MXPA96001719A - Method for increasing the trehalose content of the organisms through its transformation with the adnre of trehalosa-6-phosphate synthase / phosphatase of selaginella lepidophy - Google Patents

Method for increasing the trehalose content of the organisms through its transformation with the adnre of trehalosa-6-phosphate synthase / phosphatase of selaginella lepidophy

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Publication number
MXPA96001719A
MXPA96001719A MXPA/A/1996/001719A MX9601719A MXPA96001719A MX PA96001719 A MXPA96001719 A MX PA96001719A MX 9601719 A MX9601719 A MX 9601719A MX PA96001719 A MXPA96001719 A MX PA96001719A
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Mexico
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leu
trehalose
glu
val
gly
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MXPA/A/1996/001719A
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Spanish (es)
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MX9601719A (en
MX205414B (en
Inventor
Zentella Gomez Rodolfo
Iturriaga De La Fuente Gabriel
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Universidad Catolica De Leuven
Universidad Nacional Autonoma De Mexico
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Application filed by Universidad Catolica De Leuven, Universidad Nacional Autonoma De Mexico filed Critical Universidad Catolica De Leuven
Priority to MX9601719A priority Critical patent/MX205414B/en
Priority claimed from MX9601719A external-priority patent/MX205414B/en
Priority to EP97923309A priority patent/EP0933428A2/en
Priority to PCT/MX1997/000012 priority patent/WO1997042327A2/en
Priority to BR9710436A priority patent/BR9710436A/en
Priority to JP9539760A priority patent/JP2000509602A/en
Priority to AU29135/97A priority patent/AU727509B2/en
Publication of MX9601719A publication Critical patent/MX9601719A/en
Publication of MXPA96001719A publication Critical patent/MXPA96001719A/en
Publication of MX205414B publication Critical patent/MX205414B/en

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Abstract

The present invention relates to cloning and determination of the nucleotide sequence of a complete complementary DNA molecule encoding the bifunctional enzyme trehalose-6-phosphate synthase / plant phosphatase. The complementary DNA molecule codes for a bifunctional enzyme that synthesizes trehalose. The complementary DNA can be subcloned into appropriate vectors for expression in host cells. Transformed cells produce trehalose, in contrast to untransformed cells, which will increase tolerance to heat stress, cold, salinity and drought. This invention can be used to improve the thermotolerance and osmotolerance of plants grown in arid or semi-arid zones and will also contribute to decrease the use of irrigation water. On the other hand, the trehalose produced in transgenic plants could increase the shelf life of agricultural products, preserving them for long periods in a dehydrated state without losing their odor, taste and texture properties, once they are rehydrated. Finally, the overproduction of trehalose with bacteria, yeasts, fungi, animal cells or transgenic plants will constitute a cheap source of this product to be used as an additive to preserve various biological products or processed foods.

Description

Method to increase the trehalose content of the organisms by means of their transformation with the cDNA of the trehalose-6-phosphate synthase / phosphatase of Selaginella lepidophylla.
TECHNICAL FIELD The present invention relates to the use of recombinant DNA techniques for the genetic modification of the carbohydrate metabolism of organisms, the DNA necessary for this, the enzyme (s) involved in the synthesis of specific carbohydrates, as well as the like the modified organisms and their parts. Said organisms or their parts can be used for the production of said specific carbohydrates, or, they can be processed as foods or ingredients, having properties improved by the presence of said carbohydrates.
BACKGROUND The accumulation of solutes compatible with metabolism in various organisms such as bacteria, algae, yeast, fungi, insects, crustaceans and plants, emerged in Evolution as an adaptive mechanism to contend with water scarcity, salinity or freezing due to the properties osmoregulatory of said solutes. Among these osmotically active compounds are the polyols sorbitol, mannitol, arabitol or glycerol; amino acids such as proline or the glycine-betaine derivative; and the disaccharides sucrose and trehalose [Yancey, P.H., Clark, M.E., Hand, S.C., Bowlus, R.D. & Somero, G.N. (1982) Science 212: 1214-1222].
In addition to being osmolytes, glycerol, sucrose and trehalose are osmoprotectors since they play a key role in the structural and functional stabilization of membranes and proteins in the anhydrous state. This seems to be due to the fact that the water molecules are replaced by the osmoprotector that in this way solves the macromolecules of the cell [Clegg, J.S. (1985) The physical properties and metabolic status of Artemia cysts at low water contents: The "water replacement hypothesis", pp. 169-187. In: Membranes, metabolis and dry organisms. Leopold, A.C. (Ed.). Cornell Univ. Press, Ithaca, New York]. In particular trehalose has the best biophysical and biochemical properties as an osmoprotector [Crowe, J.H., Crowe, L.M. & Chapman D. (1984) Science 221: 701-703; Crowe, J.H., Crowe, L.M. , Carpenter, J.F. & Wistrom, A. (1987) Biochem. J. 212: 1-10].
Trehalose [O-alpha-D-glucopyranosyl- (1-l) -alpha-D-glucopyranose] is a dimer of two glucose molecules linked through their reducing groups. These reducing groups are absent in trehalose, so it does not take part in the reactions of Maillard, in which the reducing group of sugars reacts with the amino group of proteins causing darkening and change in the smell and taste of food [Nursten, H.E. (1986) Maillard browning reactions in dried foods, pp. 53-68. In: Concentration and drying of foods. Macarthy, D. (Ed.). Elsevier Applied Science, London].
Studies with liposomes using calorimetry, infrared spectroscopy, nuclear magnetic resonance and X-ray diffraction, demonstrate that trehalose is able to keep lipids in fluid phase in the absence of water and as a consequence avoids phase separation, rupture and disintegration of the membranes [Crowe, JH, Crowe, LM & Jackson, S.A. (1983) Arch. Biochem. Biophys. 220: 477-484; Crowe, J.H., Crowe, L.M. & Chapman D. (1984) Science 221: 701-703; Crowe, L.M., omersley, C, Crowe, J.H., Reid, D., Appel, L., & Rudolph, A. (1986) Biochem. Biophys.
Acta 861: 131-140; Crowe, J.H., Crowe, L.M., Carpenter, J.F. & istrom, A. (1987) Biochem. J. 2A2 .: 1-10; Lee, C.W.B., augh, J.S. & Griffin, R.G. (1986) Biochemistry 21: 3724-3737; Lis, L.J., Ta ura-Lis, W., Lim, T.K. & Quinn, P.J. (1990) Biochim. Biophys. Act 1021: 201-204]. It has been proposed that this is due to the interaction of the hydroxyl groups of trehalose with the phosphate groups of the phospholipids through hydrogen bonds and also to the trehalose-trehalose interaction that encapsulates the membranes avoiding the exit of other molecules present inside.
It has been found that trehalose is able to preserve the activity of enzymes maintained under total dehydration. For example, if the T7 DNA polymerase, T4 DNA ligase or various restriction enzymes are dehydrated in the presence of trehalose and remain in this state for several months, even at 37, 55 or 70aC, at the end of this time they recover 100% of their activity when rehydrated [Cola? o, C, Sen, S., Thangavelu, M., Pinder, S. & Roser, B. (1992) Bio / Technology l: 1007-1111].
On the other hand, carrot or tobacco cells can be cryopreserved in the presence of trehalose [Bhandal, I.S., Hauptman, R.M. & Widhol, J.M. (1985) Plant Physiol. 11: 430-432]. It has been reported that trehalose concentrations of less than 2mM are much more efficient than sucrose for cryopreservation of spinach-isolated thylakoids [Hincha, D.K. (1989) Biochem. Biophys. Acta 9 ^ 7: 231-234].
Among the organisms that synthesize trehalose are bacteria, yeasts, some fungi, spores, nematodes, larvae of crustaceans, insects and plants, particularly some of resurrection such as Myrothamnus flabellifolius and Selaginella lepidophylla [Adams, R.P., Kendall, E. & Kartha, K.K. (1990) Biochem. Systematics & Ecology 18_: 107-110; Bianchi, G., Gamba, A., Murelli, C, Salamini, F. & Bartels, D. (1993) Physiol. Plant. £ 7: 223-226; Weisburd, S. (1988) Sci. News 211: 107-110].
This last species produces at least three times more trehalose than M. flabellifolius [Müller, J., Boller, T. & Wie ken, A. (1995) Plant Science 112: 1-9]. In S. lepidophylla trehalose constitutes 80% of soluble carbohydrates, so this plant is one of the organisms with the highest content of trehalose. This plant can be cryopreserved in liquid nitrogen and, when thawed, it revives normally, as it also survives extreme vacuum, heat up to 100 ° C or high doses of ionizing radiation without suffering damage [Roser, B. (1991) Trends Food Sci. Technol. 2: 166-169; Roser, B. & Cola? O, C. (1993) New Scient. 111: 25-28; Weisburd, S. (1988) Sci. News 112: 107-110].
The biosynthesis of trehalose in Escherichia coli and Saccharomyces cerevisiae consists of two enzymatic steps catalyzed by trehalose-6-phosphate synthase, which produces trehalose-6-phosphate from glucose-6-phosphate and UDP-glucose, and by trehalose -6-phosphate phosphatase, which finally produces trehalose [Bell,. , Klaassen, P., Ohnacker, M., Boller, T., Herweijer, M., Schoppink, P., Van Der Zee & Weimken, A. (1992) Eur. J. Biochem. 2H3.? 951-959; Cabib, E. & Leloir, L.F. (1957) J. Biol. Chem. 211: 259-275; Giaver, H.M., Styrvold, O.B., Kaasen, I. & Strom, A.R. (1988) J. Bacteriol. 170: 2841-2849; Londesborough, J. & Vuorio, O. (1991) J. Gen. Microbiol. 137: 323-330]. The genes of both enzymes have already been isolated and sequenced, both bacteria and yeast [Bell, W., Klaassen, P., Ohnacker, M., Boller, T., Herweijer, M., Schoppink, P., Van Der Zee & Weimken, A. (1992) Eur. J. Biochem. 209: 951-959; From Virgilio, C, Bürckert, N., Bell, W., Jenó, P., A.R. (1993) FEMS Microbiol. Let. 1DJ7: 25-30].
The deletion of the otsA or otsB genes of E. coli, which code for trehalose-6-phosphate synthase and phosphatase, respectively, causes loss of osmotolerance and thermotolerance. In addition, the transcription of the otsA and otsB genes is induced under osmotic and caloric stress [Kaasen, I., Falkenberg, P., Styrvold, O.B. & Strom, A.R. (1992) J. Bacteriol. 124 .: 889-898; Hengge-Aronis, R., Klein, W., Lange, R., Rimmele, M. & Boos, W. (1991) J. Bacteriol. 121: 7918-7924].
In yeast S. cerevisiae, the oligomer of trehalose-6-phosphate synthase / phosphatase consists of three subunits, TPS1 (56 kD), TPS2 (100 kD) and TPS3 (130 kD), which correspond to trehalose-6 -phosphate synthase, trehalose-6-phosphate phosphatase and a polypeptide of possible regulatory function, respectively [Bell, W., Klaassen, P., Ohnacker, M., Boller, T., Herweijer, M., Schoppink, P., Van Der Zee & Weimken, A. (1992) Eur. J. Biochem. 209: 951-959; From Virgilio, C, Bürckert, N., Bell, W., Jenó, P., Boller, T. & Weimken A. (1993) Eur. J. Biochem. 212: 315-323; Thevelein, J.M. & Hohmann, S. (1995) Trends Biochem. Sci. 21: 3-10; Vuorio, O.E., Kalkkinen, N. & Londesborough, J. (1993) Eur. J. Biochem. 211: 849-861].
The activity of the holoenzyme trehalose-6-phosphate synthase / yeast phosphatase has a complex regulation and still little understood. First, the TPS1 subunit appears to be activated by dephosphorylation [Panek, A.C., de Araujo, P.S., Neto, M.V. & Panek, A.D. (1987) Curr. Genet H: 459-465]. On the other hand, it has been suggested that the inability of the yeast to grow in glucose when the TPS1 gene is inactivated by mutation, is due to the role played by the TPS1 subunit in regulating the entry of glucose into the cell [Thevelein, J.M. & Hohmann, & (1995) Trends Biochem. Sci. 21: 3-10]. This double function for TPS1, synthase and glucose sensor, raises a mechanism of metabolic regulation where a cyclic AMP-dependent kinase seems to be involved as a signal for the deactivation of TPS1 [Panek, A.C., de Araujo, P.S., Neto, M.V. & Panek, A.D. (1987) Curr. Genet 11: 459-465; Thevelein, J.M. & Hohmann, S. (1995) Trends Biochem. Sci. 21: 3-10].
There is clear genetic and biochemical evidence in the yeast of the role of trehalose in heat tolerance [De Virgilio, C, Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1994) Eur. J. Biochem. 211: 179-186; Hottiger, T., De Virgilio, C, Hall, M.N., Boller, T. & Weimken, A. (1994) Eur. J. Biochem. 219: 187-193], freezing [Oda, Y., Uno, K. & Ohta, S. (1986) App. & Environ. Microbiol. 2: 941-943] and dehydration stress [Gadd, G.M., Chalmers, K. & Reed R.H. (1987) FEMS Microbiol. Lett. A: 249-254].
The synthesis of trehalose in transgenic tobacco has been reported using the otsA and otsB genes of E. coli under the control of the 35S promoter. However, trehalose levels were 0.012% of fresh weight, which turns out to be insufficient for scaling and industrialization purposes [Goddijn, OJM, Verwoerd, TC, Voogd, E., Krutwagen, R., de Graaf, P. , van Dun, K., de Laat, A. & van den Elzen, P. (1995) Plant Physiol. 108 Supplement: 149]. Recently, the production of transgenic drought tolerant tobacco plants was reported using the yeast TPSl gene [Holmstrom, K.-O., Mntyla, E., Welin, B., Mandal, A., Palva, ET, Tunnela, OE & Londesborough, J. (1996) Nature 379: 683-684]. The synthesis of trehalose in these plants constitutes 0.08 to 0.32% dry weight, which means a low accumulation in trehalose, in order to scale the production of this disaccharide in transgenic plants.
The overexpression of heterologous genes in a given organism must take into account that the use of codons is different for distant species from the phylogenetic point of view [Fox, T.D. (1987) Ann. Rev. Genet. 21: 67-91]. As far as the expression in plant cells of yeast or bacterial genes, it could not be high due to the inability of the cell to efficiently translate these transcripts. The expression of the genes TPS1 and TPS2 of yeast, or of the genes otsA and otsB of E. coli in plant cells, could be limited. To solve this problem it has been suggested to modify the codons of TPS1 and TPS2 by site-directed mutagenesis [Londesborough, J. & Vuorio, 0., Helsinki Finland, 05422254 (Jun-06-1995)].
Another strategy to mass produce trehalose in plants would be using genes from the plants themselves. Isolation of the trehalose-6-phosphate synthase / phosphatase cDNA from the Selaginella lepidophylla resurrection plant, which produces trehalose at the highest levels reported to date for an organism (10% of dry weight vs. yeast producing 15% of dry weight) [Müller, J., Boller, T. &; Wiemken, A. (1995) Plant Science 112: 1-9], will obviate the problem cited above, when said gene is overexpressed in plants. This is due to four reasons. First, the sl-tps / p gene encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase is constitutively transcribed and correlated with the high levels of trehalose reported in S. lepidophylla, both hydrated and dry. Due to the above, it does not seem to have a mechanism of post-translational or metabolic regulation that limits its enzymatic activity, contrary to the TPS1 of yeast. Second, since sl -tps / p is a single copy gene in S. lepidophylla, the synthesis of trehalose in this plant is exclusively due to the enzyme encoded (SL-TPS / P) by said gene. So it is expected that this single gene, sl -tps / p, is capable of producing high levels of trehalose when expressed in transgenic organisms. Third, the use of sl-tps / p codons is closer to that of other plants, in contrast to E. coli or yeast. This will result in the production of considerably higher levels of trehalose in plants that overexpress the sl-tps / p gene. Finally, since the product of the sl -tps / p gene of S. lepidophylla is a bifunctional enzyme, it contains the catalytic activities of synthase and phosphatase, it is technically easier to introduce a single gene than several.
The potential use of trehalose encompasses the agri-food and pharmaceutical industry. On the one hand, the synthesis of trehalose in transgenic plants will allow them to survive conditions that limit water, excess heat, cold or salinity. This will result in an increase in the productivity of rainfed agriculture and in arid or semi-arid zones. Moreover, given the foreseeable shortage of water worldwide for the coming decades, irrigation agriculture will also be limited. Under these conditions, the decrease in the use of water would not affect the yield and productivity of the crops that synthesize trehalose. In addition, the plants or parts of them that synthesize trehalose, may be preserved in a dehydrated state for long periods without the need for refrigeration, and preserving the organoleptic properties that the consumer demands. This unprecedented increase in the shelf life of agricultural products will have a strong impact on its market as it will reduce transport and storage costs.
Secondly, the synthesis of high levels of trehalose in plants or other transformed organisms represents potentially an inexpensive source of trehalose for the industry compared to the current price of obtaining yeast trehalose equivalent to two hundred US dollars per kilogram [Kidd, G. & Devorak, J. (1994) Bio / Technol. 12: 1328-1329]. Trehalose is an additive that has the unique property of preserving food in a dehydrated state and preserving its flavor, smell and consistency properties when rehydrated. Due to the above, foods processed by incorporating trehalose into their preparation will come to be considered fresh when rehydrated. This also implies that all kinds of preservatives and preservers that are still used may be dispensed with, but that the consumer rejects them because of the harmful, possible or proven effect they represent for their health. Moreover, the fact that trehalose preserves active biomolecules implies that dehydrated foods containing it will have a nutritional value, comparable to fresh products, and higher than those dehydrated foods that lack trehalose. In addition, it should be noted that the process of dehydration in the presence of trehalose, this is air drying, represents the cheapest method on the market to dehydrate food, its derivatives or biomolecules [Roser, B. (1991) Trends Food Sci. & Tech. 2: 166-169].
The enzyme trehalase, which degrades trehalose in two glucose molecules, is found in the digestive tract of mammals and humans. Since a range of foods that are consumed daily, such as honey, mushrooms, bread, beer, wine and vinegar contain trehalose [Roser, B. & Cola? O, C. (1993) New Scient. 138: 25-28], the consumption of this disaccharide in other foods does not present a health risk [Gudand-Hoyer, E. (1994) Am. J. Clin. Nutr. 5) (suppl.): 735S-741S]. In fact, the use of trehalose as an additive to preserve food has already been approved in England [Roser, B. (1991) Eur. Biotechnol. News 111: 2], so its approval in Mexico, USA or other countries should not be a problem.
Finally, the availability of massive amounts of trehalose will allow its use as a preservative of enzymes, vaccines, hormones and other proteins and drugs in a dehydrated state without losing bioactivity.
SUMMARY OF THE INVENTION The invention described herein consists of the isolation, cloning and overexpression in transgenic organisms of a cDNA of a gene of plant origin that codes for the activity of trehalose-6-phosphate synthase / phosphatase, particularly the sl-tps / p gene encoding for the bifunctional enzyme trehalose-6-phosphate synthase / phosphatase (SL-TPS / P) of the resurrection plant Selaginella lepidophylla. Said enzyme allows high levels of trehalose to be obtained in transgenic plants.
In addition, the plants are tolerant to heat, cold, salinity and water scarcity. Subcloning said cDNA into appropriate expression vectors to transform other organisms such as bacteria, yeast and animals, will allow the synthesis of trehalose in said organisms conferring tolerance to heat, cold, salinity and water shortage. On the other hand, the sl-tps / p gene can also be used to synthesize massive quantities of trehalose in microorganisms, isolated cells and complete organisms of plants or animals, which have been transformed with said gene.
FIGURES Figure 1. Structure of the enzyme SL-TPS / P of S. lepidophylla. The scheme shows the scale size (in kD) of SL-TPS / P and its structure based on the similarity in the amino acid sequence with respect to the TPS1 and the TPS2 of yeast. The diagonal stripes and the grid denote similar regions Figure 2. Restriction map of the sl-tps / p gene cDNA. The restriction sites on this map were confirmed with the nucleotide sequence. In the upper part of the diagram are shown the cleavage sites for the restriction enzymes: Eco Rl (R), Eco RV (E), Hind III (H), Sac I (Se), Salt I (S). There are no sites for the enzymes: Bam HI, Kpn I, Pst I, Sma I, Xba I. The length of the cDNA in kb is indicated in the lower part of the diagram. The 5 'end of the cDNA is located on the left side of the scheme.
Figure 3. Schematic representation of plasmid pIBT6. The abbreviations mean: AmpR, a gene that confers resistance to ampicillin; lac Z, promoter of the β-Galactosidase gene; slps / p, trehalose-6-phosphate synthase / phosphatase cDNA; T3 and T7 are the promoters of phages T3 and T7 for in vitro transcription. Plasmid pIBT6 has an approximate size of 6150 bp.
The arrows indicate the direction of transcription.
Figure 4. Schematic representation of the pBN35 expression vector. The abbreviations mean: RB and LB, right and left borders, respectively, of the T-DNA of the Ti plasmid of Agrobacteri m tumefasciens; KanR, gene that confers resistance to Kanamycin for selection in E. coli; pNOS, promoter of the nopaline synthase gene; NPT II, gene that codes for neomycin phosphotransferase and allows to select plant cells resistant to kanamycin; pA, poly-A signal; 35S, 35S promoter of cauliflower virus; SMC, multiple cloning site. Plasmid pBN35 has an approximate size of 12 kb. The arrows indicate the direction of transcription.
Figure 5. Schematic representation of plasmid pIBT36. The abbreviations mean: RB and LB, right and left borders, respectively, of the T-DNA of the Ti plasmid of Agrobacterium tumefasciens; KanR, gene that confers resistance to Kanamycin for selection in E. coli; pNOS, promoter of the nopaline synthase gene; NPT II, gene that codes for neomycin phosphotransferase and allows to select plant cells resistant to kanamycin; pA, poly-A signal; 35S, 35S promoter of cauliflower virus; sl-tps / p, cDNA of trehalose-6-phosphate synthase / phosphatase. Plasmid pIBT36 has an approximate size of 15.2 kb. The arrows indicate the direction of transcription.
DETAILED DESCRIPTION In the following description, the cDNA encoding the bifunctional enzyme trehalose-6-phosphate synthase / phosphatase is designated as sl -tps / p; SL-TPS / P is the enzyme trehalose-6-phosphate synthase / phosphatase; sl-tps is the sequence that codes for trehalose-6-phosphate synthase; and SL-TPS is the polypeptide with trehalose-6-phosphate synthase activity.
To isolate the clone sl-tps / p, a comparison was made of the amino acid sequences of trehalose-6-phosphate synthase, deduced from the reported nucleotide sequences. The sequences used come from E. coli, EC-otsA [Kaasen, I., McDougall, J., Strom, A.R. (1994) Gene 145 .: 9-15]; Schizosaccharo yces pombe, SP-TPS1 [Blazquez, M.A., Stucka, R., Feldman, H. & Gancedo, C. (1994) J. Bacteriol. 126 .: 3895-3902]; Aspergillus niger, AN-TPS1 [Wolschek, M.F. & Kubicek, C.P. (1994) NCBI: Seq. ID 551471; not published]; Saccharomyces cerevisiae, SC-TPS1 [McDougall, J., Kaasen, I. & Strom, A.R. (1993) FEMS Microbiol. Let. 107: 25-30]; Kluyveromyces lactis, KL-GGS1 [Luyten, K., Koning W., Tesseur, I., Ruiz, MC, Ramos, J., Cobbaert, P., Thevelein, JM, Hohmann, S. (1993) Eur. J Biochem. 217: 701-713]. Based on this comparison, highly conserved regions were selected to synthesize degenerate oligonucleotides, with which the cDNA library of Selaginella lepidophylla was screened. The oligonucleotides synthesized were TPS5 '-1 (YTNTGGCCNBCNTTYCAYTAY), TPS5 '-2 (GGNTKBTTYYTNCAYAYNCCNTTYCC), TPS5 '-3 (MGNYTNGAYTAYWBNAARGGNB-TNCC), TPS3' -1 (SWNACNARRTTCATNCCRTCNCK) and TPS3 '-2 (CCRWANTKNCCRTTDATNCKNCC).
From 4x105 recombinant plated bacteriophages of a S. lepidophylla cDNA expression bank, 13 plates were obtained that hybridized with the oligonucleotides TPS5'-1, TPS5'-3 and TPS3'-2. These 13 bacteriophage isolates were plated again at different dilutions to obtain separate plates. Only in 6 cases were plates obtained that hybridized with the oligonucleotides TPS5'-2 and TPS3'-1, in addition to rehybridizing with the mixture of TPS5'-1, TPS5'-3 and TPS3'-2. A single plate of each of these 6 different isolates was used to cleave the plasmid pBluescript SK (-) from the bacteriophage. From the colonies that grew in ampicillin, plasmid DNA was extracted and cut with the enzymes Eco Rl and Xho I to isolate the corresponding insert. After carrying out a restriction mapping with various enzymes and by means of Southern hybridization, DNA fragments were identified that hybridized with the oligonucleotides in these six clones. Next, the clone that contained the largest insert (Figure 2) was selected to create deletions and determine its nucleotide sequence. This clone, pIBT6 (Figure 3), was the only one that hybridized with the oligonucleotide TPS5'-1, which corresponds to the amino-terminal end of the region of homology selected to isolate the cDNA.
The complete sequence of the insert of clone pIBT6 is 3223 bp and is called sl-tps / p (SEQ ID NO: 1). It contains an open reading frame of 2985 bp (including the stop codon) that codes for a polypeptide termed SL-TPS / P (SEQ ID NO: 2) of 994 amino acids with a molecular weight of 109.1 kD. The sequence includes 32 nuclei from the poly-A tail. In addition, sl-tps / p contains 5 'and 3' untranslated regions of 110 bp and 96 bp, respectively. All of the above confirms that sl-tps / p is a complete clone of cDNA. The context of the initiation codon in SL-TPS / P corresponds to that reported in plants [Lütcke, H.A., Chow, K.C., Mickel, F.S., Moss, K.A. , Kern, H.F. & Scheele, G.A. (1987) EMBO J. 1: 43-48].
The amino-terminal region of 600 amino acids of SL-TPS / P has an identity of 53% and similarity of 70% with the TPS1 of the yeast Schizosaccharomyces po be, which consists of 570 amino acids. These 600 amino acids constitute per se a trehalose-6-phosphate synthase which is referred to herein as SL-TPS (SEQ ID NO: 4) and sl-tps (SEQ ID NO: 3) to the region of the cDNA encoding it. On the other hand, the alignment of the deduced amino acid sequence of SL-TPS / P with the sequence of TPS2 from S. cerevisiae [De Virgilio, C, Bürckert, N., Bell, W., Jenó, P., Boller , T. & Weimken A. (1993) Eur. J. Biochem. 212: 315-323], also revealed a homology consisting of 29% identity and 52% similarity.
It should be noted that TPS1 and TPS2 of yeast share 33% identity with each other. The similarity between SL-TPS / P and TPS1 or between the first and TPS2, corresponds to regions of amino acids highly conserved between the different trehalose-6-phosphate synthases or phosphatases of yeast, fungi and bacteria (Figure 1). The percentage identity between SL-TPS / P and TPS1 or TPS2 is within the expected range, given the phylogenetic relationship between plants and fungi, for genes that code for polypeptides with the same function. For example, while the deduced sequence of the hsplOl gene from Arabidopsis thaliana when compared to that of the yeast hsplO homolog showed only 43% identity, the plant gene is able to complement the yeast mutant in that gene [Schirmer , EC, Lindquist, S. & Vierling, E. (1994) Plant Cell j =: 1899-1909].
It has been established that the sequence similarity deduced in different proteins, even from non-phylogenetically related species, is evidence of similar structure and related or identical function [Doolittle, R.F. (1995) Annu. Rev. Biochem. : 287-314]. An important criterion for determining a secondary structure and similar function between two protein sequences is the hydrophobicity pattern [Kyte, J. &; Doolittle, R.F. (1982) J. Mol. Biol. 157: 105-132]. The SL-TPS / P enzyme from S. lepidophylla and the TPSl from yeast have a nearly identical hydrophobicity pattern. For all the above mentioned it is concluded that the function of SL-TPS / P is that of a bifunctional enzyme with catalytic activities of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.
The information available to date on the function of trehalose suggests that this disaccharide plays an active role in cell protection during the drought in Selaginella. To corroborate that the sl-tps / p cDNA corresponds to an active gene, the expression pattern of said gene was determined using Northern gels, evaluating the specific transcript levels under normal conditions and water deficit. To analyze the expression of the sl-tps / p gene in a Northern gel, in each lane of the gel 2 μg of polyA * RNA extracted from micelles of S. lepidophylla hydrated, dehydrated for 2.5 hours, 5 hours and one year. As a probe, the sl-tps / p cDNA radioactively labeled with 3aP was used. In the autoradiogram, a 3.2 kb band can be observed, present both in the hydrated and dehydrated microphones at different intervals. This result demonstrates a constitutive expression of the sl-tps / p gene, which is in agreement with the observation made previously by other authors [Adams, R.P., Kendall, E. & Kartha, K.K. (1990) Biochem. Systematics & Ecology IB: 107-110] where the presence of trehalose was found at relatively comparable levels, both in hydrated and dehydrated plants. Unlike E. coli and yeast, in S. lepidophylla the expression of trehalose-6-phosphate synthase / phosphatase is not induced by the effect of osmotic or caloric stress but remains constant regardless of the water status of the plant. Therefore, the correlation in the constitutive expression of the sl-tps / p gene with the presence of trehalose in Selaginella suggests that the enzyme is not modified in its activity, in contrast to what happens in yeast where the activity of the enzyme is subject to regulation by phosphorylation and substrate [Panek, AC, de Araujo, PS, Neto, MV & Panek, A.D. (1987) Curr. Genet 11: 459-465; Thevelein, J.M. & Hohmann, S. (1995) Trends Biochem. Sci. 21: 3-10].
To determine the copy number of the sl-tps / p gene in the genome of S. lepidophylla, a genomic Southern was performed. In the gel, 20 μg of S. lepidophylla genomic DNA cut with the following restriction enzymes were loaded in each lane: Eco Rl, Xba I or Bam HI. As a probe, the sl-tps cDNA radioactively labeled with "P" was used. In the autoradiogram of this experiment, two bands were observed, one of 1.6 and another 6 kb when cutting the DNA with the Eco Rl enzyme, a band of 9 kb at the cut with Xba I, and two bands of 11 and 12 kb when digesting DNA with Bam HI This experiment suggests that the sl-tps / p gene is found as a single copy in the genome of S. lepidophylla. the sl-tps / p gene of this plant seems to be solely responsible for the synthesis of trehalose, which corresponds to approximately 80% of the soluble carbohydrates in S. lepidophylla.
To obtain plants that synthesize trehalose, the sl-tps / p s cDNA was first subcloned into the pBN35 expression vector (Figure 4), to yield the plasmid pIBT36 (Figure 5). The vector pBN35 allows to express any gene under the control of the 35S promoter of the CaMV virus of the cauliflower [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 21: 285-294] which is a strong and constitutive promoter. The plasmid pIBT36 was used to obtain tobacco plants, transformed by means of the Agrobacterium system, which upon regeneration were able to produce trehalose. This construction can be expressed in any plant that can be transformed, using the Agrobacterium system or by any other method that is known in the state of the art. From 31 transgenic plants obtained, the expression of sl -tps / p cDNA in 16 plants was detected, using Northern gels. In 10 of these plants the presence of trehalose was detected at high levels, which correlated with the activity of trehalose-6-phosphate synthase. The presence of trehalose or enzyme activity was not detected in plants not transformed or transformed only with the pBN35 vector.
Trehalose as an additive in foods and for the conservation of biomolecules and other substances of medical interest, may be obtained at a more attractive price through its mass production in transgenic organisms, such as yeasts and transgenic plants. To achieve this, it is necessary to obtain high levels of trehalose in the plant tissue, in order to reduce the costs of extraction and purification. The present invention gives rise to a strategy for the mass production of trehalose in plants which includes the transformation with sl-tps / po sl-tps cDNA (in suitable constructions) of tobacco plants, which besides being one of the Plant models more worked in molecular biology has a large leaf area that lends itself to the production of large volumes of trehalose, and potato plants, which is the crop with the highest yield of biomass per hectare, which at the same time, being the a product of natural consumption of man, would lead to trehalose with greater acceptance by both the public and by regulatory bodies. It is obvious to a person skilled in the art that the sl-tps / po cDNA of the sl-tps cDNA fragment of the present invention is not restricted to tobacco and potato plants, but can be used in the transformation of any another plant.
As already mentioned, there are serious metabolic type limitations for the production of high levels of trehalose in yeast. The present invention provides a way to obviate these problems, by using the sl-tps / po cDNA of JSI-tps in suitable constructions involving Yip or multicopy integrative vectors, taking advantage of that as already stated above, that the SL-TPS / P does not seem to have a mechanism of post-translational or metabolic regulation that limits its enzymatic activity and also that it is able to direct the production of high levels of trehalose. This will not only allow the massive production of trehalose, but also improve the yields of the yeasts in the baking and in the alcoholic fermentations of beer and wine.
The cereals that are the basis of the world diet such as rice, wheat and corn, legumes as well as other plants such as tobacco, which is used preferably in the description of the present, could be grown under unfavorable climatic conditions, through the use of the corresponding transgenic plants, capable of producing trehalose in response to cold, heat, salinity or drought, or in a constitutive way and thereby resist the adverse environmental conditions. The present invention provides a means to achieve such resistance, through the use of sl-tps / p or sl-tps cDNA in constructions containing either constitutive promoters or promoters that induce their expression only under stress. The harvested products, in addition to having been cultivated under conditions that normally would not have been achieved, may be stored for longer periods or under extreme conditions, unlike the products obtained with non-transformed plants.
The properties of resistance to stress by cold, heat and drought that the trehalose confers to the tissues that contain it, will allow, by using the cDNA of sl-tps / po of sl-tps in the transformation with vectors that include constitutive promoters , tissue-specific or organ-specific, organs or tissues are produced for reproduction, such as pollen, tubers, seeds and flowers that can be conserved for longer periods with greater viability, which would be a great help for plant breeding programs and conservation of germplasm.
MATERIAL AND METHODS OF DESCRIPTION Material The reagents used were Baker or Sigma analytical grade. The restriction and modification enzymes were Boehringer-Mannheim brand. The ZAP cDNA synthesis kit, the Uni-ZAP XR vector and the Gigapack II Gold packaging extracts were from Stratagene Cloning Systems (USA). The Sequenase Version 2.0 kit to determine the nucleotide sequence was from United States Biochemical Corporation (USA).
The resurrection plant Selaginella lepidophylla (Hook. &Grev.) Spring. was collected dehydrated in rocky soils of arid zones in the States of Morelos and Oáxaca de la República Mexican Later it was cultivated in controlled conditions (24BC and 16 h of light with 50% average humidity) in Conviron growth chambers or in the greenhouse. The plants were irrigated every third day with a volume of water of 20 ml for 2 L pots. To water stress S. lepidophylla, the whole plant or microfilar fronds were air dried by placing them on Whatman 3MM filter paper. From this moment, the dehydration time was determined.
Strains The cDNA library was plated in the E. coli XLl-Blue MRF1 strain and the SOLR strain was used to cleave the pBluescript from lambda phage as described in the manual of the "ZAP-cDNA Synthesis Kit" [Stratagene Cloning Systems , Calif., USA; # catalog 200400, 200401 and 2004029]. The strain of E. coli DH5 alpha was used to subclone and make constructions. The strain of A. tumefasciens LBA4404 was used to transform tobacco and the strain of E. coli HB101, carrying the plasmid pRK2013 [Bevan, M. (1984) Nucí Acids Res. 22: 8711-8721], was used to mobilize plasmid pIBT36 from E. coli to A. tumefasciens by means of triparental conjugation as described above [Bevan, M. (1984) Nucí. Acids Res. 22: 8711-8721].
DNA manipulation Recombinant DNA techniques such as bacterial transformation, isolation of plasmid DNA and bacteriophage lambda were carried out according to standard procedures [Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning: A laboratory manual. Second Edition. Cold Spring Harbor Laboratory Press, New York]. The labeling of radioactive fragments was carried out by the "random-priming" technique with oligonucleotides [Feinberg, A.P. & Vogelstein, B. (1983) Anal. Biochem. 112: 6-13]. buildings The expression vector pBN35 is a derivative of pBinl9 [Bevan, M. (1984) Nucí. Acids Res. 22: 8711-8721], which was constructed by subcloning 850 bp of CaMV 35S promoter from cauliflower [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 21: 285-294] between the Hind III sites and Sal I of pBinl9, and the 260 bp fragment constituting the poly-adenylation signal of the nopaline synthetase gene of T-DNA [Bevan, M., Barnes, W. & Chilton, M.-D. (1983) Nucí. Acids Res. 11: 369-385], in the Sac I and Eco Rl sites of the same vector (Figure 4).
Plasmid pIBT36 (FIG. 5) was constructed by subcloning the sl-tps / p cDNA at the Bam HI and Kpn I sites of the? BN35 expression vector.
Construction of the cDNA library of S. lepidophylla To isolate the cDNA clones, an expression bank was prepared from mRNA isolated from dehydrated S. lepidophylla microphylls for 2.5 h. , using the ZAP cDNA synthesis kit, the Uni-ZAP XR vector and the Gigapack II Gold packaging extracts, following step by step the laboratory manual "ZAP-cDNA Synthesis Kit" provided by the manufacturer [Stratagene Cloning Systems, Calif. ., USA; # catalog 200400, 200401 and 2004029]. The polyA * RNA was extracted from the microphylls of S. lepidophylla, dehydrated for 2.5 h. , according to a known method [Chomczyniski, P. & Sacchi, N. (1987) Anal. Biochem. 1 £ 2: 156-159]. The initial titre of the bank was 2 x 106 bacteriophage / ml plates and after amplification it was 1.5 x 1011 bacteriophage plates / ml.
The pBluescript SK (-) plasmid was excised from the bacteriophage by means of the technique known as "zapping" according to the "ZAP-cDNA Synthesis Kit" Laboratory Manual [Stratagene Cloning Systems, Calif., USA; # catalog 200400, 200401 and 2004029].
DNA sequencing From the selected clone, we proceeded to create consecutive deletions of the insert with the enzymes Exo III and Nuclease Sl [Henikoff, S. (1984) Gene 21: 351-359], to later determine its nucleotide sequence by the method of termination of the chain with dideoxynucleotides [Sanger, F., Nicklen, S. & Coulson, A.R. (1977). Proc. Nati Acad. Sci. USA 24: 5463-5467].
To analyze the DNA sequence, the software package of the University of Wisconsin Genetics Computer Group (UWGCG) [Devereux, J. Haeberli, P. & Smithies, O. (1984) Nucí.
Acids Res. 12: 387-395]. The hydrophobicity plots were obtained by a known program [Kyte, J. & Doolittle, R.F. (1982) J. Mol. Biol. 152: 105-132] and protein sequence alignments with the BESTFIT program of the UWGCG package.
Nucleic acid hybridization For screening the bank, the bacteriophage plates were transferred to a Hybond N * nylon membrane (Amersham Life Sciences), which was treated according to the conventional method to denature the DNA [Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning: A laboratory manual.
Second Edition. Cold Spring Harbor Laboratory Press, New York]. The filter was hybridized with the oligonucleotides, labeled with the 32P isotope by means of the polynucleotide kinase, using 6 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate) a 3 OC. Three washes of the filter were carried out, for 20 minutes each at the same temperature, under the following conditions: 6 x SSC; 4 x SSC; and 2 x SSC.
Southern and Northern blot and gel techniques were performed according to standard protocols [Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning: A laboratory manual. Second Edition. Cold Spring Harbor Laboratory Press, New York], with the following modifications. For him Southern genomic DNA was fractionated on an agarose gel at 0.8 % in TBE buffer and transferred to a nylon Hybond N * membrane (Amersham Life Sciences). The filter was hybridized using the sl-tps / p cDNA labeled with the "P" isotope as a probe, using 2 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate) at 65 ° C. filter washes, for 20 min each at the same temperature, under the following conditions: 2 x SSC, 1 x SSC, and 0.5 x SSC In the Northern a 1.2% agarose gel in MOPS-formaldehyde buffer was used and for the transfer a Hybond N * nylon membrane was also used. Hybridization conditions were in 50% formamide and 2 x SSC at 42 ° C. The three successive washes of the filter were made with 2 x SSC, 2 x SSC and 1 x SSC, respectively at 55 ° C.
Tobacco transformation The transformation of tobacco. { Nicotiana tabacum var. SR1) was carried out by the sheet disc method [Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., Fraley, R.T. (1985) Science 227: 1229-1231], using Agrobacterium tumefasciens LBA4404 containing the plasmid pIBT36. The leaf disks were cultivated in Petri dishes containing MS medium with vitamins [Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15: 473-497], hormones (0.1 ppm of NAA and 1 ppm of BAP) and antibiotics (lOOμg / ml of kanamycin and 200 μg / ml of carbenicillin) to regenerate shoots in 4 to 6 weeks. Subsequently, the shoots were transferred to Magenta bottles containing MS medium (100 μg / ml kanamycin and 200 μg / ml carbenicillin) without hormones or vitamins, for root formation in 2 to 3 more weeks. The regenerated plants were transferred to pots with soil and were grown in cultivation chambers (at 24 ° C with 16 hours of light) to obtain fertile plants after 4 to 6 weeks.
Determination of trehalose Trehalose was determined by the degradative method with trehalase [Araujo, P.S., Panek, A.C., Ferreira, R. & Panek, A.D. (1989) Anal. Biochem. 176: 432-436]. To obtain soluble sugars, 500 mg of fresh tissue or 50 mg of dry tissue (frozen in liquid nitrogen) were milled in 0.5 ml of 100 mM PBS buffer at pH 7.0, in a homogenizer for microcentrifuge tubes. 4 volumes of absolute ethanol were added and the samples boiled 10 minutes in threaded tubes to avoid evaporation. Subsequently, it was centrifuged in microcentrifuge tubes, 2 minutes at 13,000 rpm to recover the supernatant. It was reextracted again with the same volume of 80% ethanol and the tablet was dried under vacuum. The samples were resuspended in 0.250 ml of 50mM PBS at pH 6.5.
For the determination of trehalose, 10 to 30 μl of extract was taken, 4 μl (c.a. 15 mU) of trehalase was added (Sigma cat no T-8778) and incubated for 2 hrs at 30 ° C. A tube with extract but without trehalase was put as negative control and a tube with pure trehalose (Sigma cat.t. T-3663) was placed as a positive control. The volume was brought to 0.5 ml with 50 mM PBS, pH 7.0 and 0.5 μl of glucose oxidase and peroxidase from the Sigma kit was added. cat. 510-A, for the determination of glucose. It was incubated 40 min at 37 QC and the optical density was determined at 425 nm immediately. To calculate the glucose concentration, a standard glucose curve with values between 0 to 75 mM was used. The values of the tubes without trehalase from those treated with this enzyme were subtracted to calculate the amount of trehalose, taking into account that 1 mole of glucose is 1/2 mole of trehalose.
Determination of enzymatic activity, To determine the activity of trehalose-6-phosphate synthase, a reported method was followed [Londesborough, J. & Vuorio O. (1991) J. Gen. Microbiol. 137: 323-330] which basically consists of a coupled assay measuring the molar extinction of NADH at 340 nm. The reaction is performed in a volume of lOOμl containing 40 mM HEPES / KOH buffer pH 6.8, 10 mM glucose-6-phosphate, 5 mM UDP-glucose, 10 mM MgCl2 and 1 mg / ml bovine serum albumin . The reaction is incubated 10 min at 30 ° C and stopped by boiling the sample for 2 min. After cooling the tube, 900 μl containing 40 mM buffer HEPES / KOH pH 6.8, 10 mM MgCl2, 2. 5 μg / ml phosphoenolpyruvate, 0.24 mM NADH, 3.5 units pyruvate kinase and 5 units lactate dehydrogenase (Sigma Cat. No. P-0294). The disappearance of NADH at 340 nm was measured spectrophotometrically by incubating to the same conditions mentioned above. To determine the specific activity of trehalose-6-phosphate synthase, the protein concentration was measured by means of the Bradford method [Bradford, M.M. (1976) Anal. Biochem. 22: 248-254].
The following examples are to illustrate in detail the present invention and in no way constitute a limitation to apply this invention. The information of the present invention allows to produce trehalose by means of recombinant DNA techniques in cells or organisms transformed with the clone sl-tps / p of cDNA. Transformation methods and vectors suitable for use in microorganisms and plants are known to those familiar with the state of the art.
Example 1. Isolation of sl-tps / p cDNA Dehydrated Selaginella lepidophylla resurrection plants were collected from rocky soils of arid zones in the States of Morelos and Oaxaca of the Mexican Republic. Later they were cultivated in pots from 2L to 24aC with 16 hours of light and an average humidity of 50% in Conviron growth chambers. The plants were watered every third day with 20 ml of water.
To isolate the cDNA clones, an expression bank was prepared, from 5 μg of RNA isolated from 50 g of dehydrated S. lepidophylla microphylls, for 2.5 hr. After synthesizing the cDNA, it was cloned using lμg of the Uni-ZAP XR vector. The bacteriophages were packed in vitro and subsequently screened with a mixture of degenerate oligonucleotides coding for consensus regions in the trehalose-6-phosphate synthase and phosphatase of the reported sequences of E. coli and yeast. One of the isolated clones corresponds to a cDNA. { sl-tps / p) with the complete coding region. The analysis of the deduced amino acid sequence resulted in a 53% identity for trehalose-6-phosphate synthase and 29% identity for trehalose-6-phosphate phosphatase, compared to reported sequences of trehalose-6-phosphate synthase. bacteria and various yeasts. The homology of the protein encoded by sl-tps / p, called SL-TPS / P with trehalose-6-phosphate synthase, maps in the N-terminal region of the former and the homology of SL-TPS / P with trehalose -6-phosphate phosphatase is found throughout the entire sequence (Fig. 1).
Example 2. Construction of transgenic organisms The tobacco cells (Nicotiana tabacum var. SRl) were transformed by the leaf disc method, using Agrobacterium tumefasciens LBA4404 containing the plasmid pIBT36. The leaf discs were cultivated in Petri dishes containing MS medium with vitamins, 0.1 ppm NAA, 1 ppm BAP, lOOμg / ml kanamycin and 200 μg / ml carbenicillin to regenerate shoots in 4 to 6 weeks. The shoots were transferred to MS medium with 100 μg / ml of kanamycin and 200 μg / ml of cabenicillin without hormones or vitamins, for root formation in 2 to 3 more weeks. The regenerated plants were transferred to culture chambers at 24 aC and 16 hours of light to obtain fertile plants after 4 to 6 weeks.
From a total of 31 transgenic plants obtained, the expression of sl-tps / p cDNA was detected in 16 of them, using Northern gels. In 10 of these plants the presence of trehalose was found at high levels, which correlated with the activity of trehalose-6-phosphate synthase. The presence of trehalose or enzyme activity was not detected in untransformed plants or in those transformed only with the pBN35 vector.
Example 3. Improvement in the synthesis of trehalose in yeast There is clear evidence that the presence of glucose causes a decrease in the synthesis of trehalose in yeast [Thevelein, J.M. & Hohmann, S. (1995) Trends Biochem. Sci. 21: 3-10]. This seems to be due to post-translational modifications of the TPS1 and a possible negative regulation of the transcription in the gene of said enzyme [Panek, A.C., de Araujo, P.S., Neto, M.V. & Panek, A.D. (1987) Curr. Genet 11: 459-465]. The above constitutes a serious limitation in the mass production of trehalose in yeast. The following is a method to obtain high levels of trehalose in yeast, without fluctuations due to catabolic repression.
S. cerevisiae strains having auxotrophic markers such as his3, leu2, ura3, etc. are required to allow selection by transformation with the YIp integrative vectors [Orr-Weaver, T.L., Szostak, J.W. & Rothstein, R.S. (1983) Methods in Enzymol. 101: 228-245]. These vectors can be constructed by a person who is familiar with the state of the art. The integrative vector is based on the homologous recombination of the marker, present in the genome of the yeast, with 5 'and 3' fragments of the same gene, which must flank the region of DNA that it is desired to integrate into the genome of the yeast. v First, the cDNA of the sl-tps / p must be subcloned into an E. coli vector, so that the cDNA of the sl-tps / p is under the control of a strong promoter of a yeast gene that is not subject to metabolic regulation [Romans, MA , Scorer, C.A. & Clare, J.J. (1992) Yeast 1: 423-428]. The construction should also contain a transcription termination signal (ter) that is located at the 3 'end of the sl-tps / p cDNA. This sl-tps / p / ter promoter / DNA fusion is subcloned into a YIp vector containing the 5 'and 3' regions of the ura3 gene, so that the fusion is integrated between these, in addition to the marker gene leu2. The resulting construction is transformed into a strain of yeast with genotype ura3 'and Ieu2", to select the appropriate recombinants.The integration of the construction in the genome is also confirmed by Southern hybridization, according to the known restriction pattern of the fragments of DNA involved Once the recombinant strain has been characterized in a selective medium, it can be grown in a conventional medium, since the integration in the chromosome of the construction avoids the strict need for selective medium that is required when using episomal vectors for the expression of exogenous genes in yeast [Stearns, T., Ma, H. &Botstein, D. (1990) Methods in Enzymol, 1 £ 5: 280-291] This particularity in the design of this recombinant strain has an impact economic for the escalation of a biotechnological process since the selective medium is much more expensive than the conventional one.
The innovation described here is that the recombinant S. cerevisiae strain will express the enzyme SL-TPS / P at high levels with the subsequent overproduction of trehalose above what has been achieved so far by those familiar with the state of the art, since the activity of SL-TPS / P is not subject to post-translational regulation. The synthesis of trehalose at high levels in yeast will improve the performance in baking and in the alcoholic fermentations of beer and wine. In addition, the trehalose obtained by this method can be purified for other industrial uses such as food preservative or biomolecules.
Example 4. Massive synthesis of trehalose in transgenic potato and tobacco plants Genetic engineering has made it possible to express almost any gene in a heterologous organism. Transgenic plants can be used as bioreactors to mass produce compounds of commercial interest that are normally only obtained in limited quantities, such as biodegradable plastics, various carbohydrates, pharmaceutical polypeptides and enzymes for industrial use [Goddijn, O.J.M. & Pen, J. (1995) Trends Biotech. H: 379-387]. There are several methods reported to transform higher plants, including crops of greater economic importance [Walden, R. & Wingender, R. (1995) Trends Biotech. H: 324-331]. The transformation of tobacco [Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., Fraley, R.T. (1985) Science 222: 1229-1231] and Pope [Sheerman, S. & Bevan, M.W. (1988) Plant Cell Rep. 2: 13-16] is carried out efficiently using the system of Agrobacterium tumefasciens and this technique can be assembled in the laboratory by people with a mastery in the state of the art. Constructs for expression in sl-tps / p cDNA plants can be made in a vector derived from the Ti plasmid that lacks the tumorigenic genes of the T-DNA and that contains a selection marker for the transformed plants that confer , for example, resistance to kanamycin [Bevan, M. (1984) Nucí. Acids Res. 22: 8711-8721]. In addition, a suitable promoter should be selected, depending on the use that is required to give the transgenic plants. The T-DNA nopaline synthetase gene [Bevan, M., Barnes, W. & Chilton, M.-D. (1983) Nucí. Acids Res. 11: 369-385].
For example, if you plan to overproduce trehalose for industrial use, you can use a plant such as Solanum tuberosum that stores a large amount of carbohydrates in the tuber which is a specialized organ commonly known as a potato. In terms of the usable biomass of a plant, the potato represents one of the most productive crops per unit area [Johnson, V.A. & Lay, C.L. (1974) Agrie. Food Chem. 22: 558-566]. There are strong and tuber-specific promoters, such as the patatin-class 1 gene [Bevan, M., Barker, R., Goldsbrough, A., Jarvis, M., Kavanagh, T. & Iturriaga, G. (1986) Nucí. Acids Res. 14: 4625-4638; Jefferson, R., Goldsbrough, A. & Bevan, M. (1990) Plant Mol. Biol. 14: 995-1006] that could be used to produce large amounts of trehalose. The convenience of using the potato as a system to overproduce trehalose, is that the tuber of this plant is a human food so the trehalose from this organ would be easily accepted by the consumer.
Tobacco as a system to overproduce trehalose, in order to use it as an additive in dried foods, may not have market acceptance for possible contaminants, such as nicotine, that could be obtained by purifying trehalose. Trehalose overproduced in tobacco could be used to preserve biomolecules for industrial use, such as restriction and modification enzymes [Cola? O, C, Sen, S., Thangavelu, M., Pinder, S. & Roser, B. (1992) Bio / Technology 10: 1007-1111]. For the overexpression of trehalose in tobacco, the plasmid pIBT36 would be used where the sl-tps / p gene is under the control of the 35S promoter of the cauliflower virus [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 21: 285-294].
Example 5. Transgenic plants of cereals resistant to environmental stress The cereals that constitute the base of the world diet, could be cultivated in unfavorable climatic conditions, if they produce trehalose in response to cold, heat, salinity or drought. To achieve this, it is required to express the sl-tps / p cDNA under the control of promoters that are induced by any of these environmental factors [Baker, S.S., Wilhelm, K.S. & Thomashow, M.F. (1994) Plant Mol. Biol. 24: 701-713 Takahashi, T., Naito, S. & Komeda, Y. (1992) Plant J. 2: 751-761 Yamaguchi-Shinozaki, K. & Shinozaki, K. (1994) Plant Cell 1 251-264]. The synthesis of trehalose, only under stress conditions, would prevent the continuous production of trehalose (using a constitutive promoter) to divert the metabolism of carbohydrates and consequently decrease the quality and productivity of the grains. There are reports for corn transformation [D'Halluin, K., Bonne, E., Bossut, M., De Beuckeleer, M. & Lee ans, J. (1992) Plant Cell 4: 1495-1505], barley [Wan, Y. & Lemaux, P.G. (1994) Plant Physiol. 1M: 37-48], wheat [Vasil, V., Castillo, A.M. , Fro m, M.E. & Vasil, I.K. (1992) Bio / Technology 1Q: 667-674] and rice [Shimamoto, K., Terada, R., Izawa, T. & Fujimoto, H. (1989) Nature 211: 274-276]. This methodology can be implemented by a person who is familiar with the state of the art.
Example 6. Fruits of transgenic plants with longer shelf life Various fruits, such as tomato, mango and banana, mature quickly and are exposed to putrefaction before reaching the consumer. The early harvest of the fruits and their refrigerated storage or in chambers of controlled environment, has been traditionally used to avoid the aforementioned problem. However, these methods are expensive, especially if it is required to transport the fruits to distant places. In order to increase the shelf life in the tomato, a retardation in its maturation has been reported using transgenic plants that express in antisense the gene of the polygalacturonase, which intervenes in the ripening of the fruit [Smith, CJS, Watson, CF, Ray, J., Bird, CR, Morris, PC, Schuch, W. & Grierson, D. (1988) Nature 114: 724-726]. Despite this slowdown in maturation, the problem remains that after a certain time this process is carried out without necessarily the product has reached the consumer in good condition.
As an alternative to this method, it is proposed here to produce trehalose in transgenic tomato, mango and banana plants. For example, using a specific promoter of the tomato fruit [Bird, C.R., Smith, C.J.S., Ray, J.A., Moureau, P., Bevan, M.W. , Bird, A.S., Hughes, S., Morris, P.C., Grierson, D., Schuch, W. (1988) Plant Mol. Biol. 11: 651-662] could be overexpressed to SL-TPS so that trehalose accumulates specifically in said organ. The transformation and regeneration method for tomato plants [McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., Fraley, R. (1986) Plant Cell Rep. 1: 81 -84] can be carried out by anyone who knows the state of the art. The tomatoes or other fruits could be harvested mature to then be subjected whole or in parts to desiccation and preserved for long periods without the need for refrigeration. When rehydrated, the fruits would have the normal organoleptic properties that the consumer demands. In principle, the strategy described above can be implemented for other fruits as long as there is a system of regeneration and transformation for the plant of interest and that an appropriate promoter is available.
Example 7. Increase in the viability of cells, organs or plant parts involved in sexual or asexual reproduction The production of pollen with prolonged viability would be of great help in programs of plant breeding and in the preservation of germplasm. Similarly, the possibility of storing for long periods and increasing the viability of seeds, bulbs, tubers, cuttings for grafts, stakes and flowers, will have a great impact on plant breeding and conservation of germplasm. The presence of trehalose in a tissue, organ or part of the transformed plant will allow preserving said tissue, organ or part at room temperature in dehydrated state for significantly longer periods than without trehalose. To achieve this goal it is necessary to clone in a vector for plant expression the sl-tps / p cDNA under a tissue-specific or organ-specific promoter and transform the plant of interest with this construction by any of the methods reported and which are known by someone who is familiar with the state of the art. There are reported pollen-specific promoters [Guerrero, F.D., Crossland, L., Smutzer, G.S., Hamilton, D.A. & Mascarenhas, J.P. (1990) Mol. Ge. Genet 224: 161-168], tuber-specific [Bevan, M., Barker, R., Goldsbrough, A., Jarvis, M., Kavanagh, T. & Iturriaga, G. (1986) Nucí. Acids Res. 14: 4625-4638; Jefferson, R., Goldsbrough, A. & Bevan, M. (1990) Plant Mol. Biol. 14: 995-1006] and seed-specific [Colot, V., Robert, L.S., Kavanaugh, T.A., Bevan, M.W. & Beachy, R.N. (1988) EMBO J. 7: 297-302], which could be used to construct hybrid genes for the expression of trehalose in plants.LIST OF SEQUENCES Number of Sequences; INFORMATION FOR SEQ ID NO: 1 I. CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3223 base pairs (B) TYPE: nucleic acids (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear II. TYPE OF MOLECULE: cDNA for mRNA III. HYPOTHETICAL: no IV. ANTI-SENSE: no VI. ORIGINAL SOURCE: (A) ORGANISM: Sellaginella lepidophylla (D) DEVELOPMENT STATUS: adult (F) TYPE OF TISSUE: microphils VII. IMMEDIATE SOURCE: (A) LIBRARY: cDNA (B) CLONE (S): pIBT6 IX. CHARACTERISTICS (A) NAME: RBS (chromosome binding site) (B) LOCATION: from 1 to 110 base pairs IX. CHARACTERISTICS: (A) NAME: sl -tps / p (B) LOCATION: from 111 to 3095 base pairs (C) IDENTIFICATION METHOD: by similarity with known sequences (D) OTHER INFORMATION: codifies for a bifunctional enzyme, trehalose- 6-phosphate synthase and trehalose-6-phosphate phosphatase IX. CHARACTERISTICS: (A) NAME: 3 'untranslated region (trailer) (B) LOCATION: from 3096 to 3191 base pairs IX. CHARACTERISTICS: (A) NAME: site poly-A (B) LOCATION: from 3192 to 3223 base pairs AGTGGCGATG GTGGCCATGG TTTGCTGAAT TTGTATTTAG TTAAATTGTT TTTTGCTGCG 60 GGCGGGTTGT TTTTTTCTTT TCTGCTGCGC CGCGTGCGTG TGCAATACCT ATG CCT CAG 119 Met Pro Gln 1 CCT TAC CCT TCT TCC TCA TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TGA TCC Ser T Ser Ser T Wing Gly Gly 5 10 15 GGC GGC GCG GCG GCG GGC GGC GGC GGC GGC GGC GCC TCC AGC TTG CCG 215 Gly Wing Wing Wing Wing Gly Gly Gly Gly Gly Wing Wing Phe Ser Leu Pro 20 25 30 35 CCG TCG CTA GCA TCA TCG CGA CGA GTG GAG CGC CTG GTG CGT GAG AGG CAG 263 Pro Ser Leu Wing Being Ser Arg Val Glu Arg Leu Val Arg Glu Arg Gln 40 45 50 CTG CGG AAT CA AGG CAG GAG GAAT CCT GAG GAT GAG CAG CAG GCG 311 Leu Arg Asn Gln Arg Gln Glu Asp Glu Pro Glu Asp Glu Gln Gln Wing 55 60 65 CTG GAG GCG GAG GAA GCG GCG GTG GCG GCT ACC GAG GTG CCC GAT GCC 359 Leu Glu Ala Glu Glu Ala Ala Ala Ala Ala Thr Glu Val Pro Asp Ala 70 75 80 GTC GCC GCT GCC ACG CCA TCG CTC TCC GAT GAG CCG TCC AAG ATT TCC 407 Val Wing Wing Wing Thr Pro Ser Leu Ser Asp Glu Pro Ser Lys li e Ser 85 90 95 AGC GGT CGA GGC CAG CGG TTG CTT GTG GTT GCC AAT CGC CTT CCC TTG 455 Ser Gly Arg Gly Gln Arg Leu Leu Val Val Wing Asn Arg Leu Pro Leu 100 105 110 115 TCT GCC ACG AGG AAA GGC GAG ACG GAA TGG AAT TTG GAG ATG AGC GCC 503 Ser Wing Thr Arg Lys Gly Glu Thr Glu Trp Asn Leu Glu Met Ser Wing 120 125 130 GGG GGC CTT GTA AGT GCG CTT TTG GGC GTC AAG CAG TTT GAA GTC ACC 551 Gly Gly Leu Val Be Ala Wing Leu Gly Val Lys Gln Phe Glu Val Thr 135 140 145 TGG ATC GGT TGG CCT GGT GTC TAT GTA CAG GAG GAG AAG GGT GAG AAA 599 Trp lie Gly Trp Pro Gly Val Tyr Val Gln Asp Glu Lys Gly Glu Lys 150 155 160 TCG CTG CGA GGA GCT CTT GAA GAA AAG GGA TTT GTG CCC GTT CTT CTC 647 Ser Leu Arg Gly Ala Leu Glu Glu Lys Gly Phe Val Pro Val Leu Leu 165 170 175 GAC GAG GCA ACT GTT GAT CAG TAC TAC AAT GGC TAT TGC AAC AAC GTG 695 Asp Glu Wing Thr Val Asp Gln Tyr Tyr Asn Gly Tyr Cys Asn Asn Val 180 185 190 195 CTT TGG CCG CTT TTC CAC TAC ATT GGT CTT AGG CAG GAA GAC CGG CTG 743 Leu Trp Pro Leu Phe His Tyr lie Gly Leu Arg Gln Glu Asp Arg Leu 200 205 210 GCT GCC ACG CGC AGC TTA CTA TCC CAG TTT AAC GCA TAT AAA CGT GCT 791 Wing Wing Thr Arg Ser Leu Leu Ser Gln Phe Asn Wing Tyr Lys Arg Ala 215 220 225 AAT CGT TTG TTG GCG GAG GTG TTC AAT TTC TGA TGA GAG GGG GAT 839 Asn Arg Leu Phe Wing Glu Wing Val Phe Asn Phe Tyr Gln Glu Gly Asp 230 235 240 GTG TGG TGG TGC CAC GAT TAC CAT CTT ATG TTC CTC CCC AGC TAC CTC 887 Val Val Trp Cys His Asp Tyr His Leu Met Phe Leu Pro Ser Tyr Leu 245 250 255 AAG GAG AAG GAC AGC CAG ATG AAA GTC GGG TGG TTC CTC CAC ACG CCG 935 Lys Glu Lys Asp Ser Gln Met Lys Val Gly Trp Phe Leu His Thr Pro 260 265 270 275 TTC TCG TCT TCT GAG ATT TAC AGA ACG CTG CCG CTG CGG GCC CTG 983 Phe Pro Ser Ser Glu lie Tyr Arg Thr Leu Pro Leu Arg Ala Glu Leu 280 285 290 CTC CAA GGC GTC TTA GCT GCG GAT TTG GTG GGG TTC CAC ACÁ TAC GAC 1031 Leu Gln Gly Val Leu Ala Wing Asp Leu Val Gly Phe His Thr Tyr Asp 295 300 305 TAT GCA AGG CAC TTT GTT AGC GCG TGC ACA CGG ATA CTC GGG CTG GAA 1079 Tyr Ala Arg His Phe Val Ser Wing Cys Thr Arg lie Leu Gly Leu Glu 310 315 320 GGC ACT CCC GAG GGT GTC GAG GAT CAG GGG AAG AAG ACG CGA GTG GCT 1127 Gly Thr Pro Glu Gly Val Glu Asp Gln Gly Lys Asn Thr Arg Val Wing 325 330 335 GCC TTC CCC GTG GGG ATC GAC TCG GAG CGA TTT ATC GAG GCC GTA GAA 1175 Wing Phe Pro Val Gly Lie Asp Ser Glu Arg Phe lie Glu Wing Val Glu 340 345 350 355 ACT GAT GCG GTC AAG AAA CAC ATG CAA GAG CTG AGC CAG CGT TTT GCT 1223 Thr Asp Ala Val Lys Lys His Met Gln Glu Leu Ser Gln Arg Phe Wing 360 365 370 GGT CGT AAG GTT ATG TTG GGG GTG GAT AGG CTT GAC ATG ATT AAA GGA 1271 Gly Arg Lys Val Met Leu Gly Val Asp Arg Leu Asp Met lie Lys Gly 375 380 385 ATT CCA CAG AAG CTG CTA GCC TTT GAA AAA TTC CTC GAG GAG AAC TCC 1319 lie Pro Gln Lys Leu Leu Wing Phe Glu Lys Phe Leu Glu Glu Asn Ser 390 395 400 GAG TGG CGT GAT AAG GTC GTC CTG GTG CA ATC GCG GTG CCG ACT AGA 1367 Glu Tr p Arg Asp Lys Val Val Leu Val Gln lie Wing Val Pro Thr Arg 405 410 415 ACG GAC GTC CTC GAG TAC CA AAG CTT ACG AGC CAG GTT CAC GAG ATT 1415 Thr Asp Val Leu Glu Tyr Gln Lys Leu Thr Ser Gln Val His Glu lie 420 425 430 435 GTT GGT CGC ATA AAT GGA CGT TTC GGC TCC TTG ACG GCT GTT CCT ATC 1463 Val Gly Arg lie Asn Gly Arg Phe Gly Ser Leu Thr Wing Val Pro lie 440 445 450 CAT CAC CTC GAT CGC TCC ATG AAA TTT CCG GAG CTT TGT GCC TTA TAT 1511 His His Leu Asp Arg Ser Met Lys Phe Pro Glu Leu Cys Ala Leu Tyr 455 460 465 GCA ATC ACT GAT GTC CTG CTC GTG ACA TCC CTG CGT GAT GGC ATG AAC 1559 Ala lie Thr Asp Val Leu Leu Val Thr Ser Leu Arg Asp Gly Met Asn 470 475 480 CTC GTG AGC TAC GAG TTC GTT GCT TGC CAA AAG GAT AAG AAG GGC GCG 1607 Leu Val Ser Tyr Glu Phe Val Wing Cys Gln Lys Asp Lys Lys Gly Wing 485 490 495 CTT ATT CTG AGT GAG TTT GCA GGC GCT GCG CAG TCT CTG GGT GCG GGG 1655 Leu lie Leu Ser Glu Phe Wing Gly Wing Wing Gln Ser Leu Gly Wing Gly 500 505 510 515 TCT ATC CTC ATA AAT CCG TGG AAT ATA ATA G AG TCG TCC AAC GCC ATT 1703 Be lie Leu lie Asn Pro Trp Asn lie lie Glu Be As Asn Ala lie 520 525 530 GCG GAC GCT CTC AAC ATG CCA GAA GAA GAA CGG GAA GAA CGG CAT CGT 1751 Ala Asp Ala Leu Asn Met Pro Glu Glu Glu Arg Glu Glu Arg His Arg 535 540 545 CAT AAC TTC ATG CAC ATA ACT ACT CAC AGT GCT CAG GG TGG GCG GAG 1799 His Asn Phe Met His lie Thr Thr His Ser Wing Gln Val Trp Wing Glu 550 555 560 ACG TTT ATC AGC GAA CTC AAT GAT TCC ATC TTG GAA GCC CTG CGC 1847 Thr Phe lie Ser Glu Leu Asn Asp Ser lie Leu Glu Wing Glu Leu Arg 565 570 575 ACT CTG CAT ATT CCG CCT CAA TTG CCT TTG GAT AAA GCA GTC GCA AAG 1895 Thr Leu His Lie Pro Pro Gln Leu Pro Leu Asp Lys Ala Val Wing Lys 580 585 590 595 TAC TCG GAG TCA AAG AAC CGG CTA GTA ATT TTG GGC TTC AAC TCG ACT 1943 Tyr Ser Glu Ser Lys Asn Arg Leu Val lie Leu Gly Phe Asn Ser Thr 600 605 610 TTG ACC GCG CAA GTG GAA GCT CCG AGA GGT AGG GCG CCC GAC CAG ATC 1991 Leu Thr Ala Gln Val Glu Ala Pro Arg Gly Arg Ala Pro Asp Gln lie 615 620 625 AGG GAG ATG AAG ATA CGT CTT CAT CCT AGC ATA AAG GAC ATC CTC AAT 2039 Arg Glu Met Lys lie Arg Leu His Pro Ser lie Lys Asp lie Leu Asn 630 635 640 GTA CTT TGC TCT GAT CCA AAG ACG ATA ATA GTC ATC CTA AGC GGG AGC 2087 Val Leu Cys Ser Asp Pro Lys Thr Thr lie Val lie Leu Ser Gly Ser 645 650 655 GAG CGC GTG GCT CTT GAC GTA GTA TTT GGA GTC TTC GAT TTG TGG CTG 2135 Glu Arg Val Ala Leu Asp Glu Val Phe Gly Glu Phe Asp Leu Trp Leu 660 665 670 675 GCG GAA AAC GGG ATG TTT CTT CGT CAT ACT CAG GGG GAG TGG ATG 2183 Wing Wing Glu Asn Gly Met Phe Leu Arg His Thr Gln Gly Glu Trp Met 680 685 690 ACÁ ACÁ ATG CCC GAA CAT CTG AAC ATG GAT TGG TTG GAA AGT GTA CAG 2231 Thr Thr Met Pro Glu His Leu Asn Met sp Trp Leu Glu Ser Val Gln 695 700 705 TTG GTC TTT GAT TAT TTT TGT GAG AGG ACG CCA CGC TCT TTT GTC GAG 2279 Leu Val Phe Asp Tyr Phe Cys Glu Arg Thr Pro Arg Ser Phe Val Glu 710 715 720 ACC CGT GAG ACG TCT GTG TGG AAC TAT AAG TAT GCA GAT GTT GAA 2327 Thr Arg Glu Thr Ser Leu Val Trp Asn Tyr Lys Tyr Ala Asp Val Glu 725 730 735 TTC GGC AGG GTG CAG GCA CGT GAT ATG CTA CAG CAC CTG TGG ACC GGG 2375 Phe Gly Arg Val Gln Wing Arg Asp Met Leu Gln His Leu Trp Thr Gly 740 745 750 755 CCC ATA TCC AAC GCT GCC GTC GTC GTC GTG CAA GGC GGA AAG TCG GTC 2423 Pro lie Be Asn Ala Wing Val Asp Val Val Gln Gly Gly Lys Ser Val 760 765 770 GAG GTC CGC CCC GTA GGA GTC TCG AAG GGG TCT GCA ATT GAC CGG ATT 2471 Glu Val Arg Pro Val Gly Val Ser Lys Gly Ser Ala lie Asp Arg lie 775 780 785 CTA GGC GAA ATC GTG CAC AGC AAA CAC ATG ACG ATA CCC ATC GAC TAC 2519 Leu Gly Lie Val His Ser Lys His Met Thr lie Pro lie Asp Tyr 790 795 800 GTC CTT TGC ATA GGA CAC TTT TTG AGC AAG GAC GAT ATC TAC ACA 2567 Val Leu Cys lie Gly His Phe Leu Ser Lys Asp Glu Asp lie Tyr Thr 805 810 815 TTC GAA CCG GAG CTG CCA CTG CTG GAC AGG GAC TCG TCG ACG AGC 2615 Phe Phe Glu Pro Glu Leu Pro Leu Leu Asp Arg Asp Ser Ser Thr Ser 820 825 830 835 AAC GGA GGG AAA CCA CTG GGT GGA AAG CTT CCA ATA GAC CGA AAG TCT 2663 Asn Gly Gly Lys Pro Leu Gly Gly L ys Leu Pro lie Asp Arg Lys Ser 840 845 850 TCA AAG AGC TCC TCT CGC ATG AAG CCG CCA GTG TCG TCA CCA AAG TCA 2711 Ser Lys Ser Ser Arg Met Lys Pro Pro Val Ser Ser Pro Lys Ser 855 860 865 CCC GGC CGT GGA AGC GAG CAG CAG CAG CAG GCT GAG GAG GCA AGC AGA 2759 Pro Gly Arg Gly Glu Gln Gln Gln Gln Glu Glu Wing Glu Ala Ser Arg 870 875 880 TGG GAA GGA TCG TCC GTG CTG GAT CTC CAG GGA GAC AAC TAC TTC AGC 2807 Trp Glu Gly Ser Ser Val Leu Asp Leu Gln Gly Glu Asn Tyr Phe Ser 885 890 895 TGT GCA GTG GGA ACC ATG AAG AGG TCA CTA GCT CGC TAC TGC CTC ACT 2855 Cys Wing Val Gly Thr Met Lys Arg Ser Leu Wing Arg Tyr Cys Leu Thr 900 905 910 915 TCT TCA GAG GTG GTG ACÁ TTC CTG ACC TCG CTC ACÁ AGC ACÁ GTG 2903 Ser Ser Glu Glu Val Val Thr Phe Leu Thr Ser Leu Thr Ser Thr Val 920 925 930 GCA GCA GCA GCA GGG GCA GGA GCA GGA GGA AGA GCG ACG GGA TCA GGA 2951 Wing Wing Wing Wing Gly Wing Gly Wing Gly Wing Arg Wing Gly Ser Gly 935 940 945 GCA GCA GCA GCA GGA GCA GCA GCA GGA GGT GGG GAT CAT GAA GCT 2999 Ala Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Asp His Glu Ala 950 955, 960 CCA GGA TCA CCA ATC AGG AAA AGC GAT TCG TTC AAG ACG AGC GGG TGG 3047 Pro Gly Ser Pro lie Arg Lys Ser Asp Ser Phe Lys Thr Ser Gly Trp 965 970 975 CAT AGT CCA ACG CCC CGC TCT CCA AAG CTC GCT CCA TGA GTA GCG CAG 3095 Pro Thr Ser Pro His Arg Ser Leu Pro Lys Ala Pro Ala Val Gln 980 985 990 GATGGAGGGA TGGAAAAAGA CAAGCAAAGC ATCCTTGTCA AAGGCACAAG CAAAGCAAGT CGCCTTCTGA 3155 AAAAAAAAAA AAAAAAAAAA ATGCAAGCAA AGGCTAAAGC GAGGCCAAAA 3215 AAAAAAAA 3223 INFORMATION FOR SEQ ID NO: 2 I. CHARACTERISTICS THE SEQUENCE: (A) LENGTH: 994 amino acid residues (B) TYPE: amino acids II. TYPE OF MOLECULE: protein III. HYPOTHETICAL: no IV. ANTI-SENTÍDO: no V. TYPE OF FRAGMENT: complete protein VI. ORIGINAL SOURCE: (A) ORGANISM: Selaginella lepidophylla (D) DEVELOPMENT STATE: adult (F) TYPE OF TISSUE: microphils VII. IMMEDIATE SOURCE: (A) LIBRARY: cDNA (B) CLONE (S): pIBT6 IX. CHARACTERISTICS: (A) NAME: SL-TPS / P (B) LOCALIZATION: from 1 to 994 amino acids (C) IDENTIFICATION METHOD: by similarity with known sequences (D) OTHER INFORMATION: bifunctional enzymatic activity of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.
Met Pro Gln Pro Tyr Pro Ser Ser Ser Thr Ser Asn Ala Lys Glu 1 5 10 15 Wing Gly Gly Wing Wing Wing Wing Wing Gly Gly Gly Gly Wing Gly Wing Phe 20 25 30 Ser Leu Pro Pro Ser Wing Le Ser Wing Being Arg Val Glu Arg Leu Val Arg 35 40 45 Glu Arg Gln Leu Arg Asn Gln Arg Gln Glu Asp Glu Pro Glu Asp Glu 50 55 60 Gln Gln Ala Leu Glu Ala Glu Glu Ala Ala Ala Ala Ala Thr Glu Val 65 70 75 80 Pro Asp Ala Val Ala Ala Ala Thr Pro Ser Leu Ser Asp Glu Pro Ser 85 90 95 Lys lie Ser Gly Arg Gly Gln Arg Leu Leu Val Val Ala Asn Arg 100 105 110 Leu Pro Leu Ser Wing Thr Arg Lys Gly Glu Thr Glu Trp Asn Leu Glu 115 120 125 Met Ser Wing Gly Gly Leu Val Ser Wing Leu Leu Gly Val Lys Gln Phe 130 135 140 Glu Val Thr Trp lie Gly Trp Pro Gly Val Tyr Val Gln Asp Glu Lys 145 150 155 160 Gly Glu Lys Ser Leu Arg Gly Ala Leu Glu Glu Lys Gly Phe Val Pro 165 170 175 Val Leu Leu Asp Glu Wing Thr Val Asp Gln Tyr Tyr Asn Gly Tyr Cys 180 185 190 Asn Asn Val Leu Trp Pro Leu Phe His Tyr lie Gly Leu Arg Gln Glu 195 200 205 Asp Arg Leu Wing Wing Thr Arg Ser Leu Leu Ser Gln Phe Asn Wing Tyr 210 215 220 Lys Arg Ala Asn Arg Leu Phe Ala Glu Ala Val Phe Asn Phe Tyr Gln 225 230 235 240 Glu Gly Asp Val Val Trp Cys His Asp Tyr His Leu Met Phe Leu Pro 245 250 255 Ser Tyr Leu Lys Glu Lys Asp Ser Gln Met Lys Val Gly Trp Phe Leu 260 265 270 His Thr Pro Phe Pro Ser Glu lie Tyr Arg Thr Leu Pro Leu Arg 275 280 285 Wing Glu Leu Leu Gln Gly Val Leu Wing Wing Asp Leu Val Gly Phe His 290 295 300 Thr Tyr Asp Tyr Wing Arg His Phe Val Wing Wing Cys Thr Arg lie Leu 305 310 315 320 Gly Leu Glu Gly Thr Pro Glu Gly Val Glu Asp Gln Gly Lys Asn Thr 325 330 335 Arg Val Ala Ala Phe Pro Val Gly He Asp Ser Glu Arg Phe He Glu 340 345 350 Wing Val Glu Thr Asp Wing Val Lys Lys His Met Gln Glu Leu Ser Gln 355 360 365 Arg Phe Wing Gly Arg Lys Val Met Leu Gly Val Asp Arg Leu Asp Met 370 375 380 He Lys Gly He Pro Gln Lys Leu Leu Wing Phe Glu Lys Phe Leu Glu 385 390 395 400 Glu Asn Ser Glu Trp Arg Asp Lys Val Val Leu Val Gln He Ala Val 405 410 415 Pro Thr Arg Thr Asp Val Leu Glu Tyr Gln Lys Leu Thr Ser Gln Val 420 425 430 His Glu He Val Gly Arg He Asn Gly Arg Phe Gly Ser Leu Thr Wing 435 440 445 Val Pro He His His Leu Asp Arg Ser Met Lys Phe Pro Glu Leu Cys 450 455 460 Wing Leu Tyr Wing He Thr Asp Val Leu Leu Val Thr Ser Leu Arg Asp 465 470 475 480 Gly Met Asn Leu Val Ser Tyr Glu Phe Val Wing Cys Gln Lys Asp Lys 485 490 495 Lys Gly Ala Leu He Leu Ser Glu Phe Ala Gly Ala Ala Gln Ser Leu 500 505 510 Gly Wing Gly Ser He Leu He Asn Pro Trp Asn He He Glu Be Ser 515 520 525 Asn Wing He Wing Asp Wing Leu Asn Met Pro Glu Glu Glu Arlu Glu Glu 530 535 540 Arg His Arg His Asn Phe Met His He Thr Thr His Ser Wing Gln Val 545 550 555 560 Trp Wing Glu Thr Phe He Ser Glu Leu Asn Asp Ser He Leu Glu Wing 565 570 575 Glu Leu Arg Thr Leu His He Pro Pro Gln Leu Pro Leu Asp Lys Ala 580 585 590 Val Ala Lys Tyr Ser Glu Ser Lys Asn Arg Leu Val He Leu Gly Phe 595 600 605 Asn Ser Thr Leu Thr Ala Gln Val Glu Ala Pro Arg Gly Arg Ala Pro 610 615 620 Asp Gln He Arg Glu Met Lys He Arg Leu His Pro Ser He Lys Asp 625 630 635 640 He Leu Asn Val Leu Cys Ser Asp Pro Lys Thr Thr He Val He Leu 645 650 655 Ser Gly Ser Glu Arg Val Wing Leu Asp Glu Val Phe Gly Glu Phe Asp 660 665 670 Leu Trp Leu Ala Ala Glu Asn Gly Met Phe Leu Arg His Thr Gln Gly 675 680 685 Glu Trp Met Thr Thr Met Pro Glu His Leu Asn Met Asp Trp Leu Glu 690 695 700 Ser Val Gln Leu Val Phe Asp Tyr Phe Cys Glu Arg Thr Pro Arg Ser 705 710 715 720 Phe Val Glu Thr Arg Glu Thr Ser Leu Val Trp Asn Tyr Lys Tyr Wing 725 730 735 Asp Val Glu Phe Gly Arg Val Gln Wing Arg Asp Met Leu Gln His Leu 740 745 750 Trp Thr Gly Pro He Ser Asn Ala Ala Val Asp Val Val Gln Gly Gly 755 760 765 Lys Ser Val Glu Val Arg Pro Val Gly Val Ser Lys Gly Ser Ala Ala 770 775 780 Asp Arg He Leu Gly Glu He Val His Ser Lys His Met Thr He Pro 785 790 795 800 He Asp Tyr Val Leu Cys He Gly His Phe Leu Ser Lys Asp Glu Asp 805 810 815 He Tyr Thr Phe Phe Glu Pro Glu Leu Pro Leu Leu Asp Arg Asp Ser 820 825 830 Ser Thr Ser Asn 'Gly Gly Lys Pro Leu Gly Gly Lys Leu Pro He Asp 835 840 845 Arg Lys Ser Ser Lys Ser Ser Arg Met Lys Pro Pro Val Ser 850 855 860 Pro Lys Ser Pro Gly Arg Gly Ser Glu Gln Gln Gln Gln Wing Glu Glu 865 870 875 880 Wing Ser Arg Trp Glu Gly Ser Ser Val Leu Asp Leu Gln Gly Glu Asn 885 890 895 Tyr Phe Ser Cys Wing Val Gly Thr Met Lys Arg Ser Leu Wing Arg Tyr 900 905 910 Cys Leu Thr Ser Ser Glu Glu Val Val Thr Phe Leu Thr Ser Leu Thr 915 - 920 925 Ser Thr Val Wing Wing Wing Wing Gly Wing Gly Wing Gly Wing Arg Wing Thr 930 935 940 Gly Ser Gly Ala Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Asp 945 950 955 960 His Glu Ala Pro Gly Ser Pro He Arg Lys Ser Asp Ser Phe Lys Thr 965 970 975 Ser Gly Trp His Pro Pro Thr Pro Arg Pro Pro Lys Leu Pro Pro Wing 980 985 990 Val Gln INFORMATION FOR SEQ ID NO: 3 I. CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1910 base pairs (B) TYPE: nucleic acids (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear II. TYPE OF MOLECULE: cDNA for mRNA III. HYPOTHETICAL: no IV. ANTI-SENSE: no VI. ORIGINAL SOURCE: (A) ORGANISM: Selaginella lepidophylla (D) DEVELOPMENT STATE: adult (F) TYPE OF TISSUE: microphils VII. IMMEDIATE SOURCE: (A) LIBRARY: cDNA (B) CLONE (S): pIBTd IX. CHARACTERISTICS: (A) NAME: RBS (chromosome binding site) (B) LOCATION: from 1 to 110 base pairs IX. CHARACTERISTICS: (A) NAME: sl -tps (B) LOCATION: from 111 to 1910 base pairs (C) IDENTIFICATION METHOD: by similarity with known sequences (D) OTHER INFORMATION: codifies for a trehalose-6-phosphate synthase AGTGGCGATG GTGGCCATGG TTTGCTGAAT TTGTATTTAG TTAAATTGTT TTTTGCTGCG 60 GGCGGGTTGT TTTTTTCTTT TCTGCTGCGC CGCGTGCGTG TGCAATACCT ATG CCT CAG 119 Met Pro Gln 1 CCT TAC CCT TCT TCC TCA TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TGA TCC Ser T Ser Ser T Ala Gly Gly May 10 15 GGC GCT GCG GCG GCG GGG GGC GGC GGC GGC GGC GCC TTC AGC TTG GCC 215 Gly Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Phe Ser Leu Pro 20 25 30 35 CCG TCG CTA GCA TCA GCT CGA GTG GAG CGC CTG GTG CGT GAG AGG CAG 263 Pro Ser Leu Wing Ser Ser Arg Val Glu Arg Leu Val Arg Glu Arg Gln 40 45 50 CTG CGG AAT CA AGG CAG GAG GAT GAA CCT GAG GAT GAG CAG CAG GCG 311 Leu Arg Asn Gln Arg Gln Glu Asp Glu Pro Glu Asp Glu Gln Gln Ala 55 60 65 CTG GAG GCG GAG GAA GCG GCG GTG GCG GCT ACC GAG GTG CCC GAT GCC 359 Leu Glu Ala Glu Glu Ala Ala Val Ala Ala Thr Glu Val Pro Asp Wing 70 75 80 GTC GCC GCT GCC ACG CCA TCG CTC TCC GAT GAG CCG TCC AAG ATT TCC 407 Val Wing Wing Thr Pro Wing Ser Leu Ser Asp Glu Pro Ser Lys He Ser 85 90 95 AGC GGT CGA GGC CAG CGG TTG CTT GTG GTT GCC AAT CGC CTT CCC TTG 455 Ser Gly Arg Gly Gln Arg Leu Leu Val Val Wing Asn Arg Leu Pro Leu 100 105 110 115 TCT GCC ACG AGG AAA GGC GAG ACG GAA TGG AAT TTG GAG ATG AGC GCC 503 Ser Ala Thr Arg Lys Gly Glu Thr Glu Trp Asn Leu Glu Met Ser Ala 120 125 130 GGG GGC CTT GTA AGT GCG CTT TTG GGC GTC AAG CAG TTT GAA GTC ACC 551 Gly Gly Leu Val Ser Ala Leu Leu Gly Val Lys Gln Phe Glu Val Thr 135 140 145 TGG ATC GGA TGG CCT GGT GTC TAT GTA CAA GAC GAG AAG GGT GAG AAA 599 Trp He Gly Trp Pro Gly Val Tyr Val Gln Asp Glu Lys Gly Glu Lys 150 155 160 GCT CTG CGA GGA GCT CTT GAA GAA AAG GGA TTT GTG CCC GT T CTT CTC 647 Ser Leu Arg Gly Ala Leu Glu Glu Lys Gly Phe Val Pro Val Leu Leu 165 170 175 GAC GAG GCA ACT GTT GAT CAG TAC TAC AAT GGC TAT TGC AAC AAC GTG 695 Asp Glu Ala Thr Val Asp Gln Tyr Tyr Asn Gly Tyr Cys Asn Asn Val 180 185 190 195 CTT TGG CCG CTT TTC CAC TAC ATT GGT CTT AGG CAG GAA GAC CGG CTG 743 Leu Trp Pro Leu Phe His Tyr He Gly Leu Arg Gln Glu Asp Arg Leu 200 205 210 GCT GCC ACG CGC AGC TTA CTA TCC CAG TTT AAC GCA TAT AAA CGT GCT 791 Ala Ala Thr Arg Ser Leu Leu Ser Gln Phe Asn Ala Tyr Lys Arg Ala 215 220 225 AAT CGT TTG TTT GCG GAG GCT GTG TTC AAT TTC TAC CAG GAA GGG GAT 839 Asn Arg Leu Phe Wing Glu Wing Val Phe Asn Phe Tyr Gln Glu Gly Asp 230 235 240 GTG GTG TGG TGC CAC GAT TAC CAT CTT ATG TTC CTC CCC AGC TAC CTC 887 Val Val Trp Cys His Asp Tyr His Leu Met Phe Leu Pro Ser Tyr Leu 245 250 255 AAG GAG AAG GAC AGC CAG ATO AAA GTC GGG TGG TTC CTC CAC ACG CCG 935 Lys Glu Lys Asp Ser Gln Met Lys Val Gly Trp Phe Leu His Thr Pro 260 265 270 275 TTC CCC TCG TCT GAG ATT TAC AGA ACG CTG CCG CTG CGG GCC GAG CTG 983 Phe Pro Ser Ser Glu He Tyr Arg Thr Leu Pro Leu Arg Ala Glu Leu 280 285 290 CTC CAÁ GGC GTC TTA GCT GCG GAT TTG GTG GGG TTC CAC ACAC TAC GAC 1031 Leu Gln Gly Val Leu Wing Wing Asp Leu Val Gly Phe His Thr Tyr Asp 295 300 305 TAT GCA AGG CAC TTT GTT AGC GCG TGC ACA CGG ATA CTC GGG CTG GAA 1079 Tyr Wing Arg His Phe Val Wing Wing Cys Thr Arg He Leu Gly Leu Glu 310 315 320 GGC ACT CCC GAG GGT GTC GAG GAT CAG GAG AAG ACG CGA GTG GCT 1127 Gly Thr Pro Glu Gly Val Glu Asp Gln Gly Lys Asn Thr Arg Val Wing 325 330 335 GCC TTC CCC GTG GGG ATC GAC TCG GAG CGA TTT ATC GAG GCC GTA GAA 1175 Wing Phe Pro Val Gly He Asp Ser Glu Arg Phe He Glu Wing Val Glu 340 345 350 355 ACT GAT GCG GTC AAG AA CAC ATG CAG GG CTG AGC CAG CGT TTT GCT 1223 Thr Asp Wing Val Lys Lys His Met Gln Glu Leu Ser Gln Arg Phe Wing 360 365 370 GGT CGT AAG GTT ATG TTG GGG GTG GAT AGG CTT GAC ATG ATT AAA GGA 1271 Gly Arg Lys Val Met Leu Gly Val Asp Arg Leu Asp Met He Lys Gly 375 380 385 ATT CCA CAG AAG CTG CTA GCC TTT GAA AAA TTC CTC GAG GAC AAC TCC 1319 He Pro Gln Lys Leu Leu Ala Phe Glu Lys Phe Leu Glu Glu Asn Ser 390 395 400 GAG TGG CGT GAT AAG GTC GTC CTG GTG CAA ATC GCG GTG CCG ACT AGA 1367 Glu Trp Arg Asp Lys Val Val Leu Val Gln He Ala Val Pro Thr Arg 405 410 415 ACG GAC GTC CTC GAG TAC CAA AAG CTT ACG AGC CAG GTT CAC GAG ATT 1415 Thr Asp Val Leu Glu Tyr Gln Lys Leu Thr Ser Gln Val His Glu He 420 425 430 435 GTT GGT CGC ATA AAT GGA CGT TTC GGC TCC TTG ACG GCT GTT CCT ATC 1463 Val Gly Arg He Asn Gly Arg Phe Gly Ser Leu Thr Wing Val Pro He 440 445 450 CAT CAC CTC GAT CGC TCC ATG AAA TTT CCG GAG CTT TGT GCC TTA TAT 1511 His His Leu Asp Arg Ser Met Lys Phe Pro Glu Leu Cys Ala Leu Tyr 455 46 0 465 GCA ATC ACT GAT GTC CTG CTC GTG ACA TCC CTG CGT GAT GGC ATG AAC 1559 Wing He Thr Asp Val Leu Leu Val Thr Ser Leu Arg Asp Gly Met Asn 470 475 480 CTC GTG AGC TAC GAG TTC GTT GCT TGC CAA AAG GAT AAG AAG GGC GCG 1607 Leu Val Ser Tyr Glu Phe Val Wing Cys Gln Lys Asp Lys Lys Gly Wing 485 490 495 CTT ATT CTG AGT GAG TTT GCA GGC GCT GCG CAG T CTG GGT GCG GGG 1655 Leu He Leu Ser Glu Phe Ala Gly Ala Wing Gln Ser Leu Gly Wing Gly 500 505 510 515 TCT ATC CTC ATA AAT CCG TGG AAT ATA ATA GAG TCG TCC AAC GCC ATT 1703 Ser He Leu He Asn Pro Trp Asn He He Glu Ser Ser Asn Ala He 520 525 530 GCG GAC GCT CTC AAC ATG CCA GAA GAA GAA CGG GAA GAA CGG CAT CGT 1751 Ala Asp Ala Leu Asn Met Pro Glu Glu Glu Arlu Glu Glu Arg His Arg 535 540 545 CAT AAC TTC ATG CAC ATA ACT ACT CAC AGT GCT CAA GTA TGG GCG GAG 1799 His Asn Phe Met His He Thr Thr His Ser Wing Gln Val Trp Wing Glu 550 555 560 ACG TTT ATC AGC GAA CTC AAT GAT TCC ATC TTG GAA GCC GAG CTG CGC 1847 Thr Phe He Ser Glu Leu Asn Asp Ser He Leu Glu Al to Glu Leu Arg 565 570 575 ACT CTG CAT ATT CCG CCT CAA TTG CCT TTG GAT AAA GCA GTC GCA AAG 1895 Thr Leu His Pro Pro Gln Leu Pro Leu Asp Lys Ala Val Wing Lys 580 585 590 595 TAC TCG GAG TCA AAG 1910 Tyr Ser Glu Ser Lys 600 INFORMATION FOR SEQ ID NO: 4 I. CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 600 amino acid residues (B) TYPE: amino acids II. TYPE OF MOLECULE: protein III. HYPOTHETICAL: no IV. ANTI-SENSE: not V. TYPE OF FRAGMENT: fragment N-terminal VI. ORIGINAL SOURCE: (A) ORGANISM: Selaginella lepidophylla (D) DEVELOPMENT STATE: adult (F) TYPE OF TISSUE: microphils VII. IMMEDIATE SOURCE: (A) LIBRARY: cDNA (B) CLONE (S): pIBT6 IX. CHARACTERISTICS: (A) NAME: SL-TPS (B) LOCATION: from 1 to 600 amino acids (C) IDENTIFICATION METHOD: by simily with known sequences (D) OTHER INFORMATION: enzymatic activity of trehalose-6-phosphate synthase Met Pro Gln Pro Tyr Pro Ser Ser Ser Thr Ser Asn Ala Lys Glu 1 5 10 15 Wing Gly Gly Wing Wing Wing Wing Wing Gly Gly Gly Gly Wing Gly Wing Phe 20 25 30 Ser Leu Pro Pro Ser Wing Le Ser Wing Being Arg Val Glu Arg Leu Val Arg 35 40 45 Glu Arg Gln Leu Arg Asn Gln Arg Gln Glu Asp Glu Pro Glu Asp Glu 50 55 60 Gln Gln Ala Leu Glu Ala Glu Glu Ala Ala Ala Ala Ala Thr Glu Val 65 70 75 80 Pro Asp Ala Val Ala Ala Ala Thr Pro Ser Leu Ser Asp Glu Pro Ser 85 90 95 Lys Be Ser Gly Arg Gly Gln Arg Leu Leu Val Val Wing Asn Arg 100 105 110 Leu Pro Leu Ser Wing Thr Arg Lys Gly Glu Thr Glu Trp Asn Leu Glu 115 120 125 Met Ser Wing Gly Gly Leu Val Ser Wing Leu Leu Gly Val Lys Gln Phe 130 135 140 Glu Val Thr Trp He Gly Trp Pro Gly Val Tyr Val Gln Asp Glu Lys 145 150 155 160 Gly Glu Lys Ser Leu Arg Gly Wing Leu Glu Glu Lys Gly Phe Val Pro 165 170 175 Val Leu Leu Asp Glu Wing Thr Val Asp Gln Tyr Tyr Asn Gly Tyr Cys 180 185 190 Asn Asn Val Leu Trp Pro Leu Phe His Tyr He Gly Leu Arg Gln Glu 195 200 205 Asp Arg Leu Ala Ala Thr Arg Ser Leu Leu Ser Gln Phe Asn Wing Tyr 210 215 220 Lys Arg Wing Asn Arg Leu Phe Wing Glu Wing Val Phe Asn Phe Tyr Gln 225 230 235 240 Glu Gly Asp Val Val Trp Cys His Asp Tyr His Leu Met Phe Leu Pro 245 250 255 Ser Tyr Leu Lys Glu Lys Asp Ser Gln Met Lys Val Gly Trp Phe Leu 260 265 270 His Thr Pro Phe Pro Ser Ser Glu He Tyr Arg Thr Leu Pro Leu Arg 275 280 285 Wing Glu Leu Leu Gln Gly Val Leu Wing Wing Asp Leu Val Gly Phe His 290 295 300 Thr Tyr Asp Tyr Ala Arg His Phe Val Ser Ala Cys Thr Arg He Leu 305 310 315 320 Gly Leu Glu Gly Thr Pro Glu Gly Val Glu Asp Gln Gly Lys Asn Thr 325 330 335 Arg Val Wing Wing Phe Pro Val Gly He Asp Ser Glu Arg Phe He Glu 340 345 350 Wing Val Glu Thr Asp Wing Val Lys Lys His Met Gln Glu Leu Ser Gln 355 360 365 Arg Phe Wing Gly Arg Lys Val Met Leu Gly Val Asp Arg Leu Asp Met 370 375 380 He Lys Gly He Pro Gln Lys Leu Leu Wing Phe Glu Lys Phe Leu Glu 385 390 395 400 Glu Asn Ser Glu Trp Arg Asp Lys Val Val Leu Val Gln He Ala Val 405 410 415 Pro Thr Arg Thr Asp Val Leu Glu Tyr Gln Lys Leu Thr Ser Gln Val 420 425 430 His Glu He Val Gly Arg He Asn Gly Arg Phe Gly Ser Leu Thr Ala 435 440 445 Val Pro He His His Leu Asp Arg Ser Met Lys Phe Pro Glu Leu Cys 450 455 460 Wing Leu Tyr Wing He Thr Asp Val Leu Leu Val Thr Ser Leu Arg Asp 465 470 475 480 Gly Met Asn Leu Val Ser Tyr Glu Phe Val Wing Cys Gln Lys Asp Lys 485 490 495 Lys Gly Ala Leu He Leu Ser Glu Phe Ala Gly Ala Ala Gln Ser Leu 500 505 510 Gly Ala Gly Ser He Leu He Asn Pro Trp Asn He He Glu Be Ser 515 520 525 Asn Wing He Wing Asp Wing Leu Asn Met Pro Glu Glu Glu Arg Glu Glu 530 535 540 Arg His Arg His Asn Phe Met His He Thr Thr His Ser Wing Gln Val 545 550 555 560 Trp Wing Glu Thr Phe He Ser Glu Leu Asn Asp Ser He Leu Glu Wing 565 570 575 Glu Leu Arg Thr Leu His He Pro Pro Gln Leu Pro Leu Asp Lys Wing 580 585 590 Val Wing Lys Tyr Ser Glu Ser Lys 595 600

Claims (23)

1. An isolated and purified DNA fragment, characterized in that it consists of the sequence SEQ ID NO: 1 or a functionally equivalent mutation, which codes for a bifunctional polypeptide with activity of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.
2. A DNA fragment according to clause 1, characterized in that it is isolated from a plant.
3. A DNA fragment according to clause 2, characterized in that the plant from which it is isolated is preferably the Selaginella lepi dophyl 1 a.
4. A DNA fragment according to clause 1, characterized in that it encodes a bifunctional polypeptide (SL-TPS / P) with trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase activity with an amino acid sequence of SEQ ID NO: 2 or a functionally equivalent mutation of SEQ ID NO: 2.
5. An isolated and purified DNA fragment characterized in that it consists of the sequence SEQ ID NO: 3 or a functionally equivalent mutation, which codes for a polypeptide with trehalose-6-phosphate synthase activity.
6. A DNA fragment according to clause 5, characterized in that it is isolated from a plant.
7. A DNA fragment according to clause 6, characterized in that the plant from which it is isolated is preferably the Selaginella lepi dophyl 1 a.
8. A DNA fragment according to clause 5, characterized in that it encodes an active polypeptide (SL-TPS) with trehalose-6-phosphate synthase activity with an amino acid sequence SEQ ID NO: 4 or functionally equivalent mutations of SEQ ID NO. : 4.
9. A gene characterized in that it comprises in its sequence the DNA fragment of clause 4, which codes for the SL-TPS / P polypeptide or a functionally equivalent mutation.
10. A gene characterized in that it comprises in its sequence the DNA fragment of clause 8, which codes for the SL-TPS polypeptide or a functionally equivalent mutation.
11. A molecular expression vehicle characterized in that it comprises the DNA sequence of clause 1.
12. A molecular expression vehicle characterized in that it comprises the DNA sequence of clause 5.
13. A genetically modified host, characterized by containing the DNA sequence of clause 1 or a functionally equivalent mutation.
14. A genetically modified host, characterized by containing the DNA sequence of clause 5 or a functionally equivalent mutation.
15. A genetically modified host according to any of clauses 13 or 14, characterized in that said host is a plant or a part thereof, a yeast or a bacterium.
16. A genetically modified host, according to clause 15, characterized by containing high levels of trehalose.
17. A genetically modified host, according to clause 15, characterized in that it is more resistant to cold, heat and water stress, than the same unmodified host.
18. A genetically modified host, according to clause 15, characterized in that it can produce products with increased shelf life, once dehydrated.
19. A genetically modified host, according to clause 15, characterized in that it exhibits increased viability in the cells or reproductive organs.
20. An SL-TPS / P polypeptide, characterized by having an amino acid sequence SEQ ID NO: 2 or functionally equivalent modifications.
21. An SL-TPS / P polypeptide according to clause 20, characterized as being bifunctional having both trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase activity for the production of trehalose.
22. An SL-TPS polypeptide, characterized by having an amino acid sequence SEQ ID NO: 4 or equivalent functional mutations.
23. An SL-TPS polypeptide according to clause 22, characterized by having an activity of trehalose-6-phosphate synthase.
MX9601719A 1996-05-08 1996-05-08 METHOD OF INCREASING TREHALOSE IN ORGANISMS BY TRANSFORMATION OF SELAGINELLA LEPIDOPHYLIA TREHALOSE-6-PHOSPHATE SINTASE/PHOSPHATASE cDNA. MX205414B (en)

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Application Number Priority Date Filing Date Title
MX9601719A MX205414B (en) 1996-05-08 1996-05-08 METHOD OF INCREASING TREHALOSE IN ORGANISMS BY TRANSFORMATION OF SELAGINELLA LEPIDOPHYLIA TREHALOSE-6-PHOSPHATE SINTASE/PHOSPHATASE cDNA.
EP97923309A EP0933428A2 (en) 1996-05-08 1997-05-06 METHOD FOR INCREASING THE CONTENT OF TREHALOSE IN ORGANISMS THROUGH THE TRANSFORMATION THEREOF WITH THE cDNA OF THE TREHALOSE-6-PHOSPHATE SYNTHETASE/PHOSPHATASE OF SELAGINELLA LEPIDOPHYLLA
PCT/MX1997/000012 WO1997042327A2 (en) 1996-05-08 1997-05-06 METHOD FOR INCREASING THE CONTENT OF TREHALOSE IN ORGANISMS THROUGH THE TRANSFORMATION THEREOF WITH THE cDNA OF THE TREHALOSE-6-PHOSPHATE SYNTHETASE/PHOSPHATASE OF SELAGINELLA LEPIDOPHYLLA
BR9710436A BR9710436A (en) 1996-05-08 1997-05-06 Process to increase trehalose content in organisms by transforming them with selaginella lepidophylla trehalose-6-phosphate synthesis-phosphate cdna
JP9539760A JP2000509602A (en) 1996-05-08 1997-05-06 Method for increasing trehalose content in organisms by transformation with cDNA of trehalose-6-phosphate synthase / phosphatase from Seraginella lepidophila
AU29135/97A AU727509B2 (en) 1996-05-08 1997-05-06 Method for increasing the trehalose content in organisms by means of their transformation with the cDNA of trehalose- 6-phosphate synthase/phosphatase from selaginella lepidophylla

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MX9601719A MX205414B (en) 1996-05-08 1996-05-08 METHOD OF INCREASING TREHALOSE IN ORGANISMS BY TRANSFORMATION OF SELAGINELLA LEPIDOPHYLIA TREHALOSE-6-PHOSPHATE SINTASE/PHOSPHATASE cDNA.

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MX9601719A MX9601719A (en) 1997-11-29
MXPA96001719A true MXPA96001719A (en) 1998-07-03
MX205414B MX205414B (en) 2001-12-07

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IN1997CH00924A (en) 1996-05-03 2005-03-04 Syngenta Mogen Bv Regulating metabolism by modifying the level of trehalose-6-phosphate
TW466116B (en) 1997-03-04 2001-12-01 Hayashibara Biochem Lab Reduction inhibitory agent for active-oxygen eliminating activity, method for inhibiting the reduction of said activity, and composition containing said agent
WO1999023225A1 (en) * 1997-10-30 1999-05-14 Mogen International N.V. Novel high-fermenting microorganisms
WO1999024558A2 (en) * 1997-10-30 1999-05-20 Mogen International N.V. Novel high-fermenting microorganisms
HUP0102598A3 (en) * 1998-03-11 2003-03-28 Syngenta Participations Ag Expression of trehalose biosynthetic genes in plants
US7345217B2 (en) 1998-09-22 2008-03-18 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
EP1002867A1 (en) * 1998-10-15 2000-05-24 K.U. Leuven Research & Development Specific genetic modification of the activity of trehalose-6-phosphate synthase and expression in a homologous or heterologous environment
AU1768201A (en) * 1999-11-17 2001-05-30 Robert Creelman Seed trait genes
EP2175028A3 (en) * 2000-04-07 2010-07-14 BASF Plant Science GmbH Phosphatase stress-related proteins and methods of use in plants
US8426678B2 (en) 2002-09-18 2013-04-23 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
EP1375669A1 (en) * 2002-06-13 2004-01-02 Stichting Voor De Technische Wetenschappen Method for enhancing the disease resistance in plants by altering trehalose-6-phosphate levels
EP1873247A1 (en) * 2006-06-29 2008-01-02 VIB vzw Novel bifunctional trehalose synthase
CN101978065B (en) 2008-02-29 2014-07-23 孟山都技术公司 Corn plant event mon87460 and compositions and methods for detection thereof
CN103739548B (en) * 2013-12-27 2015-08-05 南京中医药大学 Compound with anti-mycotic activity and preparation method thereof and application

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