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  • 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
  • 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

• ⁇ -galactosidase ⁇ encodes a specific plant polypeptide named ⁇ -galactosidase ⁇ , is provided.
• polypeptides of the invention and methods of modifying fruit quality by employment of a polynucleotide or polypeptide of the present invention.
• compositions and size occur in the pectic fraction of the cell wall (see
• ripening include increased solubility, depolymerization, de-esterification and a
• pectin-modifying enzymes are polygalacturonase (endo- ⁇ l ⁇ 4-D-galacturonan
• ⁇ (l->4)-D-galactopyranoside is the only enzyme identified in higher plants capable of directly cleaving ⁇ (l-»4)galactan bonds, and probably
• ⁇ -galactosidase activity is thought to be important in cell wall metabolism
• ⁇ -Galactosidases are generally assayed using artificial
• substrates such as /7-nitrophenyl- ⁇ -D-galactopyranoside (PNP), 4-
• galactosidase II was active against ⁇ (l- 4)galactan. Even though they were
• galactosidase LI protein Although ⁇ -galactosidase LI, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan has been purified, cloning of the
• the modification of plant gene expression has been achieved by several methods.
• the molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal
• genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product.
• Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.
• the present invention is based on the discovery of novel DNA sequences
• TBG1, TBG2, TBG3, TBG4 RT-PCR clones
• TBG5, TBG6, and TBG7 and having the nucleic acid sequences designated SEQ ID NOs 1-7, respectively as shown in Figure 2, were identified which had
• a method for modifying cell wall metabolism which involves modifying
• Also provided by the present invention is a DNA construct including
• the present invention also relates to recombinant vectors, which include
• polypeptides or peptides by recombinant techniques.
• the present invention also provides plant cells containing DNA
• ⁇ -galactosidase gene expression ⁇ -galactosidase gene expression; and seeds produced from such plants.
• the ⁇ -galactosidase LI protein of the present invention has demonstrated
• the ⁇ -galactosidase LI protein also may be involved in cell
• the enzyme may allow any one of the cells wall.
• the ⁇ -galactosidase of the invention may be involved in conversion of
• chloroplasts green - chlorophyll
• chromoplasts red - lycopene
• the family of genes represented by the nucleotide sequences shown in Figure 2 is expected to code for a group of similar enzymes with the same type of hydrolytic activity but with different tissue and or substrate
• the ⁇ -galactosidase LI protein of the present invention as well as other
• the present invention also provides ⁇ -galactosidase enzymes for use as
• Figure 1A and IB shows a phylogenic tree based on shared amino acid sequence identity among tomato ⁇ -galactosidase clones TGB1-7 and other known plant ⁇ -galactosidase polypeptides.
• Figure 2 shows cDNA sequences [SEQ LD NOs: 1-7, respectively] for the seven ⁇ -galactosidase genes of the invention: TGB1, TGB2, TGB3, TGB4, TGB5, TGB6, TGB7.
• Figure 3 shows multiple sequence alignment of the deduced amino acid sequences of tomato fruit for cDNA clones TGB1, TGB2, TGB3, TGB4, TGB5, TGB6 and TGB7 [SEQ LD NOs: 8-16, respectively] and various plant ⁇ -galactosidase cDNA clones.
• Figure 4 shows autoradiograph of northern blot analysis of TBG expression in various plant tissues (flowers, leaves, roots and stems).
• Figure 5 shows Autoradiograph of northern blot analysis of TBG expression in fruit tissues at different stages of development.
• Figure 6 shows autogradiograph of northern blot analysis of TBG expression in fruit tissues (mature green or turning stage fruit peel, outer pericarp, inner paricarp and locular).
• Figure 7 shows autoradiograph of northern blot analysis of TBG expression in normal and mutant fruit tissues.
• Figure 8 shows autoradiograph of northern blot analysis of TBG expression in response to ethylene treatment of mature green fruit tissues.
• Figure 9 shows Western blot analysis of TBG4 expression by yeast.
• Figure 10 shows detection of ⁇ -galactosidase activity from pZBG2-l-4 expression in E. col
• Figure 11 A - ⁇ (1-4) shows the comparative results of texture measurements for fruit from tomato plants containing antisense constructs to suppress TBG4 mRNA and fruit from the parental line.
• Figures 12A - B show Northern blot analysis of TBG4 expression in transgenic fruit containing TBG4 antisense construct.
• Figure 13 shows a Binary construct used to transform plants and express TBG4 (pZBG2-l-4) in the antisense orientation.
• the present invention provides isolated nucleic acid molecules
• pZBG2-l-4 encoding ⁇ -galactosidase LI, is recorded in GenBank as Accession
• the exemplary ⁇ -galactosidase LI protein of the present invention as
• TBG3 was high.
• TBG4 was also very similar to both TBG1 and 3.
• the amino acid sequences of TBG2 and 7 were unique because several regions of amino acids
• Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
• the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods
• nucleotide sequence of a nucleic acid molecule or polynucleotide
• nucleic acid molecule described in Figure 2 [SEQ ID NO: 4] was discovered in a cDNA library derived from breaker, turning and pink fruit pericarp from
• GenBank (no. AF020390) and is provided in Figure 2 [SEQ ID NO: 4].
• the cDNA insert is 2532 nucleotides (nt) long and contains a single, long open
• the deposited cDNA which comprises about 724 amino acids, may be
• the invention further provides polypeptides having various residues deleted from the N-terminus of the complete polypeptide, including
• polypeptides lacking one or more amino acids from the N-terminus of the ⁇ -
• the mature polypeptide contains three possible N- glycosylation sites at asparagine numbers 282, 459 and 713, however the asparagine at position 713 is unlikely to be glycosylated due to the proline at
• polypeptide was 75 kD with a pi of 8.9.
• LI protein of the invention includes a leader sequence and a mature protein, as
• the present invention provides a
• nucleotide sequence encoding the mature ⁇ -galactosidase LI polypeptide
• TFG4 has been expressed in E. coli strain XLI blue MR (lacZ) (Stratagene, La Jolla, CA), as described hereinbelow (see Example).
• TBG1, 2 and 3 were predicted to have cleavable signal sequences by SignalP, but uncleavable signal sequences by PSORT.
• TBG7 was suggested to be targeted to the chloroplast by PSORT. Particular observations for each
• TBG7 248 amino acids [SEQ ED NO: 13], signal sequence not determined; TBG7:
• Tomato ⁇ -galactosidase (TBG) cDNA sequence data Five full-length and 2 partial-length cDNAs were cloned and sequenced. The DNA and deduced amino acid sequence data is presented below
• N-LINK possible N-linked glycosylation sites
• ER endoplasmic reticulum
• out secreted
• PM tethered to plasma membrane
• CHLOR chloroplast
• nucleic acid molecules of the present invention may be in
• RNA such as mRNA
• DNA including, for instance, cDNA and genomic DNA obtained by cloning or produced
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA or RNA may be the coding strand, also known as the
• sense strand or it may be the non-coding strand, also referred to as the anti-sense strand.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
• recombinant DNA molecules contained in a vector are
• isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• isolated RNA molecules include in vivo or in
• nucleic acid molecules according to the present invention further include such
• Isolated nucleic acid molecules of the present invention include DNA
• DNA molecules comprising the coding sequence for the
• isolated nucleic acid molecules of the invention include
• DNA molecules which comprise a sequence substantially different from those
• this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 2 [SEQ ED NO: 4] or a nucleic acid molecule having a sequence complementary to the above
• probes for gene mapping by in situ hybridization with chromosomes, and for
• the present invention is further directed to nucleic acid molecules
• the invention provides a polynucleotide having a nucleotide sequence representing the portion of Figure 2 [SEQ ED NO: 4] which consists of positions 1-2538 of Figure 2 [SEQ ID NO: 4].
• the invention provides additional nucleic acid molecules
• the invention provides an isolated nucleic acid
• nucleic acid molecule of the invention comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone
• nucleic acid molecules of the present invention which
• encode a ⁇ -galactosidase LI polypeptide may include, but are not limited to
• pre-, or pro- or prepro- protein sequence the coding sequence of the mature
• polypeptide with or without the aforementioned additional coding sequences.
• TBG2 is expressed before the onset of fruit ripening and continues at uniform
• TBG2 has been found to be expressed in
• TBG2 to be unaffected by ethylene.
• TBG2 is expressed in the ripening mutants rin, nor and Nr at the normal chronological time after anthesis.
• antisense orientation specifically in tomato fruit, in all tomato fruit tissues, starting before and continuing throughout the entire ripening process.
• the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or
• allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of
• Non-naturally occurring variants may be produced using art-known
• variants include those produced by nucleotide substitutions, deletions or additions.
• substitutions, deletions or additions may involve one or more nucleotides.
• the variants may be altered in coding regions,
• non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or
• galactosidase II protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
• nucleic acid molecules encoding the mature protein having the amino acid sequence shown in Figure 2 as pZBG2-l-4 or
• nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide selected from the group consisting of: (a) a nucleotide
• the present invention also relates to vectors which include the isolated
• DNA molecules of the present invention are genetically identical to
• galactosidase Et polypeptides or fragments thereof by recombinant techniques.
• the vector may be, for example, a phage, plasmid, viral or retroviral vector.
• Retroviral vectors may be replication competent or replication defective.
• the polynucleotides may be joined to a vector containing a selectable
• plasmid vector is introduced in
• a precipitate such as a calcium phosphate precipitate, or in a complex with a
• the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome
• the constructs will preferably include a translation initiating codon at the
• the expression vectors will preferably include at least one
• markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or
• ampicillin resistance genes for culturing in E. coli and other bacteria.
• appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, StrepZBG2-l-4yces and Salmonella
• fungal cells such as yeast cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, 293
• vectors preferred for use in bacteria include pQ ⁇ 70, pQE60
• pBS vectors Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• preferred eukaryotic vectors are
• leaf, flower, and stem tissues were harvested from greenhouse- grown plants and roots were harvested from seedlings grown in basal tissue
• RNA was extracted using the method described in Verwoerd et al.
• Poly(A)RNA was purified from total RNA using oligo(dT) columns (Pharmacia, Piscataway, NJ). RNA was quantified by measuring A 260 using a dual beam spectrophotometer.
• the two primers used for the first reaction were BG5 ⁇ 1 (WSNGGNWSNATHCAYTAYCC) and BG3 ⁇
• Amplification was done using AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) and standard PCR conditions using the cDNA
• the first comprised poly(A) RNA isolated from breaker, turning and pink fruit pericarp from 'Rutgers' plants.
• the cDNA synthesis and library construction was done exactly according to the manufacturers instructions for the ZAP-cDNA Gigapack LI Gold Cloning
• First-strand cDNA synthesis was primed using a poly(dT) primer and inserts were directionally cloned into the Uni-Zap XR vector using
• the second library comprised poly(A) RNA
• RT-PCR2-1 One of the clones (RT-PCR2-1) was used to screen 10 6 plaques from the
• tomato fruit cDNA libraries at low stringency hybridization at 45 °C, no
• DNA DNA gel blot analysis was done essentially as described in Smith and
• RNA (20 ⁇ g/ lane) was separated in a formaldehyde/Mops
• RNA ladder standard (GibcoBRL) was used to estimate the length of the RNAs. Probes were synthesized using a random
• RNA blots were stripped and re-probed at a reduced hybridization and
• 32 P(dATP)-labeled probe was diluted to 1-2 x 10 6
• ABI automated sequencer (Applied Biosystems, Foster City, CA). The sequencing of both cDNA insert strands was done by primer walking.
• galactosidase genes had a fruit-specific expression pattern. With the exception of TBG2, transcripts of all clones were detected in non-fruit tissues (Fig. 4).
• TBG3 Transcripts of TBG 1 , 4, 5 and 6 were detected in all the tissues tested.
• TBG7 transcript was detected in flower and stem tissues.
• TBG1 and 3 had similar expression patterns and their transcripts were
• TBG2 had a unique
• TBG4 expression pattern was similar to TBG1 and
• TBG5 had a similar expression pattern to TBG4 during the ripening stages of development, however TBG5 transcript was also detected throughout all the earlier stages of fruit development.
• TBG6 had an interesting expression pattern and its transcript was only detected at high levels in all pre-ripening
• TBG7 also had a unique expression pattern and its transcript was
• TBG7 TBG7
• TBG4 transcripts were detected in RNA samples extracted from all
• TBG4 transcript was notably more abundant in the peel.
• TBG3 and TBG5 expression patterns were unique and their transcripts
• TBG4 transcript was not detected in fruit tissue of N ' and was detected at much lower levels in rin and nor than wild type fruit tissues. Normally TBG6 transcripts are detectable at high levels throughout the early stages of fruit development but are not detectable after the mature green
• TBG6 transcripts persisted even to 50 dpp in fruit of all three mutants.
• TBG4 expression might be up-regulated by ethylene and that TBG6
• TBG4 was used first because the
• TBG4 protein was successfully affinity purified using an anti-FLAG affinity
• the affinity-purified TBG4 enzyme was shown to have ⁇ (l— >4)-D-
• Table ETI Cell wall degrading activity of TBG4 and TBG1 expressed in yeast. Removal of galactosyl residues from chelator soluble (CSP) and alkali soluble (ASP) pectin and hemicellulosic (HCF) cell wall fractions purified from tomato fruit. ⁇ _ ⁇ g galactose released enzyme substrate boiled live a TBG4 CSP 0 5
• the TBG4 ORF was cloned in-frame into the repressible/inducible bacterial expression vector pFLAG-CTC.
• the host strain XL 1 -Blue MR is a
• pZBG2-l-44 ORF were grown at 20°C and induced with EPTG, the cells
• coli ⁇ -galactosidase activity was pG ⁇ M ( ⁇ ) and the vector used as a negative
• control and for expression was pFLAG-CTC either without (°,*») or containing the pZBG2-l-4 ORF ( ⁇ ,A).
• TBG4 mRNA were up to 40% firmer [compare means of parental line #1 with
• constructs were made using the constitutively expressed 35S CaMV promoter
• cultivars Of the fresh pick cultivars one is a soft fruit large cherry tomato (cv 'Ailsa Craig'), the second is a soft fruit old breeding line (cv 'Rutgers') and
• line 1 was the parental line and lines 2-8 each represent an independent transformant containing one T-DNA copy
• fruit from transformed line 2 had fruit with a mean firmness that was 40%
• composition of fruit where TBG4 mRNA levels have been suppressed are designed to show a link between increased fruit firmness and
• TBG4 mRNA suppression TBG4 encoded enzyme activity suppression, possible cell wall modification (e.g. increased galactosyl residue content) and a decrease in free galactose levels during fruit ripening.
• the antisense transcripts appear as two bands, smaller in length than the endogenous mRNA.
• the two bands probably resulted from 1, the expected transcriptional stop signal provided by the NOS -terminator and 2, a cryptic transcriptional stop signal in the antisense TBG4 cDNA. The most
• Line 2 also had the firmest red fruit (see Figure 11A -D).
• pZBG2-l-4 is a cDNA derived from the
• transcript of the TBG4 gene which codes for ⁇ -galactosidase LI for the
• ⁇ -galactosidase IE activity remained at levels equal to mature green fruit and
• yeast expression system has both ⁇ -galactosidase activity and exogalactinase
• ⁇ -galatosidase LI is expressed during fruit ripening. Plant Physiol. 117:

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