WO1992005258A1 - Gene encodant une enzyme de l'orge - Google Patents

Gene encodant une enzyme de l'orge Download PDF

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Publication number
WO1992005258A1
WO1992005258A1 PCT/AU1991/000426 AU9100426W WO9205258A1 WO 1992005258 A1 WO1992005258 A1 WO 1992005258A1 AU 9100426 W AU9100426 W AU 9100426W WO 9205258 A1 WO9205258 A1 WO 9205258A1
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barley
sequence
glucanase
dna
isoenzyme
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PCT/AU1991/000426
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English (en)
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Geoffrey Bruce Fincher
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La Trobe University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C1/00—Preparation of malt
    • C12C1/18—Preparation of malt extract or of special kinds of malt, e.g. caramel, black malt
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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)
    • C12Y302/01073—Licheninase (3.2.1.73)

Definitions

  • This invention provides a gene encoding barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I (El); nucleotide sequences encoding for promoter and enhancer regions of said gene; procedures for the isolation of said gene; synthetic nucleotide sequences characterizing said gene, or promoter regions of said gene, or enhancer regions of said gene, or the gene encoding barley (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme II (cDNA for EII); applications/uses for said isolated gene and said synthetic nucleotide sequences, including molecular cloning of the gene encoding said isoenzyme I or the said synthetic nucleotide sequences, and preparation of "synthetic polypeptides" having said isoenzyme I or II activity, which may be modified with respect to thermal stability or other properties through protein engineering.
  • Endosperm cell wall degradation is important in the production of malt for the brewing industry, where barley is germinated under conditions designed to maximise endosperm modification while limiting seedling growth.
  • a commonly-used indicator of malt quality is the amount of material which can subsequently be extracted from the malt with hot water (malt extract) .
  • High levels of (l 3,l->-4)- ⁇ -glucan adversely affect the recovery of malt extract probably because the extent of cell wall degradation, and hence storage polymer mobilisation, is diminished.
  • (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase activity is positively correlated with endosperm modification and malt extract. Therefore the ability of a barley variety to rapidly produce high levels of (l ⁇ -3,l ⁇ -4)- ⁇ -glucanases is an important quality determinant.
  • an isolated DNA sequence which encodes for barley (1 ⁇ -3,1 ⁇ 4)- ⁇ -glucanase isoenzyme I, said isolated DNA sequence having a nucleotide sequence in accordance with Fig. 1 of the accompanying drawings, said nucleotide sequence having been submitted to the GenBank/EMBL Data Bank on 5th September 1990 and accorded accession number X56260.
  • DNA sequence which encodes for barley (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme I sai ⁇ DNA sequence having from nucleotides 3144 to 4061 of the nucleotide sequence according to Fig. 1 of the accompanying drawings, in particular, said nucleotides 3144 to 4061 modified at nucleotides 3711 to 3719, especially the substitution of cytosine with adenine at nucleotide 3712, so as to improve the heat stability of the barley (l-*-3,l-*-4)-B- glucanase isoenzyme I translated therefrom; or
  • DNA sequence which encodes for a promotor region of barley (l ⁇ -3, 1 ⁇ 4)- ⁇ -glucanase isoenzyme I said promoter region being the sequence from nucleotides 1 to 3000 of the nucleotide sequence conforming with Fig. 1 of the accompanying drawings, in particular, said nucleotides 1 to 620, preferably 1 to 480, more preferably 1 to 474, of the nucleotide sequence conforming with Fig. 1 of the accompanying drawings; or
  • a synthetic or copy DNA (c-DNA) sequence which encodes for barley (1 ⁇ 3, l ⁇ 4)- ⁇ - glucanase isoenzyme I, said DNA sequence having a nucleotide sequence confo.rming with Fig. 9 of the accompanying drawings, as well as a synthetic or copy DNA (c-DNA) sequence which encodes for barley (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme II, said DNA sequence having a nucleotide sequence confo.rming with Fig. 10 of the accompanying drawings.
  • plasmids or phages or other vectors or micro-organisms, in particular, yeasts, embodying such an isolated or synthetic DNA sequence.
  • a method for the preparation of such a plasmid or phage or other vector which comprises isolating the specified DNA sequence from barley plant material by digesting an appropriate DNA sample of the barley plant material with a restriction endonuclease; locating genomic DNA fragments carrying (l ⁇ -3,l-4)- ⁇ - glucanase sequences using fragments of a (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase copy DNA (cDNA); then excising and inserting the appropriate DNA sequence into the plasmid or phage or other vector.
  • synthetic polypeptides having barley (1 ⁇ 3,1+4)- ⁇ -glucanase activity obtained by protein engineering or recombinant DNA expression, employing such an isolate or synthetic DNA sequence, or such a plasmid or phage or other vector, or such a microorganism, in particular, yeast.
  • polypeptides having barley (1 ⁇ 3,1 ⁇ )- ⁇ -glucanase isoenzyme I or II activity, said polypeptides having an amino acid sequence in general conformity with any one of Figs.
  • a portion(s) of the respective amino acid sequence optionally has a deletion and/or substitution variant thereof and/or one or more additional amino acids directly attached to its amino terminus or carboxy terminus, or optionally is modified by glycosylation or phosphorylation or acetylation such that the resultant peptide can be used with or without further processing to provide a biological activity of barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I or II in substantial measure.
  • the indicated vector comprising the isolated or synthetic nucleotide sequence wherein the nucleotide sequence is capable of being expressed by a host containing the vector;
  • barley grains embodying such an isolated or synthetic DNA sequence introduced via such a plasmid or phage or other vector so as to enhance the degradation of barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucan; or (iii) such a synthetic polypeptide so as to enhance the degradation of barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucan.
  • nucleotide sequences set out in Figs. 1, 9 and 10 of the accompanying drawings it will be understood that the indicated sequences also embrace the sequence set out in the respective figure of drawings except for the omission or addition or substitution or rearrangement of any functionally non-critical (a) nucleotide constituents thereof, or (b) fragments thereof, or (c) multiple fragments thereof, which omission or addition or substitution or rearrangement does not significantly affect the (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme I or II activity of proteins encoded by the nucleotide sequence of in the respective figure of drawings, or which do not significantly affect the promotor region(s) of the DNA sequence, or which do not significantly affect the enhancer region of the DNA sequence.
  • two (l ⁇ -3,l ⁇ -4)- ⁇ -glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes are expressed in germinated barley grain, where they function to depolymerize the (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucans that constitute 70-75% by weight of cell walls of the starchy endosperm (Fincher 1989).
  • the purified (l ⁇ -3,l ⁇ -4)- ⁇ -glucanases differ in their apparent molecular weight, isoelectric point, carbohydrate content and thermal stability, but exhibit identical substrate specificity and share similar kinetic properties (Woodward and Fincher 1982a,b) .
  • a gene for isoenzyme El from a barley genomic library and determined the nucleotide sequence of a 4643 bp fragment.
  • the gene is located on barley chromosome 5 while the gene for (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme EII is carried on chromosome 1.
  • the isoenzyme El gene contains a single 2514 bp intron that is inserted in codon 25 of a sequence encoding a signal peptide of 28 amino acids.
  • the coding region of the mature enzyme is characterized by a high (G+C) content, which results from an extreme bias towards the use of these nucleotides in the wobble base position of codons.
  • nucleotide sequence of the gene has enabled the complete primary structure of the enzyme to be deduced; isoenzyme El shows 92% positional identity with the primary sequence of (l ⁇ -3,l ⁇ -4)- ⁇ - glucanase isoenzyme EII at both the nucleotide and amino acid level.
  • nucleotide sequences of the two genes diverge markedly in their 3 ' untranslated regions. Oligonucleotide probes specific for these 3' untranslated regions were used to define the expression sites of the two genes by Northern analysis and by amplifying specific cDNAs through the polymerase chain reaction.
  • the DNA sequences according to the invention may be isolated from barley plant material by methods known in principle to persons skilled in the art, for instance, by a method as set out below, in which DNA samples of the barley plant material are digested with a restriction endonuclease; genomic DNA fragments carrying (1 ⁇ -3,1 ⁇ 4)- ⁇ -glucanase sequences are located using fragments of a (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase copy DNA (cDNA) or synthetic oligonucleotides with sequences corresponding to portions of the cDNA or gene sequence; then the appropriate DNA sequences can be excised for insertion into a plasmid vector for storage.
  • cDNA cDNA
  • the isolated or synthetic DNA sequences according to the invention may be employed in conventional genetic engineering techniques, for example, in transferring the isolated or synthetic nucleotide sequences into yeast to remove residual ⁇ -glucan in beer production, as a result of which the invention further provides a plasmid or phage or other vector comprising the isolated or synthetic DNA sequences of the invention, as well as microorganisms containing the isolated or synthetic DNA sequences.
  • applications/uses for the isolated or synthetic nucleotide sequences according to the invention principally relate to the malting and brewing industries, for instance transferring the isolated or synthetic nucleotide sequences into barley so as to generate improved varieties; or transferring the isolated or synthetic nucleotide sequences into yeast to remove residual ⁇ -glucan in beer production; or so as to generate thermostable said isoenzymes El and EII by site-directed or general mutagenesis; o so as to enhance levels of expression by genetic engineering of the promoter sequence elements, said isoenzymes El and EII functioning to remove cell walls in the starchy endosperm of germinating barley grains, essentially for successful germination of grain, the importance of which lies in the direct correlation of (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase levels with malting quality of barleys, i.e. the greater the levels of the enzyme, the better the malting quality.
  • synthetic polypeptides having barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I activity.
  • the “synthetic polypeptides” of the present invention include barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I with the amino acid sequence recited herein but with: (i) the substitution of one or more amino acids of those in the amino acid sequences shown herein for barley (l ⁇ - 3, 1 ⁇ 4)- ⁇ -glucanase isoenzyme I, which is expected to have barley (1 ⁇ -3,1 ⁇ )- ⁇ -glucanase isoenzyme I-like activity with improved thermal stability and to be readily producible via recombinant DNA (rDNA) in bacterial or other expression systems; of (ii) various larger peptides containing the recited sequence (or such a deletion and/or substitution variant thereof) together with one or more additional amino acids directly attached
  • synthetic polypeptides means peptides produced by a technique (e.g. protein engineering or recombinant DNA expression) other than its natural production in a living plant. Accordingly, the "synthetic" polypeptides of this invention are to be distinguished from peptides produced in living plants via expression of DNA occurring naturally in those plants. As produced, such "synthetic polypeptides” are normally free from peptides of barley (and usually other plant) origin.
  • a preferred method for producing the synthetic barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I peptides of the present invention is by rDNA technology utilizing host cells such as bacteria (e.g. E ⁇ coli) or eucaryotic cells such as yeast or cultured insect cells. Modifications of the DNA sequences herein can be made to affect their efficiency of peptide production in a desired host cell.
  • host cells such as bacteria (e.g. E ⁇ coli) or eucaryotic cells such as yeast or cultured insect cells.
  • Such modifications include, but are not limited to: host-preferred codon substitution; construction of DNA coding for fusion proteins including barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme I; substitution of codons to eliminate or enhance mRNA structural features affecting their translation; and other modifications that improve production of barley (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme I in the selected host cell.
  • isolated or synthetic DNA sequences of the present invention as well as plasmids or phages or other vectors or microorganisms embodying such isolated or synthetic DNA sequences, beside synthetic polypeptides obtained by protein engineering or recombinant DNA expression, employing such vectors or microorganism, have particular use in the malting or brewing industries.
  • Barley is usually germanated under controlled conditions for about 4 days, the conditions being designed to maximize the levels of hydrolytic enzymes, e.g. (l ⁇ -3,l ⁇ -4)- ⁇ -glucanases, -amylases, proteases, etc., in the grain to minimize the amount of seedling growth (i.e. root and shoot growth), so as to limit the incorporation into the vegetative tissues of the seedling, of grain nutrients that are ultimately required by the brewing yeast.
  • hydrolytic enzymes e.g. (l ⁇ -3,l ⁇ -4)- ⁇ -glucanases, -amylases, proteases, etc.
  • Brewers crush or mill the malted barley and extract soluble sugars, oligosaccharides , peptides, amino acids etc. into hot water, which might initially be 45-65°C, in a process called mashing, which results in the loss of much of the (l ⁇ -3 ,l ⁇ -4)- ⁇ -glucanase that survives the kilning.
  • Other enzymes such as ⁇ -amylases, can degrade their substrates during the extraction period to produce a higher proportion of low molecular weight molecules that can subsequently be used by the yeast to support their growth and to provide the yeast with fermentable sugars and disaccharides for ethanol production in the beer.
  • the synthetic polypeptide of the present invention can be added at the mashing stage so as to enhance the degradation of (l ⁇ -3, l ⁇ -4, )- ⁇ -glucans .
  • the "mash" may stand or be mixed for a period of time to effect extraction, and the soluble extract may then be separated from the spent grains by a variety of methods . If the separation of soluble extract from the spent grains involves filtration or drainage of the extract through the bed of spent grains, run off times can be dramatically increased by highly viscous, residual (l ⁇ -3, l ⁇ -4, )- ⁇ -glucans. Thereafter, there are a number of steps in which the extract is boiled to precipitate proteins and to denature the enzymes .
  • Hops may be added at this stage to the extract to give the bitter flavour, and then the extract is cooled prior to fermentation.
  • precipitates may be removed by filtration or centrifugation, and residual (l ⁇ -3, l ⁇ - 4, )- ⁇ -glucans can slow down these steps.
  • the cooled extract is then usually "spiked" with a yeast culture, which grows over a period of several days, at cool temperatures, on the nutrients of the malt extract, and in the near-anaerobic conditions, the sugars and disaccharides are fermented to ethanol .
  • the yeast are removed, together with any material that precipitated during the fermentation, and this again is usually achieved by centrifugation or filtration, residual (1 ⁇ 3, l ⁇ - 4)- ⁇ -glucans also slowing down this step.
  • transgenic cells in which the introduced DNA has been stably incorporated into the chromosome of the barley cell, the selection being based on the acquired resistance of the barley cells to the antibiotic;
  • transgenic barley so produced could be re-introduced into breeding programs and the new character could be cross-bred into agronomically acceptable barleys and seed distributed to farmers, as such manipulations would probably result in some changes in the original barley and different barley varieties are bred to suit the conditions of particular local environments.
  • Barley (Hordeum vulgare, cv Clipper) was obtained from the Victorian Crops Research Institute, Horsham, Victoria, Australia and from the cereal collection of the John Innes Centre for Plant Science Research, Norwich, U.K. Other barley cultivars, hexaploid wheats (Triticu aestivum) and various aneuploid wheat stocks (Sears 1966; Sears and Sears 1978) were as described by Sharp et al. (1988).
  • barley grains were surface sterilized with 2.5% (w/v) sodium hypochlorite for 20 min at room temperature, washed thoroughly with lOmM HCl and water, and soaked in sterile water containing 100 g/ml neomycin, lOOyg/ml chloramphenicol and 100 units/ml nystatin for 16 h at room temperature. Grain was germinated on filter paper in sterile petri dishes at 22 ⁇ C.
  • Tissues were excised for RNA isolation at selected times after the initiation of germination, as follows: scutella, 1 day; aleurone layers, 3 days; coleoptiles, 6 days; young roots and root hairs, 2 days; young leaves, approximately 10 days (that is, the first leaves to emerge from the coleoptile). Mature leaves, stems and roots obtained from glasshouse-grown barley plants approximately 4 weeks after planting were washed thoroughly and surface sterilized in sodium hypochlorite before RNA isolation.
  • RNA was extracted from tissue ground to a fine powder under liquid N 2 in a guanidiniu thiocyanate buffer and purified by equilibrium Centrifugation in caesium trifluoroacetate (Okayama et al. 1987) or, alternatively, by extraction into 50mM Tris-HCl, pH9.0 (containing lOmM EDTA, 2% w/v SDS, lOmM ⁇ -mercap- toethanol, lOOmM NaCl and 0.1% w/v proteinase K), exhaustive removal of protein with phenol-chloroform and recovery of RNA by ethanol precipitation (Baulcombe and Buffard 1983).
  • RNA samples (5-10Ug) were separated on 1% (w/v) agarose gels in 2.2M formaldehyde (Sambrook et al. 1989) and transferred to nitrocellulose filters (Hybond-C Extra, .Amersham Corp.). To ensure approximately equal loadings, control gels were stained with ethidium bromide for comparison of the relative intensities of ribosomal RNA bands. Filters were prehybridized at 60"C for 4 h in 6x standard saline citrate (SSC 150mM NaCl/15mM sodium citrate, pH7.0) containing 1% (w/v) SDS, Ix Denhardt's solution (Denhardt 1966), and 0.1 mg/ml herring sperm DNA.
  • SSC 150mM NaCl/15mM sodium citrate, pH7.0 6x standard saline citrate
  • Ix Denhardt's solution Ix Denhardt's solution (Denhardt 1966)
  • Oligonucloetide probes (30-mers) were synthesized using solid phase phosphoramidite chemistry on an Applied Biosystems Model 380A DNA synthesizer and end-labelled with T4 polynucleotide kinase and [ ⁇ - 32 P]ATP (Sambrook et al. 1989). Filters were hybridized with oligonucleotide probes at 60 e C for 16 hr in prehybridization buffer containing 10% (w/v) dextran sulphate. Filters were washed exhaustively in 3 x SSC/0.1% SDS at 60"C. DNA probes were labelled to a specific activity of more than 10 9 y.dpm/Ug with [
  • DNA Isolation and Southern Analysis Isolation of genomic DNA, restriction enzyme digestion, agarose gel electrophoresis, alkaline Southern blotting to Amersham Hybond-N Plus membranes and hybridization procedures were as described by Sharp et al (1988). Near full-length (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase cDNAs were used as probes.
  • First strand cDNA was prepared from 5-10 ⁇ g total RNA primed with appropriate oligonucleotides by reverse transcriptase (Sambrook et al. 1989) in a total volume of 50 ⁇ l. After 1 hr at 42*C, 2.5 ⁇ l of the first strand cDNA reaction was mixed in a total volume of 50 ⁇ l with flanking oligonucleotides and Tag polymerase, and incubated under mineral oil in a Perkin-Elmer Cetus (Norwalk, CT, USA) DNA Thermal Cycler as follows; 94"C/2 min; 94'C/30 sec, 40*C/20 sec, 72 e C/2 min, repeated 30x; 72"C/10 min; 4*C. The polymerase chain reaction (PCR) products were examined by agarose or polyacrylamide gel electrophoresis.
  • PCR polymerase chain reaction
  • the barley genomic library was obtained from Clontec
  • the library was prepared from partially digested DNA from 7-day old seedlings of Hordeum vulgare L. (var. NK 1558) in the cloning vector EMBL3.
  • the library was plated out on a lawn of E. coli NM 538 cells and screened by hybridization of nitrocellulose filter plaque replicas (Sambrook et al. 1989) using as a probe the 734 bp Hinfl fragment of a (l ⁇ -3,l ⁇ -4)- ⁇ -glucanse cDNA (Fincher et al. 1986).
  • the probe was labelled and prehybridization, hybridization and washing procedures were essentially as described by Loi et al. (1988).
  • phage DNA was isolated and the genomic DNA insert analysed with restriction endonucleases.
  • a 3.8 kb HindiII fragment, a 4.2 kb Sail fragment, and 1.5 kb and 1.8 kb BamHI fragments were sub-cloned into the corresponding sites of plasmid pUC19 for sequence analysis.
  • RNA was extracted from scutella excised from the grain and incubated for 2 days at 25*C in lOmM CaCl 2 and 5 ⁇ M gibberellic acid (GA), and from aleurone layers after 1.5 days incubation.
  • Procedures for the synthesis of cDNA, the preparation of cDNA libraries in ⁇ gtll and screening were as described elsewhere (Doan and Fincher 1988; Hoj et al. 1989). Both libraries were screened with a cDNA encoding barley (1 ⁇ -3,1 ⁇ 4)- ⁇ -glucanase isoenzyme EII (Fincher et al. 1986).
  • the dideoxynucleotide chain termination procedure of Sanger et al. (1977) was used to sequence the (1 ⁇ -3,1 ⁇ - 4)- ⁇ - glucanase genomic clone.
  • Appropriate restriction endonuclease fragments were recovered from agarose gels with Geneclean (BIOIOI) and subcloned into pUC19 or into M13mpl8 or mpl9 for sequencing.
  • Computer analysis were performed with the Pustell Sequence Analysis Programs (International Biotechnologies Inc., New Haven, CT, USA) , the Wisconsin Genetics Computer Group package (University of Wisconsin, Madison, WI, USA; Devereux et al. 1984) and with the Staden programs (MRC Laboratory of Molecular Biology, Cambridge, UK; Staden 1986).
  • RNA preparations (5-50 ⁇ g) from young leaves and scutella were annealed with lOng end-labelled oligonucleotide specific for the 5 ' untranslated regions of the isoenzyme El gene in deionized 50% (v/v) formamide at 34 ⁇ C for 16 h after heating the incubation mixture at 85 ⁇ C for 10 min (Sambrook et al. 1989). After recovery of the RNA by ethanol precipitation, reverse transcription and polyacrylamide gel electrophoresis of the extended primers were as described by Sambrook et al. (1989).
  • the isoenzyme EII cDNA clone (Fincher et al. 1986) used to screen the library was less than full-length and did not include non-coding sequence. Additional cDNA clones were therefore isolated to obtain further sequence information of isoenzyme EII mRNAs and also for comparison of isoenzyme El mRNA with the genomic sequence.
  • a 1431 bp cDNA encoding isoenzyme El identified from the deduced protein-coding sequence, was isolated from the scutellum cDNA library and exhibited more than 98% positional identity with the genomic clone where the sequences overlap although, as discussed below, the genomic sequence is interrupted by a single large intron.
  • This isoenzyme El cDNA includes the complete coding sequence of the mRNA and non-translated 5' and 3' flanking sequences.
  • a 1229 bp isoenzyme EII cDNA obtained from the aleurone cDNA library was also identified from the deduced amino acid sequence.
  • the isoenzyme EII cDNA had associated 5' and 3' non-translated sequences, including a short poly(A) tail.
  • the sequence of the 3' region of the isoenzyme El cDNA showed 98% positional identity with a 286 bp cDNA isolated by Jackson et al. (1986).
  • the c-DNAs. encoding for isoenzymes El and EII are shown in Fig. 9 and Fig. 10, respectively.
  • the peptide encoded by the 5' exon resembles a signal peptide, having a relatively hydrophilic NH 2 -terminal region and a hydrophobic core which becomes more hydrophilic towards its COOH-terminus, where there is a helix-breaking proline residue and a large polar amino acid (glutamine) (Fig. 1, Watson 1984).
  • Fig. 1 The absence of a charged amino acid close to the NH 2 -terminus (Fig. 1) is unusual but not unique in eukaryotic signal peptides (Watson 1984).
  • the intron is typical of plant introns. Both the 5' and 3' boundary sequences at positions 619 and 3132 (Table 2) are similar to the consensus sequence for plant introns and there are stop codons in all reading frames within the 3 1 region of the intron (Fig. 1). The high (A+T) content near the intron boundaries is a characteristic of plant introns required for effective splicing of adjacent exons (Goodall and Fillipowicz 1989). The ACTAAC motif beginning 26 bp from the 3' end of the intron corresponds in both position and sequence to the consensus sequence for the branch point of type II introns of plant genes (Goodall and Fillipowicz 1989).
  • proximal promoter comprising a CAAT sequence that begins 30 bp 5' to a possible TATA box that is located, in turn, approximately 220 nucleotide pairs on the 5' side (Fig. 1; Table 2) of an ATG codon initiating the open frame of the putative exon.
  • the nucleotide sequence surrounding the ATG translation start point of this putative exon is very similar to the optimal sequence for initiation by eukaryotic ribosomes (CCACCATGG; Kozak 1986) and also is in reasonable agreement with consensus sequences derived from plant genes (Table 2). Although the initiation codon is not the first after the putative transcription start point (Fig.
  • Helix-breaking proline and glycine residues would be located close to the 3' end of this exon and therefore close to the protein processing site (von Heijne 1983) located before the fourth amino acid (isoleucine) on the large exon of the gene (Fig. 1).
  • the length of the intron between these exons would be 55 bp, which is smaller than a value of 70 to 73 bp recently reported to be the minimal functional length of plant introns; however, shorter introns have been recorded (Goodall and Filipowicz 1990).
  • the sequence analysis of the 5' part of the gene therefore implies the existence of two promoter sequences, the proximal promoter being within the large intron of the region transcribed from the distal promoter.
  • oligonucleotide primers complementary to the putative signal peptide (nucleotides 3058-3077, Fig. 1) of the proximal promoter or the 5 ' untranslated region (nucleotides 499-515, Fig. 1) of the distal promoter.
  • the secretion of the barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme El from isolated scutella appears to be enhanced by the phytohormone gibberellic acid (GA), although high levels of endogenous hormone in the tissue fragments mask the effects of added GA (Stuart et al. 1986).
  • GA phytohormone gibberellic acid
  • levels of secreted (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase enzyme (Stuart et al. 1986) and translatable mRNA (Mundy and Fincher 1986) are significantly enhanced by GA treatment. Accordingly, the promoter region of the barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme El gene (Fig.
  • the region of the gene that encodes the mature (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase enzyme has an overall (G+C) content of 67% and this is mainly attributable to a strong bias towards the use of G and C in the wobble base position of codons. Of the 306 codons in this region, only 13 have A or T in the third nucleotide position. In the two possible signal peptide regions of the gene codon usage is not strongly biased towards the use of G and C at the wobble base positions (Fig. 1).
  • the bias in codon usage may be related to mRNA stability or to translational efficiency, both of which would contribute to the high levels of expression of these genes in the aleurone cells of germinating cereal grains, where hydrolytic enzymes are required for rapid endosperm mobilization (Fincher 1989).
  • the position of the intron also marks the transition point in codon usage in the gene.
  • Barely (l ⁇ - 3)- ⁇ -glucanase genes (Hoj et al. 1989; P. Xu, J. Wang and G.B. Fincher, unpublished data)
  • a wheat (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase cDNA (D. Lai and G.B. Fincher, unpublished data)
  • wheat ⁇ -amylase genes exhibit a similar abrupt change from (A+T) to (G+C) in the wobble base position at the site of intron insertion near the 5' end of the genes.
  • the nucleotide sequence of the gene (Fig. 1) has permitted the complete primary structure of isoenzyme El to be defined.
  • the coding region for the mature enzyme encodes a polypeptide of 306 amino acids.
  • the calculated pi for the polypeptide is 8.7, which may be compared with a value of 8.5 reported for purified (l ⁇ 3,l ⁇ -4)- ⁇ -glucanse isoenzyme El and a value of greater than 10 for isoenzyme EII (Woodward and Fincher 1982a; Fincher et al 1986).
  • the primary structure of isoenzyme EII derived from the cDNA is also 306 amino acids long, but has a calculated pi value of 10.6.
  • the two protein sequences are compared in Fig. 3.
  • a 1.4kb Hinfl fragment from the coding region of the genomic clone was used to probe various RNA samples on a Northern blot.
  • the probe includes regions of similarity between the isoenzyme El and EII genes and would therefore detect transcripts from both. No expression was detected in roots, coleoptiles, stems or mature leaves but an mRNA of approximately 1550 nucleotides was detected in the RNA of aleurones from germinated grains (Fig. 5a) and of young leaves. A relatively faint signal could also be detected with scutellar RNA (Fig. 5a).
  • oligonucleotide probes complementary to the 3' untranslated region of the isoenzyme El and EII mRNAs allowed separate detection of these two types of mRNA and showed that the aleurone cells expressed both isoenzyme El and EII mRNAs whereas the scutellar and leaf RNA contained only the El type (Figs. 5b, c) .
  • oligonucleotides were designed (Fig. 6) to specifically amplify first strand cDNAs synthesized from leaf, scutellum and aleurone RNA preparations. Synthesis of first strand cDNA by reverse transcriptase was primed with oligonucleotide 12 (3347 to 3366; Figs. 1 and 6), which was complementary to part of the protein-coding region. In subsequent PCR incubations the cDNAs were amplified with oligonucleotides 13 (500 to 519, Figs.
  • PCR fragments were 362 bp for oligonucleotides 13/12 and 347 bp for oligonucleotides 14/12.
  • the primary structure of the isoenzyme El gene and its corresponding protein are typical of plant genes and proteins in terms of content, size and position of sequence motifs for transcription, RNA processing, translation (Table 2) and protein processing.
  • sequence motifs in the gene and primer extension data imply that there are two promoters. In young leaves and aleurones the distal promoter was the more active, as shown by the primer extension (Fig. 2) and PCR (Fig. 7) data.
  • the proximal promoter is expressed in specialized tissues or under physiological conditions not tested here. Until the use of the proximal promoter is investigated further, we can only speculate on the reason for the existence of two promoters.
  • HindiII fragment observed by Loi et al. (1988) and seen also in Fig. 8b has been cloned and sequenced, and corresponds to the isoenzyme El gene.
  • the gene encoding (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme El is carried on barley chromosome 5, while the isoenzyme EII gene is located on barley chromosome 1.
  • Fig. 8b Because the conserved 3.8kb Hindlll fragment (Fig. 8b) is known to be the isoenzyme El gene, and within the constraints imposed by the relatively small number of barley varieties and restriction enzymes examined, it appears that the isoenzyme El gene is conserved while the isoenzyme EII gene exhibits significant polymorphism.
  • the RFLPs may prove useful in the construction of detailed genetic maps for barley and wheat, which may in turn be of value in breeding programs through the identification of linkages between marker genes and genes of agronomic or commercial importance (Beckmann and Soller 1986; Sharp et al. 1988).
  • isoenzyme El and EII genes were differential expression of the isoenzyme El and EII genes in the scutellum and aleurone cells, as suggested previously by analysis of enzyme accumulation (Stuart et al. 1986) and which is now confirmed using specific oligonucleotide probes complementary to the 3' non- translated regions of their mRNAs (Fig. 5). Presumably, isoenzyme El and EII proteins with slightly different properties fulfil complementary functions in the various tissues examined. A third isoenzyme, designated isoenzyme EIII, was also secreted from isolated scutella (Stuart et al.
  • the two (l ⁇ -3,l ⁇ -4)- ⁇ -glucanases in germinated barley grain clearly function to depolymerize the (l ⁇ -3,l ⁇ -4)- ⁇ - glucans of the endosperm cell walls, which represent a physical barrier between hydrolytic enzymes secreted from peripheral tissues and their substrates within the cells of the starchy endosperm.
  • Northern analyses (Fig. 5) also indicate that the isoenzyme El gene is transcribed at relatively high levels in young leaves, as they emerge from the coleoptiles approximately 10 days after the initiation of germination.
  • (1 3,l ⁇ -4)- ⁇ -glucanase isoenzyme El produced at this time may be to loosen the cell wall matrix during cell expansion and elongation in growing tissues (Hoson and Nevins 1989; Sakurai and Matsuda 1978).
  • the levels of enzyme required for such loosening would be low, whereas the observed levels of isoenzyme El mRNA in young leaves is higher than in germinating grain, where extensive degradation of endosperm cell walls is achieved.
  • the numbers of residues were calculated from the molar composition (Woodward and Fincher 1982a) assuming a total of 306 residues.
  • the total number of calculated amino acids is 305 because of errors introduced by approximating individual values to the nearest integer.
  • Fig. 1 Complete nucleotide sequence and derived amino acid sequence for a 4643 bp barley genomic DNA fragment carrying the gene for d ⁇ -3, l ⁇ -4) - ⁇ -glucanase isoenzyme El. Possible TATA boxes and transcription start points are underlined for both the distal promoter (nucleotides 420 and 474) and a putative proximal promoter (2797 and 2836) , and a potential polyadenylation signal is shown (4399) . The intron boundaries are indicated by arrows, as is the NH ⁇ -terminal isoleucine residue of the mature protein. Fig.
  • proximal promoter the product was derived from 50ug scutellar RNA, is 241 nucleotides in length and is shown with nucleotide sequence generated from the isoenzyme El gene itself with the same oligonucleotide used for the primer extension. Details of oligonucleotide sequences and locations are described in the text.
  • Fig. 3 Amino acid sequence similarities between barley (l ⁇ -3,1 ⁇ -4)- ⁇ -glucanase isoenzyme El and isoenzyme EII. Only the residues of isoenzyme EII that differ from the corresponding residue in isoenzyme El are shown. The underlined residues indicate the N-glycosylation site in isoenzyme EII.
  • Fig. 4(a) Comparison of the nucleotide sequences of cDNAs encoding (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzymes El and EII in the region of the signal peptides and their 5 ' flanking sequences.
  • the vertical lines indicate nucleotide identity and standard one letter abbreviations are used to show the deduced amino acid sequences of the signal peptides. Arrows indicate the processing point of the signal peptide, adjacent to the NH 2 -terminal residue of the mature protein.
  • (b) The nucleotide sequence of the 3' untranslated region of the cDNA encoding (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme EII. Comparison of this sequence with the corresponding 3' region of the genomic clone for isoenzyme El (Fig. 1, beginning at nucleotide 4062) reveals no sequence similarity.
  • Fig. 5 Northern analyses of total RNA preparations from aleurone (A), scutellum (S), young leaves (YL), mature leaves (ML), roots (R) , coleptiles (C) and stems (St). Filters were probed with a DNA fragment that binds to both isoenzyme El and isoenzyme EII mRNA (panel a), with an oligonucleotide specific for the 3' untranslated region of isoenzyme El mRNA (panel b) , and with an oligonucleotide specific for the 3' region of isoenzyme EII mRNA (panel c).
  • Fig. 6 Diagramatic representation of the barley (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase isoenzyme El gene, showing certain restriction enzyme sites, distal and proximal promoter regions, and exons.
  • the arrows indicate the approximate positions and orientations of oligonucleotides 12, 13 and 14, which were used in PCR experiments to investigate transcriptional activity of the two potential promoters (Fig. 7). The sequences and precise locations of the oligonucleotides are described in the text.
  • Fig. 7 Agarose electrophoresis of PCR products from aleurone (A) scutellum (S) and leaf (L) .
  • first strand cDNA was primed either with oligonucleotides 13/12 to detect transcription from the distal promoter (loaded in the left lane of the two lanes for each tissue) and oligonucleotides 14/12 for transcription from the putative proximal promoter (loaded in the right lane of each pair). Little or no DNA is detected with oligonucleotides 14/12, in each case.
  • the exact positions and sequences of the oligonucleotides are indicated in the text.
  • the mobility of 344 bp and 394 bp DNA size markers are indicated.
  • Controls to check the oligonucleotides include the isoenzyme El cDNA (E), which results from transcription from the distal promoter and is 362 bp in length; as expected it has no sequence primed by oligonucleotide 14.
  • the other control DNA is a 3.8 kb Hindlll fragment of the gene that encompasses the proximal promoter but not the distal promoter (cf. Fig. 6); as observed, this cDNA should yield a PCR product of 402 bp from oligonucleotides 14/12, but has no sequence corresponding to oligonucleotide 13.
  • Fig. 8 (a) Southern analyses of wheat aneuploid line DNA cut with Dral and probed with a (1 ⁇ -3,1 ⁇ - 4)- ⁇ -glucanase cDNA.
  • each wheat aneuploid group (1 to 7) there are three lines that are nullisomic for that particular chromosome from the A, B and D genomes.
  • the absence of bands in the wheat nullisomic lines from groups 1 and 7 indicate that the (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase genes are located on these chromosomes.
  • Size markers are shown in the left lane and are 23.1 kb, 9.4 kb, 6.6 kb, 4.3 kb, 2.3 kb and 2.0 kb in length.
  • Fig. 9 Nucleotide sequence for the isolated 1436 bp c-DNA of barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme El. The putative signal peptide is highlighted; the stop codon is marked by an asterisk; and the putative polyadenylation signal is underlined.
  • Fig. 10 Nucleotide sequence for the isolated 1229 bp c-DNA of barley (l ⁇ -3,l ⁇ -4)- ⁇ -glucanase isoenzyme EII. The putative signal peptide is highlighted; the stop codon is marked by an asterisk; and the putative polyadenylation signal is underlined.

Abstract

On décrit un gène encodant une isoenzyme EI (1←3, 1←4)-β-glucanase de l'orge; des séquences de nucléotides codant pour les régions promotrices et activatrices dudit gène; des modes opératoires d'isolation dudit gène; des séquences de nucléotides synthétiques caractérisant ledit gène, les régions promotrices dudit gène, la région activatrice dudit gène, ou le gène encodant l'isoenzyme II (1←3, 1←4)-β-glucanase de l'orge. L'invention prévoit des applications/utilisations du gène isolé ou des séquences de nucléotides synthétiques ou isolées, principalement dans les domaines de l'industrie brassicole et de maltage, qui consistent par exemple à transférer le gène isolé ou les séquences de nucléotides synthétiques ou isolées dans de l'orge de manière à produire des variétés améliorées, ou à préparer des enzymes à activité (1←3, 1←4)-β-glucanase de l'orge en grandes quantités pour les utiliser comme adjuvants dans l'industrie brassicole et de maltage.
PCT/AU1991/000426 1990-09-20 1991-09-17 Gene encodant une enzyme de l'orge WO1992005258A1 (fr)

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