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WO1996037613A1 - Gene controlant la senescence chez les plantes - Google Patents

Gene controlant la senescence chez les plantes Download PDF

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Publication number
WO1996037613A1
WO1996037613A1 PCT/GB1996/001223 GB9601223W WO9637613A1 WO 1996037613 A1 WO1996037613 A1 WO 1996037613A1 GB 9601223 W GB9601223 W GB 9601223W WO 9637613 A1 WO9637613 A1 WO 9637613A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
dna
phenotype
green
stay
Prior art date
Application number
PCT/GB1996/001223
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English (en)
Inventor
Howard Thomas
Catherine Margaret Griffiths
Barbara Dorothea Hauck
Petra Hilary Dawn SCHÜNMANN
Original Assignee
Germinal Holdings Limited
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Publication date
Application filed by Germinal Holdings Limited filed Critical Germinal Holdings Limited
Priority to AU57737/96A priority Critical patent/AU5773796A/en
Publication of WO1996037613A1 publication Critical patent/WO1996037613A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention relates to a so-called "stay-green” mutation of a gene controlling leaf senescence in plant species, and the use of this nucleotide sequences comprising or derived from this gene mutation in recombinant DNA technology in the production of novel and improved plant varieties and for use identifying strains produced in conventional plant breeding which have the mutation.
  • Senescence in plants is genetically programmed, as is apparent from the fact that a senescence occurs in an orderly and timely fashion in the seasonal cycle of deciduous trees and other plants. It is therefore unlikely to be the result of the kind of random design failure that occurs in elderly animals. Genetic analysis has shown that there are senescence genes in plants.
  • genes controlling senescence fall into five broad categories, classified according to when they are turned on during the lifetime of a leaf. These categories are as follows:
  • Group 1 genes controlling the primary metabolic activities of viable cells
  • Group 3 genes which control growth and photosynthesis and which contribute to senescence by switching off;
  • Group 4 genes which make up the senescence trigger;
  • Group 5 genes which code for the•characteristic activities of senescence such as enzymes that break down cell structures.
  • Isolation and cloning of DNA which contributes to the "stay-green" characteristics would provide a useful diagnostic tool in plant breeding which would enable the identification of plants of this phenotype speedily and accurately, without the need to embark on the lengthy backcrossing processes as well as forming the basis of further recombinant processes.
  • the applicants have cloned and sequenced a DNA molecule which is obtainable from the genetic material putatively from chromosome 6 of a stay green Festuca- oliu ⁇ . hybrid and which includes the locus designated sid.
  • This clone is derived from message RNA that is highly abundant in a mutant of Festuca pratensis with a defect in its chlorophyll breakdown machines .
  • the clone or sequences derived therefrom may be incorporated into a cloning vector.
  • nucleic acid which comprises the sequence of Fig.l or an allele, variant or fragment thereof and which when expressed in a plant is capable of conferring on it a "stay-green" phenotype.
  • the nucleic acid is preferably a cDNA isolate which is obtainable from a Festuca pratensis plant exhibiting a "stay-green" phenotype, said isolate comprising nucleic acid which is associated with said phenotype and which is substantially free of material with which it is associated in nature.
  • nucleic acid isolate which is putatively obtainable from chromosome 6 of a Festuca- Lolium hybrid which has a "stay-green" phenotype, said isolate comprising nucleic acid which is responsible for producing said phenotype and which is substantially free of material with which it is associated with in nature.
  • the invention provides a nucleotide sequence- encoding a amino acid sequence as set out in Figure 2a or a fragment or variant thereof.
  • the amino acid sequence of Figure 2a has a methionine residue near one end as indicated and it is possible that this is the true initiation site.
  • a preferred nucleotide sequence of the invention encodes a fragment of the amino acid sequence of Figure 2b.
  • variant' refers to amino acid sequences which differ from the basic sequence but which have a significant homology with said sequence.
  • variants will be at least 60% and preferably at least 90% homologous with the starting sequence, such as that shown in Figure 2.
  • nucleotide sequence which encodes a fragment or a variant of the amino acid sequence of Figure 2 will selectively hybridise with the nucleotide sequence of Figure 1 or its complementary strand, preferably under stringent hybridisation conditions.
  • sequences, as well as the full length sequence will be useful as nucleotide probes for example in plant breeding procedures such as for screening for the abundance of the corresponding mRNA in genetic lines derived from the original green mutant of Festuca pratensis .
  • Suitable species which could benefit from modification of the allele responsible for the stay-green phenotype include crop species such as ryegrass, fescue and vegetables such as green peppers and flageolet beans.
  • crop species such as ryegrass, fescue and vegetables such as green peppers and flageolet beans.
  • the post-harvest quality and appearance of these crops may be beneficially affected using recombinant DNA techniques .
  • the gene encoding gem is grossly overexpressed in tissues of plants both homozygous and heterozygous for the gene. Detection of overexpression distinguishes phenotypically normal plants with one copy of gem from those that are homozygous wild-type. Conventionally, identification of gem heterozygotes necessitates additional test backcrosses which, in grasses with vernalisation and complex daylength requirements for flowering, introduces significant delays into a breeding programme.
  • the present application provides methods by which the need for test backcrosses is obviated, based on selective hybridisation of nucleic acids from test plants with probes based on the nuclotide sequences of the invention.
  • the invention provides a method for determining plants which contain DNA capable of conferring the "stay-green" phenotype comprising probing nucleic acid from a test plant with a labelled probe comprising a nucleotide sequence which encodes the amino acid sequence of Fig. 2 or a fragment thereof; and comparing the results obtained from the test with results obtained from probing, with said probe, nucleic acid from a plant known to be homozygous or heterozygous for over- expression of DNA capable of conferring the "stay-green" phenotype.
  • the method may comprise isolating RNA or DNA from a plant under test. Using this technique, plants corresponding to RNA or DNA samples giving high levels of hybridisation with gem can be selected and used. for further varietal development.
  • RNA is utilised in this method.
  • One technique which can be used in the isolation of RNA and the application to for example nylon membranes for subsequent probing by dot- or slot-blotting are described hereinafter in Example 1.
  • Suitable probes may be based upon the all or part of the nucleotide sequences of Figure 1. They may be labelled for example radioactively using conventional methods such as those described in Example 1 hereinafter. The probe is then applied to the membrane also as described in Example 1 and the abundance of gem transcipts in each RNA sample related to the binding of the radioactive cDNA as revealed by autoradiography.
  • the invention provides a method for selecting plants which contain gem sequences said method comprises isolating RNA or genomic DNA from a plant under test, where RNA is isolated, using this as a template to form cDNA, amplifying a sequence known to correspond to all or part of the nucleotide sequence of the invention and detecting the amplified DNA.
  • Amplification is suitably effected using PCR (polymerase chain reaction) techniques, using oligonuclotide primers based upon the sequence of gem in a manne known in the art.
  • Figure 1 shows a cDNA sequence of the gem clone
  • Figure 2 shows the derived amino acid sequence of the expression product of said clone
  • Figure 3 shows expression patterns of ca , encoding LHCP- 2, and the stay-green related gene gem.
  • RNA from (J)uvenile, (E)merging, (M) ature and (S)enescing leaf tissue of Fp genotypes sid ⁇ , sid YY and the hybrid between them was probed with a wheat cab cDNA and Fp gem cDNA. Prolonged exposure of the northern blot revealed a weak gem signal in the M and S tracks of YY. Genomic organisation and expression of sid
  • Chromosome preparations were made from root tip squashes of sid y y F2 hybrids .
  • Total geono ic DNA was prepared from Lm and Fp, fluorescently labelled, and used to block and detect areas of interspecific recombination respectively.
  • Introgressed Festuca DNA was shown as lighter areas on the Lm chromosome set. As the F2 hybrid from which the chromosomes were isolated was phenotypically stay-green, and as sid y is recessive, there must be two Lolium chromosomes with fluorescent segments.
  • the fluorescent labelling illustrated three such regions, of which two were clearly a pair of chromosomes, each with a distal segment of Festuca chromatin in the long arm.
  • the third segment included a centromere and was represented as only a single copy.
  • the location of the segment including the sid locus has been confirmed in another diploid of Lm and by the presence of four such chromosomes in a tetraploid nulliplex genotype.
  • the chromosome carrying sid has been putatively specified as number 6.
  • the gene was then tagged with restriction loci from the corresponding part of the linkage map. Gene expression during leaf development and senescence was compared between sid ⁇ and sid 77 Fp.
  • the forward and reverse primers for the PCR reaction was designed to hybridise to the sequences flanking the FcoRI site in ⁇ gtlO .
  • the following reaction mix was prepared: 1 X reaction buffer, 0.2mM dNTP-mix, 200 ng each of oligonucleotide primer 1 and 2, and 0.4 ⁇ l Tag polymerase (purified from Thermus aquaticus, 5000 units ml "1 ) .
  • the reaction mix was added to the DNA in a ratio of 46 ⁇ l:4 ⁇ l respectively in a 0.5 ml Eppendorf tube, and the contents were mixed gently so as not to introduce any air bubbles.
  • One drop of mineral oil was added to each sample to prevent evaporation from the samples during the reaction.
  • DNA was fractionated on a 1.5% agarose/TBE gel containing EtBr at a concentration of 0.02 ⁇ g ml "1 .
  • the gel was run at 60 V for 2.5 h.
  • DNA was transferred to Hybond N + membrane (Amersham International) by Southern blotting. After electrophoresis, the gel was incubated in 0.25 M Hcl for 15 min with gentle agitation, to denature the DNA into single strands. The gel was then rinsed in water and incubated in 0.4 M NaOH until the bromophenol blue dye turned from yellow back to blue. This was to neutralise the DNA while keeping it single stranded.
  • the bottom of a large glass dish was filled with 0.4 M NaOH, 4 sheets of Whatman 3MM paper were wetted in 0.4 M NaOH and laid over the reservoir on a glass plate with their edges in contact with the solution below. The gel was laid upside-down on top of the wicks.
  • the area to be blotted was defined with Nescofilm.
  • a membrane was cut slightly larger than the area of gel to be blotted, wetted in distilled water and then laid on top of the gel, with one edge of the membrane aligned with the base of the wells of the gel. This enabled of the size of fragments with hybridised with the radiolabelled probe to be found.
  • the DNA ladder was not blotted because it was found to hybridise strongly with the oligolabelled probe, obscuring the rest of the signal .
  • Four sheets of 3MM paper were then placed on top of the membrane, the first of which was wetted in distilled water before use followed by a nappy booster pad which had been cut in half and the two halves stacked on top of each other.
  • RNA and poly A+RNA were carried out using Northern blotting as follows :
  • a 1.5% agarose gel was prepared by melting agarose in water, using ll/15th of the final volume, cooling the mixture slightly, and then adding 1/lOth volume of 10 x MOPS buffer and l/6th volume of formaldehyde. The gel was poured and allowed to set in a fume hood.
  • the RNA samples were prepared as follows. Each sample contained 20 ⁇ g of RNA in a final volume of 10 ⁇ l, 10 ⁇ l of RNA sample buffer and 2 ⁇ l 0.5 mg ml "1 EtBr.
  • RNA ladder was treated in the same way. The gel was run at 65 V for 3 h or until the bromophenol blue dye front was about 2 cm from the bottom of the gel to ensure maximum fractionation of the total RNA extract .
  • Gel running buffer contained 1 x MOPS buffer and 8% (v/v) formaldehyde. The gel was viewed on a IJV transilluminator to check that the loading was equal in each track.
  • Hybond N membrane was cut slightly larger than the area of interest to be blotted to ensure that all the RNA would be completely transferred.
  • the RNA ladder was not blotted because it was found to hybridise strongly with an oligolabelled probe, obscuring the rest of the signal.
  • the Hybond N membrane was pre-soaked in 10 x SSC.
  • Four 15 x 26 cm sheets of 3MM filter paper were soaked in 10 x SSC and set up as wicks over a reservoir of 10 x SSC supported by a glass plate. Any bubbles were ironed out by rolling a glass pipette over the surface.
  • the formaldehyde gel was placed upside-down on the wicks.
  • RNA migrates along the bottom of the gel when electrophoresed so inverting the gel meant that there was less distance for the RNA to migrate onto the filter.
  • the area to be blotted was defined with Nescofilm and then the pre-soaked Hybond N membrane was placed on the gel with the top edge in line with the wells on the gel . This enabled the calculation of the size of any fragments which hybridised with radiolabelled probes.
  • One 15 x 10 cm sheet of 3MM filter paper presoaked in 10 x SSC was placed on top of the membrane. On top of this, four dry 15 x 10 cm 3MM filter paper sheets were placed followed by one nappy booster pad which had been cut in half and the two halves stacked on top of each other.
  • the Northern blot was dismantled and the membrane and gel were viewed on the UV transilluminator to check for complete transfer of RNA to the filter.
  • the membrane was rinsed in 2 x SSC for 15 min and baked between two pieces of 3MM filter paper at 80°C for 1 hour.
  • the prehybridisation/hybridisation buffer was prepared and prewarmed to 42°C. For one membrane the buffer contained 50% (v/v) formamide, 5 x SSPE, 0.5% (w/v) SDS and 5 x Denhardts .
  • the baked membrane was rolled up in mesh and then placed in a Hybaid bottle to which 28.5 ml of prehybridisation buffer and 1.5 ml of boiled 10 mg ml "1 herring sperm DNA were added.
  • the herring sperm DNA was used to block all sites on the membrane which might bind DNA non-specifically.
  • the membrane was left to prehybridise with rolling for at least 4 h at 42°C
  • the probe was labelled by the oligolabelling procedure using a Boehringer Random Priming Kit.
  • Purified insert DNA was labelled as follows: 25-50 ng of DNA in a final volume of 9 ⁇ l of reaction mix, 1 ⁇ l of reaction mix, 1 ⁇ l of 2 units/ ⁇ l, Klenow enzyme to make the second strand of DNA and 5 ⁇ lO Ci/ml [ ⁇ .- 32 P] dCTP.
  • the ingredients were mixed by pipetting up and down and left for 4 h at room temperature.
  • the radioactivity was monitored with a Geiger counter and the percentage incorporation of radio-labelled 32 P into the newly synthesise DNA strand was estimated.
  • Herring sperm DNA (10 mg ml "1 ) was added to the probe to give a final concentration in the hybridisation buffer of 0.5 mg ml" 1 and then boiled for 5 min to denature. Meanwhile the prehybridisation buffer was poured off the membrane and replaced with 9.5 ml of hybridisation buffer. The probe was added and the membrane was then left to hybridise overnight at 42°C.
  • the membrane was washed in 2 x SSC, 0.1% SDS at room temperature for 2 x 15 min. A further 2 x 15 min washes were carried out with 0.1 x SSC, 0.1% SDS.
  • the counts on the membrane had decreased to less than 5 counts per second, it was sealed in a plastic bag. More stringent washing at 42°C for 15 min and then 65° for 15 min with 0.1 x SSC, 0.1% SDS were tried if the counts were too high.
  • the membrane was set up for autoradiography at -80°C with intensifying screens for 1- 14 d, depending on the intensity of the signal.
  • Seeds of Festuca pratensis varieties R (wild-type) and B (stay-green mutant) were grown under an 8 hour photoperiod at 20°C.
  • the seeds were germinated in small plastic boxes on 3 sheets of tissue paper overlaid with one disc of Whatman filter paper (size 1) pre-soaked in deionised water. This was not allowed to dry out during the period of germination.
  • the seedlings were transferred to a fully hydroponic growth system and supported around the base of the stem by collars of expanded polyurethane foam.
  • Plants were harvested when 5 leaves had developed (about 50 days from the time of sowing) and transferred to the laboratory. They were dissected into four distinct parts; senescent leaf, mature leaf, emerging leaf (no ligule formed) and juvenile tissue (contained within the base of the emerging leaf) . Once dissected, the leaves were immediately immersed in liquid nitrogen. Leaves of each type were pooled together, wrapped in aluminium foil in portions of 5 grams or less, and stored at -80°C.
  • the frozen plant material was ground to a fine powder in a mortar which had been pre-cooled with liquid nitrogen. It was then decanted into centrifuge tubes containing 4ml g "1 fresh weight of buffer 1 (see below) and an equal volume of water-saturated phenol (AquaPhenol from Appligene) which had been pre-warmed to 65°C in a water bath.
  • Buffer 1 consisted of a sterile solution of 0.2M NaAc, lOmM EDTA pH 8.0 and 1% (w/v) SDS. Each tube was inverted several times to mix the contents and incubated at 65°C for 10 minutes.
  • the tubes were allowed to cool to room temperature and AnalaR chloroform was added in the ratio of 4ml g "1 fresh weight of plant tissue. The contents were mixed carefully by inverting the tubes several times and the tubes were centrifuged for 9,000g 10 min. The lower (organic) phase was discarded and a further 4 volumes of AnalaR chloroform were added to the aqeous phase and interface. After mixing and centrifuging as before, the upper (aqueous) layer was removed and transferred to another sterile centrifuge tube. Once again 4 volumes of AnalaR chloroform were added, mixed and centrifuged. The upper (aqueous) layer was removed and transferred to another sterile centrifuge tube.
  • RNA concentration was calculated on the basis of 50 ⁇ g ml "1 RNA giving an absorbance of 1 at 260nm. The purity was estimated from the ratio of the absorbance at 260nm compared with that at 280nm, a value of 2 being taken to be pure RNA.
  • a Pharmacia Quickprep mRNA Purification Kit was used to select poly A + RNA. The procedure was as follows (all buffers mentioned were in the kit) . Two oligo(dT)- cellulose spun columns were inverted several times to resuspend the matrix. The top and bottom closures were removed and each column was placed in a 15ml Corex tube to let the buffer drain through and equilibrate the column. Care was taken not to let the column dry out.
  • the columns were washed at 400 g for 2 min with 3 x 0.25 ml high salt buffer followed by 3 x 0.25 ml low salt buffer.
  • the poly A + RNA was eluted from the column into a sterile Eppendorf tube with 4 x 0.25 ml washes of elution buffer (pre-warmed to 65°C) .
  • cDNA was synthesised from 5 ⁇ g of poly A + RNA obtained in step A using a cDNA synthesis kit (Pharmacia LKB Biotechnology AB) .
  • the first strand of DNA was synthesised using reverse transcriptase with poly A + RNA as a template and oligo d(T) 12 . 1S as a primer.
  • E. coli ribonuclease H was used to nick the RNA strand of the RNA:cDNA duplex formed in the first step and E. coli DNA polymerase I utilised the nicks to replace the RNA with DNA by nick translation. Finally, Klenow enzyme was added to ensure that the ends of the cDNA were blunt .
  • the resulting double-stranded DNA (cDNA) was purified by phenol/chloroform extraction and spin dialysis, as described in the Pharmacia protocol. Purification of the cDNA by spin dialysis enabled the buffer to be changed easily to ligation buffer and allowed the cDNA to be recovered without significant dilution.
  • the cDNA from emerging leaves of B was divided in half, on half for probe labelling which was stored at -20°C and the other half for ligating with the vector, which was stored at 4'C. All of the cDNA from emerging leaves of R was stored at -20°C for probe labelling later.
  • a cDNA library was constructed from yy leaf tissue as follows :
  • Ec ⁇ RI/NotI adaptors were carried out as described in the Pharmacia protocol. Because the cDNA column effluent had been divided, 2.5 ⁇ l of EcoRl/Notl adaptors were used. For litigation to the lambda arms 'precipitation mixture 1' was followed as laid out in the protocol. The following test ligations were carried out as controls: (i) self-ligation of lambda arms and (ii) ligation using the insert provided with the lambda arms.
  • the NZY broth contained 5g NaCl, 2g MgS0 4 .7H 2 0, 5g yeast extract, lOg N-Z-Amine A (Casein enzymatic hydrolysate) per litre and was adjusted to pH 7.5 with NaOH.
  • NZY agar consisted of 15g agar per litre of NZY broth and was autoclaved before use.
  • Top agar contained 0.7g agarose per 100ml of NZY broth. Bacteria were grown from a single colony in TB broth at 37°C for approximately 4-6 h with shaking at 200rpm, centrifuged at 400g for 10 min and resuspended in half the volume of lOmM MgS0 4 .
  • TB broth contained 5g NaCl and lOg Bactotryptone per litre adjusted to pH 7.4 with NaOH and autoclaved. To this, sterile MgSO. and maltose was added to give a final concentration of lOmM and 0.2% respectively.
  • the prehybridisation/hybridisation buffer was prepared and prewarmed to 65°C.
  • the buffer contained: 50ml 20 x SSC, 10ml 50xDenhardts, 1ml 20% SDS and 139ml deionised water.
  • the baked membranes were rolled up in mesh and then placed in a Hybaid bottle (2 membranes per bottle) to which 60ml of prehybridisation buffer plus 300 ⁇ l of boiled lOmg ml "1 herring sperm DNA were added.
  • the membranes were left to prehybridise with rolling for at least 2 h at 65°C.
  • the probe was labelled by the 'oligolabelling' procedure using a Boehringer Random Priming Kit . Two probes were made from cDNA of R and B emerging tissue. For each probe 2 ⁇ l of cDNA was used.
  • the prehybridisation buffer was poured off the membrane and 30ml of hybridisation buffer was added.
  • the probe DNA was boiled for 5 min with 150 ⁇ l lOmg ml "1 herring sperm DNA and added to the hybridisation buffer.
  • the membrane was left to hybridise with rolling overnight at 65°C. The following day, the membranes were washed as outlined as before, except that a more stringent washing temperature of 65°C instead of 42°C was used.
  • the membrane was set up for autoradiography at -80"C for 1-7 d, depending on the intensity of the signal.
  • plaques which either hybridised strongly with B cDNA and not with R cDNA or plaques which hybridised strongly with B cDNA and more weakly with R cDNA were taken by removing the 'plug' of agar surrounding the plaque using a glass pasteur pipette. The isolated plaque was then placed in a screw top Eppendorf tube which contained 20 ⁇ l of chlorophorm (the chlorophorm was to prevent growth of contaminants) and 1ml SM buffer, mixed and left at room temperature for 1 h and then 4°C overnight . The isolated cDNA clone was checked for plaque purity by a second round of screening.
  • plaques were screened per 23.5 cm square petri dish. This density of plating was to ensure that the plaques were single isolates.
  • the petri dishes were pre-chilled for 30 min at 4°C and the DNA transferred onto Hybond N + membrane which had been pre-soaked in 2 x SSC and blotted between pieces of 3MM paper.
  • One piece of Hybond N + membrane was placed in contact with the plate for 1 minute. Reference marks were made on the filter and the plate to enable plaques to be selected at a later date. The membrane was removed from the plate and the DNA was denatured by leaving the membrane for 5 min
  • the prehybridisation/hybridisation buffer was prepared and prewarmed to 65°C. This contained 5 x SSC, 2.5 x Denhardts and 0.1% (w/v) SDS.
  • the baked membranes were rolled up in mesh and then placed in a Hybaid bottle (2 membranes per bottle) to which 60 ml of prehybridisation buffer containing herring sperm DNA at a concentration of 50 ⁇ g ml "1 had been added. This was to block all sites on the membrane which might bind DNA non-specifically.
  • the membranes were left to pre-hybridise with rolling for at least 2 h at ' 65°C.
  • the probe was labelled by the 'oligolabelling' procedure using a Boehringer Random Priming Kit.
  • the prehybridisation buffer was poured off the membrane and 30 ml of hybridisation buffer was added.
  • the probe DNA was boiled for 5 min to denature the DNA.
  • the probe was then added to the hybridisation buffer in the Hybaid bottle and was left to hybridise with rolling overnight at 65°C.
  • the radiolabelled DNA which was single stranded was then able to bind to any complementary sequences.
  • the membranes were washed as outlined in method 1.3 except that a more stringent washing temperature of 65°C instead of 42°C was used to ensure that all non-specifically bound DNA were removed.
  • the membrane was set up for autoradiography at 80°C for 1-7 d, depending on the intensity of the signal.
  • Plaques were selected from the agar plates. These were plaques which hybridised strongly with radiolabelled B cDNA clones which had been amplified using PCR. The plaques were taken by removing the 'plug' of agar surrounding the plaque using a glass pasteur pipette. The isolated plaque was then placed in a screw-top
  • Eppendorf tube which contained 1 ml SM buffer and 2% (v/v) chloroform (the chloroform was to prevent growth of contaminants) . This was left at room temperature for 1 h to allow phage to elute into the solution from the agar and then stored at 4°C.
  • Phage were plated with E. coli strain NM514 at a sufficient density for confluent lysis. This was done on NZY medium overnight. The following day 3 ml of SM buffer was added to each plate. The plates were left for 2 h at room temperature on a shaker in order to elute phage into the buffer. The phage lysate was then carefully pipetted from the surface of the agar and stored at 4°C.
  • the protocol used for the isolation of phage ⁇ DNA required the initial preparation of a DEAE-cellulose resin (DE52 Whatman, Clifton) . This was prepared as follows; 100 g of DE52 was placed in a beaker and several volumes of 0.05 N Hcl were added slowly, making sure that the pH did not go below 4.5. Then, with constant gentle stirring, concentrated NaOH was added slowly until the pH was 6.8. The resin was allowed to settle and once the supernatant had been decanted it was washed several times with L medium. The washing was done by gently re- suspending the resin in two volumes of L medium, allowing it to settle and decanting the excess L medium.
  • DEAE-cellulose resin DE52 Whatman, Clifton
  • the DE52 was re-suspended to make a final slurry of approximately 75% resin and 25% L medium. Then sodium azide was added to give a final concentration of 0.1% to prevent growth of microorganisms. The mixture was stored at 4°C and gently re-suspended prior to use.
  • the ⁇ DNA was extracted as follows: 0.6 ml DE52 reagent was added to an Eppendorf tube with 0.6 ml of phage lysate (the phage lysate was prepared as outlined in method 5) . This was mixed gently by inverting the tube 20-30 times. The reagent was pelleted by centrifugation at 12 OOOg for 5 min in an Eppendorf microfuge. The supernatant was removed carefully and transferred to a new microfuge tube. This was then spun to remove any remaining DE52 reagent. The DE52 reagent binds and removes the contaminating cellular DNA and RNA. Next, 800 ⁇ l of supernatant was transferred to a new microfuge tube.
  • the ⁇ DNA extracted above was digested with FcoRI for 2 h in a water bath at 37°C.
  • 2 ⁇ l 10 x EcoRI buffer Boehringer kit
  • 2 ⁇ l 10 mg ml "1 Rnase and 1 ⁇ l lOU/ ⁇ l FcoRI (boeringer kit) were added.
  • 1/4 was retained and stored at -20 * C for subcloning into the plasmid pUBSl.
  • the remaining 3/4 of the EcoRI digest was fractionated on a 1.5% agarose/TBE gel containing EtBr at a concentration of 0.02 ⁇ g ml-1.
  • the 5 ⁇ l of cDNA which had been retained after the digestion of the ⁇ DNA with EcoRI was used to ligate to the plas id pUBSl.
  • the following components were added to the cDNA: 0.85 ⁇ l EcoRI cut and phosphatased pUBSl (this had been phosphatased after digestion with EcoRI to prevent the plasmid arms reannealing) , 1 x ligase buffer and 1 ⁇ l lU/ ⁇ l T4 DNA ligase (Pharmacia) .
  • the mixture was incubated at 12°C overnight and was then stored at - 20°C until required for transformation into E. coli strain MC1022 .
  • Transformation reactions were set up as follows: to 200 ⁇ l of competant cells on ice, 5 ⁇ l of the ligation was added. This was left on ice for 30 minutes. The cells were then heat shocked at 37°C for 2 minutes to induce the uptake of the plasmid by the E. coli cells. To this, 800 ⁇ l of L Broth were added and the cells were left at room temperature for 30 minutes to allow them to express the ampicillin resistance from the plasmid. The cells were then plated onto LB agar which contained 75 ⁇ M IPTG, 0.004% XGAL and 50 ⁇ g ml-1 Ampicillin. The plates were incubated overnight at 37'C.
  • MC1022 cells are not ampicillin resistant (resistance is encoded by the plasmid) and the transformation efficiency of circular DNA is much higher than that of linear DNA.
  • Transformed MC1022 cells were selected. This was done by selecting single isolated while colonies using a sterile cocktail stick. The colony was touched with the cocktail stick and streaked out onto LB agar containing IPTG, XGAL and ampicillin as before. The cocktail stick was placed into a Sterilin tube (Sterilin Ltd.) containing 10 ml of L Broth with ampicillin at a concentration of 50 ⁇ g ml-1. The culture was incubated with shaking at 37 * C overnight and used for the isolation of plasmid DNA.
  • the overnight cultures were centrifuged at 400g (Centaur 2 MSE) for 10 min to pellet the cells.
  • the supernatant was removed and 50 ⁇ l of 25% sucrose/50 mM Tris pH 8.0 were added to resuspend the pellet.
  • This solution was then transferred to an Eppendorf tube and to this 300 ⁇ l of M-STET was added.
  • Lysozyme which had been freshly prepared at a concentration of 50 mg ml-1 in 50 mM Tris pH 8.0 was then added to the plasmid DNA to give a final concentration of 0.8 mg ml-1. This was immediately boiled for 1 min and transferred to ice. Lysozyme and Trition digest the bacterial cell wall and membrane.
  • the tubes were then centrifuged at 12 OOOg for 30 minutes at 4°C.
  • the resulting gelatinous material containing the chromosomal DNA was removed with a sterile toothpick. Two phenol/chloroform extractions were made to remove proteins and lipids and the aqueous (upper) phase was retained.
  • the DNA was then precipitated with l/10th the volume of sodium acetate and an equal volume of cold isopropanol (-20 * C) at -80 'C for 20 min.
  • the resulting precipitate was pelleted by centrifugation at 12 OOOg for 10 min at 4"C. This was washed in 70% AnalaR ethanol to remove any remaining salt, dried, resuspended in 50 ⁇ l TE pH 8.0 and stored at -20°C.
  • the library was screened for mutant- specific clones with cDNA from YY and yy tissue. Eight cDNA clones associated with leaf tissue of the mutant were selected. Southern analysis indicated that they were all versions of the same sequence. Further rounds of differential screening picked out even more versions, including one clone which is full-length, or nearly so, as indicated by insert size compared with message size on a northern blot. The size, 1.8kb, is consistent with a 60 x 10 3 Mr polypeptide, and the expression pattern revealed by northern analysis also confirmed this .
  • the cDNA cloned from sid y ⁇ Fp is referred to hereafter as gem (green-enhanced message) .
  • the abundance, in leaves at different stages of development, of mRNA hybridising to gem was revealed in the northern blot of Figure 3.
  • RNA from juvenile (J) , emerging (E) , mature (M) and senescing (S) leaf tissue of Fp genotypes sid 77 and sid” and the hybrid between them was extracted as described in Example 1 and subjected to northern analysis as described above.
  • the blot was probed with a wheat ca cDNA and Fp gem cDNA. Expression was found to be extremely differential between yy and YY and shows a strong upward trend with aging. After prolonged overexposure of the autoradiogram a very weak signal could be discerned in tracks of RNA from YY leaves at or soon after senescence initiation.
  • a solution of 60 ml 1 x TBE acrylamide mix was prepared from 54 ml 6% (w/v) acrylamide stock and 6 ml 10 x TBE which had been filtered.
  • the bottom of the gel was sealed by taking 10 ml of this mix, adding 25 ⁇ l 25% (w/v) AMPS and 100 ⁇ l TEMED (Both from Life Technologies Ltd., Uxbridge, U.K.) And pipetting the solution along the strip of 3MM paper at the bottom of the gel plates .
  • the acrylamide solution soaked the paper and seeped between the plates in the corners to seal the bottom of the gel.
  • 100 ⁇ l each of 25% AMPS and TEMED were added to the remaining 50 ml of mix.
  • the plates were held at a 30° angle to the horizontal and the solution was pipetted slowly between them from one corner using a 25ml pipette.
  • the clingflim and tissues were removed from the plates, which were then wiped to clean off all traces of acrylamide.
  • the area surrounding the comb was rinsed with water and the comb was withdrawn slowly. After washing out the slot with more water, the comb was replaced, teeth innermost, so that the teeth just touched the gel itself, reacting the wells.
  • the gel plate assembly was supported upright in the apparatus and was then ready to be loaded with samples.
  • T7 and T3 primers were synthesised at the Department of Biochemistry, UW Aberystwyth, using a 'Gene Assembler Plus' oligosynthesiser (Pharmacia Ltd., Milton Keynes, U.K.) .
  • Sequencing reactions A 20 ⁇ l sample from each plasmid miniprep to be sequences was treated first with 2 ⁇ l 10 mg ml "1 RNase at 37°C for 15 min, to digest away any contaminating RNA, and then with 5 ⁇ l 1 M NaOH, 1 mM EDTA at 37°C for 15 min to denature the DNA into single strands. The alkali was removed by spin dialysis through Sepharose CL-6B, equilibrated in 10 mM Tris pH 8.0, 0.1 mM EDTA.
  • Reaction mix for four different DNA/primer mixtures was prepared by combining 22.4 ⁇ l 10 mM Tirs pH 8.0, 0.1 mM EDTA, 4.4 ⁇ l 0.1 M DTT, 1.8 ⁇ l label mix (7.5 ⁇ M each of dCTP, dGTP and dTTP) and 2 ⁇ l 1.85 Mbq [o-- 35 S] dATP with 4 ⁇ l enzyme dilution buffer (10 mM Tris pH 7.5, 5 mM DTT, 0.5 mg ml "1 BSA) and 1.2 ⁇ l Sequenase (USB, Cambridge BioScience, U.K.) In an Eppendorf tube on ice.
  • the composition of the termination mixes was as follows . Each mix contained 908 ⁇ l 10 mM Tris pH 8.0, 0.1 mM EDTA, lO ⁇ l 5 M NaCl and 20 ⁇ l each of 5 mM solutions of dATP, dCTP, dGTP and dTTP. In addition, A-mix contained 2 ⁇ l of 5 mM dATP, C-mix contained 2 ⁇ l of 5 mM ddCTP, etc. The reactions were stopped by the addition to each well of 4 ⁇ l of formamide dye solution (98% (v/v) BRL Ultrapure formamide, 10 mM EDTA, 2 mg ml "1 xylene cyanol, 2 mg ml "1 bromophenol blue) . Loading a sequence gel
  • the samples were denatured by incubation in a 80°C water bath for 2 min and then stored on ice until they were loaded on the sequencing gel (prepared as described in section 11) .
  • the wells of the gel were rinsed with 1 x TBE to remove the urea which leached from the gel . This was done so as not to distort the migration of the samples into the gel .
  • An aliquot of 1.5 ⁇ l of each sample was loaded using a spade- ended Multi-flex pipette tip (Anachem, Luton, U.K.) On a Gilson Pipetman. Initially the tip was positioned near the bottom of the slot, and then as the sample was being dispensed, the tip was gradually withdrawn.
  • Electrophoresis and fixing of a sequencing gel Once loading had been completed, the gel was electrophoresed at 50 W for 1.5 h (short run) or 2.5h (long run) . These times corresponded to the times taken from the bromophenol blue (short run) or xylene cyanol (long run) dye fronts to run off the bottom of the gel.
  • the buffer was poured from the back panel of the gel apparatus and the gel plates were gently parted such that the gel remained on the front plate.
  • the gel complete with the plate was placed for 25 min in 21 10% (v/v) methanol, 10% (v/v) acetic acid to fix, during which time the urea dialysed out of the gel . It is important to fix 35 S sequencing gels otherwise the ure. quenches the radioactive signal.
  • the plate was removed from the fix and a 40 x 17 cm shee: of Whatman 3MM paper was lowered onto the gel from the middle outwards . Excess gel was trimmed off with a raze: bland and the 3MM paper was peeled off the plate with thf gel attached to it. This was done with extreme care.
  • the gel was covered with clingfilm and then dried down onto the paper using a slab gel drier (model 1125B, Bio- Rad Laboratories Ltd., Watford, U.K.) At 80°C for 1 h. Finally the clingfilm was removed from the dried gel and the radioactive signal was detected by autoradiography at room temperature for 1-4 d, depending on the intensity of the signal.
  • the DNA sequence was analysed on a DEC Micrc Vax 3600 computer.
  • l/4th volume 4M NaCl was added.
  • an equal volume of 13% (w/v) PEG 8000 was mixed with the samples which were then incubated on ice for 20 minutes.
  • Plasmid DNA was pelleted by centrifugation in a microcentrifuge at maximum speed and 4°C for 15 minutes. The pellets were washed in 70% (v/v) ethanol, allowed to air-dry and then resuspended in 20 ⁇ l water.
  • the primers used in the sequencing reactions were T3 and T7 primers (Promega, Southampton, UK) which anneal to the T3 and T7 promoters adjacent to the polylinker region of the plasmid; internal primers were synthesised by Oswel DNA Service (Edinburgh, UK) .
  • PCR was carried out in a total volume of 20 ⁇ l containing 9.5 ⁇ l terminator premix, l ⁇ g dsDNA template and 4 pmol primer. Each sample was overlaid with 2 drops of mineral oil. The tubes were then placed in thermal cycler preheated to 96°C and 25 cycles were run. In each cycle, DNA was denatured at 96°C for 30 seconds, primers were annealed at 50°C for 15 seconds and the second DNA strand was polymerised at 60°C for 4 minutes. After the last cycle, the samples were stored at 4°C until purification of the PCR products.
  • Polyacrylamide gel electrophoresis was carried out using an automated sequencer (model 373A, Applied Biosystems, Warrington, UK) .
  • the gel kit was cleaned and assembled according to the manufacturer's instructions.
  • Gels contained lx TBE 6% (w/v) acrylamide (19:1 acrylamide:bis-acrylamide) and 8.3M urea and polymerisation was started by adding 400 ⁇ l 9% (w/v) APS and 45 ⁇ l TEMED. After prerunning the gel for up to one hour samples were loaded and electrophoresis was carried out at 28W for 12 hours using lx TBE as running buffer.

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Abstract

L'invention concerne la mutation dite 'rester verte' d'un gène contrôlant la sénescence des feuilles dans une espèce de plantes. L'invention traite également de l'utilisation des séquences de nucléotides comprenant la mutation de ce gène, ou dérivées de la mutation de ce gène, dans la technologie de l'ADN de recombinaison. L'invention a pour objet de permettre la production de variants de plantes nouveaux et améliorés, pouvant être utilisés dans des souches d'identification produites dans la culture de plantes traditionnelles dans lesquelles se produit la mutation.
PCT/GB1996/001223 1995-05-22 1996-05-22 Gene controlant la senescence chez les plantes WO1996037613A1 (fr)

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GBGB9510321.4A GB9510321D0 (en) 1995-05-22 1995-05-22 Novel plant derived material
GB9510321.4 1995-05-22

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001061023A1 (fr) * 2000-02-16 2001-08-23 Plant Research International B.V. Reduction de la degradation in planta de produits d'une plante recombinante

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007993A1 (fr) * 1993-09-13 1995-03-23 Zeneca Limited Regulation de la senescence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007993A1 (fr) * 1993-09-13 1995-03-23 Zeneca Limited Regulation de la senescence

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOHN, I., ET AL.: "Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis", THE PLANT JOURNAL, vol. 7, no. 3, March 1995 (1995-03-01), pages 483 - 490, XP002014397 *
THOMAS H ET AL: "Crops that stay green.", ANNALS OF APPLIED BIOLOGY 123 (1). 1993. 193-219., XP000602791 *
THOMAS H ET AL: "LEAF SENESCENCE IN A NON-YELLOWING MUTANT OF FESTUCA-PRATENSIS TRANSCRIPTS AND TRANSLATION PRODUCTS.", J PLANT PHYSIOL 139 (4). 1992. 403-412., XP000603534 *
THOMAS, H.M., ET AL.: "Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum x Festuca pratensis by genome in situ hybridization", THEORETICAL AND APPLIED GENETICS, vol. 88, 1994, pages 909 - 913, XP000602640 *
VINCENTINI F ET AL: "Chlorophyll breakdown in senescent leaves: Identification of the biochemical lesion in a stay - green genotype of Festuca pratensis Huds.", NEW PHYTOLOGIST 129 (2). 1995. 247-252., XP000602676 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001061023A1 (fr) * 2000-02-16 2001-08-23 Plant Research International B.V. Reduction de la degradation in planta de produits d'une plante recombinante
EP1130104A1 (fr) * 2000-02-16 2001-09-05 Stichting Dienst Landbouwkundig Onderzoek Réduction de la dégradation des produits végétales in planta

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AU5773796A (en) 1996-12-11

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