign or natural to the organism (for example, the plant) in question. Typical examples of a GDI include genes that code for proteins and enzymes that modify metabolic and catabolic processes. For example, GDI can be a protein that has additional nutritional value for plants, such as a food or a crop. Typical examples include plant proieins that can inhibit the formation of anti-nutive factors and plant proteins having a more convenient amino acid composition (eg, a higher lysine content than the non-transgenic plant). GDI can even encode an enzyme that can be used in food processing, such as quirno ina, taunatma and alpha-galactosidase. The GDI can also encode an agent to introduce or increase resistance to pathogens. The GDI can even be a construction of opposite direction to modify the expression of natural transcripts, present in the relevant tissues. The GDI can also encode a non-natural plant compound that is beneficial to animals or to humans. For example, GDI could encode a pharmaceutically active protein or enzyme, such as any of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood coagulant factors. In this regard, the ransformed cell or organism could provide acceptable amounts of the desired compound, which would be easily recoverable, for example, from the tubers. Preferably, the GDI is a gene that encodes either a protein that has a high nutritional value, a toxin for pests, a transcription of the opposite direction, such as for patatin, ADP ~ gluco <; -ap-phosphonlase (see, for example, F.P-A-455316), alpha-amylase (see, for excerpt, EP-B-0470145), a protease of the opposite direction or a glucanase. A preferred GDI is an opposite sense sequence for the alpha-amylase gene described in EP-B-0470145. The term "organism", in relation to the present invention, includes any organism that can. activating the promoter of the present invention, such as alabaster-producing organisms (e.g., alpha -anuí asa), which include plants, algae, fungi and bacteria, as well as their cell lines. Preferably, the term denotes a plant or a cell thereof, or preferably a calyx-like, better still, a potato. The term "transgenic organism", in relation to the present invention, means an organism comprising an expressible construct according to the present invention or a product such as a construct. For example, the transgenic organism may comprise an exogenous nucleotide sequence (eg, GDI as co-described herein) under the control of a promoter according to the present invention; or a natural nucleotide sequence, under the control of a loosely inactivated (for example, truncated) promoter, according to the present invention. The terms "cell", "tissue" and "organ" include the cell, the tissue and the organ per se and when they are inside an organism. For a class / type of promoters according to the present invention, the term means cells, < - 1 tissue or tuber organ of the potato and / or cell, tissue or organ of the root of the potato and / or the cell, tissue or organ of a potato sprout and / or the cell , the tissue or organ of the stem of the potato. Preferably, the term means just or at least one cell, tissue or organ of the potato tuber. Preferably the expressible construct is incorporated into the genome of the organism. The term incorporated cubr-e preferably incorporates stable within the genome. The term "nucleotide" in relation to GDI includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably it means DNA, better yet, cDNA. The term "expression vector" means a construct capable of being expressed in v or v or in v11, The term "transformation vector" means a construct capable of being transferred from one species to another, such as, for example, a plasm from E.coli to a plant cell. Although the promoters of the present invention are not described in EP-B-0470145 or CA-A-2006454, these two documents do provide some useful fundamental comments about the types of techniques that can be used to implement the present invention. Some of these teachings are included in the following comments. The basic principle in the construction of genetically modified plants is to insert genetic information into the genome of the plant in order to obtain a stable maintenance of the inserted genetic material. There are several techniques for inserting genetic information, the two main principles being to direct the introduction of genetic information and the introduction of genetic information through the use of a vector system. A review of the general techniques found in Potryl-us (Annu Re. Plant Physiol. Plant Mol. Biol. Ti 9911 42: 205-225) and in Chpstou (Agro-Food-Indus ry Hi- T ch, March / April 1994, 17-27). Thus, in one aspect, the present invention relates to a vector system carrying a promoter or construct according to the present invention, and which is capable of introducing the promoter or construct into the genome of a plant, such as a plant of the family of the 5/29/97 Solanaceae, in particular of the genus Solanum, especially, Solan? m tuberosum. The vector system may comprise a vector, but preferably comprises two vectors; in the case of the two vectors, the vector system is usually called a binary vector system. Binary vector systems are described in greater detail in Gynheung An and co-authors (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19. A system used extensively for the transformation of plant cells with a cited promoter or construct is based on the use of a Ti plasmid from Agrobactera urn turnefaciens or a Ri plasmid from Agrobactenurn rhizogenes An and coauthors (1986), Plant Physiol., 81, 301-305 and Butchor, D.N. and coauthors (1980), Tissue Culture Methods for Plant Pathologists, eds .: D.S.

Ingra s and 3.P. Helgeson, 203-208. Various different Ti and Ri plasmids have been formed, which are suitable for the construction of the plant or the plant cell constructs described above. A non-limiting example of said plasm Ti is pGV3850. The promoter or construction of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent to a T-DNA sequence, in order to avoid interruption of the sequences immediately surrounding the edges of the T-DNA. T-DNA, at least when one of those regions seems to be essential for the insertion of the modified T-DNA into the genorn of the plant. As will be understood from the foregoing explanation, the vector system of the present invention is preferably one that contains the sequences necessary to infect a plant (e.g., the vir region) and at least an edge portion of a T sequence. -ADN, the edge part being located in the same vector as the genetic construction. Aditionally, the vector system is preferably a T-tube of Agrobactepum turne faciens or a Ri plasmid of A robacter? Rhizogenes or a derivative thereof, since these plasmids are well known and are widely used in the construction of transgenic plants, there are many vector systems that are based on these plurrules or their derivatives. In the construction of a transgenic plant, the promoter or construction can first be constructed in a microorganism where the vector can be reproduced and which is easy to manipulate before inserting into the plant. An example of a useful microorganism is. co 1, but other microorganisms having the above properties can be used. When a vector of a vector system co or the one defined above, has been constructed in E. coli, it is transferred, if necessary, to a suitable strain of Agrobacterium, for example, Agrobacterium tumefaciens. The T-plasm or host of the promoter or construction of the invention in such a manner is preferably transferred to a strain of Agrobacterium, eg, A. t? efaciens, in order to obtain an A robactenurn cell that hosts the promoter or constructor of the invention, whose DNA is subsequently transferred to the cell of the plant that is going to rnodi f c r. Direct infection of plant tissues by Agrobactepum is a simple technique that has been widely used and is described in Butcher C.N. the al (1980), Tissue Culture Methods for Plant Pathologists, ed. : D. S.

Ingrams and 3.P. Helgeson, 203-208. See also Potry us / Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Chpstou / Agro-Food-Industry Hi-Tech March / April 1994 17-27). As reported in CA-A-2006454, a large number of cloning vectors are available which contain a reproduction system in E.coli and a marker that allows the selection of the transformed cells. The vectors contain, for example, pBR 332, series pUC, sene M13 rnp, pACYC 184, etc. In that way, the construct or promoter can be introduced into a suitable restriction position in the vector. The plasmid contained for the transformation in E. coli is used. The E. coli cells are cultured in a suitable nutrient medium and then harvested and subjected to smooth. The plasmid is then recovered. As an analytical method, a sequence analysis, regeneration analysis, elephoresis and other biological-biochemical-molecular methods are generally used. After each manipulation, the sequence of AON used can be restricted and connected to the next DNA sequence. Each sequence can be cloned in the same plasmid or in a different plasmid. After each method of introducing the desired promoter or construct into the plants, other DNA sequences may be necessary. If, for example, for the transformation, the plausid Tí or the plasmid Ri of the plant cells is lacquered, at least the right limit and, frequently, however, the right and left limits of T-AÜN of plasmids Ti and Plasmid Rl, as flanking areas of the genes, can be connected. The use of T-DNA for the transformation of plant cells is being studied intensively and is well described in EP 120 516; Hoe erna, in The Bmary Plant Vector System Offset-drukkep j Kanters, B.B., Alblasserda, 1985, Chapter V; Fraley the. al, Crit. Rev. Plant Sea. , 4: 1-46 and An et al., EMBO 3. (1985) 4: 277-284. Direct infection of the plant tissues by Agrobacter um is another simple technique that can be used.

Typically, a plant that is going to be infected is injured, for example, by cutting the plant with a razor or pricking the plant with a needle or by rubbing the plant with an abrasive. The wound is then anoculated with the Agrobacterium, for example, in a solution. Alternatively, infection of a plant or of a particular part or roof of the plant, that is, on a part of a leaf, root, stem or other part of the plant, can be effected. The inoculated plant or plant part is then grown in a suitable culture medium and "grown mature plants are grown. When plant cells are constructed, they can be developed and maintained according to well-known tissue culture methods, such as by culturing the cells in a suitable culture medium, to which the necessary growth factors are supplied, such as amino acids, hormones and plants, vitamins, etc. The regeneration of transformed cells into genetically modified plants can be achieved using known methods for the regeneration of plants from cell or tissue culture, for example, by selecting transpublished germinations using an antibiotic and subculturing the germinations in a medium that contains the appropriate nutrients, plant hormones, etc. In summary, therefore, the present invention relates to a promoter and also to a construction comprising it. In particular, the present invention relates to the use of a promoter for the expression of a GDI in a cell / organ / organism, such as one or more specific tissues of a plant, in particular a dicotyledonous plant, such as a potato. More particularly, in a preferred embodiment, the present invention relates to a partially inactivated (eg, truncated) a-arnilasse type 3 promoter. The present invention also relates to the application of a set of partially inactivated gene promoters to express a GDI specifically in a tuberous tissue of a dicot, especially a potato plant. The following sample has been deposited in accordance with the Budapest Treaty, at the recognized depository The National Collect ons of Industrial and Marine Bacteria Limated (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, AB2 1RY, United United on August 26, 1994. DH5alfa-gPAmy 351 (Deposit No. NCIMB 40682). This sample shows a bacterial material of E. coli that contains the plasmid pBluescppt (see figure 7 for a general map of it) that contains a genome DNA fragment with EcoRl of 5.5 isolated from the potato (Solanum t? Berosuin) . The EcorRI 5.5 fragment contains the promoter region and a portion of the 5 'untranslated sequence of the structural gene of a potato alpha-arnase gene. The plasmid was formed by inserting the 5.5 kb EcoRl potato fragment into the polylinker of the pBS vector (Short and co-authors [1988] Nuc Acid, Res. 16: 7583-7600). The plasmid promoter can be isolated by enzyme digestion with EcoRl and then extracted using typical separation techniques (e.g., gels). The following sample has been deposited according to the Budapest treaty at the recognized depository The National Collations of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Dpve, Aberdeen, Scotland, AB2 1RY, United Kingdom on October 20, 1994.

DH5alpha-? 3K4 (Deposit No. NCIMB 40691) This sample is a bacterial material (DH5alpha-) of E. coli containing the plasmid gone p_JK4 (described below). The present invention will now be described only by way of examples, in which reference is made to the following figures: Figure 1 shows a restriction enzyme map. Figure 2 shows a potato restriction enzyme. Figure 3 shows a nucleotide sequence of a promoter according to the present invention. Figure 4 is an illustrative representation of some omissions made to the sequence of Figure 2. Figure 5 is an illustrative representation of some omissions made of the sequence of Figure 2. Figure 6 shows a sequence of sensitizer sequences. Figure 7 shows a map of pBl? EScppt K5 (2.95 Vb) and a map of pBlueScppt M13 (3.2 b). Figure 8 is a restriction enzyme map. Figure 9 shows a restriction map of? 3K4; and Figure 10 is a map of pEPL. In more detail, FIG. 1 is a restriction enzyme map of the genomic clone gPAmy 351, isolated from the potato variety Saturna, where the arrow indicates the position of the promoter-, the closed bar indicates the position of coding sequences , H = H? NdIII, E = EcoRI, S = SalI, ATG = codon of initiation of the coding sequence of alpha-a -lase and A asterisk marks the position of the EcoRI fragment of 5.5 kb. Figure 2 is a sequence map of the alpha-Amy 3 promoter where the arrow shows the degree of the sequence reactions, the position of the HE fragment is shown in B together with the sequenced part 5 'of the sequence of ornamentation. of promoter, the names of the individual fragments (see also figure 4) are given above the arrows, ATG = codon of initiation of the coding sequence of α-arnilasa and the omission fragments selected for the functional analysis are indicated using asterisks Figure 3 is a nucleotide sequence from the alpha-Amy 3 promoter, where the restriction sites are in bold type. The TATA, CCAAT and ATG sites are underlined, the position of the proposed CAP site and the untranslated leader sequence are indicated, and the nucleotide sequence of 166 base pairs sandwiched between the two highlighting lines (ie, from the position of nucleotide -857 to nucleotide position -691) is represented as SEO.ID No. 1 (see below). This 166-base pair nucleotide sequence can be transferred as the "delta" fragment or sequence. Figures 4 and 5 represent two omission senes of the alpha-Amy 3 promoter, those of Figure 5 being used for functional analysis. Figure 6 shows a series of sensitizer sequences for use with the present invention, wherein Uni = T7 sensitizer and Rev -sensitizer-T3. With more detail, the nucleotide sequence of Figure 3 is part of the promoter sequence of Figure 1 (discussed below) and part of the structural alpha-arnilase gene which, in turn, is part of the sequence of the figure. 8. Part of this core sequence is part of the sequences shown in the attached sequence lists. The nucleotide sequence of Figure 3 is repeated as Seq.lD No. 17. Figure 8 is an illustrative representation of the plasmid gPAmy351. The highlighted portion is an EcoRl -SlaT fragment isolated from potato (Solanum tuberosum), which is equal to the fragment shown in figure 1. The FcoRl-Salí fragment contains the 5.5 kb EcoRI fragment (called the Eco subclone). . 5) that is indicated by the line shown in the lower part of the drawing. The EcoRI fragment of 5.5 kb contains the promoter region and part of the 5 'unfransladada sequence of the structural gene of a potato fa-arnilasa. The following restriction enzyme sites are shown in Figure 8: E: EcoRI, Ha: HaelII, Sp: Sspl, H: HjLndlII, P: Pvul, S: SalI. In addition, the putative CAAT and TATA boxes and the ATG initiation site are displayed. The introns are shown as open bars and the exons as closed bars. The EcoRI fragment of 5.5 is cloned in a pBluescnpt M13 plasmid (shown in Figure 7) or in pBSK plasmid gone (also shown in Figure 7). For convenience, table i correlates the sequence references shown in the appended figures with the sequences shown in the attached sequence lists.

TABLE 1 SEQUENCE I.D. No, FIGURE No. / REFERENCE FIGURE 4 / Delta 4 / EH 3 4 / 8.5-E 4 4 / 9.5-7 5 4 / 8-1.7 6 4 / 7-1 7 4 / 6-15 8 4 / 6- ,! 9 4 / .., 10 4 / 5-24 11 4 / 4-1 12 4 / 4-2 13 4 / 1-8 14 4 / 1-6 .15 EH8 16 _ / -. 17 3 5 / HE 3 5 / HFP8 7 5 / HPF6 11 5 / HFP4 15 5 / EH8 In the following examples the following materials are used and the following methods are followed, respectively.

MATERIALS AND METHODS PLANT MATERIAL Root tissue was harvested from flowering potato plants (Solanum tuberosum, variety Saturna). The roots were directly floated in liquid nitrogen and portions of 10-15 g were stored at -80 ° C, until it was used.

Strains bac erian; LBA4404: contains the disarmed pTiAchd plasmid,? AL4404 in the streptomycin-resistant derivative of strain Ach5 (4) of Agrobacterium tu efaciens.

FAGOS AND PLASHIDOS lambda EMBL3: see reference (5)? BR327: see reference (6) pBS +, pBS-: see reference (7) pBSK * -, pBSK-: see reference (7) pBHOl, pBI121: see reference (8,9).

MEANS AND PLATES Broth medium L (LB): Per liter: 5 g of yeast extract, 5 g of NZ-arnide, 5 g of NaCl, 5 g of bacto-peptone. Autoclave. LB plates: LB medium plus 15 g of Bacto agar per liter. Autoclave. It is poured into plastic Petri dishes (25 ml / dish). Amp plates: As the plates LB plus 35 rng of arnpicilina per liter after treatment in autoclave. Plates AX1: Corno plates LB plus 35 rng of ampicillin, 120 rng of TPTG (Isopropyl ogalactoside), 40 mg of Xgal (dissolved in dimethylhydrazide) per liter after autoclaving. Xgal: 5-bromo-4-chloro-3-? dol-ß-D- alaco ida. Kan plates: As LB plates plus 50 mg of canarnicin per liter, after autoclaving. Half YMB: 3.1.

Per liter: 0.66 g K2HPO4 - 3H20, 0.2 g of rtgS0 «, 0.1 g of NaCl, 10.0 g of anitol, 0.4 g of yeast extract. The pH is adjusted to 7.0. It is treated in an autoclave. Medium Liquid MBa: Per liter: 4.4 g of MS salts (Baasal salt of Murashige and Skoog (10) Sigma), 20 g of sucrose. The pH is adjusted to 5.7 with NaOH.

Medium Solid MBa: Same as the liquid MBa medium plus 0.8% Difco agar. MBa co: As the average solid MBa plus 0.5 rng of t-Zeatin (trans isomer, Sigma) and 2.0 mg of 2,4 ~ D- (2,4-dichlorophenoxyacetic acid, Sigma) per liter. Medium Solid MBb: Same as the medium Solid MBa but instead of 20 g of sucrose, 30 g of sucrose per liter is added. Water: The water used in Materials and Methods was always distilled water and autoclaved before use.

ISOLATION OF GENOMIC POTATO DNA OF HIGH MOLECULAR WEIGHT In order to obtain a high molecular weight genomic DNA, a procedure essentially as described by Fischer and Goldberg (.1.1) was followed. This first includes the isolation of nuclei and then the preparation of the nuclear DNA. 10 to 15 g of Saturna root tissue was ground to a fine powder, in nitrogen, and homogenized in 100 rnl of regulator H (regulator lxH (11): 100 nl of lOxHB, 250 ml of AM sucrose, 10 tnl of 100 rnM of PMSF, 1 rnl of ß-rnerca? Toeta oi, 5 nl ile Triton X-100, 634 rnl of H2O, adjust to pH 9.5, ß-mercaptoethanol is added just before use, lOxHB: 40 mM of spermidine., 10 mM of ina sperm, 0.1 M of Na-EDTA, 0.1 M of Tris, 0.8 mM of KCl, is adjusted to pH 9.4-9.5 with ION NaOH. PMSF: fluoride le fenii et isis fonilo dissolved in ethanol). The resuspended plant material was filtered through a 70 μm nylon filter (Nitex filter, pre-regulated in lxH buffer). The resulting filtrate was poured into two centrifuge bottles (Sorvall GSA) and pellets were formed at 4000 rpm for 20 minutes at 4 ° C. The supernatant was discarded and the pellets were gently resuspended by adding 20 ml of the ixH regulator per tube and then carefully shaking the tubes. The nuclei were pelleted again at 4000 rprn for 20 minutes at 4 ° C, the supernatant was removed and the pellets were gently resuspended in 10 ml of regulator-lxH. The supernatant was pooled and 20 ml of cold lysis buffer (sis buffer: 2% Sarcosyl, 0.1 M tris, 0.04 M Na2 ~ EDTA) was added dropwise while gently stirring the solution. Immediately after the last drop of lysis buffer was added, 0.972 g of CsCl / ml was gently stirred into the solution (the solution should now be at room temperature). The resulting solution was centrifuged for 45 minutes at 10 k rpm, 4 ° C. The supernatants were carefully removed using a Pasteur pipette avoiding any scrap co protein floating on the top or altering the pellets. The volume of the supernatant was determined and 0.2 mg of ethidium bromide / nl was added. The DNA solution was gently examined in quick-seal polyalorne tubes, which were then sealed. The tubes were centrifuged in a Bec rnan VTI 65 rotor at 18 ° C and 40 k rpm for 38 hours. The genomic band was removed with ultraviolet light, with a 15-1 ü gauge needle connected to a 5 ml syringe, and gently poured into a 5 ml polymenal tube. The tube or tubes were then filled with 1.57 g / ml of CsCl solution in 50 M Tris-HCl (pH 9.5), 20 inM Na2-EDTA. 75 μl of ethidium bromide (5 mg / ml) was added.The tubes were centrifuged in the VTI 65 rotor at 18 ° C and 46 k rpm for 17 hours.The genomic band was removed under ultraviolet light in length long and ethidium bromide was extracted with isopropanol saturated with CsCl (7 to 8 times). The CsCl was removed from the DNA by dialysis in the TE regulator (lxTE: 10 ml of Tris-HCl, 1 mM of Na2-EDTA pH 8.0) at 4 ° C for 18 hours, with three changes, the DNA of high-molecular-weight genomic DNA was no longer precipitated and it was harvested at 4 ° C.

CONSTRUCTION OF A TENOMIC BANK OF POTATO The high molecular weight potato DNA was prepared from roots of the Saturn variety, as described above. The quality of DNA was tested by restriction enzyme digestion and by eiect roforesi s in gel. The genomic DNA was partially digested with Sau3A and the created fragments (9-23 kb) were inserted into the BarnHT site of the lambda vector EMBL3 (4). About 1.1x106 independent isolates were plated and amplified to form a permanent bank (12). Plate hybridization was used to discriminate the bank for the Gramil asa genes.

DISCRIMINATION OF THE BANK Essentially the discrimination of the potato genomic bank was carried out as described by references 13 and l. The pfu / rnl (ufp: plaque forming unit) of the amplified genomic bank was determined in duplicate before discrimination. Competent infection cells (PKL17 or LE392) were prepared by inoculating the cells in 30 ml of fresh L-broth containing 0.2% sucrose and 10 nM CaCl2. The cells were cultured for 4-5 hours at 37 ° C before adding 0.1 volume of cold CaCl2 and kept on ice until used. 100 μl of phage were diluted in the phage buffer to give an appropriate number of pfu (phage lug: 10 mM Tris-HCl, pH 7.5, 10 M MgCl 2, 20 mM NaCl), mixed with 25-100 μi of freshly prepared cells (depending on the actual number of cells) and incubated at 37 ° C for 15-20 minutes. The suspension was mixed with 3 ml of hot upper agar (42 ° C) of 0.8-1%, which contained 10 mM of MgCl 2 and spread to plates on dry LB plates. LB plates of 22x22 cm (dried for 3-4 hours at 37 ° C) were used to discriminate the gene bank. Each plate contained approximately 2xl05 plaques, which were mixed with 1 ml of infectious competent cells (pr-spaced as above) and incubated for 20 minutes at 37 ° C. This mixture was then added to 25 ml of hot upper agarose (42-45 ° C), 0.3%, with 10 mM of MgCl 2 and the solution was poured onto the dry, fresh LB plate. The large LB plates were incubated (not turned upside down) overnight, at 37 ° C. The phages from the plates were then transferred to Hybond N filters (A ersham) in duplicate. Plates were placed at 4 ° C for 1 to 2 hours to prevent the agarose layer from sticking to the filters. Plates were placed on ice, just before use and remained on ice when working with the filters. Two Hybond N filters and a plate for the orientation of the filters were marked. The first filter was placed on the plates for 45 seconds; then it was floated on a denaturation regulator (0.5 M NaOH, 1.5 M NaCl) for 7 minutes, with the phages looking up, then floated on the regulator-neutralization (0.5 M Tps-HCl (pH 7.4), 3 M NaCl) for 2 times 3 minutes, and finally washed in 2xSSC (IxSSC: 0.15 M NaCl, 0.015 Na ~ citrate). The filter was dried in air and the phage DNA was fixed to the membrane by UV entanglement. The second filter was laid on the same plate, after the first one, for 120 seconds and then treated again like the first one. These filters were used in the hybridization of plates after the Hybond N membrane protocol, according to the supplier's instructions (Arnersharn). The X-ray film of the first and second Hybond N membranes was oriented in such a way that the signals of both filters adjusted with each other. Positive plates were cut with a scalpel (blocks lxl c) and immersed in 1 ml of phage regulator. The phage containing the tubes was stored air-tight (pair ilm) at 4 ° C, after 2 to 3 drops of chloroform had been added. The plate containing the plates (22x22 crn) was stored by placing a piece of soaked filter paper (in chloroform) on the lid. The plates were also stored hermetically at 4 ° C, with the plates facing upwards. Additional purification of positive plates was performed by forming plates of the dilutions of the storage tube (containing lxl crn blocks) with freshly prepared cells and forming them onto plates on round LB plates with 1% hot upper agar (42 ° C) and 10 M MgCl2. New filter impressions were formed with Hybond N, following the procedure indicated above, with plates of 22x22 crn. The plates that gave positive signal were isolated by sticking the tip of a Pasteur pipette through the plate and transferring it to 500 μl of phage regulator. A new sequence of dilutions was formed, extended to plates and the respective filters hybridized until the positive plates were purified. The phages were stored in a hermetic manner to the a 4 ° C in 500 μl of phage buffer with a drop of chloroform, or the phages were isolated as such from plaque lysate. Plaque lysate material was formed as described by (14).

ISOLATION OF LAMBDA RECOMBINANT DNA Large-scale preparations followed the method described in (14), which includes forming strands of the phaco-encoding DNA on a CsCl gradient. Two versions (A, B) of a small-scale preparation were used, as follows: (A) LE392 cells were inoculated into LB with 0.2% maltose and 10 M MgCl2 and developed in 0 / N at 37 ° C. The cells were pelleted by centrifugation for 10 minutes at 4 ° C in a Sorvall centrifuge and gently resuspended in a volume of 10 M cold MgSO 4. The cells were stored at 4 ° C until they were used. Five individual plates of a plate were transferred to 500 μl of phage buffer and followed by resting for 2 to 2.5 hours at 4 ° C. After the tube vortex was formed, 100 μl of the released phage were mixed with 200 μl of freshly prepared LE392 cells. Alternately 50-100 μl of released phages from a platelet lysate were mixed with the cells. The phages and cells were incubated for 20 minutes at 37 ° C and then added to 25 nmol of LB precancelated (at 37 ° C) with 20 rnM MgSO; and 30 mM Tris-HCl, pH 7.5, and incubated by shaking 0 / N at 37 ° C. Another 10 rni of LB preheated with 20 mM MgCl 2 and 30 rnM Tris-HCl, pH 7.5 was added, and the mixture was incubated for 1 to 2 hours shaking at 37 ° C. After the clear mulch (a few drops of chloroform were added to help), the solution was centrifuged at 8000 rprn for 10 minutes at 4 ° C. The supernatant was transferred to a new tube and re-centred, if necessary, to remove cell debris. The recirculating lambda DNA was then purified using an Oiagen column, following the supplier's instructions (15).

(B) The procedure was as indicated in (A), until after the first centrifugation in the O / N culture. The supernatant was then transferred to a new tube and the DNase corresponding to 1 μg / rnl was added. The solution was incubated for 30 minutes at 37 ° C and then a volume of 20% cold PEG, 2 M NaCl, mixed in a phage regulator was added and the mixture was incubated for 1 hour on ice. The phage were pelleted by centrifugation for 20 minutes at 4 ° C, at 10 V r-prn. The PEG pellet was resuspended in 400 μl of phage buffer and transferred to a Jöndorf tube. 1 μl of RNase (10 rng / rnl) was added and incubated < - .JO for 30 minutes at 37 ° C. Then 8 μl of O was added. p of Na2-EDTA, pH 8.0, and 4 μl of 10% SDS, and the tube was incubated for another 15 minutes at 68 ° C. The mixture was allowed to reach room temperature and then an equal amount of phenol saturated with the TE-regulator (lxTE: 10 M Tris pH 7.5, 1 inM Na2EDTA) was used to extract the DNA. An equal mixture of saturated phenol-chloroform was used to extract the upper aqueous phase and a final extraction with chloroform was carried out. The upper phase was transferred to a new tube and the solution was formed with 0.3 M Na acetate and 2-3 volumes of cold ethanol were added. The DNA precipitation was obtained by storing at 0 / N at -20 ° C, centrifuging for 5 minutes and resuspending the pellet in 50-100 μl of TE buffer. The quantity and quality of recombinant phage DNA were tested by restriction enzyme digestion and electrophoresis with agarose gel (0.8-1%) (16).

PREPARATION OF PLASMID DNA The preparation of the plasmid is described in EP-B-0470145. In particular, the small scale preparation of plasmid DNA was carried out in the following manner. Bacterial strains harboring the plasmids were grown overnight in 2 ml of broth medium L (LB) with added protein (35 μg / i). The operations were carried out in 1.5 nl Eppendorf tubes and the centrifugation was carried out in an Eppendorf centrifuge at 4 ° C. The cells were harvested from the overnight culture by centrifugation for 2 minutes, washed with 1 ml of 10 mM Tns-HCl (pH 8.5), 50 M EDTA and centrifuged dur-ante for 2 minutes. The pellet was suspended in 150 μl of 15% sucrose, 50 rnM of Tns-HCi (pH 8. ^), 50 M of EDTA, revolutionizing to form a vortex. 50 μl of 4 mg / ml of lysozyme was added and the mixture was incubated for 30 minutes at room temperature and 30 minutes on ice. To 400 μl of ice cold H2O was added and the mixture was kept on ice for 30 minutes, incubated at 70-72 ° C for 15 minutes and centrifuged for 15 minutes. To the supernatant was added 75 μl of 0.5 M Na perchlorate and 200 μl isopropanol (the asopropane was stored at room temperature) and the mixture was centrifuged for 15 minutes at 4 ° C. The pellet was suspended in 300 μl of 0.3 M Na acetate and 2-3 volumes of cold ethanol were added. Precipitation was obtained by storing for 5 minutes at -80 ° C or at 0 / N at -20 ° C, centrifuging dur-ante for 5 minutes, drying the vacuum for 2 minutes and re-dissolving the pellet in 20 μl of H2O. The yield was 5-10 μg of the plasmid DNA. The large-scale preparation of the plasmid DNA was achieved by simply increasing the preparation to a small scale to ten times. Working on corex tubes of 15 rnl, the scale of all the ingredients was increased tenfold. The centrifugation was carried out in a Sorvall chilling centrifuge at 4 ° C. Only the changes with respect to the above will be mentioned in the following. After incubating at 70-72 ° C, centrifugation was performed at 17,000 rpn for 30 minutes. After adding isopropanol and after adding cold ethanol, the centrifugation was maintained for 15 minutes at 17,000 rprn. The final pellet of DNA was suspended in H2O and transferred to an Eppendorf tube and then received a small centrifugation to remove the residues. . The supernatant was adjusted to 0.3 M Na acetate and 2-3 volumes of cold ethanol were added. The pellet was resuspended in 40 μl of H2O. The yield was usually 20-28 μg of DNA. To obtain very pure plasmid DNA, 200-300 μg of isolated plasmid DNA, from the method carried out on a larger scale, was formed on a gradient of CsCl. Solid CsCl was mixed with H 2 O (1: 1 by weight / volume) and 0.2 mg / ml of ethidium bromide was added. The solution was poured into a quick-seal polylayer tube and the plasmid DNA was mixed with solid CsCl (1: 1 by weight) olumen. The tube was filled, sealed and centrifuged in a Beckman VTT 65 rotor at 15 ° C, at 48,000 rprn for 16 hours at IB hour. The centrifuge was stopped without using the brake. The plasmid DNA was extracted to bands from the tubes, using a syringe and the ethidium bromide was extracted with the isopropanol saturated with CsCl 7 to 8 times. The CsCl was removed by dialysis in 10 ml of Tris-HCl (pH 8.0), 1 rnM of EDTA for 48 hours, with three changes of regulator. The DNA was precipitated by adjusting to 0.3 M of Ha-acetate and adding 2-3 volumes of cold ethanol. Small-scale plaemide preparation, starting from E. coli, was usually followed by precipitation with LiCl to remove 'the RNA from the DNA solution. The plasmid DNA prepared on a small scale was dissolved in 100 μl of distilled water. One volume of 5 M LiCl was added and the mixture was incubated at -20 ° C for 30 minutes, followed by centrifugation at 12,000 r.p.m for 15 minutes, 4 ° C. The supernatant was transferred to a new Eppendorf tube and 2 volumes of TE buffer or water were added. Precipitation was achieved with 2.5 volumes of 96% ethanol, storing for 10 minutes at -80 ° C or O / N -20 ° C. He rushed the DNA by centrifuging for 15 minutes at 12,000 rprn, at 4 ° C, drying under vacuum for 2 minutes and re-dissolving in 10 μl of TE or water.

DIGESTION WITH RESTRAINING ENZYME The protocol followed was noted in EP-B-0470145. In particular, all the restriction endonucleases used were from Biolabs, Arnersharn or Boehringer Mannheirn and were used according to the supplier's instructions. One unit of enzyme was used per 1 μg of H "'and the incubation was carried out for 2 hours.The regulator was changed in double digests by changing the volume or adding the necessary ingredient, according to the instructions of the enzyme.

DNA MARK3E A randomly sensitized DNA marker kit (Boehpnger Mannhei) was used, following the supplier's instructions. Briefly, 2 μl of the DNA fragment (25-50 ng) was mixed with 8 μl of H 2 O and incubated at 95 ° C for 10 minutes to denature the DNA. It was centrifuged briefly and placed on ice. Then 1 μl of each of dGTP, dATP and dTTP, and 2 μl of reaction mixture and 5 μl (approximately 50 μCi of dCTP32) were added. 1 μl of the Klenow enzyme starts the reaction and the tube is incubated at 37 ° C for 30 minutes. Then it is placed on ice. The labeled DNA fragment was purified using an ELUTIP column (Schleicher a Schuell). The column was prepared by previous operation (gravity) in 3 ml of regulator with high salt content (1.0 M NaCl, 20 mM Tris-HCl (pH 7.5), l.ü inM EDIA), followed by 5 ml of regulator with low salt content (0.2 M NaCl, 20 mM Tps-HCl (pH 7.5), 1.0 M EDTA, 250 μL of low salt content was added and the marker tube and all the solution were spread over the The column was then washed with 2x400 μl of ba or salt content followed by 3x200 μl of high salt content, then the radioactive probe was heated, denatured and used in the hybridization.

TRANSFER OF SOUTHERN AND HYBRIDIZATION The DNA fragments to be transferred were fractionated on non-denaturing agarose gels (14) and transferred to Hybo dMR N or HybondM * N + nylon membrane, positively charged (Amershand Life Science) by Southern blotting (17, 18). Hybridization to Hybond ™ N nylon membranes was carried out according to the supplier's instructions (18).

THE PREPARATION OF VECTORS The preparation of vectors was carried out as described in EP-B-0470145 in the following manner: the vectors were digested (pBS - / + or pBSK - / +) with one or two restriction enzymes, extracted twice with saturated phenol (phenol was first mixed with 0.1 M Tps-HCl, then mixed twice with TE buffer (10 M Tris-HCl, pH 8, 1 rnM 2-EDTA)) and once with chloroform and precipitated with 0.3 M sodium acetate and 2.5 volumes of cold ethanol. The pellet was rinsed in 70% cold ethanol and dissolved in H2O, which gave a concentration of 25-50 ng / μl. The vectors were tested for their background before use (self-ligating with and without T-4-DNA-l asa). If necessary, the vector was treated with alkaline phosphatase (Boehpnger Mannheirn) as described by the supplier. After said treatment, the resulting pellet was resuspended in H2O to give a final concentration of 10 ng / μl.

LIGATION The phage DNA or the plasmid comprising a fragment to be subcloned with one or more restriction enzymes was digested to operate either on a 5% acplarnide gel or on an appropriate arose gel. The fragment that is subcloned from the gel was isolated either by electroelution, as described in (14) or using the GENECLEAN II equipment (BIO 101 Inc., La Dolía, California), following the supplier's instructions. Various proportions of fragment to vector (from 2: 1 to 5: 1, based on the number of molecules) were used in the ligation reaction. 1 μl (10-100 ng) of a solution containing the vector was combined with the fragment, 1 μl of T4 ligation buffer (10 × (20 M Tris-HCl, pH 7.6, 10 mM MgCl 2, 0.6 rnM was added. of ATP, 10 rnM of dithioteitol)) and 1 μl of T4 ~ DNA-lyase (Boehrmger Mannheim) to a mixture of fragment and vector, at a total volume of 10 μl. The reaction was incubated at 15 ° C 0 / N if the ligated DNA fragments had rigid ends. If the DNA had blunt ends, the incubation was carried out at room temperature for 1 hour. The ligation mixture was stored at -20 ° C if not used immediately, usually 5 μl of the ligation mixture was used for the transformation. DNA fragments treated with a DNA trimming kit (see "subcloning and sequence formation") were ligated following the team protocol to blunt DNA (Amershain).

PREPARATION OF E. COLI COMPETENT CELLS AND TRANSFORMATION This was done in accordance with the protocols set forth in EP-B-0470145, as follows: JM109 (or DH5er) cells were inoculated into 4 rnl of L-broth formed at 10 rnM MgS0 <; and 10 mM MgCl2. The cells were developed 0 / N 37 ° C. One rni of the 0 / N culture was added to 40 rnl of pre-warmed LB medium (at 37 ° C) (with 10 mM of MgSO, and 10 rnM of 4? MgCl2). The culture was shaken at 250-275 rprn for 1 to 2 hours until the OD450 reached 0.8-0.9. The cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4 ° C. The pellets were gently resuspended in 30 ml of 0.1 M cold CaCl2, another centrifugation pelleted the cells again and again suspended in 15 ml of 0.1 M cold CaCl2. The suspension was placed on ice for 20 minutes, and then centrifuged as before. Finally, the cells were gently resuspended in 3 ml of 0.1 M CaCl2 fpo and placed on ice for at least 1 hour before it was ready to be used for transformation (19). 5 μl of ligation mixture was combined with 95 μl of cold, sterile TCM (10 nM of Tris-HCl, pH 7.5), 10 mM of CaCl 2, 10 inM of MgCl 2) and 0.2 ml of the competent cells. The mixture was allowed to stand for at least '0 minutes on ice, then 5 minutes at 37 ° C (or 2 minutes at 42 ° C). The solution was transferred to 0.8 ml of L-broth, 10 mM of MgSO 4, 10 mM of MgCl 2, V was incubated for 45 minutes at 37 ° C and then formed into plates on 5 AXI plates or other plates (e.g. Arnp) at 0.2 ml / plate. The plates were left standing 10 minutes before inverting and incubated 0 / N at 37 ° C. It was dried in plastic bags with the top side down at 4 ° C.

OMISSIONS OF NUCLEASQ EXO / III MUNG BEAN A sequence of omissions of a larger, subcloned genomic fragment was carried out, using the ExoIII / Mung Bean (Stratagene) omission equipment. The subclone selected for the series of omissions was purified by forming two bands on a gradient of CsCl (see "Preparation of the DNA <the plasmid") to obtain high amounts of supercloned plasmid DNA. The generation of the omissions was carried out using the ExoIII / Mung Bean omission equipment, following the supplier's instructions. The temperature during the ExoIII treatment was 23 ° C, since at that temperature approximately 125 base pairs per minute must be eliminated.

PURIFICATION OF SENSITIZERS AFTER THE SYNTHESIS IN A DNA SYNTHETIZER The sensitizer was synthesized on a polystyrene support column (Applied Biosystens, 393 DNA / RNA Synthesizer) and eluted from le colu ne with NH.; 0H. The column was drunk and 1.5 ml of NH4OH was added to the polyesthene material in a small glass tube. The mixture was incubated at 85 ° C for 1 hour and then 5 minutes on ice. The supernatant containing the single-stranded DNA was transferred to Eppendorf tubes and the NH-0H was evaporated in a vacuum centrifuge for at least 3 hours. The pellet was resuspended in 200 μl of distilled water and precipitated with 550 μl of ethanol and 20 μl of sodium acetate. The pellet was resuspended in 200 μl of water and the precipitation was repeated with ethanol and sodium acetate. Finally, the pellet was suspended in 100-200 μl of distilled water and the DO26O was measured by means of a Gene Ouant RNA / DNA calculator (Pharmacia) of the DNA of the single filament. An OD26O of 1 corresponds to approximately 33 μg / rnl of single-stranded DNA.

SUBCLONATION AND DETERMINATION OF SEQUENCE Lambda DNA was digested with appropriate restriction enzymes and the generated fragments were isolated and split from agarose gels, using the equipment Geneclean (BIO 101, Tnc., La 3olla, California) according to the provider's instructions. Genocan DNA fragments (or fragments obtained from plasmids) were ligated into the polylinker region of the vector BlueScpbe pBS - / + (or? BSK - / +, Stretagene). After transforming the F.col 1 strain with the ligated plasmid, the reclosing subclones could be selected by forming plaques on AXI plates (they will be blences and the non-recombination clones will be blue when the vector is a pBlueScppt (107) plasmid).

Plasmid DNA, from putative subclones, was digested with appropriate restriction enzymes, subjected to gel electrophoresis and after Southern, hybrid with a properly labeled DNA probe, to verify the origin of the inserted fragment. Then, the sequence of the pBS genomic DNA subclones was determined, generated, according to the plasmid preparation protocol, indicated in EP-B-0470145. In this regard, the plasmid (double-filament template to be sequenced) was purified by the plasmid small-escele preparation method, the DNA was denatured in 0.2 M NaOH (5 minutes at room temperature), neutralized The mixture was mixed with 0.4 volumes of 5 M of ernionium etcete (pH 7.5) and then precipitated with 4 volumes of cold ethanol (5 minutes at -80 ° C) .The pellet was rinsed with cold ethanol at 70 ° C and turned to suspend in 10 μl of H 2 O. To subclone DNA fragments generated using an ExoIII / Mung Bean nuclease kit, it was first blunted or digested with a restriction enzyme, after blunting. unknown (after treatment with ExoIII / Mung Been) or with coherent ends, was obtained using DNA blunting equipment (Amersham) following the supplier's instructions.The linked omission plasmids (see "Ligation") generated Transformed DH5a competent cells and white colonies, selected on AXI plates, were analyzed by their insert by digestion with restriction enzyme, and by sequence determination. Sequence determination was obtained by means of a Seq? Enese ™ DNA sequence determination equipment from United States Biochernical Corp., following the sequence determination protocol annexed to the kit (Sequenese ^ R: Protocols per-step weight for sequence determination of DNA with Sequenaee, third edition, United States Biochem cal, Corporation PO Box 22400, Cleveland Ohio 44122). However, the following modifications were made to the suggested protocol. Instead of adding DTT, labeling mix and 35c¡dATP to the set DNA mixture, 4 rnl of 35Sequet ™ was added (DuPont). In addition to the sensitizers T3 and T7 (Strategene), all the sensitizers generated in a DNA synthesizer were used (Applied Biosystems, 392 DNA / RNA Synthesizer). 0.5 picomole of sensitizeror was used to determine the sequence of 1 picomol of piasmido. The sensitizing sequences are shown in Figure 6. The sequence determination reaction was subjected to electrophoresis on 6% or 8% denatural poly lacrylate knots, for 4 hours, 40 U, then dried by a Dryer and gel and eutorediogre io for 3 to 48 hours at room temperature. The denaturing stylet gels were formed from pre-nested polyether plamide solutions, 6 M ~ 6 and Gel-Mix 8 (GIBCO BRL, Life Technologies, Inc.) in accordance with the manufacturer's instructions.

PREPARATION OF COMPETENT AGROBACTERIUM CELLS AND TRANSFORMATION The strain LBA 4404 was maintained in YMB plates (pH 7.0) containing 100 rng / ml of rifampicin (Sigma) and 500 mg / rnl of streptomycin (Sigina). 2.5 rnl of LB medium (pH 7.4) was inoculated with the bacteria. The suspension was allowed to develop for 24 hours at 28 ° C, in an incubation shaker, at 300-340 rpm. The 1: 9 suspension was then diluted with LB and incubated for another 2 or 3 hours at 28 ° C and 300-340 rpm. When the OD was 0.5 to 1.25 rnl, aliquots of the cells were harvested in 50 ml tubes, in a centp leak with cooling at 10,000 rpm, for 5 minutes and 4 ° C. The tubes were placed on ice and the pellet was resuspended in 0.5 i of 20 rnM of CaCl2- Aliquots of 0.1 ml of the resuspended cells were quickly frozen, in cryotubes of 1 rnl, in liquid nitrogen, and stored at -80 ° C. Transformation was obtained using the freeze-decongestant method (20) as follows: An aliquot of 0.1 ml competent LBA 4404 cells was frozen on ice in CeCl2, and 1 μg of plasmid DNA was added. It was incubated to mix at 37 ° C for 5 minutes and 1 ml of LB (pH 7.4) was added. Incubation at room temperature with shaking (100 rpm) for 4 hours was followed by a rapid centrifugation at 10,900 rp a ° C, for 30 seconds. One pellet was resuspended in 100 μl of LB and formed on plates on plates (i d) containing 50 rnG / 1 yttria of cañamielna (Sigma). The plates were incubated for 48 hours at 28 ° 0 or until the colonies were of adequate size. This was the first round of selection. Only bacteria transformed with a plesmid containing the NPT II gene, which confers resistance to kanamycin, is capable of surviving on the canemicme plate. For the second round of selection, six of the colonies obtained were transferred to a YMB pLece containing 100 rng / liter of p ampicma, 500 mg / liter of streptomycin and 50 g / liter of canapucma. LBA 4404 is resistant to pfempicine and streptomycin, and the plasmid confers resistance to canarnicme. The plates were incubated at 28 ° C until the colonies reached an adequate size (approximately 4-5 days). The colonies were tested for their plasma content.

Plasmid preparations were generated from the colonies and the DNA was digested with appropriate restriction enzymes and operated on a 1% agerous gel to ensure that the piasmid and the inserted fragment had the correct dyeing. The digested DNA was obtained on a Hybond N + membrane and hybridized with a suitable radioactively labeled probe (a fragment of the plasmid or graft DNA). The storage of processed LBA 4404 was at -80 ° C. 2 ml of LB medium was inoculated containing 100 mg / liter of kidney, 500 g / liter of streptomycin and 50 mg / l of cane with bacteria and incubated at 28 ° C, for 48 hours, with shaking ( 300-340 rprn). The 1: 1 suspension was diluted with 35% sterile glycerol and aliquots were formed in cryotubes, 800 μl per tube and stored at -80 ° C.

THE TRANSFORMATION OF THE POPE A culture of the transformed bacterium LBA 4404 was prepared by inoculating 2 ml of YMB (pH 7.0) with the bacteria and incubating at 28 ° C for 24 hours. The suspension was diluted 1:10 and incubated for another 18 hours. The bacterium was centrifuged at 19,000 rph, 4 ° C for 10 minutes, and the pellet was rinsed twice with 2.5 ml of 2 M magnesium sulfate, before re-suspension in liquid MBa at an OD660 nrn of 0.5. The paper plant material used for the transformation was maintained in the middle MBA, and was added 2 μM STS (21.22). Through multiplication sprouts t > to superiors as well as nodes, if the leaves were too large they were eliminated. Five shoots were allowed to grow per container with 80 rnl of medium, at 25 ° C and for 30-35 days after the subculture, the nodes could be used for transformation. The stems of the micropropagated plants were cut just above and below the node, so that only the mornings were used. These possibly? They can be divided so that the explants have an approximate length of 4 millimeters. The explants were floated in the bacterial suspension for 30 minutes and stained dry on filter paper and transferred to co-active plates (co MBe). The explants were covered with filter paper moistened in liquid MBA and the plates were covered with cloth for 3 dies and left at 25 ° C. The explants were then washed in liquid MBA containing 800 mg / liter. Two explants were shaken by me for 18 hours, then dry spotted and transferred to the selection medium. The selection medium was MBb to which was added 500 g of canemicine, 800 ng of cerbenicilma (Duchefa), 0.1 mg of GA3 (gibberellic acid, Sigrne) and 1 rng of t-zeetma, per liter. The cerbemcil was added to put in any Agrobectena row. The explants were subcultured every three weeks.

REGENERATION OF WHOLE POTATO PLANTS The outbreaks of the explants that were harvested by subcivilization had more than 1 crn and were transferred to a solid MBA medium containing 400 rng (liter of carbetin, 2 μM of STS and 0.5 mg / liter of t-zea). After approximately two weeks, the harvests were transferred to a root formation medium, which is MBa soldered by 2 μM of added STS, and 5 μm of material was formed from 0.19 g of Na2 S2O3. -5H20 and 10.19 mg of AgN 3 were released in 7 liters of water and sterilized by filtration.After about 2 weeks, the shoots had taken root and were ready to be poured on the ground.The seedlings were rinsed in warm water to remove the waste from the environment and were planted in small pots with instantaneous sphagnum TKS 2 (Flora Gard, Alername) .The plentules were kept moist during the plentecion and they were later irrigated.The pots were placed in a plastic "tent" with 100 % humidity and 21-23 ° C, up that the seedlings had taken root in the earth. Then the store was removed and the plants were regularly watered. After four weeks of development the plants were placed in pots, using large pots (diameter of 27 cm) and transferred to a growth chamber with 16 hours of die, 22 ° C and eight hours of night, 15 ° C. When the plants had withered, the tubers were harvested.

GENERATION OF MICROTUBERCULES FROM POTATO PLANTS PROPAGADAS IN VITRO A node 5 mm below and 2 rnm above the node was cut from a node propagated in vi tro or from a selected shoot. The ho was removed and the explants were placed in a solid development medium. The medium contained per liter: 4.4 g of Murashige and Skoog (MS, (10)), balsal salts (Sigma), 60 g of sucrose, 2 rng of BAP (6-benzyl? Nopupne, Sigrna), 2 g of Gelpte (Scott Laboratories, Inc., Cerson, California). The explants were incubated for 7 days at 20 ° C with 16 hours of light / 8 hours of darkness. The plates were then wrapped in papol aluminum and kept in the dark at 20 ° C for 21-28 days. Then it was possible to harvest small tubes, one per explant. It generated buds of icrot? Bicules from the microtuberc? S, cutting them in half (from top to bottom). They were placed on a solid MBa medium and incubated in the dark for 7 days at 25 ° C, and the newly developed shoots could be analyzed by GUS.

HISTOCHEMICAL LOCATION OF ACTIVITY OF BETA-GLUCURONIDASE (GUS) The tissue was cut in small sections with a shaving vessel and placed in X-gluc (X-gluc: 5-bromo-2-chloro-3-? Ndol-i-ß-glucuromide), which is a solution of 50 g of X-gluc dissolved in a regulator with: 0.1 M of NePOü (pH 7.0), 1 rnM of K3 (F (CN), 0.1 mM of K «(F í« "N) ß. ^ H2Q, 10 rnM of Na2EDTA and 3% of sucrose (23)) to cover 1 section, the rnicrotubers were cut in half, the tubers were unrolled in pots to rebenedes delgedes, the leaves were cut into pieces of approximately 0.5 cm2 and the Stalk tissue to approximately 1 mrn thick slices The sections were incubated in X-gluc for 2-12 hrs at 37 ° C, taking care to prevent evaporation X-glα was removed and 96% ethanol was added. % to the tissue sections to remove the chlorophyll and other pigments.The incubation in ethanol was carried out overnight at 5 ° C and the following die tissue was transferred in a 2% saccharine solution and after a Nearly 1 hour was exercised in a dissecting microscope.

ISOLATION OF GENOMIC CLONES OF ot-AMILASA True cDNA clones coding for a-arnilase from potato (Solanurn tuberosum) had previously been isolated (as described in EP- 0470145). A Pstl-Sell fragment from the plasmid pAmyZ3 (EP-B-0470145) which encodes a perianth as a probe (see "DNA labeling" in Materials and Methods) was used to discriminate the potato DNA lambde side. genopuco (see "Building a genomic potato bank" in Materials and methods). Discrimination of approximately 1.6x106 '^ go was carried out as described in materials and methods. Two positive clones, gPAmy351 and gPArny331, were isolated by three rounds of purifications in place. A clone (gPArny331) was found that was unstable during the isolation of lambde DNA (see the method in Materials and methods) so that only clone gPArny351 is analyzed in detail. Shown in figure 1 is a rnape of the restriction enzyme of the insert (insert size approximately 22 kb). The map formation of the "-amylase coding part of the genomic sequence was carried out by means of Southern blotting of various digests of the clone, followed by hybridization with the PstI-Sal I fragment of? ArnyZ3. gPAmy351 contains all the promoter region of the a-ernilose gene as well as 1,270 bases of the structural gene. This covers the sequences encoding 142 amino acids corresponding approximately to 1/3 of the total amino acid sequence encoded by the α-amylase of pape of type Amy3 / 4 (407 enino acids, see EP-B-0470145).

SUBCONNECTION OF THE GENOMIC FRAGMENT THAT CONTAINS THE a-AMYLASE PROMOTER The EcoR1 fragment of a maximum of 5.5 kb was subcloned, indicated by an asterisk in figure 1, - < from the genomic clone gPAmy351, in a dephosphorylated EcoRI site of a pBS vector (see Materials and methods). This subclone was named Eco 5.5 and contains the ATG initiation codon and the upstream sequences of it (see the following paragraph for a more detailed description of the sequence). These sequences located upstream will be referred to hereinafter as the a-Arny promoter. A large-scale plasmid preparation of the Eco 5.5 plasmid was digested with EcoRI and HaelII, this creates a fragment of 1,350 pairs of kisses including the ATG start codon, as well as putative CAAT and TATA cells. As shown by others (see, for example, 24-31), the sequence region count from the ATG start codon and approximately 1,000 to 1500 base pairs upstream, which includes the entire promoter, is frequent enough to mediate the transcription of the gene at the time and place-corrector. The EcoRI-HaelII fragment was subcloned into a pBSK vector, digested with EcoRI and S al and dephosphorylated by alkaline phosphatase (see Materials and methods). This subclone EH8 was named and the genomic fragment that it carries was selected for functional analysis. The identity of the insert in the plasmid EH8 was identified by determining the sequence with the sensitizers T3 and T7 (see Figure 2B and Materials and methods).

DETERMINATION OF THE SEQUENCE OF THE a-AMILASE PROMOTER The sequence of approximately 2900 base pairs of the insert was determined in gPAmy351 (figure 1) by subcloning various fragments and using the sensitizers shown in Figure 6 (see Matepes and methods). This covers 1734 pairs of kisses upstream of the initiation codon (ATG) and 1440 pairs of current downstream kisses. The sequence map of the region upstream of the start codon ATG is shown in figure 2A and the DNA sequence is shown in figure 3. This sequence is located near the 3 'end of the insert part of gPArny351 (see Figure 1) of the 5.5 kb Eco fragment and the HindIII and EcoRI sites current upstream of the vortex codon, and from that menere, includes the Hae T? -EcoRI (EH) fragment, selected for functional analysis. The promoter sequences of a-Arny 3, from the ATG codon (A in position +1) and erriba current to position -1734 (see Figure 3) were compared with the database (EMBL) of plant sequences published (using the PC-gene program of TntelliGenetics, Inc., California) and was also compared with sequences from all organisms. There were no sequences with a significant global homology for the promoter of a-Amy 3. A cesille TATA is located at position -365. Comparing the promoter aA? N and 3 with published DNA binding sites, it was suggested that a CAP site is located 21 base pairs from the TATA box (position -344) and two CAAT cells, one at position -468. , which is 103 base pairs upstream of the TATA box, and 124 current base pair arrives from the CAP site, and another one in the position -547, 192 base pairs upstream of the TATA box and 213 pairs of bases upstream of the CAP site. The positions of the CAP site, and of the TATA and CCAAT boxes correspond well to the positions found in other eucanotic polymerase II promoters (32-33).

OMISSIONS IN THE PROMOTER OF a-AMILASA Plasma bands were formed twice or, as a result of a large escele preparation of subclone EH8 on a CsCl gradient (see Materials and methods) to obtain pure euperenroylated DNA. The operation of a sample of the plasmid DNA on the agarose gel showed that at least 85% of the preparation was supercoiled. Then the plasmid FH8 was digested with Bst XI, which creates a 3 'pendant and with BarnHI that creates a 5' pendant end, and care was taken to ensure that the digests were complete. Treatment with ExoIII / M? Ng Bean was carried out, as described in Materials and methods, and aliquots were collected at 0, 1, 2, 3, 4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 10.5 minutes. The different mixtures now contain the omissions of the plasmid EH8, and were used directly in the ligation and transformation, as explained in Materials and methods. The entire scale of subclones of omission was obtained, and these are shown in Figure 4. The sequence of all was determined with the sensitizers T3 and T7 (see Figure 8) to locate its 5 'end in proportion to the promoter. c / ~ Arny 3. Shown in figure 2A are the individual initial positions where the arrows indicate the start and degree of the sequence reactions of the subclones omitted. Three of the overshot omission subclones were selected for functional analysis], and these were 4-1, 6-15 and 8.5-E (indicated with an asterisk in figure 2B). A large-scale plasmid preparation was generated from each of the selected cloned omissions (4-1, 6-15 and 8.5-E) and clone EH8. Then they were digested with Sacl, which cuts into the pBSK- polyslinker and the blunt site (see Materials and methods) before they were digested with Sali, which cuts at the opposite site of the promoter insert. A complete plasmid (see Materials and methods) containing a ß-glueuronase gene (GUS) without promoter, completely with H dlII and blunted the HindIII sites, as described in Materials and methods, was completely digested. Subsequently, the opened plastid was digested with Salí, thereby creating a pBIlOlHandIIT Romo / Salí vector.

SUBCLONATION OF TRANSFORM Tr 1 Then, the Sacl Romo / SelI fragments obtained from clones 4-1, 6-15, 8.5-E and EH8 were subcloned into the pBHOl HindlIlRoMQ / SalI vector and the ligated plots transformed into strain AgAbactera ™ LBA4404 (see Materials and methods). The colonies obtained were tested using the restriction enzymes PstI and SalI, which were cut on each side of the insert fragment. The clones containing an anserto of the correct size were then analyzed and the sequence was determined with a designated sensibilazedor # 589 (see figure 6). Sensitizer # 589 sensitizes the GUS gene of pBHOl and allows the reading of the sequences above the GUS gene without promoter, covering the omissions of the promoter anserfades. The pBHOl plasmids containing the selected promoter ornasaones were designated EH, HFP4, HFP6 and HFP8.

In addition, a Southern blot of digested plasmids was hybridized with Pstl and Sali, with a market insert of clone EH8, which contained the largest fragment of the a-Amy 3 promoter region, to verify the origin of the inserts. To produce a smaller fragment than the one covered by 4-1, 6-15, 8.5-E or EH8, another subclone was created, subclone HE. This was obtained by digesting the EHS subclone with EcoRI and blunting the end of the Eco ^ T site followed by Ha ndlll digestion and then isolating the 288 base pair fragment containing the 3 'end promoter sequences (see figure 2B for the position of fragment HE). To subclone, this HindlII / EcoRlR "1" 0 fragment was used in a pB lOl vector, digested with SrnaT and HindIII, in the light reaction. The resulting plasmid was named HE and was transformed into strain LBA4404 of Agrobacterium (see Materials and methods). The colonies obtained on canapucine plates were tested by digestion of the plasmid purified with the restriction enzymes HaNdIII and SnaBI. The pledges of selected colonies were subjected to sequence analysis with sensitizer # 589, as explained above. In total, there were five omissions in the a-Amy 3 developer constructions, as explained in the preceding sections. They cover 1350 base pairs (EH8), 853 peres de beses (HFP4), 672 peres of bases (HFP6), 506 pairs of kisses (HFP8) and 288 base pairs (HE) of the sequences located upstream of the 5 'site of EcoRI codon ATG (see figure 2 and figure 5). They were cloned against the GUS gene without promoter of the vector pBHOl (see Materials and methods).

TRANSFORMATION OF THE POPE WITH THE PROMOTING CONSTRUCTIONS Six LBA4404 colonies containing 5 omission constructs and pBHO1 were selected and used for the transformation of Saturna stem tissue, as described in Materials and methods. As a negative control, some of Seturna's explentee were incubated with non-trending LBA4404 bacteria and the non-transformed shoots obtained from san cana icine selection plates. As a positive control, some Saturna explants were incubated with LBA4404 previously transformed with the pBT12l plasmid. pBI121 contains a GUS cassette controlled by the 35S promoter of the cauliflower moseic virus (CeMV), which is expressed constitutively in the largest part of the tissues of the plant (34-38). All the shoots regenerated after the first harvest (22 days) and the second harvest (49 days) were discarded. After 68 days there were 40 outbreaks of omission construction, 10 outbreaks of negative control and 15 outbreaks of positive control harvested and transferred to the root induction medium (see MatenaLes and methods). Cade outbreak represents, putatively, an event of individual transformation and will be referred to as lines in the future. Cade linee, if the person is transformed, represent an independent transformation event.

EXPRESSION OF GUS IN PUTATIVELY TRANSFORMED LINES LEAVES Leaves of the egenerated lines were tested, with total GUS, after the formation of the root. An expression analysis of the a-amylase genes of the present invention revealed that type 3/4 of a-apulase is expressed in the tuber, shoot, stem and root tissue, but no leaf expression was found . The GUS test of the leaves, from the putatively transformed omission lines of the transformed lobes with the pBHOl plasmid and the untransformed control lines, revealed that there was no GUS activity in any of the eils. In contrast, the GUS test of plants transformed with the pBI121 positive control plasmid showed GUS expression in the leaves, almost in all plants (see Table 1).

EXPRESSION OF GUS IN MICROTUBERCULOS AND BROTHES Tuber tubers were generated from the lines described above, as described in Meteneles and methods. These microtubers were examined for their GUS activity and the lines containing omission constructions FH8, HFP4 and HFP6 showed positive GUS staining. Again, the pBI121 control lines gave positive microtubers of GUS while the lines containing the pBHO1 plasmid showed negative microtubers. Neither my crotuberc? Of lines without transforming showed ect vided GUS, as well as the lines transformed with the constructions of omission HFP8 and HE. GUS was also enelized by the shoots generated by microtubers (see Materials and methods) and only those lines transformed with? BI1 1 (positive control) showed GUS activity. The results are summarized in table 1 shown below.

TABLE 1 EXPRESSION OF GUS Plantes Leaf Micro- Pare- Pare- Sprout Tubér-TD sheet of ex- tubéridido to acid to planteles with: plant ho or stem development developed bundled in pot in rnaceta EH 0 (36) * 7 (36) 0 (15) 0 (15) 0 (33) 0 (40) 16 (22) HFP4 0 (36) 2 (36) 0 (15) 0 (15) 0 (31) 0 (40) 15 (24) HFP6 0 (32) 5 (32) 0 (15) 0 (15) 0 (26) 0 (40) 10 (26) HFP8 0 (30) 0 (30) 0 (10) 0 (10) 0 (15) 0 (40) 0 (21) HE 0 (34) 0 (34) 0 (14) 0 (14) 0 (27) 0 (40) 0 (23) pBHOl 0 (36) 0 (36) 0 (36) 0 (35) 0 (36) 0 (40) 0 (24) PBI121 10 (15) 10 (15) 10 (10) 10 (10) 6 (6) 10 (15) 5 (5) Plantes no TD 0 (15) 0 (9) --- --- 0 (9) 0 (15) 0 (12) * The numbers in parentheses are the totel number of lines tested. (TD = transformed, EP = expente).

EXPRESSION OF GUS IN OTHER PARTS OF THE PLULES When it was generated, micro-tubes were formed at the end of the stem explants. In the upper part of the explant very often structures similar to two leaves were formed. Those stem-like explants and the upper ends perished and leafed were examined for GUS activity. As summarized in Table 1, none of the omissions containing line showed any GUS activity either in the stem-like tissue or in the leaf-like tissue of the explants. All the pBI121 lines that showed the GUS activity in their icrot? Bercle also had a GUS activity in the stem-like and leaf-like structures of the explant.

EXPRESSION OF GUS IN LEAFS, ROOTS, STEMS AND TUBERS OF LINES DEVELOPED IN MACETA The regenerated potato lines were also developed in pots in a growth chamber, at 22 ° C, 16 hours of light and 15 ° C, 8 hours of darkness) and analyzed for GUS leaves, roots, stems and tubers. None of the plant lines, except the cont-1 lines containing the construction? BI121, showed 3US activity in the leaves, as summarized in table 1 and table 2. The investigation of the expression GUS in Tubers harvested from plants developed in a pot, repeated the pattern and seen with the lines tested in the microtubers. The plants that carry one of the three constructions EHB, HFP4 and HFP6 showed GUS activity in the tubers, while the plants that carried HFP8, HE or pBHOl constructions did not show GUS activity in their tubers. Again the plants that carry the construction pBT121 had GUS activity in their tubers developed in pots, as expected. A GUS analysis of the lines mentioned in Table 2 (except for the positive control plants carrying? BI121) showed that no GUS activity was found in the root, stem or leaf tissues, even though the lines containing EH8, HFP4 and HFP6 clearly showed GUS activity both in the microtuber and in the tubular developed in a pot.

TABLE 2 EXPRESSION OF GUS IN POTATOES DEVELOPED IN MACETA Line No. Construction Tello Leaf Root Tuber Saturn control EH8 K702-15.2 0 0 0 K702-41.6 0 0 0 K702-47.3 0 0 0 K702-28.2 0 0 0 HFP4 K699-2.2 0 0 0 K699-31.5 0 0 0 K699 -44.2 0 0 0 HFP6 K700-1.5 0 0 0 K700-24.3 0 0 0 K700-38.2 0 0 0 HFP8 K703-44.6 HE K701-5.3 0 0 0 0 K701-.15.2 0 0 0 0 K701-18.2 00 00 00 0 K701-16.2 0 0 0 0 0 0 0 K701-49.2 00 00 00 0 PBI121 K661-10.4 + + - »« K661-15.3 + + + «- In conclusion, it is clear that none of the constructs of omission of the promoter to ~ Amy 3 that covers 1534? Ar-es of kisses current arrives from the start codon n ATG, leads to the expression of? n GUS gene for demes without prornotor in leaflets of plántules or in deserrolledes foliage in pot, in leaf-like tissues and in similar tissues e stem of microtuberous explants neither in roots nor shoots of plants developed in ineceta. The expression of GUS was only found in rnicrotubercles and in tubers developed in pots, which clearly shows that an of-Arny 3 promoter contains a specific element for the tubercle, clearly separable from the element or the elements that are expressed in the stem, shoot and root, located upstream from the 5 'end of the EH8 omissions. In addition, this invention also shows that the specific tuber element is located close to and commenting from the 5 'end of the HFP8 omissions and is covered by the delta sequence. This invention also shows that the stem, bud, and root expression element (s) is or is located upstream of the 5 'end of FH8, since none of the constructions EH8, HFP4, HFP6, HFP8 or EH gave expression of GUS in those tissues. Therefore, it is concluded that the elements that direct the specific expression for the root, specific for the stem and specific for the outbreak are located very upstream in the promoter 351. The applicability of the promoters is very broad. It is possible with the promoters to direct the expression of proteins to different tissues in the potato plant. It is even possible to direct ie protein expression in different tissues of other dicotyledonous plants.

PJK4 The pepe α-amylase coding sequences originate from the plasmid pAmyZ4 (see detailed description in EP-B-0470145). Briefly, pArnyZ4 encodes a precursor of a-emilesa pepe of 407 long amino acids and, edema, contains non-translational sequences 149 base pairs 5 'and 201 base pairs 3', located in the EcoRI site of the polylinker of the pBSK- plasmid . The construction of w-emilesa entit referencing? JK4, which contains the sequence shown with SEQ.I.D. No. 19, was formed using the Sacl and EcoRV fragment of? AMYZ4 and subcloning it to an appropriate plaeimido, such as the plasmid pEPL, digested with Smel and Sacl (see Figure 10). This places the antitransfer sequence downstream of an increased 35S promoter (E35S) and upstream of the DU2t terrniner. This plasmid is called pEPLZ4Sac-Eco and a HindIII fragment. With the E35S promoter containing the antitransfer potato sequence and the DU2t terninator, it was specifically amplified in a β121 digested in HindIII, thereby creating the binary plasmid pJK4, see figure 9. Other modifications of the present invention will be evident pear those who are experts in the meterie, without seizing the scope of the invention.

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. - Holters and co-authors (1978) Mol Gen Genet 163: 181-187., 21.- Linsmaier, E.U. and Skoog, F. (1965), Physiol. Plant. 18: 100-127. 22.- Chang H-H and Chang M-T (1991) Bot Bull Academia Sinica 32: 63-70. 23.- Jefferson, R.A. (1987) Plant Mol Biol Rep 5: 387. 24.- Steege, G., Nieboer, M., S? Avmg, 3. and Tempelaar, M.J. (1992) Plant Mol Biol 20: 19-30. 25.- Matton, D.P., Prescott, G., Bertrand, C, Camirand, A. and Bpsson, N. (1993) Plant Mol Biol 22: 279-291. 26.- Ohta, S., Hettop, T., Mopke, A. and Nakamura, K. (1991) Mol Gen Genet 225: 369-378. 27.- Ueda, T., Pichereky, E., Malik, V.S. and Cashmore, A.R. (1989) Plant Cell 1: 217-227. 28.- Stougeerd, 3., Sandehl, N.N .., Gron, A., Kuhle, A and Mercker, K.A. (1987), EMBO 3 6: 3565-3569. 29 .-- Stougeerd, 3., Jorgensen, J.E., Chpetensen, T., Kuhie, A. and Mercker, K.A (1990, Mol Gen Genet 220, 353-360.

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SEQUENCE LISTS (1) GENERAL INFORMATION NAME OF THE APPLICANTS: DANISCO A / S SOCIAL ADDRESS: Langebrogede 1 DK-1001 Copenegue K Dinarnerce TITLE OF THE INVENTION: PROMOTER (2a) INFORMATION FOR THE SEQUENCE I. I). 1 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solanum t? Berosum LENGTH OF THE SEQUENCE: 1.66 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: 10 20 30 40 ATAGCTTGAG GCGAAAATAT TTAATAAAAA CACTTCTTAA 50 60 70 80 TTTGTTTATA TGTTCAATTG AACATGTCCG TGATTAGAAA 90 100 110 120 ATTAAATTAA ATTCAATGAC AAATTTAATA ATTTGACACA 130 140 150 160 AAATTTATGA AAAAAATATC AAAATATAAA GAAATATTTT TTTTGA (2b) INFORMATION FOR SEQUENCE I..D. 2 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solanum tuberosum SEQUENCE LENGTH: 291 bp NUMBER OF FILAMENTS: Double TOPOLOGY: L ineal SEQUENCE: 10 20 30 40 AAGCTTCCAA TGAACC6TTG CCATGTGTCA CTGCCrATTC 50 60 70 ACCGCGAAAC ATGAATATCA CTGACGAACG ATTTCGGf-W 90 100 110 120 GGAACGAATC CAGAAAATGG ATTACTTTCT ATAAATTCCT 130 140 150 160 CGAATCTCAA CTCCATTTCG TAAAAATAAA ATTAAAAATA 170 180 190 200 TTGTTTGTTT TTGTATTTGT TTTTGTATTT CTGGTTTATG 210 220 230 240 TGG7GATCGA ATTTTCAATT TTTTTACTGG TAGTGATTCC 250 260 270 280 TACTTTTCTT CAATTGCATT TCTCCTTTTT CCATTTCACG 290 GTTGAGAATT C (2c) INFORMATION FOR THE SEQUENCE T.D. 3 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solanum tuberosum LENGTH OF THE SEQUENCE: 508 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: AATGGATTAA AAAGAAAAAA AAAACAAATA AATTGAACCG 40 GGATAAGTTG GTTGTTTAAT TGATTATTGA TTATGATCTC 80 AATTTGACAT TTTGCGCGAT CTTTCGACCT CAATTCGTAT 120 GAACTGACAC TACGCCAATG GACAGTCGCC 6TCGTCACCG 160 CCACCGCACT ATTCTCGACG CGTCGTCTAT CTCCTCCACC 200 CCACAGCCGT CAATTCCAAG CTTCCAATGA ACCGTTGCCA 240 TGTGTCACTG CCTATTCACC GCGAAACATG AATRTCACTG 280 ACGAACGATT TCGGAGCGGA ACGAATCCAG AAAATGGATT 320 ACTTTCTATA AATTCCTCGA ATCTCAACTC CATTTCGTAA 360 AAATAAAATT AAAAATATTG TTTCTTTTTG TATTTCTTTT 4U0 TGTATTTCTG GTTTATGTGG TGATCGAATT TTCAATTTTT 440 TTACTGGTAG TGATTCCTAC TTTTCTTCAA TT6CATTTCT 480 CCTTTTTTCCA TTTCACGGTT GAGAATCC 508 (2d) INFORMATION FOR THE SEQUENCE T.T). 4 TYPE OF SEQUENCE: Nuclee TYPE OF MOLECULE: DNA (genói.) ORIGINAL SOURCE: Solenum tuberos? M LENGTH OF SEQUENCE: 514 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: TTTT 04 GAAATGGATT AAAAAGAAAA AAAAAACAAA TAAATTGAAC 44 CGGGATAAGT TGGTTGTTTA ATTGATTATT GATTATGATC 84 TCAATTTGAC ATTTTGCGCG ATCTTTCGAC CTCAATTCGT 124 ATGAACTGAC ACTACGCCAA TGGACAGTCG CCGTCGTCAC 164 CGCCACCGCA CTATTCTCGA CGCGTCGTCT ATCTCCTCCA 204 CCCCACAGCC GTCAATTCCA AGCTTCCAAT GAACCGTTGC 244 CATGTGTCAC TGCCTATTCA CCGC6AAACA TGAATATCAC 284 TGACGAACGA TTTCGGAGCG GAACGAATCC AGAAAATGGA 324 TTACTTTCTA TAAATTCCTC GAATCTCAAC TCCATTTCGT 364 AAAAATAAAA TTAAAAATAT TGTTTCTTTT TGTATTTCTT 404 TTTGTATTTC TGGTTTATGT GGTGATCGAA TTTTCAATTT 444 TTTTACTGGT AGTGATTCCT ACTTTTCTTC AATT6CATTT 484 CTCCTTTTTC CATTTCACGG TTGAGAATTC 510 (2e) INFORMATION FOR THE SEQUENCE I.D. 5 TYPE OF SEQUENCE: Nuclee TYPE OF MOLECULE: DNA (GENO) ORIGINAL SOURCE: Solan? M tuberosurn SEQUENCE LENGTH: 518 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: TTTTTTTTTGA AATGGATTAA AAAGAAAAAA AAAACAAATA 40 AATTGAACCG GGATAAGTTG GTTGTTTAAT TGATTATTGA 80 TTATGATCTC AATTTGACAT TTTGCGCGAT CTTTCGACCT 120 CAATTCGTAT GAACTGACAC TACGCCAATG GACAGTCGCC 160 GTCGTCACCG CCACCGCACT ATTCTCGACG CGTCGTCT T 200 CTCCTCCACC CCACAGCCGT CAATTCCAAG CTTCCAATGA 240 ACCGTTGCCA TGTGTCACTG CCTATTCACC GCGAAACATG 280 AATATCACTG ACGAACGATT TCGGAGCGGA ACGAATCCAG 320 AAAATGGATT ACTTTCTATA AATTCCTCGA ATCTCAACTC 360 CATTTCGTAA AAATAAAATT AAAAATATTG TTTCTTTTTG 400 TATTTCTTTT TGTATTTCTG GTTTATGTGG TGATCGAATT 440 TTCAATTTTT TTACTGGTAG TGATTCCTAC TTTTCTTCAA 480 TTGCATTTCT CCTTTTTTCCA TTTCACGGTT GAGAATTC 518 (2f) INFORMATION FOR SEQUENCE I.D. 6 TYPE OF SEQUENCE: Nuclee TYPE OF MOLECULE: DNA (genornic) ORIGINAL SOURCE: Solanurn tuberosum LENGTH OF SEQUENCE: 631 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 40 AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA 80 TTTATGAAAA AAATATCAAA ATATAAAGAA ATATTTTTTT 120 TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTG 160 CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT 200 CTCAATTTGA CATTTTGCGC GATCTTTCGA CCTCAATTCG 240 TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA 280 CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC 320 ACCCCACAGC CGTCAATTCC AAGCTTCCAA TGAACCGTTG 360 CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 400 CTGACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG 440 ATTACTTTCT ATAAATTCCT CGAATCTCAA CTCCATTTCG 480 TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 520 TTTTGTATTT CTGGTTTATG TGGTGATCGA ATTTTCAATT 560 TTTTTACTGG TAGTGATTCC TACTTTTCTT CAATTGCATT 600 TCTCCTTTTT CCATTTCACG GTTGAGAATT C 631 (2g) INFORMATION FOR THE SEQUENCE I.D. 7 TYPE OF SEQUENCE: Nuclee TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solanum tuberosurn LENGTH OF SEQUENCE: 674 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Lineel SEQUENCE: ATAGCTTGAG GCGAAAATAT TTAATAAAAA CACTTCTTAA 40 TTTGTTTATA TGTTCAATTG AACATGTCCG TGATTAGAAA 80 ATTAAATTAA ATTCAATGAC AAATTTAATA ATTTGACACA 120 AAATTTATGA AAAAAATACT AAAATATAAA GAAATATTTT 160 TTTTGAAATG 6ATTAAAAAG AAAAAAAAAA CAAATAAATT 200 GAACCGGGAT AAGTTGGTTG TTTAATTGAT TATTGATTAT 240 GATCTCAATT TGACATTTTG CGCGATCTTT CGACCTCAAT 280 TCFTATFAAC TGACACTACG CCAATGGACA GTCGCCGTCG 320 TCACCGCCAC C6CACTATTC TCGACGCGTC GTCTATCTCC 360 TCCACCCCAC AGCCGTCAAT TCCAAGCTTC CAATGAACCG 400 TTGCCATGTG ACACTGCCTA TTCACCGCGA AACATGAATA 440 TCACTGACGA ACGATTTCGG AGCGGAACGA ATCCAGAAAA 480 TGGATTACTT TCTATAAATT CCTCGAATCT CAACTCCATT 520 TCGTAAAAAT AAAATTAAAA ATATTGTTTC TTTTTGTATT 560 TCTTTTTGTA TTTCTGGTTT ATGTGGTGAT CGAATTTTCA 600 ATTTTTTTAC TGGTAGTGAT TCCTACTTTT ATTCAATTGC 640 674 ATTC ATTTCTCCTT TTTCCATTTC ACGGTTGAGA (2h) INFORMATION FOR THE SEQUENCE I.D. 8 TYPE OF SEQUENCE: Nucieotide TYPE OF MOLECULE: DNA (genoric) ORIGINAL SOURCE: Soi num t? Beros? M LENGTH OF THE SEQUENCE: 687 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Lineel SEQUENCE: TTCCTTTCCT CATATAGCTT GAGGCGAA TO TATTTAATAA 40 AAACACTTCT TAATTTGTTT ATATGTTCAA TTC ACATGT 80 CCGTGATTAG AAAATTAAAT TAAATTCAAT GAC AATTT 120 ATAATTTGAC ACAAAATTTA TGAAAAAAAT ATCAAAATAT 160 AAAGAAATAT TTTTTTTGAA ATGGATTAAA AAGAAAAAAA 200 AAACAAATAA ARRGAACCGG GATAAGTTGG TTGTTTAATT 240 GATTATTGAT TATGATCTCA ATTTGACATT TTGCGCGATC 280 TTTCGACCTC AATTCGTATG AACTGACACT ACGCCAATGG 320 ACAGTCGCCG TCGTCACCGC CACCGCACTA TTCTCGACGC 360 GTCGTCTATC TCCTCCACCC CACAGCCGTC AATTCCAAGC 400 TTCCAATGAA CCGTTGCCAT GTGTCACTGC CTATTCACCG 440 CGAAACATGA ATATCACTGA CGAACGATTT CGGAGCGGAA 480 CGAATCCAGA AAATGGATTA CTTTCTATAA ATTCCTCGAA 520 TCTCAACTCC ATTTCGTAAA AATAAAATTA AAAATATTGT 560 TTCTTTTT6T ATTTCTTTTT GTATTTCTGG TTTATGTGGT 600 GATCGAATTT TCAATTTTTT TACTGGTAGT GATTCCTACT 640 TTTCTTCAAT TGCATTTCTC CTTTTTCCAT TTCACGGTTG 680 AGAATTC 687 (2l) INFORMATION FOR SEQUENCE I.D. 9 TYPE OF SEQUENCE: NucleotyD TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Soienum tuberosurn SEQUENCE LENGTH: 693 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: CACTGATTCC TTTCCTCATA TAGCTTGAGG CGARAATATT 40 TAATAAAAAC ACTTCTTAAT TTGTTTATAT GTTCAATTGA 80 ACATGTCCGT GATTAGAAAA TTAAATTAAA TTCñATGACA 120 AATTTAAT A TTTGACACAA AATTTATGAA AAAAATATC 160 AAATATAAAG AAATATTTTT TTTGAAATGG ATTAAAAAGA 200 AAAAAAAAAC AAATAAATTG AACC6GGATA AGTTGGTTGT 240 TTAATTGATT ATTGATTATG ATCTCAATTT GACATTTTGC 280 GCGATCTTTC GACCTCAATT CGTATGAACT 6ACACTACGC 320 CAATGGACAG TCGCCGTCGT CACCCCCACC GCACTATTCT 360 CGACGCGTCG TCTATCTCCT CCACCCCACA GCCGTCAATT 400 CCAAGCTTCC AATGAACCGT TGCCATGTGT CACTGCCTAT 440 TCACCGCGAA ACATG TAT CACTGACGA CGATTTCGGA 480 GCGGAACGAA TCCAGAAAAT GGATTACTTT CTATAAATTC 520 CTCG TCTC AACTCCATTT CGTAAAA T AAATTAAAAA 560 TATTGTTTCT TTTTCTATTT CTTTTTCTAT TTCTGGTTT 600 TGTGGTGATC GAATTTTCAA TTTTTTTACT GGTAGTGATT 640 CCTACTTTTC TTCAATTGCA TTTCTCCTTT TTCCATTT CA 680 CGGTTGAGAA TTC 693 (2j) INFORMATION FOR THE SEQUENCE I.D. 10 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solanum tuberosurn LENGTH OF SEQUENCE: 758 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: CTTGCGCCTT TCCCTAAATT AAGTAAAACT CTTCGCGTCA 40 TGCCTTACGC CTCCGCCTTT TAAAACACTG ATTCCTTTCC 80 TCATATAGCT TGAGGCGAAA ATATTTAATA AAAACACTTC 120 TTAATTTGTT TATATGTTCA ATTGAACATG TCCGTGATTA 160 GAAAATTAAA TTAAATTCAA TGACAAATTT AATAATTTGA 200 CACAAAATTT ATGAAAAAAA TATCAAAATA TAAAGAAATA 240 TTTTTTTTGA AATGGATTAA AAAGAAAAAA AAAACAAATA 280 AATTGAACCG GGATAAGTTG GTTGTTTAAT TGATTATTGA 320 TTATGATCTC AATTTGACAT TTTGCGCGAT CTTTCGACCT 360 CAATTCGTAT GAACTGACAC TACGCCAATG GACAGTCGCC 400 GTCGTCACCG CCACCGCACT ATTCTCGACG CGTCGTCTAT 440 CTCCTCCACC CCACAGCCGT CAATTCCAAG CTTCCAATGA 480 ACCGTTGCCA TGTGTCACTG CCTATTCAGG GCGAAACATG 520 AATATCACTG AC6AACGATT TCGGAGCGGA ACGAATCCAG 560 AAAATGGATT ACTTTCTATA AATTCCTCGA ATCTCAACTC 600 CATTTCGTAA AAATAAAATT AAAAATATTG TTTCTTTTTG T4, TATTTCTTTT TGTATTTCTG GTTTATGTG G TGATCGAATT í, ß0 TTCAATTTTT TTACTGGTAG TGATTCCTAC TTTTCTTCAA 720 TTGCATTTCT CCTTTTTCCA TTTCACGGTT GAGAATTC 758 (2k) INFORMATION FOR THE SEQUENCE I.D. 11 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solan? M tuberosurn LENGTH OF SEQUENCE: 855 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Lineel SEQUENCE: CAAATTTTGA TGTATTTTTA TAATTTTGTA TTATTATATT 40ATTTAAAAAT TTAAAGATCC ATAGGGCTTA 80 CGCCCCACGT CAAGAGGCTT GCGCCTTTCC CTAAATTAAG 120 TAAAACTCTT CGCCTCATGC CTTACGCCTC CGCCTTTTAA 160 AACACTGATT CCTTTCCTCA TATAGCTTGA GGCGAAAATA 200 TTTAATAAAA ACACTTCTTA ATTTGTTTAT ATGTTCAATT 240 GAACATGTCC GTGATTAGAA AATTAAATTA AATTCAATGA 280 CAAATTTAAT AATTTGACAC AAAATTTATG AAAAAAATAT 320 CAAAATATAA AGAAATATTT TTTTTGAAAT GGATTAAAAA 360 GAAAAAAAAA ACAAATAAAT TGAACCGGGA TAAGTTGGTT 400 GTTTAATTGA TTATTGATTA TGATCTCAAT TTGACATTTT 440 GCGCGATCTT TCGACCTC? A TTCGTATGAA CTGACACTAC 480 GCCAATGGAC AGTCGCCGTC GTCACCGCCA CCGCACTATT 520 CTCGACGCGT CGTCTATCTC CTCCACCCCA CAGCCGTCAA 550 TTCCAAGCTT CCAATGAACC GTTGCCATGT GTCACTGCCT 600 ATTCACCGCG AAACATGAAT ATCACTGACG AACGATTTCG 640 GAGCGGAACG AATCCAGAAA ATGGATTACT TTCTATAAAT 680 TCCTCGAATC TCAACTCCAT TTCGTAAAAA TAAAATTAAA 720 AATATTGTTT CTTTTTGTAT TTCTTTTTGT ATTTCTGGTT 760 TATGTGGTGA TCGAATTTTC AATTTTTTTA CTGGTAGTGA 800 TTCCTACTTT TCTTCAATTG CATTTCTCCT TTTTCCATTT 840 CACGGTTGAG AATTC 855 (21) INFORMATION FOR THE SEQUENCE I.D. 12 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solen? M tuberosum LENGTH OF THE SEQUENCE: 859 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linee! SEQUENCE: ATTTCAAATT TTGATGTATT TTTATAATTT TGTATTATTA 40 TATTATTATA CTATATTTAA AAATTTAAAG ATCCATAGGG 80 CTTACGCC C ACGTCAAGAG GCTTGCGCCT TTCCCTAAAT 120 TAAGTAAAAC TCTTCGCCTC ATGCCTTACG CCTCCGCCTT 160 TTAAAACACT GATTCCTTTC CTCATATAGC TTGAGGCGAA 200 AATATTTAAT AAAAACACTT CTTAATTTGT TTATATGTTC 240 AATTGAACAT GTCCGTGATT AGAAAATTAA ATTAAATTCA 230 ATGACAAATT TAATAATTTG ACACAAAATT TATGA1-.AAA 320 ATATCAAAAT ATAAAGAAAT ATTTTTTTTG AAATC ~ - TTA 360 AAAAGAAAAA AAAAACAAAT AAATTGAACC GGGAT -.GTT 400 GGTTGTTTAA TTGATTATTG ATTATGATCT CAATT. JACA 440 lili JUUU'J? TCTTTCGACC TCAATTCGTA TGAACTGACA 480 L 1 ALui- rt? J. GGACAGTCGC CGTCGTCACC GCCACCGCAC 520 TATTCTCGAC GCGTCGTCTA TCTCCTCCAC CCCACAGCCG 560 TCAATTCCAA GCTTCCAATG AACCGTTGCC ATGTGTCACT 600 CGCGAAACAT GAATATCACT GACGAACGAT 640 680 AACGAATCCA GAAAATGGAT TACTTTCTAT AAAT CCTCG AATCTCAACT CCATTTCGTA AAAATAAAAT 720 TAAAAATATT GTTTCTTTTT GTATTTCTTT TTGTATTTCT 760 GGTTTATGTG GTGATCGAAT TTTCAATTTT TTTACTGGTA 800 GTGATTCCTA CTTTTCTTCA ATTGCATTTC TCCTTTTTCC TGAGAATTC 840 859 u (2RN ) INFORMATION FOR THE ID SEQUENCE 13 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solanum tuberosum LENGTH OF SEQUENCE: 1.2.1.4 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: GAAGGTGATT ATACATTACG TAACATTTCT TTTAAAAATA 40 AACTTATCAT TGATCTTCAT 80 í * »r , mmf »mr ^ TAAATCTCAA AGTTATCATA TTTTATATAG_120_GTAATTTTAT TTTTACTCAT CATTGAGTGA 160 ATAATACTAG TAAGTTTTAT TTATTATTTT 200 CTTTTAGGC-3 G AATTGTAT AATATAATAA AAAATATATT 240 TTTAGAAATA ATGATTCTTT TATTATTAAA AAGTTAAGAT 280 ATTAGATTAT TTATGCTTGT ATAATAATGA ACGAAGTTTT 320 ATTTTCTATG AGTTT ATTA ATCATGTTTG TAATTATTTC 360 AAATTTTGAT GTATTTTTAT AATTTTGTAT TATTATATTA 400 TTATACTATA TTTAAAAATT TAAAGATCCA TAGGGCTTAC 440 ut.? r.-b. AAGAGGCTTG CGCCTTTCCC TAAATTAAGT 480 AA ?? V_. i - - GCCTCATGCC TTACGCCTCC GCCTTTTAAA 520 CTTTCCTCAT ATAGCTTGAG GCGAAAATAT 560 TTAATAAAAA CACTTC ^ TAA TTTGTTTATA TGTTCAATTG 600 AACATGTCCG TGATT -AA ATTAAATTAA ATTCAATGAC 640 AAATTTAATA ATTTC CA \ TTTATGA AAAAAATATC 680 AAAATATAAA GAAA TT 7TGAAATG GATTAAAAAG 720 AAAAAAAAAA CAAATAAATT VCCGGGAT AAGTTGGTTG 760 TTTAATTGAT TATTGATTAT GATCTCAATT TGACATTTTG 800 CGCGATCTTT CGACCTCAAT TCGTATGAAC TGACACTACG 840 CCAATGGACA GTCGCCGTCG TCACCGCCAC CGCACTATTC 880 TCGACGCGTC GTCTATCTCC TCCACCCCAC AGCCGTCAAT 920 TACAAGCTTC CAATGAACCG TTGCCATGTG TCACTGCCTA 960 TTCACCGCGA AACATGAATA TCACTGACGA ACGATTTCGG 1000 AGCGGAACGA ATCCAGAAAA TGGATTACTT TCTATAAATT 1040 CCTCGAATCT CAACTCCATT TCGTAAAAAT AAAATTAAAA 1080 ATATTGTTTC TTTTTGTATT TCTTTTTGTA TTTCTGGTTT 1120 ATGTGGTGAT CGAATTTTCA ATTTTTTTAC TGGTAGTGAT 1160 TTCTACTTTT CTTCAATTGC ATTTCTCCTT TTTCCATTTC 1200 ACGGTTGAGA ATTC 1214 (2n) INFORMATION FOR SEQUENCE I.D. 14 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solanurn tuberosurn LENGTH OF SEQUENCE: 1232 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: ACTATTTGAT AACATTATGA AGGTGATTAT ACATTACGTA 40 ACATTTCTTT TAAAAATATG TAAGCAAATT TACTTTTTAA 80 CTTATCATTG ATCTTCATGG TTTTGTCATA AATCTCAAAG 120 TTATCATATT TTATATAGCT ATTTGAAAGT AATTTTATTT 160 TTACTCATCA TTGAGTGATG CTTTTATTAT AATACTAGTA 200 AGTTTTATTT ATTATTTTCT TTTAGGGGTG AATTGTATAA 240 TATAATAAAA AATATATTTT TAGAAATAAT GATTCTTTTA 280 TTATTAAAAA GTTAAGATAT TAGATTATTT ATGCTTGTAT 320 AATAATGAAC GAAGTTTTAT TTTCTATGAG TTTCATTAAT 360 CATGTTTGTA ATTATTTCAA ATTTTGATGT ATTTTTATAA 400 TTTTGTATTA TTATATTATT ATACTATATT TAAAAATTTA 440 AAGATCCATA GGGCTTACGC CCCACGTCAA GAGGCTTGCG 480 CCTTTCCCTA AATTAAGTAA AACTCTTCGC CTCATGCCTT 520 ACGCCTCCGC CTTTTAAAAC ACTGATTCCT TTCCTCATAT 560 AGCTTGAGGC GAAAATATTT AATAAAAACA CTTCTTAATT 600 TGTTTATATG TTCAATTGAA CATGTCCGTG ATTAGAAAAT 640 TAAATTAAAT TCAATGACAA ATTTAATAAT TTGACACAAA 680 ATTTATGAAA AAAATATCAA AATATAAAGA AATATTTTTT 720 TTGAAATGGA TTAAAAAGAA AAAAAAAACA AATAAATTGA 760 ACCGGGATAA GTTGGTTGTT TAATTGATTA TTGATTATGA 800 TCTCAATTTG ACATTTTGCG CGATCTTTCG ACCTCAATTC 840 GTATGAACTG ACACTACGCC AATGGACAGT CGCCGTCGTC 880 ACCGCCACCG CACTATTCTC GACGCGTCGT CTATCTCCTC 920 CACCCCACAG CCGTCAATTC CAAGCTTCCA ATGAACCGTT 960 GCCATGTGTC ACTGCCTATT CACCGCGAAA CATGAATATC 1000 ACTGACGAAC GATTTCGGAG CGGAACGAAT CCAGAAAATG 1040 GATTACTTTC TATAAATTCC TCGAATCTCA ACTCCATTTC 1080 GTAAAAATAA AATTAAAAAT ATTGTTTCTT TTTGTATTTC 1120 TTTTTGTATT TCTGGTTTAT GTGGTGATCG AATTTTCAAT 1160 TTTTTTTACTG GTAGTGATTC CTACTTTTCT TCAATTGCAT 1200 TTCTCCTTTT TCCATTTCAC GGTTGAGAAT TC 1232 9.1 (2?) INFORMATION FOR SEQUENCE I.D. 15 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (GENORIAN) ORIGINAL SOURCE: Solanum tuberosum LENGTH OF SEQUENCE: 1352 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: GGCCTCACA TCAACCTTCA TAATTCTTGA ATGAATGAAT 39 GATAGACTT ATAATTTTTT AACCTATACA TATAAGAAAA 79 TTGAGAGTAA CTCAAATAAC AAGTTGTAGT ATCACATCTT 119 TACTATTTGA TAACATTATG AAGGTGATTA TACATTACGT 159 AACATTTCTT TTAAAAATAT GTAAGCAAAT TTACTTTTTA 199 ACTTATCATT GATCTTCATG GTTTTGTCAT AAATCTCAAA 239 GTTATCATAT TTTATATAGC TATTTGAAAG TAATTTTATT 279 TTTACTCATC ATTGAGTGAT GCTTTTATTA TAATACTAGT 319 AAGTTTTATT TATTATTTTC TTTTAGGGGT GAATTGTATA 359 ATATAATAAA AAATATATTT TTAGAAATAA TGATTCTTTT 399 ATTATTAAAA AGTTAAGATA TTAGATTATT TATGCTTGTA 439 TAATAATGAA CGAAGTTTTA TTTTCTATGA GTTTCATTAA 479 TCATGTTTGT AATTATTTCA AATTTTGATG TATTTTTATA 519 ATTTTGTATT ATTATATTAT TATACTATAT TTAAAAATTT 559 AAAGATCCAT AGGGCTTACG CCCCACGTCA AGAGGCTTGC 599 GCCTTTCCCT AAATTAAGTA AAACTCTTCG CCTCATGCCT 639 TACGCCTCCG CCTTTTAAAA CACTGATTCC TTTCCTCATA 679 TAGCTTGAGG CGAAAATATT TAATAAAAAC ACTTCTTAAT 719 TTGTTTATAT GTTCAATTGA ACATGTCCGT GATTAGAAAA 759 TTAAATTAAA TTCAATGACA AATTTAATAA TTTGACACAA 799 AATTTATGAA AAAAATATCA AAATATAAAG AAATATTTTT 839 TTTGAAATGG ATTAAAAAGA AAAAAAAAAC AAATAAATTG 879 AACCGGGATA AGTTGGTTGT TTAATTGATT ATTGATTATG 919 ATCTCAATTT GACATTTTGC GCGATCTTTC GACCTCAATT 959 CGTATGAACT GACACTACGC CAATGGACAG TCGCCGTCGT 999 GCACTATTCT CGACGCGTCG TCTATCTCCT 1039 CCACCCCACA GCCGTCAATT CCAAGCTTCC AATGAACCGT 1079 TGCCATGTGT CACTGCCTAT TCACCGCGAA ACATGAATAT 1119 CACTGACGAA CGATTTCGGA GCGGAACGAA TCCAGAAAAT 1159 GGATTACTTT CTATAAATTC CTCGAATCTC AACTCCATTT 1199 CGTAAAAATA AAATTAAAAA TATTGTTTCT TTTTGTATTT 1239 w ^^^^^^^^ m TTCTGGTTTA TGTGGTGATC GAATTTTCAA 1279 ^ p ^ m * ^ * ^ p GGTAGTGATT CCTACTTTTC TTCAATTGCA 1319 ^ p t ^ ^^^ p ^ f ^ ^^ TTCCATTTCA CGGTTGAGAA TTC 1352 (2p) INFORMATION FOR SEQUENCE I.D. 16 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solanum tuberosum LENGTH OF SEQUENCE: 1,734 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: 10 20 30 40 TC7TTAAG7T GTTTGCTTGA TTTTTCTTCT TCAATCTTCT 5 500 6b0u 70 80 ATATTTAAT ? CGTTTTAGCT TCAAACTTCT TCAATTTTAT 90 100 110 120 TTCAATTTAA TTCTACAAAA AAAATCTCTA TTTAGCACCA 130 140 150 160 TTCATAAAAT TCATGCTCAA AATGGGCAAA CATAAATAAT 170 1B0 190 200 AAATGTGAAG TAAATAATGG ATTAAAATAT ATATTTTTGG 210220230240 GCCTCACATC AACCTTCATA ATTCTTGAAT GAATGAATGA 250250270230 TAGACTTCAT AATTTTTTAA CCTATACATA TAAGAAAATT 29C 300,310,320 GAGAGTAACT CAAATAACAA GTTOTAGTAT CACATCTTTA 330340350360 CTATTTGATA ACAT7A7GAA GGTGATTATA CATTACGTAA 370 3SC 390,400 CATTTCTTTT AAAAATATGT AAGCAAATTT ACTTTTTAAC 410420430440 TTATCATTGA TCTTCATGGT TTTGTCATAA ATCTCAAAGT 450 460 470 480 TATCATATTT TATATAGCTA TTTGAAAGTA ATTTTATTTT 490 500 510 520 TACTCATC .-. T TGAGTGATGC TTTTATTATA ATACTAGTAA 530 540 550 560 GTTTTATTTA TTATTTTCTT TTAGGGGTGA ATTGTATAAT 570 580 590 600 ATAATAAAAA ATATATTTTT AGAAATAATG ATTCTTTTAT 610 620 630 640 TATTAAAAAG TTAAGATATT AGATTATTTA TGCTTGTATA 650 660 670 680 ATAATGAACG AAGTTTTATT TTCTATGAGT TTCATTAATC 690 700 710 720 ATGTTTGTAA TTATTTCAAA TTTTGATGTA TTTTTATAAT 730 740 750 760 TTTGTATTAT TATATTATTA TACTATATTT AAAAATTTAA 770 780 790 800 AGATCCATAG GGCTTACGCC CCACGTCAAG AGGCTTGCGC 810 820 830 840 CTTTCCCTAA ATTAAGTAAA ACTCTTCGCC TCATGCCTTA 850 860 870 880 CGCCTCCGCC TTTTAAAACA CTGATTCCTT TCCTCATATA 890 900 910 920 GCTTGAGGCG AAAATATTTA ATAAAAACAC TTCTTAATTT 930 940 950 960 GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 970 980 990 1000 AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA 1010 1020 1030 1040 TTTATGAAAA AAATATCAAA ATATAAAGAA ATATTTTTTT 1050 1060 1070 1080 TGAAATGGAT TAAAAAGPiAA AAAAAAACAA ATAAATTGAA 1090 1100 1110 1120 CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT 1130 1140 1150 1160 CTCAATTTGA CATTTTGCGC GATCTTTCGA CCTCAATTCG 1170 1180 1190 1200 TATGAACTGA CACTACGCCA ATGGACA TC GCCGTCGTCA 1210 1220 1230 1240 CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC 1250 1260 1270 1280 ACCCCACAGC CGTCAATTCC AAGCTTCCAA TGAACCGTTG 1290 1300 1310 1320 CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 1330 1340 1350 1360 CTOACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG 1370 1380 1390 1400 ATTACTT7C7 ATAAATTCCT CGAATCTCAA CTCCATTTCG 1410 1420 1430 1440 TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 1450 1460 1470 1480 TTTTGTATTT CTGGTTTATG TGGTGATCGA- ATTTTCAATT 1490 1500 1510 1520 TTTTTACTGG TAGTGATTCC TACTTTTCTT CAATTGCATT 1530 1540 1550 1560 1570 TCTCCTTTTT CCATTTCACG GTTGAGAATT CATGATTCCT 15B0 TATCAGAGGA 1590 1600 1610 1620 ATCGATCCGA TTTGACTAAT TTCACTTTTC GTCTGTATAA 1630 1640 1650 1660 ATACCAGAGT ATCTAGGTTG AGGAACGTAA TTTCAAGCTG 1670 1680 1690 1700 CGATCGGCTT TTTCCCCTGA ACGAGCAAAC 1710 1720 ACAGGTTGTG GGTTCGAGTT AGCAAGGGAC GTATAATCTC 1730 .-. ACTACAATC CATT (2q) INFORMATION FOR SEQUENCE I.D. 17 TYPE OF SEQUENCE: Nucle? gone TYPE OF MOLECULE: DNA (genomic) ORIGINAL SOURCE: Solan? m tuberos? rn LENGTH OF THE SEQUENCE: 1920 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linee! SEQUENCE 10 20 30 40 TCTTTAAGTT GTTTGCTTGA TTTTTCTTCT TCAATCTTCT 50 60 70 80 ATATTTAATT CGTTTTAGCT TCAAACTTCT TCAATTTTAT 90 100 110 120 TTCAATTTAA TTCTACAAAA AAAATCTCTA TTTAGCACCA 130 140 150 160 TTCATAAAAT TCATGCTCAA AATGGGCAAA CATAAATAAT 170 180 190 200 AAATGTGAAG TAAATAATGG ATTAAAATAT ATATTTTTGG 210 220 230 240 GCCTCACATC AACCTTCATA ATTCTTGAAT GAATGAATGA 250 260 270 280 TAGACTTCAT AATTTTTTAA CCTATACATA TAAGAAAATT 290 300 310 320 GAGAGTAACT CAAATAACAA GTTGTAGTAT CACATCTTTA 330 340 350 360 CTATTTGATA ACATTATGAA GGTGATTATA CATTACGTAA 370 .380 390 400 CATTTCTTTT AAAAATATGT AAGCAAATTT ACTTTTTAAC 410 420 430 440 TTATCATTGA TCTTCATGGT TTTGTCATAA ATCTCAAAGT 450 460 470 480 TATCATATTT TATATAGCTA TTTGAAAGTA ATTTTATTTT 490 500 510 520 TACTCATCAT TGAGTGATGC TTTTATTATA ATACTAGTAA 530 540 550 560 GTTTTATTTA TTATTTTCTT TTAGGGGTGA ATTGTATAAT 570 580 590 600 ATAATAAAAA ATATATTTTT AGAAATAATG ATTCTTTTAT 610 620 630 640 TATTAAAAAG TTAAGATATT AGATTATTTA TGCTTGTATA 650 660 670 680 ATAATGAACC AAGTTTTATT TTCTATGAGT TTCATTAATC 690 700 710 720 ATGTTTGTAA TTATTTCAAA TTTTGATGTA TTTTTATAAT 730 740 750 760 TTTGTATTAT TATATTATTA TACTATATTT AAAAATTTAA 770 780 790 800 AGATCCATAG GGCTTACGCC CCACGTCAAG AGGCTTGCGC 810 820 830 840 CTTTCCCTAA ATTAAGTAAA ACTCTTCGCC TCATGCCTTA 850 860 870 880 CGCCTCCGCC TTTTAAAACA CTGATTCCTT TCCTCATATA 890 900 910 920 GCTTGAGGCG AAAATATTTA ATAAAAACAC TTCTTAATTT 930 940 950 960 GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 970 980 990 1000 AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA 1010 1020 1030 1040 TTTATGAAAA AAATATCAAA ATATAAAGAA ATATTTTTTT 1050 1060 1070 1080 TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTGAA 1090 1100 1110 1120 CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT 1130 1140: *: D? I60 CTCAATTTGA CATTTTGCGC GATCTTT-GA CCTCAATTCG 1170 1180 1190 1200 TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA 1210 1220 1230 1240 CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC 1250 1260 1270 1280 ACCCCACAGC CGTCAATTCC AAGCTTCCAA TGAACCGTTG 1290 1300 1310 1320CTGCCTATTC ACCGCGAAAC ATGAATATCA 1330 1340 1350 1360 CTGACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG 1370 1380 1390 1400 ATTACTTTCT ATAAATTCCT CGAATCTCAA CTCCATTTCG 1410 1420 1430 1440 TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 1450 1460 1470 1480 TTTTGTATTT CTGGTTTATG TGGTGATCGA ATTTTCAATT 1490 1500 1510 1520 TTTTTACTGG TAGTGATTCC TACTTTTCTT CAATTGCATT 1530 1540 1550 1560 TCTCCTTTTT CCATTTCACG GTTGAGAATT CATGATTCCT 1570 1580 1590 1600 TATCAGAGGA ATCGATCCGA TTTGACTAAT TTCACTTTTC 1610 1620 1630 1640 GTCTGTATAA ATACCAGAGT ATCTAGGTTG AGGAACGTAA 1650 1660 1670 1680 TTTCAAGCTG CGATCGGCTT TTTCCCCTGA ACGAGCAAAC 1690 1700 1710 1720 ACAGGTTGTG GGTTCGAGTT AGCAAGGGAC GTATAATCTC 1730 1740 1750 1760 AACTACAATC CATTATGGCG CTTGATGAAA GTCAGCAGTC 1770 1780 1790 1800 TGATCCATGT AAGGTTCTCT TTTCCTTTAT ATATGCTTCA 1810 1820 1830 1840 TAATTGAGAA GGAAGACGGA GATTTGAACT TAATAAAGGC 1850 1860 1870 1880 GAAGATTTGA ACAAAATATT TTGGTATTTC ATTTAAAACT 1890 1900 1910 1920 TTACCAGTTC TAAGAGTAAA TGATTGGGAT GTGCATGTCC (2r) INFORMATION FOR THE SEQUENCE I.D. .1.8 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genic) SOURCE ORIGINAL: Soienum tuberosum LENGTH OF SEQUENCE: 1570 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: 10 20 30 40 TGTGGTGATC GAATTTTCAA TTTTTTTACT GAGTATCTAG_50_60 70 80 GTTGAGGAAC GTAATTTCAA GCTGCGATCG GCTTTTTCCC 90 100 110 120 CTGAACGAGC AAACACAGGT TGTGGGTTCG AGTTAGCAAG 130 140 150 160 GGACGTATAA TCTCAACTAC AATCCATTAT GGCGCTTGAT 170 180 190 200 GAAAGTCAGC AGTCTGATCC ATTGGTTGTG ATACGCAATG 210 220 230 240 GAAAGGAGAT CATATTGCAG GCATTCGACT GGGAATCTCA 250 260 270 280 GA TAAACATGAT TGGTGGCTAA ATTTAGATAC.-..-. GTTCCT 290300310320 GATATTGCAA AGTCTGGTTT CACAACTGCT TGGCTGCCTC 330340350360 CGGTGTGTCA GTCATTGGCT CCTGAAGGTT ACCTTCCACA 370 3B0 390 400 GAACCTTTAT TCTCTCAATT CTAAATATGG TTCTGAGGAT 410420430440 CTCTTAAAAG CTTTACTTAA TAAGATGAAG CAGTACAAAG 450460 470480 TTAGAGCGAT GGCGGACATA GTCATTAACC ACCGTGTTGG 490500510520 GACTACTCAA GGGCATGGTG GAATGTACAA CCGCTATGAT 530540550560 GGAATTCCTA TGTCTTGGGA TGAACATGCT ATTACATCTT 570580590600 GCACTGGTGG AAGGGGTAAC AAAAGCA TG GAGACAACTT 610620630640 TAATGGAGTT CCAAATATAG ATCATACACA ATCCTTTGTT 650660670680 CGGAAAGATC TCATTGACT3 GATGCGGTGG CTAAGATCCT 690700710 720 CTGTTGGCTT CCAAGATTTT CGTTTTTATT TTGCCAAAGG 730 740 750 760 TTATGCTTCA AAGTATGTAA AGGAATATAT CGAGGGAGCT 770 780 790 800 GAGCCAATAT TTGCAGTTGG AGAATACTGG GACACTTGCA 810 820 830 840 ATTACAAGGG CAGCAATTTG GATTACAACC AAGATAGTCA 850 860 870 880 CAGGCAAAGA ATCATCAATT GGATTGATGG CGCGGGACAA 890 900 910 920 CTTTCAACTG CATTCGATTT TACAACAAAA GCAGTCCTTC 930 940 950 960 AGGAAGCAGT CAAAGGAGAA TTCTGGCGTT TGCGTGACTC 970 980 990 1000 TAAGGGGAAG CCCCCAGGAG TTTTAGGATT GTGGCCTTCA 1010 1020 1030 1040 AGGGCTGTCA CTTTTATTGA TAATCACGAC ACTGGATCAA 1050 1060 1070 1080 CTCAGGCGCA TTGGCCTTTC CCTTCACGTC ATGTTATGGA 1090 1100 1110 1120 GGGCTATGCA TACATTCTTA CACACCCAGG GATACCATCA 1130 1140 1150 1160 GTTTTCTTTG ACCATTTCTA CGAATGGGAT AATTCCATGC 1170 1180 1190 1200 ATGACCAAAT TGTAAAGCTG ATTGCTATTC GGAGGAATCA 1210 1220 1230 1240 AGGCATACAC AGCCGTTCAT CTATAAGAAT TCTTGAGGCA 1250 1260 1270 1280 CAGCCAAACT TATACGCTGC AACCATTGAT GAAAAGGTTA 1290 1300 1310 1320 GCGTGAAGAT TGGGGACGGA TCATGGAGCC CTGCTGGGAA 1330 1340 1350 1360 AGAGTGGACT CTCGCGACCA GTGGCCATCG CTATGCAGTC LOO 1370 1380 1390 1400 TGGCAGAAGT AATCTTACAG CTATTCCGTT ACTTAATATA 1410 142C 1430 1440 TTAGTAGAAA TATATATGTT TTAAACCCGA GCACCTACTT 1450 1460 1470 1480 CTAACACTAG ATCCGCCTCT ACAGGCTTGG ATGGAGTGAT 1490 1500 1510 1520 GAGTTTTTTT TTCCTGTTCA TTAGACATTG CAACATGGGA 1530 1540 15"" 1560 TGTATGTTTT GTTAATAAAA GTGTTC: T '; - TCAATGCAAT 1570 GTAATAAGGG . 1 (2s) INFORMATION FOR SEQUENCE I.D. 19 TYPE OF SEQUENCE: Nucleotide TYPE OF MOLECULE: DNA (genomic) SOURCE ORIGINAL: Solanum tuberosum LENGTH OF THE SEQUENCE: 1570 bp NUMBER OF FILAMENTS: Double TOPOLOGY: Linear SEQUENCE: 20 30 40 CTTAAAAGTT AAAAAAATGA CTCATAGATC 50 60 70 80 CAACTCCTTG CATTAAAGTT CGACGCTAGC CGAAAAAGGG 90 100 110 120 GACTTGCTCG TTTGTGTCCA ACACCCAAGC TCAATCGTTC 130140150160 CCTGCATATT AGAGTTGATG TTAGGTAATA CCGCGAACTA 170180190200 CTTTCAGTCG TCAGACTAGG TAACCAACAC TATGCGTTAC 210220230240 CTTTCCTCTA GTATAACGTC CGTAAGCTGA CCCTTAGAGT 250260270 280 ATTTGTACTA ACCACCGATT TAAATCTATG CTTTCAAGGA 290 300 310 320 CTATAACGTT TCAGACCAAA CTGTTGACGA ACCGACGGAG 330 340 350 360 GCCACACAGT CAGTAACCGA GGACTTCCAA TGGAAGGTGT 370 380 390 400 CTTGGAAATA AGAG -.-. GTTAA GATTTATACC AAGACTCCTA 410 420 430 440 GAGAATTTTC GAAATGAATT ATTCTACTTC GTCATGTTTC 450 460 470 480 AATCTCGCTA CCGCCTGTAT CAGTAATTGG TGGCACAACC 490 500 510 520 CTGATGAGTT CCCGTACCAC CTTACATGTT GGCGATACTA 530 540 550 560 CCTTAAGGAT ACAGAACCCT ACTTGTACGA TAATGTAGAA 570 580 590 600 CGTGACCACC TTCCCCATTG TTTTCGTGAC CTCTGTTGAA 610 620 630 640 ATTACCTCAA GGTTTATATC TAGTATGTGT TAGGAAACAA 650 660 670 680 GCCTTTCTAG AGTAACTGAC CTACGCCACC GATTCTAGGA 690 700 710 720 GACAACCGAA GGTTCTAAAA GCAAAACTAA AACGGTTTCC 730 740 750 760 AATACGAAGT TTCATACATT TCCTTATATA GCTCCCTCGA 770 780 790 800 CTCGGTTATA AACGTCAACC TCTTATGACC CTGTGAACGT 810 820 830 840 TAATGTTCCC GTCGTTAAAC CTAATGTTGG TTCTATCAGT 850 860 870 880 GTCCGTTTCT TAGTAGTTAA CCTAACTACC GCGCCCTGTT 890 900 910 920 GAAAGTTGAC GTAAGCTAAA ATGTTGTTTT CGTCAGGAAG 930 940 950 960 TCCTTCGTCA GTTTCCTCTT AAGACCGCAA ACGCACTGAG 970 980 990 1000 ATTCCCCTTC GGGGGTCCTC AAAATCCTAA CACCGGAAGT 1010 1020 1030 1040 TCCCGACAGT GAAAATAACT ATTAGTGCTG TGACCTAGTT 1050 1060 1070 1080 AGTCCGCGT AACCGGAAAG GGAAGTGCAG TACAATACCT 1090 1100 1110 1120 CCCGATACGT ATGTAAGAAT GTGTGGGTCC CTATGGTAGT 1130 1140 1150 1160 CAAAAGAAAC TGGTAAAGAT GCTTACCCTA TTAAGGTACG 1170 1180 1190 1200 TACTGGTTTA ACATTTCGAC TAACGATAAG CCTCCTTAGT 1210 1220 1230 1240 TCCGTATGTG TCGGCAAGTA GATATTCTTA AGAACTCCGT 1250 1260 1270 1280 GTCGGTTTGA ATATGCGACG TTGGTAACTA CTTTTCCAAT 1290 1300 1310 1320 CGCACTTCTA ACCCCTGCCT AGTACCTCGG GACGACCCTT 1330 1340 1350 1360 TCTCACCTGA GAGCGCTGGT CACCGGTAGC GATACGTCAG 1370 1380 1390 1400 ACCGTCTTCA TTAGAATGTC GATAAGGCAA TGAATTATAT 1410 1420 1430 1440 AATCATCTTT ATATATACAA AATTTGGGCT CGTGGATGAA 1450 1460 1470 1480 GATTGTGATC TAGGCGGAGA TGTCCGAACC TACCTCACTA 1490 1500 1510 1520"CAAAAAAA AAGGACAAGT AATCTGTAAC GTTGTACCCT 1530 1540 1550 1560 ACATACAAAA CAATTATTTT CACAAGAACT AGTTACGTTA 1570 CATTATTCCC

Claims (15)

1. 04 NOVELTY OF THE INVENTION CLAIMS

1. - A promoter characterized in that it comprises a sequence of nucleotides corresponding to the EcoRI fragment of 5.5 kb, isolated from Solanum tuberosum, or a variant, homologue or fragment thereof.

2. A promoter characterized in that it comprises a nucleotide sequence corresponding to the 5.5 kb EcoRI fragment, isolated from Solanum tuberosum, or a variant, homologue or fragment thereof, but wherein at least a portion of the promoter is inactivated.

3. A promoter characterized in that it comprises at least one nucleotide sequence shown as Seq.l.D. No. 1, or a variant, a homologue or a fragment thereof.

4. A promoter characterized in that it comprises the nucleotide sequence of any of the sequences shown as Seq.l.D. No. 4 - 17 or a variant, a homologue or a fragment thereof. 5. A promoter characterized in that it comprises a nucleotide sequence corresponding to the EcoRI fragment of

5. 5 kb, isolated from Solanum tuberosum, or a variant, a homologue or a fragment thereof; but wherein at least the nucleotide sequence shown as Seq.l.D. is inactivated. No. i.

6. A promoter characterized in that it comprises a sequence of nucleotides corresponding to the EcoRI fragment of 5.5 kb, isolated from Solanum tuberos? M, or a variant,? N homologue or a fragment thereof; but where at least any of the sequences shown as Seq.l.D. No. 2 - 16 is inactive.

7. A construction characterized in that it comprises a promoter according to any of claims 1 to 6, cast to a GDI.

8. An expression vector, characterized in that it comprises the promoter according to any of claims 1 and 5.

9. A transformation vector, characterized in that it comprises the promoter according to claims of 1 to 6.

10. - A transformed cell or a transformed organ, characterized in that they comprise the promoter according to any of claims 1 to 6.

11.- A transgenic organism, characterized in that it comprises the promoter according to any of the claims 6 or the invention of any of claims 7 to 10.

12. A transgenic organism according to claim 11, further characterized in that the plant is a paper plant.

13. The use of the promoter as defined in claim 1, as a cold-inducible promoter.

14. - A construction characterized in that it comprises the promoter defined in claim 1 and a sequence of nucleotides encoding anti-transferase alpha-amylose.

15. The use of a promoter according to any of claims 1 to 6 to express a GDI in the tuber and / or the shoot and / or the root and / or the stem of a plant.