WO2001032886A2 - Enzyme de ramification d'amidon - Google Patents

Enzyme de ramification d'amidon Download PDF

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WO2001032886A2
WO2001032886A2 PCT/CA2000/001276 CA0001276W WO0132886A2 WO 2001032886 A2 WO2001032886 A2 WO 2001032886A2 CA 0001276 W CA0001276 W CA 0001276W WO 0132886 A2 WO0132886 A2 WO 0132886A2
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starch
wheat
endosperm
branching enzyme
plant
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PCT/CA2000/001276
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WO2001032886A3 (fr
Inventor
Monica Baga
Mingsheng Peng
Ramesh B. Nair
Anne Repellin
Ming Gao
Pierre Hucl
Graham J. Scoles
Ravindra N. Chibbar
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National Research Council Of Canada
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Priority to AU11227/01A priority Critical patent/AU1122701A/en
Priority to US10/110,777 priority patent/US7041484B1/en
Priority to CA002389390A priority patent/CA2389390A1/fr
Publication of WO2001032886A2 publication Critical patent/WO2001032886A2/fr
Publication of WO2001032886A3 publication Critical patent/WO2001032886A3/fr

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)

Definitions

  • the invention relates to the field of plant molecular biology, particularly to enzymes of starch bio-synthesis.
  • the endosperm of wheat, barley, rye and triticale contain large A-type and small B-type starch granules at maturity 1 .
  • the large A-type starch granules are more than 10 ⁇ m in diameter and lenticular in shape, while B- type starch granules are less than 10 ⁇ m in diameter and roughly spherical in shape 2 .
  • A- and B-type starch granules have significantly different chemical compositions and functional properties 3 , wheat cultivars with predominantly A- or B-type starch granules would be very useful to the food and non-food industries.
  • A-type starch granules are produced in amyloplast at about four to five days- post-anthesis (DP A), and their number increases until 12 to 14 DP A 4 . Subsequently, the A-type starch granules grow in size to an eventual diameter of from 10 ⁇ m to more than 36 ⁇ m. The number of A-type starch granules per endosperm is constant from about 15 DPA to maturity.
  • B-type starch granules are actively initiated about 14-16 DPA. Both the number and size of B-type starch granules increase until wheat grain matures. The diameter of B-type starch granules is less than 10 ⁇ m 2 .
  • the mechanisms controlling the initiation and size growth of A- and B-type starch granules are unknown. Based on the current knowledge about starch granule synthesis, several mechanisms could be proposed.
  • the initiation and size growth of A- and B-type starch granules may be controlled by different isoforms of starch synthases (SS), starch branching enzymes (SBE), and debranching enzymes (DBE). These enzymes are involved in the biogenesis of plant starch granules 5 .
  • Starch branching enzyme ( ⁇ -l,4-glucan-6-glycosyltransf erase; EC 2.4.1.18, SBE) is a key enzyme in the starch biosynthesis pathway.
  • the enzyme acts on glucose polymers and catalyses excision and transfer of glucan chains to the same or other glucan molecules. Translocated chains are attached to the polymer through ⁇ -l,6-glucosidic bonds to form branches on the ⁇ -l,4-linked glucose backbone.
  • All of the reported SBE from plants to date can be divided into two classes, SBEI and SBEII, based on their amino acid sequences 10 .
  • SBEs are in the 80-100 kDa molecular mass range and, like all enzymes of the ⁇ -amylase family, carry a ( ⁇ ) ⁇ barrel domain with four highly conserved regions at the active site 11 .
  • Analysis of plants with reduced SBEII activity and enzyme assays performed with purified SBEI and SBEII proteins suggest that the two enzyme classes differ in their enzymatic specificity 12 13 .
  • the biochemical data suggest that SBEI favours transfer of long glucan chains and acts primarily on amylose, whereas SBEII produces shorter branches and prefers amylopectin as substrate 1 15 16 .
  • SBEII produces shorter branches and prefers amylopectin as substrate 1 15 16 .
  • the exact role of the different SBE classes in the formation of the branched glucan polymers in planta is not clear. There is no previous evidence to suggest that there are SBEs specific to A- or B-type starch granules.
  • the invention provides a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention provides a DNA sequence encoding one of the starch branching enzymes that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention provides a method for increasing the concentration of A-type starch granules in endosperm of a wheat, rye, barley or triticale plant by over-expressing in the plant a gene encoding a starch branching enzyme that is bound to A-type starch granules.
  • the invention provides a method for decreasing the concentration of A-type starch granules in endosperm of a wheat, rye, barley or triticale plant by suppressing the activity of a starch branching enzyme that is bound to A-type starch granules.
  • the invention provides a method for decreasing the concentration of A-type starch granules in endosperm of a wheat, rye, barley or triticale plant by suppressing the transcription and/ or translation of a gene encoding a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention provides a method of modifying starch granule morphology in a plant expressing a gene encoding a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention provides a method for analysing a plant to determine the presence or absence of DNA encoding granule bound starch branching enzyme, comprising the steps of: providing a probe capable of hybridising with a DNA encoding a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm; exposing the probe to sequences of DNA derived from the genome of the plant; and detecting whether hybridisation with the probe has occurred.
  • the invention provides a method for analysing a plant to determine the presence or absence of transcripts encoding granule bound starch branching enzyme, comprising the steps of: providing a probe capable of hybridising with mRNA encoding a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm; exposing the probe to RNA prepared from the plant or used in in situ hybridisation analysis, and detecting whether hybridisation with the probe has occurred; providing specific primer for detection of transcripts encoding a granule bound starch branching enzyme in wheat; where detection is accomplished by RT-PCR analysis.
  • the invention provides an antibody raised to a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention provides a method for analysing a plant to determine the presence or absence of granule bound starch branching enzyme, comprising the steps of: exposing the proteins of the plant to an antibody raised to a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm; and detecting whether the antibody has bound a starch branching enzyme that is bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
  • the invention also relates to a method of genetically transforming a plant so that the plant expresses a starch branching enzyme that is bound to A-type starch granules in wheat, barley, or triticale endosperm.
  • the invention further relates to a genetically modified plant expressing a starch branching enzyme that is bound to A-type starch granules in wheat, barley, or triticale endosperm.
  • the invention also relates to a genetically modified plant having within its genome a hybrid gene, wherein the hybrid gene comprises a DNA sequence encoding a starch branching enzyme that is bound to A-type starch granules in wheat, barley or triticale endosperm, or a fragment thereof, fused to a passenger-gene.
  • Figure 1 shows a schematic alignment of pABEI and pRN60 cDNA.
  • Hatched area of pABEI coding region represents sequence encoding a putative transit peptide and horizontal arrows on the pRN60 cDNA show location of imperfect direct repeats.
  • the four black areas within the coding region represent sequences encoding the highly conserved regions of enzymes belonging to the ⁇ -amylase family 11 .
  • DNA fragments used as probes in DNA and RNA hybridisations are indicated below.
  • Figure 2 shows RNA gel analysis of Sbel expression during wheat kernel development.
  • RNA (20 ⁇ g) prepared from developing kernels harvested at different DPA. The blot was hybridised with probe 2 ( Figure 1) and estimated sizes of hybridising RNA species are shown to the left. Migration of RNA size markers is indicated to the right. B Same blot as above hybridised with a 25S rRNA DNA probe.
  • Figure 4 shows the nucleotide sequence and deduced amino acid sequence of the 4.6 kb SBEIc transcript produced in the wheat endosperm.
  • Possible polyadenylation sequence is underlined and proposed transit peptide cleavage site is indicated by an vertical arrow. Shadowed regions represent conserved sequences in enzymes belonging to the ⁇ -amylase family 11 . Start of pRN60 sequence and location of PCR primers used in the study are indicated.
  • FIG. 5 shows a schematic illustration of SBEIc precursor encoded by 4.6 kb Sbelc transcript.
  • DNA sequences corresponding to exons 1 to 14 on wheat genomic Sbel 17 are indicated. Hatched areas indicate location of predicted transit peptide and domains 1 and 2 encompass SBEI-like sequences.
  • the location of the four highly conserved regions on ( ⁇ ) ⁇ barrels of amylolytic enzymes 11 are indicated by black boxes and their sequences are shown below. Highly conserved residues are indicated by asterisks and catalytic residues present only on domain 2 are underlined.
  • SBEIc is aligned with the SBEI-like protein deduced from the wSBEI-D2 cDNA 18 and the wheat 87 kD sBE ⁇ b i9 r ⁇ - ⁇ —
  • Figure 6 shows the expression analysis of Sbelc in Eschenchia coli.
  • BE activities were determined from the BE-positive strain DH5 ⁇ and the BE-deficient strain KV832, transformed with plasmids indicated.
  • Construct pREP4-cm expresses the Lac repressor and pQE30 is a cloning vector used for construction of pQE-SBEIc.
  • the BE activity values and standard errors determined by the phosphorylase a stimulation assay 20 are expressed as ⁇ mol glucose-1 -phosphate incorporated mg protein -1 min- 1 and were determined from three separate experiments.
  • Figure 7 shows an immunoblot analysis of starch granule-bound proteins. A Analysis of starch granule-bound proteins by SDS-PAGE and silver staining. Migration of marker proteins (St) is shown to the left.
  • Tigure ⁇ shows SDS-PAGE " analysis of starch” granule proteins produced in wheat endosperm.
  • Figure 9 shows SDS-PAGE analysis of SGP extracted from wheat A- and B- type starch granules. Each lane was loaded with protein extract from 5 mg A- and B-type starch granules of five hexaploid and one tetraploid (Plenty) cultivar. Separated proteins were visualised by silver staining and migration of protein molecular weight markers (Mr) is indicated to the right.
  • Mr protein molecular weight markers
  • Figure 10 shows analysis of starch granule size distribution in wheat endosperm.
  • Figure 11 shows SDS-PAGE analysis of SGP extracted from large-size (>10 ⁇ m) and small-size ( ⁇ 10 ⁇ m) starch granules of the hexaploid wheat cultivar CDC Teal. Samples of SGP from 5 mg starch granules were from different stages of wheat endosperm development as indicated. Gel-separated proteins were visualised by silver staining and migration of protein molecular weight marker (Mr) is indicated to the right.
  • Mr protein molecular weight marker
  • Figure 12 shows immunoblot analysis of extracted SGP from wheat A- and B-type starch granules. Each lane was loaded with SGP extracted from 2 mg A- and B-type starch granules harvested from mature endosperm of the hexaploid wheat cultivar CDC Teal. To the left is shown SGP separated by SDS-PAGE and visualised by silver staining. To the right is shown immunoblot analyses of gel-separated SGP using polyclonal antisera prepared against different wheat starch biosynthetic enzymes as indicated.
  • FIG. 13 shows sub-cellular localisation of SGP-140 and SGP-145 in immature wheat kernels.
  • SDS-PAGE analysis of SGP extracted from CDC Teal pericarp starch, endosperm starch and soluble endosperm proteins were prepared from different DPA of endosperm development as indicated.
  • Samples of soluble protein [280 (10 DPA), 250 (15 DPA) or 250 (20 DPA) ⁇ g] and starch granules (5 mg) analysed were derived from the same amount of endosperm tissue.
  • Gel-separated proteins were visualised by silver staining (pericarp and endosperm starch analysis) or Coomassie blue staining (soluble endosperm analysis). Migration of molecular weight marker (Mr) is shown to the right. Below is shown immunoreactive bands formed between gel-separated SGP- 140 and SGP-145 and wheat SBEI antibodies.
  • Figure 14 shows analysis of SGP in starches from various plant sources.
  • the inventors have characterised a cDNA encoding a novel form of SBEI in wheat endosperm.
  • the encoded polypeptide was found to be preferentially associated with A-type starch granules. Isolation of a Partial SBEI cDNA Clone
  • the 346 bp 5' sequence of pRN60 cDNA did not seem to encode a transit peptide, but instead matched sequences located further downstream on the same cDNA.
  • the unusual 5' sequence carried by pRN60 lacked stop codons in frame with the downstream SBEI coding region, which suggested that the isolated cDNA could be translated from the first base, and therefore, might not represent a full-length transcript.
  • the 4563 bp SBEI cDNA Encodes a Protein With Two SBEI-like Domains
  • DNA sequence analysis of the 4563 bp Sbelc cDNA revealed an open reading frame of 1425 codons that was initiated from the 5' end of the assembled sequence and terminated at nucleotides 4278-4280.
  • the TAA stop codon was followed by a possible polyadenylation signal sequence, AATAAA, located 19 bp upstream of the polyadenylation tail.
  • Initiation of translation was assigned for the first ATG codon (nucleotides 63-65), allowing translation of 1405 codons of the open reading frame.
  • Sequence analysis of the proposed ammo-terminal region of SBEIc revealed a 50% sequence identity to transit peptides predicted from wheat Sbela and Sbelb.
  • SBEIc appeared, like the 87 kD SBEI, to be imported into plastids. Cleavage of the transit peptide was proposed to occur between amino acids Ala 6 7 and Ala 6 8 of the deduced SBEIc sequence (Ile-Ala-
  • Wheat wSBEI-D2 is an SBE-like protein predicted to be produced in wheat endosperm 18 and SBEIb is deduced N-terminal sequence of 87 kD SBEI expressed in wheat endosperm 19 . Identical amino acids are highlighted.
  • the first domain of SBEIc and the corresponding sequence on wSBEI-D2 differed from other characterised SBEI from plants at the four highly conserved regions on enzymes belonging to the ⁇ -amylase family, which include plant SBE 11 . It was especially notable that the Asp residues on regions two and four and the Glu residue on region three, all proposed to be directly involved in hydrolysis of ⁇ -1,4 glucan bonds 11 , were replaced by non-equivalent residues (Figure 5).
  • the 152 kD SBEI is Associated with Starch Granules of the Wheat Endosperm
  • the immunoblot analysis also revealed an interaction with the 92 kD protein band and several 62 to 67 kD protein bands of unknown identities. Since the 140 kD granule-bound protein corresponded reasonably well in mass to SBEIc and no SBEI corresponding in mass with SBEIc was found by immunoblot analysis of the soluble endosperm (data presented in Figure 13), the inventors reasoned that SBEIc was incorporated into starch granules. Further analysis of the granule-bound proteins using polyclonal antibodies prepared against a 87 kD wheat SBEII, revealed only an interaction with the 92 kD protein band (Figure 7B, lane ⁇ -SBEII), as previously reported by Rahman et al. (1995) 26 . Thus, isoforms analogous to SBEIc and bound to starch granules did not seem to exist for SBEII in wheat.
  • SBEIc and its isoforms are preferentially associated with A-type starch granules of wheat endosperm.
  • SBEIc Isoforms are Preferentially Associated with A-type Starch Granules in Wheat Endosperm
  • the inventors compared starch granule proteins (SGPs) localised in A- and B-type starch granules, by purifying the two granule fractions from wheat endosperm-of six wheat cultivars using-a -method previously reported 27 .
  • SGPs starch granule proteins
  • the extracted SGPs were resolved by SDS-PAGE and visualised by silver staining.
  • the 60 kD GBSSI was used as an internal standard for equal loading of proteins.
  • A-type starch granules of all wheat cultivars tested contained a polypeptide co-migrating with SBEIc of Fielder ( Figure 9).
  • Analysis of B-type starch granules from the six wheat cultivars showed a much lower abundance of the 140 and 145 kD polypeptides as compared to the A-type granules.
  • B-type granules of the cultivar Fielder only the 140 kD band was observed.
  • SGP-140 band which includes SBEIc in Fielder, and SGP-145 are preferentially associated with A-type starch granules.
  • A-type starch granules are initiated at about four to 14 DPA, whereas B-type granules are formed after 14 DPA 4 30 . After initiation, both granule types continue to grow until maturity of the endosperm 31 .
  • An image analysis of purified large-size and small-size starch granule fractions from developing endosperm of the cultivar CDC Teal showed that the growth of small starch granules formed before and after 15 DPA was significantly different (Figure 10). Prior to 15 DPA, the newly formed small starch granules grew rapidly in size to become large-size (>10 ⁇ m) starch granules ( Figure 10 A).
  • SGP-140 and SGP-145 are Pre erentially Incorporated into A-type Starch Granules Throughout Endosperm Development
  • SGP-140 and SGP-145 are preferential incorporation of SGP-140 and SGP-145 into A-type granules.
  • the preferential incorporation of SGP-140 and SGP-145 into A-type granules can be explained by synthesis of these polypeptides only during the first 15 DPA.
  • the inventors analysed the protein profiles of large-size and small-size granules isolated at different DPA ( Figure 11).
  • the large-size (>10 ⁇ m) A-type starch granules were found to show no variation in SGP-140 and SGP-145 concentration during development.
  • small-size B-type starch granules harvested after 15 DPA showed very low presence of SGP-140 and SGP-145.
  • the analyses demonstrated no significant variation in concentration of the other major granule-bound polypeptides (60, 80, 92, 100, 108 and 115 kD) for both small-size and large-size starch granules throughout endosperm development.
  • most of the A-type granule growth occurred after 15 DPA, when about 65% (w/w) of the starch in A-type granules was synthesized.
  • the constant levels of SGP-140 and SGP-145 in A-type granules strongly suggested that the two proteins were continuously incorporated into A-type granules throughout endosperm development.
  • SGP-140 plus SGP-145 in B-type granules is preferably at least about 4, more preferable at least about 5, most preferably at least about 10.
  • SBEIc SGP-140 and SGP-145 protein bands of the wheat cultivar CDC Teal have very similar N-terminal sequences as SBEIc.
  • Direct amino acid sequencing of the protein bands purified from SDS-PAGE gels suggested variation in amino acid sequence as indicated in Table I. This is likely due to presence of several polypeptides that differ slightly in sequence within the same protein band. Presence of several isoforms of SBEIc was also suggested by reverse transcription PCR analysis of transcripts produced in the cultivar Fielder.
  • SGP-140 and SGP-145 in the developing kernels SGP from pericarp and endosperm starch granules, and the soluble endosperm fraction were prepared from developing wheat kernels, and analysed by SDS-PAGE and immunoblotting ( Figure 13). The results of these analyses confirmed that SGP-140 and SGP-145 were present within the endosperm starch granules, but could not be found in the endosperm soluble fraction. Nor were SGP-140 and SGP-145 observed in pericarp starch granules harvested from 5 to 10 DPA, but could be seen as two very faint bands in pericarp granules of 15 DPA.
  • SGP-140 and/or SGP-145 Homologues Exist in Plant Species Known to Produce A- and B-type Starch Granules
  • SDS-PAGE analysis of extracted SGP from triticale, barley and rye revealed one (barley and rye) or two protein bands (triticale) with similar relative mobility as SGP-140 and SGP-145 of wheat ( Figure 14 A). -These protein bands were-also-found to react with SBEI antibodies ( Figure 14B), and thus appeared to be SGP-140 and SGP-145 homologues.
  • SBEIc encoded by the isolated cDNA differed from previously characterised SBEI isoforms by its high molecular mass and by the presence of two domains of SBEI-like sequences. Domain 1 differs from domain 2 by the lack of a 21 amino acid long peptide and a 163 residue long (-17 kD) C-terminal sequence ( Figure 5).
  • SBEI transcripts produced in the developing wheat endosperm of the cultivar Fielder suggested that there are at least three different forms of SBEIc transcripts produced. These variants would encode proteins of very similar molecular masses ( ⁇ 1 kD difference), and thus, cannot be distinguished as separate bands on one-dimensional SDS-PAGE gels.
  • Our analysis of starch granules of Triticum species suggested that variants of SBEIc also exist in both diploid (Triticum monococcum, Triticum tauschii) and tetraploid (Triticum turgidum ssp. durum) wheat (Figure 8B).
  • INDUSTRIAL APPLICABILITY SEBIc is a novel starch branching enzyme. It can be used in vitro to synthesise or modify starch. Modified starches find use in the food and beverage industries as a thickener and sweetener, as well as in industrial uses, such as the production of stiffening agents for laundering, sizing for paper and as thickening agents and adhesives 32 33 .
  • the Sbelc sequence, or fragments thereof, or complementary sequences to any of these can be used to screen plant genomes to locate genes that are homologous (i.e. which encode similar activities).
  • SBEIc in a plant can be expected to result in modification of starch granule morphology and size distribution in seed endosperm.
  • the Sbelc gene may be expressed in a plant already having a copy of this gene, in which case the expression SBEIc can be expected to increase.
  • Increase in SBEIc expression may result in increase in A-type starch granule concentration, and/ or in increase in starch granule size.
  • Cultivars having increased A-type granules would be desirable, for example, in the production of gluten, as A-type granules are more easily separated from the protein of the endosperm.
  • Wheat starch with elevated A-granule content has applications in the manufacture of biodegradable plastic film and carbonless copy paper 34 .
  • the invention also relates to homologous variants of SEQ ID NO: 1, including DNA sequences from plants encoding proteins with two SBEI-like domains, as illustrated in Figure 5, and deduced amino acid sequences of 25% or greater identity, and 40% or greater similarity, isolated and/ or characterised and/ or designed by known methods using the sequence information of SEQ ID NO:l or SEQ ID NO: 2, and to parts of reduced length that are able to function as inhibitors of gene expression by use in an anti-sense, co- suppression [Transwitch® gene suppression technology; U.S. patent no. 5,231,020, July 27, 1993; for reviews see Iyer et al.
  • homologous variant when referring to a DNA sequence, encompasses all DNA sequences encoding a protein having the same functionality as the recited sequence, as well as those having two SBEI-like domains, illustrated in Figure 5.
  • A-type starch granules Suppression of transcription and/ or translation of Sbelc, for example, by — using anti-sense approaches, would be expected to reduce the concentration of A-type starch granules. Reduction in A-type granules is desirable if the starch is going to be used as face powder, as a laundry-stiffening agent, a fat replacement or in the production of degradable plastic films 39 40 .
  • probes based on the sequence of Sbelc (SEQ ID NO: 1) or complementary sequences may be used to screen the genome of existing cultivars to find those cultivars having within their genome homologues (particularly alleles) of Sbelc, encoding SBEs that are preferentially bound to A-type starch granules.
  • Such cultivars can be chosen for crossbreeding with one-another, resulting in progeny strains having a high level of SBEIc or homologue expression.
  • cultivars having a low level of Stele-like sequences within their genome can be expected to have a low level of A-type starch granules.
  • Such cultivars could be chosen for crossbreeding with one-another, resulting in progeny strains having a low level of SBEIc expression, and a reduced content of A-type starch granules.
  • strains expressing SBEIc or homologous variants can be found using antibodies raised to SBEIc (polyclonal or monoclonal) to screen cereal varieties to find those having SBEIc or variants.
  • Antibodies to SBEIc can be produced by known methods 41 42 43 u .
  • the invention also relates to a method of genetically transforming a plant so that the plant expresses a starch branching enzyme that is bound to A-type starch granules in wheat, barley, or triticale endosperm.
  • the invention also relates to a genetically modified plant having within its genome a hybrid gene, wherein the hybrid gene comprises a DNA sequence encoding a starch branching enzyme that is bound to A-type starch granules in wheat, barley or triticale endosperm, or a fragment thereof, fused to a passenger-gene.
  • the protein encoded by the hybrid gene is preferably targeted to starch granules.
  • the passenger-gene preferably encodes a vaccine, an antibody, a pigment, a preservative, a fragrant or flavour inducing agent, a receptor, or an enzyme involved in lipid, carbohydrate or protein synthesis, degradation or modification.
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • RT-PCR reverse transcription -polymerase chain reaction
  • Probe 1 used in the library screening consisted of an 828 bp Reverse Transcription-PCR (RT-PCR) product, obtained from a reaction using 12 day old wheat kernel RNA and the Sbel -specific primers BE11 and BE12 ( Figures 1 and 4). The primers were based on sequences of previously characterised Sbel clones from wheat 17 19 . Ten of the positive clones were plaque-purified and their inserts were excised in vivo from the Uni-ZAP XRTM vector (Stratagene). The clone with the longest insert was denoted pRN60 and chosen for further characterisation.
  • RT-PCR Reverse Transcription-PCR
  • Templates for sequencing were prepared by subcloning DNA fragments into the pBluescript II SK + vector (Stratagene). DNA sequencing reactions were performed by the dye terminator cycle sequencing technique and analysed on an automated DNA Sequencer (Applied Biosystems, Foster City, CA). All reported sequences were determined on both strands and from overlapping templates. Nucleotide sequences were assembled and analysed using the Laser geneTM software (DNASTAR Inc.). Pair- wise alignments of DNA and protein sequences were calculated by the Clustal method using a ktuple value 1, gap penalty value 3 and window size 5.
  • RNA gel blot analysis was performed with 20 ⁇ g total RNA fractionated on a 1 % agarose-2.2 M formaldehyde gel, transferred to a HybondTM (Amersham) membrane, hybridised with probe 2 (nucleotides 1993 to 4209 of Sbelc; Figure 1) and washed as described by Nair et al. (1997) 60 . To assure that about the same amount of RNA was loaded onto each lane, the hybridised blot was stripped and rehybridised with a 25S ribosomal DNA probe as described 60 . Probes were radio-labelled using the RediprimeTM random primer labelling kit from Amersham. 5'-RACE
  • 5'-RACE was performed with poly(A) + RNA extracted from 12-day-old wheat endosperm following the protocol supplied with the MarathonTM cDNA Amplification Kit from Clontech.
  • the first strand synthesis was primed with the Sbel -specific BE19 primer ( Figures 3 and 4).
  • the double-stranded cDNA was ligated to the Marathon cDNA Adapter (Clontech), followed by a first round PCR amplification performed with the adapter primer API (5'-CCATCCTAATACGACTCACTATAGGGC- 3'; Clontech) and the Stel-specific primer BE25 ( Figures 3 and 4).
  • the reaction was initiated by a denaturation step at 94°C for 3 min followed, by 25 cycles of 94°C 30 sec, 62°C 20 sec and 68°C 3 min and a final 10 min extension at 68°C.
  • Products derived from the 4.8 kb Sbel transcripts were separated from shorter products derived from the 2.8 kb Sbel mRNA by agarose gel electrophoresis.
  • Products of 1.9 to 2.7 kb were gel-purified and used as a template in a nested amplification employing nested adapter primer AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3'; Clontech) and the gene-specific primer BE39 ( Figures 3 and 4).
  • the amplification conditions were 94°C 3 min, 30 cycles of 94°C 30 sec, 65°C 20 sec and 68°C 3 min, followed by a final extension at 68°C for 10 min. Amplified fragments were separated by agarose gel electrophoresis, isolated, cloned and analysed by DNA sequencing.
  • First strand cDNA used as a template in the RT-PCR reactions, was synthesised from 1.0 ⁇ g total RNA isolated from 12-day-old wheat endosperm. The RNA was primed with oligo(dT) 12 . 1 8 and reverse- transcribed in a total volume of 20 ⁇ l using SuperscriptTM II (Gibco-BRL). PCR reactions (25 ⁇ l) were performed with a 0.5 ⁇ l aliquot of the first-strand cDNA using the Long Expand TemplateTM PCR System (Boehringer Mannheim) and the primer pair BE65/BE38 ( Figures 3 and 4).
  • Reactions were initiated by a denaturation step at 94°C for 3 min, followed by 30 cycles of 94°C 30 sec, 65°C 20 sec, 68°C 2 min 30 sec and a final 10 min extension at 68°C Amplified fragments were fractionated by agarose gel electrophoresis, isolated, cloned and analysed by DNA sequencing.
  • pREP4-cm encoding the Lac repressor
  • pREP4 Qiagen
  • pKKABEI encoding the mature 87 kD wheat SBEI
  • pKK388-l bacterial expression vector
  • pRN33 60 nucleotides 317-1442 carried by a Haelll fragment were inserted into a filled-in BamHI site of the His-tag expression vector pQE31 (Qiagen).
  • the resulting construct was restricted with Kpnl and Smal, followed by introduction of nucleotides 1245-2632 located on a Kpnl-PvuII fragment, to give pQRN33.
  • the BE activity levels in cells from non-induced cultures was determined by the phosphorylase a stimulation assay 20 performed at 30°C for 30 min using two and five ⁇ g of soluble protein extract.
  • the cell extracts were prepared from cells of 1 ml culture that were lysed by sonication in 0.25 ml extraction buffer (50 mM Tris-HCl pH 7.5, 2 mM EDTA, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 15,000 x g for 20 min. Determination of protein concentration in the soluble extracts was done using the dye-binding assay (Bio-Rad).
  • the final protein extract was loaded onto a 10% preparative SDS-PAGE gel and the 87 kD SBEI band was isolated by electroelution (Model 422 Electro-eluterTM, Bio-Rad).
  • the protein eluate was concentrated using a Centriplus-30TM column (Amicon) before "" immunisation.
  • Starch granules were isolated from mature endosperm of five hexaploid wheat cultivars (Triticum aestivum L. cv. CDC Teal, McKenzie, AC Karma, AC Crystal, and Fielder), one tetraploid wheat (Triticum turgidum L. cv. Plenty) cultivar, barley (Hordeum vulgare L.), rye (Secale cereale L.), triticale (X Triticosecale Wittmack), rice (Oryza sativa L.), maize (Zea mays L.), canary seed (Phalaris canariensis L.) and potato (Solanum tuberosum L.) tubers as described by Peng et al.
  • the endosperm and pericarp fractions were homogenised with a mortar and pestle in three volumes of extraction buffer B and filtered through four layers of MiraclothTM (Calbiochem) to remove cell debris.
  • the crude starch granule fraction was pelleted by centrifugation at 15,000 x g for 30 min and further purified as described 27 .
  • the endosperm starch granules were separated into large-size (diameter >10 ⁇ m) and small-size (diameter ⁇ 10 ⁇ m) fractions and studied by image analysis as described 27 .
  • the supernatant remaining from centrifugation of the homogenised endosperm (see above) constituted the endosperm soluble fraction.
  • Protein concentration in the extract was determined using a dye-binding assay from Bio-Rad. For each endosperm fraction, the total amount of extracted soluble protein was determined.
  • Extracted total starch (10 mg) was resuspended in 150 ⁇ l of sample buffer (62.5 mM Tris-HCl pH 8.0, 10% SDS, 10% glycerol, 5% ⁇ -mercaptoethanol and 0.005% bromophenol blue), boiled for 7 min, cooled on ice for 5 min and centrifuged at 15,000 x g for 20 min.
  • sample buffer 62.5 mM Tris-HCl pH 8.0, 10% SDS, 10% glycerol, 5% ⁇ -mercaptoethanol and 0.005% bromophenol blue
  • Extracted A-type and B-type starch granules ( 50 mg) were suspended in 350 ⁇ l extraction buffer A [62.5 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 5% (v/v) ⁇ -mercaptoethanol], boiled for 15 min, cooled to room temperature, and centrifuged at 15,000 x g for 20 min. SDS-PAGE analysis of total and size fractionated starch granules was done on 10% resolving gels (30:0.135) and proteins were visualized by Coomassie blue staining and/ or silver staining (BIO-RAD).
  • SGP were extracted from 10 g A-type starch granules of CDC Teal and resolved on preparative SDS-PAGE gels.
  • the migration of SGP-140 and SGP- 145 was determined by silver staining a slice of the gel.
  • the proteins were eluted from the unstained part of the gel using an electro-eluter (Model 422 Electro-EluterTM, BIO-RAD) and elution buffer (25 mM Tris, 192 mM glycine, 0.1% SDS).
  • the eluate was dialysed for 8 h against 21 of dialysis buffer (50 mM Tris-acetate, pH 6.8, 5 mM DTT), with one buffer change.
  • the dialysed solution was concentrated to 500 ⁇ l through ultrafiltration (Amicon 100), and 200 ⁇ l of the concentrate was loaded on a preparative SDS-PAGE gel. Gel- separated proteins were blotted on a PVDF membrane, as described above. SGP-140 and SGP-145 were identified by amido black staining and subjected to N-terminal sequencing using a gas-phase protein sequencer (Applied Biosystem Model 476 A).
  • SEQ ID NO: 1 is the DNA sequence of Sbelc
  • SEQ ID NO: 2 is the amino acid sequence of SBEIc

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Abstract

L'invention concerne une enzyme de ramification d'amidon qui se lie à des granulés d'amidon de type A dans le blé, l'orge, le seigle ou le triticale. Cette enzyme n'est pas sensiblement associée aux granulés d'amidon de type B. Une séquence d'ADNc codant une isoforme de l'enzyme a été isolée du cultivar de blé Fielder, ce qui a permis de déterminer une séquence d'acides aminés dérivée.
PCT/CA2000/001276 1999-10-29 2000-10-27 Enzyme de ramification d'amidon WO2001032886A2 (fr)

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Cited By (14)

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US7521593B2 (en) 2002-05-09 2009-04-21 Commonwealth Scientific And Industrial Research Organisation Barley with altered branching enzyme activity and starch and starch containing products with an increased amylose content
US7700826B2 (en) 1999-04-29 2010-04-20 Commonwealth Scientific And Industrial Ressearch Organization Genes encoding wheat starch synthases and uses thereof
US7700139B2 (en) 2004-12-30 2010-04-20 Commonwealth Scientific And Industrial Research Organization Method and means for improving bowel health
US7790955B2 (en) 2003-10-27 2010-09-07 The Commonwealth Of Australia Commonwealth Scientific And Industrial Research Organisation Rice and products thereof having starch with an increased proportion of amylose
US7888499B2 (en) 2000-11-09 2011-02-15 Commonwealth Scientific And Industrial Research Organization Barley with reduced SSII activity and starch containing products with a reduced amylopectin content
EP2290084A2 (fr) 2003-06-30 2011-03-02 Commonwealth Scientific and Industrial Research Organization Blé avec activité modifié d'enzyme ramifiant, amidon et produits contenant de l'amidon dérivés dudit blé
US7993686B2 (en) 2004-12-30 2011-08-09 Commonwealth Scientific And Industrial Organisation Method and means for improving bowel health
US9060533B2 (en) 2010-11-04 2015-06-23 Arista Cereal Technologies Pty Limited High amylose wheat
US9357722B2 (en) 2011-11-04 2016-06-07 Arista Cereal Technologies Pty Limited High amylose wheat-II
US9752157B2 (en) 2008-07-17 2017-09-05 Commonwealth Scientific And Industrial Research Organisation High fructan cereal plants
US9826764B2 (en) 2011-02-03 2017-11-28 Commonwealth Scientific And Industrial Research Organisation Production of food and beverage products from barley grain
US10154632B2 (en) 2009-07-30 2018-12-18 Commonwealth Scientific And Industrial Research Organisation Barley and uses thereof
US10246716B2 (en) 2011-10-04 2019-04-02 Arcadia Biosciences, Inc. Wheat with increased resistant starch levels
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum

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US7700826B2 (en) 1999-04-29 2010-04-20 Commonwealth Scientific And Industrial Ressearch Organization Genes encoding wheat starch synthases and uses thereof
US8178759B2 (en) 2000-11-09 2012-05-15 Commonwealth Scientific And Industrial Research Organisation Barely with reduced SSII activity and starch and starch containing products with a reduced amylopectin content
US7888499B2 (en) 2000-11-09 2011-02-15 Commonwealth Scientific And Industrial Research Organization Barley with reduced SSII activity and starch containing products with a reduced amylopectin content
US7521593B2 (en) 2002-05-09 2009-04-21 Commonwealth Scientific And Industrial Research Organisation Barley with altered branching enzyme activity and starch and starch containing products with an increased amylose content
US7919132B2 (en) 2002-05-09 2011-04-05 Commonwealth Scientific And Industrial Research Organisation Barley with altered branching enzyme activity and starch and starch containing products with an increased amylose content
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum
EP2290084A2 (fr) 2003-06-30 2011-03-02 Commonwealth Scientific and Industrial Research Organization Blé avec activité modifié d'enzyme ramifiant, amidon et produits contenant de l'amidon dérivés dudit blé
US8188336B2 (en) 2003-10-27 2012-05-29 Commonwealth Scientific And Industrial Research Organisation Rice and products thereof having starch with an increased proportion of amylose
US9212351B2 (en) 2003-10-27 2015-12-15 Commonwealth Scientific And Industrial Research Organisation Rice and products thereof having starch with an increased proportion of amylose
US7790955B2 (en) 2003-10-27 2010-09-07 The Commonwealth Of Australia Commonwealth Scientific And Industrial Research Organisation Rice and products thereof having starch with an increased proportion of amylose
US7993686B2 (en) 2004-12-30 2011-08-09 Commonwealth Scientific And Industrial Organisation Method and means for improving bowel health
US7700139B2 (en) 2004-12-30 2010-04-20 Commonwealth Scientific And Industrial Research Organization Method and means for improving bowel health
US8501262B2 (en) 2004-12-30 2013-08-06 Commonwealth Scientific And Industrial Research Organisation Method and means for improving bowel health
US11111498B2 (en) 2008-07-17 2021-09-07 Commonwealth Scientific And Industrial Research Organisation High fructan cereal plants
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US10154632B2 (en) 2009-07-30 2018-12-18 Commonwealth Scientific And Industrial Research Organisation Barley and uses thereof
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US11304432B2 (en) 2010-11-04 2022-04-19 Arista Cereal Technologies Pty Limited Food ingredients produced from high amylose wheat
US9060533B2 (en) 2010-11-04 2015-06-23 Arista Cereal Technologies Pty Limited High amylose wheat
US10212959B2 (en) 2011-02-03 2019-02-26 Commonwealth Scientific And Industrial Research Organisation Production of food and beverage products from barley grain
US9826764B2 (en) 2011-02-03 2017-11-28 Commonwealth Scientific And Industrial Research Organisation Production of food and beverage products from barley grain
US11266171B2 (en) 2011-02-03 2022-03-08 The Healthy Grain Pty Limited Production of food and beverage products from barley grain
US10246716B2 (en) 2011-10-04 2019-04-02 Arcadia Biosciences, Inc. Wheat with increased resistant starch levels
US10246717B2 (en) 2011-10-04 2019-04-02 Arcadia Biosciences, Inc. Wheat with increased resistant starch levels
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US9357722B2 (en) 2011-11-04 2016-06-07 Arista Cereal Technologies Pty Limited High amylose wheat-II

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