WO1997042327A2 - MÉTODO PARA INCREMENTAR EL CONTENIDO DE TREHALOSA DE LOS ORGANISMOS POR MEDIO DE SU TRANSFORMACIÓN CON EL ADNc DE LA TREHALOSA-6-FOSFATO SINTASA/FOSFATASA DE SELAGINELLA LEPIDOPHYLLA - Google Patents
MÉTODO PARA INCREMENTAR EL CONTENIDO DE TREHALOSA DE LOS ORGANISMOS POR MEDIO DE SU TRANSFORMACIÓN CON EL ADNc DE LA TREHALOSA-6-FOSFATO SINTASA/FOSFATASA DE SELAGINELLA LEPIDOPHYLLA Download PDFInfo
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- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
Definitions
- 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 it, the enzyme (s) involved in the synthesis of specific carbohydrates, as well. as 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 food or ingredients, having properties enhanced by the presence of said carbohydrates.
- solutes compatible with metabolism in various organisms such as bacteria, algae, yeasts, fungi, insects, crustaceans and plants, emerged in Evolution as an adaptive mechanism to contend with water shortages, salinity or freezing due to properties osmoregulatory ⁇ of said solutes.
- osmotically active compounds are sorbitol, mannitol, arabitol or glycerol polyols; amino acids such as proline or the glycine-betaine derivative; and sucrose and trehalose disaccharides
- trehalose has the best biophysical and biochemical properties as an osmoprotector [Crowe, JH, Crowe, LM & Chapman D. (1984) Science 223: 701-703; Crowe, JH, Crowe, LM, Carpenter, JF & Wistrom, A. (1987) Biochem. J. 242: 1-10].
- Trehalose O-alpha-D-glucopyranosyl- (1-1) -alpha-D-glucopyranoside
- reducing groups are absent in the trehalose, so it does not take part in the Maillard reactions, in which the sugar reducing group reacts with the amino group of the proteins causing darkening and change in the smell and taste of food [Nursten, HE (1986) Maillard browning reactions in dried foods, pp. 53-68. In: Concentration and drying of foods. Macarthy, D. (Ed.). Elsevier Applied Science, London].
- trehalose is capable of preserving the activity of enzymes maintained under total dehydration. For example, if 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
- carrot or tobacco cells can be cryopreserved in the presence of trehalose [Bhandal, I.S.,
- Trehalose concentrations below 2mM have been reported to be much more efficient than sucrose to cryopreserve spinach-isolated thylakoids [Hincha, D.K. (1989) Biochem. Biophys Minutes 187: 231-234].
- trehalose Among the organisms that synthesize trehalose are bacteria, yeasts, some fungi, spores, nematodes, crustacean larvae, insects and plants, particularly some resurrection such as Myrothamnus flabellifolius and Selaginella lepidophylla [Adams, RP, Kendall, E. & Kartha, KK (1990) Biochem. Systematics & Ecology 18: 107-110; Bianchi, G., Gamba, A., Murelli, C., Salamini, F. & Bartels, D. (1993) Physiol. Plant Stl: 223-226; Weisburd, S. (1988) Sci. News 133: 107-110].
- trehalose-6-phosphate synthase which produces trehalose-6-phosphate from glucose-6-phosphate and UDP-glucose
- trehalose -6-phosphate phosphatase which ultimately produces trehalose
- otsA or otsB genes from E. coli, which code for trehalose-6-phosphate synthase and phosphatase, respectively, causes loss of osmotolerance and thermotolerance.
- 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. 174: 889-898; Hengge-Aronis, R., Klein, W., Lange, R., Rimmele, M. & Boos, W. (1991) J. Bacteriol. 173 .: 7918-7924].
- the trehalose-6-phosphate synthase / phosphatase oligomer 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.
- the TPS1 subunit appears to be activated by dephosphorylation [Panek, AC, de Araujo, PS, Neto, MV & Panek, AD (1987) Curr. Genet 11: 459-465].
- dephosphorylation Panek, AC, de Araujo, PS, Neto, MV & Panek, AD (1987) Curr. Genet 11: 459-465.
- 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 dried. Due to the above, it does not appear to have a mechanism of posttranslational or metabolic regulation that limits its enzymatic activity, otherwise the TPS1 of yeast.
- sl-tps / p is a single copy gene in S. lepidophylla, the synthesis of trehalc-. in this plant it is exclusively due to the enzyme coding (SL-TPS / P) for said gene.
- trehalose encompasses the agri-food and pharmaceutical industry.
- 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 temporary and arid or semi-arid agriculture.
- irrigation agriculture will also be limited. Under these conditions, the decrease in water use would not affect the yield and productivity of crops that synthesize trehalose.
- plants or parts of them that synthesize trehalose can 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 your market as it will reduce transportation and storage costs.
- Trehalose is an additive that has the unique property of preserving food in a dehydrated state and preserving its properties of taste, smell and consistency when rehydrated. Due to the above, processed foods by incorporating trehalose in their preparation will be considered fresh when rehydrated.
- the enzyme trehalose which degrades trehalose into two glucose molecules, is found in the digestive tract of mammals and humans. Since a range of foods consumed daily, such as honey, mushrooms, bread, beer, wine and vinegar contain trehalose [Roser, B. & Cola ⁇ o, C. (1993) New Scient. 123.: 25-28], the consumption of this disaccharide in other foods does not present a health risk [Gudmand-Hoyer, E. (1994) Am. J. Clin. Nutr. 5_2 (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.
- the invention described herein consists in the isolation, cloning and overexpression in transgenic organisms of a cDNA of a gene of plant origin that encodes the trehalose-6-phosphate synthase / phosphatase activity, particularly the sl-tps / p gene that encodes for the bifunctional enzyme trehalose-6-phosphate synthase / phosphatase (SL-TPS / P) of the Selaginella lepidophylla resurrection plant.
- This enzyme allows high levels of trehalose to be obtained in transgenic plants.
- plants are tolerant of heat, cold, salinity and water scarcity.
- ⁇ l-tps / p gene can also be used to synthesize massive amounts of trehalose in microorganisms, isolated cells and whole organisms of plants or animals, which have been transformed with said gene.
- FIG. 1 Structure of the SL-TPS / P enzyme of S. lepidophylla
- the scheme shows the scale size (in kD) of SL-TPS / P and its structure based on the similarity in amino acid sequence with respect to TPS1 and yeast TPS2. Diagonal stripes and the grid denote similar regions Figure 2. Restriction map of the cDNA of the sl-tps / p gene. The restriction sites on this map were confirmed with the nucleotide sequence. The cut-off sites for restriction enzymes are shown at the top of the scheme: Eco RI (R), Eco RV (E), Hind III (H), Sac I (Se), Salt I (S).
- FIG. 3 Schematic representation of plasmid pIBT6.
- the abbreviations mean: Amp, gene that confers ampicillin resistance; lac Z, promoter of the ⁇ -Galactosidase gene; sl ⁇ tps / p, cDNA of trehalose-6-phosphate synthase / phosphatase; T3 and T7 are promoters of phage T3 and T7 for in vitro transcription.
- Plasmid pIBT6 has an approximate size of 6150 bp.
- the arrows indicate the direction of the transcript.
- Figure 4. Schematic representation of the expression vector pBN35. The abbreviations mean: RB and LB, right and left borders, respectively, of the T-DNA of the Ti plasmid of Agrobacterium turne fasciens; Kan, a gene that confers resistance to Kanamycin to select in E.
- Plasmid pBN35 has an approximate size of 12 kb. The arrows indicate the direction of the transcript. Fi ⁇ ura 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; Kan * , a gene that confers resistance to Kanamycin to select in E. coli; pNOS, promoter of the nopaline synthase gene; NPT II, a 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 plBT36 has an approximate size of 15.2 kb. The arrows indicate the direction of the transcript.
- the cDNA encoding the trehalose-6-phosphate synthase / phosphatase bifunctional enzyme is referred to 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;
- SL-TPS is the polypeptide with trehalose-6-phosphate synthase activity.
- TPS5 '-3 (MGNYTNGAYTAYWBNAARGGNB-TNCC), TPS3' -1
- SWNACNARRTTCATNCCRTCNCK SWNACNARRTTCATNCCRTCNCK
- TPS3 '-2 CCRWANTKNCCRTTDATNCKNCC
- Plasmid DNA was extracted from the colonies that grew in ampieilin and cut with Eco RI and Xho I enzymes to isolate the corresponding insert. After performing 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, that clone containing the largest insert was selected ( Figure 2) to create deletions and determine their 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 homology region 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 termination codon) that codes for a polypeptide called SL-TPS / P (SEQ ID NO: 2) of 994 amino acids with a molecular weight of 109.1 kD.
- the sequence includes 32 nucleotides of the poly-A tail.
- slps / 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 cDNA clone.
- the context of initiation codon in SL-TPS / P corresponds to that reported in plants [Lütcke, HA, Chow, KC, Mickel, FS, Moss, KA,
- 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 Schizosaccharomyces por ⁇ be yeast, which consists of 570 amino acids.
- These 600 amino acids constitute per se a trehalose-6-phosphate synthase which is here referred to as SL-TPS
- the percentage of identity between SL-TPS / P and TPS1 or TPS2 is within the expected range, given the phylogenetic relationship between plants and fungi, for genes encoding polypeptides with the same function.
- the expression pattern of said gene was determined using Northern type gels, evaluating the levels of specific transcript under normal conditions and water deficit.
- Northern type gels For the analysis of the expression of the sl-tps / p gene in a Northern type gel, in each lane of the gel 2 ⁇ g of
- RNA extracted from S microphones Lepidophylla hydrated, dehydrated for 2.5 hours, 5 hours and one year.
- the ⁇ l-tps / p cDNA radiolabelled with 32 P was used.
- a 3.2 kb band can be observed in both hydrated and dehydrated microphones at different intervals. This result demonstrates a constitutive expression of the sl-tps / p gene, which is in accordance with the observation made previously by other authors [Adams, RP, Kendall, E. & Kartha, KK (1990) Biochem.
- a genomic Southern was performed. 20 ⁇ g of S genomic DNA was loaded onto each lane. lepidophylla cut with the following restriction enzymes: Eco RI, Xba I or Bam HI. As a probe, the cDNA of SL-tps radioactively labeled with 32 P was used. In the autoradiogram of said experiment, two bands, one of 1.6 and another 6 kb are observed when cutting the DNA with the enzyme Eco RI; a 9 kb band when cutting with Xba I; and two bands of 11 and 12 kb when digesting the DNA with Bam HI.
- the vector pBN35 allows to express any gene under the control of the 35S promoter of cauliflower CaMV virus [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 2_1 : 285-294] which is a strong and constitutive promoter.
- Plasmid pIBT36 was used to obtain tobacco plants, transformed through the Agrobacterium system, which when regenerated were able to produce trehalose. This construction can be expressed in any plant that can be transformed, using the Agroibacteriujn system or by any other method that is known in the state of the art.
- transgenic plants From 31 obtained transgenic plants the expression of the ⁇ l-tp ⁇ / p cDNA was detected in 16 plants, 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 vector pBN35.
- Trehalose as an additive in food and for the conservation of biomolecules and other substances of medical interest, can be obtained at a more attractive price through its mass production in transgenic organisms, such as yeasts and mass production in transgenic organisms, such as yeasts and transgenic plants.
- the present invention gives rise to a strategy for the mass production of trehalose in plants that includes the transformation with the cDNA of sl-tps / po of ⁇ l-tps (in suitable constructions) of tobacco plants, which in addition to being one of the Vegetable models more worked in molecular biology have 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 biomass yield per hectare, which at the same time, being the potato a product of natural consumption of man, would lead to trehalose with greater acceptance both by the public and by regulatory bodies.
- the present invention provides a way to obviate these problems, by using the ⁇ l-tps / po cDNA of sl -tps in suitable constructions involving Yip or multicopy integrative vectors, taking advantage of the fact that, as already stated above, that the SL-TPS / P does not appear to have a posttranslational or metabolic regulation mechanism that limits its enzymatic activity and is also capable of directing the production of high levels of trehalose. This will allow not only the mass production of trehalose, but also improve the yields of yeasts in baking and in the alcoholic fermentation of beer and wine.
- the present invention provides a means to achieve such resistance, through the use of the ⁇ l-tps / po sl-tps cDNA in constructions containing either constitutive promoters or promoters that induce their expression only under stress.
- Harvested products, in addition to having been grown under conditions where they had not normally been achieved, may be stored for longer periods or under extreme conditions unlike products obtained with non-transformed plants.
- tissue-specific or organ-specific, organs or tissues are produced for reproduction, such as pollen, tubers, seeds and flowers that can be preserved for longer periods with greater viability, which would be a great help for breeding programs and germplasm conservation.
- the reagents used were analytical grade Baker or Sigma brand. 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. It was dehydrated in rocky soils of arid areas in the States of Morelos and Oaxaca of the Mexican Republic. It was subsequently cultivated under controlled conditions (24 S C and 16 h. Of light with 50% average humidity) in Conviron growth chambers or in the greenhouse. The plants were watered every third day with a volume of water of 20 ml for 2 L pots. To subject water stress to S. lepidophylla, 2L pots. To subject water stress to S. lepidophylla, the whole plant or fronds of microphylase, they were air dried by placing them on Whatman 3MM filter paper. From this moment the dehydration time was determined.
- the cDNA bank was plated in the E. coli XLl-Blue MRF 'strain and the SOLR strain was used to cleave the pBluescript from the lambda phage as described in the "ZAP-cDNA Synthesis Kit" manual [Stratagene Cloning Systems, Calif., USA; # catalog 200400, 200401 and 2004029].
- the E. coli strain DH5 alpha was used to subclone and make constructs.
- the strain of A. tumefascien ⁇ LBA4404 was used to transform tobacco and the strain of E. coli HB101, carrying the plasmid pRK2013 [Bevan, M. (1984) Nuc ⁇ . Acids Res.
- the pBN35 expression vector is a derivative of pBinl9 [Bevan, M. (1984) Nuc ⁇ . Acids Res. 22: 8711-8721], which was constructed by subcloning the 850 bp of the 35S promoter of cauliflower CaMV virus [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 21: 285-294] between the Hind III and Sal I sites of pBinl9, and the 260 bp fragment that constitutes the poly-adenylation signal of the nopaline synthetase gene of T-DNA [Bevan, M., Barnes, W. & Chilton, M.-D. (1983) Nuc ⁇ . of T-DNA [Bevan, M., Barnes, W. & Chilton, M.-D. (1983) Nuc ⁇ .
- Plasmid pIBT36 ( Figure 5) was constructed by subcloning the sl-tps / p cDNA into the Bam HI and Kpn I sites of the pBN35 expression vector.
- an expression bank was prepared, from mRNA isolated from dehydrated S. lepidophylla microphones for 2.5 h., Using the ZAP cDNA synthesis kit, the Uni-ZAP XR vector and packaging extracts. Gigapack II Gold, 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 S. lepidophylla microphiles, dehydrated for 2.5 h. , according to a known method [Chomczyniski, P. & Sacchi, N.
- the initial titer of the bank was 2 x 10 'bacteriophage plates / ml and after amplification it was 1.5 x 10 11 bacteriophage plates / ml.
- Plasmid pBluescript SK (-) was cleaved from the bacteriophage by means of the technique known as "zapping" according to
- genomic Southern the DNA was fractionated on a 0.8% agarose gel in TBE buffer and transferred to a Hybond N * (Amersham Life Sciences) nylon membrane.
- Trehalose was determined by the degradative method with trehalose [Araujo, P.S., Panek, A.C., Ferreira, R. & Panek, A.D. (1989) Anal. Biochem 176: 432-436].
- 500 mg of fresh tissue or 50 mg of dry tissue were ground 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 were boiled for 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 with the same volume of 80% ethanol and the tablet was dried under vacuum. Samples were resuspended in 0.250 ml of 50mM PBS at pH 6.5.
- trehalose For the determination of trehalose, 10 to 30 ⁇ l of extract were taken, 4 ⁇ l (ca 15 mU) of trehalose (Sigma no. Cat. T-8778) was added and incubated for 2 hrs at 30 S C. As a control negative, a tube was placed with extract but without trehalose and as a positive control a tube was placed with pure trehalose (Sigma no. Cat. T-3663). The volume was brought to 0.5 ml with 50 mM PBS, pH 7.0 and 0.5 ⁇ l of glucose oxidase and peroxidase from Sigma no. cat. 510-A, for glucose determination. It was incubated 40 min at 37 at C and the optical density was determined at 425 nm immediately.
- 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 trehalose to be produced by means of recombinant DNA techniques in cells or organisms transformed with the sl-tps / p cDNA clone.
- the appropriate transformation methods and vectors to be used in Microorganisms and plants are known by those familiar with the state of the art.
- Selaginella lepidophylla dehydrated resurrection plants were collected from rocky soils of arid areas in the States of Morelos and Oaxaca of the Mexican Republic. Subsequently they were grown in pots of 2L at 24 S C with 16 hr of light and an average humidity of 50% in Conviron growth chambers. The plants were watered every third day with 20 ml of water.
- an expression bank was prepared, from 5 ⁇ g of mRNA isolated from 50 g of dehydrated S. lepidophylla microphiles for 2.5 hr. After synthesizing the cDNA, it was cloned using 1 ⁇ g of the Uni-ZAP XR vector. Bacteriophages were packaged in vitro and subsequently screened with a mixture of degenerate oligonucleotides encoding consensus regions in trehalose-6-phosphate synthase and phosphatase from the reported sequences of E. coli and yeast. One of the isolated clones corresponds to a cDNA ( ⁇ -tps / p) with the entire coding region.
- Tobacco cells (Nicotiana tabacum var. SRl) were transformed by the leaf disc method, using Agrobacterium tumefasciens LBA4404 containing the plasmid pIBT36.
- Leaf discs were grown in Petri dishes containing MS medium with vitamins, 0.1 ppm of NAA, 1 ppm of
- BAP 10 ⁇ g / ml of kanamycin and 200 ⁇ g / ml of carbenicillin to regenerate outbreaks in 4 to 6 weeks.
- the shoots were transferred to MS medium with 100 ⁇ g / ml of kanamycin and 200 ⁇ g / 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 S C and with 16 hours of light to obtain fertile plants after 4 to 6 weeks.
- the ⁇ l-tps / p cDNA must be subcloned into a JE vector. coli, 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 [Romanos, M.A. ,
- the construction must contain a transcription termination signal (ter) that is located at the 3 'end of the ⁇ 1-tps / p cDNA.
- This fusion, sl-tp ⁇ / p / ter promoter / DNA, is subcloned into a YIp vector containing the 5 'and 3' regions of the ura3 gene, so that the fusion is integrated between them, in addition to the leu2 marker gene.
- the resulting construct is transformed into a strain of yeast with ura3 'and leu2 genotype, to select recombinants properly.
- the innovation described here is that the recombinant S. cerevisiae strain will express the SL-TPS / P enzyme 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 SL-TPS / P activity is not subject to post-translational regulation.
- the synthesis of trehalose at high levels in yeast will improve the performance in baking and alcoholic fermentation of beer and wine.
- the trehalose obtained by this method can be purified for others 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, OJM & Pen, J . (1995) Trends Biotech. H: 379-387].
- Constructs for expression in cDNA plants of ⁇ l-tp ⁇ / p can be made in a vector derived from the Ti plasmid lacking the T-DNA tumorigenic genes and containing a selection marker for the transformed plants conferred , for example, resistance to kanamycin [Bevan, M. (1984) Nuc ⁇ . Acids Res. 22 . : 8711-8721].
- a suitable promoter should be selected, depending on the use required of transgenic plants.
- the nopalin synthetase gene of T-DNA can be used as a poly-adenylation signal [Bevan, M., Barnes, W. & Chilton, M.-D. (1983) Nuc ⁇ . Acids Res. 11: 369-385].
- tuber promoters such as the one of the patatin-class 1 gene [Bevan, M.,
- plasmid pIBT36 For overexpression of trehalose in tobacco, plasmid pIBT36 would be used where the ⁇ l-tp ⁇ / p gene is under the control of the 35S promoter of cauliflower virus [Guilley, H., Dudley, K., Jonard, G., Richards, K., & Hirth, L. (1982) Cell 21: 285-294].
- Cereals that constitute the basis of world food could be grown 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 cDNA of ⁇ -tp ⁇ / p under the control of promoters that are induced by any of these environmental factors [Baker, SS, Wilhelm, KS & Thomashow, MF (1994) Plant Mol. Biol. 24: 701-713; Takahashi, T., Naito, S. & Komeda, Y. (1992) Plant J. 2: 751-761; Yamaguchi-Shinozaki, K. & Shinozaki, K. Plant J. 2: 751-761; Yamaguchi-Shinozaki, K. & Shinozaki, K.
- Example 6 Fruits of transgenic plants with longer shelf life
- Various fruits such as tomatoes, mango and plantain, ripen quickly and are exposed to rot before reaching the hands of the consumer.
- the early harvest of the fruits and their refrigerated storage or in controlled environment chambers has been traditionally used to avoid the aforementioned problem.
- these methods are expensive, especially if it is required to transport the fruits to distant sites.
- transgenic plants that express antisense to the polygalacturonase gene, which is involved in fruit ripening [Smith, CJS, Watson, CF, Ray, J., Bird, CR, Morris, PC, Schuch, W. & Grierson, D.
- Tomatoes or other fruits could be harvested ripe to then be completely or partially dried and preserved for long periods without refrigeration. When rehydrated, the fruits would have the normal organoleptic properties that the consumer demands.
- the strategy described above can be implemented for other fruits provided that a regeneration and transformation system is available for the plant of interest and that there is an appropriate promoter.
- ORGANISM Sellaginella lepidophylla
- D STATE OF DEVELOPMENT: adult
- F TYPE OF FABRIC: microphiles
- NAME RBS (Chromosome binding site)
- NAME ⁇ l -tp ⁇ / p
- LOCATION 111 to 3095 base pairs •
- C IDENTIFICATION METHOD: by similarity with known sequences
- D OTHER INFORMATION: encodes a bifunctional enzyme, trehalose-6 -phosphate synthase and trehalose- 6-phosphate phosphatase
- Trp lie Gly Trp Pro Gly Val Tyr Val Gln Asp Glu Lys Gly Glu Ly ⁇
- ORGANISM Selaginella lepidophylla
- NAME ⁇ L-TPS / P
- LOCATION 1 to 994 amino acids
- C IDENTIFICATION METHOD: by im im i tud with known sequences
- HYPOTHETICS no IV.
- ANTI-SENSE no
- ORGANISM Selaginella lepidophylla
- D STATE OF DEVELOPMENT: adult
- F TYPE OF FABRIC: microphiles VII.
- NAME RBS (Chromosome binding site)
- B LOCATION: 1 to 110 base pairs IX.
- ORGANISM Selaginella lepidophylla
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- General Preparation And Processing Of Foods (AREA)
- Seasonings (AREA)
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97923309.5A EP0933428A1 (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 (pt) | 1996-05-08 | 1997-05-06 | Processo para aumentar o teor de trealose em organismos por meio de sua transforma-Æo com o cdna da sintese-fosfatase de trealose-6-fosfato de selaginella lepidophylla |
JP9539760A JP2000509602A (ja) | 1996-05-08 | 1997-05-06 | セラギネラ・レピドフィラ由来のトレハロース―6―リン酸シンターゼ/ホスファターゼのcDNAで形質転換することによる、生物中のトレハロース含量の増加法 |
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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MX961719 | 1996-05-08 | ||
MX9601719A MX205414B (es) | 1996-05-08 | 1996-05-08 | Metodo para incrementar de trehalosa de los organismos por medio de su transformacion con el adnc de la trehalosa-6-fosfato sintasa/fosfatasa de selaginella lepidophylla. |
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WO1997042327A2 true WO1997042327A2 (es) | 1997-11-13 |
WO1997042327A3 WO1997042327A3 (es) | 1998-01-08 |
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PCT/MX1997/000012 WO1997042327A2 (es) | 1996-05-08 | 1997-05-06 | MÉTODO PARA INCREMENTAR EL CONTENIDO DE TREHALOSA DE LOS ORGANISMOS POR MEDIO DE SU TRANSFORMACIÓN CON EL ADNc DE LA TREHALOSA-6-FOSFATO SINTASA/FOSFATASA DE SELAGINELLA LEPIDOPHYLLA |
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JP (1) | JP2000509602A (es) |
AU (1) | AU727509B2 (es) |
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WO (1) | WO1997042327A2 (es) |
Cited By (12)
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WO1997042326A2 (en) * | 1996-05-03 | 1997-11-13 | Mogen International N.V. | Regulating metabolism by modifying the level of trehalose-6-phosphate |
EP0868916A2 (en) * | 1997-03-04 | 1998-10-07 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Reduction inhibitory agent for active-oxygen eliminating activity |
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 |
WO1999046370A2 (en) * | 1998-03-11 | 1999-09-16 | Novartis Ag | Expression of trehalose biosynthetic genes in plants |
WO2000022141A2 (en) * | 1998-10-15 | 2000-04-20 | K.U. Leuven Research & Development | Specific genetic modification of the activity of trehalose-6-phosphate synthase and expression in a homologous or heterologous environment |
WO2002046442A2 (en) * | 2000-04-07 | 2002-06-13 | Basf Plant Science Gmbh | Phosphatase stress-related proteins and methods of use in plants |
EP1873247A1 (en) * | 2006-06-29 | 2008-01-02 | VIB vzw | Novel bifunctional trehalose synthase |
EP2133360A3 (en) * | 1999-11-17 | 2010-03-03 | Mendel Biotechnology, Inc. | Environmental stress tolerance genes |
US8426678B2 (en) | 2002-09-18 | 2013-04-23 | Mendel Biotechnology, Inc. | Polynucleotides and polypeptides in plants |
US8809630B2 (en) | 1998-09-22 | 2014-08-19 | Mendel Biotechnology, Inc. | Polynucleotides and polypeptides in plants |
US9228197B2 (en) | 2008-02-29 | 2016-01-05 | Monsanto Technology Llc | Corn plant event MON87460 and compositions and methods for detection thereof |
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-
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- 1997-05-06 AU AU29135/97A patent/AU727509B2/en not_active Ceased
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Also Published As
Publication number | Publication date |
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JP2000509602A (ja) | 2000-08-02 |
EP0933428A2 (en) | 1999-08-04 |
WO1997042327A3 (es) | 1998-01-08 |
MX205414B (es) | 2001-12-07 |
AU2913597A (en) | 1997-11-26 |
MX9601719A (es) | 1997-11-29 |
AU727509B2 (en) | 2000-12-14 |
BR9710436A (pt) | 1999-08-17 |
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