WO1999012950A9 - Improvements in or relating to stability of plant starches - Google Patents
Improvements in or relating to stability of plant starchesInfo
- Publication number
- WO1999012950A9 WO1999012950A9 PCT/GB1998/002665 GB9802665W WO9912950A9 WO 1999012950 A9 WO1999012950 A9 WO 1999012950A9 GB 9802665 W GB9802665 W GB 9802665W WO 9912950 A9 WO9912950 A9 WO 9912950A9
- Authority
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- WIPO (PCT)
- Prior art keywords
- plant
- starch
- sequence
- nucleic acid
- isoamylase
- Prior art date
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- 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
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2457—Pullulanase (3.2.1.41)
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- 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
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- 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
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/246—Isoamylase (3.2.1.68)
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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)
- C12Y302/01041—Pullulanase (3.2.1.41)
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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)
- C12Y302/01068—Isoamylase (3.2.1.68)
Definitions
- This invention relates to novel nucleotide sequences, nucleic acid constructs and host cells and organisms comprising said sequences, methods of altering plants using the sequences, and to products obtainable from the altered plants.
- Starch the major storage reserve in plants, consists of two main polysaccharides, amylose and amylopectin.
- Amylose is a linear polymer containing ⁇ -1,4 linked glucose units
- amylopectin is a highly branched polymer consisting of a ⁇ -1,4 linked glucan backbone with ⁇ -1,6 linked glucan branches.
- amylopectin constitutes about 75% of the starch content.
- starch synthases which elongate the glucan chains
- SBE starch branching enzymes
- Debranching enzymes hydrolyse the c -6-glucosidic linkages of ⁇ -glucan polymers and are classified into two types depending on their substrate specificities: (1) pullulanases (EC 3.2.1.41 , which are known in plants as "R- enzymes") which act on amylopectin and pullulan (a linear bacterial polymer consisting of 1-6 linked maltotriose units) but which cannot use glycogen as a substrate; and (2) isoamylases (EC 3.2.1.68) which act on amylopectin and glycogen but cannot use pullulan as a substrate.
- pullulanases EC 3.2.1.41 , which are known in plants as "R- enzymes”
- amylopectin and pullulan a linear bacterial polymer consisting of 1-6 linked maltotriose units
- isoamylases EC 3.2.1.68 which act on amylopectin and glycogen but cannot use pullulan as a substrate.
- a cDNA encoding a potato DBE was described in a second patent application (WO 96/19581) which detailed the isolation of a partial length (492 bp) cDNA clone encoding a pullulanase type DBE, as well as the effect of downregulating the expression of this gene in transgenic antisense plants.
- An unspecified number of lines were generated and some showed a decrease in debranching enzyme activity (not quantitated) which also led to a slight modification in the swelling characteristics of the starch as measured by RVA (Rapid Viscoamylograph).
- the present application relates particularly to the isolation of nucleic acid sequences encoding potato debranching enzymes (especially isoamylase type debranching enzymes) and to the uses thereof.
- the nucleic acid sequence comprises nucleotides 79-1595 of the nucleic acid sequence shown in Figure 8, or a functional equivalent thereof.
- This sequence includes a portion encoding the amino acid sequence MDWF/YNH, which portion is typically present in the nucleic acid sequences of the invention.
- Functionally equivalent nucleic acid sequences include sequences which encode substantially the same polypeptide, but which differ in nucleotide sequence from that shown in Figure 8 by virtue of the degeneracy of the genetic code.
- a nucleic acid sequence may be altered (e.g.
- the invention provides a replicable nucleic acid construct comprising the nucleic acid sequence of the first aspect defined above.
- the construct may typically comprise a selectable marker (e.g. kanamycin or hygromy cm-resistance) and may allow for transcription (and possibly translation) of the nucleic acid sequence of the invention.
- a selectable marker e.g. kanamycin or hygromy cm-resistance
- the construct will comprise a promoter (especially a promoter sequence operable in a plant and/or a promoter operable in a bacterial cell), and one or more regulatory signals (such as polyadenylation signals) known to those skilled in the art.
- Inhibition of the isoamylase gene might be expected to affect the properties of the starch present in the plant. In particular it might be expected to result in a starch having increased branching and/or shorter chain length. The obtaining of starch having such properties is a particular object of the present invention.
- the introduced sequence of the invention and the one or more additional sequences may be operably linked to a single promoter (which would ensure both sequences were transcribed at essentially the same time), or may be operably linked to separate promoters (which may be necessary for optimal expression). Where separate promoters are employed they may be identical to each other or different. Suitable promoters are well known to those skilled in the art and include both constitutive and inducible types. Examples include the CaMV 35S promoter (e.g. single or tandem repeat) and the GBSS type I or patatin promoters of potato.
- the promoter will be tissue-specific. Desirably the promoter will cause expression of the operably linked sequence at substantial levels only in the tissue of the plant where starch synthesis and/or starch storage mainly occurs.
- the operably linked promoter may be tuber-specific, such as the patatin promoter.
- the invention provides a nucleic acid sequence encoding at least a portion of a potato pullulanase type debranching enzyme, the sequence comprising substantially nucleotides 46-774 of the nucleic acid sequence shown in Figure 10, or a functional equivalent thereof.
- the term "functional equivalent” should be interpreted as explained previously, but with reference (in this aspect of the invention) to Figure 10 rather than Figure 8.
- the invention provides nucleic acid constructs, and altered host plant cells and organisms, comprising the pullulanase-type DBE nucleic acid sequence defined immediately above.
- Figure 1 shows a comparison of the known amino acid sequences of various putative isoamylase proteins.
- the shaded area indicates residues (single letter amino acid code) that are identical to die maize protein. Dashes indicate gaps introduced to maximise the alignment. Although the whole sequences were compared, only the most conserved region is shown. Sequences are identified at the right; A10906_l is an artificial sequence, A37035 is from a Pseudomonas species SMP1, HIU32761 is from Haemophilus influenzae, ISOA_PSESP is from Pseudomonas amylodermosa and ZMU18908_1 is from Zea mays (corn);
- Figures 3A and 3B show the DNA sequence (SEQ. ID. NO.s 1 and 4 respectively) and predicted intron/exon structure, including predicted amino acid sequences (SEQ. ID. NO.s 2 and 3) at the 5' and 3.' ends respectively of a partial pullulanase type DBE genomic clone (certain restriction sites are also marked);
- Figures 4A and 4B show the DNA sequence (SEQ. ID. NO.s 5 and 8 respectively) and predicted intron exon structure, including predicted amino acid sequences (SEQ. ID. NO.s 6 and 7) at the 5' and 3' ends respectively of a partial pullulanase type DBE genomic clone (certain restriction sites are also marked);
- Figures 5 A and 5B show a comparison of the 5' and 3' ends (respectively) of two partial isoamylase type DBE genomic clones recovered from potato by the present inventors (nucleotides that are identical are shown between the two sequences and dashes indicate gaps introduced to optimise the alignment);
- Figures 6 A and 6B show the DNA sequence (SEQ. ID. NO.s 9 and 11 respectively) and predicted intron exon structure, including predicted amino acid sequence (SEQ. ID. NO. 10) at the 5' and 3' ends respectively of a partial isoamylase type DBE genomic clone (certain restriction sites are also marked);
- Figure 8 shows the DNA sequence (SEQ. ID. NO. 14), restriction map and predicted open reading frame (including predicted amino acid sequence, SEQ. ID. NO. 15) of a partial length potato debranching enzyme (isoamylase) cDNA clone (pSJ132, described below); nucleotides 79-1595, including the poly(A) tail, are isoamylase, the other sequence is derived from pT7Blue vector;
- Figure 12 shows a zymogram (using a 5% polyacrylamide gel containing 1 % amylopectin) of tuber extracts from potato lines transformed with an antisense isoamylase construct in accordance with the invention.
- PDBE2 ATC AAA TCT/G A GAA nCC ATC DBE1 ATG GAT GTT GT(T/C/A) TA/TC/T AAT/C CAT
- Genomic DNA was isolated from potato leaves using a modified CTAB (cetyltrimethyl ammonium bromide) method (Murray and Thompson, 1980).
- Leaf tissue (4 gm) was frozen in liquid nitrogen and ground in a mortar and pestle and transferred to a 50 ml polypropylene centrifuge tube (Falcon) to which was added 40 ml of extraction buffer ( 2% w/v CTAB, 100 mM Tris-HCI pH 8.0, 1.4 M NaCI, 10 mM 3-mercaptoethanol and 20 mM EDTA) and incubated at 65°C for 30 min. After cooling, the mixture was split into two tubes and 15 ml of chloroform was added to each followed by gentle (end over end) mixing for 15 min at room temperature.
- CTAB cetyltrimethyl ammonium bromide
- the tubes were then centrifuged for 5 min at 5000g and the pooled aqueous phases were re-extracted with 5 ml of chloroform.
- the DNA was then precipitated by incubating on ice for 30 minutes with an equal volume of isopropanol and centrifuged for 5 minutes at 3000g. The pellet was washed once with 70% ethanol, briefly air-dried and resuspended in TE pH 8.0 containing 20 ⁇ g/ml RNase A. After incubation at room temperature for 30 minutes the DNA was extracted with 5ml of chloroform, one tenth volume of 3 M sodium acetate (NaAc) pH 5.2 was added and the DNA precipitated with two volumes of ethanol. After centrifugation and washing as above, the DNA was resuspended in 2 ml of TE buffer at pH 8.0 and estimated to be approximately 150 ng/ ⁇ l by fluorometric measurement.
- oligonucleotide primers were made corresponding to these sequences (DBE2 & DBEl respectively, see above), taking into consideration the codon usage in plant genes in order to reduce the degeneracy of the primers. These were used in a PCR reaction using first strand cDNA from leaf or tuber RNA or genomic DNA as template.
- Colonies were screened by PCR and positives were grown up and DNA isolated from a 50 ml culture by alkaline lysis and PEG precipitation. One clone of each of the 5 PCR fragments was sequenced from either end and in one direction only, therefore the sequence cannot be guaranteed 100 % correct.
- the exon sequences of the 1.8 and 1.6 kb bands (clones #43 & #34, designated pSJ134 and pSJ135 respectively) were found to be identical to pSJ98 (a pullulanase type DBE partial clone, described below) indicating that they are pullulanase type debranching enzymes.
- the two clones pSJ134 and pSJ135 are not identical, sharing 96.3% identity over the entire sequence at the 5' end, and 89.8% identity over 160 nucleotides at the 3' end, after which the two sequences diverge.
- Figure 2 A shows a comparison of the sequences of clones 134 and 135 at their 5' ends
- Figure 2B shows a comparison of their sequences at the 3' end
- Figures 3 A and 3B illustrate the presumed intron/exon structure of the 5' and 3' ends (respectively) of clone 134
- Figures 4A and 4B illustrate the presumed intron/exon structure of the 5' and 3' ends respectively of clone 135.
- two exons can be distinguished, separated by a 101 bp intron and the remainder of the exon sequences are present in the 3' end.
- Figure 5A shows a comparison of the 5' ends of these clones, whilst Figure 5B compares their 3' ends.
- Figures 6 A and 6B illustrate the presumed intron/exon structure of the 5' and 3' ends (respectively) of clone 133.
- Figures 7A and 7B illustrate the presumed intron/exon structure of the 5' and 3' ends respectively of clone 137. Only one intron can be distinguished at each end.
- the 0.8 kb clone although it had the correct primers on both ends was not a debranching enzyme but had homology to a rice Expressed Sequence Tag (RICR2232A) which encodes a 70kDa endomembrane protein.
- a new sense primer (PIAl) was designed to the isoamylase sequences just downstream of the DBEl primer and this was use in a 3 'RACE PCR to clone the 3' end of the isoamylase gene.
- Five ⁇ l of first strand cDNA and 25 pmol of each primer (PIAl and Ro) were used in a 50 ⁇ l PCR reaction (94°C 2 min followed by 30 cycles of 94°C 45 seconds, 55°C 25 seconds, 72°C 2 min).
- One ⁇ l of this reaction was then used in a second PCR amplification using PIAl and R, primers.
- a major band of approx 1.5 kb (the expected size) was amplified from both leaf and tuber cDNA templates.
- the tuber PCR fragment was purified on a 1% TAE agarose gel and then cloned in pT7Blue (clone designated pSJ132).
- the sequence and predicted ORF of this partial length clone is shown in Figure 8.
- the ORF contains 429 aa and is 77.0% identical to the maize isoamylase protein encoded by the sugary- 1 gene (data not shown).
- a plant transformation vector with the hygromycin selectable marker was constructed in which the 1.5 kb isoamylase cDNA from plasmid pSJ132 was inserted in an antisense orientation under the control of the granule bound starch synthase (GBSS) promoter. This vector (pSJ138) was then used to transform wild type and waxy potato plants.
- GBSS granule bound starch synthase
- a plant transformation vector (pSJ138) was constructed by inserting the 1.5 kb isoamylase cDNA from plasmid pSJ132 (as anXba l-Sac I fragment) in an antisense orientation under the control of the granule bound starch synthase (GBSS) promoter into the plant transformation vector pSJ39 which had been cut with the same enzymes.
- GBSS granule bound starch synthase
- pSJ39 is a modified GPTV-HYG vector (Becker et al 1992) contaimng a 0.8 kb GBSS promoter and was constructed as follows: first the BamHI site between the hygromycin selectable marker and the gene 7 polyadenylation signal in pGPTV-HYG was destroyed by cutting and filling in with klenow polymerase to create pSJ35.
- Hz ⁇ dlll-EcoRI fragment of this vector (containing the GUS gene & nos polyadenylation signal) was replaced with the Ht «dIII- EcoRI fragment (contaimng the GBSS promoter-GUS-nos poly(A) cassette) from plasmid pGB121 (obtained from R Visser, Wageningen) to create plasmid pSJ39.
- Transformed potato plants cv Desiree were produced by co-cultivation of explants with Agrobacterium tumefaciens LBA 4404 containing plant transformation vector plasmids. Two explant types were used, microtubers and leaf fragments, both produced from stock cultures, requiring different methods in culture. Stocks of plantlets were maintained of wild type untransformed Desiree or selected lines previously transformed with antisense genes coding for enzymes of the starch biosynthetic pathway by regular transfer of single node explants using Murashige and Skoog media (1962 Physiol. Plant 15, 473) solidified with 0.8% agar with the sucrose concentration reduced from 3% to 1%.
- Microtuber explants were produced by transferring nodes to the same medium with the sucrose concentration increased to 8% plus the addition of 2.5mg/l benzylaminopurine. All cultures were grown at 22°C with illumination from fluorescent tubes at 40 ⁇ E/sqm for 16 hours, except those cltures grown to produce microtubers, which were kept in darkness.
- the protocol for microtuber explants, and that for leaf explants are detailed below.
- Explants were initially precultured on the appropriate multiplication medium for two days before co-cultivation. Co-cultivation was for 10 minutes in an overnight culture of the Agrobacterium before explants were blotted on filter paper to remove excess bacteria. After blotting explants were transferred to filter paper on feeder layers and incubated in darkness at 22°C for two days.
- the feeder layers consisted of a 9cm petri dish containing the appropriate multiplication media covered by 2ml of stationary phase tobacco suspension culture cells which were in turn covered by two layers filter paper.
- Tissue culture and selection using microtuber explants was performed as follows. Microtubers were taken from stock when they were sufficiently large and mature to use. This occurred after 6-8 weeks from transfer of nodes to the high sucrose medium when they weighed more than 30mg and had started to turn from white to pink. Stocks were used for several months after tuber initiation provided they remained dormant and no new shoots or stolons had emerged from the bud end. Each tuber was cut in half longitudinally through the terminal bud and the cut end put in contact with the medium.
- the multiplication medium (Z5) used contained the salts and vitamins of Murashige and Skoog plus 3% sucrose, 0.2mg/l indole acetic acid, 5 mg/1 zeatin and 0.8% agar.
- Kanamycin was added at lOOmg/1 when NPTII was used and hygromycin at 15mg/l when HPTII was used.
- the explants were transferred to fresh medium every two weeks. Explants had two transfers on medium Z5, followed by one transfer on the same medium, except indole acetic acid and zeatin were replaced by lOmg/1 of gibberellic acid (medium MSG). At each transfer all shoots were removed and discarded. The final transfer was to hormone- free Murashige and Skoog medium with the sucrose concentration reduced from 3% to 1%. Shoots were allowed to develop at this stage. Tissue culture and selection using leaf explants was performed as follows.
- the multiplication medium used contained the salts and vitamins of Murashige and Skoog medium plus 1.6% glucose, 0.02 mg/1 naphthyl acetic acid, 2mg/l zeatin riboside and 0.8% agar. Kanamycin was added at lOOmg/l when NPTII was used and hygromycin at 5mg/l when HPTII was used. Cultures were transferred every two weeks to the same medium. After four transfers the cultures which were vitrified or showed poor shoot development were transferred to liquid Murashige and Skoog medium containing 1.6% sucrose only.
- Transformation was confirmed as follows. Shoots which developed from either tuber of leaf explants after the multiplication/organogenesis phase were transferred individually to hormone- free Murashige and Skoog medium containing 1% sucrose and 100 mg/1 kanamycin or 15mg/l hygromycin depending on the selection which was used. Only one shoot was taken from each explant unless the origin of separate shoots were well separated on the explant. Shoots which rooted on selection within two weeks were transferred to the same medium without selection and given an identifying number. When growth allowed sufficient tissue to be sampled, DNA was extracted from a few mgs of leaf and stem tissue of the culture using the method published by Edwards et al (1991 Nucleic Acids Research 19,(6) 1349) and transformation was confirmed by PCR using gene specific primers. Material was left in culture to allow individual shoots to be micropropagated. Positive shoots were micropropagated to be stored in vitro and also to provide planting material.
- Representative samples of the tubers were stored for analysis and possible regrowth and 150-200g fresh weight was taken for starch extraction.
- Washed tubers were diced and homogenised to a juice by passage through a Braun MP 75 centrifugal juicer.
- the juice was diluted with tap water to a volume of approximately 3L in a 5L conical flask. After 30 minutes the unsedimented supernatant liquid was discarded and the remaining solids washed again with a further 3L of water. After a further 30 minutes the liquid was decanted and discarded.
- the slurry of solids was poured through a 500 ⁇ m metal mesh filter which retained the bulk of the cell debris.
- the crude starch which passed through the filter was washed in the same manner until no protein foam remained and only white starch settled on the base of the flask below a clear supernatant liquid.
- the starch was harvested on filter paper using a Buchner funnel and washed on the funnel with two 500ml volumes of distiled water. Finally the starch was washed with 500ml of acetone and dried in a fume hood for approximately 30 minutes.
- the degree of branching of the starches was determined by 'H NMR spectroscopy (Gidley
- Example 3 Cloning of a pullulanase type debranching enzyme from potato.
- a PCR based cloning strategy was devised for isolating pullulanase type debranching enzymes from potato, using conserved domains within the known cloned gene sequences, of which spinach was the only plant gene (genbank SOPULSPO_l).
- the amino acid sequence DGFRFDLMG was almost completely conserved (data not shown) and this is found at aa 546-554 of the spinach protein (964 aa in total). It was hoped therefore that this would also be present in the potato gene, as the sequence DGFRFD is also completely conserved in plant starch branching enzyme genes which belong to the same superfamily of amylolytic genes.
- Peptide sequences from the purified potato debranching enzyme which were published in WO 95/04826 were also compared to the spinach sequence; of the 12 peptide sequences 10 made possible matches to the spinach sequence and of these peptide number 1 (RTLLVNLDSDDVKPEGGDNL. corresponding to aa 300-319 of the spinach sequence, identical residues underlined) was chosen to make a degenerate oligonucleotide sense primer (PDBEl;20mer,64x) to be used in PCR with a degenerate antisense primer designed to the sequence DGFRFD (PDBE2; 18mer, 16x). These primers were used in a PCR reaction with tuber or leaf first strand cDNA as template.
- cDNA Five ⁇ l of cDNA was used as template in a 50 ⁇ l PCR reaction with 50 pmol of each degenerate primer (PDBE1 & 2) under standard PCR conditions (28 cycles of 94°C 45 seconds, 48°C 25 seconds, 72°C 90 seconds).
- the PCR products were separated on a 1% agarose gel in TAE buffer.
- a strong band of the expected size (672bp) was amplified from the tuber cDNA and a much weaker band from the leaf cDNA.
- the tuber amplified band was cut out and purified by binding to DEAE paper and then cloned in the pT7Blue vector (Novagen).
- a diagram of the resulting vector is shown in Figure 11. In Figure 11, the symbols used have the same meanings as in Figure 9: pA-CaMV is the CaMV polyadenylation signal. .. This vector may then be used to transform potato plants, substantially as described in Example 2.
- zymograms are usually used to determine the presence or absence of debranching enzymes. Protein extracts were separated on native 5% polyacrylamide gels containing 1% potato amylopectin. After separation the gel was incubated for 1-3 hours in 100 mM citrate buffer pH 5.5 containing 10 mM EDTA, 1 mM acarbose, and 2.5 mM DTT. The gel was then rinsed twice in deionized water and stained with iodine (Lugol's solution). Debranching enzymes stain as blue bands against a purple background, whereas amylases appear as clear bands.
- the debranching enzyme bands in the amylopectin gel can thus be classified into isoamylase bands (which do not appear in the red pullulan containing gel) and pullulanase bands (which give clear bands in red pullulan containing gels).
- tuber extracts of ten different antisense lines were analysed in a zymogram as described above.
- the upper band with a low electrophoretic mobility is a debranching enzyme as it stains blue in the amylopectin containing gel against a purple background.
- Transformed potato line 1352 clearly lacks this isoamylase band. In this line isoamylase expression was successfully inhibited by the antisense construct.
- the lower band apparent in Figure 12 is an amylase which stains as a clear zone in a purple background. Depending on incubation time, two additional debranching enzyme bands could be detected which are of the pullulanase type and not affected in line 1352.
- RVA Rotary Visco Analyser
- Viscosity development of starches was analysed using a Rapid Visco Analyser Series 4 instrument (Newport Scientific, Sydney, Australia). For the 13 minute profile, 2 g of starch was weighed into a sample cup and 25 ml of water was added to give a final starch concentration of 7.4 % (w/v) and the analysis was performed using the standard stirring and heating protocol according to the manufacturer's instructions. Viscosity was measured in centipoise (cP).
- Figure 13 shows the viscosity profiles of tuber starches from plants transformed with the antisense isoamylase construct.
- the linear plot shows the temperature profile.
- a wild type control line 1302, unbroken thin line
- Starch from line 1352 solid thick curve
- Starch from line 1352 shows a clearly reduced peak viscosity and a significantly higher setback viscosity compared to the wild type control (1302) and starches of transformed plants with no change in isoamylase expression level
- lines 1328 - thin dotted curve; 1365 - thin dotted/dashed line; and 1369 - thin dashed line are slightly increased for line 1352.
- PCR fragment (492 bp) was isolated using primers derived from the sequence of a genomic clone which hybridised to a degenerate oligonucleotide very similar to the PDBE1 oligo which was used in the experiments described here.
- the deduced amino acid sequence of the PCR fragment did not correspond exactly to the two peptide sequences from the purified protein (RTLLVNLDSDDVKPEGGDNL, SEQ. ID. NO. 30; and RLSSAGITHVHLLPTYNFA, SEQ. ID. NO. 31; differences are shown underlined).
- the inventors have also cloned two PCR fragments (2.4 and 2.0 kb; pSJ133 and 137 respectively) from genomic DNA of potato which have significant homology to isoamylase genes. These clones differ in the size, and perhaps number, of introns and are about 96-97% identical ( Figure 4), suggesting that they are derived from different genes.
- a cDNA fragment of approx 1500 bp was obtained by 3 'RACE; this cDNA encodes 429 aa which are 77% identical to the maize isoamylase gene and the inventors are confident that this gene is an isoamylase type DBE.
- the two different isoamylase-type DBE genomic sequences isolated by die inventors relate to two different genes having slightly different functions or roles in the potato plant, and that similar multiple isoamylase-type genes exist in other plants. Different isoamylase-type genes might, for instance, be subjected to different regulatory controls. As the genomic sequences recovered are not full length genes, it is possible that the respective gene sequences may diverge in the uncloned portions.
- Both pullulanase and isoamylase genes appear to be expressed in tubers and leaves since PCR bands of the correct size can be amplified from either tuber or leaf first strand cDNA templates. The intensity of the amplified band is stronger in tubers for both DBE, possibly indicating that the expression level of both genes is higher in tubers, but this should be confirmed by northern analysis.
Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98941593A EP1009751A2 (en) | 1997-09-06 | 1998-09-04 | Improvements in or relating to stability of plant starches |
CA002302639A CA2302639A1 (en) | 1997-09-06 | 1998-09-04 | Improvements in or relating to stability of plant starches |
AU89911/98A AU8991198A (en) | 1997-09-06 | 1998-09-04 | Improvements in or relating to stability of plant starches |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9718863.5A GB9718863D0 (en) | 1997-09-06 | 1997-09-06 | Improvements in or relating to stability of plant starches |
GB9718863.5 | 1997-09-06 |
Publications (3)
Publication Number | Publication Date |
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WO1999012950A2 WO1999012950A2 (en) | 1999-03-18 |
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WO1995004826A1 (en) * | 1993-08-09 | 1995-02-16 | Institut Für Genbiologische Forschung Berlin Gmbh | Debranching enzymes and dna sequences coding them, suitable for changing the degree of branching of amylopectin starch in plants |
US5750876A (en) * | 1994-07-28 | 1998-05-12 | Monsanto Company | Isoamylase gene, compositions containing it, and methods of using isoamylases |
DE4447387A1 (en) * | 1994-12-22 | 1996-06-27 | Inst Genbiologische Forschung | Debranching enzymes from plants and DNA sequences encoding these enzymes |
DE19618125A1 (en) * | 1996-05-06 | 1997-11-13 | Planttec Biotechnologie Gmbh | Nucleic acid molecules that encode new potato debranching enzymes |
GB9716185D0 (en) * | 1997-07-31 | 1997-10-08 | Innes John Centre | Starch debranching enzymes |
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1997
- 1997-09-06 GB GBGB9718863.5A patent/GB9718863D0/en not_active Ceased
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1998
- 1998-09-04 WO PCT/GB1998/002665 patent/WO1999012950A2/en not_active Application Discontinuation
- 1998-09-04 CA CA002302639A patent/CA2302639A1/en not_active Abandoned
- 1998-09-04 AU AU89911/98A patent/AU8991198A/en not_active Abandoned
- 1998-09-04 EP EP98941593A patent/EP1009751A2/en not_active Withdrawn
Cited By (3)
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US9173394B2 (en) | 2007-09-26 | 2015-11-03 | Bayer Intellectual Property Gmbh | Active agent combinations having insecticidal and acaricidal properties |
US9574201B2 (en) | 2010-06-09 | 2017-02-21 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
US9593317B2 (en) | 2010-06-09 | 2017-03-14 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
Also Published As
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WO1999012950A3 (en) | 1999-05-06 |
EP1009751A2 (en) | 2000-06-21 |
WO1999012950A2 (en) | 1999-03-18 |
GB9718863D0 (en) | 1997-11-12 |
CA2302639A1 (en) | 1999-03-18 |
AU8991198A (en) | 1999-03-29 |
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