WO1999024588A1 - CLONE D'ADNc DU GENE ENDO-β-MANANNASE PROVENANT DE TISSUS VEGETAUX - Google Patents

CLONE D'ADNc DU GENE ENDO-β-MANANNASE PROVENANT DE TISSUS VEGETAUX Download PDF

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WO1999024588A1
WO1999024588A1 PCT/CA1998/001034 CA9801034W WO9924588A1 WO 1999024588 A1 WO1999024588 A1 WO 1999024588A1 CA 9801034 W CA9801034 W CA 9801034W WO 9924588 A1 WO9924588 A1 WO 9924588A1
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mannanase
endo
fruit
cdna clone
ripening
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PCT/CA1998/001034
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J. Derek Bewley
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University Of Guelph
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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)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
    • 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
    • C12N15/8249—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 ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase

Definitions

  • the invention relates to the preparation of a cDNA clone for endo- ⁇ -mannanase from plant tissues for controlling the ripening of fruits.
  • Tomato fruit production on an annual basis worldwide is now second only to citrus in terms to weight of crop. Approx. 60-65% of total crop weight (approx. 50 million tonnes) is used for processing (FAO Production Yearbook).
  • Cultivars suitable for mechanical harvesting for processing and fresh-market production require uniform setting and maturation of fruits; 75-90% of the fruits must be mature at the time of harvest (Lutyanenko, Genet. Sel. 55, 82-92, 1975). Slow maturing and softening of matured fruit can ensure their retention on the plants for 20-25 days without loss of marketing quality.
  • Tomato ripening is a complex process involving a number of chemical and physical changes which convert the fruit from a relatively inedible state to one of optimal quality for processing and consumption.
  • Tomato cultivars with a controlled or increased shelf life could be harvested later in the ripening process. Shipments of such fruits would be of a higher quality, be more uniform at maturity, and spoil more slowly than those in mixed shipments.
  • a large number of induced tomato mutants exist which exhibit decreased softening and prolonged shelf-lives (e.g. Nr, rin and nor) (Tigchelaar et al, Tomato Genet. Coop. 23, 33-36, 1973; Khudairi, Amer. Sci. 60, 696-707, 1972), but they are pleiotropic mutants that exhibit undesirable traits associated with fruit coloration and abnormal taste.
  • PG polygalacturonase
  • FlavrSavr tomatoes have now been withdrawn from the market, however, officially because the transformed cultivar does not meet required production and marketing requirements. An increased understanding of the ripening process has prompted a re-evaluation of the role that PG plays in fruit softening.
  • the fruit cell wall consists of protein and three major polysaccharide components, pectin, hemicellulose and cellulose (O'Neill et al, In: Methods in Plant Biochemistry 2, 415-41, 1990).
  • the hemicellulose polymers are comprised of xyloglucans, glucomannans and galactoglucomannans, which are covalently linked to pectin, and hydrogen-bonded to cellulose microfibrils.
  • Endo- ⁇ -mannanase is an important enzyme in the mobilization of cell wall reserves in seeds, and was first reported as a secreted enzyme in the endosperms of endospermic legumes (McCleary, Phytochem. 14, 1187-94, 1975). Recent studies have revealed it to be present in the seeds of more than twenty species of monocots, dicots and gymnosperms (and all cultivars or provenances thereof), usually in numerous isoforms (Dirk et al, Phytochem. 40, 1045-56, 1995). Very few studies have been conducted to determine if mannanase is present in fruit tissues, but it has been reported to be present in those of tomato (Pressey, Phytochem. 28, 3277-80, 1989), and recently confirmed
  • the present invention is a cDNA clone for endo- ⁇ -mannanase.
  • the invention also involves a method for controlling the ripening of fruit using a cDNA clone for endo- ⁇ -mannanase.
  • the invention permits for the preparation of an antisense sequence from the cDNA clone which was obtained from either a fruit such as tomatoes, melons, peaches, oranges, cucumbers and nectarines or a seed such as a tomato seed.
  • the antisense sequence can be inserted into the genome of a fruit for blocking or significantly inhibiting the expression of endo- ⁇ -mannanase. Since the fruit endo- ⁇ -mannanases are at least 60% homologous to the seed endo- ⁇ -mannanases, therefore the seed endo- ⁇ -mannanases can also be used to prepare antisense sequences useful in fruits.
  • promoters for endo- ⁇ - mannanase can be inserted into the genome of a fruit in a manner known in the art for over expressing the endo- ⁇ -mannanase gene.
  • a cDNA clone for endo- ⁇ -mannanase is provided.
  • a cDNA clone that is at least 60% homologous to a cDNA clone for endo- ⁇ -mannanase is provided. Homology refers to the fact that this cDNA clone has a nucleic acid sequence that is at least a 60% match in primary structure to that of the cDNA clone for endo- ⁇ -mannanase.
  • an expression system comprising a cDNA clone for endo- ⁇ -mannanase and a promoter for expressing same is provided.
  • an antisense sequence for endo- ⁇ -mannanase for inhibiting the expression of endo- ⁇ -mannanase is provided.
  • a method for delaying the ripening of fruit comprising the step of obtaining a cDNA clone for endo- ⁇ -mannanase produced by a fruit and inhibiting the expression of endo- ⁇ -mannanase in the fruit.
  • a method for expediting the ripening of a fruit comprising the steps of inserting a promoter for endo - ⁇ -mannanase into the genome of a fruit and overexpressing the endo- ⁇ -mannanase in the fruit.
  • Figure 1 is a complete nucleotide sequence and deduced amino acid sequence of the cDNA clone pDB-MAN. Amino acid numbering begins at the NH 2 -terminus of the mature enzyme, which is indicated by an arrow. The 65 NH 2 -terminal amino acid residues from the purified protein are underlined and a possible N-glycosylation site in this region is underlined. The putative catalytic acid (Glu-148) and nucleophile (Glu- 265) residues are also underlined. The stop codon is marked by an asterisk and a putative polyadenylation signal is underlined.
  • Figure 2 is an amino acid sequence alignment of the Trichoderma (trr), Aspergillus (asn) and tomato (man) (1— >4)- ⁇ -mannanase. The alignment was performed using the PRETTYBOX program. Amino acid numbering begins at the translation start methionine residues. Asterisks indicate the putative catalytic residues.
  • Figure 3 is a Genomic Southern blot analysis of DNA isolated from tomato seedlings (cv. Dynamo FI) cut with.YZ.aI (Xb), Hindlll (H), and ⁇ TzoI (Xh) and probed with the complete tomato cDNA.
  • Figure 4 is a Northern blot analysis of total RNA isolated from various tomato tissues. Lanes: 1 WS, whole seeds, 2 SR, seedling roots; 3 SH, seedling hypocotyls; 4 SC, seedling cotyledons, 5 ML, mature leaves; 6 MR, mature roots; 7 E, 72-h endosperm; 8 C, 72-h cotyledons.
  • Figure 5 is a table setting out the activities of endo- ⁇ -mannanase in various different fruits.
  • Figure 6 is a plot of stage of ripening versus endo- ⁇ -mannanase activity showing an endo- ⁇ -mannanase time course for ripening tomato.
  • Figure 7 is a Western blot showing the homology of endo- ⁇ -mannanase obtained from tomato seed to the endo- ⁇ -mannanases of various different fruits.
  • the c-DNA clone is for endo- ⁇ - mannanase isolated from a fruit. More specifically, the c-DNA clone is for endo- ⁇ - mannanase isolated from a fruit selected from the group consisting of tomatoes, melons, peaches, oranges, cucumbers and nectarines. Most specifically, there is provided a c- DNA clone for endo- ⁇ -mannanase isolated from a tomato seed.
  • the c-DNA clone is for endo- ⁇ -mannanase isolated from a fruit and this c-DNA clone is at least 60% homologous to a c-DNA clone for endo- ⁇ -mannanase obtained from a tomato seed.
  • Oligonucleotides corresponding to NH 2 -terminal amino acid sequences of a purified tomato (l ⁇ 4)- ⁇ -mannanase were used to screen a cDNA library which had been generated from poly(A)+ RNA of 3-d-germinated tomato seeds.
  • a cDNA library which had been generated from poly(A)+ RNA of 3-d-germinated tomato seeds.
  • One near full-length cDNA clone of 1307 bp was isolated and its complete nucleotide sequence was determined (Fig. 1).
  • the cDNA was shown to encode a tomato (1— >4)- ⁇ -mannanase because the amino acid sequence deduced from a region near its 5' end (nucleotides 88-282) is identical with the sequence of the 65 NH 2 - amino acids determined directly from the purified enzyme. A region encoding a putative signal peptide of 23 amino acid residues is observed
  • the putative signal peptide is typical of eukaryotic signal peptides (Watson, Nucleic Acids Res. 12, 1984:5145-5164).
  • the nucleotide sequence indicates that the mature enzyme has 346 amino acid residues and a calculated MR of 38 950. This can be compared with a value of 38 000 reported for the (l-»4)- ⁇ -mannanase isoform MI purified from tomato endosperm (Nonogaki and Morohashi. Plant Physiol. 110, 1996:555-559).
  • the pi calculated from the primary structure of the enzyme is 5.3; isoelectric focussing of endosperm extracts from germinated tomato seeds revealed the presence of several isoforms with pi values in the range 5.2-5.8 (Toorop et at. Planta, 200, 1996:153-158; Voigt and Bewley, Planta, 200, 1996:72-77).
  • the catalytic domains of several hundred glycosyl hydrolases have been classified into distinct families, based on similarities in amino acid sequences and hydrophobic cluster analyses (Henrissat and Bairoch, Biochom, 293, 1993:781-789).
  • the tomato (1— »4)- ⁇ -mannanase sequence determined here indicates that the enzyme is a member of family 5. According to this classification the catalytic acid of the tomato (l-»4)- ⁇ -mannanase would be expected to be Glu-148, the catalytic nucleophile would be Glu-265, and the anomeric configuration of mannosyl residues released during hydrolysis would be retained (Henrissat and Bairoch, Biochom, 293, 1993:781-789).
  • Fig. 2 The alignments presented in Fig. 2 indicate that, in comparison with the tomato enzyme, fungal (1— >4)- ⁇ -mannanases are sometimes extended at their COOH-termini. These COOH-terminal extensions presumably correspond to the protein docking domains or additional catalytic domains that have been reported in many microbial (l ⁇ 4)- ⁇ -mannanase (Gibbs et al., Appl. Environ. Microbiol. 58, 1992:3864-3867; Fanutti et al., J. Biol. Chem. 270, 1995:29312-29322). The tomato (l ⁇ 4)- ⁇ -mannanase does not appear to have multiple domains of this type (Fig. 2).
  • the (1— »4)- ⁇ -mannanase cDNA was used to investigate expression sites of the genes in several tissues, as determined by mRNA transcripts detected in Northern hybridization analyses.
  • (1— »4)- ⁇ -mannanase mRNA transcripts were only defected in the endosperm of seed 72 h after germination (Fig. 4). This is consistent with the detection of the enzyme itself in this tissue, where its synthesis is inducible by gibberellin (Toorop et at. Planta, 200, 1996:153-158; Nonogaki and Morohashi, Plant Physiol. 110, 1996:555- 559; Still and Bradford, Plant Physiol. 113, 1997:21-29; Still et al. Plant Physiol.
  • cDNA in heterologous systems should allow sufficient enzyme to be synthesized for thorough chemical and kinetic analyses, and for detailed examination of substrate specificities, catalytic mechanisms and action patterns of the enzyme. It is therefore another aspect of the present invention to provide a c-DNA clone for use in the monitoring of (l ⁇ 4)- ⁇ -mannanase gene expression, isolation of (l ⁇ 4)- ⁇ - mannanase genes from other plant species and synthesis of (1— »4)- ⁇ -mannanase through expression in heterologous systems.
  • a cDNA for endo- ⁇ -mannanase is isolated from a fruit including tomatoes, peaches, oranges, nectarines and melons or others according to the same procedure as is used for obtaining the cDNA from the tomato seed with the necessary modifications as would be apparent to a person skilled in the art. Since mannans are a major cell wall component in ripening tomato * fruits, and since the enzyme, endo- ⁇ -mannanase, which degrades them is present at the time of ripening, the activity of latter can be eliminated using anti-sense technology, and assessed as to the extent to which fruit firmness is retained. Ripening fruits of selected cultivars are assessed for endo- ⁇ -mannanase activity during fruit development and ripening.
  • the assay for the enzyme is colorimetric, and involves the incorporation of locust bean gum galactomannan into Phytagel on an assay plate. Enzyme extracts are placed in wells in the plate, incubated at 37°C and the extent of substrate breakdown assessed using Congo Red dye (Downie et al, Phytochem. 36, 829-35, 1994). The area of clearing of substrate around the wells can be accurately and quantitatively determined using Aspergillus enzyme standards. Enzyme production and visible changes to the fruit are correlated with changes in fruit firmness and skin resistance.
  • Standard tests include force-deformation using a dynamometer equipped with a flat disc, resistance to puncture using a table dynamometer with a sharp point, and damage resulting from a free-fall, usually from 70 cm (Altisent et al, Symposium on Production of Tomatoes for Processing, ISHS, pp 181-96, 1979).
  • Various points on the surface of the fruit are tested. The timing and location of the expression of the gene for endo- ⁇ -mannanase is determined in the developing fruit since this controls the appearance of the enzyme.
  • RNA Total RNA, or messenger RNA following oligo dT-column chromatography, is separated on an agarose gel and detected by northern hybridization, using a 32 P-labelled tomato seed endo- ⁇ - mannanase cDNA clone as the probe. In this way, necessary correlations are drawn between gene transcription, enzyme production and fruit ripening characteristics. Mutants defective in their ability to ripen are also used to correlate endo- ⁇ -mannanase activity with fruit softening, to determine if the rin, nor and ale genes, for example, directly or pleiotropically affect enzyme production.
  • the antisense construct is for endo- ⁇ -mannanase obtained from a fruit, more specifically, a tomato and even more specifically, a tomato seed.
  • this method comprises the steps of obtai ing a c-DNA clone for endo- ⁇ -mannanase produced by the fruit and inhibiting the expression of the endo- ⁇ -mannanase in the fruit.
  • the method further includes the steps of preparing an antisense sequence from the c-DNA clone and inserting the antisense sequence into the genome of the fruit.
  • the fruit is selected from the group including tomatoes, melons, peaches, oranges, cucumbers and nectarines.
  • the fruit is tomato.
  • the present invention also provides a method for expediting the ripening of a fruit.
  • this method comprising the steps of obtaining a cDNA clone for endo- ⁇ -mannanase and inserting a promoter for endo- ⁇ -mannanase into the genome of the fruit in order to over express the gene for endo- ⁇ -mannanase .
  • the fruit is selected from the group including tomatoes, melons, peaches, oranges, cucumbers and nectarines. In the most specific embodiment of the invention, the fruit is tomato.
  • The- present invention also embodies the use of a cDNA clone for endo- ⁇ - mannanase of a fruit for controlling the rate of ripening of the fruit.
  • this use is directed to a fruit selected from the group comprising tomatoes, melons, peaches, oranges, cucumbers and nectarines.
  • this use is specifically directed to delaying the ripening of a fruit.
  • a cDNA clone for endo- ⁇ -mannanase of a fruit to expedite the ripening of the fruit.
  • the present invention also embodies the use of an antisense sequence for endo- ⁇ -mannanase for delaying the ripening of a fruit.
  • Tomato is a crop plant with a relatively small DNA content per haploid genome, well-developed genetics and favourable tissue-culture characteristics. Efficient transformation procedures permit the introduction of foreign genes into explants and protoplasts which can be regenerated into transgenic, fertile plants (Hille et al, In: Genetic Improvement of Tomato, Monograph on Theoretical and Applied Genetics H, 283-291, 1991). Several protocols for transforming tomato have been published, including:
  • the typical vector system that is used for transformation is an Agrobacterium tumefaciens strain with a disarmed Ti plasmid and an additional plasmid carrying the gene of interest and a selectable marker gene (like NOS-NPTII-NOS for kanamycin resistance) within the T-DNA region (McKormick et al, 1986, Plant Cell Rep. 5:81).
  • the gene of interest is introduced into the plant material by cocultivating the explant and the Agrobacterium for a few days, killing the bacteria with an antibiotic and selecting shoots which regenerate from the explant on media containing the selection agent (kanamycin). DNA samples from shoots that are resistant to the selection agent are then tested to determine whether they also contain the gene of interest.
  • Tomato fruits from the transformed plants are assessed for their ability to produce endo- ⁇ -mannanase, and to transcribe the mRNA for this enzyme. Comparisons are made with the time course and quantitative expression determined for the fruits from untransformed plants. Tests for firmness and skin resistance are likewise conducted, and visible changes in fruit characteristics noted. Most facets of fruit development and ripening are unaffected, but firmness from the breaker stage is retained and endo- ⁇ - mannanase activity is eliminated or severely curtailed.
  • Seeds or tomato (Lycopersicon esculentum Mill. cv. Dynamo FI; Sandoz seeds, supplied by B. & H. Stoeff, Virginia, S. Australia) were washed in sterile distilled water to remove the Thiram seed-coat treatment and placed on two layers of Whitman No. 1 filter paper in 9-cm Petri dishes on sterile water.
  • the seedlings were harvested, surface-dried by blotting with a paper towel, frozen in liquid N 2 , and stored at -8O°C.
  • Tomato seed (1— »4)- ⁇ -mannanases form MI was purified as described by Nonogaki et al. 94, 328-334, 1995.
  • the NH 2 -terminal amino acid sequence was determined in a Hewlett-Packard G1OO5A protein sequencer (Hewlett Packard Company, Palo Alto, Calif., USA) using the Hewlett-Packard 3.0 sequencing routine, which is based on Edman degradation chemistry.
  • Experiment 3 Isolation of poly(A)+RN A.
  • the method of Prescott and Martin (Plant Mol. Biol. Rep., 4, 219-224, 1987) was used to isolate poly(A)+RNA. Seedlings were ground to a powder in liquid N 2 and suspended in a total of 7.5 ml extraction buffer (50 mM Tris-HCl buffer, pH 9, containing 150 mM LiCl, 5 mM EDTA, 5% w/v SDS) before phenol: chloroform extraction.
  • RNA was precipitated overnight at 4°C with 2 M LiCl, and after dissolving in water the poly(A)+RNA fraction was obtained by oligo(dT)-cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. 69, 1408-1412, 1972).
  • a cDNA library was prepared from 5 ⁇ g poly(A)+RNA of germinated tomato seeds using the ⁇ ZAP cDNA synthesis and cloning kit (Uni-ZAP XR; Stratagene, La Jolla. Calif, USA) according to the manufacturer's instructions.
  • the library was screened by hybridization of nitrocellulose filter plaque replicas with three degenerate oligonucleotides which were designed on the basis of NH 2 -terminal amino acid sequence data from the purified tomato seed l ⁇ 4)- ⁇ -mannanases and end-labeled with [ ⁇ PjATP.
  • the cDNA inserts of positive clones were rescued into the pBluescript SK(+) phagemid and both strands were sequenced using the dideoxynucleotide chain termination procedure (Sanger et al. Proc. Natl. Acad. Sci. 74, 5463-5467, 1977).
  • Computer analyses were effected with the University of Wisconsin Genetics Computer Group software (Devereux et al. Nucleic Acids Res., 12, 287-395, 1984) in the ANGIS suite of programs at the University of Sydney.
  • RNA samples containing, where possible, 5 ⁇ g RNA were separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred onto a Hybond-N+ (Amersham) nylon membrane, and probed with a [ 32 P]- labelled.
  • 1.1 -kb EcoRI -Xhol fragment of the (l ⁇ )- ⁇ -mannanases cDNA clone (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 nd ⁇ d., Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y., 1989).
  • the pellet was redissolved in TE buffer and extracted twice with phenol and chloroform, precipitated with 2.5 vol. ethanol and re-dissolved in TE buffer.
  • the DNA was digested with restriction endonucleases Xbal, Hindlll and Xhol and separated on a 1% agarose gel before transfer to a Hybond-N+ nylon membrane (Amersham).
  • the membrane was probed with a [ 32 P] -labelled full-length fragment of the tomato l- 4)- ⁇ -mannanases cDNA and, following hybridization, was washed progressively to 0.5 x SSC+0.1% SDS at 55°C.
  • the cDNA inserts ranged in size from 500 bp to 1.3 kb; the clone containing the largest cDNA was rescued into the pBluescript SK(+) phagemid and was designated pDB-MAN
  • the complete nucleotide sequence of the 1307-bp cDNA, together with the amino acid sequence deduced from it, are shown in Fig. 1.
  • the cDNA has an open reading frame which extends from nucleotides 19 to 1125.
  • the amino acid sequence deduced from nucleotides 88 to 282 corresponds exactly with that of the 65 NH2 -terminal amino acids determined directly from the purified tomato (1— >4)- ⁇ -mannanase isoenzyme MI (Fig. 1).
  • the cDNA encodes the tomato (l-»4)- ⁇ -mannanase.
  • a 21-bp poly(adenylic acid) tail is evident, and a putative AATAAA polyadenylation signal begins at nucleotide 1255 (Fig. 1).
  • the region of the cDNA from the codon for the NH 2 -terminal Asn residue at nucleotide 88 to the stop codon at nucleotide 1126 encodes the mature enzyme.
  • the enzyme has 346 amino acid residues, a calculated Mr, of 38 950 and an estimated pi of 5.3.
  • a single potential N-glycosylation site begins at amino acid residue 15 in the mature enzyme sequence. No myristylation or phosphorylation consensus sites could be detected.
  • Endo- ⁇ -mannanase activity was assayed using a modified gel diffusion method (Downie et al., Phytochemistry, 40, 1045-1056; Toorop et al., Planta, 200, 153-158, 1996; Still et al., Plant Physiol., 113, 21-29, 1997) based on diffusion of the enzyme through an agarose gel-Locust bean galactomannan substrate matrix from a 2 mm diameter central well.
  • a modified gel diffusion method Downie et al., Phytochemistry, 40, 1045-1056; Toorop et al., Planta, 200, 153-158, 1996; Still et al., Plant Physiol., 113, 21-29, 1997) based on diffusion of the enzyme through an agarose gel-Locust bean galactomannan substrate matrix from a 2 mm diameter central well.
  • the matrix was prepared by preheating 0.1 M citrate /0.2M Na 2 HPO 2 buffer (pH 5.0) just to boiling in a microwave or a heating plate.
  • Locust bean galactomannan substrate (LBG) (0.1% w/v) was added to the incubation buffer then stirred continuously for 2 hours on a heat plate at 80°C.
  • LBG Locust bean galactomannan substrate
  • the solution was continuously stirred overnight for 12 h at room temperature then finally centrifuged at 4000g for 15 min at 10°C. The supernatant was collected in aliquots of 50 mL.
  • a vertical cassette consisting of 0.5 mm U-frame spacer, and a cover glass plate (Pharmacia Biotech, Uppsala, Sweden) was prepared.
  • the spacer was made hydrophobic (to allow easier removal of the wet gel layer on) by spreading a few mL of repel silane (Pharmacia Biotech) over the inner face with a tissue under the fume hood.
  • repel silane Pharmacia Biotech
  • the chloride ions which result from the coating with repel silane was rinsed off with double distilled H 2 0.
  • a few mL of H 2 0 was poured on the cover glass plate and the agarose gel bond support film (Pharmacia Biotech) was placed on it with the hydrophobic side down. This facilitates filling the cassette later on.
  • the gel bond film was firmly pressed against the glass plate with a roller (Pharmacia Biotech), with the film overlapping the length of the glass pate by about 2 mm.
  • the glass plate, with the gel bond film attached, was placed on the U-frame spacer and the cassette was firmly clamped (FLEXI CLAMP, Pharmacia) together.
  • the cassette and a 10 mL pipette was prewarmed to 65°C in a ventilated oven (MEMMERT, Schwabach, Germany).
  • Type III-A agarose (Sigma Chemicals) was added to the LBG substrate solution (0.8% w/v) while still cold.
  • This agarose-substrate mixture was heated to boiling in a microwave oven (at the lowest setting) until the agarose was ' completely dissolved (rapid stirring damages the mechanical properties of agarose, which is why heating in the microwave oven is preferable).
  • the hot mixture was cooled down for 15 mins to about 65 °C in the ventilated oven then quickly dispensed into the prewarmed cassette.
  • the cassette was let stand for about 3 hours at 15°C to allow the gel to solidify before use.
  • the gel was placed in a moistened plastic box (to prevent drying) covered with plastic film then incubated for 20 h at 25°C.
  • Commercial endo- ⁇ -mannanase (Megazyme International Ltd., Wicklow, Ireland) purified from Aspergillus niger (specific activity, 38 Units/mg protein using carob galactomannan as substrate) was used as a standard.
  • a standard curve was developed in the same gel in dilutions ranging from 0.418 to 0.001045 International Units. After incubation, the gel was prewashed for 30 mins in 0.1M citrate/0.2M
  • the gel was fixed in 80% (w/v) ethanol for 10 min, destained in 0.1 M citrate/0.2 M.
  • Endo- ⁇ -mannanase activity was calculated by measuring the diameter of the hydrolyzed zones manually with sliding callipers and comparing them with a standard curve of activity using Aspergillus endo- ⁇ -mannanase. This is a routine measurement which has been employed in previous investigations of endo- ⁇ -mannanase activity (Downie et al., Phytochemistry, 40, 1045-1056, 1994; Toorop et al., Planta, 200, 153- 158, 19
  • Figure 7 shows the results of a western blot that was prepared by a method known in the art.
  • the results show binding of an antibody for the endo- ⁇ -mannanase of the tomato seed.
  • the results indicate that there exists at least a 60% homology in structure between the endo- ⁇ -mannanase of the tomato seed and the endo- ⁇ -mannanase of the fruits.
  • the tomato mannanase antisense cDNA are inserted into the intermediate vector pKYLX attached to a double CaMV35S promoter, or the super-promoter now available under licence, between the borders of the t-DNA region and mobilized into the Agrobacterium strain LB A 4404 by triparental mating with an E. coli helper strain (HB101) harbouring the mobilization plasmid pRK2013 (Figarski and Helinski, PNAS 76, 648-1652, 1979).
  • Transconjugant Agrobacterium is selected on LB plates with streptomycin and tetracyclin.
  • Agrobacterium liquid cultures are prepared for the cocultivation experiments by growing them overnight at 28°C in a shaker in LB medium containing streptomycin and acetosyringone. The cultures are diluted 1:20 before using to give a density of approximately 1 x 10 8 cells/ml.
  • Cotyledons from tomato cultivar UC82B, H722 or H902 seedlings are used in the co-cultivation procedures. Seeds from these cultivars are surface sterilized with 20% bleach and 0.1% Tween-20 for 15 minutes. The seeds are rinsed several times in sterile water and germinated on Vi MSO medium in magenta boxes. Then cotyledons from 2- week-old seedlings are removed, cut at both ends and precultured (upside down) on regeneration medium (MS 2.5 mg/1 BA, 2.5 mg/ 1 IAA) for one day.
  • the precultured cotyledons are placed into the diluted overnight Agrobacterium culture for 15-20 min and placed onto regeneration media (MS, 5 mg/1 BA, 5 mg/1 IAA) for a co-cultivation period. After two days the cotyledons are placed on regeneration medium containing 500 mg/ml carbenicillin (to kill the Agrobacterium) and 10 mg/ml kanamycin for ten days. The cotyledons are transferred to regeneration medium with higher concentrations of kanamycin (first 50 ⁇ g/ml then 75 ⁇ g/ml) in subsequent transfers at 10 day intervals. Green shoots that regenerate from the cotyledons on 75 ⁇ g/ml kanamycin are transferred to rooting medium with 100 mg/ml kanamycin. Rooted shoots are transferred to soil and grown into plants in a growth room.
  • regeneration media MS, 5 mg/1 BA, 5 mg/1 IAA
  • DNA is isolated from putative transgenics as described by Maniatis et al (eds., Molecular Cloning, a Laboratory Manual, Cold Spring Harbour Laboratory Press, New York, 1984).
  • the presence of the anti-sense mannanase gene in the selected plants is determined by Southern blotting. Success with the ACC deaminase gene is a strong indication that this technique is valid and appropriate.

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Abstract

L'invention concerne la préparation d'un clone d'ADN c pour endo-β-mannanase à partir soit d'un fruit, soit d'une graine de ce fruit. Le clone d'ADNc sert à préparer une séquence antisens servant à bloquer l'expression de l'endo-β-mannanase dans certaines variétés de fruits. Comme l'endo-β-mannanase est un enzyme qui intervient dans le mûrissement et le ramollissement des fruits, la séquence antisens permet de ralentir le mûrissement et le ramollissement qui en découle. L'invention concerne également l'emploi de promoteurs pour surexprimer le gène de l'endo-β-mannanase et donc accélérer le mûrissement. L'invention concerne un procédé permettant de ralentir le mûrissement d'un fruit en obtenant un clone ADNc pour l'endo-β-mannanase produit par le fruit et en empêchant l'expression de l'endo-β-mannanase dans le fruit. L'invention concerne également un procédé propre à accélérer le mûrissement du fruit par l'introduction d'un promoteur du gène endo-β-mannanase dans le génome du fruit.
PCT/CA1998/001034 1997-11-07 1998-11-05 CLONE D'ADNc DU GENE ENDO-β-MANANNASE PROVENANT DE TISSUS VEGETAUX WO1999024588A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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EP1138771A1 (fr) * 2000-03-30 2001-10-04 Societe Des Produits Nestle S.A. Endo-Mannanase de café
WO2010004582A1 (fr) * 2008-07-09 2010-01-14 Asis Datta Séquence polynucléotidique de ramollissement de fruit associée à une a-mannosidase et ses utilisations pour améliorer la durée de vie des fruits

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WO1994025576A1 (fr) * 1993-04-30 1994-11-10 Novo Nordisk A/S Enzyme presentant l'activite de la mannanase
WO1995010622A1 (fr) * 1993-10-12 1995-04-20 Zeneca Limited Fruit modifie contenant un transgene de galactanase
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EP0479359A1 (fr) * 1990-09-13 1992-04-08 Gist-Brocades N.V. Plantes transgéniques à contenu en carbohydrates modifié
WO1994025576A1 (fr) * 1993-04-30 1994-11-10 Novo Nordisk A/S Enzyme presentant l'activite de la mannanase
WO1995010622A1 (fr) * 1993-10-12 1995-04-20 Zeneca Limited Fruit modifie contenant un transgene de galactanase
WO1997025417A1 (fr) * 1996-01-11 1997-07-17 Recombinant Biocatalysis, Inc. Enzymes de type glycosidase

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BEWLEY, J.D., T AL.: "Molecular cloning of a cDNA encoding a (1-4)-beta-mannan endihydrolase from the seeds of germinated tomato (L. esculentum)", PLANTA, December 1997 (1997-12-01), XP002095786 *
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SOZZI, G.O., ET AL.: "Effect of a high-temperature stress on endo-beta-mannanase and alpha- and beta- galactosidase activities during tomato fruit ripening", POSTHARVEST BIOLOGY AND TECHNOLOGY, (OCT 1996) VOL. 9, NO. 1, PP. 49-63., XP002095790 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1138771A1 (fr) * 2000-03-30 2001-10-04 Societe Des Produits Nestle S.A. Endo-Mannanase de café
WO2001075084A2 (fr) * 2000-03-30 2001-10-11 Societe Des Produits Nestle S.A. Mannanase de cafe
WO2001075084A3 (fr) * 2000-03-30 2002-01-10 Nestle Sa Mannanase de cafe
JP2003529367A (ja) * 2000-03-30 2003-10-07 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム コーヒーマンナーゼ
US7148399B2 (en) * 2000-03-30 2006-12-12 Nestec S.A. Coffee mannanase
WO2010004582A1 (fr) * 2008-07-09 2010-01-14 Asis Datta Séquence polynucléotidique de ramollissement de fruit associée à une a-mannosidase et ses utilisations pour améliorer la durée de vie des fruits
US20110239325A1 (en) * 2008-07-09 2011-09-29 Asis Datta Polynucleotide sequence of fruit softening associated a-mannosidase and its uses for enhancing fruit shelf life
US8962918B2 (en) * 2008-07-09 2015-02-24 National Institute Of Plant Genome Research Polynucleotide sequence of fruit softening associated A-mannosidase and its uses for enhancing fruit shelf life

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