EP0620855A1 - Regulation des genes des plantes - Google Patents

Regulation des genes des plantes

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Publication number
EP0620855A1
EP0620855A1 EP93901841A EP93901841A EP0620855A1 EP 0620855 A1 EP0620855 A1 EP 0620855A1 EP 93901841 A EP93901841 A EP 93901841A EP 93901841 A EP93901841 A EP 93901841A EP 0620855 A1 EP0620855 A1 EP 0620855A1
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Prior art keywords
protein
plant
del
seq
amino acid
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German (de)
English (en)
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Enrico Coen
Rosemary Carpenter
Justin Goodrich
Roger Freedman
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JOHN INNES FOUNDATION
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JOHN INNES FOUNDATION
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—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 pigment biosynthesis

Definitions

  • This invention relates to the regulation of plant genes, more, particularly the regulation of genes which control the pigmentation of plants.
  • the genes of one class encode enzymes required for pigment biosynthesis and many of these genes appear to be common to different species (see Martin et al, The Plant Journal , 1 , 37-49 (1991), Sommer and Saedler, Mol . Gen . Genet . , 202, 429-434 (1986), Coen et al, Cell , 47 , 285-296 (1986) and Beld et al, Plant Mol . Biol . , 13, 491-502 (1989)).
  • the other class comprises regulators of the biosynthetic genes (see Almeida et al, Genes Dev .
  • This class includes the CI and R genes in maize, which encode products related to the myb and myc families of transcription factors respectively (see Paz-Ares et al, EMBO J . , 6 , 3553- 3558 (1987) and Ludwig et al, Proc . Natl . Acad . Sci . U . S .A . , 86, 7092-7096 (1989)).
  • the present invention is based on the isolation and characterisation of a gene designated delila. that regulates pigmentation pattern in Antirrhinum ma jus and the use of this gene to regulate the expression of one or more anthocyanin pigment genes in a plant.
  • Wild-type A. majus flowers have five red petals united to form a corolla tube with five distinct lobes.
  • the epidermal cells of the petals contain red anthocyanin pigments.
  • a recessive delila (del) mutation is known which confers a strikingly different pattern of floral pigmentation in which the corolla tubes are ivory and the lobes fully pigmented.
  • the del mutation also blocks pigmentation of the anther filaments and lower stems and reduces that of the styles, sepals, carpels and petioles (leaf stalks).
  • the wild-type del product is required in the corolla tube for normal transcript levels of many of the anthocyanin biosynthetic genes (see Almeida et al, i-bid and Martin et al ibid) .
  • pigmentation of the corolla lobes is normally unaffected by del , in certain genetic backgrounds an effect of del in the lobes is revealed suggesting that del can also act in lobes.
  • DEL deduced amino acid sequence of 644 amino acids.
  • SEQ ID NO 1 The cDNA sequence of the cloned del locus and the deduced amino acid sequence of the DEL protein are shown in SEQ ID NO 1 and the deduced amino acid sequence of the DEL protein is shown in SEQ ID NO 2.
  • the present invention provides a method for regulating the expression of one or more anthocyanin pigment genes in a plant which comprises the steps of transforming plant tissue with an expression vector comprising a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis, the said DNA segment being under the control of a promoter upstream of and operably linked thereto and regenerating from the transformed tissue plants showing altered anthocyanin pigmentation.
  • the DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 is a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2.
  • the present invention provides a plant having a DNA segment as defined above incorporated into its genome or plant propagation material (such as seeds) of such a plant.
  • the present invention provides a DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis.
  • the present invention provides the use of the DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO' s 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 or the protein encoded thereby to isolate a DNA molecule encoding a protein having the amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 from other plant species.
  • the present invention provides an expression vector comprising a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least
  • DNA segment being under the control of a promoter upstream of and operably linked thereto.
  • the invention also provides a protein which is the product of expression of the expression vector as defined above in a host cell.
  • the present invention provides a construct which comprises a transposon having cloned therein a DNA segment as defined above, the said DNA segment being under the control of a minimal promoter upstream of and operably linked thereto.
  • the present invention provides a method of trapping a promoter/enhancer which comprises the steps of introducing the construct into plant by transformation and propagating from said plant, plants having a phenotype showing altered anthocyanin pigmentation arising as a consequence of transposition of the construct.
  • the present invention provides a method for isolating a trapped promoter/enhancer from a plant which has been transformed with the construct as defined above which comprises reisolating the construct from said plant together with sequences adjacent thereto.
  • the present invention provides a method of expressing a gene of interest in a plant, which comprises transforming a cell of said plant with a first construct having said gene of interest under the control of a first promoter, which first promoter is that of an anthocyanin gene, upstream of and operably linked thereto, the said plant having incorporated into its genome a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 under the control of a second promoter upstream of and operably linked thereto, or the said plant being co-transformed with a second construct which comprises said DNA segment under the control of a third promoter, which third promoter may be the same or different to the second promoter, upstream of and operably linked thereto, or the said first construct optionally including the said DNA segment under the control of said second or third promoter upstream and operably linked thereto, or the said first construct optional
  • the present invention provides a method of expressing a gene of interest in a plant which comprises transforming said plant with a construct, which construct comprises a transposon having cloned therein a DNA segment as defined above, the said DNA segment being under the control of a minimal promoter upstream of and operably linked thereto, deriving from the transformed plant further plants having a phenotype showing altered anthocyanin pigmentation, reisolating from said plant the said construct together with sequences adjacent thereto, replacing said DNA segment in said construct with a gene of interest to form a new construct and transforming said plant with said new construct.
  • the cDNA encoding the DEL protein as shown in SEQ ID NO 1 or 2 contains a long open reading frame (ORF) starting at position +25.
  • the ORF encodes a potential protein, DEL, of 644 amino acids which shows strong homology to the products of Lc and R-S, two members of the R gene family which controls pigmentation pattern in maize.
  • Maize and Antirrhinum are taxonomically distant and belong to the monocotyledoneae and the dicotyledoneae respectively, two groups thought to have diverged about 200 million years ago at an early stage in the evolution of flowering plants. There are marked differences in morphology and pigmentation pattern between the two species.
  • the flowers of Antirrhinum are pollinated by bees and have large, vividly pigmented petals. In maize, which is wind-pollinated, the flowers are inconspicuous and there is no organ with obvious homology to petals. The organ most commonly pigmented is the seed, although the diverse alleles of the R gene family can pigment most plant tissues.
  • the plant preferably belongs to the dicotyledoneae.
  • DNA molecules encoding the DEL protein can now be obtained as required using standard techniques of cDNA cloning and/or DNA synthesis.
  • DNA molecules encoding a protein having an amino acid sequence which shows homology with the DEL protein as shown in SEQ ID NO 1 or 2 can be obtained by mutation of a DNA molecule having the sequence shown in SEQ ID NO 1 using standard techniques of recombinant DNA technology and/or by DNA synthesis.
  • DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO' s 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 or protein encoded thereby may also be used to isolate DNA molecules encoding a protein having an amino acid sequence which shows homology with the DEL protein as shown in SEQ ID NO 1 or 2 from other plant species, most preferably plant species belonging to the dicotyledoneae.
  • the DNA segment encoding the DEL protein or a protein homologous thereto will generally be incorporated in an expression vector which also includes suitable regulatory and control sequences to enable expression of the segment in a particular plant or part of a plant.
  • suitable promoters include the cauliflower mosaic virus 35S promoter and also any promoter which is expressed in epidermal cells of different plant organs, such as the promoter of a housekeeping gene or a gene for the synthesis of specific epidermal structures.
  • Plants transformed with the DNA segment encoding the DEL protein or a protein homologous thereto may be produced by standard techniques which are already known for the genetic manipulation of plants.
  • the DNA segment may be incorporated into an Agrobacterium vector and plant material may then be infected by a strain of Agrobacterium carrying this vector.
  • the DNA encoding the DEL protein or a protein homologous thereto becomes integrated into the genome of the plant tissue so that plants propagated from the tissue also carry this DNA.
  • Alternative methods for the introduction to the DNA into plant cells include precipitation onto tungsten particles and shooting using a particle gun.
  • plant pigmentation can be increased or altered by transforming plants in the manner described above with a construct including the DNA encoding the DEL protein or a protein homologous thereto under control of a suitable promoter.
  • a construct in which a regulatory sequence, such as the promoter is specific to a particular part of the plant, for example specific parts of the flower, the effect on pigmentation can be confined to that part of the plant.
  • the procedure described above can be used to enhance pigmentation of regions already pigmented in the host species or to pigment areas which are not normally pigmented in the host species.
  • One specific application of this procedure is to produce novel genetically manipulated flowering plants with a phenotype in which the flowers show a pattern or intensity of pigmentation which differs from the host species.
  • the DNA segment encoding the DEL protein or a protein homologous thereto as a promoter/enhancer trap wherein the DNA segment which is driven by a "minimal" promoter, i.e. a truncated promoter more or less deficient in cis-acting regulatory sequences, may be cloned within a transposon (which can only transpose when a second gene containing the trans-activator is also present) and the construct is then introduced into plants.
  • the transposon containing the DNA segment can then transpose to a new site near to a variety of promoters/ enhancers which can increase or activate transcription of the DNA segment.
  • transposon can subsequently be "stabilised” by crossing out the factor that activates the transposon.
  • the chimeric sequence combining the DNA segment with the trapped promoter/enhancer can be recovered by reisolating back the construct, together with adjacent sequences, from the plant and used to control the expression of any gene of interest.
  • trapped promoter/enhancer may be used to control expression of a gene of interest in two ways, namely:
  • a new construct is prepared which comprises a gene of interest under the control of an anthocyanin gene promoter upstream and operably linked thereto.
  • this new construct is transformed into a plant, wherein a promoter/enhancer has been trapped, expression is seen of the gene of interest in those cells which express delila .
  • the trapped promoter/enhancer causes expression of the DEL protein which in turn switches on the anthocyanin promoter thus causing expression of the gene of interest.
  • the DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 is excised from the construct and a heterologous gene of interest inserted in its place to form a new construct.
  • this new construct is transformed into a plant the heterologous gene of interest is expressed.
  • Antisense RNA is where a gene is expressed in the opposite sense to normal (i.e. the promoter is at the 3' end of the gene), such that the "wrong" strand of the DNA is transcribed into RNA (giving antisense RNA). This antisense RNA may form a duplex with normal sense RNA and so inactivate it.
  • Co-suppression occurs where extra copies of a gene are introduced into the genome which may result in inactivation of the endogenous gene, which may in turn cause a mutant phenotype.
  • a Ribozyme is an RNA molecule which has the property that when it hybridizes to another RNA molecule containing a particular nucleotide sequence (target sequence), it will cleave the molecule and hence inactivate it.
  • target sequence a particular nucleotide sequence
  • the nature of the target sequence depends on the critical region in the ribozyme molecule which is complementary to the target.
  • the particular RNA molecule that the ribozyme recognises can therefore be altered by changing the critical region of the ribozyme.
  • constructs can be developed with alterations in the del coding sequence which produce proteins which interfere with the functioning of delila or delila-like genes in the host species.
  • the del coding sequence or a homologue thereof can also be used as a convenient visible marker.
  • use of a DNA sequence encoding the DEL protein or a protein homologous thereto in the manner described above allows the coding sequence in question to act as a visible marker for gene expression. This can be exploited to enable easy identification of transformed cells, cells in which a particular promoter is active, cells in which gene functions have been activated or inhibited, e.g. by excision or integration of a transposon.
  • a DNA segment encoding the DEL protein or a protein homologous thereto in the manner described above may be used as a visible marker in a transgenic plant line, most particularly including dicotyledonous species, wherein a specific pigmentation pattern may be used to identify the line.
  • the del coding sequence or a homologue thereof can also be used to trans-activate or inhibit genes by placing the gene of interest under the control of a promoter, e.g. the pallida promoter of Antirrhinum , already known to be regulated by the delila gene. Plants containing such constructs, together with the del coding sequence (or an appropriate homologue thereof), would express the gene of interest only in those cells which express delila . This could be combined with the use of the del coding sequence as a visible marker so that cells expressing the gene of interest could be identified by their pigmentation phenotype.
  • a promoter e.g. the pallida promoter of Antirrhinum
  • a further possible use for the del coding sequence is in the isolation of homologous of the delila gene from various plant species.
  • Such homologous of the delila gene can be isolated using genomic or cDNA probes derived from delila clones or based on the del coding sequence as set out in SEQ ID NO 1.
  • FIGURE 1 nucleotide and predicted amino acid sequence of del cDNA.
  • FIGURE 2 Southern blots of -EcoRl - digested genomic DNA from various Antirrhinum majus plants.
  • FIGURE 3 Sequence comparisons of Del + and del-602 alleles in the region of the Tam2 insertion.
  • FIGURE 4 Northern analysis of del expression in various Antirrhinum majus flowers at different stages of development.
  • FIGURE 5 Amino acid sequence comparison of DEL protein with selected HLH proteins.
  • FIGURE 6 In situ hybridisation of medial longitudinal sections of corollas with 35 S labelled RNA probes.
  • FIGURE 7 Plasmids pBJIMM15, pBJIMM21 and pBJIMM24. 1. Isolation of the del gene
  • genomic DNA from dei-602 mutant and revertant plants was digested with restriction enzymes and probed with the various transposable elements isolated from A . majus . Because each of these elements was present in multiple copies in the genome, several bands were seen in Southern blots.
  • EcoRI digested DNA was probed with a fragment of the transposon Tam2 (Upadhaya et al., Mol . Gen . Genet . , 199, 201-207 (1985), a 5.6 kb band was consistently observed in mutants and not in revertants ( Figure 2a). This suggested that the dei-602 mutation resulted from a Tam2 insertion, and the 5.6 kb fragment was therefore cloned.
  • the resulting clone, pJAM 602 contained a 4.9 kb fragment of Tam2 with 0.7 kb of flanking DNA ( Figure 2b).
  • the flanking sequence probe A was then used to probe .EcoRI digested DNA from various genotypes: del-602 plants showed the expected 5.6 kb band; plants of the progenitor stock showed a wild-type band of 6.2 kb; and plants homozygous for a stable del allele, del-8, gave a 2.4 kb band ( Figure 2b).
  • probe A derived from the del locus reversion of the del-602 allele to wild-type should have correlated with restoration of the wild-type 6.2 kb band, and this was observed; six revertant plants obtained in the progeny of three crosses between del-602 and del-8 plants, and therefore representing at least three independent germinal reversions of the del-602 allele, all showed the 6.2 kb band, confirming that the pJAM 602 clone contained part of the del locus.
  • a clone of the Del + genomic region was obtained by screening a genomic library from the progenitor stock with probe A of pJAM 602 ( Figure 2b).
  • a comparison of the sequences flanking the Tam2 insertion in the del-602 allele with the corresponding wild-type sequences identified a direct duplication of 3 base pairs of target DNA, a length characteristic of Tam2 insertions (Upadhaya et al, ibid) ( Figure 3).
  • Figure 2(a) shows Southern blot of EcoRI-digested genomic DNA from del-602 mutant and revertant (Del + ) plants, probed with a 4.4 kb EcoRI/Hindlll fragment of the Tam2 clone pRH2, provided by Enno Krebbers and Hans Sommer. These plants were obtained in the F 2 progeny of a cross between del-602 and del-8 plants. Revertants have the presumed genotype Del + /del-8 , and mutants del-602/del-8. A restriction map of Tam2 and the origin of the probe are shown below the autoradiograph. Sites indicated are EcoRI (E), Hindlll (H) and Bgrlll (B).
  • Figure 2(b) shows Southern blot of EcoRI-digested genomic DNAs probed with fragment A of the 5.6 kb EcoRI clone, pJAM 602.
  • Lane 1 wild-type progenitor of del-602; lane 2, homozygous del-602; lane 3, homozygous del-8; lanes 4-9, revertant progeny from crosses between del-602 and del-8 plants; lanes 10-16 del mutant progeny from the same crosses.
  • a restriction map of the 5.6 kb EcoRI clone, pJAM 602, and the origin of the probe are shown below. Thick line, Tam2 sequences; thin line, flanking sequences.
  • RNA extraction and Southern blot analysis were performed as described by Coen et al., Cell , 47 , 285-296 (1986).
  • the pJAM 602 clone was obtained by digesting genomic DNA of del-602 plants with EcoRI, gel-purifying fragments in the 5-6 kb size range, ligating to ANM1149 arms and screening a library of 30,000 plaques with the Tam2 probe shown in Figure 2a.
  • the resulting clone was subcloned into Bluescript SK + (Stratagene).
  • Figure 3 shows a sequence comparison of Del + and del-
  • the target sequence, duplicated on insertion of Tam2, is boxed.
  • the pJAM 602 clone was sequenced to provide flanking sequences to the right of the Tam2 insertion.
  • 0.1 ⁇ g of genomic DNA from a del-602 plant was amplified by PCR (Saiki et al, Science , 239, 487-491 (1988), using a primer derived from sequences near the left terminus of Tarn 2 (as orientated in Figure 2b) and a second primer based on Del + genomic sequences.
  • the expected fragment of 0.3 kb was subcloned to give pJAM 122 and sequenced.
  • polyA + RNA extracted from corolla tubes was hybridised with probe A of pJAM 602.
  • ID NO 1 contained a long open reading frame (ORF) starting at position +25 with an ATG codon flanked by sequences which conformed to the consensus for initiation of translation in plants (Lüttke et al., EMBO J ., 6, 43-48 (1987).
  • the ORF encoded a potential protein, DEL, of 644 amino acids.
  • Comparison of the amino acid sequence of DEL to proteins on the PIR and SWISS databases using the FASTA program revealed a strong homology between DEL and the products of Lc (Ludwig et al., ibid) and R-S (Perrot and Cone, Nucl . Acids Res .
  • DEL also contained a highly acidic region (residues 173-319); 27 acidic and two basic residues gave an overall negative charge of -25.
  • a corresponding acidic region occurs in Lc (Ludwig et al., ibid) , but relatively little conservation in amino acid sequence was found between these regions of Lc and DEL (25% identity).
  • the conserved region near the N-terminus of DEL showed no significant homology to proteins other than those of the R gene family.
  • a del cDNA clone was used to screen a wild-type genomic library and three clones with extensive homology to del were isolated. Detailed restriction mapping indicated that they derived from independent loci, distinct from del . Therefore, A majus has a family of at least four genes related to del .
  • Pigmentation of the corolla first appeared in the lobes and in a ring at the base of the tubes, and subsequently extended throughout the tubes.
  • the strongest expression of del was seen at the base of the tubes, the region of pigmentation most greatly affected by the del mutation. Expression was also detected in the lobes, as was predicted from genetic interactions described previously (Almeida et al., ibid) . As expected, no wild- type del transcript was found in the pigmented lobes of del mutant flowers (results not shown), confirming that del expression is not required for pigment biosynthesis in the lobes.
  • del was further localised by in situ hybridisation of 35 S-labelled del RNA to sections of wild- type corollas. Signal was detected only when the antisense strand of del was used as a probe, and was strongest in the flower buds 1-6 nodes above the first fully opened flower on the inflorescence. The signal was specific to the epidermal cell layers in both tubes and lobes ( Figure 6). This corresponds to the distribution of anthocyanins, and of expression of the biosynthetic genes nivea , pallida and incolorata (Jackson, Current Biology, 1 , 99 (1991)).
  • the two epidermal layers of the petal are referred to as inner (lining the throat of the tube and contiguous surface of the lobes) and outer (lining the exterior surface of the tube and lobes).
  • signal was as intense in the outer epidermis as in the inner epidermis ( Figure 6a).
  • the outer epidermis of the tubes and lobes bore numerous multi-cellular hairs, which were unpigmented; no signal was seen in these hairs (data not shown).
  • a strong signal was also seen in both the outer and inner epidermi.
  • the signal was most abundant in the inner epidermis (Figure 6b), which also had strongest pigmentation.
  • Figure 4 shows Northern analysis of del expression.
  • the Northern blot was first probed with the del cDNA clone pJAM 121, then after autoradiography it was stripped and reprobed with a pallida ⁇ pal) cDNA clone pJAM 225, provided by C. Martin.
  • the pal gene encodes the enzyme dihydroflavonol reductase involved in anthocyanin biosynthesis, and is known to be regulated by del (Almeida et al, Genes Dev. , 3 , 1758-1767 (1989) ) .
  • RNA from wild-type flower buds (1-5 nodes above first fully open flower) dissected into three parts: the base of corolla tubes (BT), the rest of the tube (RT), and the lobes (L) as indicated above the autoradiogram, probed with the del cDNA clone pJAM 121.
  • Each lane of (b), (c) and (d) contains 10 ⁇ g of RNA, and loading appeared equal when ribosomal bands were viewed under UV illumination after staining with ethidium bromide.
  • RNA extraction and Northern analysis was carried out as described (in Coen et al., ibid) .
  • Northern blots were stripped by washing in 0.5% SDS, 0.01 % SSC for 30 minutes at 80°C.
  • Nucleotide and predicted amino acid sequence of del cDNAs is shown in Figure 1 and SEQ ID NO 1.
  • the predicted amino acid sequence of del is also shown in SEQ ID NO 2.
  • the solid triangle indicates the position of an intron within which Tam2 is inserted in the del-602 allele, namely between bases 573 and 574.
  • the region of the DEL protein with similarity to the helix-loop-helix domain of the myc family of transcription factors is underlined, it commences at residue 439 and terminates at residue 493. Residues conserved in DEL and the maize R-S gene product (after alignment) are shaded.
  • Figure 5 and SEQ ID NO's 3 to 12 show amino acid sequence comparison of DEL protein with selected HLH proteins. Alignments were made to maximise homology within the HLH domain.
  • the positions of the conserved basic region, putative amphipathic helices I and II, and the loop are shown above (Murre et al, Cell , 56, 577-783 (1989)).
  • the sequences shown are for: maize R-S
  • cDNA was synthesised from 3 ⁇ gpolyA + RNA extracted from wild-type flower buds (1-4 nodes above first fully opened flower), and cloned into the EcoRI site of ⁇ NM1149 using Amersham kits.
  • a library of 10 5 plaques was screened with probe A of pJAM 602 ( Figure 2b) and the longest clone obtained was subcloned into Bluescript SK + (Stratagene). Sequence analysis revealed that the cDNA insert lacked a poly A tail but contained a long open reading frame which terminated at an EcoRI site, suggesting that the cDNA had been cleaved at an internal site during the cloning.
  • Figure 6 shows in situ hybridisation of medial longitudinal sections of corollas with 35 S labelled RNA probes.
  • Tomato Approximately 100 tomato seed (Lycopersicon esculentum variety Money Maker) were surface sterilised using a 10% aqueous solution of bleach and sown on agar media under sterile conditions (day 0). The seeds were allowed to germinate and grow for 10 days (day 10).
  • plasmid pAL4404 Hoekema et al, Nature , 303, 179-180 (1983)
  • the plasmids can be seen in Figure 7 and are derived from the plasmid pSLJ456 (derived by J. D. Jones & C. Dean et al from pRK290 J. D. Jones & C. Dean et al P.N.A.S., 77, 7347, (1980)).
  • pBJIMM15 carries the cDNA of the del gene of Antirrhinum majus under control of the cauliflower mosaic virus 35S promoter and is terminated by the OCS gene 3' terminator sequence
  • pBJIMM21 carries a copy of the del gene derived from a genomic fragment again driven by the 35S promoter
  • pBJIMM24 carries a large genomic fragment containing both the promoter and coding sequence of del .
  • These three plasmids all also carry the NPT gene that confers kanamycin resistance and this gene is driven by the 2'1' promoter (Velten et al.
  • the tissue explants were placed on selective agar plates bearing appropriate antibiotics to kill any remaining Agrobacterium and allow only "transformed” plant tissue to regenerate.
  • the period between days 11 and 13 when the Agrobacterium is co-cultivated with the tomato tissue is to allow time for the Agrobacterium to transfer the portion of DNA from the pBJIMM plasmids that lie between the points marked "left border” and "right border” in Figure 7 into the genome of the tomato.
  • the NPT gene becomes functional confers kanamycin resistance to the tomato tissue and thereby allowing it to regenerate on agar medium containing kanamycin.
  • Tobacco The tobacco transformation procedure is very similar to that of tomato.
  • the same cultures of Agrobacterium were used, however, plants have only been regenerated from pBJIMMl ⁇ and pBJIMM21 cultures.
  • On day 0 10 ml volumes of Luria broth were inoculated with Agrobacterium cultures as previously described, in this case only cultures bearing pBJIMM15 and 24 have been used.
  • On day 2 several immature leaves of tobacco (Nicotiana tabaccum variety Samson) were harvested from plants growing in the greenhouse. These were then surface sterilized by washing in a 10% solution of bleach for five minutes and then rinsed in sterile water and cut into small tissue explants. The explants were then washed with the Agrobacterium cultures and allowed to co-cultivate for two days (days 3 to 4).
  • the pigment phenotype of the transformed tobacco was visible only in the flowers of the plant.
  • the flower petals and anther filaments of the transformants were much more intensely pigmented than similarly cultivated control plants. On sectioning this pigmentation appeared to be epidermal in the flower and both epidermal and sub- epidermal in the anther filament.
  • the phenotype was only observed in the flower as the shoots, stem and leaves of the transformants were all indistinguishable from the control plant.
  • ORGANISM Antirrhinum majus

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Abstract

L'invention concerne un procédé de régulation de l'expression d'au moins un gène de pigment d'anthocyanine d'une plante consistant à transformer le tissu de la plante avec un vecteur d'expression comprenant un segment d'ADN codant une protéine possédant la séquence d'acide aminé de la protéine DEL comme indiqué dans la description de séquence SEQ ID NO 1 ou 2, ou une protéine possédant une séquence d'acide aminé présentant une sensible homologie avec la protéine DEL comme indiqué dans la description de séquence SEQ ID NO 1 ou 2. Cette protéine est capable de réguler l'expression d'au moins un gène de plante impliqué dans la biosynthèse du pigment, ledit segment d'ADN étant sous le contrôle d'un promoteur en amont de celui-ci et lié à celui-ci et se régénérant à partir des plantes dont le tissu a été transformé et présentant une pigmentation d'anthocyanine modifiée.
EP93901841A 1992-01-09 1993-01-08 Regulation des genes des plantes Withdrawn EP0620855A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US81857092A 1992-01-09 1992-01-09
US818570 1992-01-09
PCT/GB1993/000019 WO1993014211A1 (fr) 1992-01-09 1993-01-08 Regulation des genes des plantes

Publications (1)

Publication Number Publication Date
EP0620855A1 true EP0620855A1 (fr) 1994-10-26

Family

ID=25225851

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93901841A Withdrawn EP0620855A1 (fr) 1992-01-09 1993-01-08 Regulation des genes des plantes

Country Status (4)

Country Link
EP (1) EP0620855A1 (fr)
JP (1) JPH07506000A (fr)
AU (1) AU671272B2 (fr)
WO (1) WO1993014211A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534660A (en) * 1993-04-16 1996-07-09 Dna Plant Technology Corporation Ph genes and their uses
US5646333A (en) * 1994-09-02 1997-07-08 Drexel University Plant promoter useful for directing the expression of foreign proteins to the plant epidermis
US6339185B1 (en) 1994-09-02 2002-01-15 Drexel University Plant termination sequence
JPH0970290A (ja) * 1995-02-17 1997-03-18 Suntory Ltd アシル基転移活性を有する蛋白質をコードする遺伝子
FR2768746B1 (fr) * 1997-09-23 2001-06-08 Agronomique Inst Nat Rech Promoteur specifique des petales et procede d'obtention de plantes a fleurs sans petale
GB9801598D0 (en) 1998-01-26 1998-03-25 Unilever Plc Methods and compositions for modulating flavonoid content
AU777342B2 (en) 1999-03-11 2004-10-14 Arborgen Llc Compositions and methods for the modification of gene transcription
CA2417564A1 (fr) * 2000-07-28 2002-02-07 Agriculture And Agri-Food Canada Nouveaux genes regulateurs jouant un role dans la synthese de tanin condense effectuee par des plantes
WO2002039809A2 (fr) 2000-11-17 2002-05-23 Agriculture And Agri-Food Canada Regulation de l'expression de flavonoides dans l'alfalfa par utilisation de genes de regulation du mais

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6287390A (en) * 1989-08-01 1991-03-11 Pioneer Hi-Bred International, Inc. Transcriptional activators of anthocyanin biosynthesis as visual markers for plant transformation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9314211A1 *

Also Published As

Publication number Publication date
AU3645493A (en) 1993-08-03
AU671272B2 (en) 1996-08-22
WO1993014211A1 (fr) 1993-07-22
JPH07506000A (ja) 1995-07-06

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