EP1032690A1 - Stressresistenzgen - Google Patents

Stressresistenzgen

Info

Publication number
EP1032690A1
EP1032690A1 EP98954624A EP98954624A EP1032690A1 EP 1032690 A1 EP1032690 A1 EP 1032690A1 EP 98954624 A EP98954624 A EP 98954624A EP 98954624 A EP98954624 A EP 98954624A EP 1032690 A1 EP1032690 A1 EP 1032690A1
Authority
EP
European Patent Office
Prior art keywords
plant
aldose reductase
protein
homologous protein
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98954624A
Other languages
English (en)
French (fr)
Inventor
Atilla Oberschall
Gábor HORVATH
Mária DEAK
Károlyné TÖROK
Dénes DUDITS
Atilla Feher
László SASS
Eva Hideg
Imre Vass
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BTG International Ltd
Original Assignee
BTG International Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BTG International Ltd filed Critical BTG International Ltd
Publication of EP1032690A1 publication Critical patent/EP1032690A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the present invention relates to plant cells, breeding materials, plant parts and plants in which the expression level of the aldose reductase-homologous protein is increased and, therefore, which show higher resistance to stress conditions. Plant cells, breeding materials, plant parts and plants per se are provided and these are advantageously transgenic.
  • the present invention further relates to nucleic acid , eg. DNA and RNA molecules .and vectors suitable for providing expression of aldose reductase-homologous protein in cells and processes for the preparation of plant cells overproducing aldose reductase-homologous protein.
  • the invention has particular use in reducing damage in crops caused by harmful biotic and abiotic stress conditions and for improving harvest prospects thereby.
  • plants are exposed to both abiotic (eg. photoinhibition by high light intensity, UV-B irradiation, ozone, heavy metals, high and low temperature, water deficiency, flooding and wounding) and biotic (eg. viral, bacterial and fungal infections, insect action) stress conditions.
  • abiotic eg. photoinhibition by high light intensity, UV-B irradiation, ozone, heavy metals, high and low temperature, water deficiency, flooding and wounding
  • biotic eg. viral, bacterial and fungal infections, insect action
  • Aldose reductase (EC. 1.1.1.21) and aldehyde reductase (EC. 1.1.1.2.) enzymes belong to the aldo-keto reductase superfamily. These enzymes are monomeric NADPH-dependent oxidoreductases with broad substrate specificity, ranging from aldose sugars to aromatic aldehydes (Bohren K.M. et al. 1989. J. Biol. Chem. 264: 9547-9551). Until now plant aldose reductase enzymes were isolated only from monocots. The expression of the aldose reductase-homologous gene from b? ley is induced in the late period of embryo development (Bartels, D. et al. 1991.
  • HNE Because of its reactive ⁇ - ⁇ unsaturated double bond, HNE can spontaneously form Michael adducts with the -SH groups and lysine or histidine residues of cellular proteins.
  • the aldehyde group of HNE can form Schiff bases with the ⁇ or ⁇ amino groups of proteins, thus via crosslinking it can alter their catalytic and structural properties. In nanomolar concentrations HNE can cause DNA damage while in higher, eg. micromolar, concentrations it has a cytotoxic effect.
  • HNE has an aldehyde group, a possible way of its detoxification is its reduction to alcohol, catalysed by the members of the aldo-keto reductase superfamily.
  • the object of the present invention is to produce novel, advantageously transgenic, plant types that, compared to the starting plants, ie. standard crop or ornamental lines, possess an enhanced resistance against both abiotic and biotic stress conditions and to provide a method for producing these.
  • the invention is based on the finding that, via overproduction of aldose reductase-homologous enzyme superfamily members having a general detoxificating and osmoregulatory effect, plant cells and plants regenerated from them can be made resistant to a greater extent against number of stress conditions of different origin at the same time.
  • the effect of the enzyme is correlated to its ability to reduce aldehyde groups of various substrates, eg. 4-hydroxy-nonenal (HNE) and DL- glyceraldehyde.
  • cDNA coding for an alfalfa (Medicago sativa) aldose reductase-homologous enzyme has been isolated by applying recombinant gene manipulation methods. By attaching this cDNA to a regulatory region, eg. a promoter, ensuring ectopic expression in plants, eg.
  • overproduction of the aldose reductase-homologous enzyme has been carried out in cells of transgenic plants, thereby enabling the reduction of damage in said plants exposed to stress conditions of different origin via the detoxificating and/or osmoregulatory function of the aldose reductase-homologous protein.
  • plants, plant cells, plant parts, breeding material and plant product all of which are advantageously transgenic, which as a consequence of enhanced level of the aldose reductase-homologous enzyme present therein, show an enhanced resistance against the deleterious effects of a number of, and particularly against a wide variety of, stress conditions.
  • the invention concerns plant cells that produce one or more aldose reductase-homologous superfamily enzymes capable of reducing aldehyde groups at an enhanced level and, therefore, which posses an enhanced resistance against damaging effects of stress conditions involving the production of free radicals and/or against drought. Particularly provided is resistance against the damaging effects of free radical generation.
  • Plant cells, and thus plants, according to the invention advantageously overproduce an aldose reductase-homologous protein comprising the amino acid sequence presented in SEQ ID No 1 , more preferably the alfalfa sequence in SEQ ID No 3 (MsALRh protein) or a functional variant or derivative thereof, said variant being advantageously at least 50% homologous, more advantageously at least 70% homologous and even more advantageously at least 90% homologous to the aldose reductase-homologous protein sequence represented in SEQ ID No 1, and more preferably having such homology to the alfalfa protein of SEQ ID No 3. Still more preferably the cells and plants overproduce such enzymes comprising sequences having at least 50% identity, more preferably at least 70% identity and most preferably at least 90% identity with such sequences, most preferably with SEQ ID No 3.
  • Plant cells of the invention are advantageously transgenic and are transformed by incorporation therein of a nucleic acid molecule capable of expressing an aldose reductase-homologous protein.
  • Plant cells, plant parts or plants according to the invention can also be produced via traditional non-biological mutation- selection methods wherein, for example, a chemical or UV-light is employed as the mutagenic agent and selection of plants for overproduction of aldose reductase superfamily enzyme of required activity is carried out using assays eg. as described below.
  • the level of aldose reductase expression can be determined by hybridisation (using Northern or Western blot techniques, see Example 2) or via detection of the enzymatic activity, particularly that against HNE and/or DL-glyceraldehyde eg. as described below.
  • the invention further concerns plants and plant parts comprising plant cells according to the invention.
  • Plants and plant parts according to the invention advantageously posses an enhanced level of resistance against stress conditions involving the production of free radicals, more specifically for example against treatments with herbicides and/or heavy metals and/or producing hydrogen peroxide, and/or with NaCl; and/or infections caused by viruses and/or bacteria and/or fungi; and/or drought.
  • nucleic acid sequences encoding an aldose reductase-homologous protein for use in the invention as well as recombinant nucleic acid molecules comprising such sequences.
  • this nucleic acid is recombinant, isolated, enriched and/or cell free and encodes for the proteins described above having the aforesaid homology to SEQ ID No 1 or 3.
  • This provides nucleic acid sequences coding for the aldose reductase-homologous protein, particularly for use in the invention and plant cells of the invention for use in producing plants and plant parts according to the invention.
  • nucleic acids of the invention may be designed to encode the functional variants of the reductase proteins of the invention.
  • Oligonucleotides and polynucleotides may also be used as probes and primers to identify further naturally occuring or synthetically produced reductase proteins using eg. southern or northern blotting
  • the nucleic acid is DNA or RNA, particularly cDNA or cR A and more preferably is characterised in that where it is a DNA it is a polynucleotide comprising nucleotide sequence having at least 80% identity with SEQ ID No 2 as listed in the sequence listing herein, or a sequence having degenerate substitution of codon nucleotides in that sequence, and where it is an RNA it has a complementary sequence wherein T is replaced by U.
  • the identity is 90% or more, more preferably 95% or more and most preferably 100%.
  • non-identical parts of the sequences comprise degenerate substitutions.
  • DNA or RNA is that which is capable of hybridizing with one or both strands of the nucleic acid of SEQ ID No 2, and polynucleotide and oligonucleotide fragments thereof of 15 or more contiguous bases, preferably 30 or more and more preferably 100 or more, selected from a characteristic region of the sequence with respect to nucleic acid encoding for other superfamily enzymes, under high stringency conditions, more preferably being capable of such hybridization with two or more of these polynucleotides or oligonucleotides.
  • Most suitable selections of sequences for performing these hybridizations will be selected from coding regions of SEQ ID No 2 coding for parts that are unconserved with respect to the other members of the superfamily.
  • amino acids 41 to 49 and 250 to 256 tend to be conserved within the superfamily and thus these sequences as such are unsuitable for use in selecting hybridizing sequences for selecting the preferred homologs of alfalfa type other than in a screen for superfamily members in general.
  • nucleic acid encoding for an aldose reductase homologous protein superfamily member, in the form of a vector or contract, combined in frame with a promoter, activating or otherwise regulating sequence capable of promoting its expression ectopically in plants, either constituitively or locally, eg. in vegetative or root tissues.
  • this expression is non-temporal, ie.unlike the Rocarati et al promoter, such that the plant tissue is protected from the damaging effects of free-radicals, eg. generation of HNE, at all times.
  • tissue specific regulatory regions such as tissue specific promoters
  • tissue specific promoters may be used where a specific tissue requires protection from stress related toxins and/or osmoregulation.
  • tissue specific promoters will be known to those skilled in the art, but may be exemplified by those of WO 97/20057 (incorporated herein by reference) teaching root specific promoters and those of Rocarti et al, being embryo and seed specific.
  • tissue specific promoters may be used but constuitive promoters such as CaMv35S and alfalfa (MsH3gl) (see WO 97/20058 incorporated herein by reference) will have useful application.
  • a process for producing plant cells overproducing an aldose reductase-homologous protein which comprises the transformation of a plant cell with a nucleic acid molecule according to the invention.
  • a transgenic plant or part thereof comprising recombinant nucleic acid, a vector or construct as described above.
  • Preferred plants of the fifth aspect may comprise the nucleic acid of the invention in a construct in functional association with promoter, activating or otherwise regulating sequences.
  • Preferred promoters may be tissue specific such that the resultant expression of protein, and thus its effects, are localised to a desired tisssue. Promoters with a degree of tissue specificity will be known to those skilled in the art of plant molecular biology.
  • DNA, RNA and vectors containing or encoding for these may be introduced into target cells in known fashion in the field of plant cell transformation. Particularly preferred is the method of introducing the DNA or RNA into cells, more particularly pollen cells, using techniques such as electroporation or gene gun technology.
  • tissue specific promoters, enhancers or other activators should be incorporated into the transgenic cells employed in operative relation with the DNA.
  • transgenic plant cell or plant may be produced in transgenic form incorporating the nucleic acid of the invention such that aldehyde, particularly HNE, reducing activity in the plant is altered, constituitively or ectopically.
  • aldehyde particularly HNE
  • reducing activity in the plant is altered, constituitively or ectopically.
  • Further aspects of the invention provide alfalfa homologous protein and
  • aldose reductase-homologous protein is defined in this description as an enzyme being a member of the aldo-keto reductase superfamily and similarly to other members of this family can reduce the aldehyde group of a wide variety of substrates in the presence of NADPH cofactor.
  • aldose reductase-homologous protein as used herein also includes functional variants and derivatives of the said protein.
  • a “functional variant” or a “functional derivative” of a protein is a protein the amino acid sequence of which can be derived from the amino acid sequence of the original protein by the substitution, deletion and/or addition of one or more amino acid residues in a way that, in spite of the change in the amino acid sequence, the functional variant retains at least a part of at least one of the biological activities of the original protein that is detectable for a person skilled in the art.
  • a functional variant is generally at least 50% homologous (preferably the amino acid sequence is at least 50% identical), advantageously at least 70% homologous and even more advantageously at least 90% homologous to the protein from which it can be derived.
  • a functional variant may also be any functional part of a protein; the function in the present case being particularly but not exclusively aldehyde reduction.
  • the amino acid sequence differs from SEQ ID No 1 or SEQ ID No
  • the protein comprises an amino acid sequence having 90% or more, still more preferably 95%, sequence identity with SEQ ID No 1 or SEQ ID No 3 and optimally 100% identity with those sequences.
  • homology and identity percentages can be ascertained using commercially or publically available software packages incorporating, for example, FASTA and BLASTn software or by computer servers on the internet.
  • An example of the former is the GCG Wisconsin Software package while both Genbank (see http://www.ncbi.nlm.nih.gov/BLAST) .and EMBL: (see http://www.embl-heidelberg.de/Blast2) offer internet services.
  • identity is meant that the stated percentage of the claimed amino acid sequence or base sequence is to be found in the reference sequence in the same relative positions when the sequences are optimally aligned, notwithstanding the fact that the sequences may have deletions or additions in certain positions requiring introduction of gaps to allow alignment of the highest percentage of amino acids or bases.
  • sequence are aligned by using 10 or less gaps, ie. the total number of gaps introduced into the two sequences when added together is 10 or less.
  • the length of such gaps is not of particular importance as long as the aldehyde reducing activity is retained but generally will be no more than 10, and preferably no more than 5 amino acids, or 30 and preferably no more than 15 bases.
  • Preferred parameters for BLAST searches are the default values, ie. for EMBL Advanced Blast2: Blastp Matrix BLOSUMS, Filter default, Echofilter X, Expect 10, Cutoff default, Strand both, Descriptions 50, Alignments 50.
  • BLASTn defaults are again preferably used.
  • GCG Wisconsin Package defaults are Gap Weight 12, Length weight 4.
  • the expression 'degenerative substitution' refers to substitutions of nucleotides by those which result in codons encoding for the same amino acid; such degenerative substitutions being advantageous where the cell or vector expressing the protein is of such different type to the DNA source organism cell that it has different codon preferences for transcription/translation to that of the cDNA source cell. Such degenerative substitutions will thus be host specific.
  • the expression 'conservative substitutions' as used with respect to amino acids relates to the substitution of a given amino acid by an amino acid having physicochemical characteristics in the same class.
  • an .amino acid in the SEQ ID No 1 or SEQ ID No 3 has a hydrophobic characterising group
  • a conservative substitution replaces it by another amino acid also having a hydrophobic characterising group; other such classes are those where the characterising group is hydrophilic, cationic, anionic or contains a thiol or thioether.
  • substitutions are well known to those of ordinary skill in the art, i.e. see US 5380712 which is incorporated herein by reference, and are only contemplated where the resultant protein has activity as an aldehyde reducing enzyme, particularly acting upon HNE, eg. in the presence of NADPH.
  • a protein or RNA is said to be "produced at enhanced level” or “overproduced” if the concentration of the said protein in the cell where it is produced is at least 20% higher than that in the original cell, ie. a cell of the original plant line which has now been transformed.
  • a plant, a plant tissue or a plant cell is defined as having an "enhanced level of resistance" to a harmful condition if it can withstand a 20% enhanced effect of the same harmful condition, without detectable damage, than the original plant, plant tissue or plant cell.
  • Overproduction of a molecule in a cell can be achieved via both traditional mutation and selection techniques and genetic manipulation methods.
  • ectopic expression is used herein to designate a special realisation of overproduction in the sense that, for example, an ectopically expressed protein is produced at a spatial point of a plant where it is naturally not at all (or not detectably) expressed, that is, said protein is overproduced at said point.
  • Particular types of ectopic expression will comprise eg. expression of the aldo-keto reductase protein in vegetative tissues or roots of a plant, whereas normally it may be found only temporally expressed through transcripts in embryo and/or pollen (see Roncarati et al).
  • SEQ ID No 1 Amino acid sequence shared by preferred aldose reductase- homologous enzymes to which homology of the enzymes for use in the present invention is related.
  • SEQ ID No 2 DNA including cDNA encoding for an alfalfa (Medicago satvia) aldose reductase-homologous enzyme for use in the present invention.
  • SEQ ID No 3 Amino acid sequence of an alfalfa (Medicago satvia) aldose reductase homologous enzyme for use in the present invention.
  • SEQ ID No 4 .and 5 are those of primers referred to in Example 3.
  • FIGURES Figure 1 is a comparison of the amino acid sequence of the alfalfa MsALRh protein to human, animal and plant aldose and aldehyde reductase proteins.
  • Figure 2 shows the results of Southern hybridisation of genomic DNA isolated from alfalfa. 20 ⁇ g of genomic DNA was digested with restriction enzymes indicated in the figure below each lane. The restriction fragments were separated on 1% agarose gel and blotted onto Hybond-N membrane. P-32 labelled coding region fragment of the MsALRh CDNA was used as hybridization probe.
  • FIG 3 shows the results of Northern hybridisation experiments with the MsALRh cDNA.
  • Total RNA was isolated from alfalfa A2 cell suspension treated with 150 ⁇ M ABA (abscisic acid) hormone (3a), 10% polyethylene glycol (PEG 4000, 3b) and cadmium chloride (3c), 20 ⁇ g total RNA was electrophoresed on a 1% formaldehyde-agarose gel and transferred onto Hybond-N membrane, and subsequently probed with P-32 labelled coding region of the MsALRh cDNA fragment. C in each case is untreated control.
  • Figure 4 depicts a Western hybridisation analysis of the MsALRh gene expression.
  • 4a shows the level of protein in different alfalfa tissues with longer (upper) and shorter expression time.
  • 4b shows the change of protein level during 10% PEG, 250 ⁇ M cadmium chloride and ImM hydrogen peroxide treatments.
  • Figure 5 is a map of a vector of pGEX origin (pGEX-5X-3) suitable for the expression of the cDNA encoding the enzyme.
  • the vector expresses the enzyme as a glutathion-S-transferase fusion protein in E. coli cells.
  • Figure 6 shows purification of MsALRh-GST fusion protein on glutathione- Sepharose.
  • Figure 7 shows the effect of ammonium sulphate treatment on the activity of the MsALRh-GST recombinant fusion protein.
  • Enzymatic activity on lOmM DL- glyceraldehyde substrate was measured spectrophotometrically by determining the measure of NADPH oxidation at 340 run.
  • the standard reaction mixture comprised 5 ⁇ g enzyme and 0.15 mM NADPH. Before starting the activity measurement, the enzyme was preincubated with ammonium-sulfate for 3 minutes.
  • Figure 8 shows the Western hybridisation detection of the aldose reductase- homologous protein in cell extracts isolated from the leaves of transgenic tobacco plants.
  • Figure 9a and 9b show the results of light induced fluorescence measurements of control and transgenic plant leaf discs during 20 ⁇ M paraquat (9a) and 250 ⁇ M cadmium chloride (9b) treatments.
  • Figures 10a, 10b, 10c and lOd demonstrate the results of germination tests of control (SRI) and transgenic (1/1, 1/5, 1/9) plants in control 0 conditions (10a), in the presence of 15mM hydrogen peroxide (10b), 150 ⁇ M cadmium chloride (10c) and 250mM sodium chloride (lOd).
  • Figure 11 shows results of light induced fluorescence measurements during water deficient growth of control (SRI) and transgenic (TR1/4, 1/7, 1/1, 1/5 and 1/9) plants.
  • Figure 12a shows a photograph of control and transgenic plants after 10 days of rehydration after a 35 day severe drought treatment.
  • 12b shows detection of the aldose reductase-homologous protein in cell extracts made from leaves of control and transgenic plants shown in 12a after the aforesaid drought tolerance experiment .
  • Figure 13 illustrates the photsynthetic efficiency measured as Gentry-parameter of chlorophyll fluorescence in tobacco leaves before (day 1), during (days 2-4) and after (day 8 and day 15) UV-B irradiation.
  • Full circles represent average data for plants 1/1, 1/5, 1/8, 1/9 and 1/10.
  • Empty circles represent data from plants 1/4 1/7 and control SRI.
  • the inventors demonstrate the isolation of a gene encoding an alfalfa (Medicago sativa) aldose reductase-homologous protein, in cDNA form. Its nucleotide sequence and the deduced amino acid sequence verified that the isolated cDNA codes for a novel plant aldose reductase-homologous protein that has not been identified so far.
  • the isolated aldose reductase cDNA was cloned into a plant expression vector and by the means of a generally used gene introduction method transformed tobacco plants were produced. Beyond the molecular biological characterization of the transgenic tobacco plants their increased tolerance to different stress conditions has also been shown.
  • the alfalfa cDNA library was produced as follows. Total cellular RNA was isolated from in vitro cultivated auxin shock activated alfalfa tissues, which contained dedifferentiated callus tissues and high number of somatic embryos. Stress activation (auxin shock) was performed by using 2,4- dichloro-phenoxyacetic acid (an auxin analog) applying a method known per se so as to enhance the concentration of stress induced mRNAs. The mRNA isolation method applied is detailed in Cathala et al. (1983 DNA 2: 329-335) incorporated herein by reference.
  • the mRNA molecules were then separated from the total RNA by the means of oligo-dT cellulose chromathography according to the method of Aviv, H. and Leder, P. (1972 Proc Natl. Acad. Sci. USA 69: 1408-1412) incorporated herein by reference.
  • the first cDNA strand was then synthesised on the isolated poly-A + (mRNA) fraction with an oligo-dT primer using AMV reverse transcriptase enzyme. Synthesis of the second DNA strand was done using DNA polymerase I enzyme, removal of the excess of template mRNA was performed via RNase H treatment.
  • the nucleotide sequence of about a hundred individual clones of this cDNA library (i.e. cDNA clones prepared from a stress induced mRNA isolate) was determined. Before sequencing, the inserts were subcloned into plasmid pUC 19 and the sequencing reaction was carried out on double stranded DNA using the dideoxy chain termination method with ⁇ -35-S-dATP labelling. For the sequencing reactions the T7 Sequencing Kit (Pharmacia) and Sequenase 2.0 Kit (US Biochem) were used, according to the manufacturers' protocols. Using the determined cDNA sequence, Genebank and ⁇ MBL nucleotide sequence databases were homology searched.
  • MsALRh amino acid sequence derived from the clone later designated MsALRh has homology to both human, rat and plant aldo-keto reductase proteins.
  • the length of the MsALRh cDNA is 1231 base pairs and the coding region can be located between 34 and 975 nucleotides (S ⁇ Q ID no 2). This region codes for a 313 amino acid protein.
  • the amino acid sequence of the alfalfa aldose reductase-homologous protein showed 44.3% identity with the known and best characterised monocot barley aldose reductase-homologous protein and it has 46.2% and 46.1% identity with human aldehyde reductase and pig aldose reductase enzymes, respectively (Fig. 1). It further showed a 42% amino acid identity to the dicotyledonous soybean NADPH-dependent oxidoreductase enzyme. The observed level of amino acid homology led us to conclude that the cloned alfalfa cDNA codes for a novel enzyme of the aldo-keto reductase superfamily.
  • Example 2 The amino acid sequence of the alfalfa cDNA codes for a novel enzyme of the aldo-keto reductase superfamily.
  • Genomic DNA was isolated from alfalfa cells and the purified DNA was digested with restriction endonucleases.
  • the MsALRh cDNA fragment carrying full length coding sequence was used as probe for the Southern hybridisation experiments after radioactive isotope labelling using the "random priming" method (Freinberg, A.P. and Vogelstein, B. 1983. Anal. Biochem. 137: 266-267).
  • the hybridisation was carried out in Rapid-hyb buffer (Amersham) at 65°C.
  • the result of the hybridisation (Fig. 2) showed that the aldose reductase-homologous gene has a low copy number but faint additional bands on the autoradiogram present at al restriction digestion might indicate the presence of other gene homologues in the alfalfa genome.
  • the tissue specificity experiments revealed that the alfalfa aldose reductase-homologous gene is expressed in each tissue in contrary to the barley aldose reductase-homologue, which was expressed only temporally and only in embryos.
  • the alfalfa cell suspension was exposed to different hormone and stress treatments. The results indicated that the studied gene is homoinduced.
  • Treatment with the plant stress hormone abscisic acid (ABA) significantly enhanced the RNA level 4 hours after its initiation (Fig. 3).
  • ABA abscisic acid
  • Fig. 3 The importance of this finding lies in the fact that hormone ABA is a key compound in the regulation of several genes that play important role in the adaptation to environmental stresses such as drought and low temperature.
  • lipid peroxides Treatment with polyethylene-glycol results in an osmotic stress in plant cells.
  • Expression of the MsALRh gene could be induced by such an osmotic stress as well as by cadmium-chloride treatment (heavy metal stress; Fig. 3).
  • Cadmium ion is known to induce oxidative stress beside its photosynthetic inhibitory property .and in these cases the level of lipid peroxides increases in the plant cells.
  • the decomposition products of the lipid peroxides are those lipid aldehydes that can serve as substrates of the aldo-keto reductases in the detoxification reactions.
  • a recombinant protein was produced and purified in a prokaryotic protein expression system.
  • the cDNA of the MsALRh gene was cloned into pGEX 4T-1 prokaryotic expression vector (Pharmacia) and the GST (gluthathion-S-transfarase) containing fusion protein was produced in and purified from E. coli.
  • the purified fusion protein was raised by conventional techniques. This antibody was used to analyse, by Western hybridization, the effects of different stress treatments on the production of the MsALRh protein and to detect the protein in different tissues of the alfalfa plant (Fig. 4).
  • the coding region (975 bp) of the MsALRh cDNA was PCR amplified, using
  • primerl (5'-cgaactcgagatggccacagcaatcaagttt-3') as upper and “primer2" (5'- ccgagctctacttcaccatcccagag-3 ' ) as lower primer (Xhol restriction sites in the primers are underlined) according the method of Mullis and Faloona (1987 Meth. Enzymol. 155: 335) incorporated herein by reference.
  • the PCR product was digested with Xhol restriction endonuclease and the digested fragment was cloned into the Xhol site of the pGEX 4T-1 vector (Pharmacia). The nucleotide sequence of the cloned product was verified in order to avoid PCR generated errors.
  • the transformant E. coli cells were treated with 0.5 mM isopropyl- ⁇ -D-galactopyranoside (IPTG) at 25°C for 3 hours.
  • IPTG isopropyl- ⁇ -D-galactopyranoside
  • Transformants expressing the fusion protein were identified according to the method of Smith and Johnson (1988 Gene 67: 31-40) incorporated herein by reference.
  • the MsALRh-GST fusion protein was purified on glutathione-Sepharose 4B columns following the instructions of the manufacturer's protocol (Pharmacia).
  • the produced and purified MsALRh-GST fusion protein (100-150 ⁇ g in 0.5 ml) was thoroughly mixed with the same volume of complete Freund's adjuvant (Sigma) and rabbit was immunised with this emulsion. Two subsequent immunisations were made with the emulsion of the antigen and incomplete Freund's adjuvant after 3 and 6 weeks, respectively.
  • the blood serum was checked in Western analysis. The specificity of the serum for the MsALRh protein was tested in a competition experiment. For the Western blot analysis, proteins of the alfalfa cell suspension extracts were separated on a 12% SDS-polyacrylamide gel. After separation, the proteins were transferred onto nitrocellulose membrane.
  • the blood serum raised against the fusion protein was used as first antibody (in 1 :2000 dilution); anti-rabbit-IgG antibody conjugated with peroxidase was used as second antibody in these experiments.
  • the antigen-antibody complex was detected using Super Signal R TM CL-HRP Substrate System (Pierce), a high sensitivity chemiluminescent detection method.
  • Enzymatic activity of the MsALRh-GST fusion protein on different substrates was measured photometrically: measuring the decrease of the concentration of the NADPH cofactor at 340 nm wavelength at 25°C during a 5 minutes reaction time.
  • One enzyme activity unit is expressed as the enzyme amount (in mg) which is necessary for the oxidation of 1 ⁇ mol NADPH in one minute in the presence of the substrate.
  • Aldose sugars, DL-glyceraldehyde and 4-hydroxy-nonenal (ICN Biochemicals) were used as substrates in these specificity measurements, and the results (as Km kinetic constants) are shown in Tables 1-3 below. According to our results the purified enzyme is able to reduce aldehyde substrates in the presence of NADPH cofactor. High enzymatic activities could only be measured on DL- glyceraldehyde (known as the best substrate for plant aldose reductase-homologues) and on 4-hydroxy-nonenal.
  • the MsALRh enzyme could reduce D-xylose with much less activity and showed no activity on other aldose substrates (such as D-glucose, D- galactose, D-mannose or D-ribose) even at very high (400 mM) substrate concentrations.
  • alfalfa enzyme showed very similar activity on glyceraldehyde substrate as compared to the animal aldehyde reductases. At the same time, it works less effectively in the presence of 4-hydroxy- nonenal (Table 2).
  • HNE 4-hydroxy-nonenal
  • the S0 2" ions are known to alter the enzymatic activity of rat aldose and aldehyde reductases in a different manner.
  • the resulted twofold increase in the enzymatic activity in the presence of 0.3 M ammonium-sulfate is a characteristic of the aldose reductases (Fig. 7).
  • An important property of the MsALRh enzyme is that it's activity increases by 100% even in the presence of low concentration (45 mM) of ammonium-sulfate. This result may indicate that the advantageous effect of the enzyme can be increased in the transgenic plants by ammonium-sulfate treatment.
  • MsALRh enzyme as well, as the barley aldose reductase-homologous enzyme, has also similar characteristics to both the animal aldose and aldehyde reductases but it possesses specific different characteristics, too.
  • Example 4 Insertion of the alfalfa aldose reductase-homologous (MsALRh) cDNA into tobacco plants to produce the aldose reductase protein in the vegetative organs of transgenic plants
  • the produced binary vector construct was mobilized into an Agrobacte ⁇ um strain, tobacco leaves were infected by scratching, co-cultivation was performed and kanamycin resistant plants were regenerated according to Claes et al. (1991.
  • the produced binary vector construct was mobilized into an Agrobacte ⁇ um strain, tobacco leaves were infected by scratching, co-cultivation was performed and kanamycin resistant plants were regenerated according to Claes et al. (1991. The
  • Plant J. 1: 15-26 incorporated herein by reference. Transformant plants and seeds obtained by self-pollinating were used in the germination experiments.
  • aldose reductase gene in the transgenic tobacco plants was verified by Western blot analysis using the polyclonal antibody raised against the MsALR-GST fusion protein. Ten tobacco lines were analysed (Fig. 8). Protein extracts obtained from the leaves of these lines were tested according to the western hybridisation protocol detailed previously except that the protein samples were separated on 10% SDS-polyacrylamide denaturing gel and alkaline phosphatase conjugated anti-IgG antibody was used as second antibody.
  • the antibody-protein complex was detected by BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (2,2'-di-p-nitrophenyl-3,3'-[3,3'-dimethoxi-4,4'-diphenylene]-ditetrazolium-chloride) substrates. Synthesis of considerable amount of the MsALRh protein could be detected in five of the ten examined transgenic lines, the other transgenic lines showed no expression of the MsALRh gene and no signal could be detected in the control wild type (SRI) plant, either.
  • BCIP bromo-4-chloro-3-indolyl-phosphate
  • NBT 2,2'-di-p-nitrophenyl-3,3'-[3,3'-dimethoxi-4,4'-diphenylene]-ditetrazolium-chloride
  • Transformant tobacco plants expressing alfalfa aldose reductase-homologous protein show elevated level of tolerance against different stress treatments, such as herbicide, heavy metal, hydrogen peroxide and sodium chloride
  • Transformant tobacco plants were grown in soil under controlled greenhouse conditions for 5 weeks and the water content of the soil was measured at the beginning of the experiment. The damage of the photosynthetic system was monitored during the experiment by light induced fluorescence measurements as detailed in the above experiments. Prior to the beginning of the experiment we have selected plants with similar developmental stage from the transgenic tobacco lines and the SRI control plants and leaves with the same age were chosen on each plant for the fluorescence measurements to be made at given time points during the experiment.
  • UV-B irradiation protection UV-B irradiation protection.
  • Tobacco plants were kept in a ventilated growth chamber for five test days at 26°C.
  • Photosynthetically active radiation was provided from fluorescent tubes (Tungsten 40W) at 60-80 ⁇ mol m "2 s "2 intensity from above and from the side between 8am and 6pm daily.
  • UV-B (280-320nm) irradiation was provided from UV-B 313 tubes (Q- panel, USA) at 7.5 ⁇ mol m " s " total UV-B intensity from above between 9am and 3pm daily, for 4 days starting from the second day of the test.
  • chlorophyll fluorescence measurement plants were transferred to the laboratory. Measurement was performed in the dark and was completed in ca. 45 minutes including the initial 15 minutes dark adaptation. After this plants were transferred back to the growth chamber or (during the recovery period) to the green house. All chlorophyll fluorescence measurements were performed in the afternoon between ca 3 and 5pm. Untreated plants were measured in the afternoon of the first test day, before starting UV-B irradiation at the second test day. UV-B irradiated plants were measured on the 2 nd -5 th test days, 30 minutes after cessation of irradiation.
  • Chlorophyll fluorescence was measured with a PAM Chlorophyll Fluorometer (WALZ Company, Germany) using weak ( ⁇ 1 ⁇ mol m “ s “ ) modulated red light .
  • Steady state (F s ) and maximal (F m ') fluorescence yields were measured before and upon a 0.5s saturating white light pulse (2500 ⁇ mol m “ s “ ), respectively, in 11.5 ⁇ mol m "2 s "2 actinic red light.
  • Photosynthetic efficiency was measured as the Gentry-parameter of chlorophyll fluorescence in tobacco leaves before (day 1), during (days 2-4) and after (days 8 and
  • the isolated alfalfa aldose reductase-homologous gene codes for an enzyme of novel characteristics, which can react with the toxic lipid aldehyde 4- hy droxy-nonenal .
  • Transgenic plants expressing the alfalfa aldose reductase-homologous gene were produced to make use of these and other characteristics of the enzyme and the produced plants were proven to be resistant against a wide range of harmful stress treatments.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
EP98954624A 1997-11-18 1998-11-18 Stressresistenzgen Withdrawn EP1032690A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
HU9702118A HUP9702118A3 (en) 1997-11-18 1997-11-18 Plants over producing aldose reductase homologous protein can tolerate water deficiency and resist oxidative stresses, and process for producing them
HU9702118 1998-04-21
PCT/GB1998/003464 WO1999025852A1 (en) 1997-11-18 1998-11-18 Stress resistance gene

Publications (1)

Publication Number Publication Date
EP1032690A1 true EP1032690A1 (de) 2000-09-06

Family

ID=89995761

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98954624A Withdrawn EP1032690A1 (de) 1997-11-18 1998-11-18 Stressresistenzgen

Country Status (12)

Country Link
EP (1) EP1032690A1 (de)
JP (1) JP2001523466A (de)
KR (1) KR20010032243A (de)
CN (1) CN1292823A (de)
AR (1) AR017638A1 (de)
AU (1) AU752803B2 (de)
BR (1) BR9814644A (de)
CA (1) CA2309757A1 (de)
HU (2) HUP9702118A3 (de)
NZ (1) NZ503443A (de)
WO (1) WO1999025852A1 (de)
ZA (1) ZA9810540B (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4312012B2 (ja) 2003-09-12 2009-08-12 トヨタ自動車株式会社 パラコート（登録商標）耐性遺伝子並びに維管束及びトライコーム特異的プロモーター
CN114015577B (zh) * 2021-11-03 2023-06-30 青岛科技大学 一种具有镉离子抗逆性的聚球藻株的构建方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2256856T3 (es) * 1995-04-06 2006-07-16 Seminis Vegetable Seeds, Inc. Proceso de seccion de celulas transgenicas.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9925852A1 *

Also Published As

Publication number Publication date
HUP0101283A2 (hu) 2001-08-28
ZA9810540B (en) 2000-05-18
NZ503443A (en) 2002-03-01
HU9702118D0 (en) 1998-01-28
CA2309757A1 (en) 1999-05-27
CN1292823A (zh) 2001-04-25
AR017638A1 (es) 2001-09-12
HU226743B1 (en) 2009-08-28
HUP9702118A3 (en) 2000-08-28
WO1999025852A1 (en) 1999-05-27
HUP9702118A2 (hu) 1999-07-28
BR9814644A (pt) 2000-10-03
AU1167599A (en) 1999-06-07
HUP0101283A3 (en) 2002-12-28
AU752803B2 (en) 2002-10-03
KR20010032243A (ko) 2001-04-16
JP2001523466A (ja) 2001-11-27

Similar Documents

Publication Publication Date Title
Oberschall et al. A novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses
US20200113147A1 (en) Manipulation of glutamine synthetases (gs) to improve nitrogen use efficiency and grain yield in higher plants
Tepperman et al. Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity
Tran et al. The polyphenol oxidase gene family in poplar: phylogeny, differential expression and identification of a novel, vacuolar isoform
Le Deunff et al. Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths
Jung et al. Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance
AU2019466501A9 (en) Mutant hydroxyphenylpyruvate dioxygenase polypeptide, encoding gene thereof and use thereof
Davoine et al. Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble
Feng et al. Cloning and characterization of a novel splicing isoform of the iron-superoxide dismutase gene in rice (Oryza sativa L.)
Curtis et al. Induction of dwarfism in transgenic Solanum dulcamara by over‐expression of a gibberellin 20‐oxidase cDNA from pumpkin
CN115044605A (zh) Lrrk1基因调控水稻抗坏血酸含量和耐盐性的应用
JP4998809B2 (ja) 植物の鉄欠乏耐性を向上させるポリペプチドおよびその利用
Skinner et al. Differential expression of genes encoding the light-dependent and light-independent enzymes for protochlorophyllide reduction during development in loblolly pine
AU752803B2 (en) Stress resistance gene
US8592649B2 (en) Functional expression of shuffled yeast nitrate transporter (YNT1) in maize to improve nitrate uptake under low nitrate environment
US6545202B2 (en) Transgenic plant transformed with a translationally controlled tumor protein (TCTP) gene
CA2211018C (en) Seed coat specific dna regulatory region and peroxidase
US6812338B2 (en) Peroxisomal ascorbate peroxidase gene induced by high temperature stress and a transgenic plant exhibiting thermotolerance
MX2011004216A (es) Gen at1g67330 novedoso en la eficiencia de captacion de nitrato alterada.
クボアキヒロ et al. Molecular Biological Research on Ascorbate Peroxidase and Glutathione Reductase in the Active Oxygen Scavenging System of Plants: Molecular Cloning and Analysis of Expression in Response to Air Pollutants
CN118086332A (zh) 一种小麦种子休眠基因TaPer12-3A及其应用
Labat et al. Grapevine (Vitis vinifera L.) alcohol dehydrogenase gene: An important marker of the physiological state during fruit ripening
WO1999064613A1 (fr) Gene de d-ribulose-5-phosphate-3-epimerase de la reponse aux nematodes
Ilett The characterisation of barley and wheat oxalate oxidases expressed in transgenic plants
JP2003219883A (ja) 塩ストレスによって誘導される核酸およびタンパク質

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000505

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: HIDEG, EVA

Inventor name: SASS, LASZLO

Inventor name: FEHER, ATILLA

Inventor name: DUDITS, DENES

Inventor name: TOEROK, KAROLYNE

Inventor name: DEAK, MARIA

Inventor name: HORVATH, GABOR

Inventor name: OBERSCHALL, ATILLA

RIN1 Information on inventor provided before grant (corrected)

Inventor name: HIDEG, EVA

Inventor name: SASS, LASZLO

Inventor name: FEHER, ATILLA

Inventor name: DUDITS, DENES

Inventor name: TOEROK, KAROLYNE

Inventor name: DEAK, MARIA M

Inventor name: HORVATH, GABOR

Inventor name: OBERSCHALL, ATILLA

17Q First examination report despatched

Effective date: 20031119

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040330

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1031127

Country of ref document: HK