WO1996007743A1 - Beta-galactosidase specifique au xyloglucane - Google Patents

Beta-galactosidase specifique au xyloglucane Download PDF

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WO1996007743A1
WO1996007743A1 PCT/GB1995/002098 GB9502098W WO9607743A1 WO 1996007743 A1 WO1996007743 A1 WO 1996007743A1 GB 9502098 W GB9502098 W GB 9502098W WO 9607743 A1 WO9607743 A1 WO 9607743A1
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sequence
die
plant
xyloglucan
nucleic acid
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PCT/GB1995/002098
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Sumant Chengappa
Susan Amanda Hellyer
Jacqueline De Silva
John Spence Grant Reid
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Unilever Plc
Unilever Nv
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Priority to AU33959/95A priority Critical patent/AU3395995A/en
Publication of WO1996007743A1 publication Critical patent/WO1996007743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Definitions

  • This invention relates to the nucleotide and amino acid sequences of a plant enzyme and to methods of using the same.
  • Xyloglucan-specif ⁇ c- ⁇ -galactosidase is an enzyme that catalyses the hydrolysis of terminal galactose residues from polymeric xyloglucan.
  • xyloglucan In nasturtium seeds, xyloglucan is the major component of secondary cell walls and performs a storage function. The major ⁇ - galactosidase activity reaches its peak just prior to the most rapid phase of storage xyloglucan mobilization and is thought to play an important role in this process. Hydrolysis of xyloglucan has also been implicated in growth related processes, fruit ripening and in d e generation of biologically active xyloglucan fragments (oligosaccharins).
  • Xyloglucan is the major non-cellulosic polysaccharide in the primary cell wall of dicotyledonous plants.
  • Xyloglucans are 1 ,4- 3-glucans mat are extensively substituted wi ⁇ ⁇ -l,6-xylosyl side chains, some of which are 1,2 /3-galactosylated.
  • primary cell walls some of the galactose residues are ⁇ -l,2-fucosylated.
  • the type and degree of substitution of me xyloglucan backbone varies between species.
  • Xyloglucan adapted to a storage function in seeds like nasturtium and tamarind are totally non-fucosylated.
  • Xyloglucan in the primary cell wall is found tightly hydrogen bonded to cellulose microfibrils and forms bridges between mem.
  • me xyloglucan in vitro extraction of me xyloglucan from me cell wall causes me cellulose microfibrils to collapse suggesting iiat xyloglucan is important in maintaining me spacing between microfibrils, and therefore important for wall assembly (McCann & Roberts, 1991, "Architecture of die primary cell wall", In; The Cytoskeletal Basis of Plant Grow and Form. Ed. Lloyd, C. London Academic Press Ltd, pp 109-129).
  • Xyloglucan modification occurs during different physiological processes like growm (Labavitch and Ray, 1974 Plant Physiol. 54. 105-122) and fruit ripening (Huber, 1983 J. Amer. Soc. Hort. Sci. .108, 405-409). Labavitch and Ray (1974) have shown that the mean molecular weight of xyloglucan reduces during auxin induced growm of pea epicotyl segments. Sakurai and Nevins (1993 Physiol. Plant. 89, 681-686) have demonstrated that me molecular mass of hemicellulose of red tomato fruit walls is reduced to 50% of diat in green fruits, and die decrease in average molecular mass is associated primarily with me degradation of xyloglucans.
  • xyloglucan is a storage polysaccharide that is rapidly mobilized during me process of seed germination providing a reserve energy supply for d e germinating seedling.
  • Enzymes involved in me process of metabolizing seed xyloglucan would be expected to play a crucial role in me modification and hydrolysis of xyloglucan in omer tissues. Some of these enzymes have been purified from various tissues.
  • the xyloglucan endo-transglycosylase (XET) has been purified to homogeneity and me corresponding cDNA isolated from germinating nas rtium cotyledons (Edwards et. al., 1986, de Suva et.
  • Xyloglucan endo-transglycosylase has been proposed to be responsible for enabling turgor driven cell expansion by me cleavage and reformation of xyloglucan polymers (Fry 1989, Analysis of cross-links in the growing cell wall of higher plants. In Modern Memods in Plant Analysis, New Series, vol.10 [ed. H.S. Linskens and J.S. Jackson], pp 1-42. Springer- Verlag, Berlin). Increase in the activity of XET has been demonstrated during ripening (Redgewell and Fry, 1993 Plant Physiol.
  • ⁇ -fucosidase active against xyloglucan has been purified from growing pea epicotyls, and me enzyme has been shown to inactivate a xyloglucan oligosaccharin by de-fucosylation (Augur et. al., 1993 The Plant Journal 3, 415-426).
  • the major 0-galactosidase peak active during me mobilization of xyloglucan in nasturtium cotyledons during germination has been purified to homogeneity (Edwards et. al., 1988).
  • the xyloglucan-specific ⁇ -galactosidase might be expected to perform an important role in the modification of xyloglucan in he primary cell wall during various physiological processes.
  • Galactose is a constituent of several cell wall components including pectin, hemicellulose and glycoproteins, and is found in a variety of linkages, in bom the alpha and beta conformations. Specific enzymes might be expected to hydrolyse me terminal galactosyl residues from me various polymers. Plant /3-galactosidase activities, including those tiiat catalyze the hydrolysis of terminal 0-galactosyl residues from me side chains of me pectic fraction of cell walls have been widely reported (e.g. Giannakouros et. al., 1991 Physiol. Plant 82, 413-418; Golden et. al, 1993 Phytochemistry 34, 355-360; Konno et.
  • the enzyme acts on the galactan fraction purified from d e fruit cell wall, and also catalyses die hydrolysis of galactose from xyloglucan and galactoglucomannan.
  • Two soluble isoforms of /3-galactosidase purified from musk melon fruits are active against the 5% KOH-extractable hemicellulose fraction (Ranawala et. al, 1992 Plant Physiol., 100, 1318-1325).
  • the enzyme degrades me hemicellulose extracted from green fruits to sizes similar to tiiat found in ripe fruits. Li and Andrews (1993 Plant Physiol. Suppl.
  • Gel formation occurs when about 50% of the galactose has been removed. Gel formation is dependent on die high molecular weight of die xyloglucan and occurs due to increased inter-chain interactions. The rate of hydrolysis of galactose increases linearly witii substrate concentrations up to and beyond die true solution limits of the xyloglucans. This suggests that saturation conditions can be achieved at substrate concentrations mat correspond to a hydrated solid rather than a solution, and this corresponds to die state of the xyloglucan in die cell walls.
  • die invention provides a polypeptide having xyloglucan-specific ⁇ - galactosidase activity and having substantially die sequence of amino acid residues 34 to 857 of die polypeptide sequence (Seq. ID No. 1) shown in Figure 1, or a functional equivalent thereof.
  • me polypeptide is provided in substantially pure form, free from other plant-derived components.
  • Such functionally equivalent polypeptides are intended to include precursor polypeptides which may possess the specified enzyme activity or which can be processed (e.g. by proteolysis) into a polypeptide possessing die specified activity.
  • Particular embodiments include tiiose polypeptides having signal sequences.
  • tiiose polypeptides differing from the sequence shown in Figure 1 at one or more residues where conservative substitutions have been made which do not substantially reduce the catalytic activity of die polypeptide, or those polypeptides containing deletions or additions of one or more residues which do not substantially reduce d e catalytic activity of the polypeptide.
  • tiiere will be less tiian 15, more preferably less tiian 10, and most preferably less than 5 such amino acid substitutions, although tiiose skilled in die art will appreciate tiiat die number of substitutions tiiat can be tolerated widiout substantially reducing die catalytic activity of die polypeptide will depend to a large extent on die nature of die substitution (e.g. conservative or non- conservative) and die position of the substituted residue in die molecule.
  • Such functional equivalents possess at least 60% homology with die amino acid sequence shown in Figure 1, more preferably at least 70%, and most preferably at least 80% homology.
  • die invention provides a nucleic acid sequence encoding the polypeptide defined above, or a functionally equivalent nucleic acid sequence.
  • the nucleic acid sequence comprises substantially die sequence of nucleotides 122 to 2593 of the nucleotide sequence shown in Figure 1 (Seq. ID No. 2).
  • “Functionally equivalent nucleotide sequences” are intended, in particular, to include those sequences which are capable of encoding a polypeptide exhibiting at least 70% amino acid homology, preferably at least 75%, and more preferably at least 85% homology witii me amino acid sequence of residues 34 to 857 in Figure 1, together with those nucleotide sequences which encode a polypeptide having substantially me amino acid sequence of residues 34 to 857 in Figure 1 but which differ from the nucleotide sequence shown in Figure 1 because of die degeneracy of me genetic code.
  • Such equivalent sequences are able to hybridise under standard laboratory conditions (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition) witii die complement of die sequence shown in Figure 1.
  • functionally equivalent sequences include tiiose which are die antisense equivalents of die sequence of nucleotides 122 to 2593 of Figure 1. Such antisense equivalents are therefore able to hybridise witii the sequence shown in Figure 1 and are preferably able to interfere with expression of the sense sequence at me DNA and/or mRNA level.
  • the nucleotide sequence of die invention comprises a 5' ATG start signal. It is also preferred tiiat the sequence further comprises a suitable 5' untranslated region, including a promoter, to enable expression in appropriate host cells. It is also preferred diat die sequence comprises signals to optimise expression in appropriate host cells, such as a 3' polyadenylation signal to optimise expression in eukaryotic cells.
  • the nucleotide sequence of the invention may also comprise a sequence encoding a signal peptide.
  • the nucleotide sequence of die invention comprises a sequence substantially identical to tiiat of nucleotides 1-122 and/or 2594-2778 of die sequence shown in Figure 1. However, otiier sequences embodying well known expression regulation/polypeptide processing signals may be used witii comparable facility.
  • the nucleotide sequence of die invention is comprised widiin a vector.
  • the vector is an expression vector adapted for expression in appropriate host cells.
  • the invention provides a host cell into which die sequence of die invention has been introduced.
  • the host cell is a plant cell, although otiier host cells could be employed.
  • a non- plant host such as a bacterial or a yeast cell. Culture of the bacterial or yeast host cell under suitable conditions would enable d e polypeptide to be obtained in high yield and free of otiier plant cell components.
  • the invention thus provides a method of producing die polypeptide defmed above in large amounts in isolation from other plant-derived components and in mature or in unprocessed form, comprising expressing the nucleotide sequence of die invention or a functional equivalent thereof in an appropriate host, and isolating die polypeptide so produced. Moreover, it is believed tiiat the polypeptide of me invention will prove useful in vitro in increasing the gelling properties of xyloglucan.
  • the invention tiierefore provides, in a further aspect, a method of modifying the gelling properties of xyloglucan, comprising treating xyloglucan with an effective amount of the polypeptide of me invention so as to effect a chemical change in the composition of die xyloglucan substrate sufficient to alter the gelling properties thereof.
  • Plant polysaccharides have many useful physico-chemical properties such as stabilising, suspending, thickening, gelling, coagulating, film-forming, aroma-binding and moisture regulating (Voragen et al. , 1994 "Enzymic modification of algal and plant polysaccharides", Carbohydrates in Europe 21-26). Polysaccharides extracted from plants have thus found applications as ingredients in me food industry (e.g. in spreads, dressings and ice creams). They can influence d e texmre of processed fruits and vegetables, me consistency of fruit and vegetable homogenates, the pressability and extractability of fruits and oleaginous seeds, and die filterability, clarity and cloud stability of fruit juices and nectares.
  • Tamarind gum xyloglucan
  • xyloglucan is of commercial importance as a stabilising and/or diickening ingredient. Enzymic tailoring of tamarind gum with xyloglucan-specific / 3-D-galactosidase, which removes die terminal /3-D-galactopyranosyl residues, results in an eventual increase in viscosity as die reaction proceeds, witii gel- formation occurring when about 50% of the galactose residues have been removed.
  • a gel is a fucntion of me high molecular weight of xyloglucan, with die terminal, non-reducing D-galactopyranosyl residues representing d e major stnicmral feature dete ⁇ riining d e water solubility of die molecule.
  • the selective removal of these residues does not change the degree of substitution of die (l ⁇ 4)-jS-D-glucan backbone implying that, for a xylose-substimted backbone, polymer/polymer interactions are significantly stronger than polymer/solvent interactions (Reid et al. , 1988).
  • the galactose-depleted xyloglucan has properties comparable to those of the unsubstituted (l ⁇ 4)-3-D-mannan backbone of the galactomannans (Reid et al., 1988).
  • Galactomannans with a low galactose :mannan ratio e.g. locust bean gum or "LBG”
  • LBG locust bean gum
  • undergo mixed polysaccharide interactions yielding variable rheologies (i.e. they are soluble in hot water, but insoluble or only slightly soluble in cold water, and show strong interactions witii agar, K-carrageenan and xanthan gums by which a strong gel is formed at low concentrations).
  • Galactomannans are thus effective stabilisers.
  • die invention provides a method of altering one or more characteristics of a plant or part thereof, comprising introducing into me plant an effective portion of die nucleotide sequence of the invention or a functional equivalent thereof, so as to alter the level or pattern of xyloglucan-specific (l ⁇ 4)-
  • die invention also provides a plant or part d ereof into which has been introduced an effective portion of die nucleotide sequence of die invention, or a functional equivalent thereof.
  • the sequence introduced into me plant is in the sense orientation relative to die promoter, it may result in increased levels of expression. Conversely, introduction into a plant of a sequence in the antisense orientation relative to the promoter may result in a reduction of levels of expression.
  • the phenomenon of "sense suppression" has also been well-documented in plants, wherein die presence of additional sense sequences inhibits the expression of die native gene (e.g. see Matzke & Matzke 1995 Plant Physiol. 107, 679-685). Accordingly, introduction of a sense sequence may also be used to inhibit levels of the enzyme activity in the subject plant.
  • tiiat antisense methods are mainly operable by the production of antisense mRNA which hybridises to the sense mRNA, preventing its translation into functional polypeptide, possibly by causing die hybrid RNA to be degraded (e.g. Sheehy et al., 1988 PNAS 85, 8805-8809; van der Krol et al., Mol. Gen. Genet. 220, 204-212).
  • Sense suppression also requires homology between the introduced sequence and the target gene, but the exact mechanism is unclear. It is apparent however that, in relation to both antisense and sense suppression, neither a full length nucleotide sequence, nor a "native" sequence is essential.
  • fragments of various sizes may be functional in altering one or more characteristics of a plant.
  • effective portions will comprise about 100 to 500 nucleotides (typically, 200 to 400 nucleotides), but comparatively simple trial and error can be used by tiiose skilled in the art to determine portions of other sizes (larger or smaller) which may be similarly effective in altering one or more characteristics of a plant into which tiiey have been introduced.
  • the plant into which die sequence is introduced is preferably a commercially significant plant, in which xyloglucans perform a stnicmral role and which is amenable to plant transformation techniques.
  • xyloglucans perform a stnicmral role and which is amenable to plant transformation techniques.
  • Examples of such plants include: alfalfa, apple, broccoli, cabbage, carrot, cauliflower, celery, cranberry, cucumber, eggplant, flax, grape, horseradish, kiwi, lettuce, mangoes, melon, oilseed rape, papaya, pea, peaches, pears, peppers, plum, potato, raspberry, soybean, strawberry, sugarbeat, sweet potato, tomato and walnut.
  • Figure 1 shows a nucleotide sequence in accordance witii die invention, and me deduced amino acid sequence of d e polypeptide encoded thereby.
  • 0-galactosidase was purified from germinating nasturtium seeds (Tropaeolum majus) as described (Edwards et. al., 1988 J. Biol. Chem. 263, 4333-4337).
  • the jS-galactosidase preparation was a homogeneous band of 97kDa on SDS-PAGE. On storage at -20°C, die homogenous preparation degrades to form lower molecular weight polypeptides but the intact enzyme remains the dominant polypeptide. Two separate enzyme preparations were sequence analyzed.
  • SDS-PAGE was performed by the method of Laemmli (1970 Nature, 227, 680-685) on linear 10% (w/v) acrylamide slab gels on a BioRad electrophoresis kit. Proteins were electroblotted onto PROBLOTTTM membrane (Applied Biosystems, Warrington, U.K.) as described by Matsudaira (1987 J. Biol. Chem., 262, 10035-10038) with the following adaptations. Gels were pre-run witii 50 ⁇ M glutadiione (Sigma) added to die catiiode electrode buffer. Sodium thioglycolate (O.lmM, Sigma) was added to fresh catiiode buffer for sample electrophoresis. Protein was stained witii Coomassie brilliant blue following Applied Biosystem's recommendations .
  • a preparation containing approximately 8-12 ⁇ g purified enzyme (80-120pmoles intact ⁇ - galactosidase) was brought to pH 8.5, measured witii indicator paper, using 1M Tris/HCl pH 8.5.
  • the sample (digest 1) was boiled for 5 min to denature die protein. Endoproteinase Lys-C (Sequencing grade; Boehringer Mannheim) was added at a ratio of 1:50 w/w. The incubation was left at 37 °C overnight and stored at -20 °C prior to reversed-phase chromatography.
  • a second preparation (digest 2) containing approximately 50 ⁇ g purified enzyme (500pmoles intact / 3-galactosidase) was digested as for digest 1.
  • Reversed-phase chromatography was performed at 30°C on Applied Biosytems model 130A HPLC separation system. Samples were loaded, via a 500 ⁇ l loop onto a Brownlee RP 300- C8 microbore column (250 x 1mm id; 7 ⁇ ) pre-equilibrated in 0.1 % (v/v) TFA. Flow rate was 0.1ml min 1 . Peptides were eluted witii a gradient of increasing buffer B (90% acetonitrile; 0.085% TFA) 0-70% over 70 minutes. Absorbance was monitored at 214nm and peaks were collected manually into Eppendorf mbes witii a time delay to allow for the dead space between the detector and outlet. Fractions were stored at -20°C. Prior to loading samples onto me HPLC, acetonitrile gradients were performed until a reproducible low baseline was obtained.
  • ⁇ -galactosidase appears to be readily cleaved by endoproteinase Lys-C. Selected absorbance peaks, well resolved by reversed-phase chromatography, were applied to me amino acid sequencer directly. Amino acid sequence was obtained for several peaks. The initial sequencing yields, in d e range 60-250pmoles, confirms that the data is derived from ⁇ - galactosidase. The peptide sequences obtained were used for designing oligonucleotide primers and for confirming the identity of die isolated cDNA. The sequenced peptides are boxed in the deduced amino acid sequence of die isolated cDNA ( Figure 1).
  • Degenerate deoxy-oligonucleotide primers for polymerase chain reactions were designed coding for a region within the peptide sequence obtained from fractions 3 and 11 from the first digest (Table 1).
  • the primers GF3S and GF3A were 20mers and had a degeneracy of 128.
  • One inosine residue was incorporated at the wobble base position of the first valine (V) codon.
  • the oligonucleotide primers designed from the peptide sequence obtained from fraction 11 was synthesized in two different batches, one batch (GFl lSx and GFllAx) was designed to incorporate 4 codon possibilities for die serine (S) residue and the otiier batch (GFllSy and GFllAy) was similar to die first batch except that it was designed to incorporate the otiier two codon possibilities for serine.
  • the first batch (GFllSx and GFllAx) had a degeneracy of 32
  • the second batch GFl lSy and GFllAy
  • oligonucleotide primed amplification of nasturtium genomic DNA was carried out to obtain a probe for screening of a cDNA library for a copy of the message coding for d e jS-galactosidase.
  • Nasmnium genomic DNA was isolated as reported (DellaPorta et al., 1983 Plant Mol. Biol. Rep. 1 , 19-21), and used as the template for PCR.
  • PCR was carried out in lOO ⁇ l reactions witii 100 pmol of each oligonucleotide primer, along with 1 ⁇ g of genomic DNA, 200 ⁇ M dNTPs (deoxynucleotide triphosphate), and 5 units of Taq DNA polymerase (Stratagem) in the recommended buffer conditions (10 mM Tris-Cl, pH 8.0, 50 mM KC1 and 0.01 % glycerol).
  • the reaction conditions were 92 °C for 2 min, 35 cycles of 92°C for 1 min, 40 °C for 2 min, 72 °C for 2 min, followed by a final extension of 3 min at 72 °C.
  • Products of the PCR reaction were analyzed by agarose gel electrophoresis (Sambrook et al 1989 Molecular Cloning, A laboratory Manual, 2 nd Edition. Cold Spring Harbor Laboratory Press).
  • BG3C PCR cloning vector pT7 (Novagen) utilizing the 3' A overhang incorporated into me PCR product by die Taq DNA polymerase. Plasmid DNA was purified and the double stranded DNA sequenced.
  • Double stranded DNA template preparation and sequence analysis was performed as recommended by the manufacturer of the Sequenase sequencing kit (United States Biochemicals). All DNA sequence computational analysis and database searches were performed using the DNA* ("DNAstar") DNA analysis software.
  • the deduced amino acid sequence revealed the presence of the amino acids coded for by die degenerate oligonucleotides, adjacent to which were located die predicted amino acids identified by d e sequencing of the respective peptides derived from the 0-galactosidase enzyme.
  • the two peptides are boxed in Figure 1, and are coded for by nucleotides 1274- 1297 and 1298-1339.
  • the filters were dien processed as recommended (Sambrook et al. 1989), and fixed by baking under vacuum for 2h at 80 ⁇ C.
  • the filters were incubated in 50 ml of pre- hybridization solution (6xSSC, 5x Denhardts, 100 ⁇ g/ml salmon testis DNA, and 0.5% SDS) at 55 °C for 3 hrs with gentle agitation.
  • 20ng of purified DNA (BG3C) was labelled with - 32 p (-"p usul g the SequenaseTM random primed labelling kit (United States Biochemicals).
  • Labelled DNA was separated from the unincorporated radioactive nucleotides by passage dirough a P-50 column, and added to the pre-hybridization solution at a concentration of 2xl0 5 cpm ml. Hybridization was carried out for 18 hrs witii gentle shaking at 55 °C. The filters were washed in two changes of 2x SSC solution at room temperature and two changes at 55 °C, then wrapped in saran-wrap, and exposed to x-ray film (Kodak LS). Autoradiography was carried out for 16 hrs at -80°C in cassettes witii intensifying screens. Positive plaques were identified and picked into SM.
  • the first round of screening me amplified nasmrtium oligo dT primed cDNA library using the genomic PCR fragment (BG3C) resulted in die identification of 18 putative positive clones.
  • the positive clones were plaque purified by a subsequent round of screening at which point 16 putative positive clones remained. Plaque pure phage stocks of all the positives were analyzed for the size of insert by PCR analysis and results indicated tiiat all the isolated clones were die same size, a common occurrence when screening amplified cDNA libraries.
  • GDT One of these clones (GDT) was in vivo excised witii the Bluescript phagemid from the Uni-Zap XRTM vector as described in the manufacturer's protocol (Stratagene), plasmid preparations of the isolated clones were made using Qiagen P-100 tip columns as recommended by the manufacturer and die cDNA inserts were analyzed by sequencing as described above.
  • the cDNA insert was 1566 bp and had a polyA tail at its 3' end.
  • the oligo dT primed cDNA library due to amplification, appeared to be enriched with j3-galactosidase clones with an insert size identical to die isolated cDNA clone (GDT) and hence a random hexamer primed library was syndiesized, and d e primary library (unamplified) was screened for sequences overlapping with GDT but extended at die 5' end.
  • GDT die isolated cDNA clone
  • a random primed cDNA library was syndiesized using a ZAP II cDNA syndiesis kit as described by die manufacturer (Stratagene), with a few modifications. 3 ⁇ g of a random hexamer oligonucleotide was used to prime the reverse transcription of 5 ⁇ g of poly A + RNA extracted from 12 d.a.p (days after planting) nasmrtium cotyledons as described (de Silva et al., 1993 The Plant Journal 3, 701-711).
  • Ec ⁇ RI linkers were ligated onto the ends of the double stranded cDNA product following which the cDNA was ligated into ZAP II phage arms that were digested witii EcoiXL and treated witii calf intestinal alkaline phosphatase (Stratagene).
  • the phage particles were packaged using the Stratagene Gigapack ⁇ TM packaging extract and yielded a library with a titre of 3 x 10 5 pfu. 4 x 10 4 pfu were plated onto each of 4 LB plates (150mm), and screening of die cDNA library widi 32 P labelled BG3C was performed as described above.
  • a 21 bp antisense oligonucleotide primer (BGNA) was designed 60 bp from the 5' end of the longest cDNA clone isolated by screening of the random primed library witii the ⁇ - galactosidase PCR gene fragment (BG3C).
  • This primer along with a cDNA vector based primer U19 (5'-CAAAAGGGTCAGTGCTGCA-3 ⁇ Seq. ID No. 11) was used to amplify a 356 bp (G4) fragment that was subcloned and sequenced.
  • die N-terminal sequence was identified (Boxed in Figure 1, nucleotides 122-175). Preceding the N-terminal sequence is a 33 amino acid hydrophobic region which is the putative signal peptide whose presence would be predicted for an enzyme targeted to die extracellular matrix.
  • the first strand cDNA was poly A tailed at die 3' end using terminal transferase, and die second strand was synthesized using an oligo dT-adapter primer which contained two nested primers R originate (outside) and (inside).
  • the first round of PCR was performed using RRON and BG17A (/3-galactosidase specific primer) using recommended conditions (Frohman 1990).
  • the PCR reaction was run on an agarose gel and d e DNA smear (data not shown) between 500 bp and 800 bp was electrophoresed onto DEAE nitrocellulose paper and eluted.
  • 10% of die eluted DNA was used as a template for PCR amplification using nested primers Rj and BGl 1 A, and DNA between 500 bp and 600 bp was DEAE purified as described above.
  • the purified DNA was ligated into the PCR cloning vector pT7 (Novagen), and d e colonies screened by PCR for specific inserts large enough to contain the region where die sequence of die original cDNA was in question. Selected colonies were cultured, plasmid DNA purified, and analyzed by sequencing the double stranded plasmid witii appropriate primers by Taq Dye-Deoxy terminator chemistry and analyzed on d e automated ABI 373A DNA sequencer.
  • nasmrtium j3-galactosidase cDNA To enable sense expression of the nasmrtium j3-galactosidase cDNA the 3 separate but overlapping cDNA fragments were joined toged er using a PCR based strategy. Initially d e three molecules were separately amplified by PCR using Vent polymerase (Promega). Vent polymerase has 3' ⁇ 5' exonuclease activity and was selected to avoid die incorporation of errors in the amplified cDNA molecule. The amplified fragments GDT and GRP were incorporated in the same PCR reaction and would have been expected to anneal at the overlapping region and act as primers in d e first step of amplifications, to produce a single molecule (called GRT) comprising both GDT and GRP.
  • GRT single molecule
  • XBG full length cDNA
  • the full length cDNA including C residue at position 658, is 2778 bp in length.
  • the longest open reading frame is 2571 bp (857 aa) long and codes for a 95,578Da polypeptide.
  • the mamre protein after cleavage of die 33 amino acid hydrophobic signal peptide, has a molecular weight of 91 ,833 and comprises 824 amino acids.
  • a second potential start codon (ATG) exists, which would reduce d e signal peptide to 27 amino acids.
  • the signal cleavage site has been confirmed by d e sequencing of die N-terminus of the mature enzyme.
  • the molecular weight of the mamre enzyme was estimated at 97kDa by SDS polyacrylamide gel electrophoresis (PAGE).
  • the discrepancy between die estimated molecular weight of the mamre enzyme and d e molecular weight predicted from the deduced amino acid sequence may be due to a post-translational modification like glycosylation.
  • the predicted isoelectric point (pi 6.93) is in close agreement with the estimated pi (7.1) of the mamre enzyme (Edwards et al. 1988). All the peptide sequences obtained by sequencing the N-terminus of the enzyme and peptides created by endoproteinase digestion have been located within the deduced amino acid sequence of the cDNA (Boxed in Figure 1).
  • the /3-galactosidase amino-acid sequence deduced from the cDNA has some homology with known sequences.
  • the amino acid sequence of die nasmrtium xyloglucan-specific ⁇ - galactosidase has about 53 % identity with an apple /3-galactosidase (EMBL database accession No. L 29451), 52% identity witii an asparagus /3-galactosidase (EMBL accession No.
  • GF3S AAT/C GTI GTN T A T/C A A T/C A C N GC
  • GF3A GCN GTA/G TTA/G TAN ACI ACA/G TT
  • GF3S & GF3A are 20 nucleotides long witii a degeneracy of 128)
  • Table 1 Peptide sequence and corresponding oligonucleotide sequence of fraction 3 &11 of the endo-LysC digest of die nasturtium 3-galactosidase. Underlined is die amino acid sequence utilized in the design of the degenerate oligonucleotide PCR primers. SEQUENCE LISTING
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • Lys Trp Gly His Leu Lys Glu Leu His Lys Ala He Lys Leu Cys Glu 340 345 350
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 2: GAGGAAGAAG GAATACCCAT TGATGTTTTT ATCCAAAAAA ATGAAGAAGC TTTCTTCAAT 60 AGCTACAATA CCCATTTTGC TGTTCTTGTT TCTGAATTTG TTGGTGTTTT TTTCTTCTGC 120 ATCCAATGTG ACTTATGATC GTCGTTCTCT CATCA ⁇ GAT GGCCAACGGA GGCTACTGAT 180 TTCAGCTTCT ATTCATTACC CTCGAAGTGT TCCTGGAATG TGGCCGGGGC ⁇ G ⁇ CAMC 240
  • GAACAAGCCC AAAATGTGGA CTGAGAACTG GCCTGGATGG ⁇ TAAAAC ⁇ ⁇ GGGGCTAG 840
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE ⁇ PE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention concerne un polypeptide possédant une activité de β-galactosidase spécifique au xyloglucane et comprenant la séquence de restes d'acides aminés 34-857 de la séquence représentée dans la figure, ou un de ses équivalents fonctionnels, ainsi que des séquences nucléotidiques codant ledit polypeptide.
PCT/GB1995/002098 1994-09-09 1995-09-07 Beta-galactosidase specifique au xyloglucane WO1996007743A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU33959/95A AU3395995A (en) 1994-09-09 1995-09-07 Xyloglucan-specific beta-galactosidase

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP94306635 1994-09-09
EP94306635.7 1994-09-09

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WO1996007743A1 true WO1996007743A1 (fr) 1996-03-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268197B1 (en) 1997-07-07 2001-07-31 Novozymes A/S Xyloglucan-specific alkaline xyloglucanase from bacillus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0341885A2 (fr) * 1988-05-11 1989-11-15 Zeneca Limited Tomates
EP0479359A1 (fr) * 1990-09-13 1992-04-08 Gist-Brocades N.V. Plantes transgéniques à contenu en carbohydrates modifié
WO1993017101A1 (fr) * 1992-02-28 1993-09-02 Unilever Plc Endo-1,4-beta-d-glucanase
WO1995010622A1 (fr) * 1993-10-12 1995-04-20 Zeneca Limited Fruit modifie contenant un transgene de galactanase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0341885A2 (fr) * 1988-05-11 1989-11-15 Zeneca Limited Tomates
EP0479359A1 (fr) * 1990-09-13 1992-04-08 Gist-Brocades N.V. Plantes transgéniques à contenu en carbohydrates modifié
WO1993017101A1 (fr) * 1992-02-28 1993-09-02 Unilever Plc Endo-1,4-beta-d-glucanase
WO1995010622A1 (fr) * 1993-10-12 1995-04-20 Zeneca Limited Fruit modifie contenant un transgene de galactanase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EDWARDS, M., ET AL.: "A beta-D-galactosidase from nasturtium (Tropaeolum majus L.) cotyledons", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 263, no. 9, 25 March 1988 (1988-03-25), BALTIMORE, MD US, pages 4333 - 4337 *
EMBL SEQUENCE DATABASE ENTRY L29451 RELEASE 40, 4-7-1994. Malus domestica (cv.: Granny smith) b-galactosidase-related protein mRNA *
EMBL SEQUENCE DATABASE ENTRY X77319 RELEASE 38, 2-2-1994. A.officinalis L. mRNA for beta-galacrosidase *
PIR PROTEIN SEQUENCE DATABASE ENTRY S41889 20-5-1994 *
RAGHOTHAMA, K.G., ET AL.: "Characterization of an ethylene-regulated flower senescence-related gene from carnation", PLANT MOLECULAR BIOLOGY, vol. 17, pages 61 - 71 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268197B1 (en) 1997-07-07 2001-07-31 Novozymes A/S Xyloglucan-specific alkaline xyloglucanase from bacillus

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